Mesenchymal Stem Cell-Conditioned Media Regulate Steroidogenesis and Inhibit Androgen Secretion in a PCOS Cell Model via BMP-2.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women. Previous studies have demonstrated the therapeutic efficacy of human bone marrow mesenchymal stem cells (BM-hMSCs) for PCOS; however, the regulatory mechanism remains unknown. Bone morphogenetic proteins (BMPs) secreted by BM-hMSCs may underlie the therapeutic effect of these cells on PCOS, based on the ability of BMPs to modulate androgen production and alter steroidogenesis pathway enzymes. In this study, we analyze the effect of BMP-2 on androgen production and steroidogenic pathway enzymes in H295R cells as a human PCOS in vitro cell model. In H295R cells, BMP-2 significantly suppressed cell proliferation, androgen production, and expression of androgen-synthesizing genes, as well as inflammatory gene expression. Furthermore, H295R cells treated with the BM-hMSCs secretome in the presence of neutralizing BMP-2 antibody or with BMP-2 gene knockdown showed augmented expression of androgen-producing genes. Taken together, these results indicate that BMP-2 is a key player mediating the favorable effects of the BM-hMSCs secretome in a human PCOS cell model. BMP-2 overexpression could increase the efficacy of BM-hMSC-based therapy, serving as a novel stem cell therapy for patients with intractable PCOS.

Induction of tumor suppressor KCTD11 during periovulatory period in rat ovary.

The tumor suppressor gene KCTD11 plays a critical role in cell proliferation, differentiation and invasion. The current study investigated the regulation and the spatiotemporal expression pattern of Kctd11 in the rat ovary during the periovulatory period. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration using an established gonadotropin-primed immature rat model that induces follicular development and ovulation. Real-time quantitative PCR analysis revealed that mRNA for Kctd11 was significantly induced both in theca-intersititial and granulosa cells after hCG treatment although their temporal expression patterns differed. In situ hybridization analysis demonstrated that Kctd11 mRNA expression was induced in theca-intersititial cells at 6 h after hCG, and the expression remained elevated until 12 h after hCG. Kctd11 mRNA was stimulated in granulosa cells at 6 h and reached the highest expression at 12 h. There was negligible Kctd11 mRNA signal observed in newly forming corpora lutea. In addition, the data indicate that both the protein kinase A and the protein kinase C pathway regulate the expression of Kctd11 mRNA in granulosa cells. Either forskolin or phorbol 12 myristate 13-acetate can mimic hCG induction of Kctd11 expression. Furthermore, the stimulation of Kctd11 by hCG requires new protein synthesis. Inhibition of progesterone action and the EGF pathway blocked Kctd11 mRNA expression, whereas inhibition of prostaglandin synthesis had no effect. Our finding suggest that the induction of the Kctd11 may be important for theca and granulosa cell differentiation into luteal cells.

CCL5 secreted by senescent theca-interstitial cells inhibits preantral follicular development via granulosa cellular apoptosis.

As a fundamental aging mechanism, cellular senescence causes chronic inflammation via the senescence-associated secretory phenotype (SASP). Theca-interstitial cells are an essential but little-studied component of follicle development in the ovarian microenvironment. In the present study, we observed significant cellular senescence in theca-interstitial cells and secretion of chemokine (C-C motif) ligand 5 (CCL5) by these cells during aging. Furthermore, we aimed to investigate whether and how senescence-associated secretory phenotype (SASP)-associated CCL5 may be involved in follicle development. Increased levels of CCL5 in the microenvironment of follicles attenuated preantral follicle growth, survival, and estradiol secretion. Oocyte maturation and the expression of zona pellucida 3 and differentiation factor 9 (GDF9) were also inhibited by CCL5. Granulosa cell apoptosis in follicles was promoted by CCL5, accompanied by the phosphorylation of nuclear factor-κB by CCL5 and inhibition of the PI3K/AKT pathway. These results suggest that SASP-associated CCL5 produced by senescent theca-interstitial cells may impair follicle development and maturation during ovarian aging by promoting granulosa cell apoptosis.

Regulation of follicular development and differentiation by intra-ovarian factors and endocrine hormones.

