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$&.12:/('*(0(176I am very grateful to Jehovah, the Almighty God who gave me life. He has sustained and protected
me until this very moment.
I specially thank my research supervisory committee members, Professor Masayuki Shimada, andDr. Takashi Umehara (Assistant Professor), Laboratory of Animal Production, Graduate Schoolof Biosphere Science, Hiroshima University for their innovative suggestions and guidance
throughout my doctoral study and dissertation writing. I also would love to express myappreciation to Professor Naoki Isobe and Professor Hiroyuki Horiuchi for their kind effort andconstructive comments during the preparation of my doctoral dissertation.
It is my pleasure to express sincere gratitude to the biosphere science student support team,
especially Himiko Koi for her diligence and support during my study.
I am also grateful to my wife, Ebunoluwa Adetunji, I thank you for your sacrifices and selflessness;my parents, Mr. and Mrs. R.A. Adetunji, and entire family, Adetutu, Adeola and Tejumade for
their united and untiring love and support from the onset until this moment. I appreciate their kindgesture. I cannot also forget to thank my aunt, Olayinka Oluwunmi and all my friends especiallyOgunsina sola, Adesina Adedayo and Saliu Tolulope, for their words of encouragement and forproviding moral support throughout the course of my doctoral research.
To Prof. Teruo Maeda (Ret.) and Dr. Tomoko Kawai, I say it has been wonderful having you as atutor and guide throughout my doctoral journey and I would cherish all the moment spent in thelaboratory with you. To chizuka san, Yuki san, Zhu san, Tawara san, Hibi san, Islam san and otherlaboratory members, I say thank you for your support and guidance. May Jehovah bless you all.

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1.1

The Ovary

1.2

Folliculogenesis
1.2.1

3DJH1R
11

Primordial follicle formation and2

recruitment 
1.2.2

Development of primary, preantral,2antral, and preovulatory follicles
1.3

Luteinizing hormone/choriogonadotropin4

receptor (LHCGR)
1.3.1

Mechanism of cumulus expansion,4

ovulation, and luteinization
1.4

Innate immune system and Toll-like receptors6

1.4.1

Toll-like receptor 4 and LPS7

1.4.2

Toll-like receptor 7 and 8 roles in7

Viral infection
1.4.3
1.5

Endogenous Ligands

Pelvic Inflammatory Diseases and other diseases caused by88

Bacteria and Viral infections

1.6

1.5.1

Follicular Cyst9

1.5.2

Polycystic Ovarian Cyst9

Aim of this study10i

7RSLFV3DJH1R

&KDSWHU([SHULPHQW

Study on the effect of LPS on overall ovarian health and Epigenetic dynamics of the Lhcgr andCyp19a1 gene promoter region
2.1

Introduction

13

2.2

Materials and Methods

16

2.3

Results

26

2.4

Discussion29

Abstract

33

&KDSWHU([SHULPHQW

Study on the role of Toll-like receptor 7/8 on mouse ovary

3.1

Introduction

46

3.2

Materials and Methods

47

3.3

Results

53

3.4

Discussion

57

Abstract60
&KDSWHU*HQHUDO'LVFXVVLRQ74
&KDSWHU6XPPDU\80
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ii

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7LWOHRI7DEOHVTable 1

PCR primers used for profiling the expression of genes markers for

3DJH1R
34

GC function
Table 2

PCR primers used for profiling the expression of TLR7/8 and genes

61

markers for ovarian function

iii

/LVWRI)LJXUHV
7LWOHRIWKHILJXUHVFigure 1

Structure of lipopolysaccharide (LPS). LPS is found on the cell wall

3DJH1R12

of gram-negative bacteria, such as Escherichia coli. The lipid A
region, depicted in red, elicits the immune response.
Figure 2

Expression of TLR4 (A) in granulosa cells of mice ovary at 15, 23

36

days and 24 and 48 hr after eCG, (B) Immunohistochemistry stainingof the ovary during the immature and follicle development phase(eCG 48 hr).
Figure 3

Ovarian morphology and follicular health (A) Hematoxylin-eosin

37

staining during the follicular development and ovulation phase. (B)The number of primary, secondary, and antral follicles in mice ovaryat 48 hr after eCG and/or LPS injection and hematoxylin-eosin
staining.
Figure 4

Follicular atresia and cell proliferation in ovarian follicles resulting

38

from LPS action. (A) TUNEL staining of the ovary during thefollicular development phase. (B) Immunofluorescence staining of
the ovary from mice injected with eCG with or without LPS at 48 hr
the immature.
Figure 5

Ovarian morphology and follicular health (A) Hematoxylin-eosin

39

staining of ovaries during the ovulation phase. (B) The average
number of ovulated oocyte (C, 0.1, 1 μg/kg LPS). (C) the maturation
rate (D) the average number of follicular cysts
Figure 6A

The expression of Cyp19a1, Ccnd2, Fshr, Lhcgr genes and Inhibin40

hormone related to cell proliferation, differentiation, and ovulation in
granulosa cells of mice during the follicular development phase invivo.
Figure 6B

The expression of Lhcgr, Cyp19a1 and Ccnd2 genes related to cell

41

proliferation, differentiation, and ovulation in granulosa cells of mice
during the follicular development phase cultured in vitro.
iv

7LWOHRIWKH)LJXUHVFigure 7A

The effect of LPS on the expression of EGF-like factors, progesterone

3DJH1R42

receptor (PGR)-dependent events and other targets of the LH-LH
receptor (LHR)-dependent pathway (Snap25 and Star) in granulosa
cells of mice.
Figure 7B

The effect of LPS on the expression of EGF-like factors, progesterone

43

receptor (PGR)-dependent events and other targets of the LH-LH
receptor (LHR)-dependent pathway (Ptgs2, Nrg1, Cyp11a1 andAreg) in granulosa cells of mice.
Figure 8

The effect of LPS on the expression of chemokines and cytokines

44(Chemokine ligand 5 and Interlukin-6) in the granulosa cells of mice.
Figure 9

The expression of Dnmt1 gene implicated in DNA methylation in $45

granulosa cells in vivo (B) cultured granulosa cells.
Figure 10

Kinetic changes of cytosine methylation in the (A) Lhcgr promoter

46

region, and (B) Cyp19a1 promoter region of granulosa cells of mouseovaries.
Figure 11

TLR7 expression in the ovary at different time points (A) Expression

64

of TLR7 through western blotting, where β-actin was used as control.
(B) Quantitative expression of TLR7 over β-actin in the ovary.
Figure 12

Immunofluorescence staining of the ovary during the immature,65

follicle development and ovulation phase using anti-TLR7 antibody.
Figure 13

TLR7 and TLR8 expression in the ovary at different time points (A)66

Quantitative expression in the cumulus cells. (B) Quantitative
expression in the granulosa cells.
Figure 14

The expression of inflammation-associated genes (Chemokine ligand

67

5, Interferon-gamma and Interlukin-6) in the spleen of mice.

v

7LWOHRIWKH)LJXUHVFigure 15

Effect of Resiquimod (R848) on ovarian morphology and follicular

3DJH1R
68

health (A) Hematoxylin-eosin staining of ovaries during the ovulation
phase. (B) The average number of ovulated oocytes. (C) the
maturation rate (D) fertilization rate.
Figure 16A

The effect of LPS on the expression of EGF-like factors, progesterone

69

receptor (PGR)-dependent events and other targets of the LH-LH
receptor (LHR)-dependent pathway (Ptgs2, Cyp11a1, Star and Nrg1)
in granulosa cells of mice.
Figure 16B

The effect of LPS on the expression of EGF-like factors, progesterone

70

receptor (PGR)-dependent events and other targets of the LH-LH
receptor (LHR)-dependent pathway (Snap25, Pgr, Ereg and Areg) in
granulosa cells of mice.
Figure 16C

Dose-dependent expression of Snap25, Ptgs2, Cyp11a1, Star,

71

Adamts1 and HSD3B1 in granulosa cells cultured in vitro.
Figure 17

The expression of inflammation-associated genes (Chemokine ligand

72

5 and Interlukin-6) in granulosa cells of mice ovaries.
Figure 18

Ovarian morphology and follicular health (A) The average number of73

ovulated oocyte (eCG+hCG and eCG+R848 0.1 μg/kg). (B) The
average number of ovulated oocyte (eCG+hCG+GnRH antagonist
and eCG+R848 (0.1 μg/kg) +GnRH antagonist) (C) Hematoxylineosin staining of ovaries during the ovulation phase. (D) Level ofprogesterone
Figure 19

ACE2 receptor expression in the ovary at different time points

74

vi

&KDSWHU*HQHUDO,QWURGXFWLRQ&RQWHQWV7RSLFV 

3DJH1R

1.1

The Ovary1

1.2

Folliculogenesis

1

1.2.1

Primordial follicle formation and2

recruitment 
1.2.2

Development of primary, preantral,

2

antral, and preovulatory follicles
1.3

Luteinizing hormone/choriogonadotropin receptor (LHCGR)

4

1.3.1

4

Mechanism of cumulus expansion,
ovulation, and luteinization

1.4

Innate immune system and Toll-like receptors

6

1.4.1

Toll-like receptor 4 and LPS

7

1.4.2

Toll-like receptor 7 and 8 roles in Viral

7

infection
1.4.3
1.5

Endogenous Ligands

8

Pelvic Inflammatory Diseases and other diseases caused by

8

Bacteria and Viral infections

1.6


1.5.1

Follicular Cyst

9

1.5.2

Polycystic Ovarian Cyst

9

Aim of this study10,QWURGXFWLRQ7KH2YDU\The ovary is the female reproductive organ that influences so many activities such as thedevelopment of secondary sex characteristics, regulation of the hypothalamic-pituitary-ovarian
axis as well as the production of growth factors, peptides, and steroids to mention a few (Edson etal., 2009). The ovary has two fundamental functions, and they include the production ofdevelopmentally competent oocytes and the secretion of steroid hormones which play a role infertilization and embryo implantation (Palermo, 2007). The somatic cells and germ cells which
consist of oocytes and cumulus, granulosa, theca, and stroma cells respectively are the main
components of the ovary.
)ROOLFXORJHQHVLVThe first step leading to the ovulatory stage is referred to as folliculogenesis and this describes theprocess through which a follicle is formed. A mammalian follicle comprises an oocyte that issurrounded by somatic cells such as cumulus and granulosa cells. The granulosa cells act as asource of energy and nutrient supply. Likewise, the theca cells which are somatic cells produce
steroids such as androgen which is later converted into estrogen by the granulosa cells, a steroidthat is vital for follicle development and reproductive health (Balla et al., 2003 and Tingen et al.,2009). It is the interaction of somatic and germ cells that leads to ovarian follicle maturation andthe process of maturation occurs in a stepwise manner. The process includes initiation, growth
selection, ovulation, and luteinization (Richards and Pangas, 2010).

1|P a ge 3ULPRUGLDOIROOLFOHIRUPDWLRQDQGUHFUXLWPHQW
During the early stage of fetal development, the ovaries contain a large pool of oogonia thatdevelops into the oocyte by entering the first meiotic division after which they are arrested at theprophase stage (resting phase). In mice, the formation of primordial follicles containing an oocyte
in the first prophase of meiosis with a single layer of granulosa cells occurs within the first few
days after parturition (Hirshfield, 1991). However, from this large pool, there is continuous
recruitment of primordial follicles into the growing pool but only a few develop to become
preovulatory follicles and undergo ovulation while the majority of the follicles undergo atresia(Matsuda et al., 2012 and Hsueh et al., 1994).
'HYHORSPHQWRISULPDU\SUHDQWUDODQWUDODQGSUHRYXODWRU\IROOLFOHV
Following the initial activation of primordial follicles and the cyclic recruitment or selection of afew follicles from the pool of primordial follicles for dominance and subsequent ovulation. Thereis a transition from primordial to primary follicle stage which is markedly recognizedhistologically by a change in a squamous morphology or shape of the granulosa cells to cuboidal
and they begin to express proliferating cell nuclear antigen (PCNA) (Fortune et al., 2000 and
McGee and Hsueh, 2000). During the preantral stage, follicles acquire an additional somatic cell
layer called the theca cells and undergo granulosa cell proliferation and a further increase in thesize of the oocyte. The secondary follicle contains oocytes in mid-growth stages, and it issurrounded by two or more layers of granulosa cells. Overall, the growth of the preantral follicles
is dependent on paracrine, autocrine regulatory factors, and the action of gonadotropin which
produces follicle-stimulating hormone (FSH) (Cattanach et al., 1977). Also, the bidirectional
interaction between the oocyte and the somatic structures of the follicle using growth factors suchas inhibin/activin, insulin-like growth factor and bone morphogenetic systems (BMP) also affect
2|P a gethe growth of preantral follicles (Webb et al., 1999, Knight and Glister 2001, Eppig et al., 2002).
In addition, the oocyte secretes numerous factors that mediate follicle development and ovarianfunction, which include GDF9 and BMP15 (Dong et al., 1996 and Aaltonen et al., 2000). These
oocyte secreted factors play a crucial role in granulosa cell proliferation, theca cell development,
and steroid regulation (Carabatsos et al., 1998). The formation of the antrum which is marked by
fluid-filled spaces that graduate from small spaces to form a single antral cavity signifies the
emergence of antral follicles. In mice, it takes around 15 days postpartum from the activation ofprimordial follicles to the formation of antral follicles (Hirshfield, 1991). In the matured Graafianfollicle, two functionally different granulosa cell populations can be seen, the cumulus cells whichsurround the oocyte, and the mural granulosa cells which lines the wall of the follicle. The cumulus
cells play a role in oocyte growth and developmental competence while the mural granulosa cells
are implicated in steroid production and ovulation (Balla et al., 2003). It is during the antral stage
that FSH and LH act on the granulosa and theca cells to synthesis both androgens and oestrogen.
Moreover, the ovary also produces other growth factors such as activins and inhibin to modulate
the action of FSH, hereby regulating the follicle development process when necessary (Bernard
and Woodruff, 2001). To enter the preovulatory follicle stage, follicles with higher FSHR
expression survive the reduction of FSH level in the serum. These follicles which express Lhcgr
and Cyp19a1 in granulosa cells are termed dominant follicle. The high expression of Cyp19a1
induces estrogen production in preovulatory follicles, which impacts the induction of LH surge.
The transient secretion of LH from pituitary grand acts on LH receptors expressed in granulosa
cells of preovulatory follicles to undergo the ovulation process.

