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Mitochondrial dynamics regulate Leydig cell health and integrity
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Mitochondrial dynamics regulate Leydig cell health and integrity
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Content
Copyright 2023 Samuel Kenneth Garza
Mitochondrial Dynamics Regulate
Leydig Cell Health and Integrity
By
Samuel Kenneth Garza
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
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2023
ii
Dedication
To my wife, Angela Mossler Garza, who has always been my strongest champion, unwavering
supporter, and sage confidant throughout the entirety of this journey. Throughout this
unpredictable journey, it was your love that provided clarity. For my family, who have nurtured
the seeds of my inquisitive and curious nature throughout my life and therefore supported its
vibrant bloom. You’ve helped foster a mind that dares to question and strives to understand. To
my friends, whose patience and understanding with my passion for learning deserves more than
gratitude.
iii
Acknowledgements
I would like to acknowledge and express my sincerest gratitude towards my PhD advisor,
Dr. Vassilios Papadopoulos. His unwavering patience and empathetic understanding have been
instrumental throughout my development. There were numerous moments in the beginning
when my skills fell short, and yet, he persistently offered his guidance with patience and
compassion. He not only guided me academically but also helped me to cultivate a broader
scientific perspective, an invaluable asset that will serve me for the rest of my life.
I would like to express gratitude and appreciation to Dr. Martine Culty for her support and
advocacy. Her dedication to students and relentless pursuit of inclusivity within the realm of
pharmaceutical education has been transformative in my personal journey. Had not been for her
faith in my capabilities and her unwavering support, I would not find myself in the position I am
in today. She has played a monumental role in shaping my path and empowering me in my
academic pursuits.
I am immensely grateful to the Minority Opportunities in Research (MORE) Programs at
California State University, Los Angeles for their mentorship and guidance. The values, skills, and
insights I gained from this program guided me through my academic aspirations. Their efforts to
enrich diversity in scientific research has fostered an environment of growth, learning, and
opportunity for students like myself.
I am grateful for the greater University of Southern California community, specifically the
Graduate Student Government (GSG), Undergraduate Student Government (USG), University
administrators, alumni, and the Board of Trustees. The tireless work of these individuals has been
iv
instrumental in enriching my student experience and providing invaluable opportunities for my
professional development.
Finally, I wish to extend my heartfelt gratitude to the current and former members of the
Papadopoulos and Culty labs who never hesitated to lend support throughout this journey.
Working and interacting with Dr. Yuchang Li, Dr. Lu Li, Dr. Amy Tran, Dr. Christina Lin, Dr. Melanie
Galano, Dr. Casandra Walker, Dr. Maia Corpuz, Dr. Haoyi Cui, Dr. Liting Chen, Garett Cheung,
Nidia Espinoza, Alex Zambidis, Nicole Mohajer, Christopher Victory, Elsa Sheikhpour, Amina Khan,
Priyanka Malusare, Priyadarshini Bahadure, Mahima Chandrakant Raul, Nrupa Dinesh Patel,
Hangyu Wu, Jiayi He, and Zhihang Shen enriched my time in the lab. In particular, I'd like to
express my deepest appreciation for Chantal Sottas, whose invaluable support was pivotal in the
successful execution of my animal studies. Her expertise and dedication have enriched my
research experience significantly.
v
Table of Contents
DEDICATION ................................................................................................................................... ii
ACKNOWLEDGMENTS ................................................................................................................... iii
LIST OF FIGURES ......................................................................................................................... viii
ABSTRACT ...................................................................................................................................... x
CHAPTER 1: INTRODUCTION .......................................................................................................... 1
1.1 Abstract ........................................................................................................................... 1
1.2 Introduction .................................................................................................................... 2
1.3 The role of mitochondrial dynamics in cell function ....................................................... 3
1.4 Testicular function and cell types .................................................................................... 4
1.5 Leydig cell development .................................................................................................. 5
1.6 Steroidogenesis ............................................................................................................... 8
1.7 Requirements and conditions for Leydig cell steroidogenesis ...................................... 11
1.7.1 Integrity of the plasma membrane LH receptor signal transduction cascade
responsible for sensing and responding to the blood-borne LH ....................... 12
1.7.2 Availability of sufficient amounts of the substrate cholesterol coming from
the blood or synthesized de novo ..................................................................... 12
1.7.3 Integrity of the mechanism responsible for transporting cholesterol from
intracellular stores into mitochondria ............................................................... 13
1.7.4 Availability of appropriate levels and combinations of the nuclear
transcription factors controlling the expression of proteins involved in
cholesterol transport and in testosterone biosynthesis .................................... 13
1.7.5 Maintenance of appropriate organelle structures required for optimal
testosterone formation ..................................................................................... 14
1.7.6 Appropriate spatial and temporal expression of steroidogenic enzymes ......... 14
1.8 Genetic disruptors of steroidogenesis .......................................................................... 15
1.8.1 Mutations in the LH receptor ............................................................................ 16
1.8.2 Congenital adrenal hyperplasia (CAH) ............................................................... 16
1.8.3 CYP Mutations ................................................................................................... 16
1.8.4 Mutations in the steroidogenic acute regulatory protein (STAR) ..................... 16
1.8.5 TSPO mutations ................................................................................................. 17
1.8.6 Mutations in steroidogenic factor 1 .................................................................. 17
CHAPTER 2: STRATEGIES FOR TARGETING TESTOSTERONE DEFICIENCY ...................................... 19
2.1 Abstract ......................................................................................................................... 19
2.2 Introduction .................................................................................................................. 20
2.3 Testosterone regulation and formation ........................................................................ 23
2.4 Mechanisms of Leydig cell dysfunction ......................................................................... 27
vi
2.4.1 Reductions in steroidogenic enzymes ............................................................... 28
2.4.2 Imbalanced antioxidant and reactive oxygen species production .................... 29
2.4.3 Reduced mitochondrial function of Leydig cells ................................................ 30
2.5 Endogenous targets for testosterone recovery therapy ............................................... 30
2.5.1 TSPO ligands ...................................................................................................... 32
2.5.2 VDAC1 peptides ................................................................................................. 34
2.5.3 Implantation of human Leydig-like cells ............................................................ 36
2.6 Conclusions ................................................................................................................... 37
CHAPTER 3: MITOCHONDRIAL DYNAMICS AS A TARGET OF LEYDIG CELL DYSFUNCTION ............. 39
3.1 Abstract ......................................................................................................................... 39
3.2 Introduction .................................................................................................................. 40
3.3 Materials and Methods ................................................................................................. 44
3.3.1 Cell Culture ........................................................................................................ 44
3.3.2 Animals .............................................................................................................. 44
3.3.3 Primary Leydig Cell Isolation ............................................................................. 45
3.3.4 Overexpression of Opa1 in Cultured Leydig Cells .............................................. 46
3.3.5 Mitochondrial Fusion Promoter M1 Treatment ................................................ 46
3.3.6 Immunoblot Analysis ......................................................................................... 47
3.3.7 Measurement of Steroid Hormones ................................................................. 48
3.3.8 Measurement of Cellular Respiratory Function ................................................ 49
3.3.9 Quantitative Reverse Transcription PCR (RT-qPCR) .......................................... 49
3.3.10 Mitochondrial Imaging .................................................................................... 50
3.3.11 Immunofluorescence and Confocal Microscopy ............................................. 51
3.3.12 Statistical Analysis ........................................................................................... 51
3.4 Results ........................................................................................................................... 52
3.4.1 Tspo deletion decreased mitochondrial function in MA-10 Leydig cells ........... 52
3.4.2 Increased mitochondrial fusion restores bioenergetics in TSPO-deficient
Leydig cells ........................................................................................................ 54
3.4.3 Characterization of Opa1 transfection and M1 treatment in MA-10 and nG1
Leydig cells ........................................................................................................ 56
3.4.4 Mitochondrial fusion improved mitochondrial morphology in TSPO-
deficient Leydig cells ......................................................................................... 59
3.4.5 M1 treatment improves bioenergetics and steroid formation in aged rat
Leydig cells ........................................................................................................ 61
3.5 Discussion ...................................................................................................................... 63
CHAPTER 4: EXPLORING THE RELATIONSHIP BETWEEN THE SYSTEMIC REGULATION OF
MITOCHONDRIAL DYNAMICS AND LEYDIG CELL FUNCTION ..................................................... 69
4.1 Abstract ......................................................................................................................... 69
4.2 Introduction .................................................................................................................. 70
4.2.1 Autosomal Dominant Optic Atrophy ................................................................. 72
4.2.2 Behr syndrome .................................................................................................. 72
4.2.3 Dominant optic atrophy plus syndrome ............................................................ 73
vii
4.2.4 Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
spectrum disorder ............................................................................................. 73
4.3 Materials and methods ................................................................................................. 75
4.3.1 Animals .............................................................................................................. 75
4.3.2 Mitochondrial Fusion Promoter M1 Treatment ................................................ 75
4.3.3 Purification of Leydig Cells with Magnetic Activated Cell Sorting (MACS) ........ 76
4.3.4 Immunoblot Analysis ......................................................................................... 78
4.3.5 Measurement of Steroid Hormones ................................................................. 79
4.3.6 Measurement of Cellular Respiratory Function ................................................ 79
4.3.7 Transmission electron microscopy (TEM) ......................................................... 80
4.3.8 Statistical Analysis ............................................................................................. 80
4.4 Results ........................................................................................................................... 81
4.4.1 Characterization of MACS-isolated Leydig cells ................................................ 81
4.4.2 Promoting mitochondrial fusion in Leydig cells isolated from aged Sprague
Dawley rats enhances bioenergetics ................................................................. 83
4.4.3 Administration of M1 led to a decrease in weight and testosterone levels in
rats .................................................................................................................... 85
4.4.4 Effect of M1 injection varies by tissue .............................................................. 87
4.5 Discussion ...................................................................................................................... 89
CHAPTER 5: CONCLUSIONS AND FUTURE PERSPECTIVES ON THE ROLE OF MITOCHONDRIAL
DYNAMICS IN LEYDIG CELL DYSFUNCTION AND OTHER DISEASE PATHOLOGIES ...................... 92
5.1 Summary ....................................................................................................................... 92
5.1.1 Summary of Chapter 2 ...................................................................................... 93
5.1.2 Summary of Chapter 3 ...................................................................................... 94
5.1.3 Summary of chapter 4 ....................................................................................... 95
5.2 Challenges ..................................................................................................................... 97
5.2.1 Role of mitochondrial dynamics regulating biological processes ...................... 97
5.2.2 Formulation, dosage, route of administration, pharmacodynamics, and
pharmacodynamics ........................................................................................... 97
5.2.3 Inherent discrepancies between in vitro, in vivo, and animal models .............. 98
5.3 Future Perspectives ....................................................................................................... 99
5.3.1 Contribution to the field of age-related testosterone decline .......................... 99
5.3.2 Relationship between mitochondrial contact sites and the steroidogenic
interactome ....................................................................................................... 99
5.3.3 Further exploration of the progression of age-related disease pathologies ... 100
5.4 Conclusions ................................................................................................................. 100
REFERENCES ............................................................................................................................... 102
viii
List of Figures
Figure 1. Schematic representation of the steps involved in testosterone formation .................. 7
Figure 2. Off-target effects of existing therapeutic strategies for hypogonadism ...................... 22
Figure 3. Steroidogenic InTeractomE (SITE) proteins of the Leydig cell ....................................... 26
Figure 4. TSPO deletion decreased mitochondrial function in MA-10 Leydig cells ...................... 53
Figure 5. Increased mitochondrial fusion restores bioenergetics in TSPO-deficient Leydig
cells .............................................................................................................................................. 55
Figure 6. Characterization of Opa1 transfection and M1 treatment in MA-10 and nG1
Leydig cells ................................................................................................................................... 57
Figure 7. Increased expression of OPA1 in MA-10 and nG1 after M1 treatment or Opa1
transfection .................................................................................................................................. 58
Figure 8. Mitochondrial fusion improved mitochondrial morphology in TSPO-deficient
Leydig cells ................................................................................................................................... 60
Figure 9. M1 treatment improved bioenergetics and steroid hormone formation in aged
rat Leydig cells ............................................................................................................................. 62
Figure 10. Model of Leydig cell bioenergetics and steroid formation in TSPO deficiency and
aging ............................................................................................................................................. 68
Figure 11. Characterization of MACS isolated Leydig cells .......................................................... 82
Figure 12. Promoting mitochondrial fusion in Leydig cells isolated from aged Sprague
Dawley rats enhances bioenergetics ............................................................................................ 84
ix
Figure 13. Administration of M1 led to a decrease in weight and testosterone levels in rats
..................................................................................................................................................... 86
Figure 14. Effect of M1 injection varies by tissue ........................................................................ 88
x
Abstract
The objective of this dissertation is to investigate the role of mitochondrial dynamics in
the maintenance of Leydig cell health and integrity in age-related functional decline, and the role
of mitochondrial dynamic regulation in steroid hormone formation and mitochondrial function.
Testicular Leydig cells are specialized interstitial cells which produce and maintain testosterone,
the primary androgenic steroid hormone in males. Testosterone is a sex hormone which plays a
critical role in the development and maintenance of male health and well-being. Its presence
regulates numerous physiological functions, such as the maintenance of muscle mass, bone
mineral density, mood, sexual function, and many others. The decline in testosterone levels of
aging males is a significant concern and has been linked to significant age-related morbidities and
declining health. Leydig cell functionally declines with aging, and they produce reduced levels of
testosterone. Moreover, mitochondrial function is also compromised in aged Leydig cells.
Ultimately, significant deterioration of the steroidogenic process can lead to testosterone
deficiency, known as hypogonadism.
Testosterone formation in Leydig cells is mediated by a sequential series of signaling and
metabolism events. A protein scaffold containing numerous steroidogenic proteins, dubbed the
steroidogenic interactome, is involved in the translocation of cholesterol from the cytosol into
the mitochondria. The expression of the proteins in the steroidogenic interactome is regulated
by various signaling pathways and transcription factors, which become dysregulated in aging
Leydig cells. The regulation of steroidogenesis is a complex integrated process and the
development of its dysfunction in aging is not fully understood. Herein, we’ve investigated the
role of mitochondrial dynamics in the regulation of Leydig cell steroidogenic function.
xi
We hypothesize that dysregulation of the interplay between mitochondrial dynamics and
the expression of steroidogenic proteins plays a role in the deterioration of Leydig cell health and
integrity with aging. The mitochondria are essential cellular organelles which contain numerous
steroidogenic proteins that are necessary to produce testosterone and other steroid hormones.
These dynamic organelles play a critical role in energy production, cellular metabolism, and cell
function. Mitochondria regulate their shape and structure in response to physiological stimuli
and cellular energy demands. Dysregulation of mitochondrial function has been linked with aging
and the development of numerous neurodegenerative, metabolic, and other diseases. Several
cellular signaling pathways are altered in age-related pathologies, including the regulation of
mitochondrial fission and fusion, leading to the accumulation of fragmented dysfunctional
mitochondria. Therefore, we hypothesized that Leydig cell dysfunction could be attenuated by
enhancing mitochondrial dynamics.
Three aims have been developed to address the objective of this project and the
hypothesis. First, we characterized the dysfunction that manifests in aged and steroidogenic
deficient Leydig cells. Next, we used a combination of treatments to regulate mitochondrial
dynamics in Leydig cells, resulting in the upregulation of mitochondrial fusion. Lastly, rats aged
one year were dosed with the cell-permeable mitochondrial fusion promoter 4-Chloro-2- (1- (2-
(2, 4, 6-trichlorophenyl) hydrazono) ethyl) phenol to alter systemic mitochondrial dynamics.
When taken together, these results support our hypothesis and show that the dysregulation of
mitochondrial dynamics in aged Leydig cells plays a central role in the deterioration of Leydig cell
health and integrity with aging. The dysregulation of mitochondrial dynamics has been associated
xii
with numerous chronic illnesses, and proteins involved in the regulation of mitochondrial
dynamics may be promising biological targets for the maintenance of cell health and function.
1
Chapter 1: Introduction
1
1.1 Abstract
Reduced serum testosterone, hypogonadism, is a condition which affects millions of men
around the world. This condition has been linked to numerous comorbidities, metabolic
dysfunctions, and alterations in quality of life. The production of testosterone is an indispensable
biological process with implications affecting numerous physiological processes. Cholesterol is
the precursor of all steroid hormones and its translocation from cellular stores into the
mitochondria is the rate-limiting step in steroidogenesis. Cholesterol is metabolized into several
intermediate steroid hormones by various enzymes, before being converted to testosterone.
Several systemic and cellular regulators control the rate of steroid formation, which declines with
aging. The declining production of testosterone is a biological concern of interest, as it has been
linked with numerous chronic conditions and diseases. Although the mechanisms of
steroidogenesis are well understood, the development of steroidogenic dysfunction in Leydig
cells is associated with numerous alterations in cell signaling and regulatory pathways,
complicating our understanding of the development of Leydig cell dysfunction. This chapter
explores our current understanding of the mechanisms regulating testosterone biosynthesis, as
well as genetic and environmental factors which affect its production. The existing literature
1
This chapter is derived from 1) the Andrology Handbook chapter entitled “Chapter 5: How is the synthesis of testosterone
regulated?” by Samuel Garza, Martine Culty, and Vassilios Papadopoulos; and 2) the manuscript entitled “Impact of endocrine-
disrupting chemicals on steroidogenesis and consequences on testicular function” by Cassandra Walker, Samuel Garza, Martine
Culty, and Vassilios Papadopoulos
2
evaluating the decline in testosterone formation throughout aging add to our understanding of
its decline throughout the male lifespan.
1.2 Introduction
Testosterone, the major male sexual hormone, is secreted primarily by Leydig cells of the
testis. The amount of testosterone formed by the Leydig cells reflects specific needs of the body
for cell growth, organ formation, masculinization, and maintenance of androgen-dependent
functions. During development, the differentiation of Leydig cell precursors leads to the
establishment of specific Leydig cell populations that are responsible for the formation of the
amounts of testosterone needed at various ages. In the fetus, Leydig cells are considered the
main source of testosterone essential for sexual differentiation and the prenatal masculinization
of the male urogenital system
1
. However, additional sources have emerged from studies in
mouse, where fetal Sertoli cells were shown to generate testosterone from Leydig cell-produced
androstenedione. Moreover, the existence of a “human backdoor pathway of androgen
synthesis” was recently described, in which androsterone produced from placental progesterone
may be as critical for human fetal masculinization as testosterone produced by fetal Leydig cells
and dihydrotestosterone
2
. After birth, the fetal Leydig cell population disappears and the adult
Leydig cell population, evolving in an environment free of maternal factors, develops from a small
pool of undifferentiated, self-renewing stem Leydig cells via a sequence of stages that include
Leydig cell progenitors, immature Leydig cells and adult Leydig cells. These changes reflect the
need for testosterone for development of male characteristics, puberty, and androgen-
dependent functions
1
. Changes in cell structure and gene expression are responsible for the
3
morphological and functional differences among these various cell types; the basic components
of the steroidogenic machinery are present but not used to the same extents
3
. Thus, testosterone
production changes during development are due to alterations in the cellular environment and
are designed for the optimal production of testosterone for specific biological needs.
1.3 The role of mitochondrial dynamics in cell function
Mitochondria are highly dynamic organelles that regulate numerous cellular processes.
Mitochondria produce most of the cellular energy via oxidative phosphorylation. Oxidative
phosphorylation occurs in the cristae of the mitochondria. To maintain cell health and function,
mitochondria undergo morphological changes, such as mitochondrial fusion and fission, which
alter cristae formation and the mitochondrial network
4
. The regulation of mitochondrial function
is crucial for the cell to respond to its environment.
