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The role of mitochondria in the male reproductive capacity of Caenorhabditis elegans

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The role of mitochondria in the male reproductive capacity of Caenorhabditis elegans

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Content Copyright 2020 Chia-An Yen
The Role of Mitochondria in the Male Reproductive Capacity of Caenorhabditis elegans

by

Chia-An Yen

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
in
MOLECULAR BIOLOGY

August 2020
ii
This work is dedicated to my best friend and husband, Nathan Mih, who has supported me
every step of the way.
iii
Acknowledgements
I would like to thank all the people who have helped me along this journey:
My husband, Nathan Mih, who has always been there for me even though we are not
living in the same city (from UCSD during graduate school, or from Berkeley currently). I am so
grateful to have a significant other who not only shares my love for drum and bass and animal
rescue, but also my interest in biological research.
My P.I. and mentor Dr. Sean Curran, who has been tremendously helpful and
encouraging during my graduate studies. I am very thankful for his invaluable guidance and
positive attitude in tackling scientific research.
My post-baccalaureate research mentors, Dr. Bing Ren and Dr. Danny Leung, who
provided a well-rounded research environment that broadened my perspective in science.
My undergraduate research mentor Dr. Ann Feeney, who helped start my path in
research by giving me the opportunity to intern in her lab without any prior lab experience.
The Curran lab members (past – Hans, Dana, Jackie, Akshat, Ajay, Brett, Wilber – and
present – Amy, Nicole, James, Christian, Osvaldo, and Chatrawee), who contributed to an
engaging and supportive work environment. Dana, who helped jump start my thesis project.
Hans, who just gets my weirdness and jokes (my best pal in lab). Amy and Nicole, for all the
cat-sittings and delicious baked goods.
My committee members, Dr. John Tower, Dr. Christian Pike, and Dr. Carolyn Phillips,
who have provided me with helpful comments and suggestions in research, as well as career
related advice.
My dad, Win-Yon Yen, who has always supported my interest in science and generously
provided me with the opportunity to pursue higher education in the States. My sister and
brother-in-law, Andrea Yen-Duncan and William Duncan, for being so supportive throughout my
graduate studies and hosting the best holiday getaways with the cutest mini aussie Poppy. My
iv
brother, Yu-Hsin Yen, for knowing how to make me laugh. My mom, Su-Mei Tai, for her never-
ending love and patience in raising my siblings and me.
Wilson Tien, for coming to countless DnB shows with Nathan and me (often on short
notice). Ivette Gimenez-Akasaki and Davis Akasaki, for all the fun times we have whether it’s in
LA or SD.
DJ Bryan Gee, for his monthly V Recordings Podcasts that have made late nights in the
lab a lot more fun with his superb selection in drum and bass tunes.
And last, but not least, my cats Lily, Spaghetti, Potato, and Tangerine, for their stress-
relieving (and sometimes stress-inducing hopefully in hormetic doses) furry companionship.

v
Table of Contents
Dedication ................................................................................................................................... ii
Acknowledgements .................................................................................................................... iii
List of Tables ............................................................................................................................ vii
List of Figures .......................................................................................................................... viii
Abstract...................................................................................................................................... ix
Chapter 1. Gene-diet interactions and aging in C. elegans ................................................... 1
C. elegans is a useful organism for studying dietary effects on animal physiology .................. 2
Worm dieting, obesity, and food choice .................................................................................. 3
Gene-diet interactions ............................................................................................................ 6
How are nutrients in diets sensed? ........................................................................................10
The future requires a defined diet ..........................................................................................12
Conclusions and perspectives ...............................................................................................14
Chapter 2. Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male
reproductive advantage in C. elegans .......................................................................................17
Abstract .................................................................................................................................18
Introduction ............................................................................................................................19
Results ..................................................................................................................................21
Discussion .............................................................................................................................32
Methods ................................................................................................................................36
Figures ..................................................................................................................................41
Supplemental ........................................................................................................................50
Acknowledgements ...............................................................................................................61
Chapter 3. Incomplete proline catabolism drives premature sperm aging ............................62
Abstract .................................................................................................................................63
Introduction ............................................................................................................................64
Results ..................................................................................................................................66
Discussion .............................................................................................................................72
Methods ................................................................................................................................75
Figures ..................................................................................................................................79
Supplemental ........................................................................................................................86
Acknowledgements ...............................................................................................................89
Chapter 4. Methods for assessing fertility in C. elegans from a single population ................90
Abstract .................................................................................................................................91
Introduction ............................................................................................................................92
Materials ................................................................................................................................94
Methods ................................................................................................................................97
Notes ................................................................................................................................... 103
Figures ................................................................................................................................ 105
Acknowledgements ............................................................................................................. 109
vi
Chapter 5. Redirection of SKN-1 abates the negative metabolic outcomes of a perceived
pathogen infection ................................................................................................................... 110
Abstract ............................................................................................................................... 111
Significance statement......................................................................................................... 111
Introduction .......................................................................................................................... 112
Results ................................................................................................................................ 113
Discussion ........................................................................................................................... 121
Methods .............................................................................................................................. 125
Figures ................................................................................................................................ 127
Supplemental ...................................................................................................................... 136
Acknowledgements ............................................................................................................. 147
References ............................................................................................................................. 148

vii
List of Tables
Table 1.1. Diet-gene pair induced physiological changes in C. elegans. ...................................16
Table 2.1. Key resources. .........................................................................................................37

viii
List of Figures
Figure 2.1. alh-6 fertility defects are sperm-specific. .................................................................41
Figure 2.2. alh-6 males have sperm defects on both OP50 and HT115 diets. ...........................42
Figure 2.3. Transcriptional patterns define developmental- and adult-specific consequences to
loss of alh-6 activity. ..................................................................................................................43
Figure 2.4. Loss of FAD homeostasis in alh-6 mutants leads to sperm dysfunction. .................44
Figure 2.5. Mitochondrial dynamics drive sperm quality. ...........................................................45
Figure 2.6. alh-6 and FAD function cell autonomously in the germline to regulate sperm function.
.................................................................................................................................................47
Figure 2.7. Model of alh-6 and FAD mediated male reproductive senescence. .........................49
Figure 3.1. prdh-1 mutation suppresses activation of SKN-1 and proline metabolism
deregulation in older alh-6 animals. ...........................................................................................79
Figure 3.2. prdh-1 activity is required for sperm-specific fertility defects in alh-6 mutants. .........81
Figure 3.3. Endogenous ROS drives sperm defects in alh-6 mutants. .......................................82
Figure 3.4. Increasing ETC activity through malate/fumarate supplementation alters sperm
function. ....................................................................................................................................83
Figure 3.5. Endogenous ROS drives changes in mitochondria dynamics leading to sperm
defects. .....................................................................................................................................84
Figure 3.6. alh-6 mutation accelerates male reproductive senescence. ....................................85
Figure 4.1. Reproductive output time course for wild type N2 hermaphrodites ........................ 105
Figure 4.2. Average brood size of wild type N2 hermaphrodites. ............................................. 106
Figure 4.3. Spermatids released from dissected wild type N2 males. ...................................... 107
Figure 4.4. Pseudopods are visible in Pronase activated spermatozoa released from dissected
wild type N2 males. ................................................................................................................. 108
Figure 5.1. SKN-1 activation causes redistribution of somatic lipids and activation of immune
defense genes. ....................................................................................................................... 127
Figure 5.2. Loss of Histone H3 trimethylation restricts SKN-1gf transcriptional activity and
suppresses the loss of somatic lipids. ..................................................................................... 128
Figure 5.3. Oxidative stress redirects SKN-1gf transcriptional activity while restoring somatic
lipid distribution. ...................................................................................................................... 129
Figure 5.4. Exposure to pathogens drives a rapid and SKN-1-dependent loss of somatic lipids.
............................................................................................................................................... 130
Figure 5.5. Redirection of SKN-1gf negates pathogen resistance. .......................................... 132
Figure 5.6. SKN-1 activation drives lipid utilization during Asdf. .............................................. 134
Figure 5.7. Transcriptional redirection of SKN-1 activity mitigates pleiotropic outcomes. ......... 135

ix
Abstract
Diet is the most variable aspect of life history, as most individuals have a large diversity
of food choices, varying in the type and amount that they ingest. In the short-term, diet can
affect metabolism and energy levels. However, in the long run, the net deficiency or excess of
calories from diet can influence the progression and severity of age-related diseases. An old
and yet still debated question is: how do specific dietary choices impact health- and lifespan? It
is clear that genetics can play a critical role — perhaps just as important as diet choices. For
example, poor diet in combination with genetic susceptibility can lead to metabolic disorders,
such as obesity and type 2 diabetes. Recent work in Caenorhabditis elegans has identified the
existence of diet-gene pairs, where the consequence of mutating a specific gene is only realized
on specific diets. Many core metabolic pathways are conserved from worm to human. Although
only a handful of these diet-gene pairs has been characterized, there are potentially hundreds, if
not thousands, of such interactions, which may explain the variability in the rates of aging in
humans and the incidence and severity of age-related diseases.

