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The aim of this study was to demonstrate the effects of vitamin D treatment on ultrastructural changes and AMHR2 expression in the ovary in PCOS rat model. A total of 24 female prepubertal rats were divided into 3 groups. In group 1, sesame oil was injected and used as control group. In group 2, PCOS was created by the injection of 6 mg/kg/day DHEA. In group 3, PCOS was created and 120 ng/100 g 1,25 (OH)2D3 treatment was performed. At the end of the 28th day, the blood samples were collected. The ovarian tissues were obtained for electron microscopic and immunohistochemical examinations. Serum AMH, testosterone, FSH, LH levels and LH/FSH ratios were higher in the PCOS group compared to the control group and decreased in the treatment group compared to the PCOS group. AMHR2 expression was increased in atretic and premature luteinizate antral follicles in the PCOS group compared to the control group, and decreased in the treatment group compared to the PCOS group. PCOS group electron micrographs showed degenerative changes in developing follicles, cystic follicles characterised with granulosa cell layer attenuation and thickening of the theca cell layer, and lipid accumulation in the interstitial cells. Structural changes observed in the PCOS group were improved with vitamin D treatment. As a result, there is an interaction between PCOS, AMH serum levels and AMHR2 in the ovarian follicles. Vitamin D has a positive effect on hormonal and structural changes in the PCOS group. We concluded that vitamin D supplementation may be beneficial in PCOS patients.

Background In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women. Androgen excess is observed in women with polycystic ovary syndrome (PCOS) resulting from excess endogenous androgen production, and in transgender males undergoing exogenous testosterone therapy after female sex assignment at birth. However, the molecular and morphological effects of Cyp17 overexpression and androgen excess on folliculogenesis is unknown. Methods In this work, seeking a comprehensive profiling of the local outcomes of the androgen excess in the ovary, we generated a transgenic mouse model (TC17) with doxycycline (Dox)-induced Cyp17 overexpression in a local and temporal manner. TC17 mice were obtained by a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. Results Ovaries of Dox-treated TC17 mice overexpressed Cyp17 specifically in TCs, inducing high testosterone levels. Surprisingly, TC17 ovarian morphology resembled the human ovarian features of testosterone-treated transgender men (partially impaired folliculogenesis, hypertrophic or luteinized stromal cells, atretic follicles, and collapsed clusters). We additionally assessed TC17 fertility denoting a perturbation of the normal reproductive functions (e.g., low pregnancy rate and numbers of pups per litter). Finally, RNAseq analysis permitted us to identify dysregulated genes (Lhcgr, Fshr, Runx1) and pathways (Extra Cellular Matrix and Steroid Synthesis). Conclusions Our novel mouse model is a versatile tool to provide innovative insights into study the effects of Cyp17 overexpression and hyperandrogenism in the ovary.

International audience; In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.

Background: The use of Moringa oleifera as an antioxidant should be investigated as an alternativetreatment of oxidative stress and follicular refinement in PCOS with insulin resistance.Purpose: We aimed to prove the effect of Moringa oleifera leaf extract in various dosages to decrease themalondialdehyde levels and theca cell thickness of PCOS female rat with insulin resistance.Method: Three month old Rattus norvegicus stran wistar rat weighing 100-130 grams were divided into 5groups (n = 8). PCOS model obtained by giving injection of testosterone propionate for 28 days, followedby metformin therapy and Moringa oleifera leaf extract at 250 and 500 mg/KgBW for 14 days. Then, weanalyzed levels of malondialdehyde in the blood and the thickness of theca cell.Results: Malondialdehyde levels in the PCOS control group (5.694±1.464) increased significantly (p

Gonadotropins are glycoprotein hormones that regulate development and reproduction. They are a family of structurally similar hormones likely evolved from a common ancestral encoding gene and achieved relatively high complexity in humans. Two of these molecules are the luteinizing hormone (LH) and the choriogonadotropin (hCG). They are encoded by a common gene cluster and act on the same receptor (LHCGR), however, are produced in primates and specialized for regulating unique physiological functions, such as LH for gametogenesis and hCG for pregnancy. The action of the two hormones is differentiated by LHCGR, which mediates ligand-specific intracellular signals optimized for supporting LH and hCG physiological functions. Since the two hormones are used as drugs for the treatment of infertility, the knowledge of their mode of action should be helpful for interpreting gonadotropin-related pathological conditions and optimizing their clinical treatment.

The use of Moringa oleifera as an anti-oxidant should be investigated as an alternative treatment of follicular refinement in Polycystic ovary syndrome (PCOS) with insulin resistance. We aimed to prove the effect of Moringa oleifera leaf extract in various dosages to decrease the thecacell thickness of PCOS female rat with insulin resistance. This study was a laboratory experimental research. Three month old Rattus norvegicusstrain Wistar rat weighing 100-130 g were divided into 5 groups (n= 8). PCOS model obtained by giving injection of testosterone propionate for28 days, followed by metformin therapy and Moringa oleifera leaf extract at 250 and 500 mg/kg BW for 14 days. The examination of ovariumhistology showed that leaf extract Moringa oleifera 500 mg/kg BW (0.931±0.457) significantly decreased the thickness of theca cells (P0.05)compared to the PCOS control group. The conclusion was Moringa oleifera leaf extract as an anti-oxidant proven to decrease the thickness oftheca cell of the female rat model of PCOS.

