Baicalin improves the functions of granulosa cells and the ovary in aged mice through the mTOR signaling pathway.

BACKGROUND: The mammalian follicle is the basic functional unit of the ovary, and its normal development is required to obtaining oocytes capable of fertilization. As women get older or decline in ovarian function due to certain pathological factors, the growth and development of follicles becomes abnormal, which ultimately leads to infertility and other related female diseases. Kuntai capsules are currently used in clinical practice to improve ovarian function, and they contain the natural compound Baicalin, which is a natural compound with important biological activities. At present, the role and mechanism of Baicalin in the development of ovarian follicles is unclear. METHODS: Human primary granulosa cells collected from follicular fluid, and then cultured and treated with Baicalin or its normal control, assessed for viability, subjected to RT-PCR, western blotting, flow cytometry, and hormone analyses. The estrus cycle and oocytes of CD-1 mice were studied after Baicalin administration and compared with controls. Ovaries were collected from the mice and subjected to hematoxylin-eosin staining and immunohistochemistry analysis. RESULTS: We showed that Baicalin had a dose-dependent effect on granulosa cells cultured in vitro. A low concentration of Baicalin (for example, 10 µM) helped to maintain the viability of granulosa cells; however, at a concentration exceeding 50 µM, it exerted a toxic effect. A low concentration significantly improved the viability of granulosa cells and inhibited cell apoptosis, which may be related to the resultant upregulation of Bcl-2 expression and downregulation of Bax and Caspase 3. By constructing a hydrogen peroxide-induced cell oxidative stress damage model, we found that Baicalin reversed the cell damage caused by hydrogen peroxide. In addition, Baicalin increased the secretion of estradiol and progesterone by upregulating P450arom and stAR. The results of the in vivo experiment showed that the intragastric administration of Baicalin to aged mice improved the estrous cycle and oocyte quality. Furthermore, we observed that Baicalin enhanced the viability of granulosa cells through the mTOR pathway, which in turn improve ovarian function. CONCLUSION: These results indicate that Baicalin could improve the viability of ovarian granulosa cells and the secretion of steroid hormones and thus could help to improve degenerating ovarian function and delay ovarian aging.

ß-Estradiol inhibits melatonin synthesis and melatonin receptor expression in sheep granulosa cells.

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.

Acupuncture regulates the autophagy of ovarian granulosa cells in polycystic ovarian syndrome ovulation disorder by inhibiting the PI3K/AKT/mTOR pathway through LncMEG3.

The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulation dysfunction, which are caused by the excessive autophagy of ovarian granulosa cells. Acupuncture has been shown to improve ovulation dysfunction and abnormal follicular development in PCOS patients, but its mechanism is unclear. This study hypothesized that the beneficial effects of acupuncture are the result of LncMEG3-mediated effects on the PI3K/AKT/mTOR pathway. Acupuncture (CV-4, RN-3, CV-6, SP-6 and EX-CA 1) was used to treat a rat model of polycystic ovary syndrome. Hematoxylin-eosin staining was used to observe ovarian morphology and enzyme-linked immunosorbent assay, western blotting, immunohistochemistry and real-time PCR were used to detect LH, E2, FSH, T, AMH, LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression. The ovarian morphology of 90% of the rats in the acupuncture treatment group was significantly improved after 11 consecutive days of therapy. Acupuncture also resulted in a significant decrease in serum LH, FSH, T and AMH levels and a significant increase in E2 level (P<0.01). LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression was decreased in ovarian granulosa cells after acupuncture compared with PCOS and lentiviral Intervention Group (P<0.05), while the expression of follicle stimulating hormone receptor was increased (P<0.05). These results indicate that acupuncture can down-regulate the expression of LncMEG3 and thereby inhibit the PI3K/AKT/mTOR pathway, reducing granulosa cell autophagy and normalizing their proliferation. These factors ultimately remedy abnormal follicular development. These findings suggest that acupuncture has clinical potential as a safe treatment for PCOS ovulatory dysfunction.

The major endocannabinoid anandamide (AEA) induces apoptosis of human granulosa cells.