1
Chapter 1.
Gene-diet interactions and aging in C. elegans
*This chapter is a version of a published manuscript at Experimental Gerontology.
Chia-An Yen
1
and Sean P. Curran
1,2,3
*
1. University of Southern California, Dornsife College of Letters, Arts, and Science, Department of
Molecular and Computational Biology
2. University of Southern California, Davis School of Gerontology
3. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California,
Los Angeles, CA 90089
*Correspondence to: spcurran@usc.edu
Keywords: Diet-gene pairs, C. elegans, Metabolism, E. coli
2
C. elegans is a useful organism for studying dietary effects on animal
physiology
Whether to fulfill biosynthetic deficiencies or to fuel growth and essential cellular
functions, food is essential for all organisms. Although food is universally indispensable, types of
diets are never universally equal. Despite the lack of a defined diet, recent work
in Caenorhabditis elegans is still able to demonstrate the role diet plays on organismal
physiology, as seen in studies uncovering several diet-dependent effects [1-8]. Diet can exert
immediate as well as long lasting effects on animal physiology, and remarkably, diet can also
influence the physiology of future generations [9-12]. But what can studies of C. elegans diet tell
us about human metabolism? C. elegans is an attractive model for studying metabolism and
aging for many reasons. First, it is cultured in laboratory settings using bacterial species, such
as Escherichia coli, making it easy to study the effects of different diets through the use of
different species/strains of bacteria as its food source. Second, it is characterized by short
developmental and reproductive periods and a lifespan that averages three weeks, facilitating
the rapid identification of effects that diet plays on these traits [13, 14]. Third, despite their small
1 mm size, worms are complex multicellular organisms, whose transparency allows facile visual
investigation of systems-level changes with age under different experimental conditions, such
as diet. Fourth, genetic, RNA interference (RNAi), and chemical screening approaches are well-
established in this system, facilitating fast discovery with these high-throughput approaches [15-
20]. Lastly, and perhaps most importantly, the C. elegans genome shares 60-80% of all human
genes [21-30], and many of the core metabolic pathways in humans are conserved in C.
elegans, including: the AMPK pathway [31], the TOR pathway [32-34], SREBP regulator of lipid
homeostasis [35], insulin/IGF-1 (IIS) signaling pathway [36-39], nuclear hormone receptors that
mediate energy homeostasis [40], etc. For all these reasons, C. elegans is an excellent model
for studying dietary effects on animal physiology.
3
Worm dieting, obesity, and food choice
A significant body of work exists that document how dietary restriction leads to increased
life- and healthspan across many species, including C. elegans [41-56]. However, our
understanding of how ad libitum fed – the standard dietary regimen – diets impact basal
metabolism requires further study. In addition, an examination of diets that would be classified
as “unhealthy” in the context of those basal metabolic measurements is needed to allow better
comprehension of their physiological effects on an animal and how the animal responds to diets
of varying quality.
In contrast to worms in the wild, where food of high nutritional value may not always be
present, humans living in developed countries are presented with easy and unhindered access
to food that are often highly processed and whose composition is high in fats and carbohydrates
[57]. Excess ingestion of these diets can lead to obesity [1–4]. In fact, obesity has become a
worldwide epidemic [58, 59]. Body mass index (BMI) is a measurement used to estimate body
fat content (calculated by dividing weight in kg by height in m
2
). In a report that took data from
900,000 participants from 57 different studies, it was shown that an increase in BMI is
associated with higher risks of cardiovascular diseases, high blood pressure, type 2 diabetes,
and other undesirable ailments [60]. However, the negative effects on health from increased
BMI are controversial as some studies have reported that higher BMI can be beneficial [61-64].
As such, a clearer understanding of disease risk associated with obesity combined with
information on diet is needed to better elucidate mechanisms of how organisms adapt to diets of
varying nutritional value.
How does the C. elegans diet in the wild compare to that in the lab? C. elegans are free-
living nematodes that were first isolated in soil and compost [65]. To date, C. elegans have been
found worldwide, primarily in humid temperate areas that include farmlands, woods, and
decomposing fruits and stems [66-68]. In the wild, C. elegans feeds on various soil bacteria
4
species including Bacillus megaterium, Pseudomonas medocina, Comomonas sp., other
various bacterial species, fungi, and as well as yeast, most likely as a source of cholesterol [69-
72]. When Sydney Brenner isolated C. elegans for study in the laboratory, he selected the uracil
auxotroph E. coli B strain OP50 as the laboratory diet to limit the bacterial growth on plates,
making it easier for microscopic analysis [13, 14]. Like all model organisms, a worm's dietary
composition in the wild is quite different than that in the laboratory. A worm's diet in the wild can
be a mixture of many different bacterial species, while in laboratory its diet is usually a selected
bacterial species/strain. In light of this, the use of multiple bacterial diets in studies can assist
with the realization of previously unidentified genetic pathways perhaps masked by using the
standard OP50 diet.
Even in the laboratory, diverse microbial diets have been found to have varying effects
on the rate of development and reproduction when compared to the standard OP50 diet [1].
Coonlon et al. demonstrated that different bacterial species found in soil, which are able to be
cultured in the lab and fed to worms, have divergent impacts on physiology. Their study
identified a total of 372 genes that are differentially expressed in C. elegans when exposed to
the standard laboratory diet E. coli OP50 versus the three bacterial species that they isolated
from the Konza prairie grasslands: Micrococcus luteus, B. megaterium,
and Pseudomonas species (most identical to P. fluorescens). Among these identified genes,
many are involved in metabolism, including: acdh-1 which encodes for acyl-CoA dehydrogenase
that catalyzes first step of fatty acid beta-oxidation, elo-5 which encodes for polyunsaturated
fatty acid elongase in the fatty acid biosynthesis pathway, fat-2 which encodes for delta12-
desaturase in the polyunsaturated fatty acid synthesis pathway, gei-7 which encodes for a
isocitrate lyase/malate synthase that functions in the glycoxylate cycle used in the production of
glucose from fatty acids, and cyp-37A1 which encodes for cytochrome P450 [73]. Therefore, the
bacterial diet a worm eats can elicit a metabolic response that changes according to the
available nutrients in that diet.
5
The biological basis for the changes in development rate, fitness, and lifespan that result
from the feeding of different microbes is likely complex, due to the fact that they may differ in
nutritional content and produce different metabolites that can be taken up by the worm. As such,
recent work has focused on a handful of E. coli strains that are commonly cultured and used
for C. elegans research. These include E. coli B strain OP50, E. coli K12 strain HT115, and E.
coli B and K12 hybrid strain HB101. These three diets have relatively similar overall protein and
fat content, but HB101 and HT115 both contain a much higher level of carbohydrate when
compared to OP50 [74]. Worms fed the OP50 diet show differential fat storage when compared
to HB101 and HT115. Remarkably, feeding these diets has minimal impact on lifespan [74].
This demonstrates that ingesting different bacterial diets can induce changes in a worm's
metabolic pathways to maintain homeostasis and is an example of metabolic adaptation — the
capacity to effectively utilize various diets to fuel cellular and organismal functions.
Previous studies on the effect of consumption of different bacterial species on C.
elegans have emphasized the idea that the calories are dissimilar between different diets and
that different diets can affect fat metabolism, development, fertility, and lifespan of an animal [1-
8, 73]. The measurement of food intake is itself varied among studies. Classically, pharyngeal
pumping has been used as a surrogate to estimate how much food is being ingested but recent
studies using fluorescently labeled bacteria provide a more accurate measure [75-77]. When
coupled with bacterial clearance assays [78], these two measures of “ins and outs” provide a
nice correlation of food consumption. Recently, mass-spectrometry based methods to measure
food consumption allow for higher resolution identification of ingested material [78, 79]. These
more recent measures are of particular importance as they could be adapted to quantify the
differences between ingestion of bacterial species, which can contain varied amounts of
macronutrients that are important for generating energy for cellular processes, growth, and
reproduction. In a study by Brooks et al., they found that worms fed OP50 or DA837, an OP50
derived strain, have a much higher amount of triacylglycerol (TAG) when compared to worms
6
fed HB101 or HT115. Correspondingly, these worms also have larger and more intensely
stained lipid droplets than those fed HB101 or HT115, as indicated by fixed Nile Red staining.
Therefore, the fatty acid composition of the worm reflects the levels, and likely, the type of fatty
acids in the diet.
Interestingly, C. elegans display behaviors that indicate a preference for bacterial diets
that contribute to better fitness, as measured through growth, reproduction, and lifespan [1, 73,
80]. When presented with a choice between two different E. coli strains, HB101 and DA837,
worms consistently choose the “higher quality” food HB101, which better supports growth [1,
69]. Furthermore, worms that are previously fed a higher quality diet display a strong tendency
to leave poorer diet more readily, in search of a better choice [1]. Along these lines, it was also
discovered that C. elegans exhibit a dietary choice behavior that drives them to seek out the
bacterial food source that confers the highest fitness, as measured by age-specific
development, fecundity, and lifespan [73]. Taken together, these studies reveal that, similar to
mammals, when given the choice, worms will seek out higher quality foods to support future life
history events. The evolutionary pressures underlying these behaviors are clear, but the
molecular mechanisms that drive these choices have yet to be uncovered, but will be of
significant interest.
Gene-diet interactions
It is clear that diet and genetics play important roles in the regulation of metabolism,
healthspan and lifespan [81]. However, our understanding of these interactions is limited by
studies that only query single gene mutations on one particular diet. For example, dietary
restriction (DR) is one of the most effective and well-studied environmental manipulations that
can influence the rate of aging and healthspan. The genetics underlying the effects of dietary
restriction are multifaceted [82] but several genetic loci have been identified that are central to
the response. In C. elegans, the benefits of dietary restriction require at least two transcription
7
factors: the cytoprotective cap'n'collar transcription factor SKN-1 [56, 77] and the FoxA
transcription factor PHA-4 [55]. Early work suggested that the FoxO transcription factor DAF-16
was not required for the longevity response to dietary restriction [82]. However, it was later
found that DAF-16 and AMPK are key regulators of the DR response under specific methods of
nutrient limitation [52]. As such, it is clear that genetics play an important role in the DR
response.
While the above mentioned examples define a role for specific genes in regulating
behaviors tied to the amount of food ingested, recent studies have uncovered genes that are
specific to also the type of diet ingested [6, 7, 83, 84]. These diet-gene pair interactions
emphasize the complexity of this system. Soukas et al. found that rict-1, a component of the
Target of Rapamycin complex 2 (TORC2) influences fat metabolism and lifespan in a diet-
dependent manner [2]. When fed HB101 or HT115, rict-1 mutant worms are leaner than those
fed OP50. This was surprising because HB101 and HT115 have similar overall levels of protein
and fat as OP50, while their carbohydrate levels are 3–5 times higher than that of OP50 [74].
Perhaps rict-1 mutant animals sense the three diets differently, altering their feeding behavior.
Further investigation revealed that rict-1 mutant animals spend less time on the HB101 diet
when compared to animals fed the OP50 bacteria; these mutant animals actually eat less when
fed HB101. Therefore, rict-1 seems to play a role in regulating feeding behavior when an animal
encounters diets of different qualities. This further demonstrates the importance of diet quality
and how new roles for genes can be discovered through the usage of different bacterial diets. In
addition, when compared to the wildtype animals, rict-1 mutant animals also show shortened
lifespan when fed OP50, but are long lived on HB101 [2]. This seems to be mediated through
dietary restriction since a rict-1; skn-1 double mutant is no longer long lived on HB101, and skn-
1 is known to be required for dietary restriction [56]. Soukas et al. also found that insulin
signaling through akt-1 and daf-2 is required for the shortened lifespan on OP50 because
double mutants rict-1; akt-1 and rict-1; daf-2 are no longer short-lived. Additionally, a rict-1; daf-
8
16 double mutant has an even shorter lifespan than either single mutant, indicating that insulin
signaling appears to contribute to rict-1 regulation of lifespan [2]. This study shows that the way
an animal senses food and the specific downstream signaling pathways can be important for
regulating the animal's adaptive capacity to different diets. However, we still do not know how
this signaling occurs and the nature of the specific signals from variable diets that cause rict-
1 mutant animals to have altered fat metabolism, feeding behavior, and lifespan.
Typically, researchers focus on glucose and lipids supplied by diet. However, recent
research has now added dietary amino acids to the list of regulators of animal lifespan [6-8, 85-
87]. For example, the role of the gene alh-6, a mitochondrial proline metabolism gene that
encodes for 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), in regulating lifespan on
different diets was recently discovered [6, 7]. P5CDH is a mitochondrial enzyme that is needed
to catalyze P5C to glutamate. Interestingly, alh-6 mutants were found to age prematurely when
fed the OP50 strain of E. coli, but not HT115; a direct consequence of impaired mitochondrial
function and organelle collapse [6, 7]. This represents a more recently defined diet-gene pair.
Furthermore, the reduced lifespan phenotype only occurs when worms are exposed to the
OP50 diet during the developmental period between larval stage 3 and larval stage 4 and
requires exposure throughout adulthood, suggesting that there may be critical stages in life
where diet plays a more prominent role in aging. alh-6 mutants exhibit a shortened lifespan
when proline was supplemented to the HT115 diet, but do not display a further reduced lifespan
when proline was added to the OP50 diet; this implies that the accelerated aging was caused by
the activation of the proline catabolism pathway and the accumulation of P5C, resulting in an
increase in ROS (reactive oxygen species) and altered mitochondrial morphology [6, 7]. This
suggests that specific diets have differential capacity to induce changes in organelle
morphology as we age.
Another example of a specific genetic response induced by the type of diet is found in
the tissue distribution of lipids in aged SKN-1 gain-of-function mutants (SKN-1 gf) when fed
9
specific diets. When fed the OP50 diet, SKN-1 gf animals display a lipid depletion phenotype at
the end of their reproductive period where their somatic lipid stores are mobilized to and
retained in the germline, also called age-dependent somatic depletion of fat (Asdf). Conversely,
this phenotype is not observed when the animals are fed an HT115 diet [5]. Similarly, alh-
6 mutant animals fed OP50 deplete fat rapidly through the activation of SKN-1 during acute
starvation, while those fed HT115 do not [6, 7]. Together, these studies show how genes
involved in lipid metabolism can elicit a diet-dependent physiological response in an animal.
While the above examples emphasize overall dietary intake, small changes in diet, such
as its micronutrient content, can also impact an organism. Micronutrients are vitamins and trace
elements that are used as metabolites or cofactors in metabolic pathways, which can ultimately
affect the health of an organism. Recent work done by MacNeil et al., has shown that C.
elegans develops faster while consuming a diet of Comamonas DA1877 compared to E.
coli OP50, even though the two bacterial species have similar macronutrient levels. In addition,
by just adding a small amount of Comamonas DA1877 into E. coli OP50, they were able to
recapitulate the accelerated development, suggesting that there is some signaling molecule
from the Comamonas diet that induced this change. This was later identified as vitamin B12 [4,
5]. This shows that it is important to consider the entire nutritional content of a diet, rather than
just looking at calories and macronutrient levels.
A greater appreciation and acknowledgement for the type of food sources used has
emerged in the design of experiments to help uncover genetic pathways that modulate aging
through dietary effects. This is the most prominent in feeding RNAi assays. The development of
feeding RNAi libraries in bacteria has resulted in copious usage of this tool to perform large-
scale genetic screens. Traditionally, RNAi in worms is routinely performed by feeding the E.
coli K-12 strain HT115 because they are deficient in the dsRNA specific endonuclease RNAse
III [88-90]. However, usage of the HT115 bacteria as the food source raises the question of
whether phenotypic screens, which were previously thought to be saturated, are truly penetrant.
10
Recently, the development of an OP50 strain, engineered for delivering RNAi [91], has proven
to reveal new results in experiments previously performed with the HT115 RNAi strain. A push
for using RNAi in both OP50 and HT115 background in laboratories is needed to fully flush out
the genetic pathways that underlie diet-dependent regulation of development, reproduction,
metabolism, and aging.
How are nutrients in diets sensed?
Dietary composition affects animal physiology, but the signaling pathways that are
involved in helping an animal adapt to various diets are unclear. Since diets of variable quality
can induce gene expression changes in the metabolic network, thus affecting the physiology
state and life history traits of C. elegans, worms must have a way to sense and respond to these
dietary cues. The physiological responses initiated at olfaction, and the smell of different
bacteria can impact physiological outputs including behavior and reproduction [92-94]. Worms
sense environmental stimuli through their sensory neurons, which contain specialized receptors
that recognize various types of cues, including mechanical, thermal, gustatory, and olfactory
stimuli [95]. Worms use their chemosensory amphid neurons to detect the volatile and water-
soluble compounds metabolized and produced by E. coli and exhibit either attractive or repellant
chemotaxis [96-99]. Once food is ingested, glucose levels rise, which in turn stimulates the
release of insulin-like peptides, resulting in the increased uptake of glucose by cells. Insulin
signaling is needed in both the nervous system and intestine for proper development,
reproduction, and lifespan [36, 39, 100, 101]. The insulin/IGF-1 signaling pathway was the first
longevity pathway to be discovered in C. elegans [36, 100]. Reduced expression of the DAF-
2/IGF-1 receptor or the direct downstream kinase AGE-1/PI3K inactivates the downstream
kinase signaling cascade, which results in the transcription factor DAF-16/FOXO translocating
into the nucleus to turn on its target genes, ultimately extending the lifespan of the animal [36,
11
38, 102-104]. Furthermore, mutations in the other three downstream targets of DAF-2 also
cause extension of lifespan: AKT-1, PDK-1, and SGK-1 [102, 105-108].
Although neuroendocrine signaling pathways are important for food-seeking behaviors
and for satiety [39, 97, 101, 102, 109-111], their roles in lipid storage and mobilization are less
clear. To test the role of these pathways in lipid metabolism, Watts and colleagues measured
lipid levels in the daf-2 insulin receptor mutants and daf-7/TGF- β mutants fed HB101 or OP50
diet. Like wild type animals, these mutants contained lower levels of stored fat on HB101 when
compared to on OP50. As such, the insulin/IGF and TGFβ signaling pathways do not seem to
be directly involved in the diet-induced changes in fat storage in worms. This study
identified pept-1, a gene that encodes an intestinal peptide transporter, to be essential for the
differential fat storage in worms fed different diets [74]. The signaling events following exposure
to different diets are sensed by pept-1 and result in the downstream changes in fat metabolism
and reproductive output. This study shows that some nutritional cues can be sensed directly by
the intestine. Further assessment of the roles that specific tissues, or even cells, play in
mediating diet-dependent behaviors will be of critical importance.
Recent work from Maier et al. identified nmur-1, a mammalian homolog of the
neuromedin U receptor, to be involved in the sensing of dietary cues from different food types
and the regulation of lifespan through the neurosensory system in a diet-dependent
manner. nmur-1 mutants live longer on OP50, but not on HT115 — another example of a diet-
gene pair interaction [3]. Remarkably, nmur-1 is also required for alh-6 in regulating lifespan in
a food-dependent manner [6, 7]. However, we do not currently know the downstream targets
of nmur-1 or its connection to the alh-6 pathway that allows the animal to appropriately respond
to OP50 versus HT115. It will be interesting to see if there are other neuropeptide signaling
pathways involved in the processing of different bacterial diet signals.
Nuclear hormone receptors (NHRs) are ligand-activated transcription factors that sense
environmental signals and regulate many fundamental physiological processes, including
12
metabolism, development, and reproduction [112-114]. Humans have 48 of these NHRs,
while C. elegans have an astounding number of 271 NHRs, many of which are homologs to
human NHRs but have yet to be characterized [114, 115]. Perhaps some of these NHRs are
involved in sensing certain compounds from the bacteria and activating a downstream
physiological response. The use of multiple bacterial diets in all studies will help us elucidate the
mechanisms underlying these complex diet-gene response pathways.
The future requires a defined diet
Although convenient, the use of a living organism, such as OP50, as a food source
presents additional complications when trying to define diet-gene interactions. The metabolic
status of the microbes and their pathogenicity [21] to the host are both key determinates of
host–microbe interactions that impact health and lifespan. Previous attempts to synthesize a
defined chemical diet for C. elegans have been unsuccessful for lifespan studies [41, 116-118].
Formulation of bacteria-free culture systems has always impacted developmental timing,
growth, metabolism and reproduction [41, 116, 118, 119]. As each of these biological processes
has profound impacts on lifespan, the utility of these abiotic growth systems for aging is
confounded.
In order to decipher the effects of individual macro- or micronutrient on an animal's
physiology, the development of a defined diet that is composed of known concentration of each
nutritional component is needed. The development of defined diets has helped other fields in
investigating the role of dietary components in aging. For example, a study done by Lee et al.
in Drosophila melanogaster has found that a protein-to-carbohydrate ratio of 1:16 resulted in the
flies living longer, while a ratio of 1:4 maximizes reproduction [120]. Similarly, another study
done in mice demonstrated that different ratios of macronutrients have opposing effects on
lifespan and reproduction [121-123]. Additional studies using a defined diet in flies revealed how
different concentrations of glucose and methionine can affect lifespan [124]. Moreover, recent
13
development of an entirely holidic diet for D. melanogaster resolves the prior inconsistencies in
experiments between laboratories due to the use of oligidic diet, and also allows the
manipulation of individual dietary component in nutritional studies while maintaining similar
lifespan and fecundity as before with a minimal effect on developmental rate [125]. With a
defined diet, the complicated diet-fitness response in an animal can be unraveled using
methods such as a “Geometric Framework” that focuses on first understanding the target
nutrient intake of the specific organism, changing intake ratios so as to observe how the
organism adjusts, and finally, mapping this information to organism fitness [126, 127].
Without a defined diet, the effects from any individual dietary component cannot be
separated. This problem is not limited to C. elegans; an example of the disadvantage of not
having a defined diet is highlighted in the conflicting findings between the two 20-year studies of
dietary restriction (DR) in rhesus monkeys by National Institute on Aging (NIA) and Wisconsin
National Primate Research Center (WNPRC). The study done by NIA had concluded that 30%
DR did not extend the lifespan of these animals [128], while the WNPRC study found that 30%
DR significantly increases their lifespan [129]. The problem with comparing the results of these
two studies is that the dietary composition of the food they fed the animals is very different. The
composition of the diet in the NIA study in many aspects is overall more nutritious than that of
WNPRC. NIA study's diet contains 3.9% sucrose while the WNPRC's diet contains 28.5%. The
sources of protein for the NIA study include wheat, corn, soybean, fish, and alfalfa meal, while
the WNPRC sole protein source was lactalbumin. The NIA study's diet has fat derived from soy,
wheat, corn, and fish, whereas the WNPRC study's diet contains fat from corn oil [128, 129].
Therefore, in order to better understand how a specific amount of a nutrient in a diet can affect
our physiology and even the rate we age, a chemically defined diet is needed for metabolism-
and diet-centric studies.
For worms, defining a diet is difficult because its food source, E. coli and other soil
bacteria, are living organisms, some of which are also pathogenic [130, 131]. For instance, C.
14
elegans live longer on UV-killed or non-proliferating version of the standard OP50 diet [132,
133]. Several groups have attempted to generate a bacteria-free chemically defined diet, but
most are exceptionally complicated to make and as previously stated, impact several life history
events and phenotypes [117, 134]. In addition, there is emerging evidence that the host-microbe
relationship can be symbiotic, where certain metabolites produced by the bacteria – like nitric
oxide – can be utilized by worms, and have significant impact on life- and healthspan [135]. One
study demonstrated that folate can negatively regulate the lifespan of C. elegans. This was due
to a mutation in aroD gene of the bacterial food source, which resulted in decreased folate
availability for the worms; when fed this diet, worms lived longer [136]. However, whether the
effect of decreased folate synthesis on extending lifespan is a result that acts directly on the
worm or through the bacteria remains to be determined. As such, while a defined diet will
remove the pathogenic effect of bacteria on C. elegans, further study is still needed to fully
appreciate the totality of the relationship bacteria have with their worm hosts.
Conclusions and perspectives
Although much work has been done in identifying the metabolic signaling pathways in C.
elegans and in characterizing the effects of different bacterial diets on its life history traits, the
molecular mechanisms behind diet-induced genetic responses in an organism and the
relationship between diet and lifespan remains a complicated problem to solve. Furthermore,
modernization of society entails the mass production of highly processed foods that are often
high in caloric content but low in nutritional value and the global trend of increasing incidences
of obesity; this makes understanding the interplay between dietary composition and metabolic
pathways more important than ever. Having a defined diet in laboratory studies will be helpful in
dissecting the specific molecules that contribute to aging and healthspan, but there are many
hurdles to overcome before this can be accomplished in C. elegans, since worms currently
require a “live diet” to provide important metabolites [117]. Despite what still remains to be
15
overcome, C. elegans continues to be a useful model organism for diet and aging studies for
many reasons, including its genetic homology to humans, genetic tractability, as well as the
ability to conduct large-scale experiments in a short amount of time. Complex modeling
techniques, such as those found in the field of systems biology, may help uncover complicated
genetic interactions between different pathways and differentiate a dietary response from a
pathogenic response in C. elegans to its bacterial diet, as shown in recent work by Watson et al.
[137]. Work in C. elegans isolated from the wild may help contribute to identification of new diet-
gene pairs masked by using the traditional wildtype "N2" strain that has been maintained on the
standard OP50 diet for a long time. Continuing work in dietary effects on the overall health and
aging of an organism will undoubtedly contribute to a future where diet can be more
personalized, incorporating the genetic makeup of an individual to promote better aging.
16
Gene Description Diet Physiological Impact
rict-1 [2] Component of the Target of
Rapamycin Complex 2 (TORC2)
OP50 Increased fat, shortened lifespan
HT115 Less fat, lengthened lifespan
HB101 Less fat, lengthened lifespan
alh-6 [6, 7] Proline metabolism gene: 1-
pyrroline-5-carboxylate
dehydrogenase (P5CDH)
OP50
Shortened lifespan, rapid lipid depleteion
under acute starvation
HT115
Normal lifespan, WT level of lipid
depletion under acute starvation
pept-1 [71] Intestinal peptide transporter
OP50 Normal brood size
HB101 Reduced brood size
nmur-1 [3] Mammalian homolog of the
neuromedin U receptor
OP50 Longer lifespan
HT115 Normal lifespan
skn-1 [138] Transcription factor orthologous to
mammalian Nuclear factor-
erythroid-related factor (NRF)
OP50 Asdf (+)
HT115 Asdf (-)
Table 1.1. Diet-gene pair induced physiological changes in C. elegans.
17
Chapter 2.
Loss of flavin adenine dinucleotide (FAD) impairs sperm
function and male reproductive advantage in C. elegans
*This chapter is a version of a published manuscript at eLife.
Chia-An Yen,
1,2
, Dana L. Ruter
1,2
, Christian D. Turner,
1,2
, Shanshan Pang
3
, and Sean P. Curran
1,2,4
*
1. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089
2. Department of Molecular and Computation Biology, Dornsife College of Letters, Arts, and Sciences,
University of Southern California, Los Angeles, CA 90089
3. School of Life Sciences, Chongqing University, Chongqing 401331, China.
4. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California,
Los Angeles, CA 90089
*Correspondence to: spcurran@usc.edu
Keywords: spermatogenesis, mitochondria, germ cells, reproduction, proline catabolism, alh-
6/ALDH4A1, C. elegans, senescence, aging, male-specific, FAD, flavin cofactor, riboflavin
18
Abstract
Exposure to environmental stress is clinically established to influence male reproductive
health, but the impact of normal cellular metabolism on sperm quality is less well-defined. Here
we show that impaired mitochondrial proline catabolism, reduces energy-storing flavin adenine
dinucleotide (FAD) levels, alters mitochondrial dynamics toward fusion, and leads to age-related
loss of sperm quality (size and activity), which diminishes competitive fitness of the
animal. Loss of the 1-pyrroline-5-carboxylate dehydrogenase enzyme alh-6 that catalyzes the
second step in mitochondrial proline catabolism leads to premature male reproductive
senescence. Reducing the expression of the proline catabolism enzyme alh-6 or FAD
biosynthesis pathway genes in the germline is sufficient to recapitulate the sperm-related
phenotypes observed in alh-6 loss-of-function mutants. These sperm-specific defects are
suppressed by feeding diets that restore FAD levels. Our results define a cell autonomous role
for mitochondrial proline catabolism and FAD homeostasis on sperm function and specify
strategies to pharmacologically reverse these defects.
19
Introduction
As individuals wait longer to have families, reproductive senescence has become an
increasingly prudent topic [139, 140]. Decline in oocyte quality is well-documented with age and
can result in fertility issues when older couples try to conceive [141]. Furthermore, pregnancies
at an older age pose risks for higher incidences of birth defects and miscarriages. In humans,
female reproduction ceases at an average age of 41-60, with the onset of menopause [142].
The Caenorhabditis elegans "wild type" is hermaphroditic and self-fertilizing; however, they are
capable of making and maintaining Mendelian ratios of male (sperm-only) animals in their
populations. Like humans, C. elegans experience a decline in fecundity with age by halting
oocyte production at roughly one-third of their lifespan [143]. In addition, regulators of
reproductive aging, such as insulin/IGF-1 and sma-2/TGF-  signaling, are conserved regulators
of reproductive aging from worms to humans [144]. While the majority of studies in reproductive
senescence have focused on maternal effects, male factors contribute to a large portion of
fertility complications with increasing evidence of an inverse relationship between paternal age
and sperm health [140]. In fact, studies in mammals have shown an age-related decline in
sperm quality with increased incidences of DNA damage, reduced motility, abnormal
morphology, and decreased semen volume [145-147].
Flavin adenine dinucleotide (FAD) is an important cofactor that participates in enzymatic
redox reactions that are used in cellular metabolism and homeostasis. FAD is synthesized from
riboflavin by the concerted actions of FAD synthetase and riboflavin kinase. Like humans, C.
elegans cannot synthesize riboflavin, and therefore requires dietary intake [148]. Disruption of
flavin homeostasis in humans and animal models has been associated with several diseases,
including: cardiovascular diseases, cancer, anemia, abnormal fetal development, and
neuromuscular and neurological disorders [149]; however, the link between FAD homeostasis
and fertility is undefined.
20
Several studies have documented fertility defects in C. elegans mitochondrial mutants.
Mutation in nuo-1, a complex I component of the mitochondria respiratory chain, results in
reduced brood size caused by impaired germline development [150]. Similarly, clk-1 mutation
affects the timing of egg laying, resulting in reduced brood size [151]. Both of these
mitochondrial mutations impact fertility, but their role(s) in spermatogenesis are unclear. alh-6,
the C. elegans ortholog of human ALDH4A1, is a nuclear-encoded mitochondrial enzyme that
functions in the second step of the proline metabolism pathway, converting 1-pyrroline-5-
carboxylate (P5C) to glutamate [152]. We previously revealed that alh-6(lax105) loss-of-function
mutants display altered mitochondrial structure in the muscle accompanied by increased level of
ROS in adult animals [6]. Furthermore, mutation in alh-6 results in the activation of SKN-1/NRF2
[7], an established regulator of oxidative stress response, likely through the accumulation of
toxic P5C disrupting mitochondrial homeostasis [6, 7, 153-155]. Interestingly, SKN-1 was
recently shown to respond to accumulation of damaged mitochondria by inducing their
biogenesis and degradation through autophagy [156]. Here, we identify a genetic pathway that
regulates male reproductive decline stemming from the perturbation of mitochondrial proline
metabolism leading to redox imbalance, cofactor depletion, and altered mitochondria dynamics;
all of which play a role in sperm dysfunction.
21
Results
Mutation in mitochondrial alh-6 results in diet-independent reduction in fertility
Altered mitochondrial structure and activity have been correlated with sperm dysfunction
across different species [157-160]. In addition, proper sperm function requires low levels of ROS
[161-163], although a specific role for endogenous mitochondrial derived ROS is undefined.
ALH-6/ALDH4A1, is a nuclear-encoded mitochondrial enzyme that functions in the second step
of proline catabolism, converting 1-pyrroline-5-carboxylate (P5C) to glutamate (Figure 1A). We
anticipated that mutation of alh-6 may affect the germline, based on our previous assessment of
the premature aging phenotypes in somatic cells of alh-6 mutants [6]. Using an UV-integrated
alh-6::gfp strain under its endogenous promoter, we saw that ALH-6 localizes to the
mitochondria in the germline of both hermaphrodites and males (Figure 1–figure supplement 1).
We then assessed progeny output of alh-6(lax105) hermaphrodites fed the standard OP50/E.
coli B strain diet and found a reduction in self-fertility brood size (-12.9%) (Figure 1B). Since the
somatic phenotypes of alh-6(lax105) mutants are known to be diet-dependent [6, 7], we
examined self-fertility of animals fed the HT115/E. coli K-12 strain diet to determine if the
reduced reproductive output is also dependent on the type of bacterial diet ingested.
Surprisingly, we found that the self-fertility of alh-6 animals was markedly reduced (-20.7%),
when animals were fed the HT115 diet (Figure 1C). alh-6 mutants have similar timing in their
progeny output as compared to wild type animals on both diets (Figure 1–figure supplement 2).
Since alh-6 mutants display normal development and reproductive timing, the progeny deficit is
not a result of an attenuated reproductive span which reveals the differential impact of alh-6 loss
in the soma (diet-dependent) [6] and the germline (diet-independent).
alh-6 fertility defects are sperm-specific
We noted that alh-6 mutant hermaphrodite animals laid twice as many unfertilized
oocytes as wild type animals over their reproductive-span (Figure 1D), suggesting an
22
impairment of sperm function [164-166]. It is notable that alh-6 mutant hermaphrodites lay very
few, if any, dead eggs (Figure 1D), suggesting that the loss of ALH-6 activity is not lethal. To
determine whether the reduced brood size of alh-6 mutants are due to a general loss of germ
cells or a specific defect in oocytes or sperm, we examined the mated-fertility of these animals
by mating wild type young adult (day 0-1) males to either wildtype or alh-6 mutant virgin
hermaphrodites (in wild type C. elegans, male sperm outcompetes hermaphrodite sperm >99%
of the time [167, 168] (Figure 1E). We found that the reduced fertility in alh-6 mutant
hermaphrodites is fully rescued by wild type sperm, which confirmed that oocyte quality is not
impaired but rather, alh-6 hermaphrodite sperm appears to be dysfunctional (Figure 1F).
To better assess the quality of alh-6 mutant sperm, we compared the ability of alh-6
mutant male sperm to compete against wild type hermaphrodite sperm [169]. In C. elegans wild
type animals, male sperm are larger and faster than hermaphrodite sperm, which affords a
competitive advantage [170]. To differentiate between progeny resulting from mating and
progeny that arise from hermaphrodite self-fertilization, we made use of male animals harboring
a GFP transgene such that any cross-progeny will express GFP while progeny that arise from
hermaphrodite self-sperm will not (Figure 1E). We found that wild type hermaphrodites when
mated to alh-6 mutant males have significantly more self-sperm-derived progeny as compared
to those mated to wild type males (Figure 1G). This finding indicates a competition deficit of alh-
6 male sperm resulting in this increased proportion of progeny derived from hermaphrodite
sperm, which is uncommon after mating has occurred [167]. C. elegans hermaphrodites
produce a set amount of sperm exclusively at the L4 developmental stage, before switching
exclusively to oogenesis. As such, hermaphrodites eventually deplete their reservoir of sperm
[167, 171]. To assess whether alh-6 mutant sperm are generally dysfunctional, we mated older
hermaphrodites that had depleted their complement of self-sperm and found that alh-6 mutant
males are able to produce equal numbers of progeny as wild type males when the need for
competition with hermaphrodite sperm is abated (Figure 1–figure supplement 3A); thus,
23
although alh-6 mutant sperm are impaired for competition, they remain viable for reproduction.
Similarly, older sperm-depleted alh-6 mutant hermaphrodites produced similar brood sizes when
mated to young wild type or alh-6 mutant males, which further supports a model where sperm,
but not oocytes, are defective in alh-6 mutants (Figure 1–figure supplement 3B). Taken
together, these data suggest that while alh-6 mutant male sperm remain competent for
fertilization, their competitive advantage is impaired when challenged against hermaphrodite
sperm.
Defects in mitochondrial proline catabolism impact sperm quality
Similar to mammals, the contribution of sperm to fertility in C. elegans is dictated by
distinct functional qualities, which include: sperm number, size, and motility [169, 170, 172]. We
next sought to define the nature of the sperm competition defect in alh-6 mutants by measuring
sperm number, size, and motility in alh-6 mutants compared to wild type animals. One day after
the onset of spermatogenesis (at the L4 larval stage of development), alh-6 adult
hermaphrodites have a reduced number of sperm in the spermatheca as compared to wild type
(Figure 2–figure supplement 1A), which is correlated with the reduced self-fertility observed
(Figures 1B-C). In contrast, age-matched alh-6 mutant virgin males have similar numbers of
spermatids as WT virgin males, suggesting that they have a similar rate of production (Figure
2A). We next examined sperm size in day 1 adult males and discovered that alh-6 mutant
spermatids are significantly smaller as compared to wild type (Figure 2B). To achieve motility,
C. elegans spermatids must form a pseudopod which requires protease activation [173] (Figure
2–figure supplement 1B). Sperm activation can be recapitulated in vitro by treatment of isolated
spermatids with the Streptomyces griseus protease Pronase [174]. After 30 minutes of Pronase
treatment, 80% of wildtype spermatids are fully activated, while a significantly reduced
population of alh-6 mutant spermatids mature over the same time period (Figure 2C). The
reduction in activation, as measured by the presence of a fully extended pseudopod, in alh-6
mutant spermatids is correlated with an increase in the number of cells observed at the normally
24
transient intermediate stage of spermiogenesis characterized by the presence of “spikes”
(Figure 2–figure supplement 1B-C) [174]. We observed a similar impairment in activation of alh-
6 mutant spermatids when treated with the cationic ionophore Monensin (Figure 2–figure
supplement 1D-E), except that alh-6 mutant spermatids were stalled at the “protrusion”
intermediate stage of spermiogenesis [175]. Future studies to reveal where and how
mitochondrial proline catabolism integrates into specific stages of spermiogenesis will be of
great interest [174].
Interestingly, although sperm number was the same between WT and alh-6 mutant
males on the OP50 diet, sperm number was reduced in alh-6 mutant males fed HT115 diet
compared to age-matched WT males on the same diet (Figure 2D). We also noted that
spermatids from alh-6 mutant males raised on the HT115 diet were similarly defective in size
and activation (Figure 2E-F). Taken together, although diet can influence sperm number, the
reduction of sperm size and activation are likely contributors to the reduced fertility and
competitive fitness in alh-6 mutant males; which is independent of diet.
Transcriptional signatures define temporal phenotypes of alh-6 mutant animals
We first identified alh-6 mutant in a screen for activators of the cytoprotective
transcription factor SKN-1/NRF2 using gst-4p::gfp as a reporter [6, 7]. When activated, SKN-1
transcribes a variety of gene targets that collectively act to restore cellular homeostasis.
However, this can come with an energetic cost with pleiotropic consequences [6, 7, 77, 138,
176-179]. alh-6 mutants have normal development, but display progeroid phenotypes towards
the end of the normal reproductive span [6] indicating a temporal switch in phenotypic
outcomes. We reasoned that the temporally controlled phenotypes in the alh-6 mutants could
be leveraged to identify potential mechanisms by which alh-6 loss drives cellular dysfunction.
As SKN-1 is activated in alh-6 mutants after day 2 of adulthood [6], we defined genes that
display differentially altered expression in the L4 developmental stage, when spermatogenesis
occurs, as compared to day 3 adults (post SKN-1 activation). We performed RNA-Seq analyses
25
of worms with loss of alh-6 and identified 1935 genes in L4 stage animals and 456 genes in day
3 adult animals that are differentially expressed (+/- Log2 (fold change), 0.05 FDR) (Figure 3–
figure supplement 1A-B). Notably, the gene expression changes at these two life periods had
distinct transcriptional signatures (Figures 3A-B). Because the loss of alh-6 drives
compensatory changes in normal cellular metabolism, which later in life results in the activation
of SKN-1, we expected to identify significant changes in both metabolic genes and SKN-1 target
genes. Supporting this hypothesis, the Gene Ontology (GO) terms most enriched include
oxidoreductases and metabolic enzymes in L4 stage animals (Figure 3A) and SKN-1-
dependent targets such as glutathione metabolism pathway genes in day 3 adults (Figure 3B).
Importantly, our transcriptomic analysis recapitulated the temporally-dependent phenotypic
outcomes resulting from alh-6 loss; genes in the pseudopodium and germ plasm GO terms
class displayed reduced expression in L4 alh-6 mutant animals (Figure 3A), which include
many genes in the major sperm protein (MSP) family that comprises 15% of total protein
content in C. elegans sperm and impact sperm function [180]. In contrast, genes in the muscle-
specific GO term class displayed increased expression in day 3 adults (Figure 3B), which is
activation of the SKN-1 reporter is enhanced in the muscle of alh-6 mutants [7]. Taken together,
the transcriptomic analysis of alh-6 mutants is diagnostically relevant and informative for
defining drivers of organism-level phenotypic changes in animals with altered proline
catabolism.
FAD mediates sperm functionality and competitive fitness
The strong enrichment of genes whose protein products utilize and/or bind cofactors or
co-enzymes was intriguing as the maintenance of metabolic homeostasis and the redox state of
the cell requires a sophisticated balance of multiple cofactors (Figure 4A). In fact, the proline
catabolism pathway utilizes multiple cofactors to generate glutamate from proline; PRDH-1 uses
FAD as a co-factor to convert proline to P5C while ALH-6 utilizes the reduction of NAD+ to
convert P5C to glutamate. Additionally, in the absence of ALH-6, accumulation of P5C, the toxic
26
metabolic intermediate of proline catabolism, drives the expression of pathways to detoxify P5C
(oxidoreductases, P5C reductase, etc.) (Figure 3, Figure 3–figure supplement 1C). Although
enzymes in the proline catabolism pathway utilize FAD as a cofactor, the transcriptional
signature of the alh-6 mutants includes the activation of multiple enzymes that utilize FAD,
which drove the hypothesis that FAD levels might be altered in alh-6 mutants. We measured
FAD and found a significant reduction in alh-6 mutant animals fed the OP50 diet at the L4 stage
(Figure 4B) and a similar reduction in animals fed HT115 bacteria at L4 stage (Figure 4C).
Differences in FAD levels were unremarkable in day 3 adult animals, when spermatogenesis
has long since ended (Figure 4–figure supplement 1A). Based on this finding, we predicted that
restoration of FAD levels might alleviate the sperm-specific phenotypes of alh-6 mutants.
Riboflavin is a precursor of FAD (Figure 4D) and dietary supplementation of riboflavin has been
shown to increase cellular FAD levels in wild-type animals [181, 182]. Similarly, riboflavin
supplementation to the OP50 diet of alh-6 mutants restored FAD levels to wild-type levels
(Figure 4E). We found that wild type hermaphrodites mated to alh-6 mutant males fed a
riboflavin supplemented diet produced significantly more total progeny than alh-6 males fed the
standard OP50 diet (Figure 4–figure supplement 1B). Moreover, riboflavin supplementation was
sufficient to partially restore male sperm size (Figure 4F) and also rescued the impaired
activation (Figure 4G) of male sperm in alh-6 mutants. Riboflavin supplementation increases
sperm size in WT males, but do not change sperm activation in WT males (Figure 4–figure
supplement 1C-D).
We next asked whether FAD metabolism was required for proper sperm function. FAD
can be synthesized de novo by a two-step enzymatic reaction where riboflavin is converted to
FMN by Riboflavin Kinase/R10H10.6, which is subsequently converted to FAD by FAD
Synthase/FLAD-1 (Figure 4D). We used RNA interference (RNAi) against R10H10.6 or flad-1
in wild-type male animals and measured sperm quality. Similar to alh-6 mutant sperm, RNAi
reduction of the FAD biosynthetic pathway decreased sperm size (Figures 4H, 4I, Figure 4–
27
figure supplement 1E-F) and impaired sperm activation (Figures 4J, 4K, Figures Figure 4–
figure supplement 1E-F).
NAD+ and NADH are also central adenine dinucleotide cofactors that play critical roles
in metabolism and have received recent attention as a method to combat the decline seen in
biological function with age [183]. As such, we also measured NAD and NADH levels, but found
the ratio unremarkable between wild-type and alh-6 mutant animals (Figure 4–figure
supplement 1G-I). Taken together, these data suggest that loss of alh-6 leads to a specific
decrease in cellular FAD levels and that FAD is a critical cofactor that drives proper sperm
function.
Mitochondrial dynamics regulate spermatid function
Although there is a clear and documented role for mitophagy in the clearance of paternal
mitochondria post-fertilization in C. elegans, the role(s) for mitochondrial dynamics and turnover
in sperm function prior to zygote formation are unclear. We first examined mitochondrial
dynamics in wild type spermatids by staining with the fluorescent mitochondrial-specific dye JC-
1, and noted that each spermatid on average contained multiple discernable spherical
mitochondria that are mostly not fused (Figures 5A, 5B, 5E). Previous studies in yeast and
cultured mammalian cells have shown that when cells are exposed to mild stress, the initial
response of mitochondria is to fuse in order to dilute damage [184-186].
The mitochondrial specific dye JC-1 accumulates in mitochondria in a membrane
potential-dependent manner, and as the concentration increases, its fluorescence switches from
green to red emission. The accumulation of sufficient JC-1 molecules required for red emission
is abolished by treatment with Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a chemical
inhibitor of mitochondrial oxidative phosphorylation (Figure 5–figure supplement 1A). Therefore,
a higher red-to-green fluorescence ratio in cells is indicative of healthier mitochondria species
and as such, we characterized mitochondria with red JC-1 emission in our analyses of
connectivity in spermatids. alh-6 mutant spermatids have reduced red:green JC-1 fluorescence
28
that indicates a lower mitochondrial membrane potential and an accumulation of unhealthy
mitochondria (Figure 5F) [187]. Moreover, alh-6 mutant spermatids have mitochondria that
were more interconnected (Figures 5C-E) as compared to wild type spermatids and a similar
increase in connectivity was observed when mitochondria were visualized with the membrane
potential-dependent mitochondrial dye Mitotracker Red CMXRos (Figure 5–figure supplement
1B). The increase in fused mitochondria in spermatids was also present in animals fed the
HT115 diet, which further supports a diet-independent role for alh-6 in the germline (Figure 5–
figure supplement 1C).
A connection between mitochondrial dynamics (fusion and fission) and FAD
homeostasis has not been previously described. To understand this, we perturbed FAD
biosynthesis pathway and then examined mitochondrial connectivity in spermatids. We first
reduced FAD biosynthesis with RNAi targeting R10H10.6 or flad-1, which resulted in more
connected mitochondria that resembles the increased fusion in alh-6 mutant spermatids that are
under metabolic stress (Figure 5G-H, Figure 4–figure supplement 1E-F). In addition, increasing
FAD levels by dietary supplementation of riboflavin, restored mitochondria in spermatids of alh-6
animals to more wild-type-like distributions (Figure 5I), but did not change mitochondrial
morphology in WT male spermatids (Figure 5–figure supplement 1D). Thus, the reduction of
FAD in alh-6 mutants, alters mitochondrial dynamics to a more fused and less punctate state.
Therefore, the homeostatic control of FAD level is critical to maintain proper mitochondrial
dynamics in sperm.
The role of mitochondrial dynamics in the maturation of sperm has not been studied;
however recent work has revealed that the mitochondrial fusion and fission machinery are
important for the elimination of paternal mitochondria post-fertilization [188]. FZO-1 is required
for proper fusion of the mitochondrial outer membrane while EAT-3/OPA1 regulates inner
membrane fusion. In opposition to the activities of FZO-1 and EAT-3, DRP-1 is required for
mitochondrial fission [189, 190]. The balance of this fusion and fission machinery in the upkeep
29
of mitochondrial homeostasis allows cells to respond to changes in metabolic needs and
external stress [191, 192]. RNAi of fzo-1 or eat-3 reduced mitochondrial fusion in wild-type male
sperm (Figure 5J and Figure 5–figure supplement 1E) and suppressed the enhanced fusion
observed in alh-6 mutant spermatid mitochondria (Figure 5K); indicating mitochondrial fusion of
both membranes is active in spermatids with impaired proline catabolism. We next examined
spermatids from drp-1 mutant animals and observed a greater level of mitochondrial fusion as
compared to wild type and alh-6 mutant spermatids (Figure 5L). We also observed a
synergistic level of mitochondrial fusion in spermatids derived from alh-6; drp-1 double mutants.
This finding is consistent with previous studies in yeast which reveal that defects in fusion can
be compensated for by changes in the rates of fission and vice versa [191, 192]. In support of
our model where mitochondrial dynamics act as a major driver of the sperm-specific defects in
alh-6 mutants, we discovered that loss of drp-1, which results in increased mitochondrial fusion
(like that observed in alh-6 mutants), also reduces sperm activation (Figure 5M). Moreover,
reducing fzo-1 or eat-3 does not alter activation in wild type sperm, while fzo-1 but not eat-3
RNAi restores activation in alh-6 sperm (Figures 5N-O and Figure 5–figure supplement 1E),
suggesting increased fusion mediated predominantly by fzo-1 in alh-6 sperm mitochondria is
impairing proper function. We noted that alh-6 mutant animals have an increased expression of
fzo-1 transcripts that is suggestive of a retrograde signaling response from the mitochondria
(Figure 5–figure supplement 1F). Taken together, these data support a model where loss of
mitochondrial proline catabolism induces mitochondrial stress, activating mitochondrial fusion, in
order to dilute damage to preserve functional mitochondria at the cost of sperm function. These
data also reveal a functional role for mitochondrial fusion and fission in spermatid development
and sperm function.
alh-6 and FAD are cell autonomous regulators of sperm function
Signaling between germ and somatic cells can alter function in each cell type [193-200].
In light of the differences between somatic and germline phenotypes observed in alh-6 mutant
30
animals, we performed germline specific RNAi targeting alh-6 to deduce whether the sperm
defects observed were cell autonomous. Germline specific RNAi of alh-6 in wild-type males was
not sufficient to alter sperm size (Figure 6A), but did result in diminished sperm activation
(Figure 6B,) and increased mitochondrial fusion in sperm (Figure 6C). Similarly, RNAi of alh-6
only in the soma resulted in a minor reduction of spermatid size (Figure 6–figure supplement
1A), but did not phenocopy the impairment of sperm activation as observed in alh-6 mutants
(Figure 6–figure supplement 1B). Taken together, these findings suggest that somatic
expression of alh-6 can influence spermatid size while the influence of alh-6 on spermatid
activation is cell autonomous.
Next, we restored wild-type alh-6 expression, only in the germline, in alh-6 mutant
animals, which restored sperm size in one of the two transgenic lines (Figure 6D), activation
(Figure 6E) and mitochondrial dynamics (Figure 6F), as compared to non-transgenic siblings.
We conclude that the effects of loss of alh-6 on sperm function (activation and mitochondria) are
cell autonomous because germline specific RNAi could phenocopy the sperm defects observed
in whole animal loss of alh-6, while RNAi of alh-6 only in the somatic tissues could not. In
contrast, the effect of alh-6 on sperm size is non-cell autonomous and requires somatic input
(Figure 6–figure supplement 1C-F).
Since FAD functions in a variety of essential cellular processes, we next asked if proper
sperm function required FAD homeostasis in germ cells. Similarly, we reduced R10H10.6 or
flad-1 only in the germline, which phenocopies germline knockdown of alh-6 on sperm size
(Figure 6G, J), sperm activation (Figure 6H, K), and mitochondrial fusion in sperm (Figure 6I,
L), as observed in whole animal RNAi of flad-1 or R10H10.6 (Figures 4J-K and Figures 5G-
H).These results suggest that FAD functions similarly to alh-6 in cell autonomously regulating
sperm function (activation and mitochondrial dynamics), while affecting sperm size in a cell non-
autonomous manner (Figures 4H-I). Taken together these data identify the importance of
proline catabolism and FAD homeostasis in germ cells to maintain proper sperm function. In
31
conclusion, our studies define mitochondrial proline catabolism as a critical metabolic pathway
for male reproductive health.
32
Discussion
Here we investigate the effects of disrupting mitochondrial proline catabolism through
the loss of the mitochondrial enzyme gene alh-6 and the resulting changes in FAD homeostasis,
mitochondrial dynamics, and male fertility (Figure 7). We found that alh-6 mutants show a
reduction in brood size that is sexually dimorphic; defects in sperm function but not oocytes
contribute to reduced hermaphrodite fertility. As societal factors continue to push individuals to
wait longer to have children, the increase in paternal age is inversely correlated with proper
sperm function and can give rise to fertility issues. Consequently, it is incumbent on future
studies to elucidate how restoring and maintaining functional amino acid catabolism during
aging in order to promote reproductive success.
Although C. elegans is a well-established organism for studying aging and reproduction,
with several studies describing hermaphrodite reproductive senescence, many questions
regarding the basis of male reproductive decline remain unanswered. Decades of work have
shown that exposure to pollution, toxins, xenobiotics, and other ROS-inducing compounds can
prematurely drive the loss of sperm function across species [201-203], but the impact that
normal cellular metabolism plays on sperm function and the identification of specific molecules
that can mediate sperm quality are not well-defined. In this study we characterized a new role
for mitochondrial proline catabolism and FAD homeostasis in the maintenance of proper sperm
function. Perturbation of this pathway, through mutation of alh-6/ALDH4A1, causes metabolic
stress. Consequently, this perturbation leads to reduction of cellular FAD level and increases
mitochondrial fusion in spermatids, which results in impaired sperm function and premature
reproductive senescence.
Mutation in proline dehydrogenase (PRODH) in humans results in hyperprolinemia type I
(HPI), while mutation in delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH4A1/P5CDH)
results in hyperprolinemia type II (HPII). This study reveals that in C. elegans, proline
33
catabolism impacts several functional qualities of male sperm. Loss of proline catabolism
results in smaller sperm with impaired activation, two qualities that directly impact competitive
advantage. As such, proline biosynthesis, catabolism, and steady state concentrations must be
tightly regulated, and the importance of proline in cellular homeostasis may help explain the
transcriptional responses measured in animals with dysfunctional alh-6. Our data support a cell
autonomous role for proline catabolism in sperm. However, although whole animal RNAi of alh-
6 closely phenocopies the alh-6 mutant including reduced spermatid size, germline specific
RNAi of alh-6 did not significantly reduce the size of spermatids; perhaps suggesting a partial
role for ALH-6 in somatic tissues for spermatid development, which is in line with recent studies
in C. elegans describing soma to germline signaling in sperm activation [204] . Intriguingly, the
impact of loss of alh-6 is mostly independent of diet source, unlike the somatic phenotypes
which are diet-dependent [6]. The exception is sperm number in alh-6 mutant animals on the
HT115 diet, which appears to be diet-dependent (Figure 2D). WT males have more spermatids
when fed the HT115 diet, as compared to WT animals fed OP50 diet, while alh-6 mutants have
the same number of spermatids on both diets.
Our previous work defined the age-dependent decline in function of somatic tissues,
particularly muscle in animals lacking functional ALH-6 [6, 7], which does not manifest until day
3 of adulthood. Our current study reveals that although somatic phenotypes in alh-6 mutants are
observed post-developmentally, the germline, or more specifically spermatids, are sensitive to
loss of alh-6 much earlier in development (phenotypes assayed at L4 or Day 1 of adulthood).
Reproductive senescence is a field of growing significance as the number of couples that
choose to delay having children increases. Importantly, although alh-6 mutant sperm are
impaired for competition, they remain viable for reproduction. This is similar to recent study on
comp-1, a mutation which results in context-dependent competition deficit in C. elegans sperm
[205].
34
Recent studies have focused on the role of NAD+ metabolism in cellular health, while
the impact of FAD has received less attention. FAD levels are diminished in alh-6 animals
specifically at the L4 stage when spermatogenesis is occurring. Riboflavin (Vitamin B 2) is a
precursor to the FAD and FMN cofactors that are needed for metabolic reactions in order to
maintain proper cellular function, like proline catabolism and mitochondrial oxidative
phosphorylation. Despite its importance, humans, like C. elegans, lack a riboflavin biosynthetic
pathway and therefore require riboflavin from exogenous sources [206]. Insufficient intake can
lead to impairment of flavin homeostasis, which is associated with cancer, cardiovascular
diseases, anemia, neurological disorders, impaired fetal development, etc. [206]. Our study
suggests that riboflavin and FAD play critical roles in reproduction, specifically in germ cell
development, as loss of FAD biosynthesis or loss of alh-6 specifically in the germline
recapitulates the sperm defects observed in whole animal knockdown or alh-6 mutation.
Importantly, these sperm-specific defects can be corrected by dietary supplementation of
vitamin B2, which in light of the exceptional conservation of mitochondrial homeostatic
pathways, suggest the nutraceutical role vitamin B2 could play in sperm health across species.
Our study also demonstrates that spermatids lacking alh-6 have increased mitochondrial
fusion; a perturbation at the mitochondrial organelle structure-level that contributes to the
sperm-specific phenotypes observed. In addition to prior work showing fzo-1/MFN1/MFN2 and
drp-1/DRP-1 to be important for mitochondrial elimination post-fertilization [188], our work
reveals that mitochondrial fission and fusion machinery are present and active in spermatids
and that perturbation of these dynamics can affect sperm maturation and competitive fitness.
Future work to define how alh-6 spermatids use mitophagy, which can clear damaged
mitochondria, will be of interest. In conclusion, our work identifies proline metabolism as a
major metabolic pathway that can impact sperm maturation and male reproductive success.
Moreover, these studies identify specific interventions to reverse the redox imbalance, cofactor
35
depletion, and altered mitochondria dynamics, all of which play a part in sperm dysfunction
resulting from proline metabolism defects.
36
Methods
Reagent type
(species) or
resource
Designation Source or reference Identifiers Additional information
Strain (C. elegans) N2
Caenorhabditis
Genetics Center (CGG)

