Follicular fluid exosomes regulate oxidative stress resistance, proliferation, and steroid synthesis in porcine theca cells.

Theca cells (TCs) are regulated by various factors during ovarian development. However, the role of follicular fluid exosomes in ovarian TCs has not yet been reported. In the present study, we explored the effects of follicular fluid exosomes on porcine ovarian TCs. TCs were treated with follicular fluid exosomes in vitro, and the differential gene expression profiles of TCs in the exosome and control groups were obtained via transcriptome sequencing. Differentially expressed genes were identified and found to be associated with antioxidative stress, proliferation, and steroid hormone synthesis of TCs. In addition, exosomes were found to increase antioxidative stress, proliferation, and steroid synthesis, as revealed by a higher mRNA and protein expression of GPX1, CCND1, PCNA, CYP11A1, and HSD3B1 and lower mRNA and protein expression of TNFR1 and BAX. In conclusion, we demonstrated that exosomes are essential components in regulating the physiological function of TCs.

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.

Bisphenol S enhances gap junction intercellular communication in ovarian theca cells.

Gap junction intercellular communication (GJIC) is necessary for ovarian function, and it is temporospatially regulated during follicular development and ovulation. At outermost layer of the antral follicle, theca cells provide structural, steroidogenic, and vascular support. Inter- and extra-thecal GJIC is required for intrafollicular trafficking of signaling molecules. Because GJIC can be altered by hormones and endocrine disrupting chemicals (EDCs), we tested if any of five common EDCs (bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), perfluorooctanesulfonic acid (PFOS), and triphenyltin chloride (TPT)) can interfere with theca cell GJIC. Since most chemicals are reported to repress GJIC, we hypothesized that all chemicals tested, within environmentally relevant human exposure concentrations, will inhibit theca cell GJICs. To evaluate this hypothesis, we used a scrape loading/dye transfer assay. BPS, but no other chemical tested, enhanced GJIC in a dose- and time-dependent manner in ovine primary theca cells. A signal-protein inhibitor approach was used to explore the GJIC-modulatory pathways involved. Phospholipase C and mitogen-activated protein kinase (MAPK) inhibitors significantly attenuated BPS-induced enhanced GJIC. Human theca cells were used to evaluate translational relevance of these findings. Human primary theca cells had a ∼40% increase in GJIC in response to BPS, which was attenuated with a MAPK inhibitor, suggestive of a conserved mechanism. Upregulation of GJIC could result in hyperplasia of the theca cell layer or prevent ovulation by holding the oocyte in meiotic arrest. Further studies are necessary to understand in vitro to in vivo translatability of these findings on follicle development and fertility outcomes.

Theca cells can support bovine oocyte growth in vitro without the addition of steroid hormones.

Theca cells (TCs) are essential to folliculogenesis by contributing to steroidogenesis. However, the in vitro growth (IVG) of oocytes co-cultured with TCs has not yet been examined. In the present study, we investigated the feasibility of the IVG of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) co-cultured with TCs and the developmental competence of co-cultured oocytes. OCGCs and TCs were co-cultured without steroid hormone addition for 12 days. Steroidogenesis, the viability of OCGCs, and TC numbers during co-culture were assessed every 4 days. After IVG, oocytes were matured and the nuclear status was evaluated. Some oocytes were inseminated and cultured to examine blastocyst development. During the co-culture, androstenedione production by TCs was only observed during the first 4 days (1.1 ng/well) while estradiol-17ß was continuously produced, peaking during the second 4 days (0.5 ng/well). The number of TCs decreased to ∼60% of the seeding number (4.0 × 104 cells/well) during the first 4 days, and was maintained thereafter. The majority of co-cultured OCGCs (82.7%) survived after 12-day IVG. Only a few OCGCs (6.2%) survived in the OCGC culture without TCs (p < 0.01); however, the addition of androstenedione to the culture medium markedly improved survivability to 80.1%, which was similar to that in the co-culture with TCs. In the subsequent development of oocytes derived from the co-culture, 58.3% reached metaphase II stage, 58.7% cleaved, and 17.3% developed to blastocysts, which were similar values to those of oocytes cultured with the addition of androstenedione. In conclusion, TC-produced androgen contributes to OCGC growth and the acquisition of subsequent embryonic developmental competence.

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.

Coordination of Ovulation and Oocyte Maturation: A Good Egg at the Right Time.

Ovulation is the appropriately timed release of a mature, developmentally competent oocyte from the ovary into the oviduct, where fertilization occurs. Importantly, ovulation is tightly linked with oocyte maturation, demonstrating the interdependency of these two parallel processes, both essential for female fertility. Initiated by pituitary gonadotropins, the ovulatory process is mediated by intrafollicular paracrine factors from the theca, mural, and cumulus granulosa cells, as well as the oocyte itself. The result is the induction of cumulus expansion, proteolysis, angiogenesis, inflammation, and smooth muscle contraction, which are each required for follicular rupture. These complex intercellular communication networks and the essential ovulatory genes have been well defined in mouse models and are highly conserved in primates, including humans. Importantly, recent discoveries in regulation of ovulation highlight new areas of investigation.

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.

Membrane receptor-independent inhibitory effect of melatonin on androgen production in porcine theca cells.