Mitochondrial membrane fusion is mediated by the optic atrophy 1 (OPA1) protein and allows
for the formation of an interconnected network of mitochondrial structures. OPA1 promotes the
tethering of the inner and outer membranes and enhances the formation of contact sites, a
conduit which mediates the import of cellular cargo into the mitochondria
5
. Contact sites have
been proposed to play a role in the translocation of cholesterol into the mitochondria, and
therefore, a role in steroidogenesis
6
. Interestingly, inhibition of mitochondrial fusion causes a
decline in steroid hormone formation and a decline in the expression of steroidogenic proteins,
supporting the premise that mitochondrial fusion is necessary for steroid hormone formation
7
.
4
1.4 Testicular function and cell types
Various organs comprise the male reproductive system, which include the prostate,
epididymis, penis, vas deferens, bulbourethral glands, seminal vesicles, and testes
8
. The teste,
arguably the most critical male reproductive organ, is the site of spermatogenesis and steroid
hormone production
1
. Adult males with healthy and mature reproductive systems have their
testes positioned in the scrotum along with the epididymis, and part of the vas deferens. The
testes are descended from the abdominal cavity into the scrotum through two gestational
phases, between weeks 15 and 35 in humans. This is under the control of testosterone and
insulin-like hormone 3 (INSL3), which is produced by fetal Leydig cells (FLCS)
8
. The development
of internal and external genitalia is regulated by fetal testosterone levels and its metabolite
dihydrotestosterone (DHT). Moreover, estrogens modulate FLC function
2
. The gestational
development of all male reproductive tissues is regulated by the testis. Postnatally, the adult
testis is dedicated to androgen production, the production of spermatozoa, and regulating other
non-reproductive tissues
9
. The regulation of these processes requires tightly regulated hormonal
function, specifically the production of androgens and estrogens at various stages of the male
lifespan, from developmental gestation through adulthood.
The testis comprises germ cells and several types of somatic cells. Each testis is composed of
lobules maintained by connective tissue. Each lobule contains up to four seminiferous tubules,
surrounded by the interstitium. The germinal epithelium lines the inside of the tubules and
comprises the Sertoli cells. Several layers of germ cells maintain its structure and tightly regulate
the stages of spermatogenesis
10
. Sertoli cells provide germ cell nutrients, phagocytose
byproducts from sperm production, secrete hormones including androgen-binding protein (ABP),
5
inhibin, transferrin, and anti-mullerian hormone (AMH). Additionally, Sertoli cells are responsible
for the generation, function, and maintenance of the blood-testis barrier. The blood-testis barrier
is composed of tight junction proteins which rise pre-pubertally and compartmentalize the
tubules into two distinct compartments: the basal compartment and the adluminal
compartment. The adluminal compartment is where immature spermatozoa are released
through spermiation
11
. Spermatogonial stem cells, spermatogonia, and preleptotene
spermatocytes reside within the basal compartment. Cell signaling from Sertoli cells, peritubular
myoid cells, and macrophages regulate and maintain spermatogenesis throughout the male
lifespan
10
. Spermatogenesis occurs in asynchronous waves to support a consistent production of
spermatozoa that mature as they migrate through the epididymis
12
.
Interstitial Leydig cells reside outside of the seminiferous epithelium, scattered throughout
the connective tissue. Leydig cells are highly specialized steroid producing cells which maintain
the circulating levels of testosterone in the male body. Leydig cells contain steroidogenic
enzymes that metabolize cholesterol and produce androgens which are needed for testicular
development, male genitalia differentiation, and reproductive function
2
. The production of
steroid hormones, steroidogenesis, is mediated by a comprehensive network of protein-protein
interactions
13
.
1.5 Leydig cell development
Several intermediate and essential androgens and estrogens essential for organ development
and reproductive differentiation are produced by Leydig cells (Figure 1). 5α-reductase
6
metabolizes testosterone, forming Dihydrotestosterone (DHT), which maintains male sex
characteristics. Moreover, testosterone and androstenedione are metabolized by CYP19A1 to
produce estradiol and estrone respectively. Various Leydig cell types arise throughout male
development and are characterized by different androgen and estrogen levels
14
. Leydig cell
development requires the production and balance of steroid hormones. Fetal Leydig cells (FLCs),
which arise in fetal development, produce testosterone levels that are required for the
differentiation of male genitalia and brain masculinization. These testosterone levels are
produced without stimulation, as the luteinizing hormone (LH) receptor (LHR) is expressed later
in fetal development
1
. Increasing CYP11A1, 3β-HSD, 3α-HSD, CYP17A1, and 5α-reductase
expression throughout fetal development regulates the production of specific hormones
14
. The
development of the urinary tract from the Wolffian ducts requires testosterone
15
. Fetal DHT
regulates genitalia and prostate development. Overall, androgen production arises during fetal
development, peaking before birth, and declining postnatally as the FLC population declines
1
.
Following birth, adult Leydig cells (ALCs) develop from immature Leydig cells (ILCs). Firstly,
stem Leydig cells (SLCs), which lack steroidogenic enzymes and are present in peritubular and
perivascular compartments, give rise to progenitor Leydig cells (PLCs). PLCs express CYP11A1, 3β-
HSD, CYP17A1, 5α-reductase, and 3α-HSD, which allow for the production of androstenedione
1,2
.
Next, PLCs differentiate into immature Leydig cells (ILCs) which express 17β-HSD3, a critical
enzyme which produces testosterone. At this stage, the testosterone which is produced from
ILCs becomes metabolized by 5α-reductase, producing DHT, and further, 5α-androstane-3α and
17β-diol by 3α-HSD. ALCs expressing the LHR develop from ILCs at the onset of puberty, and 5α-
reductase expression diminishes, leading to both increased steroidogenic stimulation and
7
testosterone secretion
2
. The production of testosterone through from ALCs from puberty to
adulthood results in the development of male sex characteristics and spermatogenesis.
Figure 1. Schematic representation of the steps involved in testosterone formation.
Luteinizing hormone (LH) binds to a G-protein coupled receptor leading to activation of
adenylate cyclase (AC) that produces cAMP, the major second messenger of LH action in
Leydig cells. cAMP subsequently activates the cAMP-dependent protein kinase (PKA), an
event that triggers a series of reactions including the de-esterification of cholesterol from
lipid droplets and activation of proteins, PKA substrates, involved in cholesterol transport
into mitochondria. cAMP, as well as PKA act also in the nucleus activating steroidogenic
protein and enzyme gene expression. Free cholesterol is transported and imported into
mitochondria via SITE formed to amplify the effect of LH and cAMP. This complex included
proteins such as the steroidogenesis acute regulatory protein (STAR), translocator protein
(18- kDa; TSPO), and voltage-dependent anion channel (VDAC) 1. Imported cholesterol is
metabolized to pregnenolone by the cytochrome P450 side chain cleavage (CYP11A1).
Pregnenolone is subsequently metabolized in the smooth endoplasmic reticulum by a
series of enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP17A1 and 17β-
hydroxysteroid dehydrogenase (17β-HSD) to form testosterone.
8
1.6 Steroidogenesis
Steroidogenesis is the process of cholesterol translocation and metabolism that result in
androgen formation. Cholesterol is the precursor molecule of all androgens, including
androsterone, androstanediol, 5α-dihydrotestosterone (DHT), androstenedione, and
testosterone. These androgens are required for the development and maintenance of lifelong
functions in males. As described, testosterone regulates the development of internal and
external male genitalia, sex characteristics, and spermatogenesis. The testicular Leydig cells are
the primary source of testosterone in males. Various other androgens and estrogens involved in
male development, including DHT, estrone and estradiol, are also produced by Leydig cells. All
steroid hormones originate from a precursor cholesterol molecule, which is metabolized by
numerous steroidogenic enzymes. Cholesterol may be generated either de novo intercellularly
from acetate, or imported from circulating lipoproteins. This process involves proteins from
different cytosolic, extracellular, and subcellular compartments, including multiple steroidogenic
enzymes
16
. This process relies on specific protein-protein interactions and cellular coordination
for proper biological development.
Steroidogenic function is regulated at two levels: (i) the availability and trafficking of
substrate cholesterol, and (ii) the presence of steroidogenic enzymes. The first level can be
induced by hormones; however, the second level depends on the ability of Leydig cells to carry
out the trophic cascade and protein interactions that are required for steroid hormone
formation. Systemically, steroid biosynthesis is regulated by the hypothalamic-pituitary-gonadal
(HPG) axis
17
. In this axis, the hypothalamus releases gonadotropin-releasing hormone (GnRH),
which stimulates LH secretion from the pituitary gland. LH binds to the Leydig cell LHR, a G
9
protein-coupled receptor, and activates adenylate cyclase to produce cyclic adenosine 3ʹ,5ʹ-
monophosphate (cAMP)
18
. cAMP then binds to protein kinase A (PKA), which rapidly
phosphorylates substrates, such as the Steroidogenic Acute Regulatory protein (STAR). STAR then
participates in the movement of cholesterol to the outer mitochondrial membrane. Cholesterol
from plasma and intracellular membranes is first utilized, followed by cholesterol which is freed
by cholesteryl ester hydrolase (nCEH) that becomes phosphorylated by PKA. Elsewhere,
steroidogenic factor 1 (SF-1; Nuclear receptor subfamily 5 group A member 1; NR5A1) and cAMP
response element binding protein (CREB) are activated and enhance the expression of
steroidogenic genes. Downstream of steroid biosynthesis, testosterone binds the nuclear
hormone androgen receptor (AR), which exerts effects on target tissues such as the brain,
cardiovascular system, and skeletal systems, in addition to its function reproductively
2
.
Androgens can act as positive regulators of gene expression, with the induction of AR, co-
activators, androgen-responsive elements in the promoter region of target genes enhancing
steroid biosynthesis. Conversely, androgens also act as negative regulators of steroid
biosynthesis, as testosterone inhibits the release of GnRH and LH from the hypothalamus and
pituitary, respectively.
Cholesterol transport from the plasma membrane and intracellular stores to the
mitochondria is the rate-limiting step in steroidogenesis
1
. The transport of free cholesterol to the
mitochondria is done via vesicular and non-vesicular transport. Vesicular transport is facilitated
by mitochondrial and endoplasmic reticulum interactions. STAR participates in the non-vesicular
transport of cholesterol as part of a multi-protein scaffold known as the steroidogenic
interactome (SITE) induces cholesterols translocation into the mitochondria
13
. SITE contains
10
cytosolic, mitochondrial, and endoplasmic reticulum proteins. Of these, the cholesterol-binding
protein translocator protein (TSPO) present at the outer mitochondrial membrane plays a role in
Leydig cell steroidogenic function
19
. The multi-protein SITE complex regulates steroidogenic
production, from cholesterol translocation from the outer mitochondrial membrane to CYP11A1
for metabolism
20
. Steroidogenesis is tightly regulated by numerous factors. Disruption of this
regulation can have deleterious effects, as testosterone deficiency or excess testosterone are
both detrimental. Excess testosterone that is produced or administered suppress GnRH and LH
which disrupts Leydig cell steroidogenesis. Testicular testosterone formation is essential for
spermatogenesis, and therefore there is the potential for male infertility
21
. Moreover, aromatase
converts testosterone into the estrogen 17β-estradiol, resulting in alterations to secondary sex
characteristics. Conversely, testosterone deficiency, which is typically observed in aging men and
known as hypogonadism, has been associated with numerous conditions and disruptions to
systemic functions, such as spermatogenesis
22
.
Steroidogenesis requires numerous cytochrome P450 and hydroxysteroid dehydrogenase
enzymes. Cytochrome P450 family 11 subfamily A1 (CYP11A1) is the first steroidogenic
metabolizing monooxygenase and resides in the inner mitochondrial membrane. CYP11A1
catalyzes the conversion of cholesterol via electrons provides from the electron transport chain.
CYP11A1 abundance determines steroidogenic capacity and is regulated by steroidogenic factor
1 (SF-1). Electrons are transported to CYP11A1 by ferredoxin reductase (FdR) and ferredoxin (FdX)
from NADPH to catalyze the conversion of cholesterol to pregnenolone. Pregnenolone is
passively diffused from the mitochondria to the endoplasmic reticulum for subsequent
metabolism via the Δ
4
and Δ
5
pathways. In humans, pregnenolone is primarily processed through
11
the Δ
5
pathway, where CYP17A1 hydroxylates pregnenolone to 17α-hydroxypregnenolone
2
. In
this pathway, electron transfer from flavoprotein P450 oxidoreductase (POR) is facilitated by
cytochrome b5 and phosphorylates the serine residue of CYP17A1 required for this reaction. 17α-
hydroxypregnenolone is then metabolized to dehydroepiandrosterone (DHEA) by an additional
CYP17A1 reaction via its 17,20-lyase activity. The DHEA molecule is then hydroxylated by 17β-
HSD, producing androstenediol. The final two reactions are catalyzed by 3β-hydroxysteroid
dehydrogenase (3β-HSD), where androstenediol is oxidized, forming a ketone group which
becomes isomerized to create testosterone. The metabolizing enzymes at the endoplasmic
reticulum reside in close proximity to one another for the efficient metabolism of intermediates.
In rodents, pregnenolone is primarily processed via the Δ
4
pathway: pregnenolone is metabolized
to progesterone by 3β-HSD, which is then metabolized CYP17A1 and 17β-HSD to produce
testosterone
2
.
1.7 Requirements and conditions for Leydig cell steroidogenesis
The conversion of cholesterol to testosterone is a tightly regulated process influenced by the
pituitary gonadotrophin LH. For Leydig cells to respond to LH and to function optimally, it is
critical that the integrity of proteins involved in steroidogenesis, from the LH receptor to
cholesterol transporting proteins and steroidogenic metabolizing enzymes, are maintained
13
.
There are several key points that are critical for the establishment and optimal function of the
steroidogenic machinery responsible for testosterone synthesis in the Leydig cell
13,14,23-25
:
12
1.7.1 Integrity of the plasma membrane LH receptor signal transduction cascade
responsible for sensing and responding to the blood-borne LH. The LH receptor is
a G protein-coupled receptor whose activation by LH upregulates the production of
cyclic adenosine 3’,5’-monophosphate (cAMP). This step precedes and initiates
cholesterol mobilization and activation of transcription factors that upregulate
steroidogenic genes.
1.7.2 Availability of sufficient amounts of the substrate cholesterol coming from the
blood or synthesized de novo. Leydig cells can synthesize cholesterol de novo from
acetate or source it from plasma lipoprotein, cholesterol esters, and the plasma
membrane for testosterone biosynthesis. Leydig cells can also use receptor-
mediated endocytic uptake to acquire lipoprotein-derived cholesterol (LDL, HDL).
The de-esterification of stored cholesterol provides an ample pool of substrate for
steroidogenesis.
13
1.7.3 Integrity of the mechanism responsible for transporting cholesterol from
intracellular stores into mitochondria. An initial protein scaffold known as the
transduceosome, comprising cytoplasmic and outer mitochondrial membrane
(OMM) proteins, receives cholesterol via vesicular or non-vesicular pathways and
responds to LH/cAMP. Hormone-induced proteins will join this scaffold to
accelerate cholesterol import. OMM proteins further interact with proteins
spanning the OMM and inner mitochondrial membrane (IMM), such as VDAC 1,
mediating cholesterol loading onto the first enzyme of the steroidogenic cascade,
the cytochrome P450 side chain cleavage (CYP11A1). The larger steroidogenic
complex that encompasses cytoplasmic, OMM and IMM proteins is defined as the
Steroidogenic InteracTomE (SITE). Some of the key proteins of the SITE include STAR,
TSPO, VDAC, and 14-3-3 adaptor proteins.
1.7.4 Availability of appropriate levels and combinations of the nuclear transcription
factors controlling the expression of proteins involved in cholesterol transport and
in testosterone biosynthesis. The differential expression of steroidogenic enzymes
is regulated by numerous transcription factors. These precisely regulate steroid
output and Leydig cell function, preventing either testosterone insufficiency or over
production.
14
1.7.5 Maintenance of appropriate organelle structures required for optimal
testosterone formation. Steroidogenic enzymes reside in the mitochondria and
smooth endoplasmic reticulum. The organelles’ integrity and proper function are
essential for normal steroid formation.
1.7.6 Appropriate spatial and temporal expression of steroidogenic enzymes.
Cytochrome P450 monooxygenases and de-hydrogenases are responsible for
metabolizing cholesterol to various intermediates leading to testosterone
formation. The availability of the co-factors is also necessary for steroidogenic
enzyme action.
The concepts of transduceosome and SITE have been instrumental in understanding the
regulation of Leydig cell testosterone production, as well as that of steroidogenic adrenal cortical
cells, which share the main steps of cholesterol transport and steroidogenic cascade. The
identification of the components of the SITE complex, uncovering the spatial organization and
interactions of the cytoplasmic and OMM elements, and the relationships between OMM and
IMM proteins in response to hormone, have led to a better understanding of this dynamic and
plastic network of proteins converging for optimal steroid hormone biosynthesis
13
. This process
can be altered by several factors. In aging, various components of the steroidogenic machinery
fail to function at an optimal level, leading to a decline in androgen formation. In some cases, this
can lead to significantly reduced testosterone, a condition known as hypogonadism
26
. Indeed,
the integrity of the transduceosome seems to be compromised in hypogonadism leading to
15
Leydig cell dysfunction
22
. The accumulation of fat mass, declining energy, alterations in mood,
and decreased bone mineral density are common symptoms of low testosterone levels
27-29
.
Administering exogenous testosterone, testosterone replacement therapy, is commonly used to
ameliorate many hypogonadism symptoms. However, monitoring and maintaining optimal
testosterone levels is challenging and adverse effects have been observed, including
polycythemia, peripheral edema, as well as cardiac and hepatic dysfunction
30,31
. Stimulating
Leydig cells to increase testosterone production is an active area of research with several
identified drug targets and novel chemical entities under investigation
32
. Stem cell-based therapy
to re-establish androgen producing Leydig cells in the body has also been an active area of
research for the treatment of hypogonadism
33
.
1.8 Genetic disruptors of steroidogenesis
Inborn errors in steroid biosynthesis in the testis and the adrenal cortex are linked to
mutations that can be lethal or lead to disease states such as pseudohermaphroditism,
hypogonadism, and infertility. These mutations can impact numerous stages in the steroidogenic
pathway, such as cholesterol transport, and steroid metabolism.
16
1.8.1 Mutations in the LH receptor cause either overactivation or inactivation and disrupt
the development of secondary sex characteristics. Activating mutations stimulate
Leydig cells during fetal and prepubertal stages, causing autonomous testosterone
production and early onset of puberty
34
. Antiandrogen and aromatase inhibitors are
effective at restoring normal prepubertal development. Mutations causing
inactivation of the LH receptor result in resistance to LH stimulation and Leydig cell
hypoplasia (LCH). LCH patients display varying symptoms from hypogonadism to
pseudohermaphroditism.
1.8.2 Congenital adrenal hyperplasia (CAH) is a rare heritable disorder caused by
mutations in enzymes of the steroidogenic pathway, most commonly 21-
hydroxylase, and impacts nearly 1 in 5000-18000 children worldwide. CAH is
characterized by reduced cortisol and aldosterone, and increased progesterone, 17-
OH-prog, and sex steroids resulting in early virilization of the male
35,36
.
1.8.3 CYP Mutations include rare cases of 17α-hydroxylase (CYP17A1), 3β-hydroxysteroid
dehydrogenase and 20,22-desmolase (part of CYP11A1) have been linked to altered
androgen formation and ambiguous genitalia in boys
37
.
1.8.4 Mutations in the steroidogenic acute regulatory protein (STAR), an essential
protein in cholesterol transport, cause mineralocorticoid deficiency and a lipoid
congenital adrenal hyperplasia phenotype among patients STAR mutations also
cause CAH conditions that result in the buildup of lipid droplets in Leydig cells
35
.
17
1.8.5 TSPO mutations limit the translocation of cholesterol into the mitochondria and
cause esterified cholesterol accumulations and disruptions to steroid formation
38
.
