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Fetal exposure to genistein and DEHP mixtures alters the expression of genes involved in critical testicular functions in adult rats

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Fetal exposure to genistein and DEHP mixtures alters the expression of genes involved in critical testicular functions in adult rats

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FETAL EXPOSURE TO GENISTEIN AND DEHP MIXTURES ALTERS THE EXPRESSION OF
GENES INVOLVED IN CRITICAL TESTICULAR FUNCTIONS IN ADULT RATS

BY

PRIYANKA RAVINDRA MALUSARE

A THESIS PRESENTED TO THE
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

AUGUST 2023

ii

DEDICATION
To my PI, Dr. Martine Culty, whose guidance, and unwavering support were fundamental in completing
my thesis. Your knowledge, devotion, and mentoring have helped shaped me into the researcher I am today.
I will be forever grateful for your tremendous impact and conviction in my ability, which made this journey
possible.

iii

ACKNOWLEDGEMENTS
I would like to express my heartfelt gratitude to Dr. Martine Culty, my Lab's Principal Investigator, for her
great advice, mentorship, and steadfast support during this research endeavor. Her knowledge, devotion,
and enthusiasm for scientific discovery have all played a role in defining my academic and professional
development. I am eternally grateful to her for her invaluable contributions.
I also want to thank my valued lab colleagues and friends, Chantal Sottas, Casandra Walker, Garret Cheung
and Amina Khan for their collaborative attitude, insightful talks, and unwavering support. Their
perspectives, shared information, and constructive input were critical in molding the study's conclusion.
I am grateful to my friends and family for their unwavering encouragement, understanding, and faith in my
skills. Their constant encouragement, patience, and love have provided the necessary foundation and
motivation during challenging times. This thesis reflects their collaborative efforts, and I am thankful for
their presence in both my academic and personal lives.
I am deeply indebted to my thesis committee; Dr. Vassilios Papadopoulos and Dr. Angel Tabancay for their
time, devotion, and insightful feedback. Their knowledge in their various domains has helped shape the
path of my study and ensure its rigor and intellectual brilliance.
Furthermore, I am thankful to the USC Alfred E. Mann School of Pharmacy and Pharmaceutical Sciences
for allowing me to conduct this study and for making resources and facilities accessible to me.

iv

TABLE OF CONTENTS

Dedication……………………………………………………………………………………………….….ii
Acknowledgements.......................................................................................................................................iii
List of Tables................................................................................................................................................v
List of Figures................................................................................................................................................vi
Abbreviations………………………………………………………………………………………….…...ix
Abstract.........................................................................................................................................................xi
Chapter1:Introduction....................................................................................................................................1
Chapter2:Methods........................................................................................................................................16
Chapter3:Results..........................................................................................................................................19
Chapter4:Discussion……………................................................................................................................36
References……………................................................................................................................................50

v

LIST OF TABLES:
Table 1: Different types of EDCs and their effect on the male reproductive system
Table 2:The rate of adverse reproductive effects (A) Abnormal testes and small litter and (B)Infertiliy rate
in adult rats exposed in utero to Gen and DEHP, alone or mixed, at doses corresponding to those in humans.
Gen+DEHP mixtures aggravated the effects, and the lowest dose had negative effects in F1 to F3
generations.
Table 3: In Silico search for Foxa3-intercating genes in testes. Genes related to Foxa3 were searched in
IPA in rats that were exposed to (A) 0.1 and (B) 10 mg/kg/day using RNA-seq data
Table 4: Genes that bind to Foxa3 by Chip-seq analysis which were examined using RNA-seq data in rats
exposed to Gen+DEHP at 0.1 and 10 mg/kg/day.
Table 5: IPA was used to identify differentially expressed genes associated to "steroidogenesis" in testes
exposed to Gen+DEHP at 0.1 mg/kg/day
Table 6: Foxa1 is differentially expressed in F1 and F2 generation exposed to Gen+DEHP at 10 mg/kg/day
Table 7: Microarray data was used to examine the fold change of genes associated to DNA
Table 8: RNA seq analysis of F1 PND120 Gen+DEHP and F2 PND120 Gen+DEHP at 10 mg/kg/day
Table 9: RNA seq analysis of F1 PND120 GEN+DEHP and F2 PND120 GEN+DEHP at 10 mg/kg/day
Table 10: RNA seq analysis of F1 PND120 GEN+DEHP and F2 PND120 GEN+DEHP at 10 mg/kg/day
Table 11: RNA seq analysis of F1 PND120 Gen+DEHP at 10 mg/kg/day
Table 12:RNA-Seq analysis gene expression changes in Stat3 and Jak2 in F1 and F2 rat testes

vi

LIST OF FIGURES:
Figure1: Factors affecting male fertility. FSH, follicle-stimulating hormone; LH, luteinizing hormone;
ROS, reactive oxygen species.
Figure 2: Historical milestones in understanding the mechanism of EDCs.
Figure 3: EDCs and the disruption of Hypothalamus-pituitary-testis axis.
Figure 4: Cross-section of Seminiferous tubules.
Figure 5: Critical windows for sensitivity to the effects of EDCs
Figure 6: EDC-induced epigenetic alterations.
Figure 7: Testicular Morphology in F1 and F2 Adult Rat Testis is altered by Gen and DEHP
exposure. PND120 rat testes were exposed in utero to Gen, DEHP, and Gen+DEHP at
10mg/kg/day.
Figure 8: Effect of exposure to EDC alters 5methylcytosine in PND120 rat testes.5mec was used as a marker
for DNA methylation.
Figure 9: Effect of exposure to EDC alters H3k27 methylation in PND120 rat testes.H3k27 was used as a
marker for DNA methylation.
Figure 10: Venn’s Diagrams showing the number of altered genes, common and unique between treatments,
in gene array analysis of neonatal and adult rats, exposed in utero to vehicle or 10 mg/kg/day of Gen, DEHP
or their mixture.
Figure 11: IPA analysis showing canonical pathways that are differentiated in PND120 rats at 0.1
mg/kg/day dose.
Figure 12: Foxa3 mRNA expression in F1, F2, and F3 adult rat testis.
Figure 13: RNA sequencing on rats exposed to Gen+DEHP at 0.1 and 10 mg/kg/day was analyzed using
Partek Flow.
Figure 14: Expression of Foxa1 in testes of adult rats exposed in utero to F1 GD at 0.1 and 10mg/kg/day.
Immunofluorescence analysis of Foxa1.
Figure 15: Foxa1 interaction network in Gen+DEHP at 10 mg/kg/day exposed rat testes demonstrating its
relationship to Dnmt3a.
vii

Figure 16: Expression of Foxa1 in testes of adult rats exposed in utero to F2 GD at 10mg/kg/day.
Immunofluorescence analysis of Foxa1.
Figure 17: IPA search for genes associated to the term "DNA methylation" in my dataset for testes exposed
to Gen+DEHP at 10 mg/kg/day dosage.
Figure 18: In IPA, an interaction network shows differentially expressed genes associated to Dnmt3a in F1
rats exposed in utero to Gen+DEHP at a level of 10 mg/kg/day.
Figure 19: Interaction network in IPA showing differentially expressed genes related to Tet1 in F1 rats
exposed in-utero to Gen+DEHP at 10 mg/kg/day dose.
Figure 20: Expression of Dnmt3a in testes of adult rats exposed in utero to F1 GD at 10mg/kg/day.
Immunofluorescence analysis of Dnmt3a
Figure 21: qPCR analysis of genes involved in DNA methylation (Dnmt3a, Tet1). N=5 rats per treatment
Figure 22: IPA analysis showing the differential expression of genes that are related to the term “histone
modification.”
Figure 23: Interaction network in IPA showing differentially expressed genes related to (A)Brwd1 and (B)
Ezh1 in F1 rats exposed in-utero to Gen+DEHP at 10 mg/kg/day dose.
Figure 24: qPCR analysis of genes involved in Histone modification (Brwd1). N=5 rats per treatment.
Figure 25: IPA analysis showing the differential expression of genes that are related to the term “Chromatin
remodeling.”
Figure26: Interaction network in IPA showing differentially expressed genes related to Atrx in F1 rats
exposed in-utero to Gen+DEHP at 10 mg/kg/day.
Figure 27: qPCR analysis of genes involved in chromatin remodeling (Atrx). N=5 rats per treatment.
Figure 28: Interaction network in IPA showing differentially expressed microRNAs related to
(A)Transcription and (B)RNA expression rat testes exposed in-utero to Gen+DEHP at 10 mg/kg/day.
Figure 29: mRNA expression changes in Stat3 and Gen,DEHP alone and mixture determined by qPCR
Figure 30:IPA analysis shows a canonical pathway for Stat3 and interacting genes in (A)F1 and (B)F2
generation exposed to Gen+DEHP at 10 mg/kg/day
Figure 31 : Immunofluorescence analysis of Stat3 in adult rat testes exposed in utero to Gen+DEHP at
10mg/kg/day in F2 generation Signal intensity quantified by Image J.
viii

Figure 32: Immunofluorescence analysis of Stat3 in adult rat testes exposed in utero to Gen+DEHP at
10mg/kg/day in F2 generation. Signal intensity quantified by Image J.
Figure 33: Intergenerational and Transgenerational inheritance.
Figure 34: Basic functions and knockout phenotypes of Dnmt genes.
Figure 35: Localization of different types of Dnmts in seminiferous tubules.
Figure 36: Developmental DNA methylation reprogramming.
Figure 37: Histone modifications and spermatogenesis.
Figure 38: EZH1 and EZH2 roles in the creation and maintenance of H3K27 methylation during
spermatogenesis.
Figure 39: DBP induced testosterone synthesis inhibition.

ix

ABBREVIATIONS

Gen: Genistein
DEHP :2-(diethylhexyl phthalate)
Gen+DEHP or GD: Genistein and DEHP mixture-
0.1 : 0.1mg/kg/day
10:10mg/kg/day
EDC: Endocrine disrupting chemical-
Foxa3:Forkhead Box A3
AR: Androgen Receptor
ER :Estrogen Receptor
Foxa1 :Forkhead Box A3
Dnmt3a: DNA (cytosine-5)-methyltransferase 3A
Tet1: Tet Methylcytosine Dioxygenase 1
TdG: Thymine DNA Glycosylase
Dnmt3b: DNA (cytosine-5)-methyltransferase 3B
Dnmt3l: DNA (cytosine-5)-methyltransferase 3L
DBCP: dibromochloropropane
DDT: Dichlorodiphenyltrichloroethane
PGCs: Primordial germ cells
GnRH: Gonadotropin-releasing hormone
INSL3: Insulin-like 3
PPAR: Peroxisome proliferator-activated receptors
Stat3: Signal transducer and activator of transcription 3
Jak2: Janus kinase 2
H3k27: Methylation of histone H3 on lysine 27
5mec:5-Methylcytosine
Tmeff2: Transmembrane protein with an EGF-like and two Follistatin-like domains 2
Phlpp1: PH domain and leucine rich repeat protein phosphatase 1
Arid4a: AT-Rich Interaction Domain 4A
Atrx: alpha-thalassemia/mental retardation, X-linked
Movl10l1: Moloney leukemia virus 10 protein
Brwd1: Bromodomain and WD Repeat Domain Containing 1
Ezh1: Enhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit

x

STATEMENT
Most of my thesis work was included in the poster presentation “Fetal Exposure To Genistein And DEHP
Mixtures Alters The Expression Of Genes Involved In Critical Testicular Functions In Adult Rats”
that was presented at the 45th Annual Conference of the American Society of Andrology on April 19-23,
2023 in Boston, USA.

xi

ABSTRACT
Approximately 50 million people worldwide suffer from infertility with male factors accounting for nearly
half of all cases. Exposure to endocrine disrupting chemicals (EDCs) play a role in the etiology of male
reproductive diseases in animal models, and strong associations have been reported in human studies
between exposures to EDCs and male reproductive diseases. The mechanisms by which fetal-maternal EDC
exposure damage the male reproductive system in offspring is not fully understood. They can act by EDCs
well-known actions, such as hormone mimicking and receptor binding interference in fetal reproductive
tissues, as well as alteration of placental transfer that could have broader effects on fetal development.
Moreover, studies have shown that the impact of EDCs can carry over from directly exposed fetuses (F1
generation) to their descendants (F2 and F3 generations) via epigenetic effects. Humans are exposed to
multiple EDCs throughout their life. My goal is to identify genes and functional mechanisms altered by
EDCs in testis, as read-out for disrupted reproductive function, using the rat as model, and two common
EDCs, genistein (Gen) and Di (2-ethylhexyl) phthalate (DEHP), used at doses meaningful to human
exposure. While exposure to the phytoestrogen Gen is dietary via soy products, exposure to the ubiquitous
antiandrogenic plasticizer DEHP comes from various consumer products and medical devices. In previous
studies, we found that in-utero exposure of rats to low doses of genistein and DEHP mixtures (Gen+DEHP)
altered testis development and transcriptome, and increased infertility rates. We also found that these
adverse effects are transmitted from F1 offspring exposed to the EDCs as fetuses, to their sons (F2) and
grandsons (F3), indicating inter- and transgenerational transmission. My study seeks to identify
differentially expressed genes, proteins and functioning pathways induced by Gen and DEHP fetal exposure
in testes of offspring across generations. This study gives an in-depth look at the effects of Gen and DEHP
fetal exposure on male reproductive health by identifying changes in transcription factors related to
testicular function and epigenetic processes in the testes of F1 to F3 offspring that could be linked to the
adverse effects observed in our previous studies. This work should give insights into the influence of Gen
and DEHP exposures on male infertility and reveal mechanisms involved in the negative reproductive
effects of EDC mixtures in male reproductive health. Understanding these mechanisms could help identify
new markers to assess EDCs male reproductive risk and devise ways to reduce EDCs adverse reproductive
effects.