The endocannabinoid system (ECS) plays a crucial role in human reproduction. Changes in anandamide (AEA) levels affect reproductive events and has already been suggested as biomarker of reproductive potential of male and female gametes. Although cannabinoid-receptor 1 (CB1) was already identified in human granulosa cells (hGCs) the ECS was not characterized on granulosa cells line COV434 nor the effects of AEA on GCs viability and function depicted. Therefore, the aim of this study was to characterize the ECS elements and explore the effects of AEA on both COV434 and hGCs. Our results revealed that hGCs express the full enzymatic machinery responsible for AEA metabolism as well as cannabinoid receptors. In addition, AEA induced a reduction in both COV434 and hGCs viability in a concentration and time-dependent manner. Nevertheless, the effects of AEA in cell viability was independent of either CB1 or CB2 receptors. There was no ROS release in both cell models; however, AEA induced morphological changes, presenting chromatin condensation at 72 h, and variation on mitochondrial membrane potential. Moreover, AEA induced an increase in caspase -3/-7 activities in both cell models, but in hGCs there was also an increase in caspase 8 activity. This study supports the idea that ECS balance is crucial for folliculogenesis and oocyte quality as dysregulated AEA levels may compromise female fertility.

Zearalenone Induces Apoptosis and Cytoprotective Autophagy in Chicken Granulosa Cells by PI3K-AKT-mTOR and MAPK Signaling Pathways.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in granulosa cells. This study investigated the effects of ZEA on chicken granulosa cells. The results show that ZEA at different doses significantly inhibited the growth of chicken granulosa cells by inducing apoptosis. ZEA treatment up-regulated Bax and downregulated Bcl-2 expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, resulting in chicken granulosa cells apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast with other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity. In addition, our results further show that the autophagy was stimulated by ZEA through PI3K-AKT-mTOR and MAPK signaling pathways in chicken granulosa cells.

Estrogen disorders: Interpreting the abnormal regulation of aromatase in granulosa cells (Review).

Ovarian granulosa cells (GCs) are the most important source of estrogen. Therefore, aromatase (estrogen synthase), which is the key enzyme in estrogen synthesis, is not only an important factor of ovarian development, but also the key to estrogen secretion by GCs. Disorders of the ovarian estrogen secretion are more likely to induce female estrogen­dependent diseases and fertility issues, such as ovarian cancer and polycystic ovary syndrome. Hence, aromatase is an important drug target; treatment with its inhibitors in estrogen­dependent diseases has attracted increasing attention. The present review article focuses on the regulation and mechanism of the aromatase activity in the GCs, as well as the specific regulation of aromatase promoters. In GCs, follicle­stimulating hormone (FSH) is dependent on the cyclic adenosine monophosphate (cAMP) pathway to regulate the aromatase activity, and the regulation of this enzyme is related to the activation of signaling pathways, such as phosphatidylinositol 3­kinase (PI3K) and extracellular signal­regulated kinase (ERK). In addition, endocrine­disrupting substance and other related factors affect the expression of aromatase, which eventually create an imbalance in the estrogen secretion by the target tissues. The present review highlights these useful factors as potential inhibitors for target therapy.

Determination of c-Abl tyrosine kinase and mTERT catalytic subunit of telomerase expression level during prenatal-postnatal mouse ovary-testis development.

Expression levels of genes involved in the development of germ cells vary throughout the process from bipotential gonadal period to adult gonadal formation. In mice, developments of female and male reproductive system are regulated by germ cell-specific factors and hormones, and determinative days in this regulation are very important. c-Abl is a non-receptor tyrosine kinase with cellular functions including cell proliferation, growth and development. mTERT is involved in maintaining telomerase activity and proliferation of surviving cells. We suggested that c-Abl and mTERT might be important for the healthy development of prenatal and postnatal mouse ovary and testis. We aim to demonstrate localization and expressions of c-Abl and mTERT in crucial days of ovary and testis development in prenatal and postnatal period in mouse by immunofluorescence staining and qRT-PCR, respectively. The importance of c-Abl and mTERT expressions during the healthy gonadal development is indicated in the prenatal and postnatal gonadal development. Also, protein expression levels were detected by Western Blot in only postnatal ovary and testis. Determining the functions of the c-Abl and mTERT throughout the process will be important in terms of understanding the infertility cases in the female and male with future studies.

In Search of New Therapeutics-Molecular Aspects of the PCOS Pathophysiology: Genetics, Hormones, Metabolism and Beyond.