3|P a ge/XWHLQL]LQJKRUPRQHFKRULRJRQDGRWURSLQUHFHSWRU LHCGR LHCGR is a member of the G-protein coupling receptor family and it is expressed in the thecacells of actively dividing follicles and in mural granulosa cells of preovulatory follicles (Minegishi
et al., 1990; Eppig et al., 1997). Therefore, the expression of LHR in preovulatory follicles can besaid to be a marker of the mural granulosa cell phenotype in the follicles. Also, the activation of
Lhcgr is necessary for ovulation and progesterone production (Richards, 1994). Lhcgr in thecacells stimulates the production of androgen which serves as a substrate for conversion of androgento estradiol through the action of FSH-induced aromatase in granulosa cells. To ensure theinitiation and completion of these processes, Lhcgr activates the Protein Kinase A and ERK1/2
pathways (Breen et al., 2013). In addition, PKA promotes the activation TCF3 and SF1 site/region
found in the Lhcgr promoter by increasing the phosphorylation of β-catenin that interacts withboth transcriptional factors to enhance Lhcgr transcriptional activity (Law et al., 2013). Likewise,
the Lhcgr promoter region has the SP1 binding sites whose activities not only influence theexpression of Lhcgr in both granulosa and theca cells but also serve as the main regulator of Lhcgr
gene expression (Law et al., 2013). The expression and silencing of genes are regulated by
epigenetic mechanisms. Therefore, epigenetic changes in the Lhcgr promoter region can prevent
Lhcgr transcriptional factor Sp1 from gaining access and binding to its target sequence, hereby
resulting in gene suppression (Genhard et al., 2010).
0HFKDQLVPRIFXPXOXVH[SDQVLRQRYXODWLRQDQGOXWHLQL]DWLRQ
LH directly induces follicle rupture and luteinization processes via the expression of genes in
granulosa cells. LH induces and activates progesterone production and progesterone receptor(PGR) lead to an increase in Adamts1, a downstream target that mediates follicle rupture andrelease of oocyte (Palanisamy et al., 2006). The progesterone was produced by the induction of

4|P a geseveral proteins such as Star and Cyp11a1 are induced in the ovary. However, ovulation has been
described as an inflammatory-like process because of the occurrence of follicular hyperemia, anincrease in the production of prostaglandins, implicating the activation of collagenase and otherprotease cascades. During ovulation, LH induces plasminogen activators of the urokinase-type in
the preovulatory follicles of mouse ovary which play a huge role in activating collagenase an
enzyme that is involved in tissue remodeling (Espey and Lipner, 1994). Together, these genes
support the follicle rupture and ovulation processes.
However, due to the low level of LH receptor in cumulus cells surrounding the oocyte, the processof cumulus expansion and oocyte maturation is indirectly induced by LH through EGF network(Fu et al., 2007; Fan et al., 2009). The binding of LH to LHR in mural granulosa cells leads to theaccumulation of cAMP and increased expression of secondary factors such as amphiregulin(AREG) and epiregulin (EREG) which in turn activates the ERK1/2 cascade in the cumulus cellsof preovulatory follicles (Conti et al., 2012; Park et al., 2004). This kinase acts as a mediator that
stimulates the expansion of cumulus cells and oocyte maturation due to the expression of Ptgs2,
Tnfaip6, Has2, and Ptx3. With Has2 producing hyaluronan which plays a role as the backbone of
the matrix, TNFaip6 catalyzing the formation of covalent crosslinks between the chains of serumderived inter-alpha -trypsin inhibitor and hyaluronan, Pentraxin (Ptx3) functions in stabilizing theinteraction between the cumulus matrix and serum-derived inter-alpha -trypsin inhibitor and Ptgs2
synthesizing prostaglandins required for supporting the activity of TNFaip6 (Hernandez-Gonzalez
et al., 2006). With the induction of cumulus expansion, these genes including members of the
innate surveillance system (PRRs) from the TLR family, Myd88 and Cd34, and Cd36 are expressedin cumulus cells. This shows that just as it is seen during bacterial invasion-immune cell

5|P a ge

stimulation, under ovulatory circumstances cumulus cells also prompt TLR2&4 and CD14 andother immune-related genes (Hernandez-Gonzalez et al., 2006; Shimada et al., 2008).
,QQDWHLPPXQHV\VWHPDQG7ROOOLNHUHFHSWRUVThe first responders to external invasion or infection in the host are the immune components of
the innate immune system and they include cytokines, natural killer cells, macrophages, andneutrophils. In posing an inflammatory response, the host recruits immune cells to the site of tissue
damage or microbial invasion (Tizard, 2018). This process is initiated when pattern-recognition
receptors (PRRs) on the surface or within host cells recognize or bind to pathogen-associatedmolecular patterns (PAMPs) released from damaged cells or microbial components in a distinct
and specific manner (Akira et al., 2006). Toll-like receptors which are a notable example of PRRs
are of 10 different types (TLR 1-10 inclusive) (Akira et al., 2001) and their signaling pathway isactivated from the cytoplasmic Toll-interleukin-1 receptor (TIR) domains. In this domain, an
adaptor named MyD88 which contains the TIR domain in the C-terminal portion and a death
domain in the N-terminal portion plays a huge role in signaling for all toll-like receptors (Hornget al., 2001). When MyD88 is stimulated it recruits IL-1 receptor-associated kinase (IRAK) to
TLRs by the interaction of the death domains of both molecules. This leads to the activation of aserine/threonine kinase (IRAK) associated with the IL-1 receptor which contains the TIR domainby phosphorylation. It is the interaction between IRAK and TRAF6, a member of the tumor
necrosis factor receptor-associated factor family that leads to the activation of two distinct MyD88
signaling pathways and the resultant activation of JNK and Nuclear factor-κB (NF-κB) (Fitzgerald,2001). Thereafter, NF-κB translocates into the nucleus to activate the expression of genes
associated with proinflammatory cytokines (Takeuchi and Akira et al., 2010).

6|P a ge 7ROOOLNHUHFHSWRUDQG/36
LPS is a complex glycolipid containing a hydrophilic polysaccharide and hydrophobic domain. It
is found in the outer membrane component of Gram-negative bacteria. Its hydrophobic domain
which is referred to as Lipid A is responsible for its biological activity. When a bacterial infection
occurs in a host, LPS binding proteins (LBP) detect the presence of LPS and initiates signals
through the membrane or soluble CD14 which are present on immune cells such as monocytes andmyeloid cells as well as in the plasma. However, TLR4 and MD-2 have been shown to play a hugerole in LPS signaling and have direct interaction with LPS in a manner that is distinct from CD14
(Hoshino, 1999 and Akashi et al., 2003).
 7ROOOLNHUHFHSWRUDQGUROHVLQ9LUDOLQIHFWLRQThe presence of Resiquimod and imidazoquinoline compounds in the host activate immune cells
and prompt a response through Myd88-dependent signaling cascade which eventually leads to the
production of the transcription factor NF-κB (Akira and Hemimi, 2003). TLR7 has been shown torecognize the presence of Resiquimod and imidazoquinoline by acting as an adjuvant receptor tocontrol innate and adaptive immune responses (Miller et al., 1999). Over time, plasmacytoid DC
precursors have been implicated to produce IFN-α and IFN-β in response to viral infections but inrecent time, TLR7 and TLR8 ligands like R848 have been recognized as a more potent inducer of
IFN-α. Although R837 and R848 both illicit immune response from immune cells, it has been
discovered that R848 is about 100 times more potent than its counterpart (Ekman, 2011).
(QGRJHQRXV/LJDQGV
Endogenousmolecules are derived from host tissues and cells or cell components and they activate
TLR signaling and induce sterile inflammatory responses. Examples of endogenous ligands
7|P a geinclude fibronectin, heparan sulfate, biglycan, fibrinogen, oligosaccharides of hyaluronan, andhyaluronan fragments (Chiron et al., 2008; Rifkin et al., 2005). Likewise, heat shock proteins,
purified preparations from bacterial, and humans may serve as potent activators of the innate
immune system (Tsan and Gao, 2004). Usually, these endogenous ligands and their receptors are
localized in different cellular compartments and are unable to interact physiologically. In mostcases, endogenous ligands are released from injured or dying cells through a non-conventional
lysosomal route (Pollanen et al., 2009). Likewise, endogenous TLR ligands are also referred to as
alarmins and provide early warning calls to the innate and adaptive immune systems. The innate
immune system is not only activated by PAMPs but also by DAMPs released by injured cells andthey help the immune system to sense an ongoing infection and in recruiting more immune cells
to the predilection site (Seong and Matzinger, 2004; Medzhitov, 2008). More so, endogenous TLR
ligands may be involved in the pathogenesis of some autoimmune diseases (Marshak-Rothstein,2006). Overall, TLR-mediated sterile inflammation triggered by endogenous ligands is involvedin many pathological processes.
 3HOYLF,QIODPPDWRU\'LVHDVHDQGRWKHUGLVHDVHVFDXVHGE\%DFWHULDDQG9LUDOLQIHFWLRQVThe action of LPS and R848 on the reproductive ability of an organism is disruptive and result inaberrant developments in the structure and function of an organism’s reproductive system.
Noteworthy is a myriad of pelvic inflammatory diseases that plaque women and female livestock
across the world. These diseases occur when pathogenic organisms enter the vagina or cervix andascend to other structures of the upper female reproductive tract. A Pelvic inflammatory diseaserefers to a clinical syndrome of a broad range of infectious diseases that cause inflammation andaffect the upper genital tract of a female (Brunham et al., 2015). Some of such conditions caused
by PID include pelvic pain, follicular cyst, polycystic ovarian syndrome (PCOS), endometritis,
8|P a gesalpingitis, tubal-ovarian abscess, perihepatitis, reduced pregnancy rate, and most devastatingly
sub-infertility and outright infertility (Verweij et al., 2016 and Brunham et al., 2015). Other
diseases caused by bacterial and viral infections are brucellosis, leptospirosis, urethritis, andvaginitis as well as genital herpes, virus diarrhea (BVD), Bovine leukemia virus, and porcine
reproductive and respiratory syndrome respectively.
 )ROOLFXODUF\VW
Follicular cyst refers to the failure of one or multiple secondary follicles to ovulate. These cysts
are formed in most cases by the blockage of ovulation arising from an inadequate supply of
Luteinizing hormone during LH surge and due to excess production of follicle-stimulating
hormone (FSH) during ovulation. At its peak, LH prompts the expulsion of the oocyte or follicle
rupture but when this does not take place the follicle becomes a cyst.
3RO\F\VWLF2YDULDQF\VW
Polycystic Ovarian cyst is an endocrine disorder that affects women in their perimenopausal years.
It is characterized by clinical or biochemical hyperandrogenism, amenorrhea, and polycystic
ovaries. In young girls, it is marked by an increase in gonadotropin-releasing hormone (GnRH)
pulse frequency which leads to over-secretion and an increase in ovarian androgen production(Blank et al., 2006). As a result, ovarian follicles grow too early and antral follicle development isarrested leading to PCOS (Franks et al., 2000). Due to the insulin resistance and obesity that is
associated with PCOS, it is regarded not only as a reproductive condition but also metabolic
disease.

9|P a ge $LPRIWKLVVWXG\
It is known that bacteria or viruses that invade the body elicit an immune response from the host.
More so, LPS and ssRNA viral particles have been found in the uterus of cattle and testes ofhumans where they impact negatively on the normal functioning of the reproductive system
including reduction in the synthesis of steroids such as estrogen and progesterone. Apart from
disrupting follicle growth and maturation, they also cause an increase in the level of chemokine
and cytokine, an occurrence that can be attributed to the inflammatory response posed by the host's
innate immune system. Since these pathogenic components of the cell membrane can penetrate
through the external and internal barriers of the ovary to reach the granulosa cells, any disruption
in the functioning of GC’s will affect cumulus expansion, oocyte maturation, and oocyte releaseduring ovulation. In addition, toll-like receptors 4, 7, and 8 prompt an inflammatory response fromthe host on recognizing bacteria components (LPS) and viral pathogens or agonists (Resiquimod)
respectively, leading to a cascade of reactions including the recruitment of immune cells andhormonal imbalances that affect the functioning of the cells at the predilection site. However, thedirect mechanism through which bacterial infections cause a reduction in steroid secretion andovulation of oocytes remains unclear. Likewise, there is no report on the expression of SARSCOV-2, ACE2 receptor or TLR7/8 agonist (R848) on granulosa cells and the impact on ovarianfunctioning as well as the mechanism by which it causes ovarian dysfunction.
Therefore, this study was undertaken to understand the mechanism by which LPS and ssRNA
viruses cause low reproductive performance of the female reproductive system. Likewise, for the
first time detecting the expression of ACE2 and TLR 7/8 on the granulosa cells of mouse ovary
and the mechanism by which ssRNA agonist, Resiquimod (R848) disrupt normal ovarian function.
For this, two (2) individual experiments were conducted considering the precise hypothesis.
10 | P a g e

In Experiment 1, it was hypothesized that the disruptive action of LPS on follicle development,
ovulation, and overall ovarian health is a result of the epigenetic regulatory action of LPS which
results in the methylation of DNA at the Lhcgr and Cyp19a1 gene promoter region. To clear the
hypothesis, the impact of LPS on follicle development and ovulation was studied both in vivo andin vitro when required. More so, the expression of Dnmt1 was studied both in vitro and in vivo, as
well DNA methylation analysis of the Lhcgr and Cyp19a1 gene promoter region in mice treatedwith LPS was compared with that of non-treated mice to understand the epigenetic regulatory
action of LPS.
In Experiment 2, it was hypothesized that TLR7 and 8 are expressed in the granulosa cells ofmouse ovary and R848 prompts a disruption in the normal functioning of the female reproductive
health. To clear this hypothesis, TLR7 and 8 expression level in the mRNA and protein extractsof granulosa cells of mouse ovary was examined. TLR7 immunostaining was carried out to detect
if TLR7 positive cells can be detected in the ovary of mice. Likewise, R848 a ligand of TLR7 and
8 was injected into mice and ovarian morphology and fertility parameters were studied using
super-ovulated mice.

11 | P a g e)LJXUH  Structure of lipopolysaccharide (LPS). LPS is found on the cell wall of gramnegative bacteria, such as Escherichia coli. The lipid A region, depicted in red, elicits theimmune response.



12 | P a g e([SHULPHQW
6WXG\RQWKHHIIHFWRI/36RQRYHUDOORYDULDQKHDOWKDQG(SLJHQHWLFG\QDPLFVRIWKH Lhcgr DQGCyp19a1JHQHSURPRWHUUHJLRQ&RQWHQWV

7RSLFV

3DJH1R

2.1

Introduction

13

2.2

Materials and Methods

16

2.3

Results

26

2.4

Discussion

29

Abstract33,QWURGXFWLRQInfections of the upper female genital tract result in pelvic inflammatory disease (PID), whichcauses infertility (Ross, 2002). Gram-negative bacteria, among other factors that can cause PID,
have been a menace in countries around the globe (Deb et al., 2004). According to the National
Health and Nutrition Examination Survey (NHANES) 2013-2014 cycle, the number of womenwith a reported lifetime history of PID is estimated to be approximately 5 million, representing
4.4% of sexually experienced women between 18 and 44 years old (Kreisel et al., 2017). Likewise,
Bos taurus is not exempted from bacterial infection of the genital tract, resulting in low
reproductive performance, abnormal estrus cycle, follicular cysts, and low conception rates(Sheldon and Dobson, 2004).
Gram-negative bacteria possess an outer protective cell wall that harbors several pathogenassociated molecular patterns (PAMPs) that trigger inflammation and cause other toxic effects
when they infect a host or are lysed by antibiotics (Tuomanen et al., 1985a,b and Rietschel et al.,1994). A notable example is the outer membrane component of Escherichia coli,
lipopolysaccharide (LPS), which has been found and isolated from the genital tract of cows
suffering from uterine damage, ovarian dysfunction, and infertility (Sheldon et al. 2002; Ross,
2002 and Williams, 2007). When released into circulation, LPS is recognized by its receptor, atransmembrane protein Toll-like receptor (TLR4), which is mainly expressed in immune cells suchas neutrophils and macrophages. LPS is also recognized by other coreceptors such as cluster ofdifferentiation 14 (CD14) and myeloid differentiation factor 2 (MD2) (Akira et al., 2006;
Bromfield and Sheldon, 2011). The binding of LPS to its receptor (TLR4) then triggers theinduction of NF-κB-dependent proinflammatory cytokines such as tumor necrosis factor-α (TNFα) and prostaglandins (Medzhitov and Kagan, 2006). The production of a type I interferon response
13 | P a g e

by the host is regulated by Toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing
interferon beta (TRIF) as well as TRIF-related adaptor molecule (TRAM), which are thedownstream pathways of TLR4 (Kawai et al. 1999; Yamamoto et al., 2003 a,b). TLR4 is also
expressed in ovarian granulosa cells and cumulus cells. Endogenous ligands such as hyaluronic
acid activate TLR4 in these cells during the ovulation process, which plays a substantial role inachieving successful fertilization (Shimada et al., 2006, 2008). Additionally, E. coli infection and
LPS have been observed to perturb ovarian follicle growth and to interfere with granulosa cell
estradiol production and secretion in vivo (Erin et al., 2008).
FSH acts on granulosa cells, and LH stimulates theca cells to induce follicular development with
granulosa cell proliferation and expansion of small follicles into larger follicles. In large antral
follicles, the gene expression pattern changes in a manner that allows granulosa cells to acquire
responsive ability to ovulation stimuli. The transient induction of LH acts on the LH receptor
encoded by Lhcgr which is expressed in granulosa cells to ensure oocyte maturation and successful
ovulation (Barros et al., 2010). The mechanism behind Lhcgr expression is a complex process inwhich β-catenin is reported to activate SF1 and T-cell factor (TCF) 3, which is required for FSH
to induce the expression of Lhcgr mRNA (Law et al., 2013, and Hunzicker-Dunn et al., 2012).
Interestingly, an in vitro promoter assay study showed that FSH directly upregulates Lhcgr
promoter activity; however, the mRNA level of Lhcgr is only induced in granulosa cells after morethan 24 hrs under both in vivo and in vitro conditions (Kawai et al., 2012 and Selig et al., 2013).
The late response is explained by epigenetic regulation; in the granulosa cells of small follicles, ahigh rate of DNA methylation is observed in the Lhcgr promoter region, which changes to ademethylated state in larger follicles following the injection of equine chorionic gonadotropin(eCG) (Kawai et al., 2018). However, bacterial infection or LPS, a component of the cell
14 | P a g emembrane is capable of inducing changes in histone modifications in cells and altering the hosttranscriptional framework (Hamon and Cossart, 2008). The lack of binding between thetranscriptional factor and the CpG island is due to methylation patterns because when DNA
methylation occurs it inhibits the binding of transcriptional factors. At the level of embryonic cell
division, de novo methyltransferases DNMT3A and DNMT3B, as well as DNMT1, regulates andmaintains the methylation and demethylation of genes which explains the silence and expressionof some genes at a specific stage of development (Lock et al., 1987, Mutskov and Felsenfeld, 2004
and Okano et al., 1999). While some studies have shown that LPS disrupt steroid production andovarian function, there is little information about the mechanism of this action. However, LPS has
been discovered to influence some epigenetic regulator genes (Matt et al., 2018). Therefore, wehypothesized that the low reproductive performance observed during bacterial infection is as aresult of changes in the DNA methylation status of the Lhcgr and Cyp19a1 promoter region. To
this end, we investigated the effect of bacterial infection on ovulation as well as the expression ofgenes involved in DNA methylation.






