Regulation Of Granulosa Cell Function Research Articles

The Effects of Platelet-Derived Growth Factor BB on Ovine Granulosa Cell Function.

Platelet derived growth factors (PDGF) have been shown to modulate ovarian follicular development in some species. However, relatively little is known regarding the potential role of PDGF in regulating granulosa cell function in ruminants. Oocytes and granulosa cells in growing ovine follicles express mRNA encoding both PDGFA and PDGFB and follicular atresia appeared to cause decreased expression in the granulosa cells. However, with in situ hybridization, mRNAs for PDGF receptors (PDGFRA and PDGFRB) were not detectible in the granulosa cells. Thus, it is not clear whether PDGF can regulate granulosa cell function in vitro. Therefore, our aim was to determine the effects of PDGF on thymidine incorporation and progesterone production in sheep granulosa cells. Granulosa cells were collected from ovine follicles 1-2 mm in diameter. The incorporation of 3H-thymidine was determined during a 24 hour culture (20,000 viable cells per well) with M199 media. Secretion of progesterone was measured following culture of granulosa cells (100,000 viable cells per well) in an enriched McCoys 5a media with 30 ng/ml androstenedione, 3 ng/ml ovine FSH and 1 ng/ml IGF-1. Cells were cultured for 6 days with media being replaced every 48 hrs. The concentration of progesterone was measured by RIA in the media collected at the end of the culture period. The amount of DNA at the end of the culture period was also measured using binding of Hoechst 33258 dye. Triplicate wells were included in each assay and a minimum of 3 independent granulosa cell cultures were examined. The effects of human PDGFBB (10 ng/ml) was compared to vehicle treated control cells using paired t-tests. Addition of PDGFBB increased (P<0.05) 3H-thymidine incorporation 4.33 ± 1.10 fold compared to control cultures. Progesterone concentrations in PDGFBB treated granulosa cells decreased (P<0.01) to 0.33 ± 0.03 of those observed in control cultures with no effect on DNA content (1.06 ± 0.09 fold relative to control cultures). Given that PDGFBB regulated ovine granulosa cell function even though mRNA encoding PDGF receptors could not be localized to granulosa cells by in situ hybridization, RT-PCR was used to determine if culture of the granulosa cells might induce expression of PDGF receptors. Total cellular mRNA was extracted from freshly isolated granulosa cells or those cultured for 2 days (2 pools each). One pool of freshly isolated granulosa cells and both pools of cultured granulosa cells contained both PDGFRA and PDGFRB mRNA. Therefore, PDGFs are likely regulators of granulosa cell function in sheep although differences in receptor expression requires further exploration.

The Extracellular Matrix Protein, Spondin 1, Regulates Granulosa Cell Function and May Regulate Vascularization of the Corpus Luteum.

Dramatic changes in the composition of the ovarian extracellular matrix (ECM) and cell-cell adhesion proteins occur throughout folliculogenesis. The ECM protein, Spondin 1 (SPON1), is a secreted adhesion protein best known for its ability to regulate patterning of the developing nervous system by promoting outgrowth and adhesion of neural cells. SPON1 has been previously identified in bovine follicular fluid, and was found to promote the proliferation of rat vascular smooth muscle cells. Although SPON1 is highly expressed in the human ovary, and dramatically overexpressed in ovarian cancer, a function for SPON1 in the ovary has never been studied. The objectives of our study were to: a) characterize the expression and localization of SPON1 in the mouse ovary, and b) identify what effect SPON1 may have on granulosa cell behaviour and function. Using qPCR, we found that SPON1 mRNA levels increased 5-fold in granulosa cells isolated from mice treated with PMSG for 48h compared to vehicle-treated controls, indicating that FSH increases SPON1 expression. Immunofluorescence studies localized SPON1 to regions between and surrounding both theca and granulosa cells of follicles of various sizes, from small secondary to preovulatory follicles. In the corpus luteum (CL), the pattern of SPON1 immunoreactivity was strikingly similar to that of platelet endothelial cell adhesion molecule (PECAM-1), which localizes to endothelial cells of the vasculature, suggesting that SPON1 may regulate vascularization during formation of the CL. Finally, we found that SPON1 has diverse effects when added as a recombinant protein to both granulosa cell lines and mouse primary granulosa cells. SPON1 enhanced the proliferation of KGN human granulosa cells, and in primary mouse granulosa cells, SPON1 enhanced granulosa cell viability by up to 50% compared to vehicle-treated controls, suggesting that SPON1 may regulate granulosa cell viability during folliculogenesis. Lastly, SPON1 dramatically induced aggregation of primary mouse granulosa cells, suggesting a role for SPON1 in granulosa cell adhesion, as has been reported in SPON1-treated neural cells. In summary, we have shown that the ECM protein, SPON1, regulates granulosa cell proliferation, survival, and cell-cell adhesion, and may also regulate vascularization of the corpus luteum. Given the diversity of its expression and effects on granulosa cells, we hypothesize that SPON1 plays a previously-unidentified important role in the regulation of various stages of folliculogenesis.