1

CHAPTER 1: INTRODUCTION
DISORDERS ASSOCIATED WITH THE MALE REPRODUCTIVE SYSTEM:

• Male infertility: Infertility is the failure of a couple to conceive even after a year of unprotected sexual
activity. The prevalence of infertility, which affects one in every six couples globally, has made it a
global health issue(Skakkebaek and colleagues, 2022).The study of male infertility has received a great
deal of attention during the past several years. This is significantly impacted by the general public's
greater knowledge of the issue, the global decline in the quality of healthy sperm in men, and the
psychological impact of the illness on men's mental health(Blay et al., 2020). Several variables,
including exposure to chemicals in the environment, radiation, or medicines, might affect a couple's
fertility rate. It is finally acknowledged that male-specific tumors like prostate and testicular cancer can
harm male reproductive capabilities.
• Testicular cancer: Even though testicular cancer is not given much importance when comparing it
with other types of male cancer, studies show that it is the most frequently diagnosed form of cancer in
men of reproductive age (Tvrda et al., 2015).Its non-invasive precursor form, carcinoma in situ (CIS)
also called germ cell neoplasia in situ (GCNIS) forms in early life from fetal/neonatal germ cells that
fail to differentiate. The sudden hormonal surge that occurs during puberty in men triggers CIS cells
to transform into aggressive/invasive tumor cells such as seminoma and embryonal carcinoma, and this
disease is believed to share common causes with male infertility and other male reproductive syndromes
and disorders, this might be one of the reasons for the correlation (Moorthy HK, 2008)between male
Figure1: Factors affecting male fertility. FSH, follicle-stimulating
hormone; LH, luteinizing hormone; ROS, reactive oxygen
species.(Leisegang & Dutta, 2021)
2

infertility and cancer. Among all testicular cancers, testicular germ cell cancer is the most common type
of cancer roughly affecting 9000 men annually in the US alone (Tvrda et al., 2015).
• Prostate Cancer: According to research by the center for disease control and Prevention, prostate
cancer is the most common cancer among American males, with an incidence of 1,276,106 new cases
and 358,989 fatalities(Tvrda et al., 2015; Rawla, 2019). It is the second leading cause of death in the
US, after lung cancer. The cells that line the glandular tissue of the prostate, which oversees creating
prostate fluid, are typically where the tumor begins. While there is no one-size-fits-all approach to
avoiding prostate cancer, people may lower their risk by getting frequent examinations, living a healthy
lifestyle, and limiting their exposure to risky drugs(Cozzi, 2022).
FACTORS AFFECTING MALE INFERTILITY:
• Genetic factors: They are fundamental in the emergence of both male malignancies and diminished
fertility. Y chromosome’s short arm is home to the genes that determine sex. It is also suggested that
primordial germ cells with Y chromosomal abnormalities give rise to testicular germ cell tumors(Tvrda
et al., 2015; Alberto Ferlin, 2006).Male malignancies are also affected by defects in DNA repair
pathways, which can result in genomic instability, raising the risk of cancer formation. Tumor
suppressor genes also play a role in male cancers. Both hereditary and acquired chromosomal
abnormalities are possible. Klinefelter syndrome, which accounts for 14% of instances of male
infertility, is the most common hereditary cause of azoospermia in people with aneuploid sex
chromosomes(Tvrda et al., 2015).
• Epigenetic factors: Since epigenetic processes do not alter the DNA sequence but instead induce
differences in gene expression by either activating or suppressing transcription, they are different from
genetic factors(Dada et al., 2012; Tvrda et al., 2015). The most thoroughly studied epigenetic
mechanism is DNA methylation (Tvrda et al., 2015). While Tet and TdG enzymes are responsible for
removing DNA methyl groups, the DNA methyl transferases family of enzymes, which includes
Dnmt3a and Dnmt3b adds a methyl group to the 5' end of cytosine (Gunes & Esteves, 2021).
• Environmental factors: Studies have shown that environmental pollution adversely affects semen
quality by interrupting the process of spermatogenesis, and disrupting Sertoli cells and Leydig cells,
which play a crucial role in the functioning of the normal male reproductive system, further causing
male infertility(Tvrda et al., 2015).Additionally, various natural and manmade chemicals present in the
environment have a detrimental impact on human fertility (Babakhanzadeh et al., 2020). An obvious
example of environment playing a role in male infertility is the study done on workers who were in
contact with the pesticide dibromochloropropane (DBCP). The results of these studies showed that
these workers, due to the exposure of DBCP became infertile because of either azoospermia or
3

oligozoospermia(Eaton, 1986). The external environment of an individual varies every day and from
one stage of life to another. As a result of industrialization, people's lifestyles now are significantly
different from those of past generations. Simply put, we cannot avoid chemical exposure, no matter
how hard we attempt to adjust our lifestyles, diets, and other behaviors. The reproductive system can
be impacted by environmental toxicants at any stage of life, from conception to old age, but it is most
vulnerable during fetal development, and exposure to harmful chemicals during this phase can cause
lifelong consequences. Examples of environmental stresses affecting male infertility include chemicals
such as phthalate plasticizers, exposure to radiation, elevated temperature, prolonged sitting at work,
etc. A lot of studies have shown that smoking cigarettes and alcohol consumption are directly
proportional to male infertility cases (Babakhanzadeh et al., 2020).Over the last few years, a significant
amount of epidemiological and experimental studies have been done on the effect of EDCs on human
health, further increasing awareness among humans. It has been found that EDCs are linked to harmful
effects on human health, mainly the male reproductive system (Rodprasert et al., 2019).
ENDOCRINE DISRUPTING CHEMICALS:
EDCs are defined by the World Health Organization as "an exogenous substance or mixture that alters
endocrine system functions and causes adverse health effects in an intact organism, its progeny, or
(sub)populations." (Gore, 2015) The endocrine system is critical to maintaining homeostasis in our bodies
because it uses hormones that can transport information across enormous internal distances. Chemicals
classified as "endocrine disruptors" interfere with the normal operation of the endocrine system and have a
deleterious influence on organisms(Gore, 2015). Before significant investigation, it was considered that
they worked through endocrine, androgen, progesterone, and thyroid receptors, among others. However,
extensive research in this field has revealed that the methods by which they function are wider than
previously imagined(Philippa Darbre, 2015; Guarnotta et al., 2015).Hence EDCs are compounds found in
the environment that can be either natural or manufactured that can change the hormonal system, which
allows individuals to connect with their surroundings. Food, water, plastics, personal care items, fertilizers,
papers, cutlery, cosmetics, toys, and other common products contain EDCs. Because people utilize these
items regularly, they are the ones who are exposed to the harmful consequences of these compounds.
(Darbre, Philippa, 2015)

4

A SHORT HISTORY OF ENDOCRINE DISRUPTION:

Endocrine disruption research began in the 1920s and 1940s when infertility in pigs and lambs was related
to diets high in moldy grain (mycoestrogens) and clover (phytoestrogens)(Darbre,2015). Rachel Carson's
1962 book "Silent Spring" drew a lot of attention to the use of DDT on animals(Barker, 2007).Scientists
discovered at the time that a lack of food and insects was not the sole cause of dwindling fish and bird
populations, but that DDT deposition in their bodies also played a significant impact. Rachel Carson noticed
that the buildup was hurting aquatic life's reproductive capacities(Barbara DEMENEIX et al., 2019). Even
though, she never used the term “endocrine disruption”, she was describing the phenomenon. Surprisingly,
she also predicted that in the future, the adverse effect of EDCs will also be seen in humans, which
unfortunately turned out to be true. The statistics showing an increased risk of breast cancer in girls who
were exposed to DDT during the 1960s support this hypothesis (Cohn et al., 2015).
Endocrine connection: Endocrine-disrupting chemicals interfere with the regular functioning of our
bodies hormones. Some EDCs fool function as "hormone mimics," whilst others inhibit hormones in our
bodies. In addition, a few EDCs alter our bodies' susceptibility to various hormones. As a result, they may
influence hormone function and harm human health(Barbara DEMENEIX et al., 2019).

Figure 2: Historical milestones in understanding the mechanism of EDCs EDCs(Darbre, 2015)
5

EDCS AND MALE REPRODUCTIVE HEALTH:

Male reproductive health, especially sperm count and testosterone levels, has been declining in recent years
(Tvrda et al., 2015).Subsequently, the amount of EDCs in the environment has also increased. For example,
the levels of polybrominated diphenyl ether (PBDE) have been increasing over the last 30 years, DDT,
DDE usage has been escalating in Africa and other parts of the world, and numerous EDCs like BPA, PCP,
Triclosan have also gained more significance in the environment due to their presence in commonly used
Personal care products as well as manufacturing practices(Rehman et al., 2018). This supports the argument
that exposure to EDCs can be a contributing factor to the decrease in semen quality resulting in increased
male infertility. The endocrine system is important for male reproductive health as androgens like
testosterone are not only responsible for the development of internal and external male genitalia, but also
male secondary traits, as well as helping in the process of spermatogenesis(Rehman et al., 2018). As a
result, antiandrogenic EDCs and medications can disturb the process, resulting in male reproductive
diseases. Most of the research on the impact of EDCs on the male reproductive system has focused on
fundamental seminal parameters; however, there is evidence that EDCs can also influence the level of
endocrine systems. TCS, for example, has been shown to bioaccumulate in the epididymis(Pycke &
2014).BPA has been documented to exhibit estrogenic and antiandrogenic effects. Moreover, it has been
linked to a reduction in sperm quality. Toxicological research has shown that BPA can lead to adverse
reproductive outcomes in rodents, resulting in reduced epididymal weight, daily sperm production, and
testosterone levels(Cariati et al., 2019).
Figure 3: EDCs and the disruption of Hypothalamus-pituitary-testis
axis.(Casandra Walker, 2021)
6

EDCs can negatively impact the male reproductive system by interfering with the normal functioning of
the endocrine system. Some possible mechanisms by which the EDCs impair the male reproductive system
include:(Philippa D. Darbre, 2022)
• Mimicking the action of natural hormones such as estrogen, testosterone, and thyroid, sabotages
the hormonal equilibrium and disrupts the male reproductive system.
• Interfering with hormone receptor binding
• Influencing the synthesis, metabolic, transport, and excretion of hormones thus causing hormonal
imbalance.
• Disrupting the normal growth and functioning of male reproductive organs such as the testes,
epididymis, prostate gland, and seminal vesicles can cause decreased sperm production, sperm
quality, and male infertility.
• Causing oxidative and DNA damage, epigenetic alterations leading to male infertility
The mechanism through which EDCs act on the endocrine system is done on animals and in-vitro
systems. Thus, the limited human data and in many instances inconsistent data across studies, highlight
the need for further research on the impact of these chemicals on the male reproductive system.
FETAL-MATERNAL EXPOSURE TO EDCS:
Pregnancy is a vulnerable phase for both the mother and the developing embryo. Both can be concurrently
affected by the effects of EDCs(Walker et al., 2020). Childbearing age is one of the most important times
in a woman’s life, the environment of a woman during that phase will determine the outcomes of
pregnancies in the future. Diseases like diabetes, cardiovascular, obesity, etc. can have negative effects on
the fetus, including gestational diabetes, perinatal death, and low birth weight. Hence, the best way to avoid
these outcomes is by having a comorbidity-free reproductive age(Rolfo et al., 2020). Endocrine-disrupting
chemicals can play a major role in fetal programming as the placenta is not an effective barrier against these
chemicals, additionally, the fetus might be more sensitive to these effects than the mother. For that reason,
maternal uptake of appropriate nutrients and chemicals plays a fundamental role. This is supported by
various studies on animals and humans, studying the effect of insufficient or excessive uptake of these
substances(Street & Bernasconi, 2020).
From environment to the placenta: While studying the impact of EDCs, many researchers focused on single
chemicals. For instance, on the measurement of phthalate metabolizers in the urine of pregnant mothers, it
7

was found the levels of this plasticizer present in cosmetics and personal care products were above the limit
of detection in at least one sample in 50% of the women included in the study (Arbuckle et al., 2016).
Another study focused on investigating the levels of Triclosan present in toothpaste and medical devices.
TCS was detected in all participants of the study (Pycke et al., 2014). Moreover, other studies dealt with
chemicals like paraben, PAHs, PFOAs, and other harmful chemicals in the environment. In the past few
years, the focus has shifted to studying the relationship between EDCs and the placenta. Researchers found
a range of EDCs in the placenta and their effect on fetal growth and development was directly proportional
to each other. The effect was more prominent in males. As a result, the placenta seems to play a critical role
in the impairment of the male reproductive system. It is important to understand the mechanisms by which
EDCs affect the morphology and function of the male reproductive system.
From Placenta to Newborn(Street & Bernasconi, 2020): The mechanism of how EDCs cross the placental
barrier is still largely unknown. Some studies say that the mechanism might be passive while some suggest
it to be active, it mainly depends on the specific EDC. There are multiple factors affecting the transfer of
EDCs into the fetus such as physicochemical properties of the EDCs, distribution into the tissues, and
socioeconomic factors like maternal age and race.
Table 1: Different types of EDCs and their effect on the male reproductive system:
EDCs Effect
Bisphenol A Decreased sperm count and motility(Hu et al., 2017).
Pesticides Reduced sperm quality and hormonal imbalances in men(Tavares
et al., 2013).
Phthalates Abnormal sperm quality and morphology, decreased
testosterone(Akingbemi et al., 2004; Pan et al., 2006).
Dioxins Male/female ratio of off springs, decreased sperm
concentrations(Larsen, 2006).
8

Parabens Decreased sperm motility and abnormal sperm
morphology(Jurewicz et al., 2017).
Triclosan Infertility, abnormal sperm morphology, and decreased sperm
count(Zhu et al., 2016).
THE PHYSIOLOGY OF MALE REPRODUCTIVE SYSTEM:(Gurung et al., 2023)
The male reproductive system comprises the testes, epididymis, vas deferens, prostate, scrotum, and penis.
These organs are responsible for the formation, storage, and ejaculation of sperm as well as producing
androgens for male reproduction. Spermatogonia line the basement membrane inside the seminiferous
tubule. The Leydig cells play a crucial role as they produce the most important androgen-Testosterone. The
Sertoli cells also play a key role in producing hormones like Inhibin B and Mullerian inhibiting substances.
The hypothalamus makes Gonadotropin-releasing hormone (GnRH) which regulates the release of
hormones like follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The hypothalamus-
pituitary-gonadal axis consisting of a group of the above hormones working together plays a pivotal role in
supporting and regulating sexual development and function in males(Gurung et al., 2023; Ilacqua et al.,
2018; Weinbauer et al., 2010).