1.8.6 Mutations in steroidogenic factor 1, which drives the expression of many
steroidogenic genes, may also result in testicular failure leading to disorders of sex
development
39
.
Testosterone production is also influenced by external factors such as drugs and
environmental compounds. Numerous pharmaceuticals, agricultural and industrial chemicals act
as endocrine disrupting compounds (EDCs) that can affect male reproductive functions and
health, transcriptional regulation, or androgen receptor binding EDCs exposure can occur via
ingestion, inhalation, or skin absorption. Bisphenols, perfluoroalkyls, phthalates, flame
retardants, fungicides, herbicides and parabens, as well as dietary natural compounds have been
reported to exert EDC properties on components of the steroidogenic cascade, such as LH signal
transduction, cholesterol transport, and steroidogenic enzymes
40
. The risk impact of EDCs
exposure on steroidogenesis is not fully understood, given their ability to disrupt various signaling
mechanisms within Leydig cells and other testicular cell types involved in male reproductive
functions. Studies evaluating the impact of individual EDC’s exposure on steroidogenesis may not
accurately represent the risk of EDC mixtures and their metabolites, found at detectable levels in
blood, such as phthalate and pesticide mixtures reported to induce reproductive defects in a
18
cumulative manner despite individual doses being below the no-observed-adverse-effect
levels
41
.
19
Chapter 2: Strategies for targeting testosterone deficiency
2
2.1 Abstract
Reduced serum testosterone affects millions of men across the world and has been linked to
several comorbidities, metabolic dysfunctions, and quality of life changes. The standard
treatment for testosterone deficiency remains testosterone replacement therapy. However,
limitations on its use and the risk of significant adverse effects make alternative therapeutics
desirable. Studies on the mechanisms regulating and synthesizing testosterone formation in
testicular Leydig cells demonstrate numerous endogenous targets that could increase
testosterone biosynthesis, which could alleviate reduced testosterone effects. Testosterone
biosynthesis is facilitated by a conglomerate of cytosolic and mitochondrial proteins that
facilitate cholesterol translocation into the mitochondria, the rate-limiting step in
steroidogenesis. An effective therapeutic approach would be to increase endogenous
testosterone formation by enhancing steroidogenesis in Leydig cells. Numerous ligands for
steroidogenic proteins have been developed which increase steroid hormone formation.
However, off target effects on neurosteroid and adrenal steroid formation may limit their clinical
use. First-in-class biologics, such as voltage dependent anion channel peptides and
transplantation of induced human Leydig-like cells offer advances in the development of specific
strategies that could be used to enhance endogenous steroid formation in hormone deficient
patients.
2
This chapter is derived from the manuscript “Testosterone recovery therapy targeting dysfunctional Leydig cells” by Samuel
Garza and Vassilios Papadopoulos
20
2.2 Introduction
Although some testosterone decline is normal in men of middle and advanced age, some men
have significantly decreased testosterone levels known as hypogonadism. Hypogonadism is a
condition characterized by severe testosterone deficiency and affects nearly 5 million men in the
United States
1
. While hypogonadism is most commonly associated with infertility, it has also
been correlated with other numerous conditions, such as cardiovascular disease, depression,
fatigue, reduced bone mineral density, increased body fat, metabolic syndrome, and declining
muscle mass
27,42
. Hypogonadism can be separated into two categories: primary hypogonadism
and secondary hypogonadism. Primary hypogonadal patients present depleted testosterone
levels due to a suboptimal response to luteinizing hormone (LH) stimulation; whereas secondary
hypogonadism is characterized by low LH levels or low gonadotropin releasing hormone (GnRH)
levels, leading to insufficient steroid hormone biosynthesis
43
. Moreover, primary hypogonadal
patients display increased LH, suggesting that Leydig cell mechanisms are disrupted
44
. The
primary causes of secondary hypogonadism are associated with the pituitary or hypothalamus
4
.
These can be congenital, acquired, or caused by damage to gonadotrophs[4].
Given testosterone’s essential role in spermatogenesis, hypogonadal patients suffer from
infertility
12
. Furthermore, androgen metabolites levels, such as dihydrotestosterone (DHT) and
3α-androstenediol glucuronide (3α-ADG), become imbalanced and cause alterations in
secondary sex characteristics, including muscle mass, body mass index, and facial hair
44
. Patients
may present with fatigue and declining mood, given the ability of neurosteroids to act as positive
or negative regulators of the GABA receptor
45
. There are also numerous congenital and acquired
21
origins of hypogonadism that may manifest throughout the male lifespan
46
. Therapeutic
strategies for endogenous targets to treat hypogonadism from all origins are highly sought.
Numerous exogenous treatment options are available for testosterone deficiency, however,
there are concerns regarding off-target effects (Fig. 2). Testosterone replacement therapy
(TRT)
47
and aromatase inhibitors
28
have been used to elevate serum testosterone and alleviate
symptoms of hypogonadism. TRT involves administering exogenous testosterone at appropriate
intervals, both daily-acting, intermediate acting (1-3 weeks), and long-acting (2-6 months)
47
.
However, this exogenous testosterone leads to hypothalamic-pituitary-gonadal axis (HPG)
imbalance and suppresses the release of gonadotropins
48
. This represses Leydig cell testosterone
biosynthesis, a critical driver of spermatogenesis, and leads to reduced fertility
47,48
. Moreover,
intermediate and long-acting injections may produce serious adverse events (SAEs) including
pulmonary microembolism, anaphylaxis, and polycythaemia
47,49,50
, and an increased risk of
cardiovascular disease and stroke may exist in older men receiving TRT as indicated in recent
studies
51,52
, resulting in the FDA and medical societies cautioning its use
42
. Numerous alternatives
to TRT have been considered
31
. The testosterone metabolite DHT is also used strategically to
treat hypogonadism in some countries
53
. DHT binds to androgen receptors with a greater affinity
than testosterone and provides some relief from symptoms of hypogonadism
54
. The
disadvantages of DHT are its price, increased hemoglobin, increased red blood cell count, and
inferior clinical results when compared to TRT
53,54
. Aromatase inhibitors are also used to prevent
aromatase from converting testosterone to estrogen, thereby, maintaining testosterone levels
55
.
In clinical studies with aromatase inhibitor used for hypogonadal patients, LH levels, free
testosterone, and sexual desire increased
56
. Moreover, aromatase inhibitors may be suitable for
22
hypogonadal patients with increased estrogen levels
54
. However, concerns regarding the effect
of aromatase inhibitors on bone minerals still remain after treatment with the inhibitor letrozole
led to vertebrae deformities in 45% of adolescent males with delayed puberty
57
. The selective
estrogen receptor modulators clomiphene citrate and tamoxifen are also used off-label for the
treatment of primary hypogonadism due to their ability to induce the release of GnRH by the
hypothalamus and subsequently increase the production of the gonadotropins LH and FSH by the
anterior pitutary
16
.
Figure 2: Off-target effects of existing therapeutic strategies for hypogonadism.
Numerous therapeutics that are used to treat testosterone deficiency have off-target
effects on the hypothalamic pituitary gonadal axis, adrenal gland, and testicular Leydig
23
cells. Abbreviations: GnRH, gonadotropin releasing hormone; TSPO, translocator protein;
VDAC1, voltage-dependent anion channel 1
2.3 Testosterone regulation and formation
Testosterone biosynthesis predominantly occurs in testicular Leydig cells and is tightly
regulated by the hypothalamus-pituitary-gonadal (HPG) axis, comprised of the hypothalamus,
pituitary, and testes
58
. In this system, the hypothalamus secretes GnRH which reaches and
stimulates the anterior pituitary gland to release LH. LH acts on the testicular Leydig cell LH
receptor (LHR), a G protein-coupled receptor, and initiates a signaling cascade that mobilizes
cholesterol and increases testosterone biosynthesis
58
. LHR stimulation activates adenylate
cyclase and increases cAMP production and subsequent cAMP-dependent kinase activation
1
.
Mechanistic targets inducing the production of endogenous testosterone in Leydig cells would
be most desirable. Viable drug targets should have specificity, a sustainable response, and
acceptable safety profiles.
The rate-limiting step in steroid hormone biosynthesis is cholesterol’s translocation across
the outer and inner mitochondrial membranes (OMM, IMM) into the mitochondria
1
.
Cholesterol’s translocation into the IMM results in cholesterol side chain cleavage by the
cytochrome P450 CYP11A1, producing pregnenolone
59
. This translocation is mediated through a
multi-protein scaffold termed the Steroidogenic InteracTomE (SITE)
60
. The SITE is comprised of
cytosolic and mitochondrial proteins, of which numerous have become focal points in the search
for endogenous targets that induce steroidogenesis (Fig. 3). Cytosolic SITE proteins include the
acyl-CoA-binding protein (ACBD1/DBI)
61-63
, ACBD3
60
, Sec23ip
60
, steroidogenic acute regulatory
protein (STAR)
64-68
, 14-3-3 proteins
69-71
, and the cAMP-dependent protein kinase-regulatory
24
subunit Iα (PKA-RIα), which is composed of regulatory and catalytic subunits that phosphorylate
STAR with cAMP increases
72
. OMM SITE proteins include the translocator protein (TSPO)
1
, the
voltage dependent anion channel (VDAC1)
1
, and ATPase family AAA domain-containing protein
3A (ATAD3A)
60
, while IMM SITE proteins include the cholesterol side-chain cleavage enzyme
(CYP11A1), ferredoxin (FDX) and ferredoxin reductase (FDR)
60
. The fine details of cholesterol’s
translocation across the mitochondrial membranes are not yet clear, but there are notable
protein-protein interactions that have been elucidated.
VDAC1 and TSPO are the main anchors of the cytosolic proteins to mitochondrial contact
sites
25,60
. ATAD3A bridges the mitochondrial membranes and is involved in contact site
formation, mediating access of cholesterol to CYP11A1
13,73
. The adenine nucleotide translocase
(ANT) protein interacts strongly with VDAC1 to form a contact site complex between the OMM
and IMM, which is involved for the trafficking of molecules across the mitochondrial
membranes
74
, but does not interact directly with the SITE complex as currently identified
13
. In
addition, the IMM optic atrophy 1 (OPA1) protein participates in the formation of contact sites
and mitochondrial fusion between mitochondrial membranes, a process essential for
steroidogenesis
7
. External response to hormonal stimulation initiates STAR targeting to the SITE
complex at the OMM
75
. STAR anchors to the mitochondrial SITE scaffold at VDAC1, a solute-
specific transporter to the IMM
74
, and STAR becomes phosphorylated by PKA
23
. PKA is targeted
to mitochondria by A-kinase anchoring proteins binding to the regulatory subunits to PKA, such
as ACBD3
76
, a protein that interacts with TSPO, and AKAP121
77
, leading to effective translation
and phosphorylation of STAR and conformational changes which would accelerate cholesterol
translocation and optimize steroid formation
59,64
. In response to these changes TSPO is
25
polymerized and cholesterol binding is enhanced
78
, due to TSPO’s high affinity for
cholesterol
13,79,80
. TSPO contains five transmembrane domains with separate cholesterol and
drug binding domains and is highly abundant in the OMM
81
. ACBD1/DBI is an endogenous ligand
of TSPO, involved in hormone-dependent steroid formation
60
. The polymerization of TSPO
strengthens the TSPO-VDAC1 interaction, enhancing cholesterol binding and transport
13,72,82
.
SITE optimization enhances cholesterol translocation across the mitochondrial membranes to
CYP11A1, where FDX and FDR regulate the electrons needed for side-chain cleavage by the
enzyme
13,60,81
. 14-3-3γ and 14-3-3ε are hormonally stimulated and act as negative regulators of
steroidogenesis by delaying maximal steroid hormone formation
69
. Upon hormone stimulation,
14-3-3γ interacts with STAR, limiting its activity in cholesterol transport
69
. Similarly, stimulation
also triggers 14-3-3ε binding to the VDAC1-TSPO complex and regulates cholesterol translocation
into the mitochondria by reducing the rate of transport
69
. Other intracellular regulators of
steroidogenesis include signaling molecules (PDGF, DHH) kinases (MAPK, PKG, CAMKI, AMPK),
and transcription factors (NUR77, MEF2, GATA4)
83,84
. Moreover, numerous nuclear receptors and
protein phosphorylation events are involved in steroidogenesis regulation
85,86
. Steroidogenesis
is also regulated systemically by the HPG axis
1
. It is imperative that steroid hormone synthesis is
precisely regulated, as insufficient or overproduction of steroids is detrimental
1
.
26
Figure 3: Steroidogenic InTeractomE (SITE) proteins of the Leydig cell. Cytosolic, outer
mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and endoplasmic
reticulum proteins interact to facilitate the transfer of cholesterol into the mitochondria and
production of numerous steroid hormones, including testosterone in the endoplasmic reticulum.
Abbreviations: 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-hydroxysteroid
dehydrogenase; ACBD1, acetyl coenzyme A-binding domain 1 or diazepam binding inhibitor;
ACBD3, acetyl coenzyme A-binding domain 3; ATAD3A, ATPase family AAA domain-containing
protein 3A; CYP11A1, cytochrome P450 11A1; CYP17A1, cytochrome 17A1; FDR, ferredoxin
reductase; FDX, ferredoxin; PKA, cAMP-dependent protein kinase; PKA-R, regulatory subunit;
PKA-C, catalytic subunit; Sec23ip, Sec23-interacting protein; STAR, steroidogenic acute
regulatory protein; TSPO, translocator protein; VDAC1, voltage-dependent anion channel 1
27
2.4 Mechanisms of Leydig cell dysfunction
The physiopathology of numerous diseases related to impaired steroid hormone biosynthesis
are mediated by compromised Leydig cell integrity. In aging, the integrity of Leydig cell-specific
mechanisms mediating steroid hormone biosynthesis is compromised. Whereas gene mutations
in key steroidogenic genes can lead to disease phenotypes or lethality, compromised Leydig cell
integrity can be caused by several intracellular factors:
28
2.4.1 Reductions in steroidogenic enzymes. The steroidogenic machinery tightly
regulates and maintains steroid hormone biosynthesis
1
. Declining or aberrant
expression of SITE proteins or other proteins involved in steroidogenesis can occur
at the transport, import, or conversion steps
60
. For example, STAR is constitutively
expressed in Leydig cells, mediating cholesterol transport from intracellular stores
to the mitochondria
87
, and extracellular hormonal stimulation of Leydig cells
increases STAR expression to upregulate cholesterol translocation
88
. Mutations in
Star lead to steroid hormone biosynthesis deficiency and the accumulation of lipids
in testosterone producing cells
35,36
. Moreover, decreased Star/STAR expression with
age reduces cholesterol translocation in aged Leydig cells
89
. Mutations to TSPO also
alter the ability of steroidogenic cells to import cholesterol into the mitochondria
90
.
This results in increased lipid accumulation and disruption of steroid production and
has implications for the hormone biosynthesis in the brain, adrenal glands, and
testis
20,90,91
. TSPO’s decline in aging Leydig cells showed that alterations in
cholesterol import play a role in age-related testosterone decline
23
. Other
downstream steroidogenic enzymes that are decreased in aging include CYP11A1,
HSD3B, CYP17A1, and HSD17B
22
.
29
2.4.2 Imbalanced antioxidant and reactive oxygen species production. Reactive oxygen
species (ROS) are mostly produced by the mitochondria and can compromise the
integrity of cellular machinery and structures
92
. Age-related oxidant/antioxidant
imbalances are correlated with protein, lipid, and DNA damage, linking integrity of
mitochondrial quality control to the development of age-related pathologies
93
.
Oxidant/antioxidant imbalance may arise from increased oxidant production in
Leydig cells, as mitochondrial superoxide production has been observed in aged rat
Leydig cells
94
. While the generation of ATP via the electron transport chain produces
ROS ubiquitously in mammalian cells, Leydig cells also produce ROS through
hormone biosynthesis via mitochondrial and smooth endoplasmic reticulum P450
reactions
95,96
. Changes in biosynthesis can, thus, alter ROS production. Over time,
ROS exposure damages mitochondria and compromises their function, leading to
mitochondrial dysfunction
97
. When left uncleared, dysfunctional mitochondria
produce excessive amounts of ROS which further damage cellular enzymes and
structures
93,98
. Dysfunctional mitochondrial are normally eliminated via
mitochondrial autophagy (mitophagy) and replaced by new mitochondria through
mitochondrial biogenesis
99
. However, this process, which is disrupted in
compromised Leydig cells, causing disruptions to mitochondrial function, cellular
homeostasis, and steroidogenesis
89,93
.
30
2.4.3 Reduced mitochondrial function of Leydig cells. Leydig cell steroidogenic function
and cellular bioenergetics are integrally linked to one another, as steroidogenesis
requires reliable mitochondrial membrane potential and ATP synthesis
100,101
.
Mitochondrial dynamics such as fission, fusion, biogenesis, and mitophagy are,
therefore, required for sustainable steroidogenic capacity
7
. The clearance of
dysfunction mitochondria is mediated by PINK1/PARKIN interactions
97
and the
generation of new mitochondria, mitochondrial biogenesis, is regulated by the
genes Nrf1/2 and Tfam
102
. The trafficking of molecules across the mitochondrial
membranes is mediated through a variety of mitochondrial contact sites, pores, and
transporters all of which are regulated by Mfn1/2, Opa1, and Drp1
99
. Aging leads to
a decline in these genes’ expression systemically across many tissues
103
, and the
reduction of steroidogenic capacity in aging Leydig cells in particular is driven by this
mitochondrial dysfunction
89
. When compared with healthy cells, aged Leydig cells
present depressed ATP levels, mitochondrial biogenesis, and mitophagy. Moreover,
the expression of genes regulating these mitochondrial dynamics are decreased
89
.
2.5 Endogenous targets for testosterone recovery therapy
The role of numerous SITE proteins and steroidogenic regulators have been investigated to
identify endogenous therapeutic targets that induce steroid hormone formation. Several
proteins within the cytosol and mitochondria mediate cholesterol translocation from intracellular
stores to the OMM where the SITE complex resides
13
. Rone et al. investigated the role of
31
numerous steroidogenic and mitochondrial dynamic proteins to elucidate their role in
steroidogenesis
13
. Such investigations revealed that knocking down OPA1, VDAC1, and ATAD3A
had no effect on membrane permeable steroid formation. However, VDAC1 and ATAD3A
knockdowns did reduce hormone induced steroidogenesis, suggesting that OPA1 is not critical
for hormone-induced steroidogenesis
13
. Recently it was shown that upregulating OPA1 via
pharmacological and transfection methods increased TSPO expression and steroid hormone
formation in both basal and hormone-stimulated dysfunctional Leydig cells, suggesting that
OPA1 may play a role in the regulation and formation of the SITE complex
104
. Progress has been
made in targeting SITE proteins to ameliorate testosterone decline. Studies on TSPO ligands and
14-3-3ε peptides (VDAC1 peptides) have offered potential therapeutic strategies for inducing
endogenous testosterone formation. While TSPO ligands enhance cholesterol translocation,
VDAC1 peptides are designed to block the negative regulation of steroidogenesis
70,84,105-108
.