Laboratory reference strain
(wild type)
Strain (C. elegans) SPC321 PMID: 24440036 Genotype: alh-6(lax105)
Strain (C. elegans) SPC326 PMID: 24440036

alh-6p::alh-6::gfp
Strain (C. elegans) SPC447 This paper

Genotype: alh-
6(lax105);laxEx025(pie-1p::alh-
6;myo-2p::rfp;myo-3p::rfp;rab-
3p::rfp)
Strain (C. elegans) SPC455 This paper

Genotype: alh-
6(lax105);laxEx033(pie-1p::alh-
6;myo-2p::rfp;myo-3p::rfp;rab-
3p::rfp)
Strain (C. elegans) SPC473 This paper
Genotype: alh-
6(lax105);laxEx051(pie-1p::alh-
6;myo-2p::rfp;myo-3p::rfp;rab-
3p::rfp)
Strain (C. elegans) CL2166
Caenorhabditis
Genetics Center (CGG)

Genotype: gst4-p::gfp
Strain (C. elegans) SPC223 PMID: 24440036

Genotype: alh-6(lax105);gst-
4p::gfp
Strain (C. elegans) DCL569
Caenorhabditis
Genetics Center (CGG)

Genotype: [mkcSi13(sun-
1p::rde-1::sun-1 3'UTR + unc-
119(+)) II; rde-1(mkc36) V
Strain (C. elegans) CU6372
Caenorhabditis
Genetics Center (CGG)

Genotype: drp-1(tm1108)
Strain (C. elegans) GR1948 PMID: 24684932
Genotype: mut-
14(mg464);smut-1(tm1301) V.
Chemical
compound, drug
Riboflavin Millipore Sigma R9504 Concentration used: 2.5mM
Commercial Assay
or kit
FAD
Colorimetric/Fluor
ometric Assay Kit
BioVision K357

Commercial Assay
or kit
NAD/NADH
Quantification
Colorimetric Kit
BioVision K337

Chemical
compound, drug
Pronase Millipore Sigma P8811 Concentration used: 200ug/mL
Chemical
compound, drug
eBioscience™
Monensin Solution
(1000X)
Thermo Fisher Scientific 00-4505-51 Concentration used: 100nM
Chemical
compound, drug
MitoProbe™ JC-1
Assay Kit
Thermo Fisher Scientific M34152
Concentration used: JC-1
15uM, CCCP 50uM
Chemical
compound, drug
MitoTracker™
Red CMXRos
Thermo Fisher Scientific M7512
Concentration used:
100uM dried on plate
37
Reagent type
(species) or
resource
Designation Source or reference Identifiers Additional information
Software GraphPad Prism
GraphPad Prism
(https://graphpad.com)
RRID:SCR
_015807
Version 6
Software ImageJ
ImageJ
(http://imagej.nih.gov/ij/)
RRID:SCR
_003070

Table 2.1. Key resources.