Prenatal testosterone (T)–treated sheep, similar to polycystic ovarian syndrome women, manifest reduced cyclicity, functional hyperandrogenism, and polycystic ovary (PCO) morphology. The PCO morphology results from increased follicular recruitment and persistence of antral follicles, a consequence of reduced follicular growth and atresia, and is driven by cell-specific gene expression changes that are poorly understood. Therefore, using RNA sequencing, cell-specific transcriptional changes were assessed in laser capture microdissection isolated antral follicular granulosa and theca cells from age 21 months control and prenatal T–treated (100 mg intramuscular twice weekly from gestational day 30 to 90; term: 147 days) sheep. In controls, 3494 genes were differentially expressed between cell types with cell signaling, proliferation, extracellular matrix, immune, and tissue development genes enriched in theca; and mitochondrial, chromosomal, RNA, fatty acid, and cell cycle process genes enriched in granulosa cells. Prenatal T treatment 1) increased gene expression of transforming growth factor β receptor 1 and exosome component 9, and decreased BCL6 corepressor like 1, BCL9 like, and MAPK interacting serine/threonine kinase 2 in both cells, 2) induced differential expression of 92 genes that included increased mitochondrial, ribosome biogenesis, ribonucleoprotein, and ubiquitin, and decreased cell development and extracellular matrix-related pathways in granulosa cells, and 3) induced differential expression of 56 genes that included increased noncoding RNA processing, ribosome biogenesis, and mitochondrial matrix, and decreased transcription factor pathways in theca cells. These data indicate that follicular function is affected by genes involved in transforming growth factor signaling, extracellular matrix, mitochondria, epigenetics, and apoptosis both in a common as well as a cell-specific manner and suggest possible mechanistic pathways for prenatal T treatment–induced PCO morphology in sheep.

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.

Polycystic Ovarian Syndrome (PCOS) is a complex endocrine and reproductive disorder. A main hallmark includes increased androgen production. The root of Paeonia lactiflora Pall. (Bai Shao) is used in Chinese herbal medicine for reproductive disorders, however its effects and mechanisms on ovarian theca cells has not yet been fully elucidated.The aim of this study was to evaluate effect of paeoniflorin extract (PFE), the main constituents of Bai Shao, on androgen production in ovarian theca cells.Primary murine theca cells were treated with concentrations of PFE (1-100 µg/mL) in the presence of dexamethasone (10 µM) with media-only treated cells used as the control. After 24 h, culture media was collected for biochemistry assays of testosterone and progesterone. Expression of key steroidogenic enzymes, cholesterol side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17A1) was characterized using immunofluorescence staining, immunoblotting and qRT-PCR.Dexamethasone significantly enhanced testosterone secretion (P 0.05 vs. the control cells). PFE reversed over-production of testosterone induced by dexamethasone in a dose-dependent manner. The treatment with PFE also normalized production of progesterone in dexamethasone-treated cells. Expression of CYP11A1 and CYP17A1 in the theca cells were visualised by immunofluorescence staining. All doses of PFE significantly inhibited CYP17A1 expression detected by immunoblotting, but only 100 µg/mL of PFE downregulated CYP11A1 expression and reduced CYP11A1 significantly in dexamethasone-treated theca cells.PFE may reduce over-secretion of testosterone in theca cells through downregulation of CYP17A1 and CYP11A1. These findings provide scientific evidence to treat ovarian hyperandrogenism with the root of Paeonia lactiflora Pall.

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(−KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(−KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(−KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD.

Background: This study aimed to investigate the expression and localisation of fol­licle stimulating hormone receptor/growth hormone receptor/luteinising hormone receptor (FSHR/GHR/LHR) in different tissues and examine the regulatory effects of FSHR/GHR/LHR in the reproductive organs of female yaks during luteal phase. Materials and methods: The quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry assays were utilised to analyse the expression and localisation of FSHR/GHR/LHR in different tissues on female yaks. Results: The qRT-PCR results showed that the mRNA expressions of FSHR/GHR/ /LHR were significantly different in the non-reproductive organs (p < 0.01); the highest expression level was observed in the kidney, cerebellum and lung, whereas the lower expression level was observed in the liver and spleen. Im­munohistochemistry assay results showed that FSHR/GHR/LHR were located in kidney tubules, Purkinje cells, cerebellar medulla, alveolar cells and hepato­cytes. In addition, the expression levels of FSHR and GHR were considerably higher than LHR in the reproductive organs of female yaks during luteal phase (p < 0.01). FSHR/GHR/LHR were located in cardiac muscle cells, cerebellar medulla, and theca cell lining of reproductive organs. Furthermore, the expression level of FSHR was higher than those of GHR and LHR in all examined tissues. Conclusions: Therefore, the expression and localisation of FSHR/GHR/LHR possibly helped to evaluate the effects of them in tissue specific expression on female yaks, investigate the function and mechanism of FSHR/GHR/LHR in the reproductive organs of female yaks during luteal phase. (Folia Morphol 2018; 77, 2: 301–309)

Ovarian development requires coordinate communications among oocytes, granulosa cells, and theca cells. Two Hedgehog (Hh) pathway ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), are produced by the granulosa cells and work together to regulate theca cell specification and development. Mice lacking both Dhh and Ihh had loss of normal ovarian function, which raised the question of which biological actions are specifically controlled by each ligand during folliculogenesis. By comparing the reproductive fitness, hormonal profiles, and ovarian transcriptomes among control, Dhh single-knockout (KO), Ihh KO, and Dhh/Ihh double-knockout (DKO) mice, we examined the specific roles of Dhh and Ihh in these processes. Dhh/Ihh DKO female mice were infertile because of a lack of theca cells and their steroid product androgen. Although Dhh and Ihh KO mice were fertile with normal folliculogenesis, they had decreased androgen production and alterations in their ovarian transcriptomes. Absence of Ihh led to aberrant steroidogenesis and elevated inflammation responses, which were not found in Dhh KO mouse ovaries, implicating that IHH has a greater impact than DHH on the activation of the Hh signaling pathway in the ovary. Our findings provide insight into not only how the Hh pathway influences folliculogenesis but also the distinct and overlapping roles of Dhh and Ihh in supporting ovarian development.