In a healthy female reproductive system, a subtle hormonal and metabolic dance leads to repetitive cyclic changes in the ovaries and uterus, which make an effective ovulation and potential implantation of an embryo possible. However, that is not so in the case of polycystic ovary syndrome (PCOS), in which case the central mechanism responsible for entraining hormonal and metabolic rhythms during the menstrual cycle is notably disrupted. In this review we provide a detailed description of the possible scenario of PCOS pathogenesis. We begin from the analysis of how a set of genetic disorders related to PCOS leads to particular malfunctions at a molecular level (e.g., increased enzyme activities of cytochrome P450 (CYP) type 17A1 (17α-hydroxylase), 3ß-HSD type II and CYP type 11A1 (side-chain cleavage enzyme) in theca cells, or changes in the expression of aquaporins in granulosa cells) and discuss further cellular- and tissue-level consequences (e.g., anovulation, elevated levels of the advanced glycation end products in ovaries), which in turn lead to the observed subsequent systemic symptoms. Since gene-editing therapy is currently out of reach, herein special emphasis is placed on discussing what kinds of drug targets and which potentially active substances seem promising for an effective medication, acting on the primary causes of PCOS on a molecular level.

Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†.

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3ß-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3ß-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

Salt-inducible Kinases Are Critical Determinants of Female Fertility.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3',5'-cyclic adenosine 5'-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Caspase-3 gene expression in human luteinized granulosa cells is inversely correlated with the number of oocytes retrieved after controlled ovarian stimulation.

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman's r = -0.308), the number of collected oocytes (r = -0.451), the number of mature oocytes (r = -0.526), the number of fertilized oocytes (r = -0.439) and the number of viable embryos (r = -0.443, all statistically significant at p < 0.02 level). No such associations were found with caspase-8, bax or bcl-2. These preliminary findings suggest that increased caspase-3 gene expression in granulosa cells is associated with a worse ovulatory response in humans.

Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women.

Sirtuins are a family of deacetylases that modify structural proteins, metabolic enzymes, and histones to change cellular protein localization and function. In mammals, there are seven sirtuins involved in processes like oxidative stress or metabolic homeostasis associated with aging, degeneration or cancer. We studied gene expression of sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL) from IVF patients in different infertility diagnostic groups and in oocyte donors (OD; control group). Study 1: sirtuins genes' expression levels and correlations with age and IVF parameters in women with no ovarian factor. We found significantly higher expression levels of SIRT1, SIRT2 and SIRT5 in patients ≥40 years old than in OD and in women between 27 and 39 years old with tubal or male factor, and no ovarian factor (NOF). Only SIRT2, SIRT5 and SIRT7 expression correlated with age. Study 2: sirtuin genes' expression in women poor responders (PR), endometriosis (EM) and polycystic ovarian syndrome. Compared to NOF controls, we found higher SIRT2 gene expression in all diagnostic groups while SIRT3, SIRT5, SIRT6 and SIRT7 expression were higher only in PR. Related to clinical parameters SIRT1, SIRT6 and SIRT7 correlate positively with FSH and LH doses administered in EM patients. The number of mature oocytes retrieved in PR is positively correlated with the expression levels of SIRT3, SIRT4 and SIRT5. These data suggest that cellular physiopathology in PR's follicle may be associated with cumulative DNA damage, indicating that further studies are necessary.

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway.

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.

MiRNA-335-5p negatively regulates granulosa cell proliferation via SGK3 in PCOS.

Polycystic ovary syndrome (PCOS) is a major cause of infertility in women of reproductive age. However, its exact etiology remains unknown. In this study, we sequenced miRNAs in human follicular fluid and identified 16 downregulated and 3 upregulated miRNAs in PCOS group compared with non-PCOS group. Among the differential expressed miRNAs, miR-335-5p was verified lower abundance in PCOS than non-PCOS group using quantitative real-time PCR. Besides, miR-335-5p negatively correlated with antral follicle count, anti-Müllerian hormone and total testosterone. Bioinformatics analysis identified serum/glucocorticoid-regulated kinase family member 3 (SGK3) as a potential target gene of miR-335-5p. SGK3 is involved in protein kinase B-mammalian target of rapamycin kinase (AKT-mTOR) signaling pathway and cell proliferation. Western blotting and cell counting kit-8 assays demonstrated that miR-335-5p mimic reduced, while miR-335-5p inhibitor increased, SGK3 abundance, AKT-mTOR pathway and cell proliferation in human granulosa-like tumor KGN cells. Dual-luciferase reporter assays showed that miR-335-5p binds to the 3' untranslated region of SGK3 mRNA. Furthermore, miR-335-5p was decreased and SGK3 was elevated in human granulosa cells obtained from PCOS patients as compared with non-PCOS controls. These findings suggested that miR-335-5p is involved in granulosa cells proliferation by reducing SGK3 expression, which might provide a molecular target to improve dysfunctional granulosa cells in patients with PCOS.

Role of granulosa cell mitogen-activated protein kinase 3/1 in gonadotropin-mediated meiotic resumption from diplotene arrest of mammalian oocytes.