C. elegans strains and maintenance
C. elegans were cultured using standard techniques at 20 C. The following strains were
used: wild type (WT) N2 Bristol, SPC321[alh-6(lax105)], SPC326[alh-6p::alh-6::gfp],
SPC447[alh-6(lax105);laxEx025(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)], SPC455[alh-
6(lax105);laxEx033(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)], SPC473[alh-
6(lax105);laxEx051(pie-1p::alh-6;myo-2p::rfp;myo-3p::rfp;rab-3p::rfp)]], CL2166[gst4-p::gfp],
SPC223[alh-6(lax105);gst-4p::gfp], DCL569[mkcSi13(sun-1p::rde-1::sun-1 3'UTR + unc-119(+))
II; rde-1(mkc36) V], CU6372[drp-1(tm1108)], and GR1948[mut-14(mg464);smut-1(tm1301) V].
Double and triple mutants were generated by standard genetic techniques. E. coli strains used
were as follows: B Strain OP50[13] and HT115(DE3) [F
-
mcrA mcrB IN(rrnD-rrnE)1 lambda
-
rnc14::Tn10 (DE3)][90]. For dietary supplement assays, riboflavin was added to the NGM plate
mix to final concentration 2.5mM.
RNAi-based experiments
RNAi experiments were done using HT115-based RNAi [90], which yielded similar
results as OP50 RNAi E. coli B strain as described in [207]. All strains were adapted to diets for
at least three generations and strains were never allowed to starve. All RNAi clones were
sequenced prior to use and RNAi knockdown efficiency measured. RNAi cultures were seeded
on IPTG plates and allowed to induce overnight prior to dropping eggs on them for experiments.
38
Microscopy
Zeiss Axio Imager and ZEN software were used to acquire all images used in this study.
For GFP reporter strains, worms were mounted in M9 with 10mM levamisole and imaged with
DIC and GFP filters. For sperm number, assay samples were imaged with DIC and DAPI filters
in z-stacks. For sperm size and activation assays, dissected sperm samples were imaged at
100x with DIC filter on two different focal planes for each field to ensure accuracy. For sperm
mitochondria assays, dissected sperm samples were imaged at 100x with DIC, GFP, and RFP
filters in z-stacks to assess overall mitochondria content within each spermatid.
Fertility assay
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day, synchronized L1 larvae were dropped on NGM plates seeded with
either OP50 or HT115. 48 hours later, at least ten L4 hermaphrodites for each genotype were
singled onto individual plates and moved every 12 hours until egg laying ceased. Progeny were
counted 48 hours after the singled hermaphrodite was moved to a different plate. Plates were
counted twice for accuracy.
Mated reproductive assay
Males were synchronized by egg laying, picked as L4 larvae for use as young adults for
mating experiments. Singled L4 stage hermaphrodites were each put on a plate with 30ul of
OP50 seeded in the center together with three virgin adult males. 24 hours post-mating, males
were removed, and each hermaphrodite was moved to a new plate every 24 hours until egg
laying ceased. Progeny were counted 48 hours after the hermaphrodite was moved from the
plate. For sperm competition assay, progeny with GFP fluorescence were counted from the
cohort. Plates were counted twice for accuracy.
Cofactor Measurements
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day, synchronized L1s were dropped on NGM plates with or without
39
supplement seeded with 25X concentrated OP50. FAD levels are measured following directions
in FAD Colorimetric/Fluorometric Assay Kit (K357) from BioVision. NAD/NADH levels are
measured following directions in NAD/NADH Quantification Colorimetric Kit (K337).
Sperm Number Assay
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day, synchronized L1s were dropped on NGM plates with the indicated food
source. At 48 hours (L4 developmental stage) males were isolated to new plates. 72 hours
post-drop, day 1 adult virgin male animals were washed 3x with 1xPBST, fixed with 40% 2-
propanol, and stained with DAPI for 2 hours. Samples were washed for 30 minutes with PBST,
mounted with Vectashield mounting medium, and covered with coverslip to image. Spermatids
in the seminal vesicle were counted through all planes in z-stack.
Sperm Size Assay
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35 L pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
CaCl2, 1mM MgSO4, 10mM dextrose) to release spermatids, which were immediately imaged.
Sperm Activation with Pronase and Monensin
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35 L pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
CaCl2, 1mM MgSO4, 1mg/ml BSA) supplemented with either 200 g/mL Pronase® (Millipore
Sigma) or 100nM Monensin (Thermo Fisher Scientific 00-4505-51) to release spermatids.
Another 25ul of the same solution was added and the spermatids were incubated at RT for 30
minutes for activation to occur before imaging.
Sperm Mitochondria Staining
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35 L pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
40
CaCl2, 1mM MgSO4, 1mg/ml BSA) with JC-1(Thermo Fisher Scientific M34152) added to 15 M
final concentration. Another 25ul of the same solution was added and the spermatids were
incubated at RT for 10 minutes. The slide was washed three times with 100ul SM buffer before
imaging. For carbonyl cyanide m-chlorophenyl hydrazine (CCCP) uncoupler control in JC-1
staining experiment, 50uM final concentration was used in staining solution. For staining with
MitoTracker Red CMXRos (Thermo Fisher Scientific M7512), stock solution was diluted to
100uM final concentration in M9 and 50ul of this solution was applied on top of a spot of 50uL
25X concentrated OP50 seeded on a NGM plate. Solution was allowed to dry on the plate
before L4 virgin males were moved onto the food spot. Animals were allowed to stain overnight
(18-24 hours) and dissected next day in SM buffer for spermatids to image.
RNA-Sequencing
Worms were egg prepped and eggs were allowed to hatch overnight. The next day,
synchronized L1s were dropped on NGM plates seeded with 25X concentrated OP50. 48 and
120 hours post drop, L4 animals and day 3 adult animals, respectively, were washed 3 times
with M9 and frozen in TRI Reagent at -80 C. Animals were homogenized and RNA extraction
was performed following the protocol in Zymo Direct-zol RNA Isolation Kit. RNA samples were
sequenced and analyzed by Novogene.
Statistical analysis
Data are presented as mean  SEM. Comparisons and significance were analyzed in
Graphpad Prism 7. Comparisons between two groups were done using Student’s Test.
Comparisons between more than two groups were done using ANOVA. For sperm activation
assays, Fisher’s Exact Test was used and p-values are adjusted for multiple comparisons.
*p<0.05 **p<0.01 *** p<0.001 ****<0.0001.
41
Figures

Figure 2.1. alh-6 fertility defects are sperm-specific.
(A) Proline catabolism pathway. (B-C) alh-6 hermaphrodites have reduced brood size when fed
OP50 (B) or HT115 (C) diets. (D) alh-6 hermaphrodites lay increased number of unfertilized
oocytes, but few dead embryos. (E) Mated reproductive assay scheme utilizes males to
maximize reproductive output (as in F) and can exploit males harboring GFP to differentiate
progeny resulting from self- versus male-sperm (as in G). (F) Wild type (WT) and alh-6
hermaphrodites mated with WT males yield similar number of total progeny. (G) WT
hermaphrodites mated with alh-6;gst-4p::gfp males yield more non-GFP progeny (indicating
self-fertilization) than hermaphrodites mated with WT males harboring gst-4p::gfp. Statistical
comparisons by unpaired t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies
performed in at least biological triplicate; refer to Supplemental File 1 for n for each comparison.

42

Figure 2.2. alh-6 males have sperm defects on both OP50 and HT115 diets.
(A-C) sperm phenotypes on OP50 diet. (A) Sperm quantity is similar between wild type (WT)
and alh-6 mutant day 1 adult males. (B) Spermatid size is reduced in alh-6 mutant day 1 adult
males as compared to age matched WT males. (C) Sperm activation is impaired in alh-6 mutant
day 1 adult males relative to age-matched WT males. (D-F) sperm phenotypes on HT115 diet.
(D) Sperm quantity is reduced in alh-6 mutant day 1 adult males compared to age-matched WT
males. (E) Spermatid size is reduced in alh-6 mutant day 1 adult males as compared to age
matched WT males fed HT115. (F) Sperm activation is impaired in alh-6 mutant day 1 adult
males relative to age-matched WT males fed HT115. Statistical comparisons of sperm number
and size by unpaired t-test and sperm activation by Fisher’s exact test. *, p<0.05; **, p<0.01; ***,
p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer to
Supplemental File 1 for n for each comparison.

43

Figure 2.3. Transcriptional patterns define developmental- and adult-specific
consequences to loss of alh-6 activity.
Gene Ontology (GO) term enrichment analysis of RNA-Seq data. (A) Transcriptional changes at
L4 stage are enriched for metabolism and sperm-specific genes. (B) Transcriptional changes at
day 3 adulthood are enriched for changes in glutathione activity, oxidoreductase activity, and
muscle-specific genes. All studies performed in at least biological triplicate; refer to
Supplemental File 1 for n for each comparison.

44

Figure 2.4. Loss of FAD homeostasis in alh-6 mutants leads to sperm dysfunction.
(A) Metabolic pathways utilize adenine dinucleotide cofactors to maintain redox balance in cells.
(B-C) FAD+ levels are reduced in alh-6 mutant animals fed OP50 (B) or HT115 (C) at the L4
developmental stage. (D) FAD biosynthetic pathway. (E-G) Dietary supplement of riboflavin
restores FAD level (E), sperm size (F), and sperm activation (G) in alh-6 mutants. (H-I) RNAi
knockdown of R10H10.6 (H) or flad-1 (I) in WT males reduces their sperm size compared to
L4440 vector control. (J-K) RNAi knockdown of R10H10.6 (J) or flad-1 (K) in WT males impairs
sperm activation upon Pronase treatment. Statistical comparisons of sperm size by ANOVA.
Statistical comparisons of activation by fisher’s exact test with p-value cut-off adjusted by
number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies
performed in biological triplicate; refer to Supplemental File 1 for n for each comparison.

45

Figure 2.5. Mitochondrial dynamics drive sperm quality.
(A-E) JC-1 dye stained mitochondria of WT (A-B), alh-6 mutant (C-D); (B and D) are ImageJ
detection of JC-1 stained sperm mitochondria area which are quantified in (E). (F) Mitochondria
46
in alh-6 mutant spermatids have reduced JC-1 red/green fluorescence ratio, indicating
mitochondria depolarization. (G-H) RNAi knockdown of FAD biosynthetic pathway genes,
R10H10.6 (G) or flad-1 (H) increases mitochondrial fusion in WT spermatids. (I) Dietary
supplement of FAD precursor riboflavin restores mitochondrial fusion in alh-6 spermatids to WT
level. (J-K) eat-3 or fzo-1 RNAi decreases mitochondrial fusion in both WT (J) and alh-6 (K)
mutant spermatids. (L) drp-1 mutation increases mitochondrial fusion in both WT and alh-6
spermatids. (M) drp-1 mutation significantly impairs sperm activation in both WT and alh-6
mutant spermatids. (N) fzo-1 RNAi restores sperm activation in alh-6 mutant. (O) eat-3 RNAi
reduces sperm activation in WT males but not alh-6 males. Statistical comparisons of JC-1
Red/Green FL ratio by unpaired t-test. Statistical comparisons of mitochondria fusion by
ANOVA. Statistical comparisons of sperm activation by Fisher’s exact test with p-value cut-off
adjusted by number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All
studies performed in at least biological triplicate; refer to Supplemental File 1 for n for each
comparison.

47

Figure 2.6. alh-6 and FAD function cell autonomously in the germline to regulate sperm
function.
(A-C) Germline-specific RNAi of alh-6 does not change sperm size (A), but does impair sperm
activation (B) and increases mitochondrial fusion in sperm (C). (D-F) Germline-specific rescue
of WT alh-6 in alh-6 mutant male animals increases sperm size (D) and restores activation (E)
and mitochondrial dynamics (F). Statistical comparisons of sperm size and mitochondrial fusion
in spermatids by unpaired t-test. Similarly, (G-L) germline-specific RNAi of R10H10.6 and flad-1
do not change sperm size (G,J), impair sperm activation (H,K), and increase mitochondrial
fusion in sperm (I,L). Statistical comparisons of sperm activation by Fisher’s exact test with p-
value cut-off adjusted by number of comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****,
p<0.0001. All studies performed in biological triplicate; refer to Supplemental File 1 for n for
each comparison.
48

49

Figure 2.7. Model of alh-6 and FAD mediated male reproductive senescence.

50
Supplemental

Figure 2.1 –figure supplement 1. Mitochondrial localization of ALH-6 in the germline. UV
integrated alh-6::gfp strain under its endogenous promoter reveals expression of ALH-6 in
hermaphrodite (A-B) and male (C-D) germline. a and c are DIC images while b and d are GFP
images. Scale bar for all images is 10 m.

51

Figure 2.1 –figure supplement 2. alh-6 hermaphrodite reproductive span is similar to wild type
(WT) on different diets. (A-B) Progeny output time-courses are plotted as % total progeny for
each time point. WT and alh-6 mutant have similar output on OP50 (A) and HT115 (B).
Significance indicate differences in progeny output at a particular time point done by multiple t-
tests. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological
triplicate; refer to Supplemental File 1 for n for each comparison.

52

Figure 2.1 –figure supplement 3. alh-6 fertility defects are sperm-specific. (A) Day 4 adult
WT hermaphrodites mated to either gst-4p::gfp or alh-6;gst-4p::gfp males yield similar total
brood size. (B) Day 4 adult alh-6 hermaphrodites mated to either gst-4p::gfp or alh-6;gst-4p::gfp
males yield similar total brood size. Comparisons made with unpaired t-test. *, p<0.05; **,
p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to
Supplemental File 1 for n for each comparison.

53

Figure 2.2 –figure supplement 1. (A) alh-6 hermaphrodites have reduced sperm number as
day 1 adults. (B) Spermiogenesis stages (round, spike, protrusion, pseudopod). Spermatozoa
with fully formed pseudopods are considered activated in Pronase and Monensin experiments.
(C) alh-6 male spermatids treated with Pronase are stalled at the “spikes” stage compared to
WT male spermatids. (D) alh-6 male spermatids treated with Monensin have reduced activation
compared to WT male spermatids. (E) alh-6 male spermatids treated with Monensin are stalled
54
at the “protrusion stage” compared to age-matched WT spermatids. Statistical comparisons of
sperm number by unpaired t-test and sperm activation by Fisher’s exact test. *, p<0.05; **,
p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in at least biological triplicate; refer
to Supplemental File 1 for n for each comparison.

55

Figure 2.3 –figure supplement 1. RNA-Sequencing data of WT and alh-6 hermaphrodites
at L4 and day 3 adulthood. (A) Number of genes that are significantly upregulated in alh-
6(lax105) compared to WT at L4 and Day 3 adult stages. (B) Number of genes that are
significantly downregulated in alh-6(lax105) compared to WT at L4 and Day 3 adult stages. FDR
= 0.05. (C) alh-6 mutants display increased expression of metabolic enzymes to reduce P5C
levels. All studies performed in biological triplicate; refer to Supplemental File 1 for n for each
comparison.

56

Figure 2.4 –figure supplement 1. Adenine nucleotide cofactor homeostasis is disrupted in
alh-6 mutants. (A) FAD levels are unchanged between WT and alh-6 mutant animals fed OP50
at day 3 adulthood. (B) WT hermaphrodites mated to alh-6;gst-4p::gfp males fed OP50
supplemented with 2.5mM riboflavin results in increase in total brood size compared to WT
hermaphrodites mated to non-supplemented alh-6;gst-4p::gfp males. (C) Dietary riboflavin
57
supplement increased spermatid size of WT males. (D) WT males fed OP50 diet supplemented
with riboflavin have similar % spermatid activated upon Pronase treatment as those without
riboflavin supplement. (E) R10H10.6 1 expression is modestly reduced by whole animal RNAi
via RT-PCR verification. (F) flad-1 expression is reduced by whole animal RNAi via RT-PCR
verification. (G) NAD levels are unchanged between WT and alh-6 animals at L4 and day 3
adulthood (H) NADH level is unchanged between aged matched WT and alh-6 hermaphrodites
at both L4 and Day 3 adulthood. (I) NAD+/NADH level is unchanged between aged matched
WT and alh-6 hermaphrodites at both L4 and Day 3 adulthood. *, p<0.05; **, p<0.01; ***,
p<0.001; ****, p<0.0001. Statistical comparisons done by unpaired t-test for all experiments
except for sperm activation, which is done by Fischer’s exact test. All studies performed in
biological triplicate; refer to Supplemental File 1 for n for each comparison.

58

Figure 2.5 –figure supplement 1. fzo-1 is involved in mitochondrial dynamics aberration in
alh-6 spermatids. (A) JC-1 stained alh-6 spermatid mitochondria show sensitivity to
mitochondrial uncoupler CCCP treatment. Intensity of red mitochondria species (J-aggregates)
are dissipated while that of green mitochondria species (monomers) are intensified, indicating
membrane depolarization (Note that CCCP treatment is known to cause cellular swelling). Scale
bar = 1uM. (B) alh-6 spermatid mitochondria stained with MitoTracker Red CMXRos show
59
increased fusion compared to WT male spermatids. Scale bar = 0.5uM. (C) Spermatids of alh-6
males fed HT115 diet still display increased mitochondrial fusion compared to spermatids of
age-matched WT males. (D) Riboflavin supplementation did not alter mitochondria fusion in
spermatids of WT males. (E) eat-3 and fzo-1 RNAi knockdown in WT and alh-6 mutants are
verified using RT-PCR. (F) fzo-1 expression is increased in alh-6 mutants, while eat-3
expression is not significantly increased. Statistical comparisons done by unpaired t-test. *,
p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate;
refer to Supplemental File 1 for n for each comparison.