Luteinizing hormone (LH) is a glycoprotein that regulates the function of ovarian follicular cells. Theca cells (TCs) also have a key role in follicular growth and atresia. The effects and intracellular signaling mechanisms were investigated of LH on apoptosis and steroidogenesis in goat gonadotropin-independent follicular (1.0–4.0 mm) TCs. The results indicated that LH increased androstenedione secretion and relative abundance of CYP17A1 and BCL2 mRNA in the TCs, whereas LH in combination with LY294002, a PI3K/AKT inhibitor, decreased LH-induced function. The apoptosis ratio and expression of the BAX gene in TCs were less with LH treatment, and the extent of this inhibition was decreased by suppressing the PI3K/AKT pathway. In conclusion, results of the present study indicate LH regulates apoptosis and steroidogenesis in goat TCs by activating the PI3K/AKT pathway.

Bisphenol S (BPS) is an endocrine-disrupting chemical with multiple potential mechanisms of action, including as an oestrogen receptor agonist. BPS is increasingly used in plastics and thermal receipts as a substitute for bisphenol A, which has been phased out due to concerns about human health implications. The ability of BPS to alter female reproductive function in mammals has not been widely studied, despite the importance of normal hormone signalling for female reproduction. The aim of this study was to investigate how BPS (in a wide range of doses, including very low doses) affects granulosa cell and theca cell steroid hormone production and cell viability in the bovine. Granulosa cell oestradiol production was stimulated when cells were exposed to 100 μM BPS under basal conditions, but there was no effect of BPS when cells were stimulated with follicle-stimulating hormone (FSH). Additionally, there was no effect of BPS on granulosa cell progesterone production or cell viability under basal or FSH-stimulated conditions. BPS did not affect theca cell androstenedione or progesterone production, or theca cell viability under basal or luteinizing hormone-stimulated conditions. This study suggests for the first time that BPS may alter oestradiol production by bovine granulosa cells, albeit at a concentration that is unlikely to be physiologically relevant. Further studies are needed to determine the effects of BPS on the bovine oocyte and on other functions of follicular cells.

The mycotoxin deoxynivalenol (DON) has been shown to inhibit ovarian granulosa cell function in cattle in vitro, but it is not known whether DON or its metabolite deepoxy-DON (DOM-1) affects theca cell function. The objectives of this study were to determine the effects of DON and of DOM-1 on theca cell steroidogenesis and apoptosis, and to determine the main pathways through which they act. Bovine theca cells were cultured in a nonluteinizing serum-free culture system, and challenged with DON or DOM-1 for 4 days to measure steroidogenesis and apoptosis, for 1-8 h to measure immediate-early genes, and for 5-60 min to measure phosphorylation of intracellular signaling proteins. Addition of DON decreased progesterone secretion at doses as low as 0.5 ng/ml but had no effect on testosterone secretion. Addition of DOM-1 inhibited progesterone and testosterone secretion at 0.5 ng/ml. Treatment of cells with 1 ng/ml DOM-1 increased the proportion of apoptotic cells, whereas DON had no effect. Addition of DON or DOM-1 stimulated phosphorylation of EIF2AK2, MAPK3/1, and AKT. However, DON inhibited and DOM-1 stimulated MAPK14 phosphorylation. DON increased the levels of mRNA encoding early-immediate genes EGR1, EGR3, and FOS, whereas DOM-1 was without effect. DOM-1 but not DON increased abundance of mRNA of the endoplasmic reticulum (ER) stress-related proteins, PRKRA and ATF4. We conclude that DOM-1 has a major impact on theca function in cattle, and possibly induces theca cell apoptosis through ER stress.

Background: Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. Methods: Small white follicles (1–2 mm in diameter) were treated for RNA isolation; Small white follicles (1–2 mm in diameter) and large white follicles (4–6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4–6 mm in diameter), small yellow follicles (6–8 mm in diameter), large yellow follicles (9–12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. Results: The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6–8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4–6 mm follicles relative to follicles of other sizes (P < 0.05). Conclusions: Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