Interstitial compartment: Out of the total testis volume, around 15% is a part of the interstitial
compartment, out of which 20% comprises Leydig cells. They are the most important cells in this
compartment as they are responsible for producing testosterone and INSL3. The Leydig cells start being
Figure 4: Cross-section of Seminiferous tubules(Mäkelä &
Toppari, 2017)
9

fetal Leydig cells to neonatal Leydig cells and eventually degenerate into immature Leydig cells. Fetal
Leydig cells are immature not due to their functionality but due to their embryonic origin, they originate
from stem cells and later gain the ability to produce testosterone. The structure of these cells is similar to
steroid-secreting cells as the main function is to produce testosterone(Weinbauer et al., 2010). This allows
them to regulate important processes such as sexual development and spermatogenesis. The androgens
produced by Leydig cells in interstitial cells then diffuse into the seminiferous tubules to help regulate
sperm production. Hence a decrease in intratesticular testosterone levels can be associated with defects in
spermatogenesis. In addition to Leydig cells, the interstitial compartment also contains immune cells, blood
vessels, and fibroblasts(Ilacqua et al., 2018).
Tubular compartment: This is where the process of spermatogenesis occurs. It comprises germ cells and
somatic cells such as peritubular cells and Sertoli cells. The testis of an average human has around 600
seminiferous tubules. The peritubular cells release factors that are responsible for cellular contractility
(myosin and actin) Sertoli cells are also called the support system of germinal epithelium as they make sure
that the germ cells remain inside the seminiferous tubules by forming a blood-testis barrier(Ilacqua et al.,
2018). The structure just like Leydig cells corresponds to the function of these cells. For example, the
smooth endoplasmic reticulum helps in steroid production whereas the rough endoplasmic reticulum
controls protein synthesis(Weinbauer et al., 2010).The Sertoli cells, peritubular myoid cells as well as
macrophages send signals to the Spermatogonia stem cells ensuring persistent spermatogenesis throughout
the lifespan(Casandra Walker, 2021).
Additionally, the Sertoli cells also help in the production of sperms, secreting crucial molecules such as
Inhibin B, ABP, and activin required to regulate spermatogenesis by the hormone-negative system. But the
most important function of Sertoli cells is to secret the Mullerian Inhibiting Factor necessary to inhibit the
development of female sex organs after the testis originate embryogenically. Inside the barrier created by
these cells are other cells such as primary and secondary spermatogonia while the exterior part has germinal
epithelial cells and primitive spermatogonia. Sertoli cells are also called the “biological clock” of the testis
as the increase in the number of lipid droplets in these cells is directly proportional to the age of the
individual(Griswold, 1998).

10

SPERMATOGENESIS:
Spermatogenesis is a continuous process throughout life. For that reason, the adult testis has all stages
starting from stem cells to mature sperm. This process of stem cells generating haploid spermatozoa occurs
inside the seminiferous tubules and the wall of the seminiferous tubules is the main site for sperm
production. The sperm cells then traverse into the epididymis where they undergo maturation and prepare
for ejaculation. The process of spermatogenesis takes approximately 74 days, and a new cycle starts every
16 days (Houda et al., 2022; Mäkelä & Toppari, 2017; Suede SH, 2023).
Spermatogenesis occurs in two phases:
1. Spermatocytogenesis-The primary germ cells also called the primary spermatogonium Type A are present
in the wall of the seminiferous tubules. The first step in the series of spermatogenesis is the mitosis of
primary spermatogonia type A cells into primary spermatocytes which are diploid to maintain the
chromosome number. These main spermatocytes go through meiosis I, which divides the chromosomal
number in half, resulting in secondary spermatocytes (haploid cells). These cells then undergo meiosis II
while maintaining the chromosome number and giving rise to 4 immature sperms also known as spermatids.
This process starts from the wall of the seminiferous tubules and ends in the lumen. During the whole
process, Sertoli cells provide nutrition and support to these developing gametes.= (Mäkelä & Toppari, 2017;
Suede SH, 2023).
2. Spermiogenesis: The spermatids are immature and contain various cell bodies such as mitochondria, Golgi
apparatus, and centrioles. Spermiogenesis allows the immature sperms to be converted into mature sperms.
This process is divided into 4 phases; The Golgi phase where the enzymes in the Golgi body form
acrosomes, The acrosomal phase where the acrosome condenses around the nucleus, the tail phase allows
the centrioles to be elongated to form a tail and the last stage is the maturation phase causing the loss of
excess cytoplasm. In the end, mature sperms are formed which are still nonmotile. Hence, they travel from
the seminiferous tubule to the Epididymis through the process of spermiation. In the epididymis, they gain
motility and are ready to be fertilized(Suede SH, 2023).
Hormonal control of spermatogenesis: The process of spermatogenesis begins at puberty, when the
hypothalamus, pituitary, and Leydig glands interact. The lack of these glands does not prevent
spermatogenesis, which is triggered by FSH and LH. FSH is required to activate Sertoli cells and the Blood-
testis barrier, whereas LH is required to create testosterone. External factors such as diet, alcohol, infectious
diseases, anabolic steroids, and X-ray exposure can negatively impact the rate of spermatogenesis(Darbre,
2015; Darbre, 2022).
11

EFFECT OF GENISTEIN AND DEHP ON HUMAN HEALTH:
Soy formula that has soy protein isolates is given to infants as a substitute for human milk and is also a part
of a diet for a lot of vegans as it is considered a “healthy alternative”. However, this diet does more harm
than good. Soy proteins consist of phytoestrogens which are non-steroidal, estrogenic compounds that
belong to the isoflavone class(Patisaul, 2017; Steven Jones, 2014).These isoflavones can either be
conjugated to glucose or be present in the unconjugated form. Genistein belongs to the latter. Fetal exposure
to Genistein is usually through maternal ingestion of soy-based products followed by placental transfer to
the fetus. The mechanisms of Genistein are varied, it can act by activating PPAR and estrogen, manipulation
of signaling molecules, or DNA methylation. In the case of fetal exposure, it disrupts the production of
testosterone by binding to ERα or causes aberrant prepubertal spermatogenesis as well as Leydig and Sertoli
cell development(Walker et al., 2020; Zheng et al., 2013).
Phthalates are found in various consumer products including paints, packaging, children’s toys, medical
tubing, personal care products, and pharmaceuticals (Arbuckle, 2016; Edwards et al., 2022).The most
commonly found phthalates are lower molecular weight (diethyl phthalate [DEP], dimethyl phthalate
[DMP], and dibutyl phthalate [DBP]) and higher molecular weight (di (2-ethylhexyl) phthalate [DEHP],
di-isodecyl phthalate [DIDP], diisononyl phthalate [DINP], and benzyl butyl phthalate [BBP]). (Eaton,
1986)The high molecular-weight phthalates are used as plasticizers whereas the low molecular-weight
phthalates are used in waxes, adhesives, personal care products, and pharmaceuticals. Among these, DEHP
being the cheapest is used widely in Polyvinylchlorides. It can leach out into the environment as it is not
chemically bound to the plastic. Phthalates are considered reproductive toxicants due to their anti-androgen
properties that are independent of the androgen receptor. The general population can be exposed to DEHP
through dermal, inhalation, or oral routes, however, the vulnerable population such as neonates in the ICU
are exposed to these compounds when these chemicals are released from medical equipment. Fetal exposure
can occur via maternal ingestion or inhalation of phthalates. Animal studies revealed dose-dependent
induction of testicular atrophy, alterations in spermatogenesis, sperm motility, and concentrations, they can
also disrupt the functioning of Leydig and Sertoli cells thus causing infertility. Based on mice fertility
studies, DEHP plays a crucial role in disrupting male reproduction as compared to other phthalates(Darbre,
2015; Zarean et al., 2016).
Not a lot of studies have been done on the combined exposure of these chemicals and most of the animal
studies have used doses higher than the levels found in the environment. In previous studies in our lab, we
found that fetal exposure to a mixture of the phytoestrogen Genistein (GEN) and the antiandrogenic
plasticizer di (2-ethylhexyl) phthalate (DEHP), at 0.1 mg/kg/day which corresponds to the exposure in the
general population and at 10 mg/kg/day mimicking that of the more vulnerable population such as neonates
12

in ICUs or people on vegan diets causes abnormal testicular development In adult PND120 male rats(Steven
Jones, 2014; Walker et al., 2020).Reversibility of the effects of these chemicals depends on the timing of
the exposure. For example, fetal exposure to EDCs has long-term and permanent effects in the later stages
of life. On the other hand, if the individual was exposed to these chemicals in their adult life such as
exposures to EDCs in personal care products, quitting the use of these products has been shown to reduce
the effects of EDCs in young girls facing early menstruation. However complex diseases such as cancer
which develop over the years cannot be reversible(Darbre, 2022).

EPIGENETICS AND EDCS:
Studies on the effects of exposure to EDCs focus on the short-term effects of these chemicals on human
health and ignore the persistent effects of early life exposures. Epigenetic mechanisms provide another
mechanism as most of the factors cannot alter DNA, but few environmental factors can cause epigenetic
alterations, thus modifying the genomic activity(Darbre, 2022). A growing body of evidence indicates that
exposure to EDCs during early fetal life can cause challenges such as cancer and Cognitive impairment in
the later life adulthood. For example, in 1940s-1970s, pregnant women were administered synthetic
estrogen, Diethylstilbestrol to avoid miscarriages. But later it was found that the daughters of these women
(DES daughters) were at a higher risk of infertility and rare vaginal adenocarcinoma. In addition to that,
the sons of the DES daughters also had abnormal male reproductive system. The mechanisms of how the
fetal exposure led to persistent changes in the next generation is not fully understood, however studies
suggest that epigenetic mechanisms might be playing a crucial role in this(Doherty et al., 2010). Epigenetic
alterations in simple terms can be defined as “any permanent modification in the operation of genes that
lingers even after the original trigger has ended and is not caused by an alterations in the genetic sequence
or framework” Exposure to certain chemicals during pregnancy can have adverse effects on the pregnant
women, her fetus i.e. intergenerational inheritance and also the primordial germ cells in the fetus i.e.
transgenerational inheritance(Darbre, 2022). One critical window of sensitivity to endocrine-disrupting
chemical (EDC) exposure occurs throughout human development, and the ramifications may not be
apparent until a latency phase during infancy or later into adulthood. Some repercussions may be even more
long-lasting and handed down to future generations (multigenerational/transgenerational) without the need
for additional exposure(Darbre, 2022).
13

The changes in the F2 generation are called “intergenerational” because the fetal gametes of the F2
generation were exposed to the chemicals along with in-utero exposure to the F1 generation. However, the
F3 generation is called “transgenerational” as this generation was never exposed directly to the
chemicals(Alavian-Ghavanini & Rüegg, 2018). There is growing evidence on the changes in F3
generations, for example: animal studies showed that exposure to BPA decreased the rate of fertility of
mice in the F3 generations(Manikkam et al., 2012).The two important windows for epigenetic
reprogramming are 1) During the process of embryonic reprogramming in the pre-implantation embryo and
2) During the germline specific reprograming that occurs in the fetal stage. The most widely studied
epigenetic modulations are DNA Methylation, Histone modification contributing to the DNA shape and
accessibility and noncoding RNAs which also indirectly play a role in epigenetic- DNA regulation. Despite
a large number of studies on epigenetics,
a clear understanding of the link between
epigenetics and intergenerational or
transgenerational inheritance has not
been found(Gunes & Esteves, 2021).
Getting to the root of these mechanisms
can not only help us understand the link
but also help us develop methods to
assess chemicals and their ability to
cause epigenetic alterations.