60

Figure 2.6 –figure supplement 1. Germline expression of WT alh-6 is sufficient to rescue
sperm defect. (A-B) Soma-restricted RNAi of alh-6 slightly reduces sperm size (A), but does
not affect sperm activation (B). (C-F) Whole animal RNAi of alh-6 reduces sperm size (C),
impairs activation (D), and increases mitofusion in spermatids (E). (F) alh-6 expression is
reduced by whole animal RNAi as shown in RT-PCR verification. Statistical comparisons done
by unpaired t-test for all experiments except for sperm activation, which is done by Fischer’s
exact test. All studies performed in biological triplicate; refer to Supplemental File 1 for n for
each comparison.
61
Acknowledgements
We thank N. Mih, K. Han, and L. Thomas for technical assistance; H. Dalton, A.
Hammerquist, N. Stuhr, W. Escorcia, and J. Nhan for critical reading of the manuscript; C.
Phillips for the soma-restricted RNAi strain GR1948; and D. Chavez for protocol on MitoTracker
Red staining. Some strains were provided by the CGC, which is funded by the NIH Office of
Research Infrastructure Programs (P40 OD010440). This work was funded by the NIH
(R01GM109028, R01AG058610, to S.P.C., T32AG000037 to D.L.R., and T32GM118289 to
C.D.T), and the American Federation of Aging Research (C-A.Y. and S.P.C).
Competing interests: The authors declare no competing interests.
Data availability: All relevant data are available from the authors. RNA-Seq data are deposited
in GEO database (GSE121920).
62
Chapter 3.
Incomplete proline catabolism drives premature sperm aging
*This chapter is a version of a submitted manuscript.
Chia-An Yen
1,2
and Sean P. Curran
1,2
*
1. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089
2. Department of Molecular and Computation Biology, Dornsife College of Letters, Arts, and Sciences,
University of Southern California, Los Angeles, CA 90089
*Correspondence to: spcurran@usc.edu
Keywords: spermatogenesis, mitochondria, germ cells, reproduction, proline catabolism, alh-
6/ALDH4A1, C. elegans, senescence, aging, male-specific, ROS, NAC, Vitamin C
63
Abstract
Infertility is a common health issue with rising prevalence in parents with advanced age.
Environmental stress has established negative effects on reproductive health, however, the
impact of altering cellular metabolism and its endogenous reactive oxygen species (ROS) on
fertility remain unclear. Here we demonstrate that proline catabolism, in general, promotes
larger sperm size, but the loss of proline dehydrogenase, the first committed step in proline
catabolism, is relatively benign. In contrast, disruption of alh-6, which facilitates the second step
of proline catabolism by converting 1-pyrroline-5-carboxylate (P5C) to glutamate, results in
premature reproductive senescence, specifically in males. The premature reproductive
senescence in alh-6 mutant males is caused by aberrant ROS homeostasis, which can be
countered by genetically limiting flux through the ALH-6 pathway or pharmacological treatment
with antioxidants. Taken together, our work uncovers proline metabolism as a critical
component of normal sperm function that can alter the rate of aging in the male reproductive
system.
64
Introduction
Infertility is defined as the inability for a couple to conceive within a year of unprotected
sex. There is an estimated number of 12-13% of couples in the US that struggle with infertility
[208]. While emphasis on female factors in fertility is important and their role have been
extensively studied, male factors play an equally important role in determining the outcome of
successful fertilization. Male fertility is often measured as a function of sperm quality and
quantity, since these factors are correlated with time to pregnancy and pregnancy success
[209]. As increasing number of couples wait to have children, age becomes a risk factor for
infertility problem; the increase in paternal age, much like maternal age, is also associated with
adverse gamete health, negative pregnancy outcome, and increase risk of birth defects [210].
In sexually reproducing species, sperm competition plays an important role in
reproductive fitness. In species where females mate with multiple males, a male can improve
his reproductive success if his sperm outcompetes sperm from other males in fertilizing the
oocyte of a female. This competitive edge can be achieved by males through producing large
quantities of sperm or generating higher-quality sperm [211]. In Caenorhabditis elegans,
adolescent hermaphrodites produce sperm before switching to oogenesis in adulthood. When
mated with males, hermaphrodite-derived sperm are disadvantaged when compared to the
greater size and speed of male sperm [170]. In addition to hermaphrodite-male sperm
competition, male-male sperm competition can also occur when a hermaphrodite is mated with
multiple partners. As such, sperm quality is a competitive parameter of overall fitness in
sexually reproducing species.
Mitochondria are essential for their role in fueling cellular functions. Notably, multiple
studies in humans and mice have implicated different aspects of mitochondrial function in sperm
quality including mitochondria ultrastructure, mitochondrial genome and copy number [170]
mitochondria protein levels, as well as enzyme activity of electron transport chain (ETC)
65
complexes [158]. While all these studies imply that mitochondrial integrity and activity are
important for proper sperm function, the mechanism behind this relationship remains unclear.
While cells need mitochondria to generate energy, this process generates ROS as a
natural byproduct [212]. Low levels of ROS are essential, and play an important role in cell
signaling, hypoxia adaptation, aging, autophagy, immunity, and cell differentiation [213] while
high levels of ROS can be detrimental to cellular function and can lead to cell death. In
mammals, multiple aspects of sperm function and successful fertilization including capacitation,
hyperactivation, acrosome reaction, and sperm-oocyte fusion require low levels of ROS [201].
Interestingly, many studies have found elevated ROS in sperm to be associated with increased
lipid peroxidation, increased DNA damage, and reduced sperm motility and viability; although
the source of ROS and the mechanism behind ROS-induced sperm defects are unknown [201].
Recent studies show that mitochondria-generated ROS through inhibition of the ETC results in
spermatozoa with reduced motility and increased lipid peroxidation in vitro [214, 215]. Since the
level of ROS in semen also increases with age [145], understanding ROS-mediated sperm
defects may provide insight into male reproductive senescence.
Proline plays a critical role in cellular metabolism and functions as a central amino acid
in cellular bioenergetics and redox control [216]; but has recently become recognized as a
mediator of aging and age-related conditions [6, 7, 217-219]. Glutamate generated via proline
catabolism is a two-step metabolic process where proline is first converted to P5C by proline
dehydrogenase, PRDH-1, and subsequently to glutamate by P5C dehydrogenase, ALH-6. Loss
of ALH-6 results in the accumulation of the P5C, which generates ROS and leads to a loss of
cellular integrity [6, 7, 155]. Our previous findings demonstrated that loss of alh-6 leads to the
depletion of FAD reserves and drives changes in mitochondrial dynamics, leading to sperm
dysfunction [217]. Here we investigate the role of proline metabolism in regulating sperm health
as a function of age.
66
Results
Mutations in proline dehydrogenase suppress alh-6 mutant phenotypes
Our previous study linked the loss of ALH-6 activity with impaired male reproductive
fitness in C. elegans [217]. We sought to determine if Aldh4a1, the mammalian ortholog of alh-
6, was regulated by age in the male reproductive system of mice. We examined the expression
of proline catabolism pathway genes Prodh and Aldh4a1 in young (3-month-old) and middle
aged (12-month-old) mice testes. The expression of Prodh, was similar (Figure 1A) between
12-month-old and 3-month-old mice testes, but the expression of Aldh4a1 was significantly
reduced in the older mice testes (Figure 1B). Furthermore, expression of both Prodh and
Aldh4a1 remain similar in the livers of 3-month-old and 12-month-old mice, showing that the
differential expression of Aldh4a1 but not Prodh in older mice testes is tissue-specific (Fig S1A-
B). It is established that oxidative stress increases with age in male testes due to the reduction
in expression and production of antioxidant enzymes [220, 221] Reduction of ALDH4A1 without
a corresponding reduction in PRDH1 could lead to the accumulation of P5C, a scenario that
could lead to sperm dysfunction as we previously found in worms [217]. Based on this finding,
we exploited the facile ability to genetically manipulate C. elegans to identify genetic regulators
of the stress response resulting from loss of alh-6.
We performed an ethyl methanesulfonate (EMS) mutagenesis screen to identify
suppressors of the age-dependent induction of the gst-4p::gfp reporter that defines alh-6 mutant
animals (Figure 1C) [6, 217]. One complementation group, defined by the suppressor allele
lax228, mapped to the right arm of chromosome IV between the DraI SNPs E03H12 and
Y105C5B (Figure 1D). We performed whole genome sequencing to identify non-synonymous
mutations in the exons of protein coding genes in this region and compiled a list of candidate
genes (Figure S1C) [222]. We tested each of these genes by RNAi in the alh-6(lax105);gst-
4p::gfp strain to identify RNAi clones that could phenocopy the effects of the lax228 suppressor
67
allele. RNAi of B0513.5, hereafter referred to as prdh-1 as it encodes a putative proline
dehydrogenase enzyme homologous to mammalian Prodh, was the only RNAi target in this
region that phenocopied the lax228 mutant (Figures 1E-F, S1D). Proline dehydrogenases
catalyze the first enzymatic step of proline catabolism (Figure 1G), converting proline to P5C
[152]. Next, we followed the loss of the age-dependent expression of gst-4p in the muscle while
backcrossing the alh-6(lax105);prdh-1(lax228);gst-4p::gfp strain to alh-6(lax105) mutants six
times at which time we then sequence verified the G to A transition mutation in exon 3 of prdh-1
resulting in a Valine to Methionine (V124M) substitution that defines the lax228 allele. Finally,
we performed two additional backcrosses to remove the gst-4p::gfp reporter to generate the alh-
6(lax105);prdh-1(lax228) strain SPC494; referred hereafter as alh-6;prdh-1.
Temporal regulation of mitochondrial proline catabolism pathway gene expression
We examined the expression of the proline catabolism pathway genes via RT-PCR
(Figure 1H) and confirmed a significant increase in the expression of enzymes that would
prevent the accumulation of P5C in alh-6 mutant larval stage 4 (L4) animals, confirming our
previous RNA-Seq analysis of alh-6 mutant animals [217]. At the L4 stage, when
spermatogenesis occurs, alh-6 mutant animals display a decrease in alh-6 expression, which
would further contribute to P5C accumulation. The expression of alh-6 is partially restored in
alh-6;prdh-1 double mutant animals, which notably also display increased expression of prdh-1.
The expression of pyrroline-5-carboxylate reductase (pycr-1/PYCR), which converts P5C back
to proline, and ornithine transaminase (oatr-1/OAT) which converts P5C to ornithine, were
increased in both alh-6 and alh-6;prdh-1 double mutants. Surprisingly, the expression of
pyrroline-5-carboxylate synthase (alh-13/P5CS) was also increased, however P5CS has two
enzymatic functions: glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR)
activities that impact additional nodes of cellular metabolism [223, 224]. Moreover, since proline
itself has important roles in cellular protection, the increased expression of pycr-1 might be an
important stress response, but with pleiotropic consequences as it would deplete glutamate and
68
increase an already accumulating pool of P5C. Intriguingly, alh-6;prdh-1 mutants at L4 stage
show a similar increase in expression of pycr-1, alh-13, and oatr-1 to alh-6 mutants.
Next, we measured proline metabolism genes at day 3 adulthood, when activation of the
SKN-1 reporter is observed in alh-6 animals (Figure 1I). Surprisingly and unlike developing
animals, very few of the proline catabolism pathway genes were significantly altered in the alh-6
mutants, with or without prdh-1. However, the expression of two P5C reductase genes, pycr-
1/M153.1 and pycr-4/F55G1.9 displayed temporal specificity at development and adulthood,
respectively (Figure 1H-I and Figure S1E-J). Noticeably, older alh-6 mutant animals show
increased expression of alh-6 and pycr-4, both of which can reduce P5C (Figure 1G).
Importantly, loss of prdh-1 attenuated the increased expression of both genes. Due to the
marked transcriptional responses observed at the L4 and day 3 adulthood stages in alh-6 and
alh-6;prdh-1 mutants, we next sought to understand the physiological consequence of loss of
both alh-6 and prdh-1.
Loss of cellular proline catabolism is not causal for sperm defects in alh-6 mutants
Because of the impact that loss of alh-6 has on the expression of other proline
catabolism pathway genes during development, we examined how the complete loss of proline
catabolism would impact spermatid development, which occurs at the L4 stage. Our previous
studies identified a role for alh-6 in spermatid development and as such, we examined the alh-
6;prdh-1 double mutant animals in a panel of reproduction and sperm quality assays [217]. The
reduction in spermatid size (Figure 2A) and impairment of spermatid activation (Figure 2B) in
alh-6 mutant males are both restored to wild type (WT) levels when combined with a mutation in
prdh-1. Additionally, in the context of the alh-6 single mutant hermaphrodite, loss of prdh-1
results in a trend toward increased fertility (Figure 2C), suppresses the number of unfertilized
oocytes (Figure 2D), and increases sperm count (Figure 2E). Finally, the reduced ability of alh-
6 male sperm to compete against wild type hermaphrodite sperm was abrogated in the alh-
6;prdh-1 double mutant (Figure 2F). Taken together, these results reveal that loss of flux
69
through the mitochondrial proline catabolism pathway is benign for animal reproductive fitness
but suggests instead that P5C accumulation is instrumental in driving sperm dysfunction in alh-6
animals.
Endogenous ROS drives alh-6 sperm defects
Several studies have examined the impact of exogenous exposure to ROS-inducing
electrophiles on sperm function [225, 226], but the impact of endogenously produced ROS on
sperm function remains poorly defined. Proline catabolism has been linked to mitochondrial
ROS homeostasis in somatic tissues [6, 7, 227], but not in the context of spermatogenesis. The
continuous generation of P5C by PRDH-1 should lead to redox imbalance and impairment of
normal function of germ cells as it does for somatic tissues [6, 153-155, 228]. We hypothesized
that if the sperm defects in the alh-6 mutants are a result of loss of ROS homeostasis, then
antioxidant supplementation could alleviate these phenotypes. We supplemented the diet of alh-
6 mutant males with the antioxidant N-acetylcysteine (NAC), from birth through reproductive
maturity, and re-measured the reproductive parameters of these animals. NAC supplementation
restored spermatid size (Figure 3A) and activation (Figure 3B) of alh-6 animals to WT levels,
while NAC supplementation in wild type (Figures 3C-D) or alh-6; prdh-1 double mutants
(Figures 3E-F) had no effect. Similar results were obtained using Vitamin C as an antioxidant
(Figure S2A-B). These data reveal that loss of ALH-6 activity drives redox imbalance, which
leads to defects in sperm health and function.
To further examine the role of endogenously produced ROS on gamete dysfunction, we
pharmacologically altered mitochondrial ETC activity. While ETC is essential for cellular
respiration, it can also be a major source of metabolism-generated ROS [229]. Increased
availability of ETC substrates can alter the rates of oxidative phosphorylation and electron flow
through the ETC [230]. Specifically, dietary supplementation of malate and fumarate has been
demonstrated to decrease electron flow through complex III by activation of NADH-fumarate
reductase (malate dismutation) and passing electrons to rhodoquinone instead of coenzyme Q;
70
which could increase ROS production in vivo when ETC complex inhibitors are absent [231,
232]. Supplementation of malate and fumarate did not change WT or alh-6;prdh-1 male sperm
size, but further decreased alh-6 mutant male sperm size (Figure 4A-B, S3A). Surprisingly,
malate and fumarate supplementation also decreased WT and alh-6 mutant male sperm
competition as shown in the increase in usage of hermaphrodite self-sperm (Figure 4C-D.)
Collectively, these data suggest that driving continued activation of endogenous metabolic
pathways can lead to premature sperm dysfunction; similar to that observed in alh-6 animals.
Recently, we reported that alh-6 mutant spermatids have increased mitochondrial fusion
[217]. Loss of prdh-1, which restores sperm function (Figure 2), returned spermatid
mitochondria to a more punctate and less connected structure that resembles mitochondria in
WT spermatids (Figures 5A-G). Similarly, treatment with the antioxidant NAC (Figure 5H)
returned alh-6 mutant mitochondria in spermatids to wild type levels. Furthermore, NAC
treatment does not change the fusion level of WT male spermatids (Figure 5I). Taken together,
these data reveal that antioxidant supplementation can act as a treatment to overcome
reproductive deficiencies stemming from defects in cellular metabolism.
alh-6 mutation accelerates male reproductive aging
Like most tissues, with increasing age, the reproductive system functionally declines.
Clinically, age-related decline in male fertility is diagnosed by a decline in sperm quality and
quantity [233]. Sperm from older men are reduced in number, display increased abnormalities,
and reduced in motility, which collectively diminish success in both in vivo and in vitro
fertilization (IVF) [146, 234]. In C. elegans, the decline in male fertility has been shown in aged
males where sperm number is reduced and activation in vitro is reduced [235]. In our hands, the
ability for sperm to activate with Pronase treatment [175, 217] also declines with age in wild type
males (Fig 6A). alh-6 mutant sperm at day 1 of adulthood are more similar to day 6 WT adults
and as such, experience premature reproductive senescence in the form of impaired sperm
activation capacity. Importantly, the loss of prdh-1 restored the age-related changes in
71
spermatid activation to wild type levels in alh-6 mutant male sperm, supporting our finding that
flux through the proline catabolism pathway, in the absence of P5C dehydrogenase activity, is
causal for age-related sperm dysfunction.
In order to determine if the accelerated loss of activation was causal for the fertilization
defects observed in alh-6 mutant animals, we performed sperm competition assays as before,
but with aged males mated to young hermaphrodites. As expected, WT hermaphrodites mated
with day 3 adult WT males resulted in the generation of a small fraction of non-GFP progeny
(usage of hermaphrodite sperm) that are not present in day 1 adults (Fig 2F). In contrast, sperm
from alh-6 day 3 adults was significantly impaired for competition against hermaphrodite self-
sperm resulting in higher incidence of non-GFP progeny (Fig 6B). This deficit in competitive
advantage is restored to WT levels by the loss of prdh-1. Together, these data reveal a novel
role for alh-6 in the regulation of male reproductive senescence. The loss of ALH-6 results in
metabolic changes in order to restore homeostasis, which leads to redox imbalance and
accelerated aging in the germline. Importantly, these sperm defects can be modulated by
reducing flux through the proline catabolism pathway or dietary antioxidant treatment. Finally,
the loss of competitive advantage in germ cells of animals with incomplete proline catabolism
has implications for overall fitness of the organism.
72
Discussion
Although nearly half of idiopathic infertility cases are thought to have a genetic basis with
lifestyle as contributing factors [236], their underlying mechanistic basis is largely unknown.
While the importance of maternal age in female reproductive capacity has been highlighted in
many studies [237], the role of paternal age in fertility has received less attention. Our previous
study identified flavin adenine dinucleotide (FAD) homeostasis and changes in mitochondrial
metabolism and dynamics as mechanisms that regulate male fertility in C. elegans [217]. This
study expands our understanding of how altering endogenous metabolic processes can affect
sperm aging.
It is perhaps surprising that the complete loss of mitochondrial proline catabolism (loss of
both prdh-1 and alh-6) is relatively benign. We discovered that a mutation in the upstream
proline dehydrogenase pathway gene, prdh-1, is able to suppress the sperm defects observed
in alh-6 mutants (Figure 2), revealing that the overall reduction in proline catabolism is not
causal for the observed reproductive phenotypes, but rather, continued flux through the proline
catabolism pathway without the ability to effectively convert P5C to other intermediates.
Interestingly, our analysis of gene expression changes in proline metabolism genes showed a
downregulation of alh-6 in alh-6 mutants (loss of P5C reductase), but an upregulation of prdh-1
in prdh-1;alh-6 double mutants (loss or proline catabolism) at L4 stage (Figure 1H). This is
perhaps due to a feedback mechanism from the changes in expression of other genes in the
pathway. alh-6 mutants show an increased expression of pycr-1, alh-13, and oatr-1 to stabilize
the levels of P5C in the cell. Similarly, prdh-1 expression is increased perhaps in response to
the increase in the same set of genes. Interestingly, we noted that pycr-1 and alh-13 levels are
highly reduced with age (Figure S1G, S1I). In the context of alh-6 mutants, which require higher
P5C reductase to balance the increase in alh-6 at older age, the expression of pycr-4 is
upregulated in place of pycr-1 (Figure 1I, Figure S1H). In humans, hyperprolinemia (HP) types I
73
(PRODH) and II (ALDH4A1) are both diagnosed by elevated level of proline in plasma, with
addition of high level of P5C in HPII patients. The symptoms of HPI varies in severity depending
on the degree of reduction in PRODH activity, and are characterized by neurological, auditory,
and renal defects , while symptoms of HPII are variable and characterized by neurological
defects [238]. Due to the limited number of clinically diagnosed HPI and HPII patients, there are
limited studies into the molecular basis of hyperprolinemia. Furthermore, there is a lack of
understanding in the long term effects of this syndrome, as well as any current effective
treatment for patients [239]. Interestingly, in a recent study of HPII patients, mitochondria
dysfunction in fibroblasts from muscle biopsy was suggested although limited by small sample
size (1 out of 5 patients) [240]. Although proline catabolism has not previously been shown to
have a direct role in fertility, fertility studies in primate and other species have shown that the
addition of proline in cryopreservation medium improves sperm mobility and preservation of
membrane integrity upon thawing [241].
About 30-40% of all male infertility cases are associated with increased levels of ROS
[242]. Additionally, ROS generation increases as sperm quality decreases with age [145, 146],
and may be causally linked. Our study demonstrates that the impaired sperm function stemming
from perturbation of mitochondrial proline catabolism, specifically mutation in alh-6/ALDH4A1,
leads to increased ROS and can be alleviated pharmacologically by antioxidant treatments
(NAC and Vitamin C). Future investigation to directly measure how antioxidants might alleviate
age-related loss of reproductive capacity in mammals will be of great interest.
Our studies reveal that the transcription of proline catabolism pathway genes is under
temporal regulation, such as pycr-1 and alh-13, which are expressed exclusively during
development (Figure S1E-J). C. elegans express a second isoform of PYCR, pycr-4, which is
expressed in distinct tissues [243]. Intriguingly, the expression of PRDH-1 and ALH-6 are
reduced in concert with age, which would maintain metabolic flux through the pathway.
However, while mutations in prdh-1 modestly increase prdh-1 expression and alh-6 expression,
74
perhaps in an attempt to maintain homeostasis, mutations in alh-6 result in a reduction of alh-6
and no change in prdh-1 expression, which would drive accumulation of P5C. These findings
reveal that the mitochondrial proline catabolism pathway is transcriptionally responsive, but
further studies to identify its transcriptional mediators are needed.
Our study reveals that the absence of ALH-6 drives early reproductive decline. These
animals begin adulthood with a diminished capacity for reproductive success; day 1 adult alh-6
mutant males, which should be optimal for reproduction, are phenotypically more similar to older
day 6 WT males. Surprisingly, the rate of decline for sperm activation capacity remains similar
between alh-6 mutants and WT animals. These data suggest a model where the mechanisms
to maintain homeostasis with age remain intact, although alh-6 mutants do exhibit premature
decline.
Lastly, we found that expression of Aldh4a1 in mice testes also declines with age, while
Prodh remains unchanged (Figure 1A-B). These expression changes in proline metabolism
pathway in old mice testes mirrors the genetic perturbation of alh-6 in our worm model.
Importantly, expression of both Aldh4a1 and Prodh are unchanged in old and young liver
tissues of mice (Figures S1A-B), revealing germline specificity of this age-related decline.
Although there is no prior evidence of Aldh4a1 in regulating male fertility in mice, Aldh4a1 has
been shown to be significantly downregulated in oocytes isolated from post-reproductive mice
compared to young female mice [244]. It may be interesting to investigate whether proline
metabolism plays a direct role in mammalian sperm health and whether mechanisms identified
in our studies are conserved. Taken together, our studies define an important and conserved
role for mitochondrial proline catabolism in male reproductive fitness, which establishes a new
target and approach for combating male infertility.
75
Methods
C. elegans strains and maintenance
C. elegans were cultured using standard techniques at 20°C. The following strains were
used: wild type (WT) N2 Bristol, CB4856 (HW), SPC402[alh-6(lax105);gst-4p::gfp;HW],
SPC494[alh-6(lax105);prdh-1(lax228)], SPC321[alh-6(lax105)], CL2166[gst4-p::gfp],
SPC223[alh-6(lax105);gst-4p::gfp], and SPC490[alh-6(lax105);prdh-1(lax228);gst-4p::gfp].
Double and triple mutants were generated by standard genetic techniques. E. coli strains used
were as follows: B Strain OP50 [13] and HT115(DE3) [F
-
mcrA mcrB IN(rrnD-rrnE)1 lambda
-
rnc14::Tn10 l(DE3)] [90]. For dietary supplement assays, the following were added to the NGM
plate mix to final concentration: 5mM NAC,10mM Vitamin C, 5mM malate, 5mM fumarate.
EMS mutagenesis
Ethyl methanesulfonate (EMS) mutagenesis was performed as previously described
[77]. Briefly, SPC223[alh-6(lax105);gst-4p::gfp] was mutagenized with EMS, and F2 worms with
reduced GFP expression (indicating suppression of SKN-1 activation) were selected. prdh-
1(lax228) was isolated and mapped to chromosome IV. Whole genome sequencing and
injection rescue confirmed mutant sequence identity.
Whole Genome Sequencing
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day 3000-4000 synchronized L1s were dropped on NGM plates seeded with
25X concentrated OP50. After 48 hours, L4 animals were washed 3 times with M9,
homogenized, and genomic DNA was extracted using Zymo Quick-DNA Miniprep Kit. DNA
samples were library prepped and sequenced by USC Epigenome Center Data Production
Facility. Sequencing data were analyzed using Galaxy.
76
Microscopy
Zeiss Axio Imager and ZEN software were used to acquire all images used in this study.
For GFP reporter strains, worms were mounted in M9 with 10mM levamisole and imaged with
DIC and GFP filters. For sperm number, assay samples were imaged with DIC and DAPI filters
in z-stacks. For sperm size and activation assays, dissected sperm samples were imaged at
100x with DIC filter on two different focal planes for each field to ensure accuracy. For sperm
mitochondria assays, dissected sperm samples were imaged at 100x with DIC, GFP, and RFP
filters in z-stacks to assess overall mitochondria content within each spermatid.
Fertility assay
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day, synchronized L1 larvae were dropped on NGM plates seeded with
OP50. 48 hours later, at least ten L4 hermaphrodites for each genotype were singled onto
individual plates and moved every 12 hours until egg laying ceased. Unfertilized oocytes were
counted 24 hours after the singled hermaphrodite was moved and progeny were scored 48
hours after the singled hermaphrodite was moved to a different plate. Plates were counted twice
for accuracy.
Mated reproductive assay
Males were synchronized by egg laying, picked as L4 larvae for use as young adults for
mating experiments. Singled L4 stage hermaphrodites were each put on a plate with 30 µl of
OP50 seeded in the center together with three virgin adult males. 24 hours post-mating, males
were removed, and each hermaphrodite was moved to a new plate every 24 hours until egg
laying ceased. Progeny were counted 48 hours after the hermaphrodite was moved from the
plate. For sperm competition assay, progeny with GFP fluorescence were counted and
removed from plates before non-GFP progeny were counted. For aged male GFP mated
reproductive assay, age-synchronized day 3 adult virgin males were used to mate to L4 WT
77
hermaphrodites and the protocol above was followed post-mating. All plates were counted twice
for accuracy.
Sperm Number Assay
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch
overnight. The next day, synchronized L1s were dropped on NGM plates seeded with OP50. 72
hours post-drop, day 1 adult hermaphrodite animals were washed 3x with 1xPBST, fixed with
40% 2-propanol, and stained with DAPI for 2 hours. Samples were washed for 30 minutes with
PBST, mounted with Vectashield mounting medium, and covered with coverslip to image.
Spermatids in spermathecae of both gonad arms were counted through all planes in z-stack.
Sperm Size Assay
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35mL pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
CaCl2, 1mM MgSO4, 10mM dextrose) to release spermatids, which were immediately imaged.
Sperm Activation with Pronase
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35mL pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
CaCl2, 1mM MgSO4, 1mg/ml BSA) supplemented with 200mg/mL Pronase® (Millipore Sigma) to
release spermatids. Another 25 µl of the same solution was added and the spermatids were
incubated at RT for 30 minutes for activation to occur before imaging. For aged male sperm
activation assays, individual L4 virgin males were singled onto plates for each strain and moved
every 3 days onto fresh plates. Due to high censor rate while aging males, a higher number of
males are required to start with when singling at L4 stage.
Sperm Mitochondria Staining
Males were isolated at L4 stage 24 hours before assay. For each strain, five day 1 adult
males were dissected in 35mL pH 7.8 SM buffer (50mM HEPES, 50mM NaCl, 25mM KCl, 5mM
CaCl2, 1mM MgSO4, 1mg/ml BSA) with JC-1(Thermo Fisher Scientific T3168) added to 15mM
78
final concentration. Another 25 µl of the same solution was added and the spermatids were
incubated at RT for 10 minutes. The slide was washed three times with 100 µl SM buffer before
imaging.
RT-PCR
Worms were treated with alkaline hypochlorite and eggs were allowed to hatch overnight
in M9. The next day, 3000-4000 synchronized L1s were dropped on NGM plates seeded with
25X concentrated OP50. After 48 and 120 hours, L4 animals and day 3 adult animals,
respectively, were washed 3 times with M9 and frozen in TRI Reagent at -80°C. Animals were
homogenized and RNA extraction was performed following the protocol in Zymo Direct-zol RNA
Isolation Kit. RNA samples were used to make cDNA using qScript cDNA Supermix
(Quantabio). cDNA is mixed with PerfeCTa SYBR Green FastMix (Quantabio) and primers of
target genes to analyze their expression on Biorad CFX96 Touch Real-Time PCR Detection
System. Target gene expressions were normalized to snb-1 in analysis. Statistical comparisons
of two groups were analyzed using unpaired t-test, while ANOVA was used for comparing
groups of three.
Statistical analysis
Data are presented as mean ± SEM. Comparisons and significance were analyzed in
GraphPad Prism 8. Comparisons between two groups were done using unpaired t-test.
Comparisons between more than two groups were done using ANOVA. For sperm activation
assays and mated reproductive assay with GFP reporter males, Fisher’s Exact Test was used
and p-values are adjusted for multiple comparisons for groups of 3. P-value of less than 0.05 is
considered significant (*p<0.05 **p<0.01 *** p<0.001 ****p<0.0001).
79
Figures

Figure 3.1. prdh-1 mutation suppresses activation of SKN-1 and proline metabolism
deregulation in older alh-6 animals.
(a) RT-PCR shows expression of Prodh remain the same between young and old mice testes.
(b) Expression of Aldh4a1 is decreased in old mice testes compared to young mice via RT-
PCR. (c) Cartoon depiction of EMS screen for suppressors of SKN-1 reporter activation in alh-6
mutants. (d) Mutation locus of prdh-1(lax228) in the gene is marked by arrow. Dashed lines
mark the linked loci identified by SNP mapping. (e-f) RNAi knockdown of prdh-1 suppressed the
80
activation of SKN-1 in alh-6;gst-4p::gfp animals. (e) Day 3 adult alh-6;gst-4p::gfp fed L4440
(control RNAi) fluoresces bright green in the muscle. (f) Day 3 adult alh-6;gst-4p::gfp fed prdh-1
RNAi shows dim green fluorescence in the muscle. (g) Schematic of biosynthetic and catabolic
pathways of proline in C. elegans. (h) alh-6 mutant animals show gene expression changes in
proline metabolism pathway at L4 stage. pycr-1, alh-13, and oatr-1 are upregulated in alh-6
mutant L4 animals. alh-6;prdh-1 animals show similar upregulation in pycr-1, alh-13, and oatr-1.
(i) alh-6 mutant animals show an increase in alh-6 expression and a compensatory upregulation
of pycr-4 as day 3 adults. alh-6;prdh-1 animals are similar to WT in expression of proline
metabolism genes, except for an up regulation of alh-6. Statistical comparisons of RT-PCR
results in mice testes were done by unpaired t-test and statistical comparisons of RT-PCR
results in worms were done using ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
All studies performed in biological triplicate; refer to Table S1 for n for each comparison.
81

Figure 3.2. prdh-1 activity is required for sperm-specific fertility defects in alh-6 mutants.
(a-e) prdh-1 mutation rescues reduced sperm size (a), impaired sperm activation (b), reduced
brood size (c), and increased number of unfertilized oocytes (d), reduced sperm number (e),
and sperm competition (f) in alh-6 animals. Statistical comparisons of sperm size, progeny,
unfertilized oocytes, and sperm number were done by ANOVA. Comparisons of sperm
activation and proportions of total non-GFP progeny versus GFP progeny were done using
Fisher’s exact test with adjusted p-value cutoffs. *, p<0.05; **, p<0.01; ***, p<0.001; ****,
p<0.0001. All studies performed in biological triplicate; refer to Table S1 for n for each
comparison.
82

Figure 3.3. Endogenous ROS drives sperm defects in alh-6 mutants.
(a-b) Antioxidant NAC supplement restores sperm size (a) and sperm activation (b) in alh-6
mutants. (c-d) NAC supplementation does not affect sperm size (c) or sperm activation (d) in
WT males. (e-f) NAC supplementation does not affect sperm size (e) or sperm activation (f) in
alh-6;prdh-1 males. Statistical comparisons of sperm size between WT and alh-6 were done
using unpaired t-test. Statistical comparisons of sperm size between a strain on
unsupplemented diet versus diet supplemented with NAC were also done with unpaired t-test.
Comparisons of sperm activation between WT and alh-6 or a strain on control diet versus
supplemented diet are done using Fisher’s exact test *, p<0.05; **, p<0.01; ***, p<0.001; ****,
p<0.0001. All studies performed in biological triplicate; refer to Table S1 for n for each
comparison.
83