Adiponectin, an adipokine hormone, influences glucose utilization, insulin sensitivity and energy homeostasis by signaling through two distinct receptors, ADIPOR1 and ADIPOR2. We previously reported that adiponectin and its receptors are expressed in several organs, including testes in chicken. We report herein that adiponectin gene is expressed exclusively in theca layer while ADIPOR1 and ADIPOR2 genes are expressed in granulosa and theca layers of all preovulatory and prehierarchical follicles of the chicken ovary. Estradiol and/or progesterone treatment of sexually immature chickens significantly altered expression of adiponectin and ADIPOR1 in the ovary. Using anti-chicken adiponectin-, ADIPOR1-, or ADIPOR2- antibodies, adiponectin-immunoreactive (ir) cells were found exclusively in the theca layer, and ADIPOR1-ir and ADIPOR2-ir cells were found both in theca and granulosa layers. Theca layer cells dispersed from preovulatory and prehierarchical follicles were found to synthesize and secrete a 720 kDa heavy molecular weight (HMW) isoform of adiponectin in vitro. Recombinant chicken adiponectin (rcADN) expressed in eukaryotic cells under serum-free conditions comprised primarily of the HMW isoform. Treatment of granulosa cells dispersed from 9 to 12 mm preovulatory follicle and 6 to 8 mm prehierarchical follicle with rcADN or an adiponectin receptor agonist, adipoRon, increased pERK and pACC abundance. In addition, both rcADN and adipoRon were found to significantly decrease the expression of steroidogenic acute regulatory protein gene expression in granulosa cells of preovulatory and prehierarchical follicles. In conclusion, adiponectin secreted by theca cell layer is identical in mass to circulating adiponectin. Systemic and/or theca-derived adiponectin is likely to affect proliferation, metabolism, and steroidogenesis of ovarian follicular cells.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder characterized by theca cell hyperplasia and excessive androgen production. An increasing body of evidence has pointed to a close association between PCOS and low-grade chronic systemic inflammation. However, the mechanistic basis for this linkage is unknown. Therefore, we evaluated the effects of the inflammatory agents lipopolysaccharide (LPS) and IL-1β on rat theca-interstitial cells (TICs). We found that incubation with either LPS or IL-1β elicited a dose-dependent increase in both TIC viability and androgen production. Using RNA sequencing analysis, we found that both of these inflammatory agents also triggered profound and widespread shifts in gene expression. Using a stringent statistical cutoff, LPS and IL-1β elicited differential expression of 5201 and 5953 genes, respectively. Among the genes upregulated by both LPS and IL-1β were key regulatory genes involved in the cholesterol and androgen biosynthesis pathways, including Cyp17a1, Cyp11a1, Hsd3b, and Hmgcr. This provides a molecular explanation for the mechanism of action of inflammatory agents leading to increased androgen production. Gene ontology and pathway analysis revealed that both LPS and IL-1β regulated genes highly enriched for many common functions, including the immune response and apoptosis. However, a large number of genes (n = 2222) were also uniquely regulated by LPS and IL-1β, indicating that these inflammatory mediators have substantial differences in their mechanism of action. Together, these findings highlight the potential molecular mechanisms through which chronic low-grade inflammation contributes to the pathogenesis of androgen excess in PCOS.

Androgens, although traditionally thought to be male sex steroids, play important roles in female reproduction, both in healthy and pathological states. This mini-review focuses on recent advances in our knowledge of the role of androgens in the ovary. Androgen receptor (AR) is expressed in oocytes, granulosa cells, and theca cells, and is temporally regulated during follicular development. Mouse knockout studies have shown that AR expression in granulosa cells is critical for normal follicular development and subsequent ovulation. In addition, androgens are involved in regulating dynamic changes in ovarian steroidogenesis that are critical for normal cycling. Androgen effects on follicle development have been incorporated into clinical practice in women with diminished ovarian reserve, albeit with limited success in available literature. At the other extreme, androgen excess leads to disordered follicle development and anovulatory infertility known as polycystic ovary syndrome (PCOS), with studies suggesting that theca cell AR may mediate many of these negative effects. Finally, both prenatal and postnatal animal models of androgen excess have been developed and are being used to study the pathophysiology of PCOS both within the ovary and with regard to overall metabolic health. Taken together, current scientific consensus is that a careful balance of androgen activity in the ovary is necessary for reproductive health in women.

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P < 0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P > 0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P < 0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P < 0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.

Glyphosate (GLY) is a common herbicide used worldwide but its effect on ovarian function in mammals is unknown. The aim of this study was to determine the potential endocrine disruptor effects of GLY on ovarian function evaluating cell proliferation, steroidogenesis and gene expression using bovine granulosa cells (GC) and theca cells as in vitro models. GC proliferation was impaired (P < 0.05) after exposure to GLY at 0.5, 1.7 and 5 μg ml-1 . GC progesterone production was not affected (P ≥ 0.05) at all doses tested while estradiol production was inhibited (P < 0.05) by GLY at 5 μg ml-1 . At the same concentration GLY showed no effect (P ≥ 0.05) on theca cell proliferation and steroidogenesis. At higher concentrations (0.01 and 0.3 mg ml-1 ), GLY had no significant effect (P ≥ 0.05) on GC proliferation and steroidogenesis. These studies, for the first time, suggest that GLY may affect the reproductive system in cattle via direct action on ovarian function; however, further studies will be required to understand better the mechanism of action and to determine the in vivo reproductive effects of GLY. Copyright © 2016 John Wiley & Sons, Ltd.

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or 5 to 6 (n = 8) postovulation for GC and TC RNA extraction from small (1–5 mm), medium (5.1–8 mm), and large (8.1–18 mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2) < progesterone (P4)] follicles than in large E2-active (i.e., concentrations of E2 > P4) follicles at both early (d 3–4) and late (d 5–6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r = −0.68), free IGF-1 (r = −0.63), and E2-to-P4 ratio (r = −0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.

Context: 11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. Objective: The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. Results: Cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. Conclusions: The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen.

Background Polycystic ovary syndrome (PCOS) is a heterogeneous condition with wide range of phenotype the cause of this disorder is unknown, however, increased ovarian androgen production including increased theca cell responsiveness to gonadotropin stimulation, increased pituitary secretion of luteinizing hormone, and hyperinsulinemia have been suggested. Some known risk factors are ethnicity and environmental factors including lifestyle and bodyweight. Method Relevant English language studies from January 1995 to September 2015 were identified, reviewed, synthesized and discussed extensively. Result various forms of PCOS, and the underlying mechanisms; and highlights biochemical, morphological and metabolic hallmarks of PCOS, including trends and emerging phenotypes; and presents a simplified synthesis that integrates current understanding of biochemical and metabolic endocrinology of PCOS are summarized. As no generally accepted criteria exist for its diagnosis, some existing diagnostic criteria that cut across different geographical regions are also highlighted. Conclusion Indeed, emerging evidence points that the severity of menstrual problems could serve as a predictor of the like-hood of insulin resistance in women of reproductive age, suggesting the need for routine metabolic screening and early intervention in PCOS.