15 | P a g e0DWHULDOVDQGPHWKRGV0DWHULDOVEquine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) were obtained
from Asuka Seiyaku (Tokyo, Japan). Dulbecco’s modified Eagle medium (DMEM)/F12 medium
and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA), and fetal calf serum(FCS) was obtained from Life Technologies (Grand Island, NY). Oligonucleotide poly-(dT) was
obtained from Invitrogen, and avian myeloma virus (AMV) reverse transcription was obtained
from Promega Corporation (Madison, WI). Lipopolysaccharide (LPS) (O111: B4 E. coli strain)
was obtained from Sigma-Aldrich (St. Louis, MO). Additionally, QIAamp DNA Blood Mini, Gel
extraction and EpiTect Bisulfite Kits were purchased from Qiagen (Germantown, USA). Routinechemicals and reagent were purchased from Sigma Chemical Co. (St. Louis, MO) and Nakalai
Chemical Co. (Osaka, Japan).
$QLPDOVImmature 3-week-old female C57BL/6 mice were obtained from Charles River Laboratories Japan(Yokohama, Japan). The animals were housed at the Experimental Animal Center, Hiroshima
University, under a 12-hr light/12-hr dark schedule with food and water provided ad libitum. The
animals were cared for in adherence to the National Institutes of Health Guide for the Care andUse of Laboratory Animals, as approved by Hiroshima University’s Animal Care and Use
Committee (#C18-34).


16 | P a g e7UHDWPHQWRIPLFHZLWK/36
Twenty-five-day-old female mice were injected intraperitoneal (IP) with 4 IU of eCG to stimulatefollicular growth. Thereafter, they were injected with 5 IU of hCG at 48 hr to stimulate ovulationand subsequent luteinization. LPS was injected intraperitoneal into mice at a dosage of 1 Pg/kg
BW at 23 days and ovaries were collected at 25 days. Also, LPS was injected intraperitoneal tomice at a dosage of 1 Pg/kg BW when animals were 25 days old. At 16 hr after hCG injection
following 48 hr after eCG injection, ovulated COCs were collected from the oviducts of three mice.
&ROOHFWLRQRIRYXODWHGRRF\WHDQGLQYLWURIHUWLOL]DWLRQTwenty-five-day-old female mice were injected intraperitoneal (IP) with 4 IU of eCG to stimulatefollicular growth. Thereafter, they were injected with 5 IU of hCG at 48 hr to stimulate ovulationand subsequent luteinization. LPS was injected intraperitoneal into mice at a dosage of 1 Pg/kg
BW at 23 days and ovaries were collected at 25 days. Also, LPS was injected IP to mice at a dosageof 1Pg/kg BW when animals were 25 days old. At 16 hr after hCG injection following 48 hr aftereCG injection, ovulated cumulus-oocyte-complexes (COCs) were collected from the oviducts ofthree mice that were euthanized and placed in 50 Pl of human tubal fluid (HTF) medium. One hr
before the collection of the COCs, spermatozoa was collected from the cauda epididymis of an
adult male mouse and placed in 500 Pl of HTF medium to allow for capacitation in an incubator
set at a temperature of 37oC, an atmosphere of 5% CO2 at maximum humidity. This was done
before the sperm was introduced to the HTF medium containing oocyte at a final concentration of
1,000 sperm cells per Pl. Thereafter, the sperm-oocyte-containing medium was incubated at 37oC
in an atmosphere of 5% CO2 at maximum humidity for 8 hr for fertilization to occur. After washingin HTF medium the oocytes are transferred to KSOM media and placed in the incubator at 37oC
17 | P a g e

in an atmosphere of 5% CO2 at maximum humidity for further development. The number ofovulated oocytes was scored.
3UHSDUDWLRQRISDUDIILQEORFNDQGRYDULDQVHFWLRQVWDLQLQJTo prepare paraffin block for ovarian sectioning, the ovaries collected from three mice were fixed
in 4% (w/v) paraformaldehyde (PFA) solution (Nacalai Chemical Co) overnight at 4°C, which was
followed by dehydration and embedding in paraffin. The dehydration process involved step-bystep incubation in 70, 80, 90, 95, and 100% ethanol solution for 2 hr each, and the dehydrated
ovaries were then incubated twice in xylene with each incubation period lasting for 2 hrs. Lastly,
the ovaries were incubated for 2 hr in paraffin liquid before they were appended in paraffin, andblocks were made by solidifying the liquid paraffin containing the ovaries. Thereafter, 5 μm thicksections were cut from the paraffin block by microtome and placed in water warmed up to 42 oC
before they were mounted on slides and air-dried. Thereafter, the slide containing ovaries was
deparaffinized by incubating in xylene twice for a period lasting 2 mins. The ovarian sections on
the glass slide were then rehydrated using ethanol following stepwise incubation in 100, 90, 80,
and 70% ethanol for a period lasting 1 to 2 mins each. Thereafter, the sections were washed in
distilled water and stained with eosin for 1 min followed by washing under running tap water andcounterstaining with hematoxylin for 30 secs followed by washing under running tap water. The
stained sections in the slides were then dehydrated by incubating step-wisely in 100, 90, 80, and
70% ethanol followed by incubating in xylene twice for a period lasting 2 mins. Thereafter, theovarian sections were covered by a coverslip using the multi-mount DPX solution. The slides were
airdried and visualized by an optical microscope using BZ-II application software (Keyence,
Tokyo, Japan).

18 | P a g e781(/DSRSWRVLVGHWHFWLRQ
Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay was carried out with theaid of In-situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany) as described in the
manufacturer’s protocol to detect follicular atresia and apoptosis. Deparaffinization andrehydration of the tissue sections were carried out by washing twice in xylene for 2 mins on bothoccasions followed by a chronological incubation in gradient concentration ethanol of (100, 95,
90, 80, and 70%) for 2 mins at each concentration. Thereafter, the slides were rinsed in PBS for 5
mins and the tissue sections were incubated in Proteinase K solution containing 2 μl of 50x
Proteinase K in 98 μl PBS buffer. Thereafter, the glass slide was washed twice in PBS buffer for
5 minutes each time and incubated with a blocking solution containing 3% (v/v) hydrogen peroxide
in methanol for 10minutes at a temperature of 15 to 25oC. Thereafter, the glass slide was washedtwice in PBS buffer for 5 mins each time and the area around the tissue was kept dry. After that,
50 μl of TUNEL reaction mixture was added to the tissue sections and incubated at 37 oC for 1 hrunder wet conditions devoid of light. The glass slide containing tissue sections serving as anegative control was treated with 50 μl of TUNEL label solution only and kept at the same
condition as described earlier. Also, the glass slide containing tissue sections serving as a positive
control was treated with DNase 1 recombinant grade I (50 IU/ml in 50 mM Tris-HCL, pH 7.5, 10
mMMgCl2 1 mg/ml BSA) for 10 mins at room temperature to induce DNA strand damage before
labeling procedures. After incubation, the glass slides were rinsed in PBS twice for 5 mins on bothoccasions followed by incubation with a secondary antibody for 1hr 30minutes at 37oC under wet
conditions devoid of light. Thereafter, the glass slides were rinsed in PBS in preparation for
immunohistochemistry. For this process, 50 to 100 μl of DAB substrate (DAB substrate comprises
5 μl of 20x DAB buffer and 20 μl 30% hydrogen peroxide) was added to the tissue sections and

19 | P a g e

incubated for 20 secs. The glass slides were transferred to water to stop the reaction and in
preparation for counterstaining with hematoxylin and the stained sections were then dehydrated
by incubating step-wisely in 100, 90, 80, and 70% ethanol followed by incubating in xylene twice
for a period lasting 2 mins. Thereafter, the ovarian sections were covered by a coverslip using themulti-mount DPX solution. The slides were airdried and visualized by an optical microscope using
BZ-II application software (Keyence, Tokyo, Japan).
51$H[WUDFWLRQDQGUHYHUVHWUDQVFULSWLRQ
Total RNA was obtained from the granulosa cells of mouse ovaries or cultured GCs using an
RNAeasy mini kit (Qiagen, Germantown, MD, USA) based on the manufacturer’s instructions.
For RNA extraction, the cultured or collected granulosa cells were placed in a tube and subjected
to homogenization and lysed using cell lysate solution containing RLT Buffer and 2mercaptoethanol. Thereafter, 70% ethanol was added to the cell lysate mixture and mixed properly
by pipetting. After that, the entire mixture was transferred to a Rneasy Mini spin column andcentrifuged at a speed of 8000 × g, washed with RW1 buffer followed by DNase (DNase stock 5
Pl + 35 Pl) treatment for 15 mins at room temperature to disintegrate the DNA in the cells.
Thereafter, repeated washing with RW1 and RPE was done, and RNA was extracted from thecolumn using RNA-free water. The concentration of RNA was calculated at an optical density of
260 nm with the assumption that an OD260 unit is equivalent to 40 ng/ml RNA. The purity of the
RNA extract was determined by measuring the absorbance ratio at 260/280 nm. Total RNA wasthen reverse-transcribed using 500 ng poly-Dt (deoxythymidine) and 0.25 IU of avian
myeloblastosis virus reverse transcriptase (AMVRT) (Promega) at 42 oC for 75 min and 95oC for
5 mins. The reverse transcriptase (RT) reaction mixture consisted of 7.25 Pl of 50 ng mRNA +RNase free water, 0.5Pl oligo DT (Primers of mRNA with poly A tail), 0.8 Pl dNTP (Base, ATGC),
20 | P a g e

7.2 Pl RNase free water, 4Pl RT Buffer and 0.25Pl RT Enzyme. cDNA products collected wereresolved on 2% (w/v) concentration of agarose gel.
5HDO7LPH3&5
Complementary DNA (cDNA) and primers of interest were added to Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA). Thereafter, PCR was carried out with the aidof a StepOne real-time PCR system (Applied Biosystems) using the following parameters: 10
minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C to 64°C. The
real-time PCR mixture includes 7.5 Pl Power SYBR Green PCR Master Mix, 0.5 + 0.5 Pl forward
and reverse primer respectively, 3.5 Pl ddH2O and 3.0Pl of cDNA sample. The primer sets usedin this study are shown in Table 1. In the real-time PCR, L19 served as a control for variations inconcentrations of mRNA and to ascertain reaction efficiency.
&ROOHFWLRQDQG in vitroFXOWXUHRIJUDQXORVDFHOOVSix hrs after injecting immature mice (23 days old) with eCG, granulosa cells were collected fromovaries as described by Kawai et al. (2018). The cells were cultured in DMEM/F12 medium
containing 1% (v/v) FCS in 5% (v/v) FCS-coated 96-well plates for 24 hrs at 37°C in an
atmosphere of 5% CO2 at maximum humidity. The medium described above was supplemented asfollows: (a) no supplement (control), (b) 100 ng/mL FSH (National Institute of Diabetes andDigestive and Kidney Diseases, Torrance, CA), and 10 ng/ml testosterone (T), (c) FSH, T and 0.1
μg/ml LPS, and (d) FSH, T and 1 μg/ml LPS.

21 | P a g e,PPXQRKLVWRFKHPLVWU\
5 μm thick ovarian sections mounted on glass slides were deparaffinized and rehydration of thetissue sections was carried out by washing twice in xylene for 2 mins on both occasions followed
by a chronological incubation in gradient concentration ethanol of (100, 95, 90, 80, and 70%) for
2 mins at each concentration followed by washing in PBS. Thereafter, the ovarian sections
were incubated in 2% hydrogen peroxide and 80% methanol solution for 15 min to blockendogenous peroxidase activity. Likewise, to prevent nonspecific binding the sections wereincubated in 5% normal goat serum for 1 hr at room temperature.
Subsequently,

the ovarian

sections

weretreated overnightat

4oC with mouseanti-

TLR4 monoclonal antibody (ab22048, Abcam USA) as the primary antibody at a dilution ratioof 1:500 in PBS containing 5% (w/v) BSA. On the following day, ovarian sections were washedwith PBS including 0.3% (v/v) Triton X-100 followed by incubation with horse anti-rabbit IgG(1:200) (Sigma) as the secondary antibody. Thereafter, the binding of the primary antibody wasvisualizedusingdiaminobenzidine (DAB)

for

5

to

7 mins and ovarian sections

were counterstained with hematoxylin, dehydrated, and mounted with DPX. The TLR4 labeling
was observed under an optical microscope using BZ-II application software (Keyence, Tokyo,
Japan).
,PPXQRIOXRUHVFHQFHVWDLQLQJ
For immunofluorescence staining of ovarian sections using anti- Ki67 antibody (ab15580, Abcam
USA), 5μm thick ovarian sections were deparaffinized using xylene followed by washing in PBS.
Thereafter, the ovarian sections mounted on glass slides were deparaffinized and rehydration of
the tissue sections was carried out by washing twice in xylene for 2 mins on both occasions. This22 | P a g ewas followed by a chronological incubation in gradient concentration ethanol of (100, 95, 90, 80,
and 70%) for 2 mins at each concentration, washed in PBS and boiled for 20 min in 10% citricacid (pH 6.0) for antigen activation. Thereafter, the sections were washed in PBS and nonspecificbinding was blocked using 5% (w/v) BSA in MOM Immunodetection Kit or PBS (Vector
Laboratories Inc, CA, USA). Furthermore, the ovarian sections were treated with Ki67 antibody(Abcam) with a dilution ratio of 1:200 as primary antibodies in 1% BSA (v/v) blocking solution
at 4°C overnight. On the next day, the ovarian sections were washed in PBS containing 0.3% (v/v)
Triton X-100, the staining was visualized with Cy3-conjugated goat anti-rabbit IgG (1:200)(Sigma) as a secondary antibody and counterstained using 4,6-diamidino-2-phenylindole (DAPI,
Vector Laboratories Inc).
0RUSKRORJLFDOFODVVLILFDWLRQRIIROOLFOHVThe classification of follicles was performed as described by Umehera et al. (2017). 5 μm thickovarian sections were cut from Paraffin‐embedded blocks at intervals of 30 μm and mounted
on glass slides, deparaffinized, rehydrated using ethanol followed by incubation in xylene andstaining using hematoxylin-eosin to visualize the nuclei and cytoplasm was performed as describedearlier for histological examination by light microscopy. The number of primary, secondary, andantral follicles in 12 sections per ovary was counted. Follicles with oocytes surrounded by a single
layer of cuboidal granulosa cells were categorized as primary follicles, follicles with oocytes
surrounded by two or more layers of granulosa cells with no visible antrum as secondary follicles
while follicles with antrum within the granulosa cell layers enclosing the oocyte were counted
as antral follicles. The analysis was carried out with an optical microscope using BZ-II application
software (Keyence, Tokyo, Japan).