Figure 3.4. Increasing ETC activity through malate/fumarate supplementation alters
sperm function.
(a) Malate/fumarate supplementation does not affect sperm size in WT males. (b)
malate/fumarate supplementation further decreases sperm size of alh-6 males. (c-d)
Malate/fumarate supplement reduces ability for WT spermatids to compete (c), while
exacerbating alh-6 spermatids in competitive ability (d). Comparisons of sperm size were
analyzed using unpaired t-tests. Comparisons of proportions of total non-GFP progeny versus
GFP progeny between unsupplemented diet and diet supplemented with malate/fumarate were
done using Fisher’s exact test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies
performed in biological triplicate; refer to Table S1 for n for each comparison.
84

Figure 3.5. Endogenous ROS drives changes in mitochondria dynamics leading to sperm
defects.
(a-f) JC-1 dye-stained mitochondria of WT. White dashed lines marked the outside of
spermatids. Yellow outlines the individual mitochondria in the spermatids. (a-b), alh-6 mutant (c-
d), prdh-1;alh-6 mutant (e-f) spermatids from dissected males. (g) alh-6 mutant male spermatids
have increased number of fused mitochondria, which is restored to WT levels in prdh-1;alh-6
mutants. (h) Antioxidant treatment with NAC restores mitochondrial dynamics to wild type levels
in alh-6 mutant spermatids. Comparisons of % fused mitochondria between strains were
analyzed using ANOVA, while comparisons for alh-6 mutant fed control diet to those that are fed
on NAC supplemented diet were done using unpaired t-test *, p<0.05; **, p<0.01; ***, p<0.001;
****, p<0.0001. All studies performed in biological triplicate; refer to Table S1 for n for each
comparison.
85

Figure 3.6. alh-6 mutation accelerates male reproductive senescence.
(a) The ability for sperm to activate upon Pronase treatment declines with age. alh-6 mutant
sperm shows premature decline compared to WT and alh-6;prdh-1. Statistical comparisons
between strains at each age show a significant decrease in ability to activate of alh-6 mutant
spermatid compared to WT, that is restored in alh-6;prdh-1. (b) The ability for male sperm to
compete against hermaphrodite self-sperm also declines with age, with alh-6 mutants showing
premature decline compared to WT and alh-6;prdh-1 mutant males. Statistical comparisons of
sperm activation and proportions of total non-GFP progeny versus GFP progeny in mating
assay done using Fisher’s exact test with p-value adjusted for multiple comparisons between
the groups. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in
biological triplicate; refer to Table S1 for n for each comparison.
86
Supplemental

Figure S3.1. Loss of proline catabolism is not causal for sperm defects in alh-6 mutants.
(a) Prodh expression is similar between 3 month and 12 month-old mice liver. (b) Aldh4a1
expression is similar between 3 month- and 12 month-old mice liver. (c) List of candidate genes
identified through whole genome sequencing of lax228 suppressor mutant generated from EMS
screen using alh-6;gst-4p::gfp. (d) Day 3 adult alh-6;gst-4p::gfp and alh-6;prdh-1;gst-4p::gfp
animals. (e-j) RT-PCR measurement of expression level of genes at L4 versus day 3 adult stage
including: prdh-1 (e), alh-6 (f), pycr-1 (g), pycr-4 (h), alh-13 (i), and oatr-1 (j). Statistical
comparisons of RT-PCR results were done by unpaired t-test *, p<0.05; **, p<0.01; ***, p<0.001;
****, p<0.0001. All studies performed in biological triplicate; refer to Table S1 for n for each
comparison.
87

Figure S3.2. Antioxidant vitamin C rescues sperm defects of alh-6. (a) Vitamin C dietary
supplement rescues alh-6 sperm size. (b) WT sperm size is unaffected by dietary vitamin C
supplement. Statistical comparisons of sperm size between WT and alh-6 were done using
unpaired t-test. Statistical comparisons of sperm size between a strain on control OP50 diet
versus diet supplemented with vitamin C were also done with unpaired t-test *, p<0.05; **,
p<0.01; ***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Table
S1 for n for each comparison.
88

Figure S3.3. Malate/fumarate supplement does not affect alh-6;prdh-1 sperm size. (a)
Dietary supplement of malate/fumarate supplement did not change sperm size of alh-6;prdh-1.
Statistical comparisons of sperm size between a strain on control OP50 diet versus diet
supplemented with malate/fumarate were also done with unpaired t-test *, p<0.05; **, p<0.01;
***, p<0.001; ****, p<0.0001. All studies performed in biological triplicate; refer to Table S1 for n
for each comparison.
89
Acknowledgements
We thank J. Gonzalez for technical assistance; N. Stuhr, A. Hammerquist, and C.
Duangjan for critical reading of the manuscript and members of the Curran lab for helpful
comments. Some strains were provided by the CGC, which is funded by the NIH Office of
Research Infrastructure Programs (P40 OD010440). This work was funded by the NIH
R01GM109028 and R01AG058610 to S.P.C. and the American Federation for Aging Research
(AFAR) to C-A.Y. and S.P.C.
Declarations of interests: The authors declare that they have no conflicts of interest with the
contents of this article
Author contributions: S.P.C. designed the study; C-A.Y. performed the experiments; C-A.Y.
and S.P.C. analyzed data; C-A.Y. and S.P.C. wrote and revised the manuscript.
Data availability: All data are contained within the manuscript.
90
Chapter 4.
Methods for assessing fertility in C. elegans from a single
population
*This chapter is a version of a published manuscript at Aging: Methods in Molecular Biology.
Chia-An Yen
1,2
and Sean P. Curran
1,2,3,*
1. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089
2. Department of Molecular and Computation Biology, Dornsife College of Letters, Arts, and Sciences,
University of Southern California, Los Angeles, CA 90089
3. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California,
Los Angeles, CA 90089
*Correspondence to: spcurran@usc.edu

Keywords: Fertility, Fecundity, Oocyte quality, Sperm quality, Reproductive senescence, C elegans,
Fertility assays, Spermatogenesis, Aging
91
Abstract
Reproductive senescence occurs in a wide range of species with mechanistic aspects
that are conserved from Caenorhabditis elegans to humans. Genetic and environmental factors
can influence fertility and reproductive output can impact rates of aging. The C. elegans Bristol
N2 strain commonly used in laboratories is hermaphroditic, producing a defined number of
sperm during larval development before switching exclusively to oogenesis. Here we show a
method of assaying both oocyte and sperm quality from a single population of animals.
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Introduction
Fertility is a major life history trait that can be readily assayed to define the impact of a
define experimental conditions (genetic or environmental). The disposable soma theory of
aging states that organisms age due to the trade-off of resources to satisfy the energetic
requirements of growth, reproduction, and maintenance [84, 138, 245]. Conditions that alter
reproduction can impact lifespan and multiple lifespan altering manipulations can change
reproductive output [6, 7, 77, 84, 138, 246]. As such, environmental and genetic factors that
alter the balance of these energetic requirements can have multiple effects on animal health
and longevity. As such, measurements of fertility are an indispensable component of
understanding the aging process.
Like humans, C. elegans experience a decline in fertility with age; at roughly one-third of
their lifespan [143]. In addition, regulators of reproductive aging like insulin/IGF-1 and sma-
2/TGF-  signaling have conserved roles from worms to human [144]. While the majority of
studies in reproductive senescence have focused on maternal effects, male factors contribute to
a large portion of fertility complications with increasing evidence of an inverse relationship
between paternal age and sperm health [210]. Thus, fertility measurements in assessing the
quality of gametes of both sexes are equally important.
C. elegans are androdiecious nematodes with both hermaphrodites and males.
Hermaphrodites undergo spermatogenesis at larval stage 4 (L4) to produce around 300
spermatids. After spermatogenesis hermaphrodites switch exclusively to oogenesis as they
molt into adults [167].
As sex determination is regulated by copies of the X chromosome – hermaphrodites are
XX and males are XO - males can be maintained at a mendelian ratios when mated to
hermaphrodites. Since mated hermaphrodites can produce significantly more progeny
93
compared to animals restricted to self-fertilization, a large number of F1 progeny containing both
males and females can be easily maintained.
Fertility of hermaphrodites can be tested by singling individual animals and tracking the
generation of live progeny, unfertilized oocytes, and dead eggs during their reproductive span.
Quality of oocyte or sperm can be better understood by mating males to a hermaphrodite in a
mated reproductive assay. Wild type male spermatozoa are better poised for fertilization than
wild type hermaphrodite spermatozoa due to their larger size and faster mobility, giving them an
competitive advantage in reaching an unfertilized oocyte [168, 170]. Male sperm quality can
also be determined through isolation of L4-stage virgin males and dissecting 24-hours post-
isolation for spermatid number, morphology, size, and ability to activate upon known in vitro
activators [174, 175]. Once gender isolated, males can be maintained in the absence of
hermaphrodites to assess the impact of aging on sperm function.
94
Materials
Prepare all solutions using ultrapure water (prepared by purifying deionized water, to
attain a sensitivity of 18 MΩ-cm at 25 °C). Prepare and store all reagents at room temperature
(unless indicated otherwise). Filter sterilize with 0.22uM filter or autoclave to sterilize (as
indicated).
C. elegans Culture and Synchronization
1. C. elegans strains (e.g. wild type (WT) N2 Bristol) can be obtained from the CGC.
2. Bacterial diets (e.g. Escherichia coli B/OP50) can be obtained from the CGC.
3. Nematode Growth Medium (NGM): 3g/L NaCl, 17g/L agar, 2.5g/L peptone, 1mM
MgSO4, 5mg/L cholesterol in ethanol, 25mM KH2PO4, 1mM CaCl2, 0.01875%
streptomycin.
4. 60 x 15 mm sterile petri dishes (small)
5. 100 x 15mm sterile petri dishes (large)
6. Alkaline hypochlorite treatment: bleach, NaOH, H2O
7. M9: 30g/L KH2PO4, 60g/L Na2HPO4, 50g NaCl, 1mM MgSO4
8. Rotating mixer with rotisseries that fit 15mL conical tubes
Fertility and Mated Reproductive Assays
1. Stereomicroscope
2. 70% Ethanol
3. Bunsen burner
4. Worm pick (platinum wire)
5. Mating plates: 60 x 15 mm sterile petri dishes containing NGM and 10ul OP50 spot
seeded.
Sperm Morphology, Size, and Activation Assays
1. Stereomicroscope (for dissection)
95
2. Compound microscope with 100x objectives and DIC filter (for imaging)
3. 70% Ethanol in H2O (v/v)
4. Bunsen burner
5. Worm pick (platinum wire)
6. 25 gauge needles or scalpel
7. Unseeded plates: 60 x 15 mm sterile petri dishes containing just NGM (no OP50)
8. Microscope slide
9. Grease pen
10. Coverslips
11. Vaseline
12. 1M HEPES (pH 7.8)
13. 5M NaCl
14. 2M KCl
15. 1M CaCl2
16. 1M MgSO4
17. pH Meter
18. Bovine Serum Albumin (BSA, Millipore Sigma) purified by heat shock fractionation. Store
at 4 C.
19. Sperm Medium Buffer (SM Buffer): 50mM HEPES, 50mM NaCl, 25mM KCl, 5mM CaCl 2,
1mM MgSO4, 1mg/mL BSA, pH 7.8. Store at 4 C.
20. Pronase (Millipore Sigma P8811). Lyophilized in powder form are typically stored at -
20 C.
21. SM Buffer + pronase: SM Buffer with 200 g/mL Pronase. Make fresh each time.
22. Plastic container that can be sealed
23. Parafilm
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24. Paper towel
DAPI Staining for Sperm Count
1. 10x Phosphate Buffered Saline (PBS): In 500mL of deionized water add 25.6g
Na2HPO4·7H2O, 80g NaCl, 2g KCl, 2g KH2PO4. Bring up to volume to 1L with H2O.
Sterilize by autoclave.
2. 40% isopropanol in H2O (v/v).
3. DAPI (4′,6-diamidino-2-phenylindole) staining solution (1mg/mL stock)
4. Triton X-100
5. Shaker/mixer for 1.5mL tubes
6. Centrifuge for 1.5mL tubes
7. Compound microscope with 40-63x objectives, DAPI and DIC filters and camera for
fluorescent image acquisition.
8. Microscope slide
9. Coverslip
10. Vectashield mounting medium

97
Methods
C. elegans were cultured using standard techniques at 20 C (Note 1). Worm strains
should adapt to diets for at least three generations without experiencing starvation prior to
experiments. Age-matched hermaphrodites and males are used for all experiments.
Expanding a single population of worms up for assays (prior to all assays below)
1. Mate 10 L4-stage hermaphrodites to 30 young adult males on small NGM plates with a
small spot of OP50 seeded in center for 24 hours.
2. On the next day, move mated hermaphrodites to a large seeded NGM plate and allow
them to lay progeny for 1 day before removing them.
Wait 5 days for F1 population with both males and hermaphrodites to become adults and
have mated with each other (Note 2).
3. Treat gravid adults with alkaline hypochlorite treatment to obtain eggs [247]. Allow eggs
to hatch overnight in M9 buffer for synchronization (Note 3).
4. On the next day, drop synchronized larval stage 1 (L1) larvae on a large seeded NGM
plates (Note 4).
5. 48-hours later, L4-stage virgin hermaphrodites or males for each genotype or condition
can be isolated for each of the assay below.
Fertility Assay
Make sure you have enough plates for moving hermaphrodites until the end of
reproduction (egg laying ceases). This will require two moves per day (every 12 hour) for 6
days.
1. Single 12 L4-stage hermaphrodites for each genotype/condition onto individual small
seeded NGM plates.
98
2. Move individual hermaphrodite every 12 hours until egg laying ceases. Average
reproductive output over reproductive span of wild type hermaphrodite fed standard
OP50 (Figure 1).
3. After moving each hermaphrodite to the next plate, score the previous plate or timepoint
for number of unfertilized oocytes (Note 5).
4. 24-hours after moving the hermaphrodite to the next plate, count the original plate for
number of unhatched or dead eggs.
5. 48-hours post moving hermaphrodite to the next plate, count the original plate for
number of progeny by picking off worms and burning them (Note 6). Check plate again
next day for any worms you might have missed.
6. Number of total oocytes can be calculated by summing up total progeny, unfertilized
oocytes, and dead eggs. Average brood size of wild type hermaphrodite (Figure 2)
Mated Reproductive Assay
Make sure you have enough plates for moving hermaphrodites until the end of
reproduction (egg laying ceases). This will be 1 move every 24 hours for 9-10 days. Mating
plates are made with NGM poured into small plates and seeded with 10ul OP50 food spot in the
center.
1. For just this experiment, a separate alkaline treatment of the plates containing gravid
hermaphrodites and males need to be done a day earlier to have males that are day 1
adults for mating to L4-stage hermaphrodites that are from the other preparation (Note
7).
2. The day prior to experiments, age-matched L4-stage virgin males from this separate
treatment were moved to a small seeded NGM plate day without hermaphrodite (Note
8).
3. Set up mating of 20 individual L4-stage hermaphrodite and young adult (or age of
interest) male in a 1:1 ratio on NGM plate seeded with small 10ul OP50 spot (Note 9).
99
4. Move individual hermaphrodite every 24 hours until egg laying ceases. Plot progeny
number every 24-hours to assess reproductive output over time.
5. Verify successful mating even by presence of male progeny on the plates (Note 10).
6. After moving each hermaphrodite to the next plate, score the previous plate or timepoint
for number of unfertilized oocytes (Note 5).
7. 24-hours after moving the hermaphrodite to the next plate, count the original plate for
number of unhatched or dead eggs.
8. 48-hours post moving hermaphrodite to the next plate, count the original plate for
number of progeny by picking off worms and burning them (Note 6). Check plate again
next day for any worms you might have missed.
9. Number of total oocytes can be calculated by summing up the number of progeny,
unfertilized oocytes, and dead eggs.
Sperm Morphology and Size
1. The day prior to experiments, put L4-stage virgin males onto a small seeded NGM plate
day without hermaphrodite (need 5 worms for each replicate of an experimental
condition). (Note 8).
2. During the day of experiment, start by preparing slides for performing dissections. Using
a grease pen, draw a small circle with its diameter about 1/3 the width of standard slide.
This grease circle keeps the buffer and worms in place during dissection. Let the circle
dry for at least 2-hours (Note 11).
3. Make SM buffer following recipe in materials and pH to 7.8. Filter sterilize and store at
4 C. Warm up to room temperature before use. Note that spermatids are sensitive to pH
and temperature changes.
4. Move day 1 (or age of interest) adult males onto an unseeded plate. Allow worms to
crawl on the plate to get rid of OP50 that are adhered to their body (Note 12).
5. Pipette 35uL of SM Buffer into the middle of grease circle on prepared slide.
100
6. Using a worm pick without any food, pick up one male at a time and move it into the drop
of SM Buffer inside grease circle. Do this for 5 males.
7. Dissect males near the tail end (where the seminal vesicle is located) using sterile 25
gauge needles or scalpel to release spermatids. (Note 13).
8. Put vaseline along the edges of a coverslip and place coverslip down gently (the side
with vaseline) to cover the area with grease circle. Image spermatids at 100x on a
compound microscope with DIC filter for morphology and size (Figure 3).
Sperm Activation
1. The day prior to experiments, put L4-stage virgin males onto a small seeded NGM plate
day without hermaphrodite (need 5 worms for each replicate of an experimental
condition). (Note 8).
2. During the day of experiment, start by preparing slides for performing dissections. Using
a grease pen, draw a small circle with its diameter about 1/3 the width of standard slide.
This grease circle keeps the buffer and worms in place during dissection. Let the circle
dry for at least 2 hours (Note 11).
3. Make SM buffer as in step 3 of Methods 3.4 (Note 14). Pronase is a mixture of several
nonspecific endo- and exoproteases that triggers the differentiation of round immobile
spermatids into mobile spermatozoa in a process called spermiogenesis. Dissolve
Pronase to a final concentration of 10mg/mL in water. Dilute to 200ug/mL in SM Buffer
prior to experiment (Note 15).
4. Make a humid chamber by placing a wet paper towel on the bottom of plastic container.
Layer a piece of parafilm on top of paper towel and close lid to retain moisture (Note 16).
5. Move day 1 (or age of interest) adult males onto an unseeded plate. Allow worms to
crawl on the plate to get rid of OP50 that are adhered to their body (Note 12).
6. Pipette 35uL of SM Buffer + Pronase into the middle of grease circle on prepared slide.
101
7. Using a worm pick without any food, pick up one male at a time and move it into the drop
of SM Buffer inside grease circle. Do this for 5 males.
8. Dissect males near the tail end (where the seminal vesicle is located) using sterile 25
gauge needles or scalpel to release spermatids. (Note 17).
9. Add 25ul more SM Buffer + Pronase onto slide (Note 18) and incubate slide in the humid
chamber assembled earlier in step 4 for 30 min at room temperature.
10. Put vaseline along the edges of a coverslip and place coverslip down gently (the side
with vaseline) to cover the area with grease circle. Image spermatids at 100x on a
compound microscope with DIC filter. Take two images for each field: one focal plane in
focus and the other a little higher (Note 19). Differentiation of spermatids into
spermatozoa upon Pronase treatment (Figure 4).
Sperm Number
1. Make 1x PBST - Dilute to 10x PBS to 1x and add in Triton X-100 to final conc of 0.01%
(Note 20).
2. The day prior to experiments, put about 30-40 L4-stage virgin males onto a small
seeded NGM plate day without hermaphrodite (Note 8). Need at least 10 for each
sample/condition.
3. The day of experiment, wash day 1 (or age of interest) adult males into a 1.5mL tube
with 1xPBST.
4. Spin down at 560 x g for 1 min and remove supernatant to 100uL mark on the tube.
5. Wash 1-3x with 1mL 1xPBST (Note 21).
6. Spin down at 560 x g for 1 min and remove supernatant to 100uL mark on the tube.
7. Add in 600uL of 40% isopropanol and incubate on shaker for 3 min to fix worms.
8. Spin down at 560 x g for 1 min and remove super natant to 100ul mark on the tube.
9. DAPI binds to DNA and allows visualization of nuclei in cells (Note 22). Make DAPI
staining solution: Dilute 1mg/mL DAPI stock solution to 10ug/mL in 40% isopropanol.
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10. Add in 600ul of DAPI staining solution and incubate in the dark for 2-hrs.
11. Remove supernatant to 100uL mark.
12. Add in 600uL of 1xPBST and incubate for 30 min in the dark to remove excess dye.
13. Mount samples on slide with Vectashield mounting medium.
14. Gently cover with coverslip and seal with nail polish.
15. Image at 40-63x with DIC and DAPI filter. Make sure you image all the spermatids in
each animal (Note 23).

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Notes
1. NGM plates are made and seeded using standard techniques [13].
2. F1 population should be roughly 50% males due to mating.
3. Incubate at least 12-hours to allow all animals to hatch. Do not exceed 24-hours.
4. Drop synchronized L1 stage animals directly onto food to allow animals to resume
development at the same rate. Animals dropped on side of food will be asynchronous.
5. Unfertilized oocytes are brown colored and circular. Eggs are shelled and oval.
6. Waiting 2 days allow progeny to develop to a size that is easy to be spotted and picked
off for counting.
7. Young adult virgin males are needed to mate readily to L4-stage hermaphrodites as they
molt into adults.
8. Allow hermaphrodites to crawl on OP50 lawn for 5-10 minutes and burn them off prior to
moving males onto the plates. The scent of hermaphrodite encourages males to stay on
OP50 lawn instead of crawling onto the side of plates and dying from dehydration.
9. Small area of food keeps worm in a small area to maximize chance of mating.
10. Male progeny usually can be spotted in the first two plates (first 48-hours). For those
plates with no male progeny, censor animals as not mated.
11. Can speed this up by drying slides in a fume hood. 2-hours is the minimum time required
for grease circle to dry completely. You can do this the day before experiment.
12. Prod worms gently or move to another unseeded plate as needed. Too much bacteria
can cause sperm cells to lyse.
13. Dip needle or scalpel in 70% ethanol and flame needle/scalpel between each sample to
sterilize. Allow to cool before use).
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14. 10mM dextrose can be used in place of BSA in SM Buffer. In our hands, sperm
activation by Pronase does not work as well with dextrose supplemented SM Buffer
compared to BSA.
15. Dissolve Pronase in solution fresh each time. Do not re-use solution. For example of
dilution in SM Buffer: 20uL of 10mg/mL Pronase should be added to 980uL of SM Buffer.
The low volume of water added to SM Buffer is negligible in altering the concentration of
contents of SM Buffer. If concerned, dilute 10mg/mL stock Pronase in SM Buffer.
16. A sealed container with wet paper towel on the bottom and a parafilm separating slide
from paper towel is used for humid chamber.
17. Dip needle or scalpel in 70% ethanol and flame needle/scalpel between each sample to
sterilize. Allow to cool before use
18. This helps with evaporation of liquid and the sensitivity of spermatids to these changes.
Evaporation makes spermatids appear irregular in shape instead of circular.
19. This helps with identifying activated versus non-activated spermatids since pseudopods
can be hard to see at only one plane field.
20. Spermatids are sensitive to pH levels. HEPES is used because of its buffering property.
21. Wash 1-2 more time depending on how cloudy the tubes look. Careful to not remove
worms accidentally when taking out supernatant from washes.
22. DAPI is a blue fluorescent dye that binds to AT regions of DNA and is readily excited by
violet (405 nm) laser line.
23. Spermatids have compact nuclei that is distinguishable from earlier stages of
spermatogenesis.