Figure 5: Critical windows for sensitivity to the effects of
EDCs.
Figure 6: EDC-induced epigenetic alterations(Nettore et al.,
2021).
14

OBJECTIVE:

Previous studies in our lab showed that in-utero exposure to Gen, DEHP and Gen+DEHP mixture at doses
mimicking general population as well as in susceptible populations led to abnormal development of testis
in adult PND120 male rats(Steven Jones, 2014). Rat model was used as it is the most commonly used model
for multigenerational reproduction studies and other studies have also shown the effect of EDCs in germ
cells from human and neonatal rats. At the basis of my project is our finding that Gen+DEHP fetal exposure
in F1 resulted in long term reproductive effects in F1 offspring, but also in their F2 and F3 descendants,
suggesting epigenetic involvment.

Table 2:The rate of adverse reproductive effects (A) Abnormal testes and small litter and (B)Infertiliy rate in
adult rats exposed in utero to Gen and DEHP, alone or mixed, at doses corresponding to those in humans.
Gen+DEHP mixtures aggravated the effects, and the lowest dose had negative effects in F1 to F3 generations.
A
B
15

My aim is to:
1. Inverstigate the effects of in-utero exposure to low doses of Gen+DEHP mixture on testicular
trasncriptome in F1 to F3 generation adult rats, using microarray and RNA-seq analysis.
2. To identify candidate genes and functional pathways altered by Gen+DEHP exposure, specifically
focusing on transcription factors Foxa1 and Foxa3, DNA methyl transferase Dnmt3a and signaling
pathway regulator Stat3
3. To elucidate the mechanisms linking their downregulation to adverse reproductive effects of endocrine
disrupting chemicals (EDCs) in male reproductive health.
SIGNIFICANCE: Many investigations on the effect of EDC exposure on the male reproductive system
have been conducted using dosages that are far too high to be relevant to humans(Sharma, 2020). However,
our work used doses that are equal to human exposure by calculating the dosage using a method that
accounts for the body surface area of both humans and rats(Reagan-Shaw, 2008). I am studying the effects
fo Gen and DEHP because exposure to Gen varies in different diets but still it is shown to be present higher
in soy-based diets. Because of the research being done on the impacts of DEHP, it is currently used less in
childcare products, but there has been no change in the usage of DEHP in medical equipment and
commercial products.My research focuses mainly on stydying the effects of these chemicals on male
infertilityIn my research, I will not only look at the impacts of these chemicals on F1 generation, but also
on F2 and F3 generation, in order to understand the intergenerational and transgenerational effects of GEN
and DEHP on infertility. This is intriguing since prior research has demonstrated that EDC exposure can
trigger epigenetic changes in future generations who were not exposed to EDCs directly.

16

CHAPTER 2:METHODS:(adapted from Steven Jones, 2014)
1. Animal Treatment And Tissue Collection:
Animal care and tissue collection were carried out in our lab in the manner previously described. Pregnant
Sprague-Dawley rats were timed and moved to a casein/cornstarch-based, phytoestrogen-free diet (casein
diet) from Charles Rivers Laboratories (Saint-Constant, QC, Canada) (Teklad diet, Envigo, Indianapolis,
IN, USA).from 2 days before gavage to weaning, to avoid dietary exposure to genistein. The rats were kept
on a 12L:12D photoperiod with free access to food and water and were cared for in accordance with
methods authorized by the McGill University Health Centre Animal Care Committee and the Canadian
Council on Animal Care. Pregnant rats were treated by gavage from gestational day 14 to parturition with
either vehicle (corn oil) alone or containing GEN, DEHP, or GEN + DEHP mixtures at the doses of 0.1 and
10 mg/kg/day, encompassing exposure levels found in the general population to those measured in
vegetarian/vegan women and more susceptible populations such as hospitalized neonates exposed to DEHP
via medical equipment and fed soy formula. Doses were adjusted to changes in dam weights. Offspring
were weighed and euthanized at PND120. Adult F1 male progeny were mated with unexposed females to
produce F2, and the process was repeated to produce F3. The testes were collected, weighed, and either
fixed in 4% paraformaldehyde or snap-frozen for gene and protein expression analyses.

Microarray analysis
Control (Corn oïl)
Genistein
DEHP
Mixture Genistein + DEHP
➢ 0.1 mg/kg/day: close to exposure levels of the
general population
➢ 10 mg/kg/day: dose at the high end of human
exposure
Sacrifice
0.1 Or 10 mg/kg/day
Testis Collection
RNA extraction
qPCR analysis
Immunohistochemical
Analysis
Exposure window
RNA-seq analysis
17

2. RNA Extraction and Quantitative Real-Time PCR:
RNA was extracted from testes using a Nucleospin XS Kit and digested with DNase I (Takara Bio, San
Jose, CA, USA). Complementary DNA was synthesized using the transcription synthesis kit (Roche
Diagnostics, Indianapolis, IN, USA). Quantitative real-time PCR (qPCR) was performed as previously
described with a LightCycler 480 using SYBR Green Supermix (BioRad, Hercules, CA, USA) and a Master
Mix kit (Roche Diagnostics). Glyceraldehyde-3-phosphate dehydrogenase was used as reference to
normalize gene expression. A minimum of 3–8 male offspring from different litters were assessed in
triplicate. The comparative Ct method was used to calculate relative gene expression. Primers specific for
the genes of interest were designed with the NCBI Primer Design Database.
3. Whole Transcriptome RNA Sequencing:
The USC Norris Molecular Genomics Core did transcriptomic RNA sequencing. Qiagen total RNA
isolation kit was used. (Germatown, MD, USA) All prep Extraction kit following the manufacturer’s
protocol (Qiagen Cat. No. 80284). Libraries were simultaneously prepared using an Illumina Truseq
Stranded mRNA Library Preparation kit (Illumina Cat. No. 20020594; San Diego, CA, USA).
Transcriptomic RNAseq libraries were sequenced on an Illumina Nextseq500 at a rate of 25 million reads
per sample with a length of 2 75 reads. Partek Flow was used to trim, standardize, and analyze the data.
Gene lists were developed to identify differentially expressed genes between Gen, DEHP, and the
combination of Gen and DEHP.
4. Statistical Analysis:
The statistical analysis was performed using one-way ANOVA with unpaired two-tailed Student’s t-test for
qPCR data analysis, using the statistical analysis functions in GraphPad Prism 7.04 program (GraphPad
Inc. San Diego, CA, USA). Because the two EDCs used were not expected to have identical effects, the
statistical significance of each control-EDC combination for qPCR analysis was determined using an
unpaired two-tailed Student's t-test. The gene array analysis was performed on three independent N (one
offspring/dam) per treatment condition, using ANOVA application from the bioinformatics Partek
platform. For the qPCR analysis, the results are presented as mean ± SEM of fold changes relative to vehicle
control. Experimental points were performed in triplicate for each sample, from 3 to 8 rats from different
dams per treatment condition. Asterisks indicate a significant change relative to control, with p-values
≤0.05 considered to be statistically significant.
5. Immunofluorescence (IF):
Slides were first dewaxed and rehydrated using Cortisol and Trilogy (Cell Marque, Rocklin, CA, USA)
solution. Following treatment with Dako Target Antigen Retrieval Solution (DAKO, Troy, MI, USA), the
sections were incubated with PBS containing 10% BSA and 10% donkey serum for one hour to block non-
18

specific protein interactions. The sections were then incubated with Sulfotransferase Family 1E Member
1(SULT1E1) (Cat # 12522-1-AP,1:200;), goat anti Chemokine ligand 13(CXCL13) (Cat # AF470,1:50;
R&D), Anti-Forkhead box protein-A1 (FOXA1)( Catalog #PA5-27157,1:100, ThermoFischerScientific),
Anti-DNA methyl transferase 3 alpha (DNMT3A) polyclonal antibody- (Catalog# PA3-
16557,1:100;Thermofisher scientific), Anti-Signal transducer and activator of transcription 3 Stat3
(D3Z2G) Rabbit mAb (Catalog #12640,1:100;Cell signaling technology) diluted in PBS containing 10%
BSA, 0.1% Triton-X, and donkey serum overnight at 4 °C. Then, the slides were incubated with a
fluorescent goat anti-rabbit Alexa Fluor 488 (ThermoFischer, Waltham, MA, USA) diluted in PBS
containing 1% BSA for one hour at room temperature. Primary antibodies for 5-methylcytosine
(Epigentek), and H3K9me3(Millipore) were used at a dilution 1:100. The secondary antibodies were Biotin
Goat Anti-Rabbit (BD Pharmingen) and Biotin Goat Anti-Mouse (BD Pharmingen). Nuclear staining was
performed using nuclear DAPI anti-fade and mounting medium (Vector Labs, Newark, CA, USA), cover
slipped, and then imaged. The immunofluorescence signals were examined on an Olympus microscope
with the proper filters. The IF analysis was carried out on 2-3 separate offspring per treatment, with sample
images provided.

19

CHAPTER 3: RESULTS

Our lab showed that F1 rats that were exposed to Gen, DEHP and Gen+DEHP showed abnormal testis
morphology In PND3 and PND120 rats.(Steven Jones, 2014) We can observe in the figure 7 that for the F1
generation, exposure to Gen and DEHP dosages of 10mg/kg/day affected the morphology of the testis. For
DEHP and Gen+DEHP exposed rats, there was a change in the morphology of seminiferous tubules,
increased interstitial space, and a pronounced Sertoli-only phenotype. Even though F2 rats were not directly
exposed to the EDCs, but resulted from the fertilization of oocytes by sperm derived from fetal germ cells
present in the testes of F1 fetuses, the consequences were more significant. Furthermore, exposure to Gen
and DEHP resulted in infertility in some rats due to a lack of germ cells. This shows that EDC exposure
can influence both the F1 and F2 generations, and that epigenetics may play a role in displaying this
phenotype.

F1
Ge
ner
atio
n

F2
Figure 7: Testicular Morphology in F1 and F2 Adult Rat Testis is altered by Gen and DEHP
exposure. PND120 rat testes were exposed in utero to Gen, DEHP, and Gen+DEHP at
10mg/kg/day. (A) In utero exposure of F1 rats to Gen, DEHP, and Gen+ DEHP. (B) Adult
F1 male offspring mated with unexposed female rats to produce F2 generation, which then
differentiated into spermatozoa.

20

5methylcytosine was chosen as a marker to investigate the involvement of epigenetics in intergenerational
EDC-induced testicular changes. At 0.1 and 10 mg/kg/day dosages, I found changes in DNA methylation
patterns in PND120 F1 rat testes. The signal was reduced in rats treated to Gen at 0.1 and 10 mg/kg/day
dosages. DEHP exposure at both doses resulted in enhanced germ cell sloughing inside the lumen. Exposure
to Gen+DEHP at 0.1 mg/kg/day in F2 generation resulted in diminished signal, but exposure to Gen+DEHP
mixture at 10 mg/kg/day in the same generation led to infertility in some of the rat testes. This implies that
Gen, DEHP alone, or in combination, can affect DNA methylation in the testis.

Figure 8: Effect of exposure to EDC alters 5methylcytosine in
PND120 rat testes.5mec was used as a marker for DNA
methylation. F0 Generation was gavaged with
Control(vehicle), Gen, DEHP and GEN+DEHP at 10
mg/kg/day. Change in DNA methylation was observed in F1
Generation. More dramatic effects were observed in F2
Generation which was exposed as germ cells. Yellow arrow:
Germ cell sloughing, Black arrow: Reduced signal.
F1 PND120 Control
Gen+ DEHP
F1
F2
F1
5mec used as a
marker for DNA
methylation.
F2
F1 PND120 Control

H3k27 used as a
marker for Histone
modifications.
Gen DEHP Gen+ DEHP
Figure 9: Effect of exposure to EDC alters H3k27 methylation in
PND120 rat testes.H3k27 was used as a marker for DNA
methylation. Fo generation was gavaged with Control(vehicle), Gen,
DEHP and Gen+DEHP, changed in histone methylation was
observed in F1 generation. More dramatic effects were observed in
F2 generation which was exposed as germ cells. Yellow arrow:
Germ cell sloughing, Black arrow: Reduced signal.

DEHP Gen

21

In Figure 9, H3k27 methylation patterns in PND120 rat testes are similarly changed. Exposure to Gen at
0.1 and 10 mg/kg/day doses resulted in diminished signal, as shown in 5mec methylation patterns.
Sloughing was more pronounced in rats given DEHP at 10mg/kg/day dosages. The morphology of adult
PND120 rats was likewise altered by Gen and DEHP exposure, as shown by increased interstitial space and
reduced signal.

The goal of this experiment was to determine the genes that were differentially expressed in PND120 rats
exposed to Gen, DEHP and Gen+DEHP roles at 10 mg/kg/day dose. The Venn diagrams reveal that more
genes were upregulated than downregulated, suggesting that DEHP alone may have a significant influence
on gene expression at both ages. Interestingly, adult exposure to the Gen+DEHP combo resulted in distinct
expression alterations in several genes, clearly demonstrating a "cocktail effect." The number of genes
downregulated or upregulated in DEHP alone and the Gen+DEHP combo was greater than in Gen alone.

Figure 10: Venn’s Diagrams showing the number of altered genes, common and unique
between treatments, in gene array analysis of neonatal and adult rats, exposed in utero to
vehicle or 10 mg/kg/day of Gen, DEHP or their mixture. Cut off in fold changes at 1.25x
and 0.75x were applied. Genes presenting expression changes with P Value < 0.05 in at
least one treatment were retained for the analysis.