Potential role of follicle‐stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH-induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin-primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 >control) that was partly attributed to the increased secretion of pro-angiogenic factors VEGF and PDGF-B. This is further supported by the evidence that pre-treatment with inhibitor of VEGF receptor-2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2-day aortic ring culture period suppressed microvessel growth in GCCM-treated groups, and also inhibited the FSH + TGFβ1-GCCM-stimulated release of matrix remodeling-associated gelatinase activities. Interestingly, pre-treatment of AG1296 at late stage suppressed GCCM-induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF-B, and that in turn up-regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development.

Identification of genes targeted by FSH and oocytes in porcine granulosa cells

In the mammalian ovarian follicle maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa cells and the cumulus cells. These cells are regulated by follicle-stimulating hormone (FSH), originating from the pituitary, and paracrine factors derived from the oocyte. To gain insight into the mechanisms involved in the regulation of granulosa cell function, this study aimed to identify genes in mural granulosa cells that are regulated by FSH and oocyte secreted factors using the pig as a model organism. Mural granulosa cells were collected from 3–6 mm follicles from sow ovaries and cultured in serum free medium in the presence or absence of FSH and/or isolated cumulus oocyte complexes (COCs). FSH significantly increased both the metabolic activity and progesterone production of granulosa cells, while the presence of COCs reversed these FSH effects. Expression levels of mRNA in the absence/presence of FSH and COCs were analyzed on porcine specific microarrays representing 11,300 genes. Both previously identified and novel FSH target genes as well as some oocyte affected genes were found. Expression of inhibitor of DNA binding protein 2 and 3, ID2 and ID3, was decreased by FSH but increased by COCs, as validated by quantitative PCR. These proteins function as dominant negative basic helix loop helix (bHLH) transcription factors and since all regulated genes contain the consensus E-box sequence that can bind bHLH factors, our data suggest that FSH and COCs may regulate granulosa cell function by tuning the activity of bHLH factors, through ID2 and ID3.

VEGF modulates the effects of gonadotropins in granulosa cells

Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8 mm or 9 to 14 mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100 ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1 ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles ( P = 0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P = 0.045); whereas higher doses of VEGF had no effect on proliferation ( P = 0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation ( P = 0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles ( P = 0.9). Granulosa cells from 9- to 14-mm follicles responded to 1 ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 ( P = 0.003) but higher VEGF doses had no effect ( P = 0.9). The PTGS2 response to 1 ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation ( P = 0.003) and PTGS2 expression ( P < 0.01) in granulosa cells from 9- to 14-mm follicles were abolished ( P = 0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating (3)H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating (3)H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive alpha-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

Serotonin may alter the pattern of gonadotropin-induced progesterone release of human granulosa cells in superfusion system.