23 | P a g e*HQRPLF'1$H[WUDFWLRQGenomic DNA was obtained from mouse granulosa cells utilizing the QIAamp DNA Blood Mini
Kit (Qiagen, Germantown, USA) based on the manufacturer’s instructions. Granulosa cells
collected from mouse ovaries were homogenized and treated step wisely with proteinase K andbuffer AL. Thereafter, ethanol was added to the mixture and mixed properly by vortexing. The
mixture was then transferred into a DNeasy Mini spin column and centrifuged at ≥ 6000 × g for
60 seconds and the flow-through in the 2 ml collection tube was discarded. Buffer AW1 was added
to the column followed by centrifugation at ≥ 6000 × g for 1 minute and buffer and buffer AW2
was added followed by centrifugation at 20,000 × g for 3 minutes and the flow-through in the 2ml
collection tube was discarded on both occasions. The DNA in the column was eluted by adding
buffer AE to the column followed by centrifugation at 20,000 × g. The concentration of the DNA
was calculated from the optical density at 260 nm with the assumption that the OD260 unit isequivalent to 50 ng/ml DNA. The purity of the DNA was estimated by measuring the absorbanceratio at 260/280nm.
%LVXOILWHVHTXHQFHDVVD\Genomic DNA (1 mg) and 0.3 M NaOH (Nacalai) were added to the sodium bisulfite reaction mix
from the EpiTect Bisulfite Kit (Qiagen), and then the mix was placed in a heating block set at 37°C
for 20 minutes. Bisulfite DNA conversion of samples was carried out using the following
parameters: 5 minutes at 95°C, 25 minutes at 60°C, 5 minutes at 95°C, 85 minutes at 60°C, 5
minutes at 95°C, 175 minutes at 60°C, 5 minutes at 95°C, and 120 minutes at 60°C. Thereafter,
purification of bisulfite-converted DNA was performed as described by Kawai et al. (2018) using
Lhcgr

and

Cyp19a1(MeDip)primersets

asfollows;

forward,5′-

24 | P a g e

GTGAGAGGGGAGGGTTGGAG-3′,
CACCA-3′;

Forward-andreverse,5′-TTCAAGACCAACATTACCAAGGATTATTTGGATAATTATTATGGTTTT

andReverse-TAAAAATAAAATCCAATCTTCC, respectively.
*HOH[WUDFWLRQDQG*HQH&ORQLQJThe PCR band containing the PCR product of the target gene was excised and extracted using Gel
Extraction Kit (Qiagen, USA) according to manufacturer instructions. The extract was cloned into
TOPO TA cloning vector (Invitrogen) and incubated at room temperature for 1 hr. Thereafter, theDNA and vector mixture was transferred to E.coli competent cells and incubated in ice for 30 mins
at room temperature. For heat shock transformation of competent cells, the mixture was placed ina water bath at 42oC for 40 secs and transferred to ice. In the recovery phase, SOC media wastransferred into the mixture and placed in an incubator with a shaker for 1hr. This was followedby plating on Petri dishes containing LB agar with ampicillin and sprayed with X-gal. The petri
dish was then incubated at 37oC for 16 to 24 hr. Thereafter, white colonies were picked up andtransferred individually to LB medium and placed in an incubator with a shaker at 37 oC for 24hr
for amplification of colonies. After that, the cloudy LB medium was collected and purified using
Qiagen Spin Miniprep Kit (Qiagen, USA) according to manufacturer instructions Then EcoR1
enzyme and 10xH buffer were added to the extract and incubated at 37oC for 1hr and the sample
was loaded in 2% agarose gel to confirm the presence of the target gene. Thereafter, the extract isprepared for the sequence analysis.
'1$VHTXHQFHDQDO\VLVThe sequence analysis was performed using BigDye Terminator version 3.1/1.1 Cycle Sequencing
Kit (Applied Biosystems). Sequence analysis was performed on more than five colonies for each
25 | P a g e

treatment. DNA nucleotide sequence was determined using the 3130/3130xl Genetic Analyzer(Applied Biosystems).
6WDWLVWLFDODQDO\VLV
All data from three replicates were analyzed by either a Student’s t-test or a one-way analysis ofvariance followed by a Tukey post hoc test (StatView; Abacus Concepts, Berkeley, CA). The
percentage of methylation of CpG sites in the LHCGR promoter region was analyzed by the
Quantification tool for Methylation Analysis (http://quma.cdb.riken.jp/). ). In some studies, dataobtained from three different cultures were analyzed by two‐way ANOVA (Statview; Abacus
Concepts, Inc.). All values are presented as the mean ± SEM. Treatments were consideredsignificantly different from one another at P < 0.05.
5HVXOWV7/5H[SUHVVLRQDQGHIIHFWRI/36RQRYDULDQPRUSKRORJ\DQGKHDOWK
To investigate the induction of TLR4 at different time points, we collected granulosa cells fromthe ovary of mice at 15 and 23 days as well as at 24 and 48 hr after eCG injection. There was asignificant difference in the expression of TLR4 at 24 and 48 hr after eCG (Figure 2A), suggestingthat TLR4 is highly expressed in larger follicles compared to smaller follicles found in immaturemice. To determine the effect of endotoxin exposure on ovarian morphology, we first collectedovaries from mice treated with and without LPS before eCG injection; and mice injected with
eCG- and/or LPS and carried out hematoxylin and eosin staining. Likewise, compact granulosa
cells were observed in antral follicles of both the control and LPS-treated groups, and there wasno damage to the follicles (Figure 3A). There was a significant difference between the secondary
follicles, while there was no significant difference in the number of antral follicles in the control
26 | P a g eand 1 μg/kg dosage LPS group (Figure 3B). However, in ovaries collected from mice before eCG
injection, there was no significant difference in the number of follicles at each developmental stage(Figure 3A). To evaluate follicular atresia, immunohistochemistry-based TUNEL assay was
carried out, and the number of observed atretic follicles in ovaries from mice treated with LPS
and/or eCG injection were similar (Figure 4A).
 (IIHFWRI/36RQRYDULDQPRUSKRORJ\GXULQJWKHRYXODWRU\SKDVH
When mice were injected with eCG and treated with hCG 48 hrs later, a higher number of corpus
lutea was observed in the control group than in the LPS-treated group (Figure 5A). The total
number of ovulated COCs in the oviduct of eCG-primed mice and the LPS-treated group (0.1 and1 μg/kg) was significantly lower than the control group that was injected eCG without LPS (Figure
5B). In addition to preventing the release of preovulatory follicles during ovulation as shown in
Figure 5A, LPS injection also results in a lower proportion of matured oocyte compared to thecontrol group suggesting that oocyte maturation and development was disrupted by endotoxin
action (Figure 5C).
7KHHIIHFWRI/36RQWKHLQGXFWLRQRI(*)OLNHIDFWRUVSURJHVWHURQHUHFHSWRU 3*5 
GHSHQGHQWHYHQWVDQGRWKHUWDUJHWVRIWKH/+/+UHFHSWRU /+5 GHSHQGHQWSDWKZD\
To analyze the induction of Ptgs2, Nrg1, Cyp11a1 and Areg, Snap25 and Star mRNA in the
granulosa cells of the ovary, granulosa cells were isolated from ovaries of eCG-primed mice at
48hr; 4 and 8hr after hCG injection (Figure 7). The expression of Ptgs2 and Star were induced
within 4hr after hCG. In the LPS treated groups Ptgs2, Star, and Cyp11a1 were significantly
reduced suggesting the role of LPS in regulating EGF-like factors, Pgr, and other LH-target genes
involved in the ovulation process.
27 | P a g e(IIHFWRI/36RQFHOOSUROLIHUDWLRQDQGGHYHORSPHQWLQWKHPRXVHRYDU\The outcome of anti-Ki67 antibody immunoflourence staining in eCG-primed and LPS-treatedmouse is shown in Figure 4B. The Ki67 positive cells were similar in both groups. We investigated
the responsive ability of large and small follicles to TLR4 and observed positive TLR4 cells in
antral follicles while TLR4 positive cells were undetected in smaller follicles of immature mice
(Figure 2B).
(IIHFWRI/36RQWKHH[SUHVVLRQRIJHQHVLQJUDQXORVDFHOOVTo explore the effect of LPS on the expression of genes associated with follicle development invivo, Lhcgr, Cyp19a1, Ccnd2, and Fshr, expression was significantly lower in granulosa cells(Figure 6A) from mouse ovaries in the LPS-treated groups than that in the control group. However,compared with the control group, there was no significant difference in the expression of Inhibin,
Ccnd2, and Fshr (Figure 6A). In the in vitro data, Lhcgr and Cyp19a1 expression in granulosa
cells cultured with FSH+LPS were significantly lower than that of the control group (FSH alone),and there was no significant difference in the expression of Ccnd2 (Figure 6B).
7KHHIIHFWRI/36RQWKHLQGXFWLRQRIFKHPRNLQHVDQGF\WRNLQHVTo analyze the induction of Il6 and Ccl5 mRNA in the granulosa cells of the ovary, granulosa cells
were isolated from ovaries of eCG-primed mice at 48 hr, 4 and 8 hr after hCG injection (Fig 8).
LPS stimulated the production of chemokines and cytokines such as Ccl5 and Il6 (Figure 8). In
the LPS treated group there was a significant increase in the expression of Il6 and Ccl5 at the 4hr
time point after hCG compared to the control group.

28 | P a g e(IIHFWRI/36DFWLRQRQWKHH[SUHVVLRQRIDNA (cytosine-5)-methyltransferase 1in vivo
DQGin vitro
A significant reduction of DNA (cytosine-5)-methyltransferase 1 Dnmt1) was observed in
granulosa cells of mice injected with eCG. The reduction was not observed in granulosa cells of
LPS-treated mice, where the level of expression was similar to that of mice before eCG injection(Figure 9A). In vitro study also revealed that Dnmt1 was significantly higher when granulosa cells
were cultured in a medium containing 1 μg/ml LPS and FSH compared to the medium containing
FSH (Figure 9B).
 (SLJHQHWLF G\QDPLFV RI /36 RQ '1$ PHWK\ODWLRQ DW WKH Lhcgr DQG Cyp19a1 JHQH
SURPRWHUUHJLRQThe injection of eCG to immature mice at a 48hr time point significantly resulted in thedemethylation of the Lhcgr and Cyp19a1 promoter region. Following the coinjection IP of eCG
and LPS at the same time point, the decrease in the rate of methylation was not detected. Also,
there was no significant difference observed in the methylation rate between the eCG and LPS
coinjected group at 48 hr and the immature group (Fig 10A and B).
 'LVFXVVLRQ
An acute increase in LPS in the circulatory system causes serious systemic inflammation, which
is known as sepsis, and sepsis triggers the release of proinflammatory cytokines. A chronic highlevel increase in LPS also causes chronic inflammatory diseases such as inflammatory bowel
disease and autoimmune and cardiovascular diseases (Suffredini et al., 1989). LPS injection did
not induce any negative changes to the phenotype of the ovary or morphology at each stage of theovarian follicle. During the follicular development phase, the growth in diameter of small follicles
29 | P a g e

developing into antral follicles increases rapidly due to FSH stimulation (Matzuk and Lamb, 2002).
It has also been reported that granulosa cell proliferation results in changes in the function of
granulosa cells of the preovulatory follicle (Cuiling et al., 2005). To this end, normal
differentiation of granulosa cells was determined by incremental changes in the expression patternsof genes such as Lhcgr and Cyp19a1. However, following LPS treatment, our data demonstrated
that the induction of Lhcgr and Cyp19a1 was not observed. Granulosa cells that express a low
level of Lhcgr do not acquire the ability to respond to ovulation stimulus, the endogenous LH surge,
or exogenous hCG, which are required to induce ovulation (Kawai et al., 2016). In fact, the numberof ovulated oocytes was significantly lower in LPS-treated mice than in control mice that were
injected superovulation treatment. This condition mimics the follicular cyst, which is marked by anormal growth process from the primordial to the preovulatory follicle stage and is followed by
the inability to release oocytes during the ovulatory phase (Garverick, 1997). This suggests thatbacterial infection can cause follicular cyst in the ovary. While some studies have shown that
endotoxin suppresses ovulation and reproductive performance (Erin et al., 2008 and Suzuki et al.,2001), there is little information about the mechanism that influences this occurrence. In contrast
with smaller follicles, preovulatory follicles were highly impacted by the LPS-TLR4 pathway
because the expression of TLR4 was significantly induced in granulosa cells at 24 and 48 hr aftereCG injection.
While the expression of Lhcgr and Cyp19a1 was suppressed by LPS, Ccnd2 and Fshr levels werenormal in granulosa cells, suggesting that selective activation of transcription factors occurred.
Although these genes are induced by the PKA-dependent signal transduction pathway, their target
transcription factors are different. Ccnd2 transcription is directly regulated by CREB, while Lhcgr
and Cyp19a1 are activated by Sp1 and Sf-1 respectively. Moreover, it is known that the expression

30 | P a g e

of Sp1 is ubiquitous (Li et al., 2004), suggesting that the downregulation of Lhcgr and Cyp19a1
expression might be caused by the inability of Sp1 and Sf1 to access and bind to promoter
sequences in both promoter regions. Our previous study reported that Lhcgr expression is highly
associated with the demethylation of its promoter region in the granulosa cells of mouse ovaries(Kawai et al., 2018). During embryonic cell division, the de novo methyltransferases DNMT3A
and DNMT3B initiate CpG island methylation at regions of silent chromatin, and the replicationof methylation patterns is maintained by DNMT1 and by other features within the DNA domain(Okano et al., 1999). Strikingly, the expression level of Dnmt1 was significantly decreased in
granulosa cells from mouse ovaries injected eCG in vivo and those cultured with FSH in vitro.
Likewise, the report that LPS influences epigenetic regulation during neuroinflammation in mice
(Matt et al., 2018) aligns with the result of our study, which shows that the coinjection of eCG and
LPS and the inclusion of LPS in the culture medium upregulate Dnmt1. In concert with the data,
the rate of DNA methylation in the Lhcgr promoter region was also affected by FSH and/or LPS.
This suggests that in addition to the LPS-induced transient activation of inflammatory responses
in both immune cells and granulosa cells, LPS is also capable of causing a long-lasting impact on
gene expression patterns due to changes in DNA methylation status. This abnormal epigenetic
regulation by LPS in granulosa cells causes follicular cysts, which dramatically decrease female
fertility.
In conclusion, the present study demonstrated that the decrease in the induction of Lhcgr andCyp19a1 due to LPS is a result of the epigenetic regulatory action of LPS. The immunologicalresponse of the host to LPS or bacterial infection prevents the preovulatory follicles from
responding to the LH surge, which is required for ovulation. Moreover, ovarian dysfunction and

31 | P a g epolycystic ovaries, as well as other PIDs that are characterized by bacterial infection in humans
and animals, are closely connected to the methylation of the Lhcgr promoter region.

32 | P a g e

$EVWUDFW
Lipopolysaccharide (LPS) is an endotoxin released by gram-negative bacteria, and it can passthrough the protective physical barriers and tight junctions of the basement membrane. LPS has
also been isolated from the genital tract of animals suffering from uterine damage and ovariandysfunction. This study provides direct molecular evidence about the mechanism through which
endotoxins cause reproductive disorders, such as follicular cysts and ovarian dysfunction.
Granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG and
LPS injection intraperitoneal. Normal large antral follicles were observed in ovaries obtained fromeCG and LPS coinjected mice, and the morphology of the ovaries was similar to that observed in
the control group (eCG-injected mice). These antral follicles were not deemed atretic because few
TUNEL-positive cells were observed. However, the granulosa cells of large antral follicles did notacquire the ability to respond to hCG stimulation. The number of ovulated oocytes was
significantly lower in LPS-injected mice after superovulation compared to mice that were notexposed to LPS. The low reactivity was caused by the limited expression of the Lhcgr, which
encodes the LH receptor in granulosa cells as well as an LPS-induced increase in the level of
Dnmt1 expression both in vivo and in vitro. The methylation rate of the Lhcgr promoter region
was significantly higher in granulosa cells obtained from the LPS treatment group compared withthe control group. Together, these findings demonstrated that the decrease in the expression of
Lhcgr due to LPS was a result of the epigenetic regulatory action of LPS. Our studies suggest thatovarian follicular cysts that is characterized by bacterial infection in humans and animals, is closely
connected to the level of methylation of the Lhcgr promoter region.