Primordial germ cell migration and homing within the gonadal ridge during early embryo development requires oocyte-secreted polypeptide, growth factors, growth and differentiation factors (GDFs), bone morphogenetic proteins, stem cell factor (SCF), and basic fibroblast growth factor (bFGF). During embryogenesis, the germ cells migrate into developing gonads and undergo intra-ovarian development which involves the contact of primordial germ cells with other cells. Further follicular development and differentiation is tightly regulated by hormones and by intraovarian regulators. Maturation of cumulus-oocyte complexes and ovulation are directly controlled by FSH and LH and requires activation of mitogen-activated protein kinase in granulosa cells. The selection of dominant follicles is driven by a series of proliferation and apoptotic events. Together, the available data suggests that follicular development is regulated both by systemic and local factors.

Experimental polycystic ovarian syndrome is associated with reduced expression and function of P2Y2 receptors in rat theca cells.

Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 µM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS.

Histological characteristics of ovarian follicle atresia in dairy cows with different milk production.

Follicle atresia in mammals is a universal phenomenon characteristic by degenerative morphological changes in granulosa and theca cells. The unfavourable effect of milk production in relation to fertility has been studied starting from the 70s of the last century; however, there is no unambiguous and persuasive data on association of ovarian atresia with milk yield of dairy cows. The aim of this study was to define histological signs of ovarian follicle atresia in dairy cows in relation to their milk production. The ovaries were recovered from slaughtered Holstein dairy cows assigned into two groups according to average level of annual milk production: Group 1 (n = 25)-low (≤8,000 kg/year) and Group 2 (n = 23)-high (≥8,000 kg/year). Atresia of antral follicles was evaluated on the basis of histopathological image (staining with basic fuchsine and toluidine blue) of nonovulated follicles, classified into five categories: an initial atresia, cystic atresia, obliterated atresia, atresia with luteinization of the granulosa and follicle structures of the fibrous body-corpus fibrosum. We found that the histopathological image of follicle atresia in groups of low-milk- or high-milk-producing cows is essentially similar. Prevalent form of atresia in follicles of all experimental cows was the formation of fibrous bodies and obliterated atresia. The occurrence of fibrous bodies was significantly higher (55.44%) in low-milk-producing cows compared with high-milk-producing cows (34.61%). In the same way, the higher incidence of obliterated atresia was recorded in ovarian follicles from cows with the lower milk production (36.96%) compared to the cows with the higher milk production (25.48%). In contrast, ovaries from lower milk-producing cows showed lower (p < 0.05) incidence of initial (p < 0.001) and cystic (p < 0.05) follicle atresia than ovaries from the higher milk-producing cows. Our results show that cows in the higher lactation group showed more initial and cystic atresia, what may adversely affect the fertility of dairy cows.

The function of high-density lipoprotein and low-density lipoprotein in the maintenance of mouse ovarian steroid balance.

The membrane proteins, low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SR-BI, gene name Scarb1), are lipoprotein receptors that play central roles in lipoprotein metabolism. Cholesterol bound in high-density lipoprotein (HDL) and LDL is transported into cells mainly by SR-BI and LDLR. The relative contribution of LDL and HDL to the steroidogenic cholesterol pool varies among species and may vary among tissues within one species. To investigate which of these pathways is more important in the supply of cholesterol in mouse ovary, we utilized immunohistochemistry, western blotting, RNAi, and RT-PCR as well as Ldlr-/- mice to explore the uptake of HDL and LDL in the ovary. Our data demonstrate that both SR-BI and LDLR are present in the interstitial cells, thecal cells, and corpora lutea (CLs), and their expression fluctuates with the development of follicles and CLs. The intracellular cholesterol concentration was significantly decreased when Ldlr or Scarb1 was silenced in luteal cells. Furthermore, Ldlr-/- mice had lower progesterone and estrogen levels compared to wild-type mice, and when Ldlr-/- mice were treated with the inhibitor of de novo cholesterol synthesis, lovastatin, serum progesterone, and estrogen concentrations were further reduced. These results demonstrate that both LDLR and SR-BI play important roles in importing cholesterol and that both HDL and LDL are crucial in steroidogenesis in mouse ovaries.

Molecular assessment, characterization, and differentiation of theca stem cells imply the presence of mesenchymal and pluripotent stem cells in sheep ovarian theca layer.