The objective of this study was to determine apoptotic cell localization in preantral and antral follicles of porcine ovaries. Additionally, the proportion of cells undergoing apoptosis was also compared between delayed puberty gilts and normal cyclic gilts. Ovarian tissues were obtained from 34 culled gilts with age and weight of 270.1 ± 3.9 days and 143.8 ± 2.4 kg, respectively. The gilts were classified according to their ovarian appearance as 'non-cyclic' (n = 7) and 'cyclic' (n = 27) gilts. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay was used to determine apoptotic cell expression in different compartments of the ovarian tissue sections. All apparent preantral (n = 110) and antral (n = 262) follicles were evaluated using image analysis software. It was found that apoptotic cells were expressed in both granulosa (22.2%) and theca cell layers (21.3%) of the follicles in the porcine ovaries. The proportion of apoptotic cells in the granulosa layer in the follicles was positively correlated with that in the theca layer (r = 0.90, p 0.001). Apoptosis did not differ significantly between preantral and antral follicles in either granulosa (27.8% and 26.4%, p 0.05) or theca cell layers (28.6% and 26.5%, p 0.05). The proportion of apoptotic cells in non-cyclic gilts was higher than cyclic gilts in both granulosa (31.7% and 22.6%, p 0.001) and theca cell layers (34.8% and 20.2%, p 0.001). This study indicated that apoptosis of the granulosa and theca cell layers in the follicles was more pronounced in the ovarian tissue of delayed puberty gilts than cyclic gilts. This implied that apoptosis could be used as a biologic marker for follicular development/function and also that apoptosis was significantly associated with anoestrus or delayed puberty in gilts, commonly observed in tropical climates.

BACKGROUND: Insulin-like peptide 3 (INSL3) is a member of the relaxin family of neohormones which has evolved to address specifically mammalian aspects of reproduction related to viviparity and internal fertilization. It was originally identified as a major product of testicular Leydig cells and has proved to be an important biomarker of Leydig cell functional capacity. However, INSL3 is also produced by theca interna cells of growing antral follicles and is secreted into the bloodstream in phases corresponding to the number and health of the follicles. Moreover, gene silencing experiments have shown that INSL3 is essentially required for androstenedione synthesis, which is the major steroid precursor for the granulosa cells of antral follicles to produce oestrogens. Knockout studies in mice confirm that loss of INSL3 or its receptor in females leads to partial infertility, with reduced follicle numbers, ovulations and litter size. Circulating INSL3 concentration corresponds to the reproductive lifespan, beginning with puberty and declining at the menopause, and thus may contribute to the physiology of other organ systems, particularly those relevant for hormone replacement strategies. SEARCH METHODS: A literature review was carried out by exhaustive searching of literature databases (PubMed and Google Scholar) with the search terms INSL3, RLF, Ley-IL and RXFP2. OBJECTIVE AND RATIONALE: We present the first comprehensive review of INSL3 and its specific receptor RXFP2, and their roles in the context of female reproductive physiology. Moreover, we highlight the potential involvement of INSL3 in female reproductive pathology, such as PCOS, its clinical application as a valuable biomarker of reproductive processes, and its potential for therapeutic interventions. OUTCOMES: In the female mammal, INSL3 is largely produced by the theca interna cells of growing antral follicles during the follicular phase of the menstrual (oestrous) cycle. Within the follicle, INSL3 acts via its G-protein-coupled receptor, RXFP2, in an autocrine/paracrine manner to orchestrate and drive the production of the major steroid precursor androstenedione and its conversion by granulosa cells into oestrogens. These in turn create a positive feedback loop promoting the expression of more theca cell INSL3. This is countered by the follicular production of bone morphogenetic proteins and by the LH surge. Thus, the activity of the theca cell INSL3-RXFP2 system effectively determines the production of estradiol within an antral follicle through the follicular phase. INSL3 is also secreted into the circulation where it acts as a valuable biomarker to monitor the growth of antral follicles; it is consequently increased in PCOS and decreased in women with premature ovarian failure (POF). As an endocrine factor, INSL3 may also influence bone metabolism and kidney function. Additionally, INSL3 or its analogues may prove valuable as an adjunct in hormone replacement therapy or to monitor or influence IVF protocols. WIDER IMPLICATIONS: The INSL3-RXFP2 system represents a new regulatory pathway essential for the proper functioning of growing antral follicles. We still know very little about its involvement in pathologies such as PCOS or POF, and its role as a new biomarker of female function needs to be explored more widely to improve diagnosis and treatment of ovarian dysfunction. We need to examine how INSL3 might be used to improve IVF protocols and outcomes. Opportunities should also be investigated in regard to the systemic application of INSL3 as a rejuvenant therapy, with positive effects on bone or kidney function, and possibly also for fertility regulation. Most research to date has involved animal models; this now needs to be extended to include more human studies.

Introduction The methylation level of promoters is one of the most studied and well-known epigenetic mechanisms that programs the amount of gene expression. Over expression of steroidogenesis genes via epigenetic control can result in hypetandrogenism, which is the main endocrine aspect of polycystic ovarian syndrome (PCOS). Aims In the present study we aimed to determine and compare the promoter methylation levels of three steroidogenic genes, CYP17, GATA6 and StAR, in theca cells of prenatally androgenized (PNA) rats to those of controls. Materials and methods Pregnant Wistar rats in the PNA group received 5 mg free testosterone, dissolved in 500 ml solvent, subcutaneously injected on day 20 of pregnancy, while controls were injected with 500 ml of solvent only. Theca cell samples, taken from the ovaries of eight to ten female offspring of both the PNA and control groups, were measured for promoter methylation levels of the aforementioned genes, using the bisulfite sequence PCR (BSP) method. Key findings Although the promoters of all three genes were slightly hypomethylated in the PNA group, the differences observed were not significant compared to the control group. The methylation of −520 and −822 positions, in the GATA6 and the StAR promoter respectively, were significantly decreased in the PNA group. Significances The results of this study suggest that alterations in the steroidogenesis pathway after exposure to excess androgen may be a result of changes in the pattern of the methylation of the relevant genes.