33 | P a g e

7DEOH3&5SULPHUVXVHGIRUSURILOLQJWKHH[SUHVVLRQRIJHQHPDUNHUIRU*&¶VIXQFWLRQ
Gene

Forward Primer

Reverse Primer

Size (bp)Lhcgr

5’-ACTGGTGTGGTTTCAGGAATT-3’5’-CCTAAGGAAGGCATAGCCCAT-3’

244

Cyp19a15′-TTGCACCCAAATGAGGACAG-3′5′-CTTCACTGGTCCCCAACACA-3′

290

Ccnd2

5′-TCACCACGTGTTCCCAAGAC-3′5′-AGCGAATTCCCTCCATCAGA-3′

243

Nrg1

5’ -AGAACCGGCTGTCTGCTTTT -3’

5’ –TAGAGCTCCTCCGCTTCCAT -3’

373

Star

5' -GCAGCAGGCAACCTGGTG -3'

5' -TGATTGTCTTCGGCAGCC -3'

249

Ccl5

5’- ATATGGCTCGGACACCATA -3’

5’- GGGAAGGGTATACAGGGTCA -3’

242

Dnmt1

5’- GTGGCAAGAAGAAAGGCAAG -3’5’- GCCCCCTTGTGAATAATCCT-3’

233

Il6

5’- CCGGAGAGGAGACTTCACAG -3’5’- GGAAATTGGGGTAGGAAGGA -3’

421

Cyp11a15’-GGGAGACATGGCCAAGATGG-3’5’-CAGCCAAAGCCCAAGTACCG-3’

279

Fshr

5′-TGGCCATTACTGGGAACACC-3′5′-TGCCAAAGATGGGGAAGAGA-3′

261

Areg

5' -TTACTTTGGCGAACGGTGTG -3'

5' -TGTGCAGTCCCGTTTTCTTG -3'

231

Ptgs2

5' -TGTACAAGCAGTGGCAAAGG -3'

5' -GCTGTGGATCTTGCACATTG -3'

431

Snap255’- GAGATGCAGAGGAGGGCTGAC -3’

5’- GCTGGCCACTACTCCATCCTG -3’

309

L19

5’-GGCATAGGGAAGAGGAAGG-3’5’-GGATGTGCTCCATGAGGATGC-3’

199

Inha

5’- TTGGAGGCAGACGCCTTATT -3’

5’- CGGTCTCTGCTGCTCCTTTT-3’

175

34 | P a g e

%$

0hr (immature)eCG 48hr

TLR4

6

5

Fold induction

4

3

2

1

0
Day15

eCG 0hreCG24heCG48h)LJXUH  Expression of TLR4 (A) in granulosa cells of mice ovary at 15, 23 days and 24 and 48 hr after eCG, (B) Immunohistochemistry staining of the ovary during
the immature and follicle development phase (eCG 48 hr). In (A) the value of granulosa cells from mice without eCG at 15 days was set as 1, and the data arepresented as fold induction. Expression levels of TLR4 was normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three
replicates. If a significant difference was observed at each time points compared with the 15 days group the comparison analysis was performed by a Student's t‐test,*P < 0.05. In (B) the ovaries were collected from immature mice and from mice injected with eCG after 48 hr. The ovarian sections were stained with anti-TLR7
antibody and digital images were captured using BZ-9000 microscope (Keyence).

35

$

eCG
LPS immature

C immature

No of follicles

%

10
9
8
7
6
5
4
3
2
1
0

C

1 μg/kg LPS

b

b

a

C 48hrLPS 48hraaa

PRIMARY

SECONDARYANTRAL)LJXUH  Ovarian morphology and follicular health (A) Hematoxylin-eosin staining during the follicular development. The ovaries were collected fromimmature mice and mice injected with or without eCG for 48 hr while the LPS group were injected with LPS intraperitoneally and digital images were captured
using BZ-9000 microscope (Keyence). (B) The number of primary, secondary and antral follicles in mice ovary 48 hr after eCG and/or LPS injection andhematoxylin-eosin staining. The number of follicles were evaluated in 12 sections per ovary. Data are summarized as mean ± standard error of mean (SEM) of
three mice for each group. Different alphabets (a and b) within the same graph differ significantly (p < 0.05)

36

$

C immature

LPS immature

eCG
C 48hrLPS 48hr%

Control

DAPI

Merge

eCG +LPS (1 μg/kg)
Merge
DAPI
)LJXUH  Follicular atresia and cell proliferation in ovarian follicles resulting from LPS action. (A) TUNEL staining of the ovary during the follicular development
phase. The ovaries were collected from immature mice and injected with or without eCG and/or LPS for 48 hr. (B) Immunofluorescence staining of the ovary frommice injected with eCG with or without LPS at 48 hr using anti-Ki67 antibody. In (A) and (B) digital images were captured using BZ-9000 microscope (Keyence).

37

$

hCG

LPS 16 hr

C 16 hr

&

%

9
8

a

45

C

40

b

35

b

30
25
20
15
10
5
0
0

0.1μg/kg

1μg/kgoocyte maturation rate

90%

LPS (1μg/kg)

No. of follicular cyst

50

'

80%
70%
60%
50%
40%
30%

7
6
5

4
3
2

20%
1

10%

0

0%
MATUREDNOT MATUREDC

LPS (1μg/kg)

LPS)LJXUH  Ovarian morphology and follicular health (A) Hematoxylin-eosin staining of ovaries during the ovulation phase. The ovaries were collected from immaturemice and injected with eCG and/or LPS followed by hCG injection and digital images were captured using BZ-9000 microscope (Keyence). (B) The average number ofovulated oocyte (C, 0.1, 1 μg/kg LPS). (C) the maturation rate (D) the average number of follicular cysts. Data are summarized as mean ± standard error of mean (SEM)of three mice for each group. Different alphabets (a and b) within the same graph differ significantly (p < 0.05).

38

Fold induction$

Cyp19a1

Ccnd2

100
90
80
70
60
50
40
30
20
10
0

18
16
14
12
10
8
6
4
2
0
im

eCG 48hr

70
60
50
40
30
20

10
im

LPS (1μg/kg)

eCG 48hr

LPS (1μg/kg)
eCG

eCG

Lhcgr

Fold induction

Fshr

8

35

7

30

6

25

5

20

4

15

3

10

2

5

1

0

0
im

eCG 48hr

LPS (1μg/kg)
eCG

Inhibin0
im

eCG 48hr

LPS (1μg/kg)

im

eCG 48hr

eCG+LPS 48hr

eCG

)LJXUH $ The expression of Cyp19a1, Ccnd2, Fshr, Lhcgr and Inhibin in granulosa cells of mice at 23 days, and mice injected intraperitoneally witheCG and/or LPS for 48 hr. The value of granulosa cells from mice without eCG at 23 days was set as 1, and the data are presented as fold induction.
Expression levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If asignificant difference was observed at each time points compared with the immature group the comparison analysis was performed by a Student's t‐test,*P < 0.05.

39

%

Lhcgr (in vitro)

Cyp19a1 (in vitro)

Ccnd2 (in vitro)

1.2
1.2

1.4

1

1.2

Fold induction

1
0.8

1

0.8

0.8

0.6
0.6

0.6

0.4

0.4
0.4

0.2

0.2

0

DMEM+FSH+T4 +0.1μg/mL LPS
1μg/mL LPS
-

0.2

0

+
+
-

+
+

0

+
-

+
+
-

+
+

+
-

+
+
-

+
+

)LJXUH % The expression of Lhcgr, Cyp19a1 and Ccnd2 in granulosa cells cultured in vitro. GC’s were collected from 6h eCG primed mouseovaries and cultured in 1% FCS supplemented DMEM/F12 media for 24 hr treated with or without FSH and Testosterone (T) and/or 0.1 and 1
μg/mL of LPS. The value of granulosa cells from mice without any treatment was set as 1, and the data are presented as fold induction. Expression
levels of mRNAs were normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If a significant
difference was observed at each time points compared with the immature group the comparison analysis was performed by a Student's t‐test, *P <
0.05.

40

Snap25

($)

Star

250

450
400

Fold induction

200

350
300

150

250
200

100

150
100

50

50
0

0
C

Lps(1ug/kg)

0C

Lps(1ug/kg)

4hrc

Lps(1ug/kg)

0C

Lps(1ug/kg)

4hr)LJXUH $ The expression of Snap25 and Star in granulosa cells of mice injected intraperitoneally with eCG, with or without hCG and/or
LPS. The value of granulosa cells from mice injected with eCG only (0) was set as 1, and the data are presented as fold induction. Expression
levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If asignificant difference was observed at each time points compared with the immature group the comparison analysis was performed by aStudent's t‐test, *P < 0.05.

41

Nrg1

3000

1.2

2500

1

2000

0.8

Fold induction

Fold induction

Ptgs2

1500

1000
500

0.6
0.4
0.2
0

0
C

Lps(1 μg/kg)

C

0

C

Lps(1 μg/kg)

Lps(1 μg/kg)

04hr

Cyp11a1

4

Fold induction

Fold induction

5

3
2
1
0
Lps(1 μg/kg)
0

Lps(1 μg/kg)

4hr

Areg

6

C

C

C

Lps(1 μg/kg)
4hr

200
180
160
140
120
100
80
60
40
20
0

C

Lps(1 μg/kg)
0C

Lps(1 μg/kg)
4hr)LJXUH % The expression of Ptgs2, Nrg1, Cyp11a1 and Areg in granulosa cells of mice at 23 days, and mice injected intraperitoneally with
eCG with or without hCG and/or LPS. The value of granulosa cells from mice injected with eCG only (0) was set as 1, and the data arepresented as fold induction. Expression levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of themean (SEM) of three replicates. If a significant difference was observed at each time points compared with the immature group the comparison
analysis was performed by a Student's t‐test, *P < 0.05.

42

CCL5

IL6
3

12



8

2.5

Fold induction

Fold Induction

10

2

1.5

6
4

1

0.5

2
0

0
C

Lps(1μg/kg)

C

Lps(1μg/kg))LJXUH  The expression of chemokines and cytokines in the granulosa cells of mice at 23 days, and mice injected intraperitoneallywith eCG and/or LPS at 48 hr. The value of granulosa cells from mice injected with eCG only (0) was set as 1, and the data arepresented as fold induction. Expression levels of the mRNA was normalized to those of L19. Values are given as mean ± standard errorof the mean (SEM) of three replicates. If a significant difference was observed at each time points compared with the immature groupthe comparison analysis was performed by a Student's t‐test, *P < 0.05.

43

$

%

Dnmt1 (in vivo)

Dnmt1 (in vitro)

1.6

2.5

Fold induction

Fold Induction

1.4
1.2
1
0.8
0.6

2
1.5
1
0.5

0.4
0.2

0

0
im

eCG 48hr

LPS (1μg/kg)
eCG

DEMEM+FSH+T4 +0.1 μg/mL LPS
1 μg/mL LPS
-

+
+
-

+
+

)LJXUH  The expression of Dnmt1 gene implicated in DNA methylation in $ granulosa cells of immature mice and mice treated with eCG (control group) and coadministration of eCG and LPS (treatment group) for 48 hr (B) cultured granulosa cells after 6 hr of eCG injection in the presence of 1% FCS. The control group(FSH+T) and the treatment groups (FSH+T+ LPS (0.1 and 1 μg/ml). The value of granulosa cells from mice without eCG (immature) (A) and the value of
granulosa cells cultured with FSH+T (B) were set as 1, and the data are presented as fold induction. Expression levels of genes were normalized to those of L19.
Values are given as mean ± standard error of the mean (SEM) of three replicates (P < 0.05).

44

60%
50%
40%
30%
20%
10%
0%
0h

48 hr
eCG

48 hr+LPS

The rate of methylated cytosine in the
Cyp19a1 promoter region

The rate of methylated cytosine in the
Lhcgr promoter region

Lhcgr promoter region

Cyp19a1 promoter region
120
100
80

60
40
20
0
0h

48 hr

48 hr+LPS

eCG)LJXUH  Kinetic changes of cytosine methylation in the Lhcgr promoter region in granulosa cells of mouse ovaries injected at eCG
0 hr and 48 hr time-point with and without LPS. Sequence analysis was carried out on over five colonies per treatment group. The
studies were carried out in triplicate. Values are given a mean ± standard error of the mean (SEM). Significant changes wereobserved between eCG primed mice at 48 hr (*P < 0.05).

45

([SHULPHQW
6WXG\RQWKHUROHVRI7ROOOLNHUHFHSWRULQPRXVHRYDU\&RQWHQWV

7RSLFV

3DJH1R

3.1

Introduction

46

3.2

Materials and Methods

47

3.3

Results

53

3.4

Discussion

57

Abstract60,QWURGXFWLRQInfections of viral origin are notable causes of diseases of the reproductive system in both animaland human populations. The human papillomavirus (HPV), SARS-CoV-2, Hepatitis B&C virus,
Porcine reproductive and respiratory syndrome (PRRS), and Mumps orthorubulavirus have beenreported to cause abnormality in sex hormones and reduction in sperm quality, stillborns andoverall low reproductive performances (Batiha et al., 2020).
When entering a host cell membrane, the glycoprotein component on the virus surface binds withthe virus-specific receptor, internalizes the virus (Hoffman et al., 2020; Wang et al., 2020) andallows the infection of the host cell. The fusion of the virus and target cell membrane, affords thevirus the opportunity to release its genome and using the host cell organelles to replicate its RNA
releasing new mature virion to other cells of interest (Boopathi et al., 2020). To ensure viral-cellfusion, host cell receptors which are specific to a particular organism are expressed in the cell
infected by the virus (Griffiths et al., 2020). Specific receptors that mediate the entry of SARS
COV-2 and PRRS are angiotensin-converting enzyme 2 (ACE2) receptor and scavenger receptor
CD163 respectively (Rahmandad et al., 2020; Van Gorp et al., 2008).
Generally, viral particles of RNA virus can be recognized by both TLR7 and 8 in the infected
cells. These receptors are localized on endoplasmic reticulum (ER) and trigger the activation of
the signaling cascade and induce type 1 interferon (IFN) and pro-inflammatory cytokines (Jurk etal., 2002; Pockros, 2002). TLR7 and TLR8 like other TLRs family, such as TLR2 and TLR4
contain Toll–interleukin 1 (IL-1) receptor (TIR) domain in their cytoplasmic region that associateswith Myd88, an adapter molecule that plays a significant role in recruiting IL-1 receptor-associatedkinase (IRAK) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to the TLRs
(Akira et al., 2001). Also, the Myd88 signaling pathway leads to the activation of NF-κB
46 | P a g e

transcription factors and c-Jun NH2 terminal kinase (Jnk) mitogen-activated protein kinases(MAPKs).
In chapter 2, TLR2 and TLR4 were expressed in ovarian granulosa cells and the activation
suppressed Lhcgr expression. Since toll-like receptors 7/8 in the ovary can prompt an immune
response in the presence of viral pathogens (Svingerud et al., 2012; Welter, 2014), it was
hypothesis that RNA viral infection in ovarian cells can directly damage reproductive performance.
In addition, RNA viruses have been detected in reproductive organs or reproductive fluid andstudies on TLR7/8 ligand (R848) have shown a strong suppression of oocyte fertilization (Pileri
and Mateu, 2016; Batiha et al., 2020; Umehara et al., 2020). Therefore, a time dependent
expression of TLR7/8 and the mechanism by which TLR7/8 agonist (R848) affect the structureand function of the mouse ovary were examined.
0DWHULDOVThe materials were described in Chapter 2 of Experiment 1. Additionally, Resiquimod (R848),
horseradish peroxidase (HRP) labeled-progesterone and Standard solutions of progesterone wasobtained from Sigma-Aldrich (St. Louis, MO). TLR7 antibody was obtained from Bioworld
Technologies (Bloomington, MN).
$QLPDOVImmature 3-week-old female C57BL/6 mice were obtained from Charles River Laboratories Japan(Yokohama, Japan). The animals were housed at the Experimental Animal Center, Hiroshima
University, under a 12-hr light/12-hr dark schedule with food and water provided ad libitum. The
animals were cared for in adherence to the National Institutes of Health Guide for the Care and47 | P a g e