The ability of ovarian theca stem cells to differentiate into oocyte and theca cells may lead to a major advancement in reproductive biology and infertility treatments. However, there is little information about function, growth and differentiation potential of these immature cells. In this study adult sheep theca stem cells (TSCs) characteristics, and differentiation potential into osteocyte-like cells (OSLCs), adipocyte-like cells (ALCs), theca progenitor-like cells (TPCs), and oocyte-like cells (OLCs) were investigated. TSCs were isolated, cultured, and compared with mesenchymal stem cells (MSCs), fibroblast cells (FCs), and pluripotent embryonic ovarian cells (EO). Adherent TSCs were morphologically similar to FCs. Cell cycle analysis showed high proliferation capacity of TSCs. TSCs were positive for the mesenchymal cells surface markers, and also expressed POU5F1. Differentiation potential of TSCs into OSLCs and ALCs were confirmed by alizarin red and oil red staining respectively. OSTEOCALCIN and COL1 were expressed in OSLCs. ALCs were positive for PPARα and LPL. TPCs expressed theca specific genes (GLI2, GLI3, PTCH1, CYP17A1, 3ß-HSD and LHR) and secreted testosterone, dehydroepiandrostenedione (DHEA), androstenedione, progesterone and estradiol. Lipid droplets in these steroid cells were viewed by oil red staining. OLCs expressed oocyte-specific marker genes including, ZP3, ZP2, GDF9, SYCP3, PRDM1, STELLA, FRAGILIS, DAZL, as well as POU5F1, and showed separated sphere structure. Our results indicated that TSCs derived from ovarian follicles contain MSCs and pluripotent stem cells (PSCs) that can be differentiated into lineages of mesenchymal origin and are capable of differentiation into TPCs and OLCs under in vitro conditions.

Expression of apelin and apelin receptor (APJ) in porcine ovarian follicles and in vitro effect of apelin on steroidogenesis and proliferation through APJ activation and different signaling pathways.

Apelin was thought to be an adipocyte-specific hormone, but recent studies indicate a link between apelin and female reproductive function. Using real-time PCR, immunoblotting, immunohistochemistry and ELISA, we demonstrated expression of apelin and its receptor (APJ) in ovarian follicles of different sizes from mature pigs. Apelin concentration in the follicular fluid, and expression of both apelin and APJ, increased with follicular growth; greatest values were found in large follicles. Immunohistochemistry revealed the positive staining for apelin and APJ in membranes of granulosa, than theca cells. Furthermore, we observed strong expression of apelin in oocytes and APJ in the zona pellucida. The effect of apelin (0.02, 0.2, 2 and 20 ng/ml) on basal and IGF1- and FSH-induced steroid hormone (progesterone [P4], and estradiol [E2]) secretion, steroidogenic enzyme (3ßHSD and CYP19A1) expression and cell proliferation (Alamar blue) was determined. Apelin was found to increase basal steroid secretion, but decrease IGF1- and FSH-induced steroid secretion, and 3ßHSD and CYP19 expression. Apelin also increased cell proliferation and the phosphorylation level of 5'-monophosphate-activated protein kinase (AMPK), phosphatidyl inositol 3' kinase/Akt (Akt) and extracellular signal-regulated kinases (ERK1/2). AMPKα was involved in the action of apelin in P4 production, and MAPK/ERK and Akt/PI3 mediated the proliferative effect of apelin. However, these effects on steroid secretion and cell proliferation were abolished when cultured in the presence of ML221, an APJ antagonist. In conclusion, apelin appears to regulate ovarian follicular functions such as steroidogenesis and proliferation via APJ activation and different signaling pathways.

Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells.

The Notch signaling pathway regulates cell proliferation, differentiation and apoptosis involved in development of the organs and tissues such as nervous system, cartilage, lungs, kidneys and prostate as well as the ovarian follicles. This study aimed to investigate the mRNA expression and localization of NOTCH2, as the key factor in Notch signaling pathway. This was determined by PCR, real-time PCR and immunohistochemistry. Additionally, the effects of inhibiting Notch signaling pathway with different concentrations (5µM, 10µM and 20µM) of N-[N-(3, 5-Difuorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling pathway, on ovine granulosa cells was determined in vitro by detecting estradiol production using enzyme linked immunosorbent assay and expressions of the genes related to the cell cycle and apoptosis using real-time polymerase chain reaction (PCR). NOTCH2, the key member of Notch signaling pathway, was found in ovine follicles, and the expression of NOTCH2 mRNA was highest in the theca cells of the follicles in medium sizes (3-5mm in diameter) and granulosa cells of the follicles in large sizes (>5mm in diameter). Immunohistochemical results demonstrated that NOTCH2 protein was expressed in granulosa cells of preantral follicles, in both granulosa cells and theca cells of antral follicles. Compared with DAPT-treated groups, the control group had a higher number of granulosa cells (P<0.05) and a higher estradiol production (P<0.05). Compared with the control group, the mRNA abundances of HES1, MYC, BAX, BCL2 and CYP19A1 in DAPT-treated groups was lower (P<0.05), respectively; whereas, the expression of CCND2, CDKN1A and TP53 mRNA showed no remarkable difference compared with control group. Collectively, Notch signaling pathway could be involved in the ovine follicular development by regulating the growth and estradiol production of granulosa cells.