Serotonin plays a hormonal function in several nonneuronal peripheral tissues, such as the ovaries. Our aim was to investigate whether there is a modulatory action of serotonin on gonadotropin-induced steroid secretion of human granulosa cells. In granulosa cell culture, serotonin was administered alone or in combination with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Also, granulosa cells were transferred into a dynamic superfusion apparatus and challenged by FSH and LH alone or along with serotonin. Estradiol and progesterone concentrations of samples were measured by radioimmunoassay. As expected, administration of FSH, LH, and serotonin alone resulted in a significant estradiol and progesterone release in cell culture, as well as a significant increase in progesterone release in dynamic superfusion system. In cell culture, co-administration of serotonin with gonadotropins had no additive effect on gonadotropin-induced secretion of progesterone, while it further augmented that of estradiol. In superfusion system, when gonadotropins were added along with serotonin, the increase in progesterone release was markedly less, while peaks of hormone response were remarkably prolonged compared to challenges by LH and FSH alone. The observed effects of serotonin on gonadotropin-induced steroid release of granulosa cells may reveal further details about the regulation of granulosa cell function.

317EMBRYONIC DEVELOPMENT AFTER HOLDING BOVINE OOCYTES IN UNDILUTED FOLLICULAR FLUID FOR A 6-HOUR PREMATURATION PERIOD

Follicular fluid has been implicated in follicular growth, initiation of steroidogenesis, regulation of granulosa cell function and oocyte maturation. Although individual components of the follicular fluid have been analyzed, it is still unclear how undiluted follicular fluid may affect subsequent in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and embryo development. The objective of this study was to simulate recovery of bovine oocytes as if under field conditions to determine the effect of holding these oocytes in follicular fluid for a 6-h holding period before performing IVF. Ovaries were obtained from a local abattoir and transported to the laboratory at ∼22°C in a 0.9% saline and antibiotic solution. At the laboratory, ovaries were rinsed with an ethanol solution and randomly allotted to four treatment groups. Follicles ranging in size from 2 to 9mm were aspirated using a 20-gauge needle attached to a sterile plastic syringe. Dominant follicles were not included. Oocytes harvested from the ovaries in Treatment 1 (Control) were placed directly into a standard laboratory maturation medium consisting of TCM-199 supplemented with LH, FSH, fetal bovine serum, estradiol and gentamicin for 22h. Standard laboratory IVF was then performed with frozen-thawed semen from a fertile bull. In Treatment 2, 3mL of pooled follicular fluid was dispensed into a 6-mL conical centrifuge tube and co-incubated with the harvested oocytes at room temperature (22°C) for 6h. Oocytes recovered from the ovaries in Treatment 3 were placed into 3mL of Ringer’s lactate solution for a 6-h holding period at 22°C. Oocytes obtained from the ovaries in Treatment 4 were placed into a mixture of 2mL of Ringer’s lactate plus 1mL of the same pooled follicular fluid and were held for 6h at 22°C. After being held for a 6-h period, oocytes were recovered from each centrifuge tube and were placed into IVM medium for 22h and then subjected to standard IVF. Embryo development in CR1aa culture medium was assessed at 72, 168 and 216h post-insemination in each treatment group. In summary, no significant difference was detected between the standard IVF procedure and oocytes held in follicular fluid 6h prior to IVF (Treatment 1 v. 2). Also, follicular fluid apparently had a positive effect on the oocytes over that of holding oocytes 6h in Ringer’s lactate alone, as indicted by a significantly greater rate of blastocyst development (Treatment 3 v. 4). In conclusion, it should not be overlooked that bovine oocytes, aspirated under field conditions, could be held up to 6h in 22°C undiluted bovine follicular fluid until they can be delivered to a full-service IVF laboratory.

Murine granulosa cell morphology and function are regulated by a synthetic Arg–Gly–Asp matrix

Extracellular matrix (ECM) proteins are established regulators of granulosa cell survival, morphology, and differentiation. In this study, the roles of ECM adhesion peptide density on murine granulosa cell adhesion, morphology, and steroid secretion were probed using synthetic matrices. The synthetic matrix was fabricated from the polysaccharide alginate, which does not inherently support cell adhesion but can be modified with controlled densities of adhesion peptides (10 −4 to 2×10 −1 ng/cm 2). GRM02, a murine granulosa cell line, and primary murine granulosa cells were cultured on alginate matrices modified by coupling of synthetic peptide sequences containing the Arg–Gly–Asp motif common to ECM proteins. Cells cultured on these peptide-modified surfaces (0.02, 0.2 ng/cm 2) attached and spread, with morphologies specific to the peptide identity and density. Additionally, progesterone and estradiol secretion was a function of peptide density, with up to threefold increases compared to controls. These results indicate that the density and identity of adhesion peptides regulate granulosa cell function. This system provides a mechanism to examine the granulosa cell-ECM interactions that occur during follicle maturation.