105
Figures

Figure 4.1. Reproductive output time course for wild type N2 hermaphrodites
106

Figure 4.2. Average brood size of wild type N2 hermaphrodites.
107

Figure 4.3. Spermatids released from dissected wild type N2 males.
108

Figure 4.4. Pseudopods are visible in Pronase activated spermatozoa released from
dissected wild type N2 males.
109
Acknowledgements
This work was funded by the NIH R01GM109028 and R01AG058610 to S.P.C. and the
American Federation of Aging Research (C-A.Y. and S.P.C).
110
Chapter 5. Redirection of SKN-1 abates the negative
metabolic outcomes of a perceived pathogen infection
*This chapter is a version of a published manuscript at Proceedings of the National Academy of
Sciences.
James D. Nhan
1,2
, Christian D. Turner
1,2
, Sarah M. Anderson
3
,

Chia-An Yen
1,2
, Hans M. Dalton
1,2
, Hilary
K. Cheesman
3
,

Dana L. Ruter
4
, Nandhitha Uma Naresh
5
, Cole M. Haynes
5
, Alexander A. Soukas
6
, Read
Pukkila-Worley
3,*
, and Sean P. Curran
1,2,7,
*
1. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089,
United States
2. Department of Molecular and Computation Biology, Dornsife College of Letters, Arts, and Sciences,
University of Southern California, Los Angeles, CA 90089, United States
3. Program in Innate Immunity, Division of Infectious Diseases and Immunology, University of
Massachusetts Medical School, Worcester, MA 01655, United States
4. Biology Department, Integrative Program for Biological and Genome Sciences, University of North
Carolina, Chapel Hill, NC 27599, United States
5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School,
Worcester, MA 01655, United States
6. Center for Human Genetic Research and Diabetes Unit, Department of Medicine, Massachusetts
General Hospital, Boston, MA 02114, United States
7. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California,
Los Angeles, CA 90089, United States
*Correspondence to: spcurran@usc.edu (S.P.C.), read.pukkila-worley@umassmed.edu (R.P.-W.)
Keywords: SKN-1, pathogen, H3K4me3, WDR-5, p38, innate immunity, lipids, germline, soma, C.
elegans, Pseudomonas, Asdf
111
Abstract
Early host responses towards pathogens are essential for defense against infection. In
Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during
xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results
in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs
health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas
aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores.
Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets,
which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects
SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In
addition, activating p38/MAPK signaling in the absence of pathogen, is sufficient to drive SKN-1-
dependent loss of somatic fat. These data define a SKN-1 and p38-dependent axis for
coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional
redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic
consequences of aberrant transcriptional activity.
Significance statement
The transcription factor SKN-1, the C. elegans ortholog of mammalian NRF2, mediates
cytoprotective responses to diverse stresses to restore cellular homeostasis. We have
discovered that pathogen exposure drives the rapid loss of somatic lipids in a SKN-1 dependent
manner. The activation of innate immunity genes by SKN-1 facilitates resistance to pathogen-
derived toxins, but this occurs at the expense of organismal lipid homeostasis, which impairs
organismal health later in life. Importantly, this program is malleable and the loss of lifelong
health can be restored, albeit at the cost of acute pathogen resistance. These data define a
new physiological basis for the decline in health associated with pathogen infection.
112
Introduction
The transcription factor SKN-1, the C. elegans ortholog of mammalian NRF2, mediates
cytoprotective responses to diverse stresses to restore cellular homeostasis [248]. For
example, SKN-1 induces the transcription of phase II detoxification genes during oxidative
stress, promotes adaptation to proteotoxic and metabolic stress, and drives the induction of
innate immune effector genes during pathogen exposure [249, 250].
Although SKN-1 activation in response to stress facilitates survival, when left unchecked,
aberrant activation of SKN-1 and its mammalian ortholog NRF2 can have negative pathological
outcomes in worms [6, 138] and humans [251, 252]. Specifically, constitutive activation of SKN-
1 shortens lifespan [6, 77] and causes a reorganization of fat from the soma to the germline,
termed age-dependent somatic depletion of fat (Asdf) [138]. Thus, although the activation of
cytoprotective transcription factors is obligatory for maintaining homeostasis when organisms
encounter stressful environments, the inability to turn off or control these transcriptional
responses can be detrimental [6, 77, 251].
Here, we show that redirecting activated SKN-1 by modulating the epigenetic landscape
abolishes negative metabolic outcomes associated with its aberrant activation, but at the cost of
increased innate immune function. Specifically, abolishing H3K4me3 epigenetic marks directed
SKN-1 activity away from innate immunity targets, thus, reestablishing pathogen sensitivity
while also restoring health-promoting, age-dependent outcomes, including homeostatic
distribution of lipids, restoration of stress resistance, and increased lifespan.
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Results
SKN-1 activation drives the post-developmental expression of innate immunity pathway
genes
Our previous studies established that activation of SKN-1 results in the age-dependent
loss of somatic lipids [138]. We demonstrated this finding by activating SKN-1 using
environmental factors, loss of a negative regulator, or in a skn-1(lax188) gain-of-function (gf)
mutant (skn-1gf). To define the extent of transcriptional dysregulation in skn-1gf mutants at the
time of somatic lipid depletion, we performed RNA sequencing (RNA-seq) on day 2 adult
worms, when these animals begin to display the Asdf phenotype [138] (Figs 1A-1C; SI
Appendix, Fig. S1). The skn-1gf mutants display dysregulation of 1,986 genes (1376
upregulated and 610 downregulated) as compared to wild type (Dataset S1).
Interestingly, genes induced in skn-1gf mutants were strongly enriched for immune and
pathogen response by Gene Ontology (GO) analysis (Figs 1D-E; Dataset S1), in addition to
oxidative stress response and xenobiotic detoxification (Figs 1D-1F; Dataset S1). Of note, the
major SKN-1-reponsive genes were represented in this dataset [7, 77, 176, 252, 253] (Dataset
S1). The transcription profile of day 2 adults was distinct from previous transcriptional analyses
of skn-1gf mutants during development [77], which do not display GO-term enrichment for
innate immunity and pathogen-related genes (Fig. 1G; Dataset S1). This finding suggests that
the age-related negative outcomes of skn-1gf mutants, like the loss of somatic fat at day 2 of
adulthood, may stem from an increase in expression of innate immune response genes.
H3K4 methylation by WDR-5 is required for SKN-1-dependent loss of somatic lipids
We performed chromatin immunoprecipitation (ChIP) followed by qPCR of SKN-1 and
SKN-1gf protein at day 2 of adulthood and observed enrichment of SKN-1gf relative to wild-type
SKN-1 at the promoters of several genes, which we identified by RNA-seq (Fig. 1; Dataset S1);
selecting representative genes from the innate immunity, oxidative stress, and metabolism GO
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classes, including: endu-2, dod-24, W06H8.2, clec-66, F17A4.9, and acs-14 (Fig. 2A; Dataset
S1). We hypothesized that manipulating epigenetic modifications to restrict the transcriptional
activity of the skn-1gf mutants could mitigate the pleiotropic effects of SKN-1 constitutive
activation. Methylation of Histone H3 at lysine 4 (H3K4me) is an established effector of
transcriptional activity and several conserved protein complexes regulate H3K4 di- and tri-
methylation states [254]. With this in mind, we screened a panel of epigenetic chromatin
modifiers by RNA interference (RNAi). We found that reducing the expression of wdr-5 or rbbp-
5–two SET1/MLL-like proteins that influence the H3K4me3 state–restored somatic fat in skn-1gf
mutant animals (SI Appendix, Fig. S2). This finding suggests that H3K4me3 mediates
physiological outcomes in the skn-1gf mutant.
We confirmed this genetic relationship in wdr-5lf(ok1417);skn-1gf double mutants at day
2 of adulthood; noting that the wdr-5lf mutant had wild-type lipid distribution between the soma
and germline and that skn-1gf does not impact H3K4me3 levels in the wdr-5lf background (Figs
2B-2D; Figs S3A-S3B). In support of the molecular connection between SKN-1gf activity and
H3K4me3 epigenetic marks, the loss of wdr-5 attenuated the shortened lifespan [77] phenotype
of skn-1gf mutants (SI Appendix, Fig. S3C). As previously documented, the skn-1gf(lax188)
allele enhances resistance to hydrogen peroxide exposure when reproduction begins (SI
Appendix, Fig. S3D), but this resistance is lost when reproduction ceases, as somatic lipids are
depleted (SI Appendix, Fig. S3E) [138]. Loss of wdr-5 restored resistance to acute oxidative
stress in the skn-1gf mutant background at this later stage of life. Recent studies have linked
oxidative stress [255] and lipid mobilization [256] to cold stress tolerance. Similar to the
oxidative stress responses, skn-1gf mutant animals at day 2 of adulthood were more sensitive
to exposure at 2 C than wild-type animals and this sensitivity was suppressed in the absence of
wdr-5 (Figs S3F-S3G); thus, cold stress resistance is associated with the abundance of somatic
lipids. It should be noted that both wdr-5lf and wdr-5lf;skn-1gf worms develop at a slightly slower
rate as compared to wild type and skn-1gf animals (SI Appendix, Fig. S3H). However, even if
115
examined 24 hours later, at day 3 of adulthood, wdr-5lf;skn-1gf continue to display a
suppression of Asdf (SI Appendix, Fig. S3I). Taken together, these data reveal the importance
of H3K4me3 marks for SKN-1-dependent metabolic and stress responses.
Loss of H3K4 tri-methylation impacts SKN-1 activation of innate immunity genes
Next, we performed RNA-Seq analysis on wdr-5lf;skn-1gf double mutants to measure
the impact of loss of H3K4me3 on SKN-1 transcriptional activity (Fig. 2E; Dataset S2). The
genes with the most significant reduction in expression, when compared to skn-1gf mutants,
were the pathogen-resistance GO-term class of genes (Fig. 2F; SI Appendix, Fig. S4A and
Dataset S2). Surprisingly, the expression of oxidative and redox homeostasis genes remained
largely unchanged (Fig. 2G; SI Appendix, Fig. S4B and Dataset S2). These data revealed that
H3K4me3 can influence the transcriptional focus of constitutively activated SKN-1 between
classes of target genes and that redirecting the transcriptional focus away from pathogen
response genes, but not oxidative stress genes, can alleviate the negative metabolic outcomes
of deregulated SKN-1 transcriptional activity (SI Appendix, Fig. S4C).
Redirection of activated SKN-1 from innate immunity genes abates metabolic
dysfunction
Our finding that oxidative stress gene targets were generally unaltered in the wdr-5lf;skn-
1gf double mutants was unexpected, given the established role of H3K4me3 as a
transcriptionally activating epigenetic mark and the impact its loss has on skn-1gf stress
responses. Our previous study has shown that acute exposure to hydrogen peroxide can
induce a loss of fat phenotype in wild type animals [138]. We wanted to test how continuous
expression of oxidative stress response genes would affect fat levels in an age-dependent
manner. To test this, we used a subliminal amount of the superoxide-generating oxidant
paraquat (75 M), in order to chronically induce an oxidative stress response, and found no
apparent change in somatic or germline lipid pools in wild-type animals (SI Appendix, Fig. S5A).
Unexpectedly, this treatment suppressed the Asdf phenotype of skn-1gf animals, restoring their
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somatic lipid stores (Figs 3A-3C; SI Appendix, Fig. S5B). We next compared the transcriptional
profiles of skn-1gf mutant animals treated with paraquat to vehicle treatment (Fig. 3D; Dataset
S3). Exposure to paraquat maintained, and for some genes enhanced, the expression of SKN-
1-dependent antioxidant pathway genes (Fig. 3E; SI Appendix, Fig. S6A; and Dataset S3) but,
remarkably, paraquat treatment reduced the expression of pathogen response genes that are
activated in skn-1gf (Fig. 3E; SI Appendix, Fig. S6B; and Dataset S3). Consistent with this
finding, chromatin immunoprecipitation revealed that the association of SKN-1gf with the
promoter regions of innate immunity genes was markedly reduced (Fig. 3G); utilizing dod-24
and endu-2 as representative reporters of innate immunity and SKN-1 responsive genes (Fig.
1E) [77, 253, 257, 258] . Importantly, association of SKN-1gf with the promoter of the oxidative
stress genes gst-4 and W06H8.2, previously identified as SKN-1-responsive to oxidative stress
[259], was enhanced with PQ treatment (Fig. 3G). SKN-1 regulates transcriptional targets that
perform highly diverse functions [77, 253, 260-263], but these data reveal that even when
activated, the protective responses regulated by SKN-1 can be refined to suit the current need
to restore homeostatic balance (Fig. 6C). Moreover, our discovery reveals the important finding
that the focus of activated transcription factors, as probed using SKN-1gf mutants, can be
interrupted and redirected to improve health.
Pathogen exposure drives the rapid loss of somatic lipids
Because C. elegans feed on bacteria, they represent an established model to study
host-microbe interactions [264]. Although live bacteria are routinely used as a food source, C.
elegans can be maintained on dead bacteria and the loss of colonization and proliferation of
bacterial cells in the intestine can increase lifespan [133]; supporting the notion that, compared
to other microbial diets, the standard OP50 E. coli diet is modestly pathogenic [133, 265]. With
this in mind, we examined lipid distribution in skn-1gf animals raised on dead bacteria and
observed suppression of Asdf (Fig. 4A). We also examined animals moved to plates with 5-
fluoro-2′-deoxyuridine (FUdR)-treated bacteria, which inhibits bacterial proliferation, and
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observed a similar suppression of Asdf (Fig. 4A). Taken together, these data suggest that the
mobilization of lipids from the soma to the germline at the end of reproduction in our skn-1gf
animals require an interaction with live and proliferating bacteria.
Based on this finding, we explored whether immune activation itself was sufficient to
drive lipid redistribution. In C. elegans, the p38 MAPK PMK-1 pathway is a canonical regulator
of innate immune defenses [266-268]. Following pathogen exposure, NSY-1/MAPKKK (the C.
elegans ASK1 homolog) phosphorylates a p38 MAPK cascade with one of the final targets
being SKN-1 [249, 250, 269]. We first examined the lipid distribution of animals harboring a
nsy-1gf allele, which drives constitutive immune activation [270]. Interestingly, nsy-1gf animals
mobilize lipids from the soma to the germline in an age-dependent manner; just like the Asdf
phenotype observed in skn-1gf mutants (Figs 4B-4C; SI Appendix, Fig. S7A). Importantly, this
loss of fat phenotype requires SKN-1 activity as both skn-1(zu135) loss-of-function (lf) allele and
skn-1 RNAi suppressed lipid redistribution in the nsy-1gf mutant background, thus confirming
the placement of SKN-1 downstream of NSY-1/MAPKKK signaling for changes in metabolic
homeostasis (Fig. 4D; SI Appendix, Fig. S7B). Taken together with the transcriptome profiling
data of the skn-1gf mutants, these data indicate that activation of SKN-1 by the NSY-1-PMK-1
pathways and the subsequent transcription of innate immunity target genes, drives the loss of
somatic lipids.
To determine whether physiological activation of innate immune defenses in the context
of pathogen infection is sufficient to drive changes in metabolic homeostasis, we exposed wild-
type and skn-1gf mutant animals to the opportunistic human pathogen Pseudomonas
aeruginosa, which also infects and kills nematodes [130, 264, 271, 272]. Post-developmental
exposure to P. aeruginosa resulted in the rapid depletion of somatic lipids (Figs 4E-4F; SI
Appendix, Fig. S8A). Importantly, exposure to the virulence-attenuated pseudomonal mutant
gacA or rhlR [130, 273, 274], which do not robustly activate C. elegans immune defenses [275],
did not cause loss of somatic fat (Fig. 4F; SI Appendix, Fig. S8B-S8C). We previously
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demonstrated that the loss of somatic lipids in skn-1gf mutants was mediated by lipid transport
from the soma to the germline by vitellogenins [138]. Similarly, vit-5 RNAi treated animals
displayed reduced somatic fat loss when exposed to Pseudomonas aeruginosa, indicating a
shared mechanism of lipid loss between skn-1gf mutants and pathogen exposure (SI Appendix,
Fig. S8D). In addition, loss of somatic lipids during infection with P. aeruginosa was accelerated
in skn-1gf mutants (SI Appendix, Fig. S8E) and delayed in skn-1 RNAi treated animals (SI
Appendix, Fig. S8F-8G). It is noteworthy that the genes with the largest changes in expression
in wild-type animals exposed to pathogens are also activated in skn-1gf mutant animals on non-
pathogenic bacteria, (e.g., fmo-2, clec-71, cpt-4, sodh-1, clec-60, cyp-34A4, F53A9.8, C34C6.7,
T07G12.5, M60.2, ugt-18, Y65B4BR.4 (for full list see Dataset S1) [258, 266, 276]. Taken
together, these findings demonstrate that the immune response to pathogenic bacteria involves
SKN-1-dependent redistribution of somatic lipids and provides evidence of a sophisticated
relationship between these previously unconnected essential aspects of organismal
homeostasis.
Somatic lipid abundance is associated with pathogen sensitivity
Our data suggest that skn-1gf animals would potentially display enhanced resistance to
pathogen exposure. The effects of pathogens on host physiology are a result of the presence of
the microbe itself and the toxins it secretes [277]. Exposure of wild-type animals to P.
aeruginosa can result in “slow-killing” of C. elegans, which reflects the colonization of C.
elegans intestine and subsequent lethal infection. Altering the media on which the P.
aeruginosa are grown prior to C. elegans exposure results in hyperproduction of phenazine
toxins, which causes rapid intoxication of nematodes, an assay which is also called “fast-killing”
[271, 272]. Although each of the modes of killing have been well-studied, several aspects of
pathogenesis remain unanswered, but it is clear that it is multifactorial. We challenged wild-type
and skn-1gf animals to the fast-kill and slow-kill models of pathogen-induced death.
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Interestingly, skn-1gf mutants were resistant to P. aeruginosa fast killing (Fig. 5A; SI Appendix,
Fig. S9A), but surprisingly the effect in the slow-killing scenario was unremarkable (Fig. 5B).
The pathogen resistance phenotype of skn-1gf animals was suppressed in the absence
of wdr-5, which is consistent with our findings that loss of H3K4me3 epigenetic marks on
chromatin diminishes the transcriptional output of SKN-1-associated pathogen defense genes.
Collectively, these findings support a new biological framework for host-pathogen responses
where the presence of a pathogen drives SKN-1 activation of pathogen defense genes (Fig.
5C). This program enables resistance to pathogen-derived toxins, but occurs at the expense of
organismal lipid homeostasis, which impairs organismal health later in life. Importantly, this
program is malleable and the loss of lifelong health can be restored, albeit at the cost of acute
pathogen resistance, by dampening SKN-1 activity at these specific gene targets.
Activated SKN-1 drives preferential utilization of diet-derived and short unsaturated
lipids
Several events that immediately precede somatic lipid depletion in animals with SKN-1
activation have been described [138]; however, a full account of the metabolic state once
somatic lipids have been depleted is lacking. We applied a tandem transcriptomic and lipidomic
assessment of animals at the peak of somatic lipid depletion (Day 2 of adulthood). We
examined our RNA-seq data (Datasets S1-S4) for changes in the expression of lipid metabolism
genes in animals with activated SKN-1 that was dampened in the absence of H3K4me3 or when
SKN-1 was redirected by PQ treatment. Several metabolism genes fit these criteria (Fig. 6; SI
Appendix, Fig. S10), including mitochondrial and peroxisomal B-oxidation genes acox-1.3, acs-
7, and acs-14 (Fig. 6A); lipolysis genes lipl-1 and lipl-3 (Fig. 6B); and lipid binding genes like
lbp-7 (Fig. 6C). We also measured a reduction in several lipid biosynthesis genes, including
cpt-6 and fat-5 whose expression is restored when SKN-1 is redirected (SI Appendix, Fig.
S10A-S10B). These data reveal that SKN-1 activation induces a metabolic state where lipid
120
utilization pathways are activated while de novo biosynthesis is suppressed; both of which could
contribute to the observed depletion of somatic lipids.
Our previous studies of L4-stage skn-1gf mutant animals identified the differential
abundance of multiple lipid species as compared to age-matched wild-type animals before Asdf
occurs [138]. To connect the transcriptomic analysis with the lipid signature of animals with a
depletion of somatic lipids, we used quantitative Nile Red staining of lipids [278] and gas
chromatography coupled to mass spectrometry (GCMS) [279]. At day 2 of adulthood, skn-1gf
animals have 60% less total fat compared to wild-type animals (Fig. 6D; SI Appendix, Fig.
S10C) and we identified several classes of lipids with differential abundance in skn-1gf mutants
as compared to wild-type animals (Figs 6E-6H). Specifically, bacterial diet-derived
cyclopropane fatty acids (Fig. 6E) and synthesized 16:1 and 18:2 species (Fig. 6F) were all
reduced. There was also a modest increase in monomethyl branched-chain fatty acids (Fig. 6G)
and a more significant increase in synthesized mono- and poly-unsaturated fat species of longer
lengths, including eicosatetraenoic acid 20:4(n-3) lipid species (Figs 6F and 6H). The relative
abundance of these lipids in skn-1gf mutants is an important biomarker as it defines the lipid
species that are a hallmark of Asdf and also which lipid species are lost in response to SKN-1
activation.
Taken together, our data identify redirection of an activated cytoprotective transcription
factor, like SKN-1, as a powerful mechanism to abate pleiotropic consequences of unchecked
transcriptional activity, including: age-dependent loss of lipid homeostasis that normally
accompanies pathogen infection and chemosensitivity, respectively (Fig. 7).
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Discussion
We report a mechanism by which the pleiotropic outcomes stemming from the activation
of SKN-1 can be ameliorated by redirecting transcriptional activity. Modulation of transcriptional
outputs, rather than inactivation of the transcription factor, provides a new model for the
treatment of pathologies stemming from transcriptional dysregulation.
Importantly, we report the rapid loss of somatic lipid stores as one of the earliest
documented pathological symptoms of pathogen exposure, which is dependent on SKN-1
activity. Although early responses to pathogens are essential for optimal health, the long-term
effects of these responses are uncharacterized. Our data reveal that SKN-1 activation depletes
somatic lipids while providing resistance to pathogen infection. However, aberrant SKN-1
activation ultimately impedes oxidative stress resistance and shortens lifespan. Dampening the
innate immunity axis of SKN-1 activation increases lifespan, restores oxidative stress
resistance, and reestablishes the healthy distribution of lipids, but compromises the ability to
survive pathogen challenge.
We previously found that depletion of the monounsaturated fatty acid oleate drives the
redistribution of somatic lipids in skn-1 gain-of-function mutants, or Asdf phenotype [138]. Of
note, oleate is also necessary for the pathogen-mediated induction of host defense genes and
resistance to diverse bacterial pathogens [280]. Taken together with data from this study that
immune activation during pathogen infection drives the Asdf phenotype, these studies
emphasize that oleate sufficiency is a key marker of health, depletion of which triggers
protective reallocation of somatic fat and suppression of immune defenses, thereby allowing
energy reserves to be devoted to the preservation of evolutionary fitness. Oleate is also
required for proper immune defenses in plants [281-283], which suggests that cyto-protective
re-distribution of host lipids and energy stores is evolutionarily ancient.
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Our previous work showed that diet can influence loss of somatic lipids in the context of
our skn-1gf worms, as the bacteria E. coli OP50/B leads to fat loss but not E. coli HT115/K-12
[138]. This was not the case for exposure to P. aeruginosa, as virulence attenuated strains of
the pathogen did not lead to depletion of somatic lipids. Thus, some aspect associated with P.
aeruginosa virulence drives somatic lipid depletion, which is partially dependent on SKN-1, as
RNAi against skn-1 attenuates the lipid depletion. Our observation that animals with reduced
SKN-1 activity eventually lose somatic fat following exposure to P. aeruginosa indicates the
presence of additional factors that mediate pathogen-dependent fat loss. Additionally, we
observed that wild-type animals exposed to P. aeruginosa displayed a rapid loss of somatic
lipids–about 4-6 hours for fat loss to occur. The speed of this response is intriguing as the "slow-
killing" model requires bacteria to colonize the intestine and during the first 24 h of exposure
there are neither disease symptoms nor appreciable mortality. Moreover, SKN-1gf mutant
animals, which are already primed for an innate immune response, deplete somatic lipids even
more rapidly than wild-type animals and loss of SKN-1 attenuates the somatic lipid depletion in
animals with constitutive activation of p38 signaling. As these host-pathogen interactions are
mediated, at least in part, by intestinal epithelial cells [258, 268, 270, 284], the loss of lipids in
this tissue is one of the first detectable physiological responses to pathogen exposure.
H3K4 methylation marks are epigenetic signatures of active gene expression, but they
are not essential for all transcriptional activity [285] and their deposition at loci after transcription
to maintain an active transcriptional state has been documented [286]. Our transcriptomic
analysis revealed that loss of H3K4me3 resulted in reduced expression of certain genes but not
others. For instance, in the skn-1gf animals the loss of H3K4me3 marks suppressed the
expression of innate immune targets, but not oxidative stress response genes. The observation
that loss of H3K4me3 marks has differential effects on the expression of specific stress
response genes could indicate a role for these marks for the recruitment of additional
transcriptional regulators beyond SKN-1. Additionally, H3K4me3 can also facilitate
123
transcriptional re-initiation, which can impact total transcriptional output of specific loci [287]. It
should be of note that our RNAseq was performed on whole worms, which precludes tissue
specific resolution of H3K4me3-sensitive targets. Nevertheless, our study identifies that the
maintenance of H3K4me3 epigenetic marks is critical for the regulation of lipid homeostasis and
pathogen resistance. Our previous study indicates a relationship between the Asdf phenotype
and a reallocation of lipids to the germline to increase reproductive output. Loss of WDR-5
activity can impact reproduction, but the loss of wdr-5 exerts effects on progeny output at
temperatures higher than those used in this study [288].
In contrast to acute hydrogen peroxide exposure, chronic exposure to a low dose of
paraquat (a superoxide-producing agent) suppressed Asdf. Our discovery that paraquat
exposure refines the transcriptional focus of activated SKN-1 suggests that transcriptional
redirection is a powerful approach to curb the negative pleiotropies of unregulated
transcriptional activity. It remains possible that low dose exposure to paraquat reduces the
pathogenicity of the OP50 E.coli diet. Exposure to much higher concentrations of paraquat can
result in reduced E. coli B growth; however treatment with 75uM only slightly impaired OP50
viability (SI Appendix, Fig. S5C-S5D). Intriguingly, E. coli K-12 strains that do not induce Asdf
[138] are resistant to the effects of paraquat [289], which support further examination of dietary
effects on SKN-1-dependent pathogen responses. Nevertheless, our results reveal that the
induced expression of oxidative stress response genes does not drive the metabolic pleiotropies
observed in skn-1gf animals. The shared suppression of pathogen response genes suggests
the activation of immune responses drives the Asdf phenotype in skn-1gf mutants (Dataset S4).
Moreover, the similarities in phenotypes between wild-type worms exposed to pathogens and
our skn-1gf worms support our transcriptional data that it is the expression of innate immunity
genes in our mutant worms that is driving its associated pleiotropies. Importantly, our data also
reveal that once activated, SKN-1 remains responsive and can be redirected away from
pathogen response genes to alleviate negative health outcomes. Determining whether the
124
ability to redirect other transcription factors, like NRF2, is a generalizable approach to treat
diseases with aberrant transcription is an exciting avenue for future investigation.
125
Methods
C. elegans and bacterial strains used and culturing methods
Worms were grown following standard culture protocols at 20 C, unless otherwise noted.
The following strains were used wild-type (WT): N2 Bristol strain, SPC227: skn-1(lax188),
RB1304: wdr-5lf(ok1417), SPC415: wdr-5lf(ok1417); skn-1gf(lax188), RPW43: nsy-1(ums8),
LG340: skn-1(zu135) and SPC425: nsy-1gf(ums8); skn-1(zu135).
Oil Red O (ORO) Staining
ORO staining lipids was performed as previously described [84, 138, 278, 290]. In brief,
synchronized worms were collected using 1 mL of 1X PBS + 0.01% Triton X-100 (PBST) into a
microcentrifuge tube and allowed to gravity settle. Supernatant was aspirated and washed three
additional times with 1 mL PBST. After the final wash, supernatant was removed until 100 L
was left and then 600 L of 60% isopropanol was added, and samples were rocked for 3
minutes at room temperature. Samples were spun down at 25xg and supernatant was removed
until 100 L was left. Worms were then stained for 2 hours with 600 L of ORO working
solution (0.5g of ORO in 100mL of 100% isopropanol) while rotating at room temperature and
de-stained in 600 L of PBST for 30 minutes. All staining was done on synchronous
populations of day 2 adult animals (120 hours post-feeding starting from a synchronous L1
population), unless otherwise stated.
Asdf Quantification
ORO-stained worms were placed on glass slides and a coverslip was placed over the
sample. Worms were scored, and images were taken using a Zeiss microscope at 10X
magnification. Fat levels of worms were placed into 3 categories: Non-Asdf, Intermediate, and
Asdf. Non-Asdf worms display no loss of fat and are stained a dark red throughout most of the
body (somatic and germ cells). Intermediate worms display significant fat loss from the somatic
tissues, with portions of the intestine being clear, but ORO-stained fat deposits are still visible
126
(somatic < germ cells). Asdf worms have had most, if not all, observable somatic fat deposits
depleted (germ cells only).
Hydrogen Peroxide (H2O2) Stress Survival Assay
Acute exposure to H2O2 was performed as previously described [138, 207]. In brief,
age-synchronized worms were exposed to 10 mM H2O2 in M9T at room temperature while
rotating for 20 minutes. Samples were washed in 1 mL of M9T three times and then dropped
directly on the OP50 bacterial lawn on an NGM plate. Scoring of dead or alive worms was done
24 hours post H2O2 exposure. Worms were considered dead either when they were
unresponsive to gentle prodding from a platinum-tipped wire or when they showed signs of
internal larval hatching.
Pathogen Exposure Experiment
An overnight culture of P. aeruginosa strain PA14 (WT), PA14 gacA, or PA14 rhIR
mutant was seeded on 6 cm “slow-killing” media plates, prepared as previously described (25).
Synchronized L1 stage C. elegans of the indicated genotypes were grown to the Day 0 adult
stage then transferred to seeded plates and incubated at 20°C for indicated time points. Plates
were incubated at 20°C to slow the kinetics of the P. aeruginosa-induced Asdf phenotype.
Worms were then collected and stained for fat and scored for fat levels as previously described
[130, 291]. P. aeruginosa “fast kill” pathogenesis assays were conducted as previously
described [271, 272]. Late L4 animals obtained from timed egg lays were used.
127
Figures