In mammals, preovulatory oocytes are encircled by several layers of granulosa cells (GCs) in follicular microenvironment. These follicular oocytes are arrested at diplotene arrest due to high level of cyclic nucleotides from encircling GCs. Pituitary gonadotropin acts at the level of encircling GCs and increases adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and activates mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. The MAPK3/1 disrupts the gap junctions between encircling GCs and oocyte. The disruption of gap junctions interrupts the transfer of cyclic nucleotides to the oocyte that results a drop in intraoocyte cAMP level. A transient decrease in oocyte cAMP level triggers maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggers meiotic resumption from diplotene arrest in follicular oocyte. Thus, MAPK3/1 from GCs origin plays important role in gonadotropin-mediated meiotic resumption from diplotene arrest in follicular oocyte of mammals.

Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice.

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.

Vitamin D3 regulates steroidogenesis in granulosa cells through AMP-activated protein kinase (AMPK) activation in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder in reproductive-aged women. Hormonal abnormality caused by steroidogenesis disturbances appears to be the main culprit of the clinical picture in PCOS. Vitamin D3 could regulate steroidogenesis in granulosa cells, but the mechanism of action of vitamin D3 on steroidogenesis remains unknown. AMP-activated protein kinase (AMPK) has a modulating role in steroid hormone production. We investigated the effect of vitamin D3 on steroidogenesis in cultured granulosa cells of dehydroepiandrosterone-induced PCOS mice and studied the involvement of AMPK signalling pathway in the current process. Immunoblotting assay showed that vitamin D3 could increase phosphorylation of AMPK alpha and acetyl-CoA carboxylase, main substrate of AMPK. Vitamin D3 and 5-aminoimidazole-4-carboxamide-1-ß-D-riboside or Aicar (AMPK activator) not only reduced gene expression of steroidogenic enzymes (P450scc or Cyp11a1, StAR, Cyp19a1 and 3B-HSD), but also reduced production of progesterone and 17B-estradiol assessed by radioimmunoassay. Pretreatment with compound C (AMPK inhibitor) decreased APMK phosphorylation and eliminated the effects of vitamin D3 and Aicar on steroidogenic enzymes expression and estradiol and progesterone production. This study showed that vitamin D3 has the main role in regulating of steroidogenesis in granulosa cells of mouse polycystic ovary through activation of the AMPK signalling pathway. SIGNIFICANCE OF THE STUDY: Polycystic ovarian syndrome (PCOS) is an endocrine disorder of women in reproductive age. This disorder is partly related to disruption in steroidogenesis pathway and dysregulation of estradiol and progesterone production in granulosa cells of polycystic ovaries. Previously, we have shown that vitamin D3 could modulate steroidogenesis pathway in PCOS granulosa cells. In this study, we investigate the molecular mechanism of vitamin D3 in regulation of steroidogenesis pathway. We have shown that vitamin D3 has a modulating role in steroidogenesis pathway of granulosa cells by regulation of AMP-activated protein kinase (AMPK) as an underlying molecular mechanism in mouse polycystic ovary.

Activated receptor tyrosine kinases in granulosa cells of ovulating follicles in mice.

Successful ovulation requires the actions of gonadotropins along with those mediated by growth factors binding to their receptor tyrosine kinases (RTKs). There are several growth factors such as epidermal growth factor family ligands and interleukins that play a role during ovulation initiated by the preovulatory surge of luteinizing hormone (LH). The aim of this project was to analyze growth factor signaling pathways induced by LH in mouse granulosa cells. Immature female mice were treated with equine chorionic gonadotropin (eCG) followed 48 hr later by human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. We performed protein array analysis where we identified higher phosphorylation of insulin-like growth factor 1 receptor (IGF1R), the fibroblast growth factor receptor 2 (FGFR2) and ephrin receptor B1 (EPHB1) in granulosa cells at 4 hr post-hCG compared to 0 hr hCG (p < 0.05). We report both a significant increase in transcript abundance (p < 0.05) and the phosphorylation level (p < 0.05) of the IGF1R in granulosa cells at hCG4h. The mRNA abundance of the Fgfr2 and Ephb1 receptors remained unaltered upon hCG treatment. Nonetheless, transcript abundance of the fibroblast growth factor 2 (Fgf2) ligand was elevated at hCG4h (p < 0.01). Based on these results we conclude that the preovulatory LH surge activates signaling pathways of IGF1R through increase in the expression of the Igf1r gene in granulosa cells of ovulating follicles in mice. The LH surge also appears to activate FGFR2 IIIc and EPHB1 signaling, although further investigation is required.