22

IPA research revealed that when rat testes were exposed to 10mg/kg/day doses of Gen, DEHP, and
Gen+DEHP mix, epigenetic pathways such as DNA double strand break repair were considerably differed,
suggesting that EDCs cause epigenetic modifications that eventually result in infertility.
Previously, in our lab we found that Foxa3, a transcription factor is downregulated by Gen+DEHP dose
and was identified in Leydig, Sertoli and germ cells. (Walker et al., 2023) Transgenerational inheritance is
another component of epigenetics. Parallel to the results in the F1 generation, we saw a similar impact in
F2, although this appeared to have recovered in F3, suggesting that epigenetics is at work.This might imply
that exposure to Gen+DEHP has an effect on Foxa3 downregulation and can be passed down across
generations, corroborating our objective of studying the function of epigenetics in this process. The changes
from F1 to F2 have an intergenerational effect. In contrast, the F3 generation has a transgenerational impact.
• F1: Foxa3 expression decreased in Gen+DEHP-treated rats, and the decline was more pronounced in
DEHP-only exposed rat testes.
• F2: GD is reduced by all treatments, with the largest statistically significant reduction occurring
between GD and Gen. DEHP and GD had a lower decline.
• F3: There is no statistically significant difference between treatments.

Figure 11: IPA analysis showing canonical pathways that are differentiated in PND120 rats at 0.1
mg/kg/day dose.

Top canonical pathways in Gen, DEHP and Gen+DEHP at 10 mg/kg/day dose
23

GENES INTERACTING WITH FOXA3:

I used IPA to do an in-silico search and discovered Foxa3 target genes that differentiated in my dataset. It
was interesting to see that the genes were downregulated in the 0.1 mg/kg/day dosage but increased in the
10 mg/kg/day dose. Foxa1 was one of the intriguing genes. The target genes are found in Leydig, Sertoli,
and germ cells. Furthermore, the majority of them are related to "steroidogenesis" and can impact
testesterone synthesis via modifying steroidogenesis.

Figure 12: Foxa3 mRNA expression in F1, F2, and F3 adult rat testis.
Table 3: In Silico search for Foxa3-intercating genes in testes. Genes
related to Foxa3 were searched in IPA in rats that were exposed to (A)
0.1 and (B) 10 mg/kg/day using RNA-seq data.

24

Chip-seq analysis assisted me in identifying Foxa3 targets in adult rat testes. The differential expression of
these genes was then investigated using RNA sequencing data. I discovered 18 differently expressed genes.
At 0.1 and 10 mg/kg/day, Cxcl13 was dramatically downregulated in the Gen+DEHP combo. Tmeff2 and
Phlpp1 were, on the other hand, increased in the dataset at both 0.1 and 10 mg/kg/day dosages. However,
these genes were not changed in the Gen and DEHP alone.
Table 4: Genes that bind to Foxa3 by Chip-seq analysis which
were examined using RNA-seq data in rats exposed to
Gen+DEHP at 0.1 and 10 mg/kg/day.
FOXA1
Table 5: IPA was used to identify differentially expressed genes
associated to "steroidogenesis" in testes exposed to Gen+DEHP at 0.1
mg/kg/day.

Figure 13: RNA sequencing
on rats exposed to
Gen+DEHP at 0.1 and 10
mg/kg/day was analyzed
using Partek Flow.

25

Another important gene that was differentially expressed in the dataset was Foxa1(Table 3). IPA analysis
showed that this gene interacts with Foxa3 and is also related to steroidogenesis. Previous studies have
shown that Foxa1 binds to the androgen receptor in the prostate(Sahuet al.,2011).RNA-sequencing revealed
that there was a higher number of differentially expressed genes in the GD treatment group at 0.1 mg/kg/day
dose.

I used immunofluorescence to corroborate the RNA-Seq results, and I discovered that Foxa1 was in fact
downregulated in F1 Gen+DEHP at 10mg/kg/day. It was also found mostly in Leydig cells. This confirms
our idea that Foxa1 and Foxa3 may interact because Foxa3 was also detected in high concentrations in
Leydig cells. According to the RNA-seq results, Foxa1 was likewise downregulated in the F2 generation.
The reduction in Foxa1 in F2 PND120 Gen+DEHP (10mg/kg/day) compared to F2 control is confirmed by
immunofluorescence. Furthermore, we discovered that Foxa1 interacts to the promoter area of Dnmt3a, a
DNA methylation enzyme. I used IPA to identify the other genes associated with DNA methylation that
are distinct in my dataset.

Foxa1 FC GD 0.1 P value FC GD 10 P value
F1 generation -2.33E+00 0.007 -1.10E+00 0.897
F2 generation -1.04E+00 0.882 1.59E+00 0.197
F1 PND120 Gen+DEHP (10mg/kg/day) F1 Control
Figure 14: Expression of Foxa1 in testes of adult rats exposed in utero to F1 GD at 0.1 and
10mg/kg/day. Immunofluorescence analysis of Foxa1.
Table 6: Foxa1 is differentially expressed in F1 and F2
generation exposed to Gen+DEHP at 10 mg/kg/day
Figure 15: Foxa1 interaction
network in Gen+DEHP at 10
mg/kg/day exposed rat testes
demonstrating its relationship to
Dnmt3a.
26

To investigate the relevance of epigenetic modifications following in-utero exposure to Gen+DEHP at 10
mg/kg/day, I used IPA to identify the genes in my dataset that are associated to DNA methylation and are
also affected. Dnmt3a, Arid4a, Dnmt3l, Atrx, and Mov10l1 were the most changed genes. These genes have
been linked to spermatogenesis. (Figure 17)

I performed an IPA analysis on genes related to Dnmt3a.Microarray
results show that genes involved in DNA methylation, such as Dnmt1,
Dnmt3l, and Dnmt3b, are downregulated in F1 generation rat testes
exposed to Gen+DEHP at 0.1 mg/kg/day.

Gene C vs GD 0.1-FOLD
CHANGE
Dnmt1 -1.003
Dnmt3b 1.140
Dnmt3l -1.093
DNA methylation
Methylation of DNA [de novo
DNA methylation, DNA
methylation,...]
Arid4a, Arid4b, Asz1, Atf7ip,
Atrx, Baz2a, Bend3, Beta-
estradiol, Bhmt, Bisphenol A,
CBX1, Cbx3, Cbx5, Cdkn1a,
Commd3-bmi1, Ctcf, Ctcfl,
Dcaf1, Dnmt1, Dnmt3a,
Dnmt3b, Dnmt3l, Ezh2, Fos,
Gatad2a,, Igf2, IGF2R, Kdm1b,
Kmt2a, Kmt2e,mis18a,
Mov10l1
Methylation of DNA fragment
[DNA fragment methylation]
Dnmt3a, Dnmt3b, Dnmt3l,
Phosphatidylserine
Methylation of genomic DNA
[genomic DNA methylation]
Agi-6780, Atrx, Dnmt1,
Dnmt3a, Dnmt3b, Idh2, N-(4-
hydroxynaphthalen-1-yl)-4-
methyl-3-
nitrobenzenesulfonamide, S-
adenosylhomocysteine
DNA Replication,
Recombination, and Repair
Agi-6780, Atrx, Dnmt1,
Dnmt3a, Dnmt3b, Idh2, N-(4-
hydroxynaphthalen-1-yl)-4-
methyl-3-
nitrobenzenesulfonamide, S-
adenosylhomocysteine, TCL1A
Table 7: Microarray of genes
associated to DNA methylation.
F2 Control F2 PND120 Gen+DEHP (10mg/kg/day)
Figure 16: Expression of Foxa1 in testes of adult rats exposed in utero to F2 GD at 10mg/kg/day.
Immunofluorescence analysis of Foxa1.
Figure 17: IPA search for genes associated to the term "DNA methylation" in my dataset for testes
exposed to Gen+DEHP at 10 mg/kg/day dosage.

27

I used IPA to design an interaction network for genes associated to Dnmt3a using the RNA-seq dataset.
Tet1 was the only gene that showed a substantial increase in expression. During embryonic development,
DNA methyltransferase 3a (Dnmt3a) is responsible for de novo DNA methylation at unmethylated
cytosines, whereas Tet1 is involved in DNA demethylation. Upregulation of these genes may reflect
alterations in DNA methylation patterns as a result of in-utero exposure to GD mixture at a dosage of 10
mg/kg/day. Figure 24 depicts the genes that interact with Tet1 and are distinguished in the RNA-seq dataset.

I used immunofluorescence to evaluate protein expression and discovered that it was downregulated in F1
PND120 rats exposed to a Gen+DEHP mixture at 10 mg/kg/day. The downregulation was most noticeable
in germ cells and Leydig cells.
Figure 18: In IPA, an interaction network shows differentially expressed genes associated to Dnmt3a in
F1 rats exposed in utero to Gen+DEHP at a level of 10 mg/kg/day.
Figure 19: Interaction network in IPA
showing differentially expressed genes
related to Tet1 in F1 rats exposed in-
utero to Gen+DEHP at 10 mg/kg/day
dose.
Control PND120 Gen+DEHP (10
mg/kg/day)
Merge Dnmt3a
Figure 20: Expression of Dnmt3a in testes of adult rats
exposed in utero to F1 GD at 10mg/kg/day.
Immunofluorescence analysis of Dnmt3a
28

In RNA seq data, I discovered that Foxa1 and Dnmt3a were downregulated in F1 PND120 Gen+DEHP
combination at 10 mg/kg/day dosage, but Foxa1 was upregulated and Dnmt3a was downregulated in F2
generation under the same conditions as the control. Tet1 was increased in F1 PND120 Gen+DEHP
combination at 10 mg/kg/day dosage, however it was downregulated in F2 generation. Foxa1 works
together with the promoter of Dnmt3a, and Dnmt3a works with Tet1 to maintain DNA methylation patterns
by performing opposing tasks. The evidence presented above shows that these genes may be involved in
epigenetic changes caused by in-utero exposure to a Gen+DEHP cocktail at a level of 10 mg/kg/day.

F1
gen
F2
gene
F3
gene
Table 8: RNA seq analysis of F1 PND120
Gen+DEHP and F2 PND120 Gen+DEHP at 10
mg/kg/day
Figure 21: qPCR analysis of genes involved in DNA methylation (Dnmt3a, Tet1). N=5 rats per treatment (.
IPA interaction Network shows genes related to (A) Dnmt3a and (B) Tet1 that are differentially expressed
in F1 rat testes exposed in utero to 10 mg/kg/day of Gen+DEHP. One sample t-test showed that the
difference in gene expression between all control and Gen+DEHP samples was statistically
significant(p<0.05)
29

qPCR research revealed that Dnmt3a was downregulated in F1 and F2 generations exposed to Gen+DEHP
at 10 mg/kg/day. Tet1, which has the opposite function of Dnmt3a, was shown to be elevated in F1 and F2
generations subjected to the same dosage.
OTHER EPIGENETIC MODIFICATIONS:
Histone Modifications:
In figure 9, we saw that In-utero exposure to Gen, DEHP and Gen+DEHP mixture alters histone
methylation patterns in adult PND120 rat testis. Figure shows the genes that are related to various histone
modification patterns in my dataset.

Among the genes associated with the term "histone modifications," Brwd1 and Ezh1 were the most
differentially expressed in my dataset.

Histone modifications
DNA Replication,
Recombination, and Repair
Atrx, Brd2, Caf-1, Chaf1a,
Daxx, Grwd1, H1-8, H2ab3,
H4c1, H4c11, H4c12, H4c13,
H4c14, H4c15, H4c2, H4c3,
H4c4, H4c5, H4c6, H4c8, H4c9,
Hist1h2ap, Hist1h3a, Histone
H3, Histone H4
Cellular Assembly and
Organization
Atrx, Brd2, Caf-1, Chaf1a,
H3c4, H3c6, H3c7, H3c8, H4-
16, H4c1, H4c11, H4c12,
HISTONE, Histone H3, Histone
H4, Hmgb1, HMGB1, Hmgb2
(Includes Others), IL2,
Smarca2, Smarca4, Smarca5,
Smyd3, Sox9, Spty2d1
Histone acetylation
Actl6a, Brd8, Dmap1, Ep400,
Epc1, Ing3, Meaf6, Morf4l1,
Naa40, Ruvbl1, Ruvbl2, Trrap,
Yeats4

GD at 10
mg/kg/day
P
value
F1 1.02E 0.795
F2 -1.05E+00 0.644
Table 9: RNA seq analysis of F1 PND120 Gen+DEHP and F2 PND120 Gen+DEHP at 10 mg/kg/day

Figure 22: IPA analysis showing the differential expression of genes that are related to the term
“histone modification.”

30

I created an interaction network of these two genes to look at the genes interacting with Brwd1 and Ezh1
that are connected to histone methylation. I discovered that these genes interact with additional genes
involved in epigenetic changes that are not necessarily connected to Histone modifications. (Figure 22)
Furthermore, I used qPCR to assess Brwd1 mRNA expression and discovered that it is highly elevated in
F1 generation exposed to Gen+DEHP at 10 mg/kg/day, but it is downregulated in F2 generation (Figure
24). The F3 generation was not distinguished, implying that transgenerational inheritance had little
importance.

