Mechanisms by which Bacterial and Viral Pathogens cause Ovarian dysfunction

概要

Doctoral Thesis

Mechanisms by which Bacterial and Viral Pathogens cause
Ovarian dysfunction

(Summary)
Adedeji Olufemi Adetunji

Graduate School of Biosphere Science
Hiroshima University

September 2021

Infertility is a major cause for concern especially in parts of the world that place a high value
on childbearing. 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.
1. Study on the effect of LPS on overall ovarian health and Epigenetic dynamics of
the Lhcgr and Cyp19a1 gene promoter region
Lipopolysaccharide (LPS) is an endotoxin and a component of the cell membrane of gramnegative 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 and Cyp19a1 promoter regions. To clear this hypothesis,
granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG
and LPS injection intraperitoneally. 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 treated with 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 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 of ovulated oocytes was significantly lower in
LPS-injected mice after superovulation compared to mice that were not exposed to LPS. It was
also observed that the low reactivity was caused by the limited expression of the LHCGR gene,
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 with the control group. Together, these findings demonstrated that the decrease
in the expression of Lhcgr and Cyp19a1 as well as 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.
2. Study on the roles of Toll-like receptor 7/8 in mouse ovary
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 expressed in mouse ovaries and TLR7/8 agonist (R848) causes a negative impact on the
structure and functioning of the ovary. To clear this hypothesis, the mechanism by which
TLR7/8 causes ovarian dysfunction was studied by injecting TLR7/8 agonist (R848) to eCGsuperovulated immature mice and 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 mice. Also, in mice treated with R848, the number of
matured oocytes and fertilization rate was reduced. More so, R848 was capable of causing 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 and in vivo studies. For the first
time, R848 was reported to prompt the luteinization of follicles. It was also 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, the injection of only R848 to superovulated mice caused the
luteinization of follicles and a dose-dependent increase in progesterone synthesis when
granulosa cells were cultured in vitro. These suggests that 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 of these viruses.
3. Conclusion
In conclusion, the present study demonstrated that the decrease in the induction of Lhcgr and
Cyp19a1 due to LPS is a result of the epigenetic regulatory action of LPS. The immunological
response 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
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. In addition,
R848 suppresses oocyte maturation and fertilization and 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. This study provides a novel
contribution to the field of mammalian ovarian biology. It is also essential to understanding the
etiology of ovulation disorder and the mechanism of action of infectious diseases in a bid to
improve the efficacy and safety of ART in animal management systems and in developing
therapeutic treatment.

Keywords: ovulation, luteinization, oocyte maturation, follicular cyst and ovary