Effects of nitric oxide and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells

Objective To determine the effects of nitric oxide (NO) and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells. Design Basic research study. Setting University research laboratory. Animal(s) Primary cell culture of granulosa cells obtained from estrogen-treated, immature Sprague-Dawley female rats. Intervention(s) Functionally immature rat granulosa cells were incubated for 48 hours with media alone; FSH; forskolin; the NO generator DETA/NO; an inhibitor of soluble guanylyl cyclase (ODQ); and/or a cell-permeable cGMP analog. Main outcome measure(s) Media concentrations of inhibin A were measured by solid-phase immunosorbent assay. Inhibin α and βA subunit mRNA levels were determined by Northern and slot blot analyses. Result(s) Whereas FSH caused a 20-fold increase in inhibin A levels compared with untreated granulosa cells, the NO generator DETA/NO significantly inhibited FSH-stimulated inhibin A concentrations. Similarly, cotreatment with FSH plus dibutyryl cGMP significantly attenuated inhibin A concentrations, compared with those in cells treated with FSH alone. Incubation with forskolin (FSK) stimulated inhibin A levels sevenfold, whereas cotreatment with FSK plus DETA/NO or FSK plus dibutyryl cGMP effectively decreased inhibin A concentrations. The effects of NO on inhibin A levels were not prevented by cotreatment with an inhibitor of soluble guanylyl cyclase. In addition, there was no influence of DETA/NO or dibutyryl cGMP on inhibin subunit mRNA levels. Conclusion(s) These findings indicate that NO and cGMP can attenuate inhibin A concentrations through actions at one or more post-FSH receptor sites. These influences may reflect inhibition of inhibin A secretion, rather than gene expression and protein synthesis. In addition, NO decreases inhibin A concentrations through both cGMP-dependent and -independent pathways. These results suggest local roles for NO and cGMP in the regulation of granulosa cell function.

The effect of catecholamines, acetylcholine and histamine on progesterone release by human granulosa cells in a granulosa cell superfusion system

There are experimental data demonstrating the presence and actions of various neurotransmitters in the ovary, thus supporting the view that they might play a role in intraovarian regulatory mechanisms,although their exact function in the regulation of ovarian hormone secretion is unclear. The objective of the present study was to investigate the direct action of catecholamines, acetylcholine and histamineon progesterone secretion of human granulosa cells in a superfused cell system. Human granulosa cells were isolated from preovulatory follicular fluid using a Percoll gradient centrifugation method. Approximately2 × 106 cells were mixed with Sephadex G-10 and were transferred into two chambers of the superfusion apparatus. The system was perfused with a culture medium and test materials were addedto the system at a dose of 100 pmol/ml. The progesterone concentration of samples was measured using an 125I radioimmunoassay. Administration of epinephrine (adrenaline), norepinephrine (noradrenaline),dopamine and histamine had no effect on progesterone release. However, acetylcholine produced a significant progesterone release, which could be blocked by atropine. The observed effect of acetylcholineon progesterone release of superfused human granulosa cells may reflect a physiological role of acetylcholine in the regulation of granulosa cell function during the menstrual cycle.

Effect of basal lamina on progesterone production by chicken granulosa cells in vitro — influence of follicular development

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90–95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with β-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F 1, mature), third largest (F 3, growing), fifth to seventh largest (F 5–7, growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F 1 follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F 3) granulosa cells than by differentiated (F 1) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F 1) and differentiating (F 5–7) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F 3) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F 5–7) granulosa cells was enhanced, whereas progesterone production by differentiated (F 1) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F 5–7) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F 1) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F 3) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.

Thecal cell-granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor, hepatocyte growth factor, and Kit ligand during ovarian follicular development.