Since hagfishes are considered the most primitive vertebrate known, extant or extinct, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproduction. However, little information is available on the endocrine regulation of the gonadal function in the hagfish. Based on EST analysis of the testis of the brown hagfish (Paramyxine atami), P450 side chain cleavage (CYP11A), which is the first and essential enzyme for steroidogenesis in jawed vertebrates, was cloned. The deduced amino acid sequence of hagfish CYP11A shows high identity to other animal forms especially in two functional domains, adrenodoxin binding domain and heme-binding domain. In the phylogenetic analysis, hagfish CYP11A forms a clade with the vertebrate CYP11A. Following the real-time PCR analysis, CYP11A mRNA expression levels were clearly correlated to the developmental stages of gonads in both sexes of the brown hagfish. By in situ hybridization, CYP11A mRNA signals were found in the theca cells of the ovarian follicles and Leydig cells and the tubule-boundary cells of the testis. These molecular and histological evidences are suggesting that CYP11A plays functional roles as a steroidogenic enzyme in gonadal development. Moreover, native GTH purified from hagfish pituitary stimulated the transcriptional levels of CYP11A in the organ-cultured testis in vitro, clearly suggesting that the steroidogenic activity of the hagfish is under the control of the pituitary GTH. It is suggested that vertebrates, during their early evolution, have established the pituitary-gonadal reproductive system.

We reported previously that fibroblast growth factor 9 (FGF9) acts as an anti-differentiation factor, stimulating proliferation of granulosa (GC) and theca (TC) cells while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TC were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. Following treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cut-off and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements up-regulated and 191 elements down-regulated by FGF9. The Ingenuity Pathway Analysis (IPA) was utilized to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions, and unveiled interesting relationships among genes related to cell proliferation (e.g., CCND1, FZD5 and MYB), antioxidation/cytoprotection (e.g., HMOX1 and NQO1) and steroidogenesis (e.g., CYP11A1 and STAR). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development.

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.

Di-2-ethylhexyl phthalate (DEHP) is an endocrine disruptor used in industry as an additive to polyvinyl chloride-based products. Pregnant dams were gavaged with oil, 1, 20, 50, or 300 mg of DEHP/kg/day from gestational day 14 until birth in order to characterize the effects of DEHP in the adult female offspring. In utero exposure to DEHP resulted in reduced estrogen levels at proestrus. Theca cell layer thickness was decreased starting at 50 mg DEHP/kg/day dose. Follicle-stimulating hormone levels were significantly increased at proestrus and estrus. F1 reproduction using a known breeder was not affected. F3 generation showed a decreased pregnancy rate and weight, and increased litter size in the animals exposed to 20 mg DEHP/kg/day. The data presented herein suggest that in utero exposure to DEHP targets the theca cell layer and decreases the estrus cycle steroid surge, but despite these effects, does not cause infertility.

Insulin-like peptide 3 (INSL3) and its specific receptor RXFP2 are both expressed by theca interna cells of the growing antral follicle where they form an essential regulatory element in the production of the steroid precursor androstenedione. Using primary cultures of bovine theca cells from the mid follicular phase together with steroid agonists and antagonists we have examined how ovarian steroids modulate INSL3 expression. Transcript analysis shows that these cells express estrogen receptors α and β, androgen and progesterone receptors, besides the orphan nuclear receptors SF1 and nur77. Whereas, exogenous androgens have little or no effect, the androgen antagonist bicalutamide stimulates INSL3 production. In contrast, estrogen receptor agonists, as also progesterone, are stimulatory. Importantly, estrogen receptor signaling is convergent with the protein kinase A signaling pathway activated by LH, such that the estrogen receptor antagonist can inhibit the mild stimulatory effect of LH, and vice versa the PKA antagonist H89 blocks stimulation by estradiol. A significant finding is that the major steroid metabolite androstenedione appears to act predominantly as an estrogen and not an androgen in this system. Transfection of INSL3 gene promoter-reporter constructs together with various steroid receptor expression plasmids supports these findings and shows that steroid action uses non-classical pathways not requiring canonical steroid-responsive elements in the proximal promoter region. Together, the results indicate that increasing estrogens in the follicular phase stimulate a feedforward loop driving INSL3 signaling and thereby promoting steroidogenesis in the growing antral follicle until the LH surge which effectively switches off INSL3 expression.

Ovarian theca cells play an indispensable role in ovarian follicular development and hormone secretion. miR-26a-5p was reported to be differentially expressed in mature and immature chicken ovaries in our previous study; however, the role of miR-26a-5p in regulating ovarian follicle function is still unclear. In this study, we demonstrated that the expression dynamics of TNRC6A mRNA in either chicken ovaries or follicles showed an opposite trend compared with that of chicken miR-26a-5p expression. miR-26a-5p inhibited TNRC6A mRNA expression by directly targeting its 3'-untranslated region in cultured chicken theca cells. Overexpression of miR-26a-5p promoted chicken follicular theca cell proliferation in vitro. Furthermore, overexpression of miR-26a-5p and knockdown of TNRC6A significantly upregulated the antiapoptotic BCL-2 gene. Taken together, this study revealed the expression dynamics of miR-26a-5p and TNRC6A in chicken ovaries and ovarian follicles and the relationship between the expression of miR-26a-5p and TNRC6A in chicken ovarian theca cells. These results suggest that miR-26a-5p facilitates chicken ovarian theca cell proliferation by targeting the TNRC6A gene.