32
2.5.1 TSPO ligands: Engagement of the OMM protein TSPO via a drug ligand-induced
activation stimulates steroid hormone production in vitro and in vivo in rats and
mice
20,81,105,106,109,110
. TSPO possesses high affinity for cholesterol binding, which
leads to its subsequent translocation to the IMM for side-chain cleavage by CYP11A1
producing pregnenolone
111,112
. TSPO’s C terminus plays a key role in the uptake of
cholesterol from the cytosol and translocation into the mitochondria
113,114
, and
disruption of the protein within steroidogenic cells disrupts mitochondrial
cholesterol transport and steroid formation
112,115
. Steroidogenesis and TSPO
expression correlate with one another as shown by disruption of steroidogenesis
with TSPO’s age-related decline in vivo and its ablation in vitro
23,115
. Moreover,
transfection of TSPO into TSPO-disrupted cells restores steroid formation,
demonstrating its indispensable role in steroidogenesis
115
. Numerous studies have
shown that drug ligands targeting TSPO produce enhanced steroid levels in both
MA-10 tumorigenic Leydig cells and isolated primary Leydig cells, as well as
increased serum testosterone levels
105,106,109
. However, serum LH levels may also
become increased following TSPO drug ligand treatment likely due to an effect of
the ligand on brain TSPO
116,117
, suggesting that using this target may enhance
testosterone biosynthesis by either stimulating the Leydig cell steroidogenic
machinery and/or by elevating LH release
105
. TSPO specific ligands are also known
to increase glucocorticoid and corticosteroid levels
20
and have been shown to affect
neurosteroid production
118-120
. Accordingly, the use of TSPO ligands as a therapeutic
approach to treat neurological and psychiatric disorders have also been
33
investigated
121
. Similarly, the use of TSPO ligands may also induce anxiolytic-like
responses, as ligand treatment has been shown to counteract panic attacks in
rodents
122
. While molecular entities targeting TSPO elevate serum testosterone
levels, adrenal steroids and neurosteroids are also affected. Therefore, TSPO ligands
have been proposed as therapeutic agents for the regulation of steroid hormones
in the testis and brain. However, this lack of specificity remains an issue, as TSPO is
expressed in numerous tissues.
34
2.5.2 VDAC1 peptides: New insights into the role of 14-3-3ε in the regulation of
steroidogenesis have made it a promising therapeutic target. 14-3-3 proteins
regulate target proteins by altering activity, post-translational modifications, and
subcellular localization
24
. LHR stimulation initiates the translocation of 14-3-3ε to
the OMM
69
and its recruitment to the TSPO-VDAC1 complex at Ser167 on VDAC1.
There it competes with TSPO for VDAC1 binding and thus reduces cholesterol
import
84
. Blocking the interaction between 14-3-3ε and VDAC1 using cell-
penetrating peptides induces steroid formation in vivo and ex vivo
108
. Aghazadeh et
al. fused a component of the HIV transcription factor 1 (TAT) with the predicted
Ser167 binding motif on 14-3-3ε, creating a cell permeable VDAC1 peptide, TAT-
VDAC1 containing Ser167 (TVS167), which competed with 14-3-3ε for VDAC1
binding
107
. This reduced negative regulation of steroidogenesis by blocking the 14-
3-3ε binding to VDAC1, which led to increased steroidogenesis in vitro and in vivo.
Given the homologous mechanisms of 14-3-3ε between species, the TAT-based
peptide offers a promising approach in humans. Although TAT peptides penetrate
indiscriminately and 14-3-3ε is found in numerous tissues, function is tissue
specific
107
. TVS167 treatment did not significantly increase corticosterone levels in
rats treated with the compound, demonstrating specificity to testicular Leydig
cells
108
. Additionally, the action of the TVS167 peptide induced steroidogenesis
independent of LH and would offer a major improvement in safety when compared
to TRT
123
. The minimal bioactive sequence of the peptide was recently identified,
and we ultimately generated bioactive stable peptide derivatives that can be
35
administered orally and induce T formation in normal and hypogonadal animal
models (manuscript in preparation). Moreover, they demonstrate safety, efficacy,
and target specificity
70,84,107,108
. In summary, these first-in-class biologics make an
excellent candidate for treatment of diseases caused by Leydig cell dysfunction over
other pharmacologic or biologic strategies.
36
2.5.3 Implantation of human Leydig-like cells: The generation of transplantable
testosterone-producing cells offers another alternative for treating pathologies
related to Leydig cell dysfunction. Previously, it was shown that mesenchymal stem
cells (MSCs) were able to differentiate into testosterone producing Leydig cells,
suggesting that healthy Leydig cell populations could be transplanted into
hypogonadal patients
124
. However, MSC isolation produces limited cell numbers and
reduces the clinical application of this method. Recent developments have revealed
human Leydig-like cells (hLLCs) can be generated from human induced pluripotent
stem cells (hiPSCs), which are highly expandable in cell culture
33,125
. Li et al.
demonstrated that hLLCs producing steroidogenic gene expression, steroidogenic
enzymes, and testosterone could be generated by differentiating early
mesenchymal progenitors from hiPSCs while overexpressing steroidogenic factor 1
(SF-1) in culture with dibutyryl-cAMP (dbcAMP), recombinant desert hedgehog, and
human chorionic gonadotropin (hCG)
33
. Given their clinical viability, the
implantation of hLLCs would represent a monumental step forward in treating
diseases related to Leydig cell dysfunction. This strategy could restore testosterone
levels by replenishing testosterone-producing-cell populations in the testicular
environment, leading to the production of endogenous testosterone formation.
37
2.6 Conclusions
Testosterone deficiency impacts the quality of life and wellbeing for millions of men
worldwide, with only limited treatments having undesirable off-target effects
31
. New
understanding of the molecular interactions producing testosterone has laid the foundation for
the development of novel therapeutic strategies (Table 1).
Table 1: Treatment options available for testosterone deficiency and their use in other
indications. Identification of the hormonally regulated multiprotein SITE complex
13
and the
deeper understanding of hormonal stimulation and cholesterol translocation from cytosolic
stores across the OMM and into the IMM for side chain cleavage demonstrates numerous
38
therapeutic targets for various indications related to hormone insufficiency (Table 1)
31,54
.
However, their effects on neurosteroids, adrenal steroids, and the HPG axis have remained a
barrier to safe and efficacious treatment of testosterone deficiency. Apart from VDAC1 peptides,
existing strategies have lacked specificity for testicular Leydig cells and, therefore, have raise
concerns regarding off-target effects. VDAC1 peptides are first-in-class biologics that offer a novel
approach for rescuing intratesticular and serum testosterone formation in hormonally mediated
diseases
107,108
. These therapeutics could be used to restore endogenous testosterone formation
and restore well-being for millions of aging men worldwide.
There are additional mechanisms to uncover. The movement of cholesterol between the
mitochondrial membranes, the relationship between aging and the Leydig cell oxidative
environment, and age-dependent protein-protein interactions remain elusive and are active
areas of research
60
. With more information we may determine the cause of reduced testosterone
and develop interventions that may maintain Leydig cell function. Moreover, targeting the
molecular deteriorations that differ between aging Leydig cells and other aging steroidogenic
tissues could lead to additional testis-specific strategies.
39
Chapter 3: Mitochondrial dynamics as a target of Leydig cell dysfunction
3
3.1 Abstract
The mitochondrial translocator protein (18 kDa; TSPO) is a high affinity cholesterol-binding
protein that is an integral component of the cholesterol trafficking scaffold responsible for
determining the rate of cholesterol import into the mitochondria for steroid biosynthesis.
Previous studies have shown that TSPO declines in aging Leydig cells, and that its decline is
associated with depressed circulating testosterone levels in aging rats. As yet, however, TSPO’s
role in the mechanistic decline in Leydig cell function is not fully understood. To begin to address
the role of TSPO depletion in Leydig cell function, we first examined mitochondrial quality in Tspo
knockout mouse tumor MA-10 nG1 Leydig cells compared to wild-type MA-10 cells. Tspo deletion
caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial
fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1)
overexpression resulted in restoration of mitochondrial function and mitochondrial morphology
as well as in steroid formation in TSPO-depleted nG1 Leydig cells. Leydig cells isolated from aged
rats form less testosterone than Leydig cells isolated from young rats. Treatment of aging Leydig
cells with M1 improved mitochondrial function and increased androgen formation, suggesting
that aging Leydig cell dysfunction may stem from compromised mitochondrial dynamics caused
by the age-dependent Leydig cell TSPO decline. These results, taken together, suggest that
3
This chapter is derived from the manuscript “Mitochondrial dynamics, Leydig cell function, and age-related testosterone
deficiency” by Samuel Garza, Liting Chen, Melanie Galano, Garett Cheung, Chantal Sottas, Lu Li, Yuchang Li, Barry R. Zirkin, and
Vassilios Papadopoulos
40
maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain
or restore testosterone levels with aging.
3.2 Introduction
Testicular Leydig cells are the main sites of testosterone production in men and contribute to
the maintenance of circulating testosterone levels
1
. Testosterone biosynthesis is essential for the
development, function and maintenance of the male reproductive system
1
. At the approximate
age of thirty in humans, testosterone levels begin to decline at a rate of 0.4-2% annually and can
lead to testosterone deficiency, known as hypogonadism
1,26,126
. Due to the importance of
testosterone in biological systems, hypogonadism is accompanied by a number of conditions
including decreased muscle mass, fat mass accumulation, mood changes, fatigue, metabolic
syndrome and others
127
.
The first step in Leydig cell testosterone production involves the metabolism of cholesterol
by the inner mitochondrial membrane enzyme cytochrome P450 side chain cleavage (CYP11A1).
The transfer of cholesterol from intracellular reserves into mitochondria, which is the rate-
limiting step in steroidogenesis, is essential for this process
128
. Possessing a high affinity for
cholesterol, the 18-kDa translocator protein (TSPO) is an essential outer mitochondrial
membrane protein that is abundant in steroidogenic cells
19
. TSPO contains a binding domain
known as the cholesterol recognition amino acid consensus motif with a strong affinity for
cholesterol
19
. Coordinating with a variety of cytosolic and mitochondrial membrane proteins
which form the Steroidogenic InteracTomE (SITE)
60
, TSPO participates in the targeting of
cholesterol to CYP11A1
13,60,129
. Although TSPO expression declines in aging Leydig cells
23
, it is not
41
yet known how this decline may be linked to the development of hypogonadism. Furthermore,
the mechanisms mediating the transport of cholesterol from the outer to the inner mitochondrial
membrane are not fully understood.
Lipids are transported through subcellular membranes via three mechanisms: vesicular
trafficking, facilitated diffusion with soluble lipid transfer proteins, and diffusion across
membrane contact sites. The vesicular trafficking network precludes mitochondria vesicle
transport
130
, and therefore cholesterol trafficking to the mitochondria is likely mediated by
transfer proteins at mitochondrial membrane contact sites
131,132
. Contact sites have long been
identified as a possible route for mitochondrial cholesterol transport
6,133,134
. Contact sites
between mitochondrial membranes are involved in molecular transport and are formed by
proteins localized to the outer and inner mitochondrial membranes
135
. One such mitochondrial
membrane pore protein, the voltage-dependent anion channel (VDAC1), provides passage for a
variety of molecules into the mitochondria
136
and forms a complex with TSPO
13
. Interestingly,
TSPO depletion in Leydig cells disrupts the VDAC/tubulin interaction, suggesting that TSPO
influences mitochondrial pore stability
132
.
Mitochondria are highly dynamic organelles constantly undergoing fusion and fission
throughout their life cycle
137
. These alterations in mitochondrial structure impact a number of
functions, including cellular bioenergetics
4
, mitochondrial degradation
138
, and oxidative stress
92
.
Moreover, mitochondrial fusion is needed for steroidogenesis and declines with aging
139
.
Mitochondrial fusion is mediated through the mitochondrial contact site and cristae-organizing
system (MICOS) complex
140
. Interactions among MICOS proteins in the inner membrane space
bring together the inner and outer mitochondrial membranes
4
. One such protein, the
42
mitochondrial membrane GTPase optic atrophy 1 (OPA1), narrows cristae junctions and increases
contact site formation
5
. Opa1 mRNA is spliced to produce long and short isoforms which promote
limited activity on their own, but together their co-expression improves fusion
141
. Opa1 deletion
results in fewer and more fragmented cristae among mitochondria and a reduction in complex
IV subunits, disrupting mitochondrial respiration
142
. OPA1 was thought to be involved in
steroidogenesis, but its depletion in the tumorigenic MA-10 mouse Leydig line failed to disrupt
hormone biosynthesis and brought into question its involvement
13
. However, redundancies in
the MICOS system and regulation of mitochondrial dynamics remain
4
. Nevertheless, TSPO
expression may influence mitochondrial fusion, as its insertion into the T-cell Jurkat cell line led
to numerous increases in MICOS- related genes
143
. Furthermore, Tspo deletion depolarizes
mitochondrial membrane potential in MA-10 Leydig cells
132
, which destabilizes mRNA cleavage
of the Opa1 isoform producing long OPA1
144
. Along with TSPO decline, imbalanced mitochondrial
fusion dynamics have been observed in aged and dysfunctional Leydig cells
89
. TSPO’s elimination
in MA-10 Leydig cells causes aberrant morphology in mitochondrial contact sites, suggesting that
TSPO influences mitochondrial integrity
132
. However, studies examining mitochondrial dynamics
in aged and dysfunctional Leydig cells are limited.
TSPO is an integral protein in health whose activation can regulate cell death and promote
protective and restorative responses
145
. Despite numerous studies of TSPO’s role in
mitochondrial function
146
, no studies have revealed why its decline in Leydig cells leads to
mitochondrial dysfunction or how this relates to reduced steroid formation. To study the
potential role of TSPO in the regulation of mitochondrial function, we utilized a TSPO knockout
(KO) nG1 MA-10 sub-cell line
132
, isolated Leydig cells from rats containing a Tspo deletion
147
, and
43
Leydig cells isolated from aging rats with reduced TSPO levels
23
. In an effort to restore Leydig cell
mitochondrial function and given TSPO’s association with bioenergetics in Leydig cells, we
promoted mitochondrial fusion and thus contact site formation using two approaches:
overexpression of Opa1 and treatment with the mitochondrial fusion promoter, 4-Chloro-2- (1-
(2- (2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (M1).
44
3.3 Materials and Methods
3.3.1 Cell Culture: MA-10 cells were grown in Dulbecco’s modified Eagle medium/F-12
medium + Glutamax, with 5% heat-inactivated fetal bovine serum (Sigma-Aldrich,
St. Louis, MO, USA), 2.5% heat-inactivated horse serum, and 1%
penicillin/streptomycin at 37 °C and 3.5% CO
2
. The MA-10-derived TSPO mutant
cells, nG1
132
, were cultured in this medium supplemented with 400 µg/mL of G418.
All cell culture supplies were from Gibco Inc. (Billings, MT, USA) unless specified
otherwise.
3.3.2 Animals: Young (2 months old), middle aged (6.5 months old) and old (12-16.5
months old) Sprague-Dawley rats (n=4 for each group) that carry Tspo deletion
mutation (SD-Tspo em5Vpl; RAT5) were generated using zinc finger nuclease
technology (ZFN), as we previously reported
147
. These and control rats were
maintained according to protocols approved by the Institutional Animal Care and
Use Committee of the University of Southern California (Protocol # 20791). Rats
were killed by decapitation. Trunk blood was collected, and plasma was separated
by centrifugation at 2000 g for 15 min, stored at -80°C, and used for determination
of circulating testosterone levels.
45
3.3.3 Primary Leydig Cell Isolation: Leydig cells were isolated from young (2 months old),
middle-aged (6.5 months old) and old (12-16.5 months old) control and Tspo mutant
rats using isosmotic continuous Percoll (Gibco Inc.) gradients generated by
centrifugation, as previously described with minor modifications
148
. Leydig cells
were isolated from two rats from each age group and the experiments were
repeated three times unless noted otherwise. Briefly, testes were perfused,
decapsulated, and dissociated using 0.25 mg/mL collagenase shaken at 80
cycles/min at 34°C for 15 min. Following dissociation, the supernatant was collected
and centrifuged at 800x g for 20 minutes. Resuspended pellets were placed into a
Percoll density gradient and centrifuged at 14,000 rpm for 45 minutes at 4 °C. The
Leydig cell-enriched fraction was isolated and placed atop a BSA density gradient
and centrifuged at 50x g for 10 minutes, yielding an 85% pure Leydig cell solution.
Leydig cell purity was determined by 3β-hydroxysteroid dehydrogenase chemical
reaction, as described previously
148
. In brief, Leydig cell fractions were incubated for
30 min at 37°C with the substrate dehydroepiandrosterone (100 g/ml; Sigma-
Aldrich, St. Louis, MO, USA) in 0.07M phosphate buffer (pH 7.2) containing 1 mg/ml
nicotinamide, 6 mg/ml g-NAD and 1.5 mg/ml nitro blue tetrazolium (Sigma-Aldrich).
46
3.3.4 Overexpression of Opa1 in Cultured Leydig Cells: Cultured cells were transfected
with OPA1-7ΔS to produce overexpression of Opa1 isoform 7ΔS1. The Pclbw-
opa1(isoform 7DeltaS1)-myc bacterial plasmid was a gift from David Chan
144
(Addgene plasmid #62846; RRID:Addgene_62846, Addgene, Watertown, MA, USA).
The bacterial plasmid was extracted using the Zyppy Plasmid Miniprep Kit (Zymo
Research, Irvine, CA, USA) and the EndoFree Plasmid Maxi Kit (Qiagen,
Germantown, MD, USA). Plasmid sequence was verified using next generation
sequencing (Genewiz, South Plainfield, NJ, USA). Plasmids were transfected into
cultured cells using Lipofectamine 3000 and Opti-MEM in accordance with
manufacturer protocols. Transfected cells were grown for two days at 37 °C with
3.5% CO 2. Overexpression of OPA1-7ΔS was confirmed using immunocytochemistry,
immunoblot, and qRT-PCR.
3.3.5 Mitochondrial Fusion Promoter M1 Treatment: Mitochondrial Fusion Promoter M1
(4-Chloro-2- (1- (2- (2,4,6-trichlorophenyl) hydrazono) ethyl) phenol, Sigma-Aldrich)
was dissolved in DMSO (10 mg/mL) and diluted to appropriate concentrations using
cell media. Cultured and isolated Leydig cells were treated with 10 µM M1 for 12
hours. The concentration of M1 used was chosen based on previous studies
149,150
.
Changes in mitochondrial respiration, steroid hormone production, gene
expression, and protein expression were measured subsequently.
47
3.3.6 Immunoblot Analysis: Protein was extracted using RIPA buffer supplemented with
protease inhibitor. Protein concentration was measured using the Pierce BCA
Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed using 1 µg/µL of purified
protein on a 4%-20% Tris-glycine gradient gel (Bio-Rad, Hercules, CA, USA). Protein
bands were electro-transferred to a polyvinylidene fluoride membrane and blocked
with 5% BSA for 30 minutes. The OPA1 mouse monoclonal antibody clone 1E8-1D9
that reacts with mouse, rat and human protein was from ThermoFisher Scientific
(product #MA5-16149). TSPO protein expression was assessed using an affinity
purified rabbit anti-peptide antibody raised against the mouse TSPO C-terminal
sequence as previously described
151
. This antiserum detects TSPO in human, mouse
and rat tissues and cells
152
. Membranes were incubated with primary antibody
overnight at 4°C at 1:1000 dilution and secondary antibody at 1:5000 for one hour
at room temperature. Membranes were then quenched with Radiance Peroxide and
Radiance Plus (Azure Biosystems, Dublin, CA, USA) and subsequently imaged with
an Azure c600 system (Azure Biosystems). Membranes were then stripped with
Restore Western Blot Stripping Buffer (ThermoFisher Scientific) and incubated with
a housekeeping antibody for normalization. Anti-sera against beta-actin (Danvers,
MA, USA), alpha tubulin (Sigma-Aldrich), and GAPDH (Proteintech, Rosemont, IL,
USA) were used as controls.
48
3.3.7 Measurement of Steroid Hormones: MA-10 wild-type and nG1 cells (1x10
4
) were
plated on 96-well plates for 24 hours. Media was removed, wells were washed with
phosphate-buffered saline, and media treatments were added. Cells were treated
with 50 ng/mL human chorionic gonadotropin (hCG; National Hormone and Peptide
Program, Harbor-UCLA Medical Center Torrance, CA, USA) or control media and
incubated for 2 hours at 37 °C. Media were collected for steroid measurement using
the Progesterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA), and cells were
lysed with 0.1 N sodium hydroxide for subsequent protein measurement using the
Bradford Protein Assay (Avantor, Radnor, PA, USA). Similarly, isolated primary
Leydig cells (1x10
5
) from wild-type young and aging rats and from the Tspo mutant
RAT5 were collected in microfuge tubes, placed in media, and shaken at 80
cycles/min at 34°C for 2 hours. Testosterone measurement was performed using
the Testosterone ELISA Kit (Cayman Chemical). Plasma testosterone levels were
measured using the same ELISA kit.