Interactions between mesenchymal-derived thecal cells and epithelial-derived granulosa cells are essential for follicular development in the ovary. These mesenchymal-epithelial cell interactions are in part mediated by keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and Kit ligand (KL). This study investigates the hypothesis that thecal cell-derived growth factors (e.g. KGF and HGF) regulate granulosa cell function, and granulosa cell-derived growth factors (e.g. KL) regulate thecal cell function. Gonadotropin regulation of this cell-cell interaction is also examined. Sensitive quantitative RT-PCR assays were used to analyze gene expression of KGF, HGF, and KL in the ovary. Thecal cell-derived KGF and HGF stimulated KL expression in bovine granulosa cells. Granulosa cell-derived KL stimulated KGF and HGF expression in bovine thecal cells. These results suggest that thecal and granulosa cells interact in a positive feedback loop mediated by KGF, HGF, and KL. Previous studies have suggested that gonadotropins (i.e. FSH and LH) regulate locally produced growth factor expression in the ovary. Treatment of bovine granulosa cells with FSH and hCG (a LH agonist) directly stimulated KL expression. The LH agonist hCG was also found to stimulate both KGF and HGF expression in thecal cells. The actions of gonadotropins on follicular development may in part be indirectly regulated by KL, KGF, and HGF expression. A novel positive feedback loop was identified between thecal cells and granulosa cells that is mediated by KL, KGF, and HGF. Thecal cell-derived KGF and HGF can stimulate granulosa cell-derived KL expression, and KL, in turn, can stimulate thecal cell-derived KGF and HGF expression. Combined observations support the hypothesis that mesenchymal-epithelial cell interactions between thecal and granulosa cells can play a significant role during ovarian follicular development and mediate gonadotropin actions.

Regulation of follicle-stimulating hormone receptor.

The acquisition of follicle-stimulating hormone (FSH) receptors during folliculogenesis is believed to be a key event in follicle development. We have examined the effects of FSH and activin on FSH receptor mRNA in cultured rat granulosa cells. Treatment of granulosa cells with FSH resulted in transient suppression of the FSH receptor mRNA levels 2-6 h after treatment, with subsequent recovery at 24 h. We could not detect a similar effect on FSH receptor mRNA by 8-bromoadenosine 3,5-cyclic monophosphate, which continuously stimulated FSH receptor mRNA over a similar time course. On the other hand, stimulation of the protein kinase C (PKC) pathway with phorbol myristate acetate mimicked the time course of the effects of FSH on the levels of FSH receptor mRNA. Taken together, these results suggest that the cAMP cascade may increase the mRNA levels of FSH receptor and, at the same time, the other cascade, PKC, may decrease FSH receptor mRNA levels. To further investigate the role of activin in the regulation of granulosa cell function, we studied the effect of activin on FSH receptor mRNA levels. Compared to the control, treatment with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum circa 4-fold increase at 24 h. Treatment of granulosa cells with activin (20-300 ng/ml) for 24 h increased FSH receptor mRNA in a dose-dependent manner to a maximum circa 4-fold increase at concentrations of 100-300 ng/ml. Although follistatin alone had no detectable effect on FSH receptor mRNA levels, combination of follistatin (0-200 ng/ml) with activin (100 ng/ml) caused a significant reduction in the levels of activin-induced FSH receptor mRNA in a dose-dependent manner.

Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion, steroid metabolism, and responsiveness to growth factors

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compared BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [3H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [3H]-progesterone to 5 alpha-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [3H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-beta when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-beta displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues.

Insulin-like growth factor binding protein-3 inhibits porcine granulosa cell function in vitro

Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) effects on basal, insulin-like growth factor I (IGF-I)-, and follicle-stimulating hormone (FSH)-stimulated progesterone (P4) secretion and [3H]aminoisobutyric acid (AIB) uptake by primary porcine granulosa cells (MDGs) and MDGs that have been passaged once (MDGp1) were assessed. Cells were treated concurrently or were preincubated with rhIGFBP-3 followed by treatment. rhIGFBP-3 had no effect on MDG or MDGp1 cell numbers after 24 h. Cotreatment with rhIGFBP-3 inhibited P4 secretion after treatment with FSH, IGF-I, and FSH plus IGF-I. FSH did not stimulate [3H]AIB uptake. However, the IGF-I-stimulated increase in [3H]AIB uptake was completely prevented by concurrent treatment with IGFBP-3. Preincubation of MDGp1 cells with IGFBP-3 dose dependently inhibited FSH- and IGF-I-stimulated P4 secretion. This inhibition was associated with increased cell association of the binding protein and increased IGF-I binding to the cells. These results indicate that IGFBP-3 is inhibitory to a variety of crucial functions in porcine granulosa cells, supporting a role for it in the regulation of granulosa cell function.