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8–22 mm) follicles and treated with various hormones in serum-free medium for 24 h or 48 h. Melatonin caused a dose-dependent inhibition (P 0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Recent reports have demonstrated that follicular atresia is initiated or caused by granulosa cell apoptosis followed by theca cell degeneration in mammalian ovaries, but the mechanism of follicular atresia is still to be elucidated. Therefore, our present study was designed to examine our hypothesis that the changes of follicular microenvironment induce the granulosa cell apoptosis during pocrine follicular atresia in vivo. We firstly isolated intact porcine antral follicles and identified them into three groups, healthy follicles (HF), early atretic follicles (EAF) and progressed atretic follicles (PAF) through morphology and histology. To further confirm their status, we detected hormone levels in follicular fluids and the expression level of apoptosis gene Bax in granulosa cells. The rate of progesterone (P) and estradiol (E2) was increased with the expression of Bax , indicating hormone can be used as a marker of granulosa cell apoptosis or follicular atresia. Finally, we analyzed the expression level of hormone receptor genes in granulosa cells and their relationship with follicular atresia. In PAF, the expression of Progesterone receptor ( PGR ) was increased significantly while estradiol receptor ( ER ) had no notable changes, which suggesting the increased-PGR accelerated the effect of P-stimulated granulosa cell apoptosis. The dramatic increasing of androgen receptor ( AR ) expression in PAF and the obvious increase of tumor necrosis factor-α receptor ( TNFR ) in EAF indicated that there are different pathways regulating granulosa cell apoptosis during follicular atresia. Together, our results suggested that different pathways of granulosa cell apoptosis was induced by changing the follicular microenvironment during follicular atresia.

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation and leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS on steroid production in bovine theca cells at different stages of follicular development. Theca cells isolated from pre- and post-selection follicles (PRFs, 8.5 mm in diameter, respectively) of bovine ovaries were exposed to LPS under luteinizing hormone (LH) conditions, estradiol (E2) conditions or both conditions in vitro. Bovine theca cells expressed the LPS receptor gene complex: Toll-like receptor 4 (TLR4), CD14 and MD2. LPS suppressed progesterone (P4) and androstenedione (A4) production with downregulation of steroidogenic enzyme transcripts when theca cells were stimulated with LH. By contrast, LPS did not affect P4 or A4 production when theca cells were stimulated with E2. P4 and A4 production in theca cells from PRFs was suppressed by LPS as early as at 48 h of culture, whereas the effect of LPS on theca cells from POFs was observed at 96 h of culture. The results demonstrate that LPS inhibits steroid production in theca cells under LH conditions. Moreover, theca cells from POFs showed a slower response to LPS compared with that of theca cells from PRFs, which might imply a distinct effect of LPS on follicles at different developmental stages. These findings suggest a possible mechanism of ovarian dysfunction and subsequent infertility in cows with endometritis.

Ovarian cancer is a highly lethal gynecological cancer, and its causes remain to be understood. Using a recently identified tumor suppressor gene, GT198 (PSMC3IP), as a unique marker, we searched for the identity of GT198 mutant cells in ovarian cancer. GT198 has germ line mutations in familial and early onset breast and ovarian cancers and recurrent somatic mutations in sporadic fallopian tube cancers. GT198 protein has been shown as a steroid hormone receptor coregulator and also as a crucial factor in DNA repair. In this study, using GT198 as a marker for microdissection, we find that ovarian tumor stromal cells harboring GT198 mutations are present in various types of ovarian cancer including high and low grade serous, endometrioid, mucinous, clear cell, and granulosa cell carcinomas and in precursor lesions such as inclusion cysts. The mutant stromal cells consist of a luteinized theca cell lineage at various differentiation stages including CD133(+), CD44(+), and CD34(+) cells, although the vast majority of them are differentiated overexpressing steroidogenic enzyme CYP17, a theca cell-specific marker. In addition, wild type GT198 suppresses whereas mutant GT198 protein stimulates CYP17 expression. The chromatin-bound GT198 on the human CYP17 promoter is decreased by overexpressing mutant GT198 protein, implicating the loss of wild type suppression in mutant cells. Together, our results suggest that GT198 mutant luteinized theca cells overexpressing CYP17 are common in ovarian cancer stroma. Because first hit cancer gene mutations would specifically mark cancer-inducing cells, the identification of mutant luteinized theca cells may add crucial evidence in understanding the cause of human ovarian cancer.

Sex determination in mammals requires interaction between the transcription factor GATA4 and its cofactor FOG2. We have recently described the function of both proteins in testis development beyond the sex determination stage; their roles in the postnatal ovary, however, remain to be defined. Here, we use gene targeting in mice to determine the requirement of GATA4 and FOG2 in ovarian development and folliculogenesis. The results from this study identify an essential role of the GATA4 protein in the ovarian morphogenetic program. We show that in contrast to the sex determination phase, which relies on the GATA4–FOG2 complex, the subsequent regulation of ovarian differentiation is dependent upon GATA4 but not FOG2. The loss of Gata4 expression within the ovary results in impaired granulosa cell proliferation and theca cell recruitment as well as fewer primordial follicles in the ovarian cortex, causing a failure in follicular development. Preantral follicular atresia is observed within the few follicles that develop despite Gata4 deficiency. The depletion of the follicular pool in GATA4 deficient ovary results in the formation of ovarian cysts and sterility.

Objective To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. Design In vivo and in vitro studies. Setting Research laboratory. Animal(s) Immature Sprague-Dawley female rats. Intervention(s) In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 μg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. Main Outcome Measure(s) Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. Result(s) In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star , Cyp11a1 , and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. Conclusion(s) These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary.