49
3.3.8 Measurement of Cellular Respiratory Function: Cultured cells (1x10
4
) and isolated
primary Leydig cells (3x10
4
) were plated onto Seahorse XF Cell Culture Microplates
overnight (Agilent Technologies, Santa Clara, CA, USA). Cell media were replaced
with Agilent Seahorse XF DMEM Medium supplemented with 1 mM glucose, 1 mM
pyruvate, and 2 mM glutamine and incubated at 37 °C in a non-CO
2
incubator. Cells
were evaluated using the Seahorse XF Cell Mito Stress Test Kit or the Seahorse XF
Real-Time ATP Rate Assay Kit according to the manufacturer’s specifications. Briefly,
working solutions of Oligomycin (2.5 µM), FCCP (2.0 µM) and Rot/AA (0.5 µM) were
prepared and loaded into a sensor cartridge that had been hydrated in Seahorse XF
Calibrant at 37 °C in a non-CO2 incubator overnight. The assay was performed using
the Seahorse XFe96 Analyzer with templates designed in Wave 2.6.1.
3.3.9 Quantitative Reverse Transcription PCR (RT-qPCR): RNA was isolated from
harvested cells using the RNAqueous Micro Kit (ThermoFisher Scientific) and
transcribed to cDNA using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa
Bio Inc., Shiga, Japan) according to the manufacturer’s specifications. Real-time PCR
was performed using the qTOWER
3
on a protocol designed in the qPCRsoft software
(Analytik Jena, Jena, Germany). Gene expression was normalized to β-actin and
expressed as fold-change. The primer sequences were as follows: TSPO F: 5’-
GGGCCTCCGGTGGTATGCTA-3’; TSPO R: 5’-GACTATGTAGGAGCCATACCCCAT-3’;
OPA1 F: 5’-AAGTGGATTGTGCCTGACTTT-3’; OPA1 R: 5’-TTTTCCTCATTCGGAGTTCG-
3’
50
3.3.10 Mitochondrial Imaging: Cells were imaged by transmission electron microscopy
(TEM) at the USC Core Center of Excellence in Nano Imaging. Cells were primarily
fixed using 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M HEPES, and 0.115 M
Sucrose. After washing with 0.1 M cacodylate, cells were placed in a secondary
fixative containing 1% osmium tetroxide. Cells were then stained with uranyl
acetate and dehydrated with a series of 30-100% of EtOH washes. The dehydrated
cells were transitioned to a microfuge tube using propylene oxide and infiltrated
using increasing concentrations of polybed 812 epoxy resin. The cell-containing-
block was sectioned using the Leica EM UC6 Ultramicrotome (Leica Biosystems,
Nussloch, Germany) and examined on the FEI Talos F200C G2 Biological
Transmission Electron Microscope (ThermoFisher Scientific).
51
3.3.11 Immunofluorescence and Confocal Microscopy: Cultured cells were fixed in 4%
paraformaldehyde in phosphate-buffered saline for ten minutes at room
temperature. After washing with phosphate-buffered saline, cells were incubated in
0.1% triton for ten minutes at room temperature. After washing and blocking with
5% horse serum, cells were incubated with anti-TSPO and anti-OPA1 antibodies at
1:400 dilution, or anti-TOMM20 antibodies (Abcam; product #ab56783) at 1:4000
overnight at 4 °C. After washing, samples were incubated with appropriate Alexa
Flour dyes 1:400 dilution at room temperature for 30 minutes, and subsequently
stained with DAPI. Digital confocal images were captured with a Zeiss laser scanning
700 series confocal microscope wavelength 400-750 nm (Zeiss, Jena, Germany) at
the USC School of Pharmacy Translational Research Laboratory. Images were
processed and quantified using the ImageJ software. Mean fluorescence intensity
was measured and represented as a percentage difference from the MA-10 control.
3.3.12 Statistical Analysis: Data from experiments performed in triplicate are expressed
as the mean ± standard error of the mean. GraphPad Prism (v.7; GraphPad Software,
San Diego, CA, USA) was used for graphical presentation and statistical analysis.
Analysis was performed using a student’s t-test or ANOVA with multiple
comparisons where appropriate. Results were considered statistically significant at
p < 0.05.
52
3.4 Results
3.4.1 Tspo deletion decreased mitochondrial function in MA-10 Leydig cells: Previous
studies have shown that Tspo deletion in MA-10 Leydig cells alters mitochondrial
integrity and the ability of the cells to produce progesterone. A major objective of
the present study was to determine the quantitative relationship between TSPO and
mitochondrial function. Cells were grown in Seahorse plates to assess mitochondrial
function in TSPO deficient nG1 Leydig cells compared to wild-type MA-10 Leydig
cells. As seen in Figure 4A, basal cellular respiration, mitochondrial proton leak,
maximal cellular respiration, ATP production, and spare respiratory capacity were
significantly decreased in nG1 cells. The nG1 oxygen consumption rate was
decreased over the MA-10 cells. The alterations seen in our results represent
declining regulation of bioenergetic machinery. Mitochondrial ATP is essential to
steroidogenic capacity and supports steroidogenesis. Evaluating the ATP production
rate using Seahorse 96-well plates revealed significant decreases in both the
mitochondrial and glycolytic ATP production rates, and therefore a significant
decline in total ATP production (Figure 4B). To determine if the alterations in ATP
production rate alter steroidogenesis, we conducted an enzyme-linked
immunosorbent assay (ELISA) to measure steroid production. As seen in Figure 4C,
although both wild-type and nG1 MA-10 cells responded to hCG treatment, both
basal and hormone-stimulated progesterone formation showed significant declines
in nG1 cells.
53
Figure 4: TSPO deletion decreased mitochondrial function in MA-10 Leydig cells. (A)
Mitochondrial stress test in MA-10 and nG1 Leydig cells with oxygen consumption quantified
using Agilent Wave. (B) ATP rate assay in MA-10 and nG1 Leydig cells. (C) Basal and hormone-
stimulated (50 ng/ml hCG) progesterone formation after two-hour treatment in MA-10 and nG1
Leydig cells measured via ELISA. Data are presented as mean ± SEM (n = 3). *p < .05 **p < .01
***p < .001 by two-tailed student's t-test. Antimycin A, Antimycin A1b; FCCP, trifluoromethoxy
carbonylcyanide phenylhydrazone; OCR, oxygen consumption rate.
MA-10
nG1
MA-10 + hCG
nG1 + hCG
0
20
40
60
100
200
300
400
500
Progesterone
(ng/ml/µg protein)
Progesterone Formation in
MA-10 and nG1 Leydig Cells
MA-10 nG1
0.0
0.5
1.0
1.5
Basal Progesterone
Formation
Fold Change
✱✱✱
MA-10 nG1
0.0
0.5
1.0
1.5
hCG Stimulated (50 ng/ml)
Progesterone Formation
Fold Change
✱✱
MA-10 nG1
0.0
0.5
1.0
1.5
hCG Stimulated (50 ng/ml)
Progesterone Formation
Fold Change
✱✱
MA-10
nG1
0
5
10
15
20
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱✱✱
***
MA-10
nG1
0.0
0.5
1.0
1.5
2.0
Mitochondrial Proton Leak
OCR (pmol/min/µg protein)
✱✱
MA-10
nG1
0
2
4
6
8
Basal Cellular Respiration
OCR (pmol/min/µg protein)
✱✱✱✱
***
MA-10
nG1
0
5
10
15
20
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱✱✱
***
MA-10
nG1
0
5
10
15
Spare Respiratory
Capacity
OCR (pmol/min/µg protein)
✱✱✱✱ ***
MA-10
nG1
0
5
10
15
20
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱✱✱
***
MA-10
nG1
0
2
4
6
ATP Production
OCR (pmol/min/µg protein)
✱✱✱✱ ***
MA-10
nG1
0
5
10
15
20
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱✱✱
***
A
MA-10 nG1
0
10
20
30
40
50
Mitochondrial ATP
Production Rate
ATP Production Rate
(pmol/min)
✱✱✱✱ ***
MA-10 nG1
0
10
20
30
40
Glycolytic ATP
Production Rate
ATP Production Rate
(pmol/min)
✱✱✱✱ ***
MA-10 nG1
0
10
20
30
40
50
Percentage of ATP Derived
from Glycolysis
% Glycolysis
✱✱✱✱
***
MA-10 nG1
0
20
40
60
80
Total ATP Production Rate
ATP Production Rate
(pmol/min)
✱✱✱✱ ***
MA-10 nG1
0
20
40
60
80
Percentage of ATP Derived from
Oxidative Phosphorlyation
% Oxidative
Phosphorylation
✱✱✱✱ ***
B
MA-10 nG1
0.0
0.5
1.0
1.5
Basal Progesterone
Formation
Fold Change
✱✱✱
MA-10 nG1
0.0
0.5
1.0
1.5
hCG Stimulated (50 ng/ml)
Progesterone Formation
Fold Change
✱✱
C
54
3.4.2 Increased mitochondrial fusion restores bioenergetics in TSPO-deficient Leydig
cells: The mitochondria of Leydig cells are highly active in fusion and fission, the
interplay of which is integral to mitochondrial turnover and the transport of
molecules into and from the mitochondria. To investigate the relationship between
mitochondrial fusion and function in Leydig cells, we induced mitochondrial fusion
by treatment of MA-10 cells with a cell-permeable phenylhydrazone, M1, which has
been shown to promote mitochondrial tubular network formation, increase OPA1
expression, and increase ATP levels. As seen in Figure 5A, treatment with M1
resulted in increased mitochondrial functions and increased steroid biosynthesis.
The oxygen consumption rate and hormone production of the M1-treated nG1 cells
reflected that of the control MA-10 cells, but MA-10 cells treated with M1 showed
decreased mitochondrial function (Figure 5A). To follow up on this, mitochondria
were stained with TOMM20 to assess the mitochondrial population in M1-treated
cells. Confocal images showed an excessive buildup of mitochondria in M1-treated
MA-10 cells, whereas nG1 cells treated with M1 showed mitochondrial populations
like that of control MA-10 cells (Figure 5B). Given that M1 increases the levels of the
mitochondrial protein OPA1, cells were transfected with Opa1. Opa1 transfection
increased mitochondrial function and hormone-stimulated steroidogenesis in nG1
(Figure 5C).
55
Figure 5: Increased mitochondrial fusion restores bioenergetics in TSPO-deficient Leydig cells. (A)
Mitochondrial bioenergetics and steroid hormone formation in MA-10 and nG1 Leydig cells after
12-h treatment with M1. (B) Mitochondrial density in basal and M1-treated MA-10 and nG1 cells.
(C) Bioenergetics and steroid hormone formation in MA-10 and nG1 after OPA1 transfection.
Data are presented as mean ± SEM. *p < .05 **p < .01 ***p < .001 by two-tailed student's t-test.
Scale bar, 0.5 μm.
MA-10
MA-10 + M1
nG1
nG1 + M1
0
1
2
3
4
5
ATP Production
OCR (pmol/min/µg protein)
✱✱✱✱ ✱✱✱ ***
MA-10
MA-10 + M1
nG1
nG1 + M1
0
2
4
6
8
10
Basal Oxygen
Consumption
OCR (pmol/min/µg protein)
✱✱✱ ✱✱✱✱ ***
MA-10
MA-10 + M1
nG1
nG1 + M1
0
5
10
15
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱✱ ✱✱
MA-10
MA-10 + M1
nG1
nG1 + M1
0
2
4
6
8
Spare Respiratory
Capacity
OCR (pmol/min/µg protein)
✱ ✱
MA-10
MA-10 + M1
nG1
nG1 + M1
0
1
2
3
4
Mitochondrial Proton
Leak
OCR (pmol/min/µg protein)
✱✱ ✱✱✱✱
MA-10
MA-10 + M1
nG1
nG1 + M1
0.0
0.5
1.0
1.5
Basal Progesterone
Formation
Fold Change
✱
MA-10
MA-10 + M1
nG1
nG1 + M1
0.0
0.5
1.0
1.5
hCG Stimulated (50 ng/ml)
Progesterone Formation
Fold Change
✱
A
Basal M1
MA-10 nG1
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
5
10
15
20
Spare Respiratory Capacity
OCR (pmol/min/µg protein)
✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
5
10
15
20
ATP Production
OCR (pmol/min/µg protein)
✱✱✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
1
2
3
4
5
Mitochondrial Proton Leak
OCR (pmol/min/µg protein)
✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
10
20
30
Maximal Cellular
Respiration
OCR (pmol/min/µg protein)
✱✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
5
10
15
Basal Oxygen
Consumption
OCR (pmol/min/µg protein)
✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0.0
0.5
1.0
1.5
2.0
Basal Progesterone
Formation
Fold Change
✱✱
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0.0
0.5
1.0
1.5
hCG Stimulated (50 ng/ml)
Progesterone Formation
Fold Change
✱✱
0 20 40 60 80
0
10
20
30
40
Oxygen Consumption Rate
Time (minutes)
OCR (pmol/min/µg protein)
MA-10
MA-10 OPA1
nG1
nG1 OPA1
C
B
56
3.4.3 Characterization of Opa1 transfection and M1 treatment in MA-10 and nG1 Leydig
cells: Imbalanced fusion dynamics may compromise the integrity of the
steroidogenic protein scaffold essential for cholesterol transport. Therefore, we
assessed how the expression of proteins in the steroidogenic scaffold is altered in
response to increased mitochondrial fusion. OPA1 overexpression was visualized in
Opa1-transfected Leydig cells. Immunoblots revealed overexpression of OPA1 in
transfected MA-10 and nG1 cells, as well as significant increases in Opa1 and Tspo
gene expression levels in transfected MA-10 cells (Figure 6A). Immunoblot analysis
of steroidogenic-related proteins revealed increases in CYP11A1 and VDAC, but not
in the bioenergetic proteins adrenodoxin (ADX) or ATP complex V beta subunit
(ATPB) (Figure 6B), suggesting that bioenergetic proteins are not upregulated
despite greater ATP production (Figure 5). M1 treatment resulted in increases in
opa1 and tspo gene expression in MA-10 cells (Figure 6C). TSPO gene expression
increased slightly in M1-treated MA-10 cells, but other proteins involved in steroid
formation did not (Figure 6D). To further address the relationship between TSPO
levels and the treatment of MA-10 cells with M1 or transfection with Opa1,
immunochemical analyses were conducted to relate OPA1 and TSPO expressions.
Increases were seen in OPA1 and TSPO levels after treatment with M1 or
transfection with Opa1 (Figure 7A). nG1 samples also showed increased
fluorescence for TSPO in M1-treated and Opa1-transfected samples (Figure 7B),
similar to the results found with MA-10 cells.
57
Figure 6: Characterization of Opa1 transfection and M1 treatment in MA-10 and nG1 Leydig cells. (A)
Representative of three independent immunoblots for OPA1 and gene expression quantification for Opa1
and Tspo in OPA1 transfected MA-10 and nG1 Leydig cells. (B) Immunoblot for steroidogenic-related
proteins TSPO, CYP11A1, VDAC, and ATP-related proteins ADX, ATPB in OPA1 transfected cells. (C)
Immunoblot for OPA1 and gene expression for Opa1 and Tspo quantification in M1-treated MA-10 and
nG1 Leydig cells. (D) Immunoblot for proteins TSPO, STAR, CYP11A1, VDAC, ADX, ATPB in M1-treated cells.
Data are presented as mean ± SEM. **p < .01 by two-tailed student's t-test.
OPA1
GAPDH
MW (kDa)
120 -
100 -
75 -
37 -
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0
1
2
3
4
OPA1/GAPDH
Fold Change
OPA1
***
**
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0.0
0.5
1.0
1.5
5
10
15
20
25
30
OPA1
Fold Change
✱✱ ✱✱
Opa1
Tspo
CYP11A1 50 -
18 -
31 -
30 -
18 -
14 -
37 -
TSPO
VDAC
STAR
ATPB
ADX
GAPDH
MW (kDa)
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0.0
0.5
1.0
1.5
TSPO/GAPDH
TSPO Expression
***
n.d. n.d.
OPA1
GAPDH
100 -
37 -
MA-10 MA-10 M1 nG1 nG1 M1 MW (kDa)
MA-10
MA-10 OPA1
nG1
nG1 OPA1
0.0
0.2
0.4
0.6
OPA1 Expression
Density of OPA1/GAPDH
opa1 tspo
CYP11A1 50 -
18 -
31 -
32 -
18 -
14 -
37 -
TSPO
VDAC
STAR
ATPB
ADX
MA-10 MA-10 M1 nG1 nG1 M1 MW (kDa)
MA-10
MA-10 M1
nG1
nG1 M1
0.0
0.5
1.0
1.5
TSPO/GAPDH
TSPO Expression
**
n.d. n.d.
A
B
C
D
opa1
tspo
58
Figure 7: Increased expression of OPA1 in MA-10 and nG1 after M1 treatment or Opa1 transfection. (A)
Representative of three independent immunofluorescence stainings for OPA1 and TSPO in OPA1
transfected and M1-treated MA-10 and nG1 Leydig cells. (B) Percent fluorescence intensity relative to the
mean intensity of MA-10. Data are presented as mean ± SEM. *p < .05 **p < .01 ***p < .001 by two-tailed
student's t-test.
B
A
MA-10
MA-10 M1
MA-10 OPA1
nG1
nG1 M1
nG1OPA1
0
1
2
3
4
%FluorescenceIntensty
(relative to control)
OPA1
✱✱✱✱ ✱
✱✱✱
MA-10
MA-10 M1
MA-10 OPA1
nG1
nG1 M1
nG1OPA1
0
1
2
3
4
%FluorescenceIntensty
(relative to control)
OPA1
✱✱✱✱ ✱
✱✱✱
MA-10
MA-10 M1
MA-10 OPA1
nG1
nG1 M1
nG1OPA1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
%FluorescenceIntensty
(relative to control)
✱✱✱✱ ✱✱✱
MA-10
MA-10 M1
MA-10 OPA1
nG1
nG1 M1
nG1OPA1
0
1
2
3
4
%FluorescenceIntensty
(relative to control)
OPA1
✱✱✱✱ ✱
✱✱✱
DAPI OPA1 TSPO Overlay
MA-10 MA-10 M1 MA-10 Opa1 nG1 nG1 M1 nG1 Opa1
5 µm 5 µm 5 µm 5 µm
5 µm 5 µm 5 µm 5 µm
5 µm 5 µm 5 µm 5 µm
5 µm 5 µm 5 µm 5 µm
5 µm 5 µm 5 µm 5 µm
5 µm 5 µm 5 µm 5 µm
59
3.4.4 Mitochondrial fusion improved mitochondrial morphology in TSPO-deficient
Leydig cells: With the knowledge that OPA1 regulates aspects of mitochondrial
cristae dynamics, we hypothesized that structural deviations seen in nG1 cells might
improve by upregulating fusion. Dysfunctional mitochondria have aberrant cristae
structures are replaced via mitochondrial biogenesis. Using transmission electron
microscopy imaging to visualize mitochondrial morphology, we found that nG1
mitochondrial morphology was restored after M1 treatment or Opa1 transfection
(Figure 8). MA-10 cells presented healthy mitochondria with the expected cristae
morphology (black arrows), while dysfunctional mitochondria were prevalent in nG1
cells (red arrowheads). Fusion upregulation via M1 and Opa1 overexpression
reduced the number of dysfunctional mitochondria in nG1 cells. Cristae prevalence
and structure (yellow double-sided arrows) were also restored in fusion-
upregulated nG1 cells. The restored mitochondrial and cristae structure indicates
the greater respiratory capacity and bioenergetic function, consistent with the
findings in Figure 5.