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (P<0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200µM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P<0.05). Only the oocytes' cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.

Thecal cell sensitivity to luteinizing hormone and insulin in polycystic ovarian syndrome.

This study examined whether a defect of steroid synthesis in ovarian theca cells may lead to the development of PCOS, through contributions to excess androgen secretion. Polycystic ovarian syndrome (PCOS) is one of the leading causes of infertility worldwide affecting around 1 in 10 of women of a reproductive age. One of the fundamental abnormalities in this syndrome is the presence of hormonal irregularities, including hyperandrogenemia, hyperinsulinemia and hypersecretion of luteinizing hormone (LH). Studies suggest that insulin treatment increases progesterone and androstenedione secretion in PCOS theca cells when compared to insulin treated normal theca cells. Furthermore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Our examination of primary thecal cultures showed an increase in both the expression of the steroidogenic enzyme CYP17 and androgen secretion in PCOS theca cells under basal conditions, when compared to non-PCOS cells. This was increased significantly under treatments of LH and insulin combined. Our results support the previous reported hypothesis that a dysfunction may exist within the PI3-K pathway. Specifically, that sensitivity exists to physiological symptoms including hyperinsulinemia and hyper secretion of LH found in PCOS through co-stimulation. The impact of these findings may allow the development of a therapeutic target in PCOS.

Characterization of annexin A2 in chicken follicle development: Evidence for its involvement in angiogenesis.

Annexin A2 (ANXA2) is a calcium-dependent, phospholipid-binding protein found in various cells and tissues. ANXA2 plays multiple roles in regulating cellular functions and is often over-expressed in different types of tumors including ovarian cancer. Others and we previously found that ANXA2 was up-regulated in the ovaries of hens with higher laying rate, indicated that ANXA2 is involved in avian follicle development. In this study, we found that ANXA2 mRNA expression increased during chicken ovary maturation and follicle development. In the pre-ovulatory follicles, ANXA2 expression level was significantly higher in theca cells than granulosa cells. In theca cells, ANXA2 expression could be stimulated by follicle-stimulating hormone (FSH) and estrogen but not luteinizing hormone (LH) or progesterone. The core promoter regions control the basal and FSH-induced ANXA2 gene expression were identified. Forced expression of ANXA2 could induce the expression of angiogenic factors and receptors in theca cells. Furthermore, ANXA2 overexpression resulted increased vascular endothelial growth factor A (VEGFA) secretion and theca cell proliferation. Current study not only provides the first evidence of expression and regulation of ANXA2 in chicken ovary, but also suggests that ANXA2 is involved in follicular angiogenesis and contributes to successful follicle development and ovulation.

Obesity-induced infertility and hyperandrogenism are corrected by deletion of the insulin receptor in the ovarian theca cell.

Women with polycystic ovary syndrome (PCOS) exhibit elevated androgen levels, oligoanovulation, infertility, and insulin resistance in metabolic tissues. The aims of these studies were to determine the role of insulin signaling in the development and function of ovarian theca cells and the pathophysiologic effects of hyperinsulinism on ovarian function in obesity. We disrupted the insulin receptor (IR) gene specifically in the theca-interstitial (TI) cells of the ovaries (Cyp17IRKO). No changes in reproductive development or function were observed in lean Cyp17IRKO female mice, suggesting that insulin signaling in TI cell is not essential for reproduction. However, when females were fed a high-fat diet, diet-induced obesity (DIO) wild-type (DIO-WT) mice were infertile and experienced increased circulating testosterone levels, whereas DIO-Cyp17IRKO mice exhibited improved fertility and testosterone levels comparable to those found in lean mice. The levels of phosphorylated IRS1 and CYP17 protein were higher in the ovary of DIO-WT compared with DIO-Cyp17IRKO or lean mice. Ex vivo studies using a whole ovary culture model demonstrated that insulin acts independently or additively with human chorionic gonadotropin to enhance androstenedione secretion. These studies reveal the causal pathway linking hyperinsulinism with ovarian hyperandrogenism and the infertility of obesity.