Use of Laboratory Animals, as approved by Hiroshima University’s Animal Care and Use
Committee (#C18-34).
7UHDWPHQWRIPLFHDQGFROOHFWLRQRIRYDULHVFXPXOXVFHOOVDQGJUDQXORVDFHOOVTwenty-three-day-old female mice were injected intraperitoneal (IP) with 4 IU of eCG to stimulatefollicular growth. Thereafter, they were injected with 5 IU of hCG and/or R848 at 48 hr to stimulate
ovulation and subsequent luteinization. Ovaries were collected at immature (0 hr), 24, 48 hr aftereCG injection and 8 and 16 hr time points after hCG. Granulosa cells were collected at immature(0h), 24, 48 hr after eCG injection, and 8 and 16 hr time points after hCG.
3UHSDUDWLRQRISDUDIILQEORFNDQGRYDULDQVHFWLRQVWDLQLQJTo prepare paraffin block for ovarian sectioning, the ovaries collected from three mice were fixed
in 4% (w/v) paraformaldehyde (PFA) solution (Nacalai Chemical Co) overnight at 4°C, which was
followed by dehydration and embedding in paraffin. The dehydration process involved a step-bystep incubation in 70, 80, 90, 95, and 100% ethanol solution for 2 hrs each, and the dehydrated
ovaries were then incubated twice in xylene with each incubation period lasting for 2 hrs. Lastly,
the ovaries were incubated for 2 hrs in paraffin liquid before they were appended in paraffin, andblocks were made by solidifying the liquid paraffin containing the ovaries. Thereafter, 5 μm thicksections were cut from the paraffin block by microtome and placed in water warmed up to 42oC
before they were mounted on slides and air-dried. Thereafter, the slide containing ovaries was
deparaffinized by incubating in xylene twice for a period lasting 2 mins. The ovarian sections on
the glass slide were then rehydrated using ethanol following a stepwise incubation in 100, 90, 80,
and 70% ethanol for a period lasting 1 to 2 mins each. Thereafter, the sections were washed in
distilled water and stained with eosin for 1 min followed by washing under running tap-water and
48 | P a g e

counterstaining with hematoxylin for 30 secs followed by washing under running tap water. The
stained sections in the slides were then dehydrated by incubating step-wisely in 100, 90, 80, and
70% ethanol followed by incubating in xylene twice for a period lasting 2 mins. Thereafter, theovarian sections are covered by a coverslip using the multi-mount DPX solution. The slides were
airdried and visualized by an optical microscope using BZ-II application software (Keyence,
Tokyo, Japan).
,PPXQRIOXRUHVFHQFHVWDLQLQJ
For immunofluorescence staining of ovarian sections using anti-TLR7 antibody (Bioworld
Technologies, Bloomington, MN), 5 μm thick ovarian sections were deparaffinized using xylene
followed by washing in PBS. Thereafter, the ovarian sections mounted on glass slides weredeparaffinized and rehydration of the tissue sections was carried out by washing twice in xylene
for 2mins on both occasions followed by a chronological incubation in gradient concentration
ethanol of (100, 95, 90, 80, and 70%) for 2 mins at each concentration then washed in PBS
and boiled for 20 min in 10% citric acid (pH 6.0) for antigen activation. Thereafter, the sections
were washed in PBS and nonspecific binding was blocked using 5% (w/v) BSA in MOM
Immunodetection Kit or PBS (Vector Laboratories Inc, CA, USA). Furthermore, theovarian sections were treated with TLR7 antibody with a dilution ratio of 1:200 as primary
antibodies in 1% BSA (v/v)

blocking solution at

4°C overnight. On the next

day,

the

ovarian sections were washed in PBS containing 0.3% (v/v) Triton X-100, the staining was
visualized with Cy3-conjugated goat anti-rabbit IgG (1:200) (Sigma) as a secondary antibody andcounterstained using 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc).

49 | P a g e([WUDFWLRQRISURWHLQDQGZHVWHUQEORWWLQJFor extraction of protein, the ovary was lysed using 1 mM EDTA solution containing complete
phosphatase and protease inhibitors (Roche, Indianapolis, IN). Then sonication was done at >10
kHz ultrasonic frequencies, the lysates were centrifuged at 10000 × g for 10mins and the protein
extracts were carefully collected as supernatant. The concentration of the protein collected wasmeasured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Life Science Group,
California, USA) based on the manufacturer’s protocol at an absorbance of 750 nm in a
spectrophotometer (Bio-Rad, California, USA). The concentration of the protein was determinedby analyzing the standard curve which was standardized using Bio-Rad protein standard solution.
Western blotting was carried out as described by Kawai et al. (2016). Protein extracts wereseparated by 725 % SDS-PAGE run at 25 mA or 100 V using vertical electrophoresis for about
1hr 30mins. The protein in the gel was transferred to a nitrocellulose membrane (PVDF) (GEBioscience, Newark, NJ, USA). 5% Bovine Serum Albumin dissolved in Tris-buffered saline(TBS) containing 0.05% (v/v) Tween-20 (TBST) to block non-specific binding sites. Thereafter,
the membrane was incubated with primary antibodies (TLR7 or Beta-actin) diluted in 5% Bovine
Serum Albumin at a concentration of 1:500 and 1:1000, v/v antibody respectively and stored
overnight at 4oC. After that, the membrane was washed in TBST for 2 hrs and the membrane wasincubated in secondary antibody (HRP-conjugated anti-rabbit IgG antibody) (Cell Signaling
Technology Catalogue number 7074) or HRP-conjugated anti-mouse IgG antibody for beta-actin
at 1:5000, v/v antibody) (Cell Signaling Technology Catalogue number 70746). After repeatedwashing in TBST, enhanced chemiluminescence (ECL) detection was carried out using ECL kit(GE Bioscience) based on manufacturer’s instructions, and the membrane was placed in (Bio-rad

50 | P a g e

ChemiDoc Touch Imaging System to detect protein bands at appropriate exposure times. The
detections were carried out in the three replicated samples and quantitative analysis was performed
with the aid of ImageJ software (http://imageJ.nih.gov/ij/).
51$H[WUDFWLRQDQG573&5
RNA extraction and RT-PCR analysis were carried out as described in Experiment 1 of Chapter 2.
5HDOWLPH3&5
The quantitative real-time PCR was carried out as described by Umehara et al. (2017). The
procedure was explained in detail in Experiment 1 of Chapter 2. The primers used in thisexperiment were summarized in Table 3.
&ROOHFWLRQRIRYXODWHGRRF\WHDQGLQYLWURIHUWLOL]DWLRQTwenty-five-day-old female mice were injected intraperitoneal (IP) with 4 IU of eCG to stimulatefollicular growth. Thereafter, they were injected with 5 IU of hCG at 48 hr to stimulate ovulationand subsequent luteinization. Also, R848 was injected IP to mice at a dosage of 0.1Pg/kg BW at
4h after hCG. At 16 hr after hCG injection, ovulated COCs were collected from the oviducts ofthree mice that were euthanized by needle puncture under the microscope and placed in 50Pl ofhuman tubal fluid (HTF) medium. One hr before the collection of the COCs, spermatozoa werecollected from the cauda epididymis of an adult male mouse and placed in 500 Pl of HTF medium
to allow for capacitation in an incubator set at a temperature of 37oC and atmospheric condition of
5% CO2 at maximum humidity before the sperm was introduced to the HTF medium containing
oocyte at a final concentration of 1,000 sperm cells per Pl. Thereafter, the sperm-oocyte-containing

51 | P a g e

medium was incubated at 37oC in an atmosphere of 5% CO2 at maximum humidity for 8 hrs for
fertilization to occur. After washing in HTF medium the oocytes are transferred to KSOM media
and placed in the incubator at 37oC and atmospheric condition of 5% CO2 at maximum humidity
for further development. The number of oocytes that were ovulated, matured, and fertilized wasscored.
&ROOHFWLRQDQG FXOWXUHRIJUDQXORVDFHOOV
44 hrs after injecting immature mice (23 days old) with eCG, granulosa cells were collected fromovaries as described by Kawai et al. (2018). The cells were pre-cultured in DMEM/F12 medium
containing 1% (v/v) FCS in 5% (v/v) FCS-coated 96-well plates for a preculture period of 4 hrs
followed by a culture period of 4 hr at 37°C in an atmosphere of 5% CO2 at maximum humidity.
The medium described above was supplemented as follows: (a) no R848 (control), (b) 10 nM R848.
0HDVXUHPHQWRISURJHVWHURQH
Media levels of progesterone was analyzed by a competitive enzyme immunoassay as describedby Isobe and Nakao, (2003). 100 Pl of secondary antibody in carbonate buffer (1:100 dilution) andincubate for 2 hrs at 20oC and wash with PBS-T. Place 150 Pl of assay buffer in each well andincubate for 30 mins at 20oC for Blocking and wash twice with PBS-T. Also, 50 Pl of sample
which was extracted by ether method were placed in the ELISA plate. After the standard andsamples have been placed in the ELISA plate, 50 Pl of HRP-P4 (1:50,000) and 50 Pl of P4-ab(1:100,000) was added to each well and incubated for 3 hrs at 20oC. 150Pl of TMB solution wasadded to each well and incubated for 30 mins at 20oC and the reaction is stopped using 30 Pl of
2N H2SO4. Thereafter the absorbance was read using the multilabel reader at 450 nm (2030
52 | P a g e

Multilabel Reader ARVO×4, Perking Elmer, Waltham, MA, USA). The level of progesterone wasnormalized through specific standard curve plotting against the standards.
6WDWLVWLFDODQDO\VLVAll data from three replicates were analyzed by either Student’s t-test or a one-way analysis ofvariance followed by a Tukey post hoc test (StatView; Abacus Concepts, Berkeley, CA). All
values are presented as the mean ± SEM. Treatments were considered significantly different fromone another at P < 0.05.
5HVXOWV([SUHVVLRQ7/5DQGLQWKHPRXVHRYDU\The presence of TLR7 and 8 were analyzed by immunofluorescence, RT-PCR, and western
blotting.

To detect the expression of TLR7 by immunofluorescence during the follicle

development and ovulatory phase, ovaries were collected from eCG and hCG hormone-injected
mice at immature, hCG (0 hr), 8 and 16 hr points. The result of Western blotting analysis of TLR7
is shown in Figure 11. The level of TLR7 in the ovary was highly expressed after hCG injection
at 8h point (Figure 11B). These results indicate that viral pathogens have a dominant effect during
the ovulatory phase. In Figure 12, the strong TLR7 positive signals were detected in mouse ovaries
at hCG 0 and 8 hr compared to the immature and 16 hr groups. By RT-PCR, both Tlr7 and Tlr8
were expressed in spleen and were used as positive control (Figure 13B). More so, there was atime dependent difference in the expression of Tlr7 and Tlr8 in granulosa cells with 8h point having
the highest level of expression (Figure 13B). However, Tlr7 and Tlr8 expression were detected in

53 | P a g e

cumulus cells but no time dependent difference in the level of expression was observed (Figure
13A).
(IIHFWRI5HVLTXLPRG 5 RQWKHH[SUHVVLRQRILQIODPPDWLRQDVVRFLDWHGJHQHVTo assess the effect of R848 on the spleen organ, the expression of inflammation-associated genes
was studied. Spleen was collected from mice injected eCG and hCG with or without R848 at 16
hr time point, and RNA was extracted from both the control and treatment group (0.1 μg/kg R848).
It was observed that there was a significant increase in Il6 and Ifnγ in the treatment group comparedto the control group. However, a drastic reduction was observed in the Ccl5 gene for the treatmentgroup (Figure 14).
(IIHFWRI5HVLTXLPRG 5 RQRYDULDQPRUSKRORJ\DQGPDWXUDWLRQUDWHQXPEHURIRYXODWHGRRF\WHVDQGIHUWLOL]DWLRQUDWH
To evaluate the effect of TLR7- and TLR8-agonist (R848) on ovarian health, the ovaries werecollected from mice injected eCG and hCG with or without R848 (injected 4 hr after hCG) at 16
hr time point and the ovarian morphology was checked after Hematoxylin-eosin staining.
Luteinization of follicles can be observed in both the control and treatment groups (Figure 15A).
The number of the ovulated oocyte in the control and R848 administered groups were similar(Figure 15B). However, the number of the matured ovulated oocytes in the R848 group decreased
compared with the control group (Figure 15C). More so, the fertilization rate was significantly
reduced in the treatment group compared to the control group (Figure 15D).

54 | P a g e (IIHFW RI 5 RQ WKH LQGXFWLRQ RI (*)OLNH IDFWRUV SURJHVWHURQH UHFHSWRU 3*5 
GHSHQGHQWHYHQWVDQGRWKHUWDUJHWVRIWKH/+/+UHFHSWRU /+5 GHSHQGHQWSDWKZD\
To assess the effect of R848 on follicle development, ovulation, and steroid synthesis in granulosa
cells, the expression of EGF-like factors, progesterone receptor (PGR)-dependent events, andgenes associated with LH-LH receptor (LHR)-dependent pathway were studied. Granulosa cells
were collected from mice injected with eCG and hCG with or without R848 and RNA wasextracted from both the control and treatment groups (0.1 μg/kg R848). It was observed that theexpression of Ptgs2, Ereg, Areg, Pgr were significantly increased at the 4hr point, whereas Snap25,
Star were significantly increased at the 8 hr point and Cyp11a1 was significantly increased at the
16 hr point (Figure 16A and B). However, Nrg1 was not significantly different at all the time points.
(Figure 16A). In the in vitro data, a dose-dependent increase was observed in the expression of
Cyp11a1, Star, HSD3β1, Adamts1, Snap25 and Ptgs2 between the 1 and 10 nM R848 comparedto the control group at 4hr point (Figure 16C).
(IIHFWRI5RQWKHH[SUHVVLRQRI&KHPRNLQHDQGF\WRNLQH
To evaluate the effect of Chemokine and cytokine in granulosa cells, the expression of Il6 andCcl5 was studied Granulosa cells were collected from mice injected with eCG and hCG with orwithout R848 and RNA was extracted from both the control and treatment groups (0.1 μg/kg R848).
It was observed that the expression of Il6 was significantly increased at the 4hr time point, whereas,in Ccl5 no significant difference was observed (Figure 17).

55 | P a g e5LQMHFWLRQVLQJXODUO\LQGXFHVOXWHLQL]DWLRQRIIROOLFOHV
Since R848 can cause an over-secretion of epidermal growth factors and progesterone receptor
target genes when coinjected with hCG (Figure 17), only R848 was injected at 48h point following
eCG injection. Figure 18A shows that in the R848 treatment group ovulation of oocytes wasobserved but at an amount significantly lower than the control group. However, the possibility that
ovulation was induced by endogenous GnRH cannot be ruled out. Therefore, GnRH-antagonist
was injected every 24 hr to both groups. In this case, there was no ovulation of oocytes in the R848
and GnRH antagonist treatment groups unlike in the hCG and GnRH antagonist group where
ovulation of oocytes were observed (Figure 18B). Hematoxylin-eosin staining of the ovarycollected from the only R848 and GnRH antagonist group show that corpus luteum was formed
and oocytes were trapped in the follicles (Fig 18C). Figure 18D confirms the supposition that R848
causes the luteinization of follicles, as the level of progesterone in the media increased in the groupcontaining 10 nM R848.
([SUHVVLRQRI$&(LQPRXVHRYDU\
To detect SARS-CoV-2 receptors in the mouse ovary and granulosa cells respectively. cDNAwasisolated from granulosa cells and mouse ovaries after 48hr of eCG injection. The receptors weredetected by PCR amplification and visualized through gel electrophoresis. To evaluate theexpression of the angiotensin-converting enzyme 2 (ACE2) receptor in granulosa cells, thegranulosa cells were collected from mice injected eCG and hCG at 0, 4, 8, and 16 hr time points.
ACE2 receptor expression was not significantly different at 0,4 and 16 hr time points except for
8h which had the highest level of expression (Figure 19).