60
Figure 8: Mitochondrial fusion improved mitochondrial morphology in TSPO-deficient Leydig cells.
Mitochondrial morphology in MA-10 and nG1 after fusion treatment under TEM. The number of healthy
mitochondria (black arrows) and compromised mitochondria (red arrowheads) are shown. Mitochondrial
cristae presence and consistency (yellow double-sided dashed arrows) are shown after fusion treatment
in cells. Scale bar, 0.5 μm. TEM, transmission electron microscopy.
No Treatment M1 Opa1
nG1 MA-10
MA-10 nG1
Opa1 M1 No Treatment
61
3.4.5 M1 treatment improves bioenergetics and steroid formation in aged rat Leydig
cells: To evaluate whether the upregulation of mitochondrial fusion enhances
steroidogenic output and mitochondrial function, Leydig cells were isolated from 2-
month-old and 1-year-old rats and treated with M1. Basal respiration, maximal
respiration, and ATP production were significantly higher in the Leydig cells of the
2-month-old rats. Leydig cells isolated from 1-year-old animals had significant
increases in maximal respiration and spare capacity. Basal respiration and ATP
production were increased at this age, albeit not significantly (Figure 9A). Serum
testosterone and testosterone produced from isolated Leydig cells showed declines
over the rat lifespan (Figure 9B). Leydig cells from rats aged 1 year produced
significantly increased levels of testosterone after M1 treatment (Figure 9C),
suggesting a role for mitochondrial fusion in the maintenance of steroidogenic
function. Tspo mutant RAT5 animals produced less testosterone in rats of 2, 6.5, and
16.5 years of age when compared to the WT. Testosterone levels were greater in
M1-treated 16.5-month-old WT and Tspo-mutant animals (Figure 9D).
62
Figure 9: M1 treatment improved bioenergetics and steroid hormone formation in aged rat Leydig cells.
(A) Mitochondrial bioenergetics in M1-treated 2-month-old and 1-year-old rat Leydig cells. (B)
Testosterone levels across rat lifespan in serum and isolated Leydig cells; 2, 6.5, and 16.5 months of age.
(C) Testosterone levels in from 2-month and 1-year-old (n = 2) Leydig cells treated with M1 for 12 h. (D)
Temporal testosterone levels produced from isolated Leydig cells of WT and Tspo RAT5, with M1-treated
16.5-month-old isolated Leydig cells. Data are presented as mean ± SEM. *p < .05 by two-tailed student's
t-test.
A
WT
WT M1
0
10
20
30
Basal Respiration
OCR (pmol/min)
✱✱✱
WT
WT M1
0
5
10
15
Proton Leak
OCR (pmol/min)
WT
WT M1
0
20
40
60
Maximal Respiration
OCR (pmol/min)
✱✱
WT
WT M1
0
10
20
30
Spare Capacity
OCR (pmol/min)
WT
WT M1
0
5
10
15
20
ATP Production
OCR (pmol/min)
✱
WT
WT M1
0
5
10
15
20
25
Basal Respiration
OCR (pmol/min)
WT
WT M1
0
2
4
6
8
Proton Leak
OCR (pmol/min)
WT
WT M1
0
10
20
30
40
Maximal Respiration
OCR (pmol/min)
✱
WT
WT M1
0
5
10
15
20
ATP Production
OCR (pmol/min)
WT
WT M1
0
5
10
15
20
Spare Capacity
OCR (pmol/min)
✱
2 Months Old
6.5 Months Old
16.5 Months Old
0
50
100
150
1000
1100
1200
5000
6000
7000
Rat Serum Testosterone
Testosterone (pg/ml)
2 Months Old
6.5 Months Old
16.5 Months Old
0
20
40
60
80
Testosterone Production of
Isolated Primary Leydig Cells
Testosterone (ng/million cells)
WT
WT M1
0
20
40
60
80
Basal Testosterone
Production in 2 Month Old
M1 Treated Isolated Primary Leydig Cells
Testosterone (ng/million cells)
WT
WT M1
0
10
20
30
40
Basal Testosterone
Production in 1 Year Old
M1 Treated Isolated Primary Leydig Cells
Testosterone (ng/million cells)
✱
B
WT
WT M1
0
10
20
30
40
Basal Testosterone
Production in 1 Year Old
M1 Treated Isolated Primary Leydig Cells
Testosterone (ng/million cells)
✱
WT
WT M1
0
20
40
60
80
Basal Testosterone
Production in 2 Month Old
M1 Treated Isolated Primary Leydig Cells
Testosterone (ng/million cells)
C
D
WT
WT M1
0
5
10
15
20
ATP Production
OCR (pmol/min)
63
3.5 Discussion
Alterations in cholesterol transport and TSPO expression may play critical roles in age-related
testosterone decline. Aging Leydig cells become TSPO deficient compared to young cells
23,106
, and
this could disrupt the translocation of cholesterol into the mitochondria; the rate-limiting step in
steroidogenesis
153
. TSPO is integrally involved in the translocation of cholesterol into the
mitochondria for steroid biosynthesis and its depletion in Leydig cells leads to functional
declines
91,132,147,154,155
. Given the impact of TSPO depletion on steroid production and
mitochondrial integrity
132
, we tested the effect of increasing mitochondrial fusion on steroid
biosynthesis by upregulating OPA1 via drug treatment and Opa1 overexpression. Our rationale
for these approaches came from previous studies showing that OPA1 expression reduces
mitochondrial dysfunction and regulates cristae remodeling
156
. Therefore, we anticipated
upregulation would improve Leydig cell function. We discovered that OPA1 upregulation indeed
restored steroid biosynthesis and Leydig cell function in TSPO-depleted cells. In these studies, we
used both transient transfection and pharmacological means to induce mitochondrial fusion. The
two approaches resulted in consistent findings, increasing our confidence that enhanced
mitochondrial fusion ameliorates dysfunction in TSPO deficient Leydig cells. Consistent with
these findings, previous studies reported that the activation of TSPO with a TSPO drug ligand led
to the stabilization of mitochondrial of mitochondrial architecture during inflammation stress in
cells of the colon, and to reduced cell death
157
.
It should be noted that that there have been studies based on the knockdown Opa1 that have
suggested that Opa1 may not be critical for steroidogenesis
13
. While OPA1 may not be
64
indispensable for steroid formation, it might be the case that compensatory mechanisms may be
involved in mitochondrial remodeling for cholesterol transport into the mitochondria. This is
supported by the finding that there are large number of steroidogenic proteins in the
steroidogenic interactome (SITE), and numerous MICOS complex proteins
5,13
. Interestingly, OPA1
protein expression fold change increase in MA-10 transfected cells was higher than in nG1
transfected cells, suggesting that TSPO stabilizes mitochondrial fusion dynamics. This is
consistent with previous findings, as nG1’s disrupted membrane potential
132
likely results in
destabilization of Opa1 mRNA cleavage, limiting fusion capacity
144
. The loss of mitochondrial
membrane potential destabilizes mRNA cleavage which produces the long isoform of OPA1
144
.
The deteriorated membrane potential in nG1 may disrupt the fusion potential in M1 treated nG1
cells, as co-expression of the long and short OPA1 isoforms enhance fusion.
Moreover, our results identify a relationship between OPA1 and TSPO expression.
Immunoblot, qPCR, and immunocytochemistry staining studies revealed increases in TSPO
expression after M1 treatment or Opa1 transfection, suggesting that OPA1 may regulate TSPO
expression. However, it may be that TSPO regulates OPA1, as our data showed depressed
OPA1/Opa1 expression in nG1 samples. Opa1 mRNA cleavage is compromised when
mitochondrial membrane potential is disrupted, which may explain the dynamics between these
two proteins seen in our results given TSPO’s influence on membrane potential. Puzzlingly, there
was an increase in TSPO fluorescence in nG1 samples after Opa1 transfection. OPA1
overexpression may be influencing transcription factors regulating TSPO expression, amplifying
residual expression. MA-10 cells have an aberrant chromosome number. Previous studies
demonstrated that extinction of one allele of Tspo gene in the constitutively steroid producing
65
R2C Leydig cell line abolished TSPO expression
115
. It is possible that OPA1 may drive Tspo
expression from the remaining alleles.
The role of OPA1 in disease progression is well known. Variations in Opa1 expression have
been implicated in a number of diseases including dominant optic atrophy, deafness, and
spinocerebellar degeneration
158
. These disruptions manifest reductions in oxygen consumption,
cell viability, and ATP synthase assembly
159
. Given OPA1’s role in mitochondrial function, we were
not surprised that increasing OPA1 expression improved function in TSPO-deficient Leydig cells.
However, we were intrigued to find that increased OPA1 levels improved steroid hormone
formation in Leydig cells. It may be the case that increasing OPA1 levels improve mitochondrial
contact site presence and cristae integrity, increasing the efficiency of cholesterol binding and
transport. Similarly, Opa1 overexpression increased TSPO and VDAC levels in MA-10 Leydig cells,
suggesting that mitochondrial fusion may regulate expression of SITE proteins. Given the
cholesterol binding properties of TSPO, these increases may enhance cholesterol translocation
into the mitochondria. The cholesterol recognition amino acid consensus is oriented such that
the presence of a binding interface between TSPO and VDAC is likely
160
. Previously, modeling
VDAC structures revealed a likely shift among backbone amides in the presence of cholesterol
161
and reasonable binding orientations for cholesterol in specific VDAC sites have also been
identified
162
. Taken together, our results indicate a relationship between mitochondrial fusion
and steroidogenic capacity.
The formation of mitochondrial cristae, the main sites of oxidative phosphorylation, is
regulated by OPA1
163
. OPA1 modulates oxidative phosphorylation by oligomerizing and
narrowing cristae junctions, leading to ATP synthase dimerization
156
. Morphologically, curvature
66
is induced which physically narrows distance between the electron transport chain and enhances
ATP synthesis efficiency
164
. Electron microscopy revealed disruptions to mitochondrial
morphology and declining cristae formation in TSPO deficient Leydig cells. Given morphological
and proton gradient irregularities previously identified in TSPO deficient Leydig cells
132
, we
anticipated a decline in ATP synthesis. As expected, ATP production was significantly decreased
in TSPO KO Leydig cells, which was restored with OPA1 overexpression, along with steroid
formation. While declines in respiration are seen in quiescent cells, alterations in proton leak
impact the coupling efficiency and reactive oxygen species production in cells
165
. Much of the
proton leak is attributed to the adenine nucleotide translocase (ANT), which collaborates with
oxidative phosphorylation machinery to regulate mitochondrial respiration
165,166
Given that
steroid synthesis by CYP11A1’s side chain cleavage of cholesterol requires electrons from
complex III and IV of the electron transport chain
167
, cristae improvements may enhance electron
availability for steroid biosynthesis. Inhibition of mitochondrial ATP reduces all steps in the
steroidogenic pathway and produces reduced steroid output
168
. Narrowing of cristae junctions
enhances mitochondrial ATP formation and likely provides electrons for side chain cleavage. It is
possible that TSPO depletion’s impact on ATP synthesis may disrupt the flow of electrons for
cholesterol side chain cleavage.
A confounding result of the present studies is that there was declining mitochondrial
respiration in the M1 treated MA-10 cells. One possible explanation could involve the
physiological role of mitochondrial fusion in cells exposed to selective stresses. Hyperfusion of
mitochondria is mediated through OPA1 and creates an excessive interconnected tubular
network in response to a selective stressor
141,169
. Indeed, confocal imaging revealed excessive
67
and connected mitochondrial populations in the MA-10 cells treated with M1, a characteristic
commonly observed in hyperfusion
169
. In addition to increased mitochondrial fusion, treatment
may be increasing stressor signaling. Interestingly, this trend was not observed in M1 treated
isolated primary rat Leydig cells. Primary Leydig cells had improved mitochondrial function after
M1 treatment. Moreover, testosterone biosynthesis was enhanced in isolated Leydig cells from
WT and Tspo-mutant RAT5 animals aged 16.5 months. This may suggest that TSPO is not directly
involved in mitochondrial fusion, but it may play a role in the integrity of the contact site complex.
Nevertheless, increased fusion resulted in enhanced steroid hormone formation. This could be
related to the inherent differences between the tumorigenic cell line and primary Leydig cells
168
.
A noteworthy difference between these models is that MA-10 Leydig cells derive a greater
proportion of their ATP from glycolysis. As such, MA-10 Leydig cells employ a larger partition of
glycolytic ATP for cholesterol transport when compared to primary cells
168
. Furthermore, primary
cells utilize mitochondrial ATP for ER enzymatic reactions that are absent in the tumorigenic
Leydig cells
168,170
. These inherent differences demonstrate the need for thorough investigation
using various models.
A sound therapeutic strategy to combat primary hypogonadism requires a comprehensive
understanding of the translocation of cholesterol into the mitochondria. TSPO decline in aging
Leydig cells is correlated with cellular dysfunction and suboptimal steroid biosynthesis
production. This decline is likely the result of insufficient cholesterol trafficking into the
mitochondria. Findings indicating that mitochondrial fusion is essential for steroidogenesis
139
support the idea that mitochondrial contact sites may act as a conduit for steroidogenesis
6
. Our
model suggests that TSPO depletion leads to declining mitochondrial cristae integrity which
68
causes depressions in mitochondrial function and steroidogenesis in Leydig cells (Figure 10). The
declining function may be ameliorated by increased fusion, as contact sites likely play a role in
regulating steroid hormone formation in addition to mitochondrial function. Encouragingly,
increased fusion in isolated primary Leydig cells from aged rats improved testosterone
production, revealing a potentially novel strategy to approach hypogonadism.
Figure 10: Model of Leydig cell bioenergetics and steroid formation in TSPO deficiency and aging.
(A) Healthy Leydig cells possess tight cristae junctions highly active in steroidogenesis and ATP
production. Electrons easily flow for cholesterol side chain cleavage. (B) Declining TSPO (Tspo
deletion and reduced expression in aging) expression destabilizes the formation of mitochondrial
contact sites and cristae formation, resulting in declining ATP production and empty
mitochondrial matrices. (C) Increasing mitochondrial fusion in these dysfunctional Leydig cells
mediates cristae stabilization caused by TSPO deficiency and improves morphology and
bioenergetics.
69
Chapter 4: Exploring the relationship between the systemic regulation of mitochondrial
dynamics and Leydig cell function
4
4.1 Abstract
Decreased expression of mitochondrial steroidogenic interactome (SITE) proteins and
diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play
a role in maintaining adequate T levels. Optic atrophy 1 (OPA1) protein regulates mitochondrial
dynamics and cristae formation, and its expression influences androgen biosynthesis. Increasing
OPA1 expression in dysfunctional Leydig cell models restored mitochondrial function, increased
SITE protein levels, and recovered androgen production to levels similarly seen in healthy Leydig
cells, suggesting a tight relationship between cristae formation and steroidogenic capacity. This
relationship suggests that mitochondrial dynamics may be a promising target to ameliorate
diminished T levels in aging males. We used twelve-month-old rats to explore the relationship
between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from rats were
treated with the cell-permeable mitochondrial fusion promoter 4-Chloro-2- (1- (2- (2, 4, 6-
trichlorophenyl) hydrazono) ethyl) phenol (M1) which enhances mitochondrial tubular network
formation. In parallel, rats were treated with 2 mg/kg/day M1 for six weeks. Body weight was
measured daily, and blood serum was taken weekly. At the end of the six weeks, Leydig cells were
isolated. Steroid production was measured by ELISA, and protein expression was evaluated by
immunoblot analysis. Mitochondrial morphology was assessed via transmission electron
microscopy (TEM). Leydig cells isolated from M1-treated rats showed enhanced mitochondrial
4
This chapter is derived in part from the poster “Mitochondrial Dynamics: A Promising Therapeutic Target for Aging Leydig Cell
Dysfunction” presented by Samuel Garza at the 48
th
annual conference of the American Society of Andrology
70
tubular network formation, improved mitochondrial function, and produced higher T levels,
compared to controls. It has been shown previously that T levels in aged rats are significantly
lower when compared to young rats. Moreover, it has been shown previously that aged rats there
express decreased levels of SITE and mitochondrial dynamic proteins compared to young rats,
suggest dysregulation of mitochondrial integrity leading to reduced steroidogenic capacity.
Promotion of mitochondrial fusion using M1 enhanced Leydig cell mitochondrial integrity and
androgen formation,. However, in vivo treatment of aged rats with M1 failed to re-establish
testosterone levels to that of young rat; the further reduction of testosterone levels and increase
in apoptosis observed a toxic effect of M1 on the testis in vivo. The M1 toxicity seemed to be
tissue-specific. These data suggest that mitochondrial dynamics may provide a promising
therapeutic target for Leydig cell dysfunction although compounds devoid of toxic effects should
be identified.
4.2 Introduction
The relationship between aging and testosterone decline has been well studied. Numerous
alterations in the cellular environment have been associated with this decline, including
reductions in the expression of steroidogenic enzymes, imbalanced antioxidant levels, reactive
oxygen species production, and reduced mitochondrial function
1
. Despite these findings, a better
understanding of the role of mitochondrial dynamics in the age-related functional decline of
Leydig cells is needed. Mitochondrial dynamics play a central role in maintaining the cellular
integrity and allow for versatility in meeting cellular demands
99
. The capacity of the mitochondria
to balance bioenergetics and cellular stressors is mediated in part through mitochondrial
71
dynamics and more research is needed to understand the interplay between aging,
hypogonadism, and mitochondrial function.
The optic atrophy 1 (OPA1) is a key protein in regulating the mitochondrial membrane
dynamics
141
. The dynamin-related GTPase located within the inner membrane space of the
mitochondria bridges the gap between the inner and outer membranes. This feature is essential
for numerous functions including bioenergetic regulation, contact site formation and
transport
159
. Interestingly, OPA1 expression levels decline with aging; a characteristic thought to
contribute to the development and progression of age-related pathologies
156
. Mutations in Opa1
have been associated with to conditions affecting juveniles and adolescents
158
:
72
4.2.1 Autosomal Dominant Optic Atrophy
171
is a condition which causes progressive
vision loss. In this condition, the Opa1 mutation compromises regulation of
mitochondrial dynamics in retinal ganglion cells. These specialized cells require high
levels of energy and regulation of mitochondrial bioenergetics is indispensable. The
compromised regulation of mitochondrial dynamics causes high levels of stress in
reginal ganglion cells, leads to cell death, and ultimately optic nerve degeneration.
Autosomal dominant optic atrophy’s onset becomes apparent early in life, as the
optic nerve rapidly deteriorates.
4.2.2 Behr syndrome
172
is a neurological disorder which leads to vision loss, muscle
spasms, compromised motor skills, and intellectual disability. This developmental
disorder compromises mitochondrial bioenergetics and leads to ataxia which
significantly stunts development and leads to health deterioration over time.
Although Behr syndrome has typically been associated with Opa3 mutations,
growing evidence suggests Opa1 mutations may play a role in its development,
contributing to the body of evidence establishing the role of mitochondrial dynamic
regulation and age-related pathologies.
73
4.2.3 Dominant optic atrophy plus syndrome
173
compromises vision, similar to
autosomal dominant optic atrophy syndrome discussed above, as well as hearing,
neurological development, and other tissues and organs. In dominant optic atrophy
plus syndrome, systemic regulation of mitochondrial dynamics is compromised,
affecting cellular metabolism and bioenergetics, causing tissue decline seen in
similar mitochondrial disorders.