Figure 5.1. SKN-1 activation causes redistribution of somatic lipids and activation of
immune defense genes.
(A) Normal lipid distribution in wild-type animals (representative image from n=873) as
compared to (B) somatic depletion of fat (Asdf) in animals with activated SKN-1 (representative
image from n=759) that occurs with age (C). (D) Gene Ontology (GO) term enrichment analysis
of differentially-expressed genes from RNA-seq of Day 2 adult skn-1gf mutant animals as
compared to age-matched wild-type (WT) controls. Analysis of the mRNA reads of the indicated
genes related to (E) innate immunity and pathogen resistance and (F) oxidative stress and
xenobiotic responses; see Dataset S1 for all RNA-seq measurements. (G) Venn diagram of
gene expression changes with at least two-fold change in skn-1gf(lax188) mutants during
development (L4 stage) and day 2 of adulthood. See also Dataset S1 and S4 for all RNA-seq
measurements. Scale bar = 50 m. ****P < 0.0001.
128

Figure 5.2. Loss of Histone H3 trimethylation restricts SKN-1gf transcriptional activity
and suppresses the loss of somatic lipids.
(A) ChIP-qPCR reveals SKN-1gf enrichment at the promoter of target genes relative to wild-type
SKN-1. (B-C) Oil-Red-O staining of lipid stores in C. elegans. (B) wdr-5lf(ok1417) mutants
display normal distribution of lipids across tissues (representative image from n=415). (C) The
loss of somatic lipid stores observed in SKN-1gf mutant animals are restored in wdr-
5lf(ok1417);skn-1gf(lax188) double mutants (representative image from n=892). (D)
Quantification of lipid distribution across tissues. All experiments were performed in a minimum
of three biological replicates and lipid distribution was assessed in at least 300 animals. (See
also SI Appendix, Fig. S3 for all measurements). (E) Volcano plot of differentially expressed
genes in skn-1gf compared to WT (red), genes altered by loss of wdr-5lf (green), and innate
immunity genes (black); changes in immune genes analyzed by unpaired, non-parametric, t-test
(Mann-Whitney). (F-G) Analysis of the mRNA reads of the indicated genes related to (F) innate
immunity and pathogen resistance and (G) oxidative stress and xenobiotic responses. See
Dataset S2 and S4 for RNA-seq measurements. Scale bar = 50 m. *P < 0.05; **P <0.01;
****P < 0.0001.
129

Figure 5.3. Oxidative stress redirects SKN-1gf transcriptional activity while restoring
somatic lipid distribution.
(A-C) The absence of somatic lipids in SKN-1gf mutants treated with vehicle (control) (A) is
restored in animals exposed to 75 µM paraquat (PQ) (B) (representative image from n=237);
arrows indicate somatic lipids. (C) Quantification of lipid distribution across tissues. All
experiments were performed in a minimum of biological triplicate and lipid distribution was
assessed in at least 300 animals. (See also SI Appendix, Fig. S5 and Dataset S3 for all
measurements). (D) Volcano plot of differentially expressed genes in skn-1gf compared to WT
(red), genes altered by PQ exposure (blue), and innate immunity genes (black); changes in
immune genes analyzed by unpaired, non-parametric, t-test (Mann-Whitney). (E-F) Analysis of
the mRNA reads of the indicated genes related to (E) oxidative stress and xenobiotic responses
and (F) innate immunity and pathogen resistance. See also Dataset S3 and S4 for RNA-seq
measurements. (G) ChIP-qPCR reveals PQ treatment redirection of SKN-1gf activity is
associated with a loss of recruitment at innate immunity gene promoters. Scale bar = 50 m.
*P < 0.05; ***P < 0.001; ****P < 0.0001.
130

Figure 5.4. Exposure to pathogens drives a rapid and SKN-1-dependent loss of somatic
lipids.
131
(A) Somatic lipid depletion requires live bacteria. (A) Bactericidal treatment of OP50 bacteria
with UV+Antibiotics (“Killed”) or FUdR reduces Asdf in skn-1gf animals relative to mock-treated
bacteria. (B-C) Constitutive activation of the p38 MAPK pathway in nsy-1gf(ums8) mutant
animals display age-dependent somatic depletion of fat (Asdf) at 144 hours post feeding;
representative image (B) and quantification of population (C), n = 254. (D) Lipid reallocation in
nsy-1gf(ums8) mutant animals requires skn-1. (E-F) Exposure of 72 hours post fed animals to
P. aeruginosa results in the rapid loss of somatic lipid stores; representative image (E) and
quantification of population (F), n = 292. All experiments were performed in a minimum of
biological triplicates and lipid distribution was assessed in at least 300 animals. Scale bar =
50 m. ****P < 0.0001.
132

Figure 5.5. Redirection of SKN-1gf negates pathogen resistance.
(A-B) Pathogen sensitivity assays. Wild-type (black), skn-1gf(lax188) mutants (red), wdr-
5lf(ok1417) mutants (blue), or wdr-5lf(ok1417);skn-1gf(lax188) double mutants (green) were
exposed to P. aeruginosa (A) “fast-kill” or (B) “slow kill” pathogen stress plates. Activated SKN-
133
1 supports resistance to pathogenic P. aeruginosa specifically on fast-kill plates, which is
abolished in the absence of H3K4me3. (C) Model for the impact of SKN-1gf on physiological
responses to P. aeruginosa secreted factors “fast killing,” but perhaps not on colonization “slow
killing.” All experiments were performed in a minimum of biological triplicates and lipid
distribution was assessed in at least 300 animals. Scale bar = 50 m. ****P < 0.0001.
134

Figure 5.6. SKN-1 activation drives lipid utilization during Asdf.
(A-C) Analysis of the mRNA reads of the indicated genes related to lipid utilization: (A) lipid
beta-oxidation and (B) lipolysis; and (C) lipid binding, which collectively reveal a scenario where
activated SKN-1 drives lipid utilization while suppressing de novo synthesis (see also SI
Appendix, Fig. S10). (D) Fixed Nile Red staining reveals skn-1gf(lax188) mutants have a ~60%
reduction in total fat (n=30). (E-H) GCMS analysis of total fatty acids in the triglyceride fraction
of wild-type (black) and skn-1gf(lax188) mutant (red) animals fed an OP50-diet at day 2 of
adulthood when Asdf is most prominent, two-tailed t-test. See also Datasets S1 thru S4 for all
RNA-seq measurements. *P < 0.05; **P <0.01; ***P < 0.001; ****P < 0.0001.
135

Figure 5.7. Transcriptional redirection of SKN-1 activity mitigates pleiotropic outcomes.
In the presence of pathogens, SKN-1 is activated (yellow arrow) and induces the expression of
several classes of target genes (e.g., oxidative stress and pathogen resistance). The induction
of pathogen resistance genes drives the depletion of somatic lipids with age that leads to poor
health outcomes. Transcriptional redirection (green arrows) does not completely turn off all
SKN-1 activity (red arrow), but rather focuses the gene targets that are activated. Redirection of
activated SKN-1 away from pathogen resistance genes and toward oxidative stress genes
abates the loss of metabolic homeostasis.
136
Supplemental

Fig. S5.1. Categorization of fat levels for wild-type and skn-1gf worms. Representative
images of lipid level distribution for ORO stained wild-type (A) and skn-1gf (B) worms, along
with population of worms scored and proportion of population displaying each phenotype at 120
hours post feeding. At this time point, skn-1gf worms display a strong loss of somatic fat
phenotype, while a majority of wild-type worms retain fat throughout their body. The criteria for
lipid level categorization is described in the methods. All experiments were performed in a
minimum of three biological replicates and lipid distribution was assessed in at least 300
animals. Scale bar = 50μm.
137

Fig. S5.2. RNAi screen of chromatin modifiers effect on Asdf levels in skn-1gf. RNAi
screen of skn-1gf worms done using OP50 RNAi clones. Chromatin modifiers associated with
H3K4me3 complex, wdr-5 and rbbp-5, suppressed the Asdf phenotype in skn-1gf worms. The
RNAi screen was performed in biological triplicate and 50 worms were scored for each 100
experiment. Two independent wdr-5 RNAi clones tested positive. set-2 RNAi animals were
developmentally delayed and asynchronous at time of scoring.
138

Fig. S5.3. Loss of wdr-5 suppresses pleiotropic phenotypes of skn-1gf animals. (A)
Representative images of lipid level distribution for ORO stained wdr-5lf;skn-1gf worms, along
with population of worms scored and proportion of population displaying each phenotype at 120
hours post feeding. Loss of wdr-5 suppresses fat loss in skn-1gf worms. (B) wdr-5lf abolishes
H3K4me3 chromatin marks. (C) skn-1gf worms have a shortened lifespan, which is partially
rescued by loss of wdr-5. Graph shows the combination of three biological replicates of the
lifespan assay with >50 worms in each replicate (after censoring – WT n=127; skn-1gf n=241;
139
wdr-5lf; skn-1gf n=304). (D-E) skn-1gf mutants are resistant to oxidative stress early in
adulthood (80hpf) (D) that is lost later in life (120hpf) (E). This increased sensitivity is abolished
in the absence of WDR-5. (F-G) Worms at 48 hours post feeding display similar survival levels
following cold stress assay except for wdr-5lf;skn-1gf, which has an increased survival
percentage, possibly due to the slower growth rate of the double mutant and reflecting the
younger developmental stage of the animal at the time of the assay (F). (G) At 144 hours post
feeding, skn-1gf worms display a decreased survival to cold stress, which may be due to the
loss of fat phenotype seen during this age. This decreased survival is suppressed by the wdr-
5lf(ok1417) mutation. (H) wdr-5lf(ok1417) animals reach egg laying adulthood at a slower rate
(less than 24 hours) than wild type animals. (I) wdr-5lf;skn-1gf display suppressed levels of
Asdf. ****P<0.0001 Fisher’s exact two-tailed test used to compare stress survival percentage
and log-rank test for lifespan analysis. All experiments were performed in a minimum of three
biological replicates and lipid distribution was assessed in at least 300 animals. Scale bar =
50μm.
140

Fig. S5.4. Comparison of transcript levels in wdr-5lf;skn-1gf relative to skn-1gf animals at
120 hours post feeding using RNA-seq. (A-B) RNA-seq analysis of wdr-5lf;skn-1gf worms
reveals genes associated with innate immune responses (A) are downregulated in wdr-5lf;skn-
1gf worms when compared to skn-1gf worms, but oxidative stress genes (B) remain at the
same. This suggests that the pleiotropic consequences suppressed in the wdr-5lf;skn-1gf
double mutant may be due to the suppression of innate immune response genes. (C) Model of
the impact of loss of Histone H3 trimethylation evokes on the transcriptional activity of SKN-1
and physiological responses. **P<0.01, ***P<0.001. ****P<0.0001
141

Fig. S5.5. Representative images of fat level categorization for WT and skn-1gf worms
treated with paraquat. Representative images of lipid level distribution for ORO stained (A)
wild-type and (B) skn-1gf worms treated with 75 μM paraquat (PQ), along with the population of
worms scored and proportion of population displaying each phenotype at 120 hours post
feeding. Exposure to 75 μM of PQ results in a relatively unchanged lipid level distribution in wild-
type worms, while skn-1gf worms show a suppression of the Asdf phenotype and an overall
increase in lipid levels compared to untreated skn-1gf worms. (C-D) PQ treatment does not
abolish OP50 bacteria growth. OP50 was inoculated in either LB (C) or LB + 75uM PQ (D) and
grown overnight with shaking. Cultures were then diluted and 5μl plated on LB and allowed to
grow to assess culture viability by colony count. All experiments were performed in a minimum
of three biological replicates and lipid distribution was assessed in at least 200 animals. Scale
bar = 50μm.
142

Fig. S5.6. Comparison of transcript levels in skn-1gf worms that were untreated or
treated with paraquat at 120 hours post feeding. RNA-seq analysis of skn-1gf worms treated
with paraquat (PQ) reveals that oxidative stress 150 genes (A) remain unchanged or increased
when compared to untreated skn-1gf worms. Read counts for genes associated with the innate
immune response (B) are significantly lower in PQ-treated skn-1gf worms compared to
untreated skn-1gf worms. This trend in gene expression remains consistent with wdr-5lf;skn-1gf,
another suppressor of the Asdf phenotype in skn-1gf worms. (C) Depiction of overall change in
transcriptional focus of SKN-1gf activity in response to oxidative stress. *P<0.05, **P<0.01,
****P<0.0001
143

Fig. S5.7. nsy-1gf worms display a similar age-dependent fat loss as skn-1gf worms. (A)
Representative images of lipid level distribution for ORO stained nsy-1gf worms, along with the
population of worms scored and the proportion of the population displaying each phenotype at
144 hours post feeding. Constitutive activation of nsy-1, a MAP3K involved in the innate
immune pathway, causes worms to undergo a similar age-dependent depletion of lipids in
somatic tissues at 144 hours post feeding. (B) This loss of fat at 144hpf is skn-1 dependent as
RNAi of nsy-1gf worms for skn-1 seems to result in more non-Asdf worms when compared to
L4440 RNAi. All experiments were performed in a minimum of three biological replicates and
lipid distribution was assessed in at least 250 animals. ****P<0.0001 Fisher’s exact two-tailed
test used to compare survival percentage. Scale bar = 50μm.
144

Fig. S5.8. Exposure to the pathogen Pseudomonas aeruginosa results in SKN-1
dependent fat loss and is attenuated by non-virulent strains. Representative images of the
lipid level distribution for wild-type worms exposed for 4 hours to P. aeruginosa (A) and 2 other
non-virulent mutants gacA (B), or rhIR (C). Worms were stained with ORO 72 hours post
feeding. The population of worms scored and the percentage displaying each phenotype are
provided. Wild-type worms exposed to pathogens exhibited a similar fat loss phenotype to that
seen in skn-1gf and nsy-1gf worms. This phenotype is not observed in wild-type worms exposed
to non-virulent mutant pathogens. (D-E) The loss of somatic lipids in response to P. aeruginosa
exposure is attenuated in vit-5 RNAi treated animals (D) and enhanced in SKN-1gf mutants (E).
(F-G) RNAi of skn-1 delays somatic fat loss in response to pathogen. At least two biological
replicates were performed and a minimum of 100 animals were analyzed in each replicate.
*P<0.05, ****P<0.0001 Fisher’s exact two-tailed test used to compare non-Asdf to Asdf worms.
145

Fig. S5.9. Adult animals are resistant to P. aeruginosa fast-killing. Wild type (black), skn-
1gf(lax188) mutants (red), wdr-5lf(ok1417) mutants (blue), or wdr-5lf(ok1417);skn-1gf(lax188)
double mutants (green) were exposed to Pseudomonas aeruginosa “fast kill” as young adults
(YA). As previously described, post-developmental animals are less sensitive to P. aeruginosa
fast-kill exposure as compared to larval stage 4 (L4) animals.
146

Fig. S5.10. Metabolic phenotypes of SKN-1gf activity and subsequent redirection. (A-B)
Analysis of the mRNA reads of the indicated genes related to metabolism. (C) Representative
images of Nile Red (NR) stained wildtype and skn-1gf(lax188) mutants reveals a 60% reduction
in total lipids at day 2 of adulthood. See also tables S1 thru S4 for all RNA-seq measurements.
*P < 0.05; ***P < 0.001; ****P < 0.0001 by two-tailed t-test. Scale bar = 50μm.
147
Acknowledgements
We thank the Caenorhabditis Genetics Center, funded by NIH Office of Research
Infrastructure Programs (P40 OD010440) for providing some strains, and WormBase. We also
thank Melanie Trombly for comments on the manuscript. This work was supported by NIH
grants R01GM109028 (S.P.C.), R01AG058610 (S.P.C.), R01AI130289 (R.P.-W.),
T32AG000037 (J.N., D.L.R., and H.M.D.), T32GM118289 (C.D.T.), T32AI007349 (S.M.A.),
F31AG051382 (H.M.D.), R01AG058256 (A.A.S.) and P30DK040561 (A.A.S.), and research
support from the American Federation for Aging Research (C.-A.Y. and S.P.C.).
Competing Interests: The authors declare no competing interests.
Author Contributions: S.P.C. designed the experiments and wrote the initial manuscript.
J.D.N. carried out the majority of the experiments, with assistance from C.D.T., S.M.A., C.-A.Y.,
H.M.D., D.L.R., H.K.C., A.A.S., R.P.-W. and S.P.C. The pathogen experiments were optimized
by S.M.A., H.K.C., N.U.N, C.M.H. and R.P.-W. R.P-W., J.N. and S.P.C. revised the manuscript.
Data and materials availability: Sequencing data are deposited to the Gene Expression
Omnibus (accession no. GSE123531). All other data needed to evaluate the conclusions are
presented in the main text or supplementary materials.
148
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Abstract (if available)

Abstract Exposure to environmental stress is clinically established to influence male reproductive health, but the impact of normal cellular metabolism on sperm quality is less well-defined. Here we show that impaired mitochondrial proline catabolism, reduces energy-storing flavin adenine dinucleotide (FAD) levels, alters mitochondrial dynamics toward fusion, and leads to age-related loss of sperm quality (size and activity), which diminishes competitive fitness of the animal. Loss of the 1-pyrroline-5-carboxylate dehydrogenase enzyme alh-6 that catalyzes the second step in mitochondrial proline catabolism leads to premature male reproductive senescence. Reducing the expression of the proline catabolism enzyme alh-6 or FAD biosynthesis pathway genes in the germline is sufficient to recapitulate the sperm-related phenotypes observed in alh-6 loss-of-function mutants. These sperm-specific defects are suppressed by feeding diets that restore FAD levels. Our results define a cell autonomous role for mitochondrial proline catabolism and FAD homeostasis on sperm function and specify strategies to pharmacologically reverse these defects.

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The therapeutic effects of dihydromyricetin (DHM) on anxiety disorders

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Physiology of the inner ear: the role of the biophysical properties of spiral ganglion neurons in encoding sound intensity information at the auditory nerve

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Creator Yen, Chia-An (author)

Core Title The role of mitochondria in the male reproductive capacity of Caenorhabditis elegans

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 07/29/2020

Defense Date 06/11/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aging,alh- 6/ALDH4A1,Asdf,C. elegans,diet-gene pairs,E. coli,FAD,fecundity,fertility,fertility assays,flavin cofactor,germ cells,germline,H3K4me3,innate immunity,Lipids,male-specific,metabolism,mitochondria,NAC,OAI-PMH Harvest,oocyte quality,p38,pathogen,proline catabolism,Pseudomonas,reproduction,reproductive senescence,riboflavin,ROS,senescence,SKN-1,soma,sperm quality,Spermatogenesis,vitamin C,WDR-5

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Curran, Sean (committee chair), Phillips, Carolyn (committee member), Pike, Christian (committee member), Tower, John (committee member)

Creator Email c33yen@gmail.com,chiaanye@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-349835

Unique identifier UC11665922

Identifier etd-YenChiaAn-8809.pdf (filename),usctheses-c89-349835 (legacy record id)

Legacy Identifier etd-YenChiaAn-8809.pdf

Dmrecord 349835

Document Type Dissertation

Rights Yen, Chia-An

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA