Undifferentiated Granulosa Cells Research Articles

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2 in regulating lipid metabolism and progesterone secretion of goose granulosa cells

Accumulating evidence shows that granulosa cells within both mammalian and avian ovaries have the ability to synthesize fatty acids through de novo lipogenesis and to accumulate triglycerides essential for oocyte and ovarian development. However, very little is known about the exact roles of key genes involved in the lipid metabolic pathway in granulosa cells. The goal of this study was to investigate the differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2, which are recognized as the rate-limiting enzymes catalyzing the last step of triglyceride biosynthesis, in regulating lipid metabolism and steroidogenesis in granulosa cells of goose follicles at different developmental stages. It was observed that the mRNAs encoding DGAT1 and DGAT2 were ubiquitous in all examined granulosa cell layers but exhibited distinct expression profiles during follicle development. Notably, the mRNA levels of DGAT1, DGAT2, FSHR, LHR, STAR, CYP11A1, and 3βHSD remained almost constant in all except for 1–2 follicles within the 8−10 mm cohort, followed by an acute increase/decrease in the F5 follicles. At the cellular level, siRNA-mediated downregulation of DGAT1 or DGAT2 did not change the amount of lipids accumulated in both undifferentiated- and differentiated granulosa cells, while overexpression of DGAT2 promoted lipid accumulation and expression of lipogenic-related genes in these cells. Meanwhile, we found that interfering DGAT2 had no effect but interfering DGAT1 or overexpressing DGAT2 stimulated progesterone secretion in undifferentiated granulosa cells; in contrast, interference or overexpression of DGAT1/2 failed to change progesterone levels in differentiated granulosa cells but differently modulated expression of steroidogenic-related genes. Therefore, it could be concluded that DGAT1 is less efficient than DGAT2 in promoting lipid accumulation in both undifferentiated- and differentiated granulosa cells and that DGAT1 negatively while DGAT2 positively regulates progesterone production in undifferentiated granulosa cells.

Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle-stimulating hormone receptor signaling.

Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GCs) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Before recruitment focus is directed toward the inhibition of FSHR-signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR messenger RNA in cultured, undifferentiated GC using small interfering RNA facilitated FSH-induced cAMP formation, STAR expression, and progesterone production. Furthermore, overexpression of bovine βARR1 and G-protein-coupled receptor kinase2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment.

Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation.

Is the genome-wide response of human cumulus cells to FSH and insulin-like growth factors (IGFs) comparable to the response observed in undifferentiated granulosa cells (GCs)? FSH actions in human cumulus cells mimic those observed in preantral undifferentiated GCs from laboratory animals, and approximately half of the regulated genes are dependent on the simultaneous activation of the IGF1 receptor (IGF1R). Animal studies have shown that FSH and the IGFs system are required for follicle growth and maturation. In humans, IGF levels in the follicular fluid correlate with patients' responses toIVF protocols. The main targets of FSH and IGFs in the ovary are the GCs; however, the genomic mechanisms involved in the response of GCs to these hormones are unknown. Human cumulus cells isolated from IVF patients were cultured for 48 h in serum-free media in the presence of vehicle, FSH, IGF1R inhibitor or their combination. Discarded cumulus cells were donated to research by reproductive-aged women undergoing IVF due to non-ovarian etiologies of infertility at a university-affiliated clinic. The effect of FSH and/or IGF1R inhibition on cumulus cell function was evaluated using Affymetrix microarrays, quantitative PCR, western blot, promoter assays and hormone level measurements. The findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes known to be markers of the preantral to preovulatory differentiation of GCs. These results also demonstrate that ~50% of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and IGFs in humans. This novel approach will allow for future mechanistic and molecular studies on the regulation of human follicle maturation. Data set can be accessed at Gene Expression Omnibus number GSE86427. Experiments were performed using primary human cumulus cells. This may not represent the response of intact follicles. Understanding the mechanisms involved in the regulation of GC differentiation by FSH and IGF in humans will contribute to improving treatments for infertility. The project was financed by the National Instituted of Health grant number R56HD086054 and R01HD057110 (C.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare.

Regulatory Mechanisms Underlying the Expression of Prolactin Receptor in Chicken Granulosa Cells

Prolactin (PRL) has both pro- and anti-gonadal roles in the regulation of avian ovarian functions through its interaction with the receptor (PRLR). However, neither the pattern of expression of PRLR nor its regulatory mechanisms during follicle development have been clearly defined. The objective of the present study was to investigate mechanisms of PRLR expression in chicken granulosa cells. Levels of PRLR transcript were highest in the stroma and walls of follicles < 2 mm in diameter and progressively declined with the maturation of follicles. In preovulatory follicles, PRLR was expressed at higher levels in granulosa than theca layers. FSH exerted the greatest stimulatory effect on PRLR and StAR expression in cultured granulosa cells of the 6–8 mm follicles but this effect declined as follicles matured to F1. In contrast, LH did not alter the expression of PRLR in granulosa cells of all follicular classes but increased levels of StAR in F2 and F1 granulosa cells. Both non-glycosylated- (NG-) and glycosylated- (G-) PRL upregulated basal PRLR expression in granulosa cells of the 6–8 mm, F3 or F1 follicles but had little effect in F2 follicles. Furthermore, FSH-stimulated PRLR expression was reduced by the addition of either isoform of PRL especially in F2 granulosa cells. These results indicate that PRLR is differentially distributed and regulated by FSH or PRL variants independently or in combination in the follicular hierarchy. By using activators and inhibitors, we further demonstrated that multiple signaling pathways, including PKA, PKC, PI3K, mTOR and AMPK, are not only directly involved in, but they can also converge to modulate ERK2 activity to regulate FSH-mediated PRLR and StAR expression in undifferentiated granulosa cells. These data provide new insights into the regulatory mechanisms controlling the expression of PRLR in granulosa cells.

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary.

ABSTRACTFas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Expression and regulation of prolactin-like protein messenger RNA in undifferentiated chicken granulosa cells

Prolactin-like protein (PRL-L; LOC417800) is a homolog of PRL in non-mammalian vertebrates and can act as a functional ligand of PRL receptor (PRLR). Despite its widespread expression in extrapituitary tissues, mechanisms of regulation of PRL-L in the chicken ovary remain unknown. In this study, we first examined PRL-L expression in chicken ovarian developing follicles. PRL-L transcript levels were highest (P<0.05) in follicular walls of <2mm follicles and progressively declined during follicle maturation. Undifferentiated granulosa cells of 6–8mm follicles had higher (P<0.05) PRL-L mRNA levels than differentiated granulosa cells of F3, F2 or F1 follicles. In cultured undifferentiated granulosa cells, levels of PRL-L transcript were increased (P<0.05) by follicle stimulating hormone (FSH) treatment while were not altered by the addition of luteinizing hormone (LH). In addition, 10ng/ml non-glycosylated (NG-) and 1ng/ml glycosylated (G-) PRL increased (P<0.05) but at higher levels (100 or 1000ng/ml) both showed no effects on PRL-L expression. Furthermore, 100ng/ml NG-PRL enhanced (P<0.05) FSH-induced PRL-L expression, whereas the effects of G-PRL were not significant. These results suggest that PRL-L mRNA is differentially expressed in the follicular hierarchy and its high abundance in undifferentiated granulosa cells is under the regulation of FSH or PRL variants independently or in combination. Moreover, in undifferentiated granulosa cells we also provide evidence for a positive role for PKA, PKC and PI3K signaling while a negative role for ERK2 in mediating FSH stimulation of PRL-L transcription.

The Hippo/MST Pathway Member SAV1 Plays a Suppressive Role in Development of the Prehierarchical Follicles in Hen Ovary.

The Hippo/MST signaling pathway is a critical player in controlling cell proliferation, self-renewal, differentiation, and apoptosis of most tissues and organs in diverse species. Previous studies have shown that Salvador homolog 1 (SAV1), a scaffolding protein which functions in the signaling system is expressed in mammalian ovaries and play a vital role in governing the follicle development. But the exact biological effects of chicken SAV1 in prehierarchical follicle development remain poorly understood. In the present study, we demonstrated that the SAV1 protein is predominantly expressed in the oocytes and undifferentiated granulosa cells in the various sized prehierarchical follicles of hen ovary, and the endogenous expression level of SAV1 mRNA appears down-regulated from the primordial follicles to the largest preovulatory follicles (F2-F1) by immunohistochemistry and real-time RT-PCR, respectively. Moreover, we found the intracellular SAV1 physically interacts with each of the pathway members, including STK4/MST1, STK3/MST2, LATS1 and MOB2 using western blotting. And SAV1 significantly promotes the phosphorylation of LATS1 induced by the kinase of STK4 or STK3 in vitro. Furthermore, SAV1 knockdown by small interfering RNA (siRNA) significantly increased proliferation of granulosa cells from the prehierarchical follicles (6–8 mm in diameter) by BrdU-incorporation assay, in which the expression levels of GDF9, StAR and FSHR mRNA was notably enhanced. Meanwhile, these findings were consolidated by the data of SAV1 overexpression. Taken together, the present results revealed that SAV1 can inhibit proliferation of the granulosa cells whereby the expression levels of GDF9, StAR and FSHR mRNA were negatively regulated. Accordingly, SAV1, as a member of the hippo/MST signaling pathway plays a suppressive role in ovarian follicle development by promoting phosphorylation and activity of the downstream LATS1, may consequently lead to prevention of the follicle selection during ovary development.

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

Vasoactive intestinal peptide promotes differentiation and clock gene expression in granulosa cells from prehierarchal follicles.

Vasoactive intestinal peptide (VIP) signaling via cyclic adenosine monophosphate (cAMP) is reported to stimulate steroidogenesis in ovarian granulosa cells from a variety of vertebrate species, including the domestic hen. Prior to follicle selection in the hen ovary (i.e., cyclic recruitment) follicle-stimulating-hormone (FSH)-induced cAMP signaling is absent within the granulosa layer until immediately following follicle selection. As a consequence, granulosa cells remain in an undifferentiated state and are unable to initiate FSH-induced steroidogenesis. VIP receptors (VPAC1 and VPAC2), like the FSH receptor, are G protein-coupled receptors, so we predicted that VIP signaling in granulosa cells is also absent in follicles that have not yet been selected into the preovulatory hierarchy. Initial studies established that mRNA encoding VPAC1 and VPAC2 are expressed within the granulosa cells throughout follicle development. Nevertheless, undifferentiated granulosa cells from prehierarchal (6-8 mm) follicles do not accumulate cAMP in response to a 4-hr incubation with chicken VIP; the capacity for such receptor signaling is attained only following selection within actively differentiating granulosa cells. VIP treatment did, however, increase expression of mRNA encoding the Gallus circadian clock protein, BMAL1-but only in granulosa cells collected from selected follicles. These findings provide evidence that, at follicle selection, the acquisition of VIP-induced cAMP cell signaling helps initiate and promote the differentiation of of granulosa cells. Furthermore, we propose that VIP signaling may regulate BMAL1 expression and, thus, a daily rhythmicity within granulosa cells of preovulatory follicles. Mol. Reprod. Dev. 83: 455-463, 2016. © 2016 Wiley Periodicals, Inc.

Vascular endothelial growth factor and angiopoietins during hen ovarian follicle development

Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P>0.05) relative to developmental stages surrounding follicle selection (6–8mm versus 9–12mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6–8mm) follicles and actively differentiating GC from selected (9–12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk.

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.

Bone Morphogenetic Protein 4 Supports the Initial Differentiation of Hen (Gallus gallus) Granulosa Cells

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6-8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for bone morphogenetic protein 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ~80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (10-100 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated protein kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.

Follicular stage-dependent regulation of apoptosis and steroidogenesis by prohibitin in rat granulosa cells

BackgroundFollicular growth and atresia are tightly regulated processes, which involve the participation of endocrine, autocrine and paracrine factors at the cellular level. Prohibitin (PHB) is a multifunctional intracellular protein playing an important role in the regulation of proliferation, apoptosis and differentiation. Here we examined the expression of PHB and its regulation by FSH in vitro and studied the role of PHB in the regulation of apoptosis and steroidogenesis in response to the apoptosis inducer staurosporine (STS) and to FSH, respectively.MethodsUndifferentiated and differentiated granulosa cells were collected from diethylstilbestrol (DES)- and equine chronic gonadotropin (eCG)-primed immature rats, respectively and then cultured with various treatments (FSH, adenovirus infection, STS) according to experimental design. The apoptosis rate, the production of estradiol and progesterone, and the expression of distinct proteins (PHB, caspase-3, phospho- and total Akt) were assessed.ResultsPHB is anti-apoptotic and its action is dependent on the differentiated state of the granulosa cells. Data from gain- and loss-of-function experiments demonstrate that PHB inhibited STS-induced caspase-3 cleavage and apoptosis in undifferentiated granulosa cells, but was ineffective in differentiated cells. In contrast, PHB suppresses FSH-induced steroidogenesis and this response is evident irrespective of the differentiated state of granulosa cells.ConclusionThese findings suggest that PHB regulates granulosa cell apoptosis and steroidogenesis in a follicular stage-dependent manner and that the dysregulation of PHB expression and action may be relevant to ovarian dysfunction.

The fate of granulosa cells following premature oocyte loss and the development of ovarian cancers

This review examines the importance of the epithelial origin of granulosa cells and their possible contribution to the development of ovarian cancers in three animal models. We hypothesise that undifferentiated granulosa cells, devoid of their germ cell regulator, retain their embryonic plasticity and may give rise to ovarian cancers of epithelial origin. Dazl-KO and FancD2-KO mice and BMP15-KO sheep are animal models in which germ cells or oocytes are lost at specific stages of follicular formation or growth, leaving behind clusters of residual, but healthy somatic cells. A common feature in Dazl- and Fancd2-KO animals following germ cell/oocyte loss is the presence of sex cords and intraovarian epithelial ducts or tubules. In Dazl-KO mice, cord/tubule-like structures, OSE invaginations and clusters of steroidogenic cells became increasingly prominent with age, but these abnormal structures remained benign. In Fancd2-KO mice, the formation of sex-cords and intraovarian tubules lead to the formation of tumours with multiple phenotypes including luteomas, papillary cysts and malignant carcinomas (e.g. adenocarcinomas). In BMP15-KO sheep, oocytes die as follicles start to grow, leaving 'nodules' containing granulosa cells with a capacity to respond to follicle stimulating hormone and synthesize inhibin. Thereafter, these nodules coalesced and a range of benign solid or semi-solid tumour phenotypes developed. We conclude that premature loss of oocytes, but not granulosa cells, leads to tumour formation with multiple phenotypes. Moreover, the severity of tumour development is linked to both the specificity of the mutation and the timing of oocyte loss relative to that of follicular formation.

Bone Morphogenetic Protein 6 (BMP6) Promotes Fsh-Responsiveness and Anti-Müllerian Hormone (AMH) mRNA Expression in Undifferentiated Granulosa Cells from Hen Prehierarchal Follicles.

Bone Morphogenetic Protein 6 (BMP6) Promotes Fsh-Responsiveness and Anti-Müllerian Hormone (AMH) mRNA Expression in Undifferentiated Granulosa Cells from Hen Prehierarchal Follicles.

Effect of resistin on granulosa and theca cell function in cattle

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 (1–5 mm) 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.

Role of Prohibitin in Regulating FSH-Induced Steroidogenesis During Follicular Development.

With the advances in gonadotropin research, treatment of human infertility with gonadotropin preparations has become a common practice. It plays critical roles in stimulating ovarian cell proliferation, differentiation and suppressing apoptosis, which ultimately induces ovarian follicle growth and ovulation. The action of gonadotropin during follicular development is however complex and is stage-dependent, thus making optimization of gonadotropin treatment for various ovarian disorders often difficult. Prohibitin (PHB) is an intracellular protein important for cell cycle regulation, apoptosis and cellular differentiation. Ovarian PHB is up-regulated by equine chorionic gonadotropin (eCG) in vivo. Although PHB prevents cell apoptosis induced by the alkaloid staurosporine in undifferentiated granulosa cells (from primarily preantral/early antral follicles), whether the expression and action of PHB are similarly regulated in differentiated granulosa cells (large antral and preovulatory follicles) are unknown. We hypothesize that PHB is an important regulator of granulosa cell fate and plays a role in regulating steroidogenesis during follicular development. Our objective is to better understand the function and regulation of PHB during follicular development, especially during FSH-induced antral follicle growth. Using granulosa cells from eCG-primed immature rats, we observed that (1) FSH up-regulates granulosa cell PHB expression in a follicular stage-dependent manner in vitro. (2) Akt content and its phosphorylation are required for the regulation of PHB by FSH. (3) Exogenous PHB significantly reduces FSH-stimulated estradiol and progesterone production in granulosa cells. (4) cyp11a1 (p450 side-chain cleavage enzyme) mRNA and protein expression in granulosa cells are down-regulated by exogenous PHB. (5) The mRNA levels of other steroidogenic enzymes, such as cyp19 (aromatase) and Steroidogenic acute regulatory protein (StAR), are also down-regulated by exogenous PHB. Conclusion: This study demonstrates that granulosa cell PHB is regulated by FSH in a follicular stage-dependent manner and that PHB regulates FSH-induced steroidogenesis in granulosa cells. Our findings raise the possibility that the dysregulation of PHB expression and function may be linked to infertility associated with compromised follicular growth and differentiation (e.g. Polycystic ovarian syndrome) and that PHB may be a therapeutic target for the treatment of infertility. Further experiments are required to test these possibilities.Supported by Canadian Institutes of Health Research (CIHR). (poster)

1089 Does EGFR gene deregulation and PI3KCA mutations predict response to chemoradiation in squamous cell anal cancer (SCAC)?

Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P > 0.05) relative to developmental stages surrounding follicle selection (6–8 mm versus 9–12 mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6–8 mm) follicles and actively differentiating GC from selected (9–12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12 mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk.

Toll-like receptor signaling in hen ovarian granulosa cells is dependent on stage of follicle maturation

The recent identification of toll-like receptor (TLR) signaling within ovarian granulosa cells has broad implications for ovarian physiology. Functions of TLRs within granulosa cells of the laying hen are of particular interest due to the method of transovarian transmission of Salmonella enteritidis, which results in egg contamination. This study utilized hen granulosa cells to evaluate the expression and function of Gallus TLR-signaling at distinct stages of follicular maturity. Data presented herein demonstrate the presence of TLR2, TLR4, and TLR15 mRNAs in undifferentiated granulosa cells from prehierarchal follicles and differentiated granulosa cells from preovulatory follicles, together with mRNAs encoding adaptor proteins and signaling components required for TLR signaling gene. Treatment with lipopolysaccharide (LPS) or LH, in vitro, led to the differential regulation of TLRs based on the stage of follicle maturation, with the largest (F1) follicle granulosa cells having the most rapid response. Furthermore, treatment with LPS resulted in attenuation of agonist-induced progesterone synthesis in undifferentiated, but not differentiated, granulosa cells. Additionally, undifferentiated granulosa cells were significantly more sensitive to LPS-induced apoptosis than differentiated granulosa cells from the F1 follicle. Together, these data provide evidence for a complete and functional TLR signaling pathway in hen granulosa cells, with effects on steroidogenesis and cell viability dependent upon stage of maturation. These differences may reflect the susceptibility of granulosa cells at early stages of maturation to undergo apoptosis in response to select pathogenic stimuli, thus attenuating transovarian transmission, whereas granulosa cells from preovulatory follicles are comparably resistant to LPS-mediated apoptosis.