Excessive secretion of androgens including androstenedione and testosterone in theca cells frequently causes female infertility in mammals. Melatonin is a potent inhibitor of androgen production in gonadal cells of several species in a membrane receptor-dependent manner. However, the function of melatonin in steroidogenesis of porcine theca cells remains unclear. Here we report that melatonin inhibits androgen biosynthesis independently of its membrane receptors in pigs. Using flow cytometry, immunofluorescence and RT-PCR we showed that the vast majority of cells isolated from the theca layer of antral follicles are indeed theca cells. Furthermore, we demonstrated that of the two of melatonin membrane receptors encoded in the porcine genome, theca cells exclusively express melatonin receptor 1B. Cell counting analysis indicated that different concentrations of melatonin did not alter the normal viability and proliferation of theca cells. Additionally, hormone radioimmunoassay and qPCR respectively showed that a high concentration of melatonin significantly repressed both androgen production and expression of steroidogenic genes involving StAR, CYP11A1, HSD3ß and SET (P < 0.05), but did not impair progesterone production. Interestingly, these effects were not reversed by N-acetyl-2-benzyltryptamin, a melatonin membrane receptor antagonist. Overall, these results demonstrate that melatonin inhibits androgen production in porcine theca cells independently of its membrane receptor.

Histological and immunohistochemical investigations of ovarian interstitial glands during non-breeding season in camels (Camelus dromedarius).

The aim of this was to investigate the histology and immunohistochemistry of interstitial glands during non-breeding season in camel ovaries. A total of 21 mature, non-pregnant and apparently healthy camels aged between 8 and 12 years were slaughtered. The ovaries were removed within 15 min, cleaned from adipose tissue, weighted and examined grossly. The histological preparation was made, and then, the blocks were cut at 3-5 microns thickness and stained by H&E for histological examinations. Moreover, some sections were stained with Sudan Black for lipid detection. Immunohistochemical analysis of paraffin-embedded ovarian tissues was performed to detect the localization of S-100, vimentin, progesterone receptors (PR) and oestrogen receptors (ER). Immunoreactive signals were detected using UltraVision Detection System. The results revealed that the interstitial glands were located in the cortical region and they were arranged in various arrangements either single, in couple or in groups rich in lipid droplet. All interstitial gland arrangements were enclosed by connective tissue capsules containing fibroblasts and collagenous fibres separated them from the surrounding ovarian structures. Both interstitial glands and their surrounding CT were penetrated by several blood vessels. There was a strong immunoreactive signal for S-100 in the nuclei of interstitial cells, and no signals were detected either in cells of the interstitial glands or their connective tissue with PR. We could conclude that the interstitial gland is distinct in ovary of camel and further studies are needed to elucidate its rule in steroid synthesis.

Ovarian Follicular Theca Cell Recruitment, Differentiation, and Impact on Fertility: 2017 Update.

The major goal of this review is to summarize recent exciting findings that have been published within the past 10 years that, to our knowledge, have not been presented in detail in previous reviews and that may impact altered follicular development in polycystic ovarian syndrome (PCOS) and premature ovarian failure in women. Specifically, we will cover the following: (1) mouse models that have led to discovery of the derivation of two precursor populations of theca cells in the embryonic gonad; (2) the key roles of the oocyte-derived factor growth differentiation factor 9 on the hedgehog (HH) signaling pathway and theca cell functions; and (3) the impact of the HH pathway on both the specification of theca endocrine cells and theca fibroblast and smooth muscle cells in developing follicles. We will also discuss the following: (1) other signaling pathways that impact the differentiation of theca cells, not only luteinizing hormone but also insulinlike 3, bone morphogenic proteins, the circadian clock genes, androgens, and estrogens; and (2) theca-associated vascular, immune, and fibroblast cells, as well as the cytokines and matrix factors that play key roles in follicle growth. Lastly, we will integrate what is known about theca cells from mouse models, human-derived theca cell lines from patients who have PCOS and patients who do not have PCOS, and microarray analyses of human and bovine theca to understand what pathways and factors contribute to follicle growth as well as to the abnormal function of theca.

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.

Control of Oocyte Growth and Development by Intercellular Communication Within the Follicular Niche.

In the mammalian ovary, each oocyte grows and develops within its own structural and developmental niche-the follicle. Together with the female germ cell in the follicle are somatic granulosa cells, specialized companion cells that surround the oocyte and provide support to it, and an outer layer of thecal cells that serve crucial roles including steroid synthesis. These follicular compartments function as a single physiological unit whose purpose is to produce a healthy egg, which upon ovulation can be fertilized and give rise to a healthy embryo, thus enabling the female germ cell to fulfill its reproductive potential. Beginning from the initial stage of follicle formation and until terminal differentiation at ovulation, oocyte and follicle growth depend absolutely on cooperation between the different cellular compartments. This cooperation synchronizes the initiation of oocyte growth with follicle activation. During growth, it enables metabolic support for the follicle-enclosed oocyte and allows the follicle to fulfill its steroidogenic potential. Near the end of the growth period, intra-follicular interactions prevent the precocious meiotic resumption of the oocyte and ensure its nuclear differentiation. Finally, cooperation enables the events of ovulation, including meiotic maturation of the oocyte and expansion of the cumulus granulosa cells. In this chapter, we discuss the cellular interactions that enable the growing follicle to produce a healthy oocyte, focusing on the communication between the germ cell and the surrounding 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.