56 | P a g e'LVFXVVLRQInfectious organisms of viral origin can circumvent the barriers of the cell membrane as they lackany unique foreign features that distinguish them from the host, however, TLRs have aptly
developed strategies for recognizing viral particles (Gregory, 2007). More so, inherent in the hostcells are receptors that bind with viral components and allow the uptake of viruses into host cells,
where they replicate and cause degenerative outcomes (Zhou et al., 2018). In recent times, several
viruses were reported to affect fertility and reproductive performance (Ljubojevic & Skerlev, 2014,
Monavari et al., 2013; Dondero et al. 1996; Xu et al., 2006 and Ma et al., 2020). Therefore,
Experiment 2 was aimed to study the expression of TLR7/8 and specific endogenous receptors,
and the mechanism by which R848 affect the structure and function of the mouse ovary.
The first phase of every viral infection is receptor recognition. In the result of the present
experiment, Angiotensin-converting enzyme 2 (ACE2) receptors were detected in granulosa cellsof mouse ovaries. This suggests that, since SARS-CoV-2 and its receptor ACE2 have a highbinding affinity, there is a possibility that the virus transmissibility will be high in the host cell (Liet al., 2005c). Likewise. the expression of TLR7 and 8 in the granulosa cell of mouse ovary showsthat toll-like receptors are present on the cell membranes and can detect or recognize the presenceof ssRNA viruses. More so, it also demonstrates its capacity to pose an inflammatory response toviral entry leading to the activation of NF-κB transcription factors through the MyD88 signaling
pathway.
Although ACE2 receptor was expressed in the different time points, it was highest at 8h point after
hCG injection. At the same time point, TLR7 expression was highest, demonstrating that viralpathogens have a dominant influence during the ovulatory phase.
57 | P a g e

Also, it was observed that TLR7/8 agonist (R848) induced an increase in the expression of
immune genes such as IL6 and interferon-gamma which are related to the TLR7/8 signaling
pathway. These results demonstrate that R848 induces immune responses in the spleen andovaries of mice, suggesting that TLR7/8 signaling and the involvement of Myd88 may be
responsible for the host immune response to viral invasion. Moreover, it has been reported thatepidermal growth factors such as amphiregulin and epiregulin mediate the action of LH by
activating the expression of EGF receptors which are present on granulosa cells and cumulus
cells (Park et al., 2004). It is the binding of the EGF domain with its receptor that activates the
ERK1/2 pathway which results in COC expansion and oocyte maturation (Yamashita et al.,2007). In this study, when R848 was co-injected with hCG, it led to the over-secretion of EGFlike factors and target

genes

involved

in progesterone synthesis.

Usually, theinjectionof hCG in superovulated mice prompts oocyte maturation, cumulus expansion, ovulation of
matured oocyte, and luteinization while genes such as Snap25, Pgr, Ereg, Areg, Ptgs2, Cyp11a1,
Star, and Nrg1 were expressed at optimum levels. In the present study, the number ofmatured oocytes in the group co-injected with hCG and R848 led to the ovulation of mainly
immature oocytes unlike the control group with a marked decrease in fertilization rate. This may
beattributed

to

the

impaired expression

of

the

progesterone

receptor

gene

and

disruption of important mediators of LH actions in granulosa and cumulus cells leads to theimpairs oocyte maturation (Breen et al., 2013). Likewise, the induction of IL-6 has been reported
to have a direct impact on the oocyte by causing negative effect on meiotic maturation (Bromfieldand Sheldon, 2011).
In controlled ovarian stimulation, the injection of LH/hCG is required to initiate LH surge (RonEl et al., 2000). LH stimulates cAMP formation and the activation of PKA signaling pathway
58 | P a g e

which initiates the expression of genes such as Star and progesterone receptor that facilitate theproduction of progesterone (Hickey et al., 1988). However, the action of R848 in inducing theterminal differentiation of GCs to luteal cells is not dependent on LH surge. Rather, R848 directly
stimulate inflammatory reaction that leads to an increase in the induction of inflammation andluteinization associated genes. As in LH signaling, R848 induces Snap25 via the progesterone
pathway as well as other progesterone receptor target genes such as Adamts1 and genes responsible
for cholesterol synthesis leading to the production of progesterone necessary for luteinization.
During this period there is a repression in Cyp19a1 expression and a shift in granulosa cells
steroidogenesis from the formation of estrogen to a progesterone producing cell by the increase in
Cyp11a1 and Hsd3b1 (Fan et al., 2009).
Therefore, it was hypothesized that R848 can mimic the action of hCG hormone in prompting theovulationandluteinizationoffollicles.

To

this

end, only

R848

was

injected in superovulated mice to study its role in the ovulation process. It was observed that
R848 afforded the development of follicles into Graafian follicles, induced luteinization andprompted a dose-dependent increase in progesterone secretion resulting in the formation of corpus
luteum, however, it did not prompt ovulation of oocyte when co-injected with GnRHantagonist to suppress endogenous LH increase (Diedrich et al., 1994). It can be concluded
that R848 suppresses oocyte maturation and fertilization and prompts the irreversible transition toa luteal cell phenotype without inducing ovulation by causing a decline in cell cycle activators andan increase in cell cycle inhibitors culminating in luteinization.

59 | P a g e

$EVWUDFWInfections of viral origin are notable causes of diseases of the female reproductive system in bothanimal and human populations. The presence of receptors for these viruses allows for virusinternalization in the host cell and an inflammatory response from the host to virus detected bytoll-like receptors. This study highlights the presence of endogenous receptors for viruses ofinterest and mechanism by which TLR7/8 agonist, R848 cause low reproductive performance ofthe ovary. It was established that ACE2 and CD163 which are receptors for SARS-CoV-2 and
PRRS are expressed in mouse ovaries. The expression of toll-like receptor 7 and ACE2 receptor
was highest at 8h time point, indicating that viral pathogens greatly influence the ovulatory phase.
It was also observed that R848 increased the production of cytokine and chemokines in the spleen
and ovaries of mice. In mice treated with R848, the number of matured oocytes and fertilization
rate was reduced. More so, R848 causes a dramatic increase in the expression of genes associatedwith progesterone receptor and epidermal growth factors. Although R848 did not induce ovulation,
it retarded oocyte maturation and prompted the irreversible transition to a luteal cell phenotype by
causing a decline in cell cycle activators and an increase in cell cycle inhibitors culminatingin luteinization.

60 | P a g e
7DEOH3&5SULPHUVXVHGIRUSURILOLQJWKHH[SUHVVLRQRI6$56&297/5VDQGJHQHPDUNHUVIRU*&¶VIXQFWLRQ*HQHV
Adamts1)RUZDUG3ULPHU

5HYHUVH3ULPHU

6L]H ES 

5’–CAGTACCAGACCAGACCTTGTGCAGACCTT-3’ 5’–CACACCTCACTGCTTACTTACTGGTTTGA-3’

HSD 3B1

5’–TGGGGAGAGAAGTCCATTCA-3’

5’–GGAGCCCCCATTCCTTACTA-3’

196

Nrg1

5’ -AGAACCGGCTGTCTGCTTTT -3’

5’ –TAGAGCTCCTCCGCTTCCAT -3’

373

StaR

5' -GCAGCAGGCAACCTGGTG -3'

5' -TGATTGTCTTCGGCAGCC -3'

249

CCL5

5’- ATATGGCTCGGACACCATA -3’

5’-GGGAAGGGTATACAGGGTCA-3’

242

Il6

5’- CCGGAGAGGAGACTTCACAG -3’5’- GGAAATTGGGGTAGGAAGGA -3’

421

Cyp11a15’-GGGAGACATGGCCAAGATGG-3’5’-CAGCCAAAGCCCAAGTACCG-3’

279

Areg

5' -TTACTTTGGCGAACGGTGTG -3'

5' -TGTGCAGTCCCGTTTTCTTG -3'

231

Ptgs2

5' -TGTACAAGCAGTGGCAAAGG -3'

5' -GCTGTGGATCTTGCACATTG -3'

431

Snap255’- GAGATGCAGAGGAGGGCTGAC -3’

5’- GCTGGCCACTACTCCATCCTG -3’

309

L19

5’-GGCATAGGGAAGAGGAAGG-3’5’-GGATGTGCTCCATGAGGATGC-3’

199

Tlr7

5’-GGAAATTGCCCTCGATGTTA-3’

5’-CAAAAATTTGGCCTCCTCAA-3’

237

TLR8

5’- GAAGCATTTCGAGCATCTCC-3’5’-GAAGACGATTTCGCCAAGAG-3’

188

ACE2

5’-CCTGTTCCGATCATCTGTTG-3’5’-GACACATTTTGGGGTGAGGT-3’

153



61 | P a g e

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Im

24h

48h

8h

16h

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TLR7

*

48h

8h

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TLR7

β-actin

Relative intensity

8
6
4
2

0
im

24h

eCG

16h

hCG

)LJXUH  TLR7 expression in the ovary at immature, 24 and 48 hr after eCG injection and 8 and 16hr following hCG injection, (A)Expression of TLR7 through western blotting; where β-actin was used as control. (B) Quantitative expression of TLR7 over β-actin in theovary. For reference, the data for the immature group was set as 1 and the data was expressed as relative intensity. Data are summarized asmean ± standard error of mean (SEM) of three mice for each group. Different alphabets (a, b and c) within the same graph differ significantly(p < 0.05).

62

hCG

immature
0hr

8hr

16hr

)LJXUH . Immunofluorescence staining of the ovary during the immature, follicle development and ovulation phase. The ovaries were collected from immature mice and mice
injected with or without eCG for 48 hr followed by hCG. The ovarian sections were stained with anti-TLR7 antibody and DAPI and digital images were captured using BZ-9000
microscope (Keyence).

63

TLR7

100
90
80
70
60
50

70
60
50
40

TLR8

40
30
20
10
0

30

20
10
0
im

eCG 24h

hCG 0hhCG 8hhCG 16himeCG 24h

hCG 0hhCG 8hhCG 16h)LJXUH  TLR7 and TLR8 expression in the ovary at immature, 24 and 48 hr after eCG injection and 8 and 16 hr following hCG injection, (A)Quantitative expression in the granulosa cells. For reference, the data for the immature group was set as 1 and the data was presented as fold induction.
Data are summarized as mean ± standard error of mean (SEM) of three mice for each group. If a significant difference was observed at each time points
compared with the immature group the comparison analysis was performed by a Student's t‐test, *P < 0.05.

64

IL6

IFNγ
1.4

12

1.2

10
8
6
4
2

1.4
1.2

1
0.8
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0.4

0.1ug/kg R848

0.8
0.6
0.4

0

0
C

1

0.2

0.2

0

CCL5

1.6

Fold induction

14

Fold induction

1.6

Fold induction

16

C

0.1ug/kg R848

C

0.1ug/kg R848

)LJXUH  The expression of IL6, IFNγ and CCL5 in the spleen of mice injected with Resiquimod (R848) at 16 hr after hCG. The value of mRNA from the control groupwas set as 1, and the data are presented as fold induction. Expression levels of the mRNAs was normalized to those of L19. Values are given as mean ± standard error of themean (SEM) of three replicates. If a significant difference was observed between the control and treatment groups the comparison analysis was performed by aStudent's t‐test, *P < 0.05.

65

$

%
eCG+hCGC 16hrNo of ovulated oocyte

0.1 μg/kg R848 16hr
45
40
35
30
25

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eCG +hCG&

eCG +hCG+ R848 0.1 ug/kg'35

Maturation rate

Fertilization rate

30
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85

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80

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75

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70

0

65
MATUREDCNOT MATURED
R848 (0.1μg/kg)

C

R848 (0.1 μg/kg))LJXUH  Ovarian morphology and follicular health (A) Hematoxylin-eosin staining of ovaries during the ovulation phase. The ovaries were collected from mice injected witheCG and/or Resiquimod (R848) followed by hCG injection and digital images were captured using BZ-9000 microscope (Keyence). (B) The average number of ovulated oocyte(0.1 μg/kg R848). (C) the maturation rate (D) fertilization rate. Data are summarized as mean ± standard error of mean (SEM) of three mice for each group. Different alphabets (aand b) within the same graph differ significantly (p < 0.05).
66

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20
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16
14
12
10
8
6
4
2
0

600

Fold induction

Fold induction

500
400
300
200
100
0

hCG 0hr

hCG 4hr

hCG 8hrC

hCG 16hr

hCG 0hr

hCG 4hr

R848

C

Star

hCG 16hr

hCG 8hr

hCG 16hr

R848

Nrg1

80

6

70

5

60

Fold induction

Fold induction

hCG 8hr

50
40

30
20
10

4
3
2
1

0
hCG 0hr

hCG 4hr

hCG 8hrCR848

hCG 16hr0
hCG 0hr

hCG 4hrC

R848)LJXUH $ The expression of Ptgs2, Cyp11a1, Star and Nrg1 in granulosa cells of mice injected intraperitoneally with eCG with or without hCG and/or
Resiquimod (R848). The value of granulosa cells from mice injected with eCG only (hCG 0 hr) was set as 1, and the data are presented as fold induction.
Expression levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If a significant
difference was observed at each time points compared with the immature group the comparison analysis was performed by a Student's t‐test, *P < 0.05.

67

%
450

Snap25

400

Pgr
300
250

300

Fold induction

Fold induction

350
250
200
150
100
50
0

150
100
50
0

hCG 0hr

hCG 4hrC

hCG 8hr

hCG 16hr

hCG 0hr

R848

hCG 4hrCEreg

hCG 8hr

Fold induction

800
600
400
200
0

hCG 16hr

R848

Areg

300

1000

Fold induction

200

250

200
150
100
50
0

hCG 0hr

hCG 4hrC

hCG 8hr
R848

hCG 16hr

hCG 0hr

hCG 4hrC

hCG 8hr

hCG 16hr

R848)LJXUH % The expression of Snap25, Pgr, Ereg and Areg in granulosa cells of mice injected intraperitoneally with eCG with or without
hCG and/or R848. The value of granulosa cells from mice injected with eCG only (hCG 0 hr) was set as 1, and the data are presented as
fold induction. Expression levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of the mean
(SEM) of three replicates. If a significant difference was observed at each time points compared with the immature group the comparison
analysis was performed by a Student's t‐test, *P < 0.05.

68&

Cyp11a1 (in vitro)

Fold induction

Fold induction

4
3
2

1
0

3

2.5

2.5

2

Fold induction

*

5

2
1.5
1
0.5

1
0.5

HSD 3B (in vitro)

30

10 nM R848

C

Fold induction

1.5
1

0

2
1.5
1

0
C

1 nM R848

10 nM R848

C

1 nM R848

1 nM R848

10 nM R848

Ptgs2 (in vitro)

0.5

0.5

C

*

2.5

2.5

10 nM R848

Snap25(in vitro)

3

*

2

1 nM R848

Fold induction

1 nM R848

*

1.5

0
C

Fold induction

Adamts1 (in vitro)

StaR (in vitro)

6

10 nM R848

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
C

1 nM R848

10 nM R848

)LJXUH & Dose-dependent expression of Snap25, Ptgs2, Cyp11a1, StaR, Adamts1 and HSD3b1 in granulosa cells cultured in vitro. GC’s were collectedfrom 48 hr eCG primed mouse ovaries and cultured in 1% FCS supplemented DMEM/F12 media for 4h treated with or without R848 (1 and 10 nM). The
value of granulosa cells cultured with DMEM/F12 were set as 1, and the data are presented as fold induction. Expression levels of genes were normalized
to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If a significant difference was observed at each time
points compared with the DMEM/F12 or control group, the comparison analysis was performed by a Student's t‐test, *P < 0.05.

69

IL6

CCL5

40

2.5
2

30

Fold induction

Fold induction

35

25

20
15
10

1.5
1
0.5

5
0

0

hCG 0hr

hCG 4hr

hCG 8hrCR848

hCG 16hr

hCG 0hr

hCG 4hr

hCG 8hrC

hCG 16hr

R848)LJXUH  The expression of chemokines and cytokines in in granulosa cells of mice injected intraperitoneally with eCG with or without hCG and/or
R848. The value of granulosa cells from mice injected with eCG only (hCG 0 hr) was set as 1, and the data are presented as fold induction. Expression
levels of the mRNA was normalized to those of L19. Values are given as mean ± standard error of the mean (SEM) of three replicates. If a significant
difference was observed at each time points compared with the immature group the comparison analysis was performed by a Student's t‐test, *P < 0.05.