F1
F2
F3
Figure 23: Interaction network in IPA showing differentially expressed genes
related to (A)Brwd1 and (B) Ezh1 in F1 rats exposed in-utero to Gen+DEHP at
10 mg/kg/day dose.
A
B
Figure 24: qPCR analysis of genes involved in Histone modification (Brwd1). N=5 rats per
treatment. One sample t-test showed that the difference in gene expression between all
control and Gen+DEHP samples was statistically significant(p<0.05)
31

Chromatin Remodeling:
After exploring my RNA-seq dataset for genes associated with chromatin remodeling, I discovered that
Atrx was dramatically downregulated. The Atrx interaction network revealed additional genes connected to
Atrx that were either elevated or downregulated in the microarray.

Atrx was downregulated in F1 generation exposed to Gen+DEHP at 10 mg/kg/day while it was upregulated
when exposed to the same treatment in F2 generation in the RNA-seq dataset.

Gene
(Atrx)
GD at 10
mg/kg/day
P
value
F1 -1.052 0.500
F2 1.33 0.175
Chromatin remodeling
Affects Remodeling of
chromatin
ARID1A, ATRX, BAZ1A,
BAZ1B, BAZ2A, BAZ2B,
BCOR, BPTF, BRDT, CD28,
CD3, CEBPG, CECR2, CHD1,
CHD1L, CHD3, CHD4, CHD7,
CHD8, CHRAC1, DAXX,
EOMES, EZH1, FOXA1,
GATA3, GATAD1,
GATAD2A, SOX9, SUV39H2,
SWI-SNF, TAF6L, TBR1
Decreases Remodeling
of chromatin
beta-estradiol, cisplatin,
CVT-313, diethylstilbestrol,
genistein, HISTONE,
histone deacetylase,
IL2RA, mature microRNA,
mifepristone, mir-30,
NANOG,
Increases Remodeling of
chromatin
FOXA1, GATA3, Hdac,
HISTONE, Histone h3,
HMGN1, HSF1, ionomycin,
KDM1A, Nfat (family),
NR3C1, POU2F1, PRC2,
RARA, RB1, REL, SMARCA4,
SMARCAD1, STAT5a/b,
STAT6, SWI-SNF, TBX21,
TNRC6A
Figure26: Interaction network in IPA showing
differentially expressed genes related to Atrx in F1
rats exposed in-utero to Gen+DEHP at 10 mg/kg/day.
Table 10: RNA seq analysis of
F1 PND120 GEN+DEHP and F2
PND120 GEN+DEHP at 10
mg/kg/day

Figure 25: IPA analysis showing the differential expression of genes that are related to the term
“Chromatin remodeling.”
32

qPCR study of Atrx gene expression revealed that testis exposed to Gen-DEHP mixture had the greatest
drop from control for F1 and F2 generation. This implies that EDC exposure can modify chromatin
remodeling processes, resulting in epigenetic changes. For the same dosage, there was no change in gene
expression in the F3 generation.
Role of MicroRNAs In Epigenetic Alterations:

In terms of epigenetics, miRNAs have been shown to play several important roles:
1)They can bind to and regulate the expression of epigenetic factors such as DNA methyltransferases
(DNMTs) and histone-modifying enzymes
2)miRNAs can also target and regulate the expression of other genes that are involved in epigenetic
processes, such as transcription factors and signaling molecules(Chuang & Jones, 2007). In addition, recent
F1 F2
F3
Figure 28: Interaction network in IPA showing differentially expressed microRNAs related to
(A)Transcription and (B)RNA expression rat testes exposed in-utero to Gen+DEHP at 10 mg/kg/day.
Figure 27: qPCR analysis of genes involved in chromatin remodeling (Atrx). N=5 rats per
treatment. One sample t-test showed that the difference in gene expression between all
control and Gen+DEHP samples was statistically significant(p<0.05)

33

research has shown that miRNAs themselves can be regulated by epigenetic modifications such as DNA
methylation and histone modifications(Barbu et al., 2021). In my dataset, I found various microRNAs that
were differentially expressed at Gen+DEHP at 10 mg/kg/day (Table 9) suggesting that these genes might
be playing a role in epigenetic alterations induced by EDC exposure in adult PND120 rats.

OTHER GENES DIFFERENTIATED AFTER IN-UTERO EXPOSURE TO GEN+DEHP AT 10
MG/KG/DAY:
Stat3 is differentially expressed in F1 and F2 generation of rat testes exposed to Gen+DEHP at 10
mg/kg/day.

Stat3 and Jak2 were elevated in the F1 generation but downregulated in the F2 generation. These genes are
involved in the formation of germ cells and the differentiation of spermatogonia. IPA generated a canonical
pathway that identifies the genes in my dataset that interact with Stat3 in both F1 and F2 generation and are
differentially expressed.
Gene Symbol Fold (GD/C)
Mir202 2.02
Mirlet7e 1.14
Mir101b 1.1
Mir107 1.07
Mir294 1.03
Mir146b 0.98
Mir708 0.9
Mir326 0.9
Mir328 0.89
Mir207 0.88
Mir295-1 0.88
Mir99b 0.86
Mir330 0.83
Mir150 0.81
STAT3 Fold change GD
vs C at 10
mg/kg/day
P value
F1
generation
1.13 0.003
F2
generation
-2.64 0.0067
JAK2 Fold change
GD vs C at
10 mg/kg/day
P value
F1 generation 1.03 0.640
F2 generation -2.27 0.002
Table 11: RNA seq analysis of F1 PND120
Gen+DEHP at 10 mg/kg/day

Table 12 :RNA-Seq analysis gene expression changes in Stat3 and Jak2 in F1 and F2 rat testes

34

Another interesting observation was that Stat3 is regulated by G-Alpha signaling in F1 generation exposed
to Gen+DEHP at 10 mg/kg/day whereas it is regulated by Jak/Stat pathway in F2 generation exposed to
Gen+DEHP at 10 mg/kg/day.

Figure 29: mRNA expression changes in Stat3 and Gen,DEHP
alone and mixture determined by qPCR
Figure 30:IPA analysis shows a canonical pathway for Stat3 and interacting genes in
(A)F1 and (B)F2 generation exposed to Gen+DEHP at 10 mg/kg/day
A B
35

I used immunofluorescence to demonstrate the distinction of Stat3 and Jak2 when treated with Gen+DEHP
at 10 mg/kg/day, Stat3 was shown to be downregulated in F1 generations of rats exposed to Gen+DEHP at
10mg/kg/day, but it was increased in F2 generations subjected to the same dosage. This shows that
epigenetic changes may be involved in the differentiation of Stat3 expression in these two generations.

F1 PND120
GEN+DEHP at
10 mg/kg/day F1 Control
Figure 32: Immunofluorescence analysis of Stat3 in adult rat testes exposed in utero to
Gen+DEHP at 10mg/kg/day in F2 generation. Signal intensity quantified by Image J.
Figure 31: Immunofluorescence analysis of Stat3 in adult rat testes exposed in utero to
Gen+DEHP at 10mg/kg/day in F2 generation Signal intensity quantified by Image J.
F2 Control
F2 PND120
GEN+DEHP at
10 mg/kg/day
36

CHAPTER 4: DISCUSSION:
Previously our lab showed that rat testis development is disrupted by simultaneous in utero exposure to the
phytoestrogen genistein and the antiandrogenic plasticizer di-(2-ethylhexyl) phthalate(Steven Jones,
2014).Our lab also identified a transcription gene Foxa3 to be downregulated on in-utero exposure to Gen
and DEHP in adult rat testis(Walker et al., 2023). Gen, a phytoestrogen found in Soy products consumed
by vegans and newborns given baby soy formula, and DEHP, a plasticizer found in medical equipment and
other consumer items such as cosmetics, have both been extensively examined as endocrine disruptors
(Gore, 2015).The male and female reproductive systems are sensitive to these substances because they rely
on hormones to operate normally. A lot of research papers look at the effect of individual EDCs on the male
reproductive system, but relatively few look at the combined effect of many EDCs (Cariati et al., 2019;
Zarean et al., 2016; Zheng et al., 2013).The studies that do, usually use a high dose which is not relevant to
the human population. For that reason, previously in our lab, it was proven that in-utero exposure to Gen
and DEHP at low doses increases the risk of infertility by altering gene expression in adult PND120
rats(Walker et al., 2020). The goal of my study was to investigate the effects of in-utero exposure to low
doses of Genistein and DEHP mixtures on testicular transcriptomes in F1 to F3 adult rats, using microarray
and RNA-seq analysis. I also identified candidate genes and functional pathways altered by Gen+DEHP
exposure, specifically focusing on transcription factors Foxa1 and Foxa3, DNA methyltransferase Dnmt3a,
and signaling pathway regulator Stat3. This helped me to elucidate the mechanisms linking their
downregulation to adverse reproductive effects of endocrine disrupting chemicals (EDCs) in male
reproductive health.
ROLE OF EPIGENETICS:
Exposure to EDCs is now recognized to have impacts not just in adults, but also in future generations. To
define the word "transgenerational," during in-utero exposure, the fetal gametes that give rise to the F2
generation are directly exposed to EDCs; consequently, the F1 and F2 generations are intergenerational,
while all subsequent generations are transgenerational(Martini, 2020). Increasing evidence shows that
environmental chemicals can cause functional changes in the genome allowing the disorders to be carried
forward to future generations(Gunes & Esteves, 2021). For example, studies found a risk between sex-
specific disorders in grandchildren and the food that was consumed by their grandmothers(Michel
Tournaire, 2018).Another study found a link between risk of obesity in boys and the cigarette smoking by
their fathers(Manikkam et al., 2013).
37

EDCs belong to one of the classes of these chemicals that can cause epigenetic alterations further affecting
male reproductive system. While this study might not be new, this area is still underexplored and needs
more research.
The two types of inheritance are:
1. Intergenerational inheritance: EDCs are directly exposed to the germ cell, causing epigenetic alterations.
If these modifications continue after conception, they can alter the phenotypic and increase the risk of a
given disease in the offspring relative to its parents or grandparents. For example, if a pregnant woman (Fo
generation) is exposed to a dangerous chemical, the kid (F1) generation and grandchildren (F2) generation
are at risk of carrying the repercussions of these toxins(Van Cauwenbergh et al., 2020).
2. Transgenerational inheritance: The phenomena in which harmful effects are passed on to subsequent
generations who have not been exposed to these toxins. If the mother was exposed to the hazardous
substances before to conceiving, the consequences in the F2 generation can be considered transgenerational;
however, if the exposure occurred in utero, the F3 generation will be the first to be considered
transgenerational(Van Cauwenbergh et al., 2020).
In my study, I studied the effects of in-utero exposure to Gen+DEHP in adult rat testes and how these EDCs
can affect the future generations inter or trans-generationally. EDCs can cause epigenetic alterations on
DNA methylation, histone modifications or microRNA expression. One of the most important factors while
considering these alterations is the timing of the exposure. For example, exposure during early development
or during prenatal period can transfer the effects in future generations. Another famous example of
transgenerational inheritance is the DES tragedy. DES was given to women to prevent miscarriages for
over 20 years. Later, the DES daughters developed a rare type of reproductive cancer which made them ban
the drug. Further investigation revealed that the reason for the higher incidence of cancer was the inter and
transgenerational inheritance of DES in f1 and F2 generations of the DES mothers(Feroe A, 2017; Montjean
et al., 2022).
Figure 33: Intergenerational and Transgenerational
inheritance(Breton et al., 2021)
38