4.2.4 Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
spectrum disorder
174
produces numerous symptoms including muscle
deterioration, encephalopathy, lactic acidosis, and stroke-like episodes. The
disorder has been linked with several mitochondrial DNA mutations, and it is unclear
whether Opa1 mutations directly cause symptoms. However, the similar clinical
presentation of this disorder suggest Opa1 may play a part in its development. The
decline in T production coincides with the declining mitochondrial function of the
steroidogenic Leydig cells
89
. Leydig cells the protein complex associated with
mitochondrial contact sites are indispensable for healthy steroidogenesis, and their
expression declines with aging
60
. This protein complex mediates the translocation
of cholesterol into the mitochondria for steroidogenesis. Steroidogenesis requires a
conglomerate of proteins, collectively known as the steroidogenic interactome
(SITE), containing numerous mitochondrial proteins.
These protein-protein interactions are responsible for cholesterol
translocation into the mitochondria for steroid biosynthesis. Importantly, the
expression of these SITE proteins is reduced in aged Leydig cells, suggesting that
74
compromised protein-protein interactions are likely responsible for reduced
cholesterol delivery and T production. Moreover, data suggest that the regulation
of mitochondrial contact sites plays a role in the expression of key steroidogenic
proteins. Mitochondrial fusion, which produces and maintains contact sites
135
,
deteriorates with aging, suggesting that age-related T decline may be a result of
declining contact site integrity
6,13
. Fusion has been shown to be an integral
requirement of steroidogenesis, and its inhibition diminishes steroid formation
7
. In
my previous chapter it was shown that enhancing mitochondrial fusion in
dysfunctional Leydig cells produces functional gains and an improvement of
mitochondrial function. The data herein investigate the role of mitochondrial fusion
in age-related T decline. Enhancing fusion in isolated aged primary Leydig cells
produces functional gains like those found in previous studies, whereas a six-week
interperitoneally injection of the mitochondrial fusion promoter M1 resulted in
decreased T formation, suggesting Leydig cell toxicity. The juxtaposition between
the isolated primary Leydig cell results and the in vivo administration findings
suggests a calibrated and targeted enhancement of mitochondrial fusion in Leydig
cells is needed.
75
4.3 Materials and methods
4.3.1 Animals: Sprague-Dawley rats (n=4 for each group) aged nine months to one year
were maintained according to protocols approved by the Institutional Animal Care
and Use Committee of the University of Southern California (Protocol #20791). Rats
were killed by decapitation. Trunk blood was collected, and plasma was separated
by centrifugation at 2000 g for 15 min, stored at -80°C, and used for determination
of circulating testosterone levels.
4.3.2 Mitochondrial Fusion Promoter M1 Treatment: Mitochondrial Fusion Promoter M1
(4-Chloro-2- (1- (2- (2,4,6-trichlorophenyl) hydrazono) ethyl) phenol, Sigma-Aldrich)
was dissolved in DMSO and diluted to 2 mg/kg/day for each animal. Animals were
separated into three groups (n=4 for each group): untreated (control) or treated
with either DMSO (DMSO control) or 2 mg/kg/day M1 (M1) daily for six weeks. The
concentration of M1 used was chosen based on previous studies (35, 36). At the end
of the treatment animals were sacrificed and testes, liver, adrenal and heart tissues
collected. Changes in testosterone levels and protein expression assessed by
immunoblotting.
76
4.3.3 Purification of Leydig Cells with Magnetic Activated Cell Sorting (MACS): Testes
were collected from rats after euthanasia, washed and placed in ice-cold phosphate
buffer saline (PBS). Testes were decapsulated and the testicular milieu was digested
in 1 mg/ml collagenase in DMEM/F12 medium containing 0.1% bovine serum
albumin (BSA) for 30 minutes at 34°C in a slow shaking incubator at 90 cycles/min.
The resulting supernatant was filtered with a 70 µM pore nylon mesh. Cells were
then washed with DMEM/F12 medium and isolated with magnetic activated cell
sorting (MACS). The Leydig cell suspension was suspended in Ca+2 Mg+2 free Hank’s
Balanced Salt Solution (HBSS) containing 0.1% BSA. Leydig cells were then isolated
following the manufacturer’s protocol. Briefly, Leydig cell-containing pellets were
suspended in ice-cold IMag buffer at 2x107 cells/ml with prolactin receptor (PRLR)
antibody (1:150) for 30 minutes at 4°C. The labeled cell suspension was washed
twice with IMag buffer and labeled with anti-mouse IgG1 magnetic beads (1:20) at
4°C for 30 minutes. Labeled Leydig cells were positively selected using using the
manufacturer’s protocol: the cell suspension was placed into the IMag Cell
Separation Magnet holder (BD Biosciences, US) for 8 minutes. The supernatant was
then removed, the positive fraction was resuspended in IMag buffer, and the
resuspended cells were placed in the magnet holder for 4 minutes. This
resuspension was performed two more times to purify the cell population.
Purification was verified using two methods: staining for 3β-hydroxysteroid
dehydrogenase and Flow Cytometry. A 3β-hydroxysteroid dehydrogenase chemical
reaction, was performed as described previously (34). In brief, Leydig cell fractions
77
were incubated for 30 min at 37°C with the substrate dehydroepiandrosterone (100
g/ml; Sigma-Aldrich, St. Louis, MO, USA) in 0.07M phosphate buffer (pH 7.2)
containing 1 mg/ml nicotinamide, 6 mg/ml g-NAD and 1.5 mg/ml nitro blue
tetrazolium (Sigma-Aldrich). Separately, Flow Cytometry was performed after cells
were incubated with a PE-conjugated goat anti-mouse IgG fluorescent secondary
antibody in darkness for 1h. PRLR+ cells were analyzed via Flow Cytometry using the
Fortessa X20 (BD Biosciences).
78
4.3.4 Immunoblot Analysis: Protein was extracted using RIPA buffer supplemented with
protease inhibitor. Protein concentration was measured using the Pierce BCA
Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed using 1 µg/µL of purified
protein on a 4%-20% Tris-glycine gradient gel (Bio-Rad, Hercules, CA, USA). Protein
bands were electro-transferred to a polyvinylidene fluoride membrane and blocked
with 5% BSA for 30 minutes. The OPA1 mouse monoclonal antibody clone 1E8-1D9
that reacts with mouse, rat and human protein was from ThermoFisher Scientific
(product #MA5-16149). TSPO protein expression was assessed using an affinity
purified rabbit anti-peptide antibody raised against the mouse TSPO C-terminal
sequence as previously described (37). This antiserum detects TSPO in human,
mouse and rat tissues and cells (38). Membranes were incubated with primary
antibody overnight at 4°C at 1:1000 dilution and secondary antibody at 1:5000 for
one hour at room temperature. Membranes were then quenched with Radiance
Peroxide and Radiance Plus (Azure Biosystems, Dublin, CA, USA) and subsequently
imaged with an Azure c600 system (Azure Biosystems). Membranes were then
stripped with Restore Western Blot Stripping Buffer (ThermoFisher Scientific) and
incubated with a housekeeping antibody for normalization. Anti-sera against beta-
actin (Danvers, MA, USA), alpha tubulin (Sigma-Aldrich), and GAPDH (Proteintech,
Rosemont, IL, USA) were used as controls. Protein expression levels were measured
using the ImageJ software, in which the density of the protein band of interest was
compared with the GAPDH level for that particular band.
79
4.3.5 Measurement of Steroid Hormones: Cells were treated with 50 ng/mL human
chorionic gonadotropin (hCG; National Hormone and Peptide Program, Harbor-
UCLA Medical Center Torrance, CA, USA) or control media and incubated for various
time points at 37 °C. Media were collected for steroid measurement using the
Testosterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA). Similarly, blood
serum was collected and evaluated using the same Testosterone ELISA Kit.
4.3.6 Measurement of Cellular Respiratory Function: Cultured cells (1x10
4
) and isolated
primary Leydig cells (3x10
4
) were plated onto Seahorse XF Cell Culture Microplates
overnight (Agilent Technologies, Santa Clara, CA, USA). Cell media were replaced
with Agilent Seahorse XF DMEM Medium supplemented with 1 mM glucose, 1 mM
pyruvate, and 2 mM glutamine and incubated at 37 °C in a non-CO2 incubator. Cells
were evaluated using the Seahorse XF Cell Mito Stress Test Kit or the Seahorse XF
Real-Time ATP Rate Assay Kit according to the manufacturer’s specifications. Briefly,
working solutions of Oligomycin (2.5 µM), FCCP (2.0 µM) and Rot/AA (0.5 µM) were
prepared and loaded into a sensor cartridge that had been hydrated in Seahorse XF
Calibrant at 37 °C in a non-CO2 incubator overnight. The assay was performed using
the Seahorse XFe96 Analyzer with templates designed in Wave 2.6.1.
80
4.3.7 Transmission electron microscopy (TEM): Cells were imaged by TEM at the USC
Core Center of Excellence in Nano Imaging. Cells were primarily fixed using 2.5%
glutaraldehyde, 2% paraformaldehyde, 0.1 M HEPES, and 0.115 M Sucrose. After
washing with 0.1 M cacodylate, cells were placed in a secondary fixative containing
1% osmium tetroxide. Cells were then stained with uranyl acetate and dehydrated
with a series of 30-100% of EtOH washes. The dehydrated cells were transitioned to
a microfuge tube using propylene oxide and infiltrated using increasing
concentrations of polybed 812 epoxy resin. The cell-containing-block was sectioned
using the Leica EM UC6 Ultramicrotome (Leica Biosystems, Nussloch, Germany) and
examined on the FEI Talos F200C G2 Biological Transmission Electron Microscope
(ThermoFisher Scientific).
4.3.8 Statistical Analysis: Data from experiments performed in triplicate are expressed as
the mean ± standard error of the mean. GraphPad Prism (v.7; GraphPad Software,
San Diego, CA, USA) was used for graphical presentation and statistical analysis.
Analysis was performed using a student’s t-test or ANOVA with multiple
comparisons where appropriate. Results were considered statistically significant at
p < 0.05.
81
4.4 Results
4.4.1 Characterization of MACS-isolated Leydig cells: Our previous studies isolated
primary Leydig cells using isosmotic continuous Percoll (Gibco Inc.) gradients
generated by centrifugation and subsequent BSA gradients
148
. It was recently
demonstrated that Leydig cells could be isolated from adult rats using magnetic-
activated cell sorting
175
, saving a considerable amount of time in experimental
procedures. Evidence suggests the prolactin receptor is a unique Leydig cell surface
marker
176,177
, which was targeted in our MACS protocol. This cell sorting technique
was used to obtain a cellular suspension that produced testosterone under both
basal and hormone stimulated conditions (Figure 11A). The obtained cells were
incubated with a PE-conjugated antibody and flow cytometry analysis revealed
more than 90% of isolated cells expressed PRLR (Figure 11B). Additionally,
steroidogenic enzymatic activity was confirmed after staining for 3β-hydroxysteroid
dehydrogenase (Figure 11C), an enzyme specific to steroidogenic cells.
82
Figure 11: Characterization of MACS isolated Leydig cells. (A) Time course testosterone formation
levels of MACS isolated cells. (B) Flow cytometry analysis for PRLR+ cells. (C) Staining for 3β-
hydroxysteroid dehydrogenase enzymatic activity. Data are presented as mean ± SEM. *p < .05, **p.
< .01, and ***p < .001 by ANOVA.
15 min
30 min
1 hour
2 hours
4 hours
0
20
40
60
80
100
120
Testosterone
(ng/million cells)
Isolated Leydig Cell
Testosterone Formation
15 min
30 min
1 hour
2 hours
4 hours
0
200
400
600
Testosterone
(ng/million cells)
hCG Stimulated (50 ng/ml)
Testosterone Formation
*** *** ***
*** *** *** *
3β-Hydroxysteroid Dehydrogenase Stain
Control Stain
15 min
30 min
1 hour
2 hours
4 hours
0
20
40
60
80
100
120
Testosterone
(ng/million cells)
Isolated Leydig Cell
Testosterone Formation
15 min
30 min
1 hour
2 hours
4 hours
0
200
400
600
Testosterone
(ng/million cells)
hCG Stimulated (50 ng/ml)
Testosterone Formation
*** *** ***
*** *** *** *
3β-Hydroxysteroid Dehydrogenase Stain
Control Stain
3β-Hydroxysteroid Dehydrogenase Stain
Control Stain
3β-Hydroxysteroid Dehydrogenase Stain
Control Stain
A
B
C
+ -
83
4.4.2 Promoting mitochondrial fusion in Leydig cells isolated from aged Sprague Dawley
rats enhances bioenergetics: To investigate the relationship between mitochondrial
fusion and function in aged Leydig cells, we induced mitochondrial fusion ex vivo by
treating isolated Leydig cells with a cell-permeable phenylhydrazone, M1, which has
been shown to promote mitochondrial tubular network formation, increase OPA1
expression, and increase ATP levels
149,150
. Treatment with M1 resulted in increased
mitochondrial functions and increased steroid biosynthesis (Figure 12A), like our
findings in chapter 3. The oxygen consumption rate and hormone production of the
M1 treated cells showed improved cellular function. Like our previous findings
178
,
we hypothesized that mitochondrial structure would improve after M1 treatment.
Dysfunctional mitochondria accumulate with aging and present abnormal shape
and structure. Using TEM to visualize mitochondrial morphology, we found that
untreated aged Leydig cells had fewer healthy mitochondria when compared to the
M1 treated Leydig cells (Figure 12B). M1 treated isolated primary Leydig cells
presented a plethora of mitochondria with healthy cristae structure (Figure 12C).
84
Figure 12: Promoting mitochondrial fusion in Leydig cells isolated from aged Sprague Dawley rats
enhances bioenergetics. (A) Oxygen consumption, ATP production, and steroid hormone
production of MACS isolated Leydig cells. (B) TEM imaging of MACS isolated Leydig cells
highlighting mitochondrial morphology. (C) Characteristics of mitochondrial health,
mitochondrial biogenesis and mitochondrial size, for MACS isolated mitochondria. Data are
presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by Student’s T Test.
0 20 40 60 80
0
10
20
30
40
Oxygen Consumption Rate
Time (minutes)
OCR (pmol/min/1000 cells)
Basal
M1
0
2
4
6
OCR (pmol/min/1000 Cells)
ATP Production
*** *
Basal
M1
0
20
40
60
80
Testosterone Production in
Isolated Leydig Cells
Testosterone
(ng/million cells)
*
Basal
M1
0
20
40
60
80
Testosterone Production in
Isolated Leydig Cells
Testosterone
(ng/million cells)
*
Isolated Primary Rat LCs M1-Treated Isolated Primary Rat LCs
A
B
C
WT M1
0.00
0.05
0.10
0.15
0.20
Number of Mitochondria/µM
2
Mitochondrial Biogenesis
WT M1
0.00
0.05
0.10
0.15
0.20
0.25
Average Mitochondrial Area/µM
2
Mean Mitochondrial Size
***
***
85
4.4.3 Administration of M1 led to a decrease in weight and testosterone levels in rats:
Throughout the study, serum and weight measurements were taken to assess the
impact of a daily intraperitoneal injection of M1 over the span of six weeks. The M1
treated animals showed declining weight over the six weeks and a decreasing trend
in circulating testosterone levels (Figure 13A). Figure 12 showed that isolated
primary Leydig cells treated with M1 ex vivo after isolation and were shown to
produce increased levels of testosterone. However, when M1 was administered
daily, testosterone levels and weight gradually declined over the course of the 6-
week study, when compared with the untreated animals. Furthermore, analysis of
the isolated primary Leydig cells’ protein expression showed increased markers of
apoptosis (CASPASE3) and decreased expression of steroidogenic proteins
(CYP11A1, TSPO, STAR, VDAC) (Figure 13B). Collectively, these data show declining
Leydig cell health when animals are injected with M1 for six weeks.
86
Figure 13: Administration of M1 led to a decrease in weight and testosterone levels in rats. (A)
Rat weight and serum testosterone levels throughout the duration of the six-week M1 injection
study. (B) Representative immunoblots and comparative protein expression levels in control and
M1 injected rat Leydig cells after the completion of the study. Data are presented as mean ± SEM.
*p < .05, **p. < .01, and ***p < .001 by Student’s T Test.
Week 0
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
0
500
1000
1500
Serum Testosterone of M1 Injected Rats
Testosterone (ng/ml)
Control
M1
Week 0
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
0
200
400
600
Weight (g)
Rat Weight Over Time
Control
M1
Control M1
0.0
0.5
1.0
1.5
2.0
VDAC1
Relative Protein Expression
STAR 30 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
STAR
Relative Protein Expression
GAPDH 37 kDa
Control M1
0.0
0.5
1.0
1.5
CYP11A1
Relative Protein Expression
CYP11A1
GAPDH
50 kDa
37 kDa
STAR
GAPDH
30 kDa
37 kDa
Control M1
0
1
2
3
STAR
Relative Protein Expression
VDAC1
GAPDH
35 kDa
37 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
VDAC1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
OPA1
Relative Protein Expression
OPA1
GAPDH
81 kDa
37 kDa
Effect of M1 injection varies by tissue
42
Leydig cells Adrenal
***
*
Control M1
0.00
0.05
0.10
0.15
Relative Protein Expression
CASPASE 3
Caspase 3
GAPDH
32 kDa
37 kDa
*
TSPO
GAPDH
18 kDa
37 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
CYP11A1
GAPDH
50 kDa
37 kDa
Control M1
0
1
2
3
4
CYP11A1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
Relative Protein Expression
CASPASE 3
Caspase 3
GAPDH
32 kDa
37 kDa
TSPO
GAPDH
18 kDa
37 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
***
Control M1
0.00
0.05
0.10
0.15
Relative Protein Expression
CASPASE 3
Control M1
0
1
2
3
4
CYP11A1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
Control M1
0
1
2
3
STAR
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
OPA1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
VDAC1
Relative Protein Expression
B
30
37
B
GAPDH
STAR
B
18
37
B
GAPDH
TSPO
B
32
37
B
GAPDH
CASPASE3 B
50
37
B
GAPDH
CYP11A1
B
81
37
B
GAPDH
OPA1
B
35
37
B
GAPDH
VDAC1
B
32
37
B
GAPDH
CASPASE 3
B
50
37
B
GAPDH
CYP11A1
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
OPA1
GAPDH
81
37
Control M1
0.0
0.5
1.0
1.5
OPA1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
OPA1
Relative Protein Expression
Control M1
0.00
0.05
0.10
0.15
Relative Protein Expression
CASPASE 3
Control M1
0
1
2
3
4
CYP11A1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
VDAC1
Relative Protein Expression
35
37 GAPDH
VDAC1
A
B
87
4.4.4 Effect of M1 injection varies by tissue: Previous studies using M1 have showed
beneficial effects in a cardiovascular disease model and their relative healthy
counterparts after injection with M1
149
. Following these previously established
injection methodologies, protein levels of adrenal, liver, and heart samples were
evaluated. Immunoblots of protein extracted from the adrenal gland, another
steroidogenic tissue, showed decreased markers of apoptosis and increased
expression of some steroidogenic proteins (Figure 14A). Although the adrenal gland
produces steroids, this production does not affect circulating testosterone levels.
This data shows increased health and increased steroidogenic capacity in adrenal
cells. Analysis of protein expression levels in liver samples showed increased levels
of apoptotic markers and decreased expression of mitochondrial proteins (Figure
14B). These markers show declining liver health and cell integrity after the injection
study. We were interested in evaluating the effect on previously evaluated tissues
and examined the expression levels of these markers in protein extracted from
heart samples. The expression of apoptotic markers decreased in heart samples
(Figure 14C). Collectively, the assessed protein expression levels show that the
effect of systemic M1 treatment varies at the tissue level, with some tissues showing
beneficial health effects (adrenal, heart) and deleterious effects in others (testis,
liver).
88
Figure 14: Effect of M1 injection varies by tissue. Representative immunoblot and protein
expression values in adrenal (A), liver (B), and heart (C) samples after six-week injection with M1.
Data are presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by Student’s T Test.