Inhibitory effect of transferrin on progesterone production in the granulosa cell of humans in vivo and porcine granulosa cell in vitro.

We evaluated the effect of transferrin on the regulation of granulosa cell function in humans by evaluating the production of progesterone (P) in the preovulatory phase in vivo, and in cultured porcine granulosa cells in vitro. Twenty-five women treated for in vitro fertilization and embryo transfer had their serum levels of 17 beta-estradiol (E2) and P determined on the day of administration of human chorionic gonadotropin. Transferrin concentrations were also determined in ovarian follicular fluid. In an in vitro study, porcine granulosa cells were cultured in the presence of follicle-stimulating hormone (FSH) and transferrin. Serum levels of P showed a significant negative correlation with those of transferrin (r = -0.53, p < 0.01), whereas serum levels of E2 did not (r = 0.14). When the subjects were divided into two groups by serum P concentration (low P < 1 ng/ml, high P > or = 1 ng/ml) serum concentrations of transferrin were significantly increased in the group with the low versus the high level of p (p < 0.01). Production of P by porcine granulosa cells was suppressed by transferrin in the presence of various concentrations of FSH. Increasing the dose of transferrin significantly suppressed the production of P by those cells in a dose-dependent fashion. The production of P during the preovulatory phase may be suppressed by transferrin in the granulosa cells.

Endocrinology: Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness

In order to study the effects of follicle-stimulating hormone (FSH) on differentiation of granulosa cells, a well-defined and validated in-vitro culture system is indispensable. In this study, pooled follicular aspirates were stimulated in vitro with FSH and luteinizing hormone (LH) for 2, 4 and 6 days, either immediately after plating or after 7 days of preincubation. Cultures were assayed for progesterone and oestradiol production. Fresh cells displayed very high basal progesterone production which could be stimulated with LH but not FSH. After preincubation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol. When cultured on human fibronectin-coated wells, similar basal but higher progesterone concentrations after stimulation were observed. In comparison with serum-free media, addition of Serum-Plus resulted in higher basal and stimulated progesterone concentration, possibly due to the presence of serum factors. This study demonstrates firstly that after 7 days preincubation, cultures gained responsiveness to FSH but remained responsive to LH during 4 days of stimulation. This suggests a persisting differentiated cell population in vitro. Secondly, the use of human fibronectin extracellular matrix and serum promotes steroid production, either due to factors promoting cell growth and function or to availability of steroid precursors. Therefore one has to be cautious with interpretation of data obtained from this widely used culture system, employing highly differentiated cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function.

The effect of histamine on progesterone and estradiol secretion of human granulosa cells in serum-free culture.

The aim of this study was to explore the direct action of histamine on progesterone and estradiol secretion of human granulosa cells cultured in serum-free medium. Human granulosa cells were isolated from preovulatory follicular fluid aspirated from 17 women (32 +/- 3 years old, mean +/- SD) undergoing in vitro fertilization treatment at the University Women's Hospital of Tübingen. Progesterone and estradiol production was measured in the presence and absence of histamine, terfenadine or cimetidine using radioimmunoassays. Statistical analysis of the data was performed by analysis of variance and Newmann-Keul tests. Histamine stimulated a dose-related increase in estradiol secretion with a maximal stimulatory effect at 10(-3) mol/l. This response was blocked specifically by the H1-receptor antagonist terfenadine. Progesterone production in response to histamine stimulation was independent of dose at the limit of significance. The specific H2-receptor antagonist cimetidine did not block the stimulatory effect of histamine. We suggest that histamine has a direct stimulatory effect on steroid production of granulosa cells mediated via the H1-receptor. This effect may have a physiological role in the regulation of granulosa cell function during the menstrual cycle.