TYPES OF EPIGENETIC MECHANISMS:
The major epigenetic mechanisms are DNA methylation, Histone modifications and non-coding RNAs.
1. DNA Methylation
DNA methylation changes gene expression without changing the coding sequence. Because it is one of the
simplest approaches to explore, it is the most commonly studied epigenetic mark. The addition of a methyl
group to the carbon group at the 5th position of cytosine, which is found adjacent to Guanine nucleotides,
is what DNA methylation is all about in general. CpG islands are non-randomly dispersed CpGs across the
genome. This additive inhibits transcription by changing chromatin or disrupting the interface between
DNA and DNA binding, resulting in a reduction in gene expression. Because DNA methylation is stable,
changes in methylation patterns that occur in an individual's early childhood might persist throughout their
lifespan(Feroe A, 2017).Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3c are the four primary DNA methylation
enzymes in mammals. Dnmt1 recognizes hemi-methylated DNA and methylates it back to its original
epigenetic pattern. Dnmt3a and Dnmt3b, on the other hand, are required during the preliminary stages of
development because they do de novo methylation during differentiation and early development. Dnmt3l
has homology with Dnmt3b and Dnmt3a but lacks enzymatic activity; yet, as a cofactor of Dnmt3c, it is
still important in male reproduction. This has been demonstrated in investigations where animals lacking
Dnmt3l and Dnmt3c are infertile and exhibit defective spermatogonia differentiation(Lombó & Herráez,
2021).
Tet enzymes remove the methyl group from the DNA, resulting in oxidation of this 5methylytosine, which
is then repaired by thymine DNA (TdG). In my investigation, we discovered that Dnmt3a was
downregulated in both the F1 and F2 generations, whereas Tet1 was upregulated in the F1 generation but
downregulated in the F2 generation. Because these two have opposing activities and must work in tandem,
upregulation of one gene and downregulation of the other at the same time can disrupt normal functioning
if these genes play critical roles in spermatogenesis. Epigenetic patterns are changed twice throughout an
individual's early development to allow for proper cell differentiation. Simply put, the first event happens
immediately after fertilization, when the fertilized egg begins to generate new cell types in order to produce
a new human. This is accomplished by DNA demethylation, histone modification, and ncRNAs, which aid
in "resetting" the zygote genome and guiding it through the embryogenesis process to grow into an adult.
The second step takes place in primordial germ cells (PGC), which produce mature germ cells (egg or
sperm). The epigenetic marks on imprinted genes in the PGCs are erased and changed depending on the
sex of the embryo for future generations' successful reproduction. It does, however, carry an epigenetic
imprint from the parents, allowing expression from a single parent and proper combination of both parent
alleles by epigenetically silencing the paternal or maternal allele. Because animals have two copies of every
39

gene, and both maternal and paternal genes have the same function, one of the parent alleles must be
silenced. As a result, these epigenetic changes persist in the majority of cells throughout life. Controlling
gene expression is referred to as genomic imprinting. Complex epigenetic networks contribute to the
endocrine system's cellular homeostasis. Thus, by altering this balance, EDCs might produce epigenetic
damage. Even while epigenetic modifications throughout childhood are deleterious in maturity, they are
nonetheless necessary because they drive processes such as bas cell differentiation and proliferation. It is
critical to investigate the relationship between epigenetic changes and downstream gene expression in order
to relate EDC exposure to epigenetic toxicity. Statistically, 53 of the 90 investigations conducted before
this foiled found relationships between DNA methylation, histone modification, and downstream gene
expression of MircroRNA. As a result, these alterations are a good indicator for investigating the impact of
EDC exposure. In rats, EDCs can have long-term impacts on Dnmt expression through numerous
generations. One research, for example,
discovered differences in anxiety behavior
following prenatal BPA exposure. These
were associated with an increase in Dnmt
expression and a reduction in the expression
of another GABAergic enzyme (Jacobs et al.,
2017). Both the enzymes Dnmt3a and
Dnmt3b are involved in DNA de novo
methylation. Figure 34 depicts the function of several kinds of Dnmts. DNA demethylation erases DNA
methylation patterns in PGCs, and reestablishment occurs during early germ cell development. Dnmts such
as Dnmt3a, Dnmt3b, and Dnmt3l carry out the reestablishment. This process takes place in the gonocytes,
where processes including de novo and maintenance methylation collaborate to form new methylation
marks on the DNA(Uysal et al., 2016).
Figure 34: Basic functions and knockout phenotypes of Dnmt
genes(Uysal et al., 2016)
40

In mice, Dnmt3a expression is seen in significant quantities in spermatogonia but not in round or elongating
spermatids. It was found in spermatogonia, spermatocytes, primary and secondary spermatocytes, and
round spermatids in humans(Uysal et al., 2016).The same was seen in my study, where the expression of
Dnmt3a was decreased in spermatogonia after exposure to Gen+DEHP(Uysal et al., 2016). As Dnmt3l lacks
enzymatic activity, this enzyme was known to indirectly regulate genomic imprinting and spermatogenesis
by being involved in DNA de novo methylation. Dnmt3l has an Atrx domain which interacts with N terminal
of histone H3 protein. Dnmt3l must be bound to H3 histone via Atrx in order to recognize the target CpG
island that will be methylated during spermatogenesis. Therefore, spermatogenic abnormalities may result
from the absence of these genes(Uysal et al., 2016).As spermatogenesis is a process that includes
spermatogonia, meiosis and differentiation stages, DNA methylation is a crucial epigenetic mechanism
required for normal spermatogenesis. Studies have shown that abnormal disruption of Dnmts and DNA
methylation can be linked to spermatogenesis impairment(Rajender, 2011). In rats it is seen that the
expression of Dnmt3a is higher in newborn pups and decreases gradually after birth(Cisternas et al., 2020).
Study showed that in mice, when the testis germ cells were exposed to high levels of Bisphenol, it showed
an increase in global DNA methylation levels. At the same time, hypomethylation of H3k9me3 was
observed(Cariati F, 2020). The two enzymes: Dnmt3a and Tet are responsible for changing the DNA
methylation patterns during gametogenesis where Tet plays a role in DNA demethylation while Dnmt3a
helps in DNA remethylation. According to studies, Tet1 can inhibit Dnmt3a action by converting the base
5-methylcytosine(5mC) to 5-hyroxymethylcytsoine(5hmC), making it impossible for Dnmt3a to methylate.
This reduces the chances of DNA methylation and increases that of DNA demethylation.
Figure 35: Localization of different types of Dnmts in seminiferous
tubules(Uysal et al., 2016)

41

In contrast to this, Dnmt3a can draw Tet1 to particular region such as CpG islands and promotes it’s activity
there(Chao, 2022). Hence it is important for these two genes to work synergistically to regulate epigenetic
landscapes by complementary binding. For example, deletion of Tet1 can cause increase in protein
accessibility for Dnmt3a whereas deletion of Dnmt3a had a varied effect on Tet1 binding. Thus, Tet1 acts
as an anchor protein to regulate DNA methylation(Gu et al., 2018). In my dataset, in-utero exposure to
Gen+DEHP at 10 mg/kg/day in adult rat testis caused upregulation of Tet1 and downregulation of Dnmt3a
in F1 generation. Upregulated Tet1 can result in increased amounts of conversion of 5mC to 5hmC, further
lowering DNA methylation levels. The DNA might be more hypomethylated and affect epigenetic patterns
and gene expression. For instance, genes that were previously silenced might become active again due to
hypomethylation caused by Tet1 overexpression. On the other hand, downregulation of Dnmt3a can cause
decrease in de novo DNA methylation and impact silencing of certain genes. The usual epigenetic landscape
ay thus be affected by overexpression of Tet1 and under expression of Dnmt3a. These events can eventually
have an effect on spermatogenesis resulting in infertility or other reproductive problems(Gu, 2018)
Foxa1 and Dnmt3a: Another intriguing discovery in my study was that Foxa1, a member of Foxa family
which is a target of Foxa3 and is also involved in steroidogenesis was discovered to be lowered by in-utero
exposure to Gen+DEHP at 10 mg/kg/day. Furthermore, Studies found that transcription factor Foxa1 binds
to the promoter region of Dnmt3a and is responsible for transcriptional activation of Dnmt3a.Thus, Foxa1
is an important factor in epigenetic alterations. Additionally, it can also promote active demethylation via
inducing Tet1 expression at transcriptional levels and interacting with Tet1 directly. Hence, Foxa1 plays a
role both in demethylation and remethylation in different conditions causing regulation of DNA
methylation(Wang et al., 2020).Further research stated that Foxa1 physically interacts with Tet1 and control
DNA demethylation. Foxa1 interacts with Tet1 and promotes DNA de-methylation followed by H3k27
acetylation at Foxa1-targer enhancers which facilitate Foxa1 recruitment and forms a positive loop. Sertoli
cells, Leydig cells as well as germ cells are known to express Foxa1(Behr et al., 2007). Foxa1 also controls
Figure 36: Developmental DNA methylation
reprogramming(Greenberg, 2019)
42

expression of genes that are required for meiotic development of germ cells. In Sertoli cells, it controls
androgen receptor signaling whereas in Leydig cells, it regulates gene expression of genes that are needed
for steroidogenesis(Kim et al., 2021). Thus reduced Foxa1 may hinder the production of testosterone further
altering spermatogenesis and male fertility. In a study done on human breast cancer cells, researchers found
the mechanism by which Foxa1 might be affecting gene methylation and controlling gene
expression(Seachrist et al., 2021).This suggests that Foxa1 plays a role in male infertility by altering DNA
methylation patterns and controlling gene expression.
2. Histone Modifications:
During spermatogenesis, the chromatin arrangement is completely transformed in male germ cells. The
stage of spermatogenesis where in the spermatids go through the process of spermiogenesis, that is the stage
when nucleosome acetylation increases in round spermatids the most. Hyperacetylation causes chromatin
relaxation and improves accessibility, further allowing histones to be removed freely. The highly compacted
structure of chromatin is one of the characteristics of chromatin. In this structure, tiny proteins called as
protamine replace 90% of the histones.

Hence the appropriate number of histones in mature sperm are important. Studies show that mice deficient
in protamine are linked to infertility(R., 2006). Post translational modifications such as acetylation,
methylation, phosphorylation and ubiquitination all play an important role in regulation of cellular
processes such as spermatogenesis and sperm maturation. They can influence the accessibility of DNA by
adding or removing groups from histones. For example, histone acetylation involves transfer of acetyl group
on lysine which removes the positive charge on lysine causing chromatin relaxation and making the
promoter region more accessible to transcription proteins(Feroe A, 2017; Luense et al., 2016).Low doses
of Bisphenol A (0.001-0.1 g/ml) increased the levels of H3k9me3 and H3k27me which are histone
Figure 37: Histone modifications and
spermatogenesis.(Chioccarelli et al., 2020)
43

modifications important for normal spermatogenesis. In contrast to DNA methylation, histone
modifications alter epigenetics by tightening or loosening DNA packing. If the chromatin is tightly packed,
it is called as a heterochromatic state where the transcription factors are not able to bind to the prompter
regions and reduces the transcriptional activity. On the other hand, when the chromatin is loosely packed
or relaxed, it is termed as euchromatic state which is a more active state and allows better accessibility(Feroe
A, 2017), The enzymes involved in histone acetylation are histone lysine methyltransferases (HKMTs),
whereas in histone demethylations are the lysine demethylases (KDMs)(Walport et al., 2018). Studies show
that H3k27 and H3k9 when monomethylated can cause gene activation whereas trimethylated H3k27 and
H3K9 can lead to gene repression. HATs are responsible for histone acetylation and HDACs play a role in
erasure of acetyl groups. These modifications are added and removed by a wide range of histone-modifying
enzymes, including histone acetyl transferases, which add acetyl groups; histone deacetylases (HDACs),
which remove acetyl groups; histone methyl transferases, which add methyl groups; and histone
demethylases, which remove methyl groups(Jacobs et al., 2017). These histone modifications interact to
create a histone code that regulates the genome's transcription state. Lysine methylation at histone tails is
another method. Methylation, acetylation, and ubiquitination all play a role in the chemical conversion of
lysine to serine in histone tails. Methylation of H3k9 causes gene silencing, whereas methylation of H3k4
activates genes(Gunes & Kulac, 2014). The two genes in my dataset that were related to spermatogenesis
and histone modifications were Brwd1 and Ezh1
Bromodomain-containing proteins have been shown to interact with chromatin's hyperacetylated lysine
residues. Brwd1 is a protein with a bromodomain that has been reported to modify gametogenesis. Histone
modifications play a key role in the activation of transcription for the haploid genome in spermatids following
meiosis. One specific example is the promoter region of the "protamine domain," containing genes like Prm1,
Prm2, and Transition protein 1. When germ cells go through meiosis and become spherical spermatids, the
promoter region of these genes undergoes major epigenetic alterations. These histone modifications are
essential for the transcriptional activation of these genes, which have been associated in the paternal genome
packing during sperm maturation (Wang T, 2019). Acetylation of histones H3 and H4 in protamine domain
promoters and coding areas increases in tandem with transcriptional activity. The vast majority of infertility
mutations affect both sexes and are found in genes involved in primordial germ cells, meiosis, or the endocrine
system(Matzuk & Lamb, 2008). Brwd1 stands out since it appears to regulate several pathways in sperm and
oocytes(Philipps, 2008).Bromodomains are 110-amino acid motifs that are recognized for their ability to
detect acetyl-lysine residues(Pattabiraman et al., 2015). This relationship is essential for an array of biological
functions, including chromatin remodeling and transcriptional activation. Infertile mice of both sexes lack
Brwd1 (bromo- and WD-containing protein-1)(Philipps, 2008). Reduced Brwd1 levels could lead to
chromatin structure to be deformed, gene expression to be reduced, and the regulatory networks required for
44

spermatogenesis to be compromised. This might affect germ cell development, meiotic division, and the
formation of mature sperm. Males may experience reduced sperm production, low sperm quality, or even
complete infertility as a result(Philipps, 2008). According to the results of RNA Seq data, Brwd1 was
upregulated in F1 adult rat testes subjected to a combination of in-utero exposure to Genistein and DEHP at
10mg/kg/day. Furthermore, for F2, Brwd1 decreased significantly. If Brwd1 expression increases in the F1
generation and then decreases in the F2 generation, it may influence spermatogenesis. This was also
corroborated by qPCR data which revealed comparable trends. In developing sperm cells, increased Brwd1
expression in the F1 generation may result in altered chromatin remodeling and transcriptional activation.
This might affect gene expression patterns, chromatin structure, and spermatogenesis. Individual regulatory
mechanisms regulated by Brwd1 and the genes with which it interacts throughout this time period would
define the precise consequences(Philipps, 2008).Brwd1 expression in the F2 generation, on the other hand,
may interfere with Brwd1's usual involvement in chromatin remodeling and transcriptional activation. This
might lead to further alterations in gene expression and chromatin structure in developing sperm cells,
potentially affecting spermatogenesis. Brwd1 expression or activity changes can have a significant influence
on spermatogenesis. Brwd1 is involved in chromatin remodeling and transcriptional activation, both of which
are linked to epigenetic modulation.
Ezh1 is a gene which has been identified as being involved in the Polycomb repressive complex 2 in the testes
and catalyzing histone H3 lysine 27 methylation(Mu et al., 2017). Ezh1 was shown to be highly expressed in
adult testis tissues, notably mitotic germ cells and pachytene
spermatocytes. Ezh1 knockout mice showed meiotic arrest and
spermatocyte loss in the seminiferous tubules(Mu et al., 2017).Ezh1
regulates gene expression and directs cell fate decisions during
spermatogenesis. It is found in high concentrations in germ cells,
particularly spermatogonia and early meiotic cells(Yuan). Ezh1-
mediated H3k27me3 deposition is involved in controlling the
expression of certain genes and contributing to the normal
spermatogenesis.
Ezh1 was increased in F1 generation exposed in-utero to Gen+DEHP
at 10 mg/kg/day in my data. If Ezh1 levels are high, it can have a
considerable epigenetic influence on spermatogenesis. Ezh1, a histone
methyltransferase, catalyzes the addition of methyl groups to histone H3 at lysine 27, resulting in the
formation of the restrictive histone mark H3K27me3. When Ezh1 levels are high, there is more H3K27me3
in the chromatin because Ezh1-mediated histone methylation is more active. This might result in a more
Figure 38: EZH1 and
EZH2 roles in the creation and
maintenance of H3K27
methylation during
spermatogenesis.(Mu et al.,
2017)
45