The influence of ovarian stromal/theca cells during in vitro culture on steroidogenesis, proliferation and apoptosis of granulosa cells derived from the goat ovary.

Early follicular development is closely related to oocyte-granulosa cells-ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis-related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.

Paracrine regulation of theca androgen production by granulosa cells in the ovary.

OBJECTIVE: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs). DESIGN: Ex vivo and in vitro experimental study. SETTING: University. ANIMAL(S): Immature female Sprague Dawley rats. INTERVENTION(S): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or ß-glycan antibody. MAIN OUTCOME MEASURE(S): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription-polymerase chain reaction. RESULT(S): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, ß-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent. CONCLUSION(S): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs by virtue of FSH seem to be required.

Ovarian regulation of neuromedin U and its local actions in the ovary, mediated through neuromedin U receptor 2.

Neuromedin U (NMU) was originally identified as an anorexigenic peptide that modulates appetite as well as energy homeostasis through the brain-gut axis. Although growing evidence has linked NMU activity with the development of female reproductive organs, no direct expression of and function for NMU in these organs has been pinpointed. Using a superovulated rat model, we found that NMU is directly expressed in the ovary, where its transcript level is tightly regulated by gonadotropins. Ovarian microdissection and immunohistochemical staining showed clearly that NMU is expressed mainly in theca/interstitial cells and to a moderate extent in granulosa cells. Primary cell studies together with reporter assays indicated the Nmu mRNA level in these cells is strongly induced via cAMP signaling, whereas this increase in expression can be reversed by the degradation message residing within its 3'-untranslated region, which recruits cis-acting mRNA degradation mechanisms, such as the gonadotropin-induced zinc finger RNA-binding protein Zfp36l1. This study also demonstrated that NMUR2, but not NMUR1, is the dominant NMU receptor in the ovary, where its expression is restricted to theca/interstitial cells. Treatment with NMU led to induction of the early response c-Fos gene, phosphorylation of extracellular signal-regulated kinase 1/2, and promotion of progesterone production in both developing and mature theca/interstitial cells. Taken as a whole, this study demonstrates that NMU and NMU receptor 2 compose a novel autocrine system in theca/interstitial cells in which the intensity of signaling is tightly controlled by gonadotropins.

Expression and effect of fibroblast growth factor 9 in bovine theca cells.

Fibroblast growth factor 9 (FGF9) protein affects granulosa cell (GC) function but is mostly localized to theca cell (TC) and stromal cell of rat ovaries. The objectives of this study were to determine the 1) effects of FGF9 on TC steroidogenesis, gene expression, and cell proliferation; 2) mechanism of action of FGF9 on TCs; and 3) hormonal control of FGF9 mRNA expression in TCs. Bovine ovaries were collected from a local slaughterhouse and TCs were collected from large (8-22 mm) follicles and treated with various hormones in serum-free medium for 24 or 48 h. FGF9 caused a dose-dependent inhibition (P<0·05) of LH- and LH+IGF1-induced androstenedione and progesterone production. Also, FGF9 inhibited (P<0·05) LH+IGF1-induced expression of LHCGR, CYP11A1, and CYP17A1 mRNA (via real-time RT-PCR) in TCs. FGF9 had no effect (P>0·10) on STAR mRNA abundance. Furthermore, FGF9 inhibited dibutyryl cAMP-induced progesterone and androstenedione production in LH+IGF1-treated TCs. By contrast, FGF9 increased (P<0·05) the number of bovine TCs. Abundance of FGF9 mRNA in GCs and TCs was several-fold greater (P<0·05) in small (1-5 mm) vs large follicles. Tumor necrosis factor α and WNT5A increased (P<0·05) abundance of FGF9 mRNA in TCs. In summary, expression of FGF9 mRNA in TCs is developmentally and hormonally regulated. FGF9 may act as an autocrine regulator of ovarian function in cattle by slowing TC differentiation via inhibiting LH+IGF1 action via decreasing gonadotropin receptors and the cAMP signaling cascade while stimulating proliferation of TCs.

Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary.

Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5  mm), pre-ovulatory follicles (6.0-7.0  mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.

Metformin: direct inhibition of rat ovarian theca-interstitial cell proliferation.

OBJECTIVE: To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK). DESIGN: In vitro experimental study. SETTING: Academic medical center laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 µg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). MAIN OUTCOME MEASURE(S): Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. RESULT(S): Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. CONCLUSION(S): Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).