Poor responders are a heterogeneous population, with some patients displaying a diminished ovarian reserve and others a poor ovarian reserve with preserved granulosa cell function. Androgen and LH/hCG supplementation has been advocated for poor responders, mainly those >40 years old. Although androgens synergistically act with FSH to support folliculogenesis, and ovarian androgen secretion declines with age, there is still no evidence that androgen therapy is actually effective to improve ovarian FSH sensitivity. The main reason seems to be that theca cell function has not been appropriately assessed in patients at risk of poor response. The definition of theca insufficiency is hampered by methodologic shortcomings in routine bioassays. Provocative tests for theca cells might help to identify those patients who could benefit from androgen supplementation. The lack of data regarding theca cells in these patients might contribute to explaining the absence of evidence for a positive effect of androgen therapy.

Many studies have suggested the important role of estrogen in ovarian differentiation and development of vertebrates including chicken. Cytochrome P450 aromatase, encoded by CYP19A1, is a key enzyme in estrogen synthesis, but the mechanism of CYP19A1 regulation in chicken remains unknown. Here, we found that CYP19A1 was only expressed in the theca cell layers of chicken ovary follicles. Steroidogenic factor 1 (SF-1, also named as nuclear receptor subfamily 5 group A member 1, NR5A1), a potential regulators, was expressed in both the theca cell layers and granulosa cell layers. Forkheadbox L2 (FOXL2), another potential regulator, was only expressed in the granulosa cell layers. Using luciferase assays in vitro, we found that SF-1 could activate the promoter of CYP19A1 by binding to the nuclear receptor half-site (5'-TCAAGGTCA-3') from -280 to -271 base pairs. FOXL2 did not activate the promoter of chicken CYP19A1 gene in either 293T or DF-1 cells. Overexpression of SF-1 in DF-1 cells upregulated aromatase expression, but FOXL2 could not. Taken together, our results indicated that SF-1 activates CYP19A1 mRNA expression via a conserved binding site in chicken ovary, but FOXL2 may not affect the expression of CYP19A1.

Interleukin 8 (IL-8) is a chemoattractant associated with ovulation in the mammalian ovary. This chemokine is also involved in the recruitment and activation of neutrophils. Using bovine tissue, we examined the possible role of IL-8 in steroid production by theca cells of the large ovarian follicles. IL-8 promoted progesterone production and stimulated StAR expression in cultured theca cells. The inhibitor of p38 did not disturb the P4 production and StAR expression in IL-8-treated theca cells. On the other hand, the inhibitor of MEK disturbed the P4 production and expression of StAR in theca cells treated with IL-8. These results suggest that IL-8 is associated with progesterone production in bovine theca cells via the MEK pathway.

The present study is designed to investigate the cellular expression and immunolocalization of three different nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) subunits in the porcine ovary. Our results showed that in the fetal and neonatal pigs, all three isoforms of NOS were mainly localized in the oocyte and showed the expression of gradual increase in the granulosa cell and theca cell with the growing follicle. In addition, subunits of the sGC, sGC α1 and β1 were mainly expressed in the granulosa cell in precious studies. The bioactivity of total NOS, eNOS, iNOS and nNOS was detected in the ovary and were higher at prenatal stages compared to postnatal stages. However, the activities of nNOS were no different between prenatal stages and postnatal stages. Taken together, our findings suggested that the NOS/sGC pathway may be involved in the follicular formation and development in the porcine ovary.

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated. In the present study, we examined the roles of THY1 and integrin beta3 in theca cell layer formation during mouse folliculogenesis. The localization pattern of THY1 and integrin beta3 in adult mouse ovary was investigated immunohistochemically. The strongest THY1 signal was observed in theca cell layers from secondary to preantral follicles, at which time theca cells have begun to participate in follicle formation. Integrin beta3 also localized to the theca cell layer of secondary to preantral follicles and showed a localization pattern similar to that of THY1. Moreover, the role of THY1 in theca cell layer formation was examined using a follicle culture system. When anti-THY1 antibody was added to this culture, no theca cell layers were formed, and the granulosa cells were distanced from each other. Because a THY1 signal was not observed in ovaries at stages earlier than prepuberty, THY1 localization also appeared to be affected by mouse development. This possibility was examined by determining the effect of administering follicle-stimulating hormone, luteinizing hormone, and 17beta-estradiol to 7-day-old mice on THY1 localization in the ovary 3 days later. Only follicle-stimulating hormone induced a THY1 signal in 10-day-old mouse ovaries. No THY1 signal was observed in untreated 10-day-old ovaries. In conclusion, THY1 might play a role in cell adhesion via binding to integrin beta3 in mouse ovaries. The present results suggest that THY1 localization may be affected by follicle-stimulating hormone in mouse ovaries.

Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1-5mm) and/or large (8-22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.

Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20β,21-trihydroxy-4-pregnen-3-one (20β-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20β-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20β-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20β-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20β-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.

Purpose Create a 3-Dimensional artificial human ovary to mature human oocytes. Methods Theca and granulosa cells were isolated from antral follicles of reproductive-aged women, seeded into micro-molded gels and self-assembled into complex 3D microtissues. Immunohistochemistry and live-dead staining confirmed theca cell identity and cellular viability at one week respectively. Placement of granulosa cell spheroids or cumulus-oocyte complexes into theca cell honeycomb openings resulted in creation of an artificial human ovary. Oocytes from this construct were assessed for polar body extrusion. Results Theca and granulosa cells self-assembled into complex microtissues, remaining viable for one week. At 72 h after artificial human ovary construction, theca cells completely surrounded the granulosa spheroids or COCs without stromal invasion or disruption. Polar body extrusion occurred in one of three COCs assessed. Conclusions An artifical human ovary can be created with self-assembled human theca and granulosa cell microtissues, and used for IVM and future oocyte toxicology studies.