compact chromatin structure and increased silencing of target genes(Shen et al., 2008). Overexpression of
Ezh1 may result in widespread transcriptional silencing of normally expressed genes, potentially affecting
various cellular processes involved in spermatogenesis. The epigenetic effects of Ezh1 overexpression can
affect gene expression patterns, cell fate determination, and chromatin architecture.
3. Chromatin remodeling:
During spermatogenesis, chromosome remodeling is required for normal sperm cell development and
maturation. It comprises dynamic changes to the structure and organization of chromatin, allowing for precise
regulation of gene expression and the formation of transcriptional pathways specific to certain cell
types(Christina Rathke, 2014).
Functions of chromatin remodeling:
• In the early stages of meiosis, chromatin remodeling is required for chromatin reprogramming in germ cells.
Histones disappear, which results in a more accessible chromatin structure. Because of this remodeling,
particular genes vital to meiotic development and the formation of haploid gametes may be activated and
silenced.
• As spermatids grow into spermatozoa, chromatin undergoes an essential remodeling from histone-based to
protamine-based. The bulk of histones are replaced by protamines, which are small, basic proteins that
contribute in the compaction of DNA into a highly condensed and stable form. Through this chromatin
remodeling process, the paternal genome must be securely packed and insulated from DNA damage.
• Chromatin remodeling also plays a function in the epigenetic regulation of gene expression during
spermatogenesis. It comprises acetylation, methylation, and phosphorylation of histones, which can affect
DNA accessibility and the attraction of transcriptional regulators. These changes regulate the expression of
genes involved in sperm maturation, meiotic progression, and germ cell differentiation in a dynamic manner.
Atrx is one of the genes that was differentially expressed in my dataset and was related to spermatogenesis. In
humans, 80% of Atrx mutations cause genital abnormalities. Atrx staining in Leydig cells is considerable,
and the fact that Atrx patients with male pseudo-hermaphroditism fail to produce normal quantities of
testosterone suggests that Atrx is important in Leydig cell steroidogenesis(Tang, 2011). It contributes to
DNA methylation at these locations by interacting with Dnmt3b (DNA methyltransferase 3B). Atrx deficiency
causes a lack of DNA methylation, which affects the structure of heterochromatin and resulting in aberrant
gene expression patterns. Specific histone variants, such as H3.3, are deposited at distinct chromosomal
regions and are linked to Atrx. Studies revealed that The Atrx protein was present in all testicular cells at
embryonic day 14.5 of the mouse testis, with Sertoli cells having the greatest levels of expression. They
46

observed that Atrx protein expression remained constant in Sertoli cells throughout sexual development and
maturity in the mouse testis but decreased in germ cells after E17.5. Atrx is found in the nuclei of Sertoli and
peritubular myoid cells, Leydig cells, and early germ cell stages, but not in post-meiotic or larger pachytene
spermatocytes (Bagheri-Fam, 2011). Atrx has been found to colocalize with chromatin in adult testis and to
act in both somatic and germ cells. The actual mechanism of action linking Atrx to previously discovered
anomalies, however, remains uncertain. Atrx and transcription factors specific to the testis may connect and
collaborate to alter chromatin(Dyer et al., 2017).
In my dataset, Atrx was known to be downregulated in F1 generation exposed to Gen+DEHP at 10 mg/kg/day
while it was restored in F2 generation exposed to the same dose. This may change the accessibility of DNA
and higher-order chromatin structure, resulting in changes in gene expression patterns. The interaction of
Atrx with DNA methyltransferases results in the establishment and maintenance of DNA methylation
patterns. Atrx deficiency may result in a loss of DNA methylation at specific genomic regions, particularly
repetitive repeats. This may result in epigenetic dysregulation and altered gene expression patterns.
Stat3:
The transcription factor Stat3 is essential for mammalian spermatogenesis because it allows for the
coordinated differentiation of stem and progenitor spermatogonia(Kaucher AV, 2012). Some of the key roles
of Stat3 in spermatogenesis include:
1. Stat3 controls the differentiation and maturation of germ cells. It is expressed in spermatogonia and
spermatocytes, and activation of this gene is essential for healthy maturation of germ cells(Kaucher AV,
2012). Stat3 signaling facilitates meiosis progression and the conversion of spermatogonia to
spermatocytes.
2. Sertoli cells, or somatic cells within seminiferous tubules, are required for spermatogonia proliferation.
Stat3 signaling in Sertoli cells is required for both proper function and the maintenance of the
spermatogenic niche. Sertoli cell Stat3 activation regulates the release of many growth factors, hormones,
and cytokines that are essential for germ cell survival, proliferation, and differentiation(Nagasawa, 2018).
3. During spermatogenesis, apoptosis is a tightly regulated process that removes undesired or damaged germ
cells. Apoptosis inhibition in developing germ cells aids in their survival and maturity. Stat3 signaling
makes this feasible. Stat3 activity in germ cells and Sertoli cells contributes to the anti-apoptotic signaling
cascades essential for optimal spermatogenesis(Al Zaid Siddiquee & Turkson, 2008).
4. Inflammation and immunological responses may have an effect on spermatogenesis. Stat3 signaling is
important in the modulation of immune responses in the testes. It assists in the regulation of cytokines
47

and mediators that cause inflammation, hence maintaining immunological homeostasis in the testicular
microenvironment(Huang et al., 2016).
5. The blood-testis barrier (BTB), a unique junctional barrier formed by Sertoli cells that isolates the
seminiferous tubules from the circulation, must be preserved. The BTB, which is crucial for good
spermatogenesis by providing a safe environment for germ cell proliferation, is keeping its integrity and
activity via Stat3 signaling(Alves-Silva et al., 2021).

Stat3 was activated via the G-alpha signaling pathway in the F1 generation subjected to Gen+DEHP at 10
mg/kg/day, although it was elevated in this generation. Stat3 can influence the transcription of target genes by
migrating into the nucleus and binding to specific DNA sequences when it works through this method. This
may have an impact on a variety of biological processes, including cell division, survival, differentiation, and
immune responses. Stat3 activation by G-alpha signaling may considerably enhance cell fate determination
and differentiation during development(Liu et al., 2006). Stat3 activation can aid in the formation of
spermatogenesis by encouraging the differentiation of spermatogonia into spermatocytes. Increased
Stat3 activity can help germ cells survive and develop. Stat3 activation can promote germ cell survival and
population number by inducing the expression of anti-apoptotic genes and advancing the cell cycle.
Stat3 overexpression can influence germ cell differentiation during spermatogenesis. It can help in the meiotic
progression of spermatocytes and the development of spermatogonia into spermatocytes. This may aid in the
correct growth and maturation of germ cells(Al Zaid Siddiquee & Turkson, 2008; Kaucher AV, 2012). Sertoli
cells, which are found within the seminiferous tubules, are important in the development of germ cells.
Figure 39: DBP induced testosterone synthesis inhibition. (Qi
Wang, 2023)
48

Increased production of growth factors, hormones, and cytokines, which are essential for germ cell
development and differentiation, can occur from upregulating Stat3, an enzyme which can influence Sertoli
cell activity(Huang et al., 2016). Stat3 activation may alter gene expression patterns during spermatogenesis,
which also influences chromatin remodeling and epigenetic alterations. The overexpression of Stat3 may
affect germ cell development and function by changing histone modifications, DNA methylation, or other
epigenetic markers that influence gene expression and chromatin structure(Zhang et al., 2005).
In F2 generation, Stat3 is shown to function through Jak-Stat pathway. The Jak-Stat system is activated when
ligands, such as cytokines or growth factors, bind to their corresponding cell surface receptors. This binding
creates receptor dimerization and activates Jaks that are associated with it. Because active Jaks
phosphorylate tyrosine residues, signal transducer and activator of transcription (Stat) proteins can bind to
the receptor's cytoplasmic domain. Once recruited to the receptor complex, the Jaks phosphorylate the Stat
proteins. Phosphorylated Stat proteins travel from the cytoplasm to the nucleus after dimerization. Stat
dimers, as transcription factors, influence gene expression after they enter the nucleus by binding to specific
DNA sequences known as Stat response elements (SREs) located in target gene promoters. The
transcription of repressed or activated target genes can be altered by active Stat proteins and affect
spermatogenesis(Lachance C, 2011). Stat3 must be activated in order for the Sertoli cells, which act as
supporting cells for the seminiferous tubules, to function effectively. In my dataset, Stat3 was downregulated
in F2 generation exposed to the Gen+DEHP dose at 10 mg/kg/day. A disruption in the Jak-Stat pathway
produced by Stat3 downregulation may impact germ cell growth and sperm production. If Stat3 regulation
moves to the Jak-Stat pathway in the F2 generation of spermatogenesis, it indicates that the upstream
signaling channel that activates Stat3 has altered(Lachance C, 2011). Unlike G-alpha signaling, Stat3
activation via the Jak-Stat pathway may result in transcriptional regulation of a distinct set of target genes.
As a result, gene expression patterns in spermatogenesis-related germ cells and supporting cells may be
changed. Stat3-targeted genes can influence a variety of biological activities, including immune regulation,
cell proliferation, differentiation, and survival. As a result of the alteration in Stat3 regulation between
generations, a transgenerational epigenetic or genetic modification may have occurred. This alteration may
modify the epigenetic environment and gene expression patterns in germ cells, which may have an influence
on spermatogenesis not only in the F2 generation but also in subsequent generations. Overall, switching to
the Jak-Stat pathway for Stat3 regulation in the F2 generation can affect spermatogenesis across generations
by altering gene expression patterns, immunological modulation, and signaling dynamics.

49

CONCULSION:
In the present study, I found that in-utero exposure of rats to Genistein and DEHP mixtures alters the
transcription factor Foxa1, which was reported to regulate Dnmt3a, possibly driving epigenetic changes in
testis. Exposure to Genistein and DEHP during fetal development induces epigenetic alterations, including
altered DNA methylation, Histone modifications and Chromatin remodeling in the adult rat testes. Fetal
exposure to Genistein and DEHP mixtures influences the expression of genes involved in cell survival,
proliferation, and differentiation, which could disrupt testis development. The genes and pathways
identified in this study might play a role in the adverse reproductive effects of fetal exposure to Genistein
and DEHP mixtures and deserve further studies. Elucidating the mechanisms leading to their disruption
should help understanding the role they play in the EDC-driven adverse reproductive effects.

50

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Abstract (if available)

Abstract Approximately 50 million people worldwide suffer from infertility with male factors accounting for nearly half of all cases. Exposure to endocrine disrupting chemicals (EDCs) have been shown to play a role in the etiology of male reproductive diseases in animal models, and strong associations have been reported in human studies between exposures to EDCs and male reproductive diseases. Humans are exposed to multiple EDCs throughout their life. While exposure to the phytoestrogen genistein is dietary via soy products, exposure to the ubiquitous antiandrogenic plasticizer DEHP comes from various consumer products and medical devices. In previous studies, we found that in-utero exposure of rats to genistein and DEHP mixtures (Gen+DEHP) altered testis development and transcriptome, and increased infertility rates. This study gives an in-depth look at the effects of endocrine disrupting chemicals (EDCs) on male reproductive health. The processes through which EDCs damage the male reproductive system are investigated, including hormone mimicking and receptor binding interference. The effects of fetal-maternal EDC exposure on placental transfer and babies are being investigated. Additionally, this study seeks to give useful insights into the influence of Gen and DEHP on male infertility and reveal potential epigenetic alterations produced by EDC exposure across generations by employing doses that mimic human exposure and using intergenerational and transgenerational evaluations. My goal is to identify genes and pathways altered in rat testes after in-utero exposure to low doses of Gen+DEHP.

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Asset Metadata

Creator Malusare, Priyanka Ravindra (author)

Core Title Fetal exposure to genistein and DEHP mixtures alters the expression of genes involved in critical testicular functions in adult rats

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2023-08

Publication Date 06/16/2023

Defense Date 06/15/2023

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

Tag DEHP,genistein,male infertility,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Culty, Martine (committee member), Papadopoulos, Vassilios (committee member), Tabancay, Angel (committee member)

Creator Email pmalusar@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113170768

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