Control M1
0.0
0.5
1.0
1.5
2.0
VDAC1
Relative Protein Expression
STAR 30 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
STAR
Relative Protein Expression
GAPDH 37 kDa
Control M1
0.0
0.5
1.0
1.5
CYP11A1
Relative Protein Expression
CYP11A1
GAPDH
50 kDa
37 kDa
42
Adrenal
*
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
Relative Protein Expression
CASPASE 3
Caspase 3
GAPDH
32 kDa
37 kDa
TSPO
GAPDH
18 kDa
37 kDa
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
B
32
37
B
GAPDH
CASPASE 3
B
50
37
B
GAPDH
CYP11A1
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
TSPO
Relative Protein Expression
OPA1
GAPDH
81
37
Control M1
0.0
0.5
1.0
1.5
OPA1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
OPA1
Relative Protein Expression
Control M1
0.00
0.05
0.10
0.15
Relative Protein Expression
CASPASE 3
Control M1
0
1
2
3
4
CYP11A1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
VDAC1
Relative Protein Expression
35
37 GAPDH
VDAC1
Liver Heart
CASPASE3
GAPDH
32
37
RIPK1
GAPDH
78
37
VDAC1
GAPDH
35
37
OPA1
GAPDH
81
37
MFN2
GAPDH
86
37
CASPASE3
GAPDH
32
37
RIPK1
GAPDH
78
37
VDAC1
GAPDH
35
37
OPA1
GAPDH
81
37
MFN2
GAPDH
86
37
Control M1
0.0
0.5
1.0
1.5
2.0
Relative Protein Expression
CASPASE 3
Control M1
0.0
0.5
1.0
1.5
2.0
RIPK1
Relative Protein Expression
Control M1
0
1
2
3
VDAC1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
OPA1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
MFN2
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
Relative Protein Expression
CASPASE 3
Control M1
0
1
2
3
RIPK1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
2.5
VDAC1
Relative Protein Expression
Control M1
0.6
0.8
1.0
1.2
1.4
OPA1
Relative Protein Expression
Control M1
0.0
0.5
1.0
1.5
2.0
MFN2
Relative Protein Expression
CASPASE3 RIPK1
VDAC1
OPA1
MFN2
CASPASE3 RIPK1
VDAC1 OPA1
MFN2
*** ***
***
*
**
*
**
B
C
Adrenal
A
89
4.5 Discussion
Age-related testosterone decline has been extensively studied and numerous findings
have enhanced our understanding of Leydig cell health and integrity. Declining Leydig cell
function has been associated with a number of cellular changes, including a reduction in the
expression of steroidogenic enzymes, increased antioxidant levels, and declining mitochondrial
function. Mitochondrial dynamics may play a role in regulating this decline, as dynamic control is
indispensable for cellular function and adaptability. Expression of key steroidogenic proteins
were previously shown to increase after promoting mitochondrial membrane fusion.
When MACS-isolated Leydig cells were treated with M1, the results suggested beneficial
effects for cell health and steroidogenic output, similar to that of the findings in chapter 3,
suggesting that Leydig cell health and function may be regulated by modulating mitochondrial
dynamics. After the treatment, the aged Leydig cells showed higher levels of mitochondrial
bioenergetics and testosterone formation. The improved oxygen consumption indicated that the
mitochondria of treated Leydig cells were more active and versatile to meet cellular energy
demands, and in addition have an increased capacity for steroid hormone formation. These
findings highlight the strong relationship between mitochondrial dynamics and Leydig cell
function. Specifically, promoting mitochondrial fusion may produce a significant improvement to
Leydig cell health, function, and longevity in aging models. However, these findings were not
emulated in our six-week injection study, as the rats produced slightly lower levels of
testosterone. It is also worth mentioning that M1 treated rats lost weight over the course of the
study.
90
The discrepancy between these two studies, specifically the beneficial effects seen in the
isolated model being in stark contrast to these results, is insightful, nonetheless. Ex vivo
treatment promoted mitochondrial membrane fusion and led to mitochondrial biogenesis,
improved mitochondrial function, and enhanced steroid hormone output. Overall, the health and
integrity of Leydig cells increased in an isolated model. There could be several reasons which
could describe this dichotomy. One possibility is that the injection may have had unintended
consequences when administered systemically in our animal model. While compounds that
modulate mitochondrial function may be beneficial when used in an isolated model, the systemic
administration may have had unintentional off-target effects that may have altered or disrupted
other physiological functions, processes, and systems. Figure 14 strengthens this suggestion, as
the effect of the treatment had either beneficial or deleterious effects depending on tissue type.
Moreover, data in chapter 3 showed that healthy MA-10 Leydig cells may suffer from hyperfusion
of mitochondria; a possibility that might be the case for some tissue samples shown here.
Another consideration is that the long-term exposure to the compound may produce negative
side effects; a possibility that may not have been observed in previous studies. In this case,
exploring the effects of long-term exposure to M1 in the MA-10 Leydig cells would be a valuable
investigation. Finally, the dosage and route of administration used could have a significant
impact. It is possible that differences in pharmacokinetics and pharmacodynamics could
significantly alter course of the compound. Thorough dosage and PK/PD studies are needed to
understand these possibilities. At present, we do not currently know how M1 is metabolized and
whether there are toxic metabolites which are generated from its metabolism. Although there is
91
a beneficial effect on testosterone formation when M1 is added directly to Leydig cells, this is not
observed when M1 is injected systemically.
Mitochondrial fusion has emerged as a promising therapeutic strategy and target for
improving cell health and function. Mitochondrial dysfunction is a hallmark of numerous
pathologies and implementing strategies which are able to tightly regulate, and control
mitochondrial health would significantly improve our ability to treat patients. Promoting
mitochondrial fusion may be one approach to enhance cell health and well-being, but more
investigations are warranted. Our findings suggest that fusion promoters could potentially
enhance the productivity of aged Leydig cells. However, more work is needed to better
understand how regulation of mitochondrial dynamics may be detrimental to Leydig cell health
and function.
92
Chapter 5: Conclusions and Future perspectives on the role of mitochondrial dynamics in Leydig
cell dysfunction and other disease pathologies
5.1 Summary
The objective of this project was to better understand the role of mitochondrial dynamics in
Leydig cell function, particularly regarding the development of age-related testosterone decline.
We’ve addressed this objective using relevant cell and animal models. Firstly, we characterized
how promoting mitochondrial fusion affects dysfunctional MA-10 Leydig cells, finding that this
approach restored mitochondrial function. Next, Leydig cells that were obtained from Sprague
Dawley rats were treated with M1 to promote mitochondrial fusion, which affirmed our previous
findings that this strategy improved mitochondrial function and steroid hormone formation.
Lastly, aged Sprague Dawley rats were injected with M1 to explore the relationship between
Leydig cell function and mitochondrial dynamics further. Interestingly, our final investigation
yielded results suggesting this strategy negatively affected Leydig cell function and steroid
hormone formation in vivo, which could be caused by unintended consequences of systemic M1
administration. Overall, this project contributed to the body of evidence suggesting the
significant role of mitochondrial fusion in steroid hormone formation. Moreover, this project
provided a greater understanding of the role of mitochondrial dynamics in Leydig cell aging.
93
5.1.1 Summary of Chapter 2: Severe testosterone decline, hypogonadism, affects nearly
five million men in the United States and is associated with numerous conditions
such as infertility, cardiovascular disease, and numerous others. Therapies to treat
this condition include testosterone replacement therapy and aromatase inhibitors.
Both strategies have negative side effects and alternative solutions to address
hypogonadism are desired. Numerous endogenous targets have been identified and
proposed to enhance steroid hormone biosynthesis.
TSPO is a crucial steroidogenic protein at the outer mitochondrial membrane.
TSPO’s high affinity for cholesterol binding has piqued significant research interest
and its role in steroidogenesis is widely accepted. TSPO ligands have been shown to
increase testosterone levels. However, these ligands may be non-specific, as they
also stimulate the production of adrenal steroids and neurosteroids. More recently,
the use of VDAC1 peptides has shown significant therapeutic potential. These
peptides block the 14-3-3ε and VDAC1 interaction and alter the regulation of
steroidogenesis. TVS167, a VDAC1 peptide, demonstrated Leydig cell specificity and
the induction of steroidogenesis. Another therapeutic strategy is the implantation
of human Leydig-like cells, which can produce steroid hormones. These cells are
differentiated from human induced pluripotent stem cells.
Studies evaluating mitochondrial dynamics as a potential therapeutic target for
the treatment of hypogonadism are limited. Previously, mitochondrial contact sites
were proposed to play a role in cholesterol transport, and mitochondrial fusion was
found to be essential for steroidogenesis. Moreover, deterioration of mitochondrial
94
function in aged and dysfunctional Leydig cells suggest a role of mitochondrial
dynamics in regulating steroidogenesis. This project evaluated these ideas and
found that promoting mitochondrial fusion enhances Leydig cell function and
steroid hormone production.
5.1.2 Summary of Chapter 3: The declining expression of key steroidogenic proteins with
aging is liked with alterations in cholesterol transport and hormone biosynthesis.
This results in the disruption of cholesterol translocation and steroidogenic function.
Most notably, TSPO decline is linked with declining mitochondrial function and
steroid biosynthesis. Given TSPO’s importance in mitochondrial function, we were
interested in mitochondrial dynamics as a target to address mitochondrial
dysfunction in TSPO deficient cells.
To enhance mitochondrial fusion in TSPO-deficient cell models, we first treated
the TSPO deficient nG1 Leydig cells with the mitochondrial fusion promoter, M1.
This treatment enhances mitochondrial membrane fusion by increasing the
expression of the mitofusion protein OPA1. These investigations showed that
inducing mitochondrial fusion enhanced mitochondrial function and
steroidogenesis in the nG1 cells. Moreover, expression of steroidogenic enzymes
increased. Next, we used a transient transfection to overexpress OPA1 and enhance
mitochondrial fusion. As expected, this strategy also increased mitochondrial
function and hormone biosynthesis. The expression of some steroidogenic enzymes
also increased with this treatment.
95
Previously, OPA1 was thought to not play a role in steroidogenesis, as its
elimination had not had an impact on steroid formation in MA-10 Leydig cells.
However, it is possible that other mitochondrial dynamic proteins may have
compensated for OPA1’s elimination. In our studies, pushing mitochondrial
membrane fusion further showed promising improvements to dysfunctional Leydig
cells, further showing that mitochondrial fusion plays a role in regulating
steroidogenic function.
5.1.3 Summary of chapter 4: Mitochondrial dynamics appear to play a regulatory role in
maintaining steroidogenic integrity. Given our previous studies demonstrated that
enhanced mitochondrial membrane fusion increased mitochondrial function,
steroid output, and expression of steroidogenic enzymes, we were interested in
exploring this relationship in an animal model.
First, we isolated Leydig cells from aged rats and treated them with M1 to
stimulate mitochondrial fusion. As expected, these Leydig cells had enhanced
bioenergetics and steroid hormone formation. This reaffirms our suggestion that
the relationship between mitochondrial dynamics and steroidogenesis can be
regulated to control steroid hormone formation. Specifically, mitochondrial fusion
as a target might allow us to significantly improve Leydig cell health, function, and
longevity in aging.
Next, we injected rats aged one year with 2 mg/kg/day of M1 to evaluate how
steroidogenic function may be affected by this therapeutic strategy. The results
showed that rats produced slightly lower testosterone levels and exhibited weight
96
loss. Moreover, the effect of M1 treatment varied by tissue. There are numerous
reasons these findings contrast previous investigations. It is possible that the
treatment had unintended effects systemically, long-term effects on Leydig cell
function, dosage effects, or that pharmacodynamic and pharmacokinetic altered
the effectiveness of our treatment strategy.
97
5.2 Challenges
5.2.1 Role of mitochondrial dynamics regulating biological processes: Mitochondrial
dynamics regulate numerous cell processes and functions which systemically
influence cell and tissue function. Mitochondrial fusion, fission, mitophagy, and
mitochondrial biogenesis regulate cell function and allow for adaptability to
changing conditions that the cell may face. Given how interconnected these
processes are, a systemic treatment designed to treat mitochondrial dysfunction
may have detrimental effects on other biological systems. Dysregulation of these
processes may cause cell-specific dysfunction and affect tissue function.
5.2.2 Formulation, dosage, route of administration, pharmacodynamics, and
pharmacodynamics: Studies using promoters of mitochondrial fusion in animals are
limited. Therefore, the most suitable form of delivery, excipients, and chemical
structure have not been identified or optimized for use in animals. This information
could be used to select a route of administration which would optimize absorption,
distribution, metabolism, and excretion. Another limitation is the information
available regarding the dosage of M1 used. A suitable dosage may need to be
adjusted based on factors of the animal profile, such as age, sex, weight, and organ
function.
98
5.2.3 Inherent discrepancies between in vitro, in vivo, and animal models: The highly
controlled environment of the preliminary MA-10 studies lacks the complexity of
systems within an animal that affects the intended properties of the drug. This is a
universal challenge for all studies that work vertically towards the development of
a novel therapeutic. The effect of the treatment changed as we transitioned from
this cell model to the animal model, highlighting complexities to consider in future
studies.
99
5.3 Future Perspectives
5.3.1 Contribution to the field of age-related testosterone decline: The gradual decline
in testosterone levels with aging is normal, but can be more pronounced in some
individuals, leading to testosterone deficiency, hypogonadism. Given testosterone
plays a role in maintaining muscle mass, sexual function, and overall vitality, its
decline has been associated with a number of age-related conditions. At the same
time, dysregulation of mitochondrial dynamics is also a hallmark of age-related
conditions, including Leydig cell function. This dissertation explores the relationship
between the two, finding that dynamics may be a promising target for Leydig cell
dysfunction.
5.3.2 Relationship between mitochondrial contact sites and the steroidogenic
interactome: Mitochondrial contact sites have long been thought to play a role in
cholesterol’s translocation for steroidogenesis. The expression of SITE proteins
declines with aging, which may be related to declining regulation of mitochondrial
dynamics. Our results suggest that the expression of SITE proteins is closely
regulated by mitochondrial fusion, as our investigations enhancing fusion ultimately
led to increased expression of key steroidogenic enzyme.
100
5.3.3 Further exploration of the progression of age-related disease pathologies:
Dysregulation of mitochondrial dynamics has been associated with the
development and progression of age-related pathologies, including
neurodegenerative diseases, cardiovascular diseases, metabolic disorders, and
numerous forms of cancers. However, it’s role in these conditions is multifaceted
and not well understood. Our studies contribute to the body of evidence which
suggests mitochondrial dynamics may be a promising target for numerous
pathologies. Developing therapeutics that selectively target the mitochondrial
dynamics of a specific cell type may offer promising strategies for the treatment and
prevention of age-related diseases.
5.4 Conclusions
Alterations of Leydig cell function and cholesterol transport have long been understood
as a hallmark of male hypogonadism. Aged Leydig cells present reduced TSPO levels when
compared to younger healthy Leydig cells. TSPO’s decline is has implications on cholesterol
transport into the mitochondria, a critical step in the steroidogenic process. Moreover, TSPO’s
role in Leydig cell bioenergetics is well understood. We became interested in exploring the
relationship between the functional decline of Leydig cells and bioenergetics, and we began to
explore the relationship between mitochondrial fusion and steroidogenic capacity and were
pleased to find that enhancing fusion improves Leydig cell function and steroidogenic output.
Mitochondrial fusion has become recognized as a promising therapeutic target for cell
health and function. Dysfunctional mitochondria have been associated with several diseases
101
which manifest in different ways. Focusing on this ubiquitous mitochondrial dysfunction could
significantly improve our ability to treat patients. While mitochondrial fusion may hold promise
as a disease target, more research is needed to determine potential as well as its limitations.
102
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Abstract (if available)
Abstract
The objective of this dissertation is to investigate the role of mitochondrial dynamics in the maintenance of Leydig cell health and integrity in age-related functional decline, and the role of mitochondrial dynamic regulation in steroid hormone formation and mitochondrial function. Testicular Leydig cells are specialized interstitial cells which produce and maintain testosterone, the primary androgenic steroid hormone in males. Testosterone is a sex hormone which plays a critical role in the development and maintenance of male health and well-being. Its presence regulates numerous physiological functions, such as the maintenance of muscle mass, bone mineral density, mood, sexual function, and many others. The decline in testosterone levels of aging males is a significant concern and has been linked to significant age-related morbidities and declining health. Leydig cell functionally declines with aging, and they produce reduced levels of testosterone. Moreover, mitochondrial function is also compromised in aged Leydig cells. Ultimately, significant deterioration of the steroidogenic process can lead to testosterone deficiency, known as hypogonadism.
Testosterone formation in Leydig cells is mediated by a sequential series of signaling and metabolism events. A protein scaffold containing numerous steroidogenic proteins, dubbed the steroidogenic interactome, is involved in the translocation of cholesterol from the cytosol into the mitochondria. The expression of the proteins in the steroidogenic interactome is regulated by various signaling pathways and transcription factors, which become dysregulated in aging Leydig cells. The regulation of steroidogenesis is a complex integrated process and the development of its dysfunction in aging is not fully understood. Herein, we’ve investigated the role of mitochondrial dynamics in the regulation of Leydig cell steroidogenic function.
We hypothesize that dysregulation of the interplay between mitochondrial dynamics and the expression of steroidogenic proteins plays a role in the deterioration of Leydig cell health and integrity with aging. The mitochondria are essential cellular organelles which contain numerous steroidogenic proteins that are necessary to produce testosterone and other steroid hormones. These dynamic organelles play a critical role in energy production, cellular metabolism, and cell function. Mitochondria regulate their shape and structure in response to physiological stimuli and cellular energy demands. Dysregulation of mitochondrial function has been linked with aging and the development of numerous neurodegenerative, metabolic, and other diseases. Several cellular signaling pathways are altered in age-related pathologies, including the regulation of mitochondrial fission and fusion, leading to the accumulation of fragmented dysfunctional mitochondria. Therefore, we hypothesized that Leydig cell dysfunction could be attenuated by enhancing mitochondrial dynamics.
Three aims have been developed to address the objective of this project and the hypothesis. First, we characterized the dysfunction that manifests in aged and steroidogenic deficient Leydig cells. Next, we used a combination of treatments to regulate mitochondrial dynamics in Leydig cells, resulting in the upregulation of mitochondrial fusion. Lastly, rats aged one year were dosed with the cell-permeable mitochondrial fusion promoter 4-Chloro-2- (1- (2- (2, 4, 6-trichlorophenyl) hydrazono) ethyl) phenol to alter systemic mitochondrial dynamics. When taken together, these results support our hypothesis and show that the dysregulation of mitochondrial dynamics in aged Leydig cells plays a central role in the deterioration of Leydig cell health and integrity with aging. The dysregulation of mitochondrial dynamics has been associated with numerous chronic illnesses, and proteins involved in the regulation of mitochondrial dynamics may be promising biological targets for the maintenance of cell health and function.
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Creator
Garza, Samuel Kenneth
(author)
Core Title
Mitochondrial dynamics regulate Leydig cell health and integrity
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Degree Conferral Date
2023-08
Publication Date
06/30/2024
Defense Date
05/17/2023
Publisher
University of Southern California
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age-related testosterone decline,aging,androgens,cell signaling,Gerontology,Hormones,hypogonadism,Leydig cell,Leydig cells,mitochondria,mitochondrial dynamics,mitochondrial function,Molecular Pharmacology,OAI-PMH Harvest,primary hypogonadism,reproductive biology,steroid,steroidogenesis,steroids,tested,testes,Testis,testosterone
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Papadopoulos, Vassilios (
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Samuelkg@usc.edu,Sgarza2@outlook.com
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Tags
age-related testosterone decline
androgens
cell signaling
hypogonadism
Leydig cell
Leydig cells
mitochondria
mitochondrial dynamics
mitochondrial function
primary hypogonadism
reproductive biology
steroid
steroidogenesis
steroids
tested
testes
testosterone