70

$

&

%
No. of ovulated oocyte

No. of ovulated oocyte

50
45
40
35
30
25
20
15
10
5
0

60

CL

CL

50

CL

o

40

CL

30

CL

20

10
eCG +hCG

eCG +R848 (0.1ug/kg)'
5

eCG+0.1 μg/kg R848+GnRH antagonistCLNot ovulated

0eCG +hCG+GnRH Antagonist eCG +R848(0.1ug/kg)+GnRH
antagonist

CL
CL
o

Level of Progesterone (ng/ml)

4
3
2
1

0
0nM

1nM

10nM

)LJXUH  Ovarian morphology and follicular health (A) The average number of ovulated oocyte (eCG+hCG and eCG+R848 0.1 μg/kg). (B) The average number of ovulated
oocyte (eCG+hCG+GnRH antagonist and eCG+R848 (0.1 μg/kg) +GnRH antagonist) (C) Hematoxylin-eosin staining of ovaries during the ovulation phase. (D) Levels ofprogesterone during lutea phase in vitro. The ovaries were collected from mice injected with eCG followed by R848 injection and digital images were captured using BZ-9000
microscope (Keyence) (CL- Corpus Luteum, O-oocyte). Progesterone level was measured using ELISA. Data are summarized as mean ± standard error of mean (SEM) of three
mice for each group. If a significant difference was observed between the groups in each graph the comparison analysis was performed by a Student's t‐test, *P < 0.05.

71

ACE2
60

Fold induction

50
40
30

20
10
0
hCG 0hhCG 4hhCG 8hhCG 16h)LJXUHACE2 receptor expression in the ovary at different time points

72

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
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




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73 | P a g e

Pelvic inflammatory disease describes the infection of the upper region of the female reproductive
tract such as fallopian tubes, uterus, and ovaries by pathogenic microorganisms. When thesemicrobes enter into the cervix, they alter the cervicovaginal environment and disrupt the normal
microbiome of the vagina and in most cases, the overgrowing organism or the original pathogenic
microbe ascend to the upper reproductive tracts (Westrom and Eschenbach, 1999). During thisprocess they cause damage resulting from the inflammation of reproductive structures, leading toovarian dysfunction and a sequelae to infertility (Brunham et al., 2015).
A notable cause of abnormal reproductive outcomes is ovarian follicular cyst. In this condition,
one or more mature follicles fail to ovulate following LH surge, however, the follicles further growinstead of undergoing atresia (Isobe, 2007). Follicular cysts have been associated with theinefficiencies of hypothalamus-pituitary function which regulate the occurrence and magnitude
and timing of the LH surge (Garverick, 1997). However, the injection of hCG, a hormone withsimilar function as LH does not induce the ovulation process of all mature oocyte (Garverick,1997), suggesting that not only the dysfunction of pituitary gland but the abnormal response ability
to LH in granulosa cells of large antral follicles are factors required to induce follicular cyst. It has
been known that infection with many kinds of bacteria including Chlamydia trachomatis and N.
gonorrhea causes reproductive dysfunctioning (Scholes et al., 2012). The presence or infusion of
LPS have also been reported to cause ovulation anomalies and ovarian follicular cyst (Peter et al.,1989). In the present study, it was demonstrated that LPS interfered with the ovulation of matured
Graafian follicle causing follicular cyst, indicating that bacterial infection is one of the factors to
induce follicular cyst. Because LPS affects the whole body via the vascular system, follicular cyst
results not only from the direct infection of the reproductive tract but from infections affecting

74 | P a g eother organs. Therefore, infection of bacteria origin needs to be examined in the diagnosis ofovulation disorder not only in human infertility care but also in livestock animals.
The present study is the first study to understand the mechanisms by which LPS induces follicular
cyst at the molecular level. The study shows that the induction of follicular cyst is associated withabnormal epigenetic regulation in granulosa cells of preovulatory follicles. DNA methylation, 5methylcytosine content of DNA, is an epigenetic mechanism that plays a fundamental role in theregulation of gene expression, especially during differentiation (Reik, 2007). Methylation caninhibit gene expression by inhibiting transcription factor binding to promoter region of genes.
Therefore, the demethylation in the promoter region is involved in the transcription of the genes.
In granulosa cells, LPS did not change the morphology, proliferation, and cell survivability;
however, FSH-induced cell differentiation to express Lhcgr was selectively suppressed by LPS.
The DNA methylation status in Lhcgr promoter region was maintained at higher level in granulosa
cells even when eCG was injected with LPS. In other reports, the role of LPS in inhibiting
osteoblastic differentiation of fibroblasts derived from human periodontal ligament by inducing
DNA hypermethylation of RUNX2 have also been established (Uehara, et al., 2014). However,the present study for the first time demonstrates that LPS downregulated the expression level of
DNMT1, an enzyme that copy DNA methylation pattern from parent DNA strand to daughter
strand in FSH-stimulated granulosa cells. Thus, the LPS-induced abnormal epigenetic regulation
may be observed in not only Lhcgr promoter region but also other genes, which impacts on large
scale gene expression patterns not only in granulosa cells, but in other cell types infected withbacteria or stimulated by LPS.
Bacterial infections are recognized by TLR2 and TLR4 while viral infections are detected by other
TLRs family. However, in most cases the TLRs family adhere to the same downstream pathway.
75 | P a g e

Additionally, infections resulting in ovarian dysfunction, poor pregnancy outcomes, and neonatal
health are not only caused by pathogenic microbes of bacteria origin but also by viruses (Coyneand Lazear, 2016; Racicot and Mor, 2017). In recent times, the role of viruses in the disruption ofmale and female reproductive health has become the front burner ((Racicot and Mor, 2017). Many
families of viruses have a strong tropism for the male reproductive system, especially the testis,and about thirty different species can affect semen quality (Osburn, 1994; Salam and Horby, 2017).
In addition, some of these viruses can impair fertility and can be sexually transmitted (Gimenes,2014). Regarding the male reproductive health, viral infections reach the testis through thebloodstream, causing damage by direct harmful effects on target organs or indirectly through proinflammatory cytokines (Guazzone, 2009). The contamination of male reproductive organs andspermatozoa leads to male sub-fertility or infertility resulting from signaling deficiencies intesticular cells that may be aggravated by pro-inflammatory mediators. In the female, these
inflammatory genes play a role in steroidogenesis and interfere with LH action during the processof oocyte maturation, ovulation and luteinization (Bornstein, 2004). However, there is no report toshow that the viral infections are found in the reproductive organs and that viral infections directly
causes damages to the reproductive performances of both male and female. The present study isthe first to show that TLR7 and TLR8, receptors for RNA virus were expressed in granulosa cells
after LH surge and the stimulation induced ovulation process in part; however, TLR7 and TLR8
pathway strongly suppressed oocyte maturation.
More so, it is known that LH surge is required for ovulation and luteinization of follicles. However,there are other processes such as gonadotropin-induced ovulation that mimic the action of LH.
This is due to a specific degree of homology between both hormones (Ma et al., 2015). In
experiment 2, R848, a ligand for TLR7 and TLR8 prompts the expression of inflammatory genes,

76 | P a g e

epidermal growth factors and progesterone receptor target genes that are involved in cumulus
expansion, ovulation and luteinization. However, its inability to induce oocyte maturation andcompletion of the ovulation process leading to the release of oocyte may be attributed to the timingof R848-induced progesterone synthesis which represses Cyp19a1 expression and causes a shift
in granulosa cell orientation. Thus, it is one possibility that RNA virus infection would induce
luteinization process before LH surge with follicle rapture, but without oocyte maturation. The
occurrence of premature luteinization of granulosa cells before LH surge causes poor ovarian
response to controlled ovarian hyperstimulation and an impaired receptivity of the endometrium
leading to reduced pregnancy rates (Fanchin et al., 1993; 1997). In conditions where Cyp11a1 are
upregulated with an abrupt repression in Cyp19a1 there will be a decline in natural and IVF
pregnancy outcome because of the impairment of the normal growth and maturation of the oocyte
which is a direct reflection of the physiological status of granulosa cells. Overall, the findings ofthis study will be useful in understanding that women and livestock exposed to bacterial and viral
infections not only stand the risk of developing follicular cyst conditions but also highly
susceptible to premature luteinization. The different phenotypes between virus infection andbacterial infection are possibly explained by the different timing of TLRs expression in granulosa
cells. The downstream pathway of the TLRs involved in detecting bacterial and viral pathogens
are mostly common, suggesting that epigenetic abnormality in granulosa cells of preovulatory andperiovulatory follicles in pathogen-infected female would induce different transcription pattern.
The abnormal transcriptome might suppress successful ovulation process. The new information
reinforces the need to implement livestock management practices that prevent exposure of animals
to microbes. Furthermore, this understanding regarding luteinization of follicles resulting from77 | P a g e

viral infection is believed to be useful in the diagnosis of reproductive diseases and improving
pregnancy outcomes in both livestock and human.
In conclusion, this study suggests that LPS induces the methylation of the CpG island in the
Cyp19a1 and Lhcgr promoter regions. More so, pathogenic microbes of viral origin have a similareffect as their bacteria counterpart and can cause infertility. TLR7/8 agonist R848 mimics LH
action by induces luteinization of follicles resulting in the formation of corpus luteum. Attention
has been drawn to care of the female gender during the ovulatory phase in a bid to facilitate
conception due to the susceptibility to viral infections at that time point. Over the years, many
bacteria and viruses have consistently caused economic losses to farmers and many women have
tried conceiving but to no avail. However, with the presence of host-cell receptors that allow theentry of viruses, there is the possibility of the use of the mouse model approach to study thesemicrobes and the impact of inflammatory responses posed by immune cells on reproductivefunctioning. Furthermore, given that bacterial and viral pathogens can cause failure of the female
reproductive organ, research focused on improving the efficacy and safety of ART and naturalpregnancy outcomes should explore the impact of ssRNA viruses.

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78 | P a g e

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79 | P a g e

6XPPDU\Infertility is a major cause for concern especially in parts of the world that place a high value onchildbearing. Besides, sub-fertility or infertility often causes heavy losses to breeders and dairy
farmers as they require the animals to get pregnant for milk production and herd expansion.
Infectious diseases of bacteria or viral origin induces inflammatory responses from the host,
however, the mechanism by which they impact negatively on reproductive health is unknown.
These studies aim to determine the deleterious effect of infectious diseases on ovarian functioning
at a cellular and molecular level using the mouse model approach.
 6WXG\RQWKHHIIHFWRI/36RQRYHUDOORYDULDQKHDOWKDQG(SLJHQHWLFG\QDPLFVRIWKH
Lhcgr DQGCyp19a1JHQHSURPRWHUUHJLRQ
Lipopolysaccharide (LPS) is an endotoxin and a component of the cell membrane of gram-negative
bacteria, and it can pass through the protective physical barriers and tight junctions of the basement
membrane. Apart from having been isolated from the genital tract of animals suffering from uterine
damage and ovarian dysfunction, LPS action affects the epigenetic signature of genes regulating
and inducing a cycle of methylation in the DNA around the promoter region. In Experiment 1, it
was hypothesized that bacterial infection changes the DNA methylation status of the Lhcgr andCyp19a1 promoter regions. To clear this hypothesis, granulosa cells and ovaries were collectedfrom immature mice treated with eCG or with eCG and LPS injection intraperitoneal. More so,
granulosa cells were cultured in DMEM/F12 with or without supplement and the addition of LPS(0.1 and 1 μg/ml LPS). Likewise, the expression of DNMT1 in granulosa cells of mice treatedwith LPS was investigated (in vivo and in vitro) and the epigenetic dynamics of LPS on DNA
methylation was studied. In the results, Normal large antral follicles were observed in ovaries
obtained from eCG and LPS coinjected mice, and the morphology of the ovaries was similar to

80 | P a g ethat observed in the control group (eCG-injected mice). These antral follicles were not deemed
atretic because few TUNEL-positive cells were observed. However, the granulosa cells of large
antral follicles did not acquire the ability to respond to hCG stimulation. Also. the number ofovulated oocytes was significantly lower in LPS-injected mice after superovulation compared tomice that were not exposed to LPS. It was also observed that the low reactivity was caused by thelimited expression of the LHCGR gene, which encodes the LH receptor in granulosa cells as wellas an LPS-induced increase in the level of Dnmt1 expression both in vivo and in vitro. The
methylation rate of the LHCGR promoter region was significantly higher in granulosa cells
obtained from the LPS treatment group compared with the control group. Together, for the firsttime these findings demonstrated that the decrease in the expression of Lhcgr and Cyp19a1 as wellas the induction of chemokines is due to bacterial infection/LPS which leads to ovarian follicular
cysts in humans and animals. More so, the decrease in the induction of Lhcgr and Cyp19a1 due to
LPS exposure is a result of the epigenetic regulatory action of LPS.
6WXG\RQWKHUROHVRI7ROOOLNHUHFHSWRULQPRXVHRYDU\
Viral particles and TLR7/8 agonists (Resiquimod) are detected by the host through Pattern
Recognition Receptors (PPRs) and they induce type 1 interferon (IFN) and pro-inflammatory
cytokines. The presence of receptors for specific pathogen allows for entry and transfer of elements
across the host plasma membrane. In experiment 2, it was hypothesized that TLR7/8 are expressedin mouse ovaries and TLR7/8 agonist (R848) causes a negative impact on the structure andfunctioning of the ovary. To clear this hypothesis, the mechanism by which TLR7/8 causes ovariandysfunction was studied by injecting TLR7/8 agonist (R848) to eCG-superovulated immature miceand culturing granulosa cells in DMEM/F12 medium with and without R848 (1 and 10nM dosage).
It was observed that R848 increased the production of cytokine and chemokines in the spleen of
81 | P a g e

mice. Also, in mice treated with R848, the number of matured oocytes and fertilization rate was
reduced. More so, R848 causes a dramatic increase in genes associated with progesterone receptor
and epidermal growth factors such as Areg, Ereg, Cyp11a1, Star, PgR, Snap25 in both in vivo andin vivo studies. For the first time, R848 was reported to prompt the luteinization of follicles. It wasalso established that ACE2 and CD163 which are receptors for SARS-CoV-2 and PRRS are
expressed in mouse ovaries. Although TLR7/8 and ACE2 receptor was expressed in the different
time points, it was highest at 8h point which demonstrates that the influence of viruses is highest
during the ovulatory phase. In addition, for the first time this study shows that the injection of only
R848 to superovulated mice caused the luteinization of follicles and a dose-dependent increase inprogesterone synthesis when granulosa cells were cultured in vitro. These suggeststhat R848 prompts the irreversible transition to a luteal cell phenotype without inducing
ovulation by causing a decline in cell cycle activators and an increase in cell cycle
inhibitors culminating in luteinization. Also, the presence of receptors for SARS-CoV-2 virus and
PRRS in mouse also provides novel opportunities for understanding the mechanism of action ofthese viruses.
&RQFOXVLRQIn conclusion, the present study demonstrated that the decrease in the induction of Lhcgr andCyp19a1 due to LPS is a result of the epigenetic regulatory action of LPS. The immunologicalresponse of the host to LPS or bacterial infection prevents the preovulatory follicles from
responding to the LH surge, which is required for ovulation. Moreover, ovarian dysfunction andpolycystic ovaries, as well as other PIDs that are characterized by bacterial infection in humans
and animals, are closely connected to the methylation of the Lhcgr promoter region. In addition,
R848 suppresses oocyte maturation and fertilization and prompts the irreversible transition to a
82 | P a g e

luteal cell phenotype without inducing ovulation by causing a decline in cell cycle activators andan increase in cell cycle inhibitors culminating in luteinization. This study provides a novel
contribution to the field of mammalian ovarian biology. It is also essential to understanding the
etiology of reproductive diseases and the mechanism of action of infectious diseases in a bid toimprove the efficacy and safety of ART in animal management systems and in developing
therapeutic treatment.



83 | P a g e

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