Interactive actions of prostaglandin and epidermal growth factor to enhance proliferation of granulosa cells from chicken prehierarchical follicles

The interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cells was investigated in prehierarchical small yellow follicles (SYF) of laying hens. The granulosa layers were dispersed into single cells by 12.5 microg/ml collagenase. After 16 h pre-incubation in 0.5% fetal calf serum-supplemented medium, the medium was replaced with serum-free medium. Immunocytochemical staining showed that granulosa cells expressed EGF and its receptor, and their expression was increased by PGE(1) (1-100 ng/ml) or forskolin (10(-7) to 10(-5)M) treatments. EGF receptor was also induced by its ligand EGF. The specific prostaglandin synthase inhibitors SC560 (for COX-1) and NS398 (for COX-2) suppressed EGF-stimulated increase of the granulosa cell number. Furthermore, the effect of EGF was confirmed by the immunocytochemical staining of the proliferating cell nuclear antigen in granulosa cells. Though EGF promoted the expression of both COX-1 and COX-2, the rescue experiment indicated that combined treatment of PGE(1) showed better rescuing effect on NS398 inhibition than SC560 at 10(-6)M, which implies COX-2 plays the predominant role in mediating EGF action. The above results indicate that reciprocal stimulation of intracellular PG and EGF production may enhance proliferation of granulosa cells, hence to facilitate development of chicken prehierarchical follicles.     

R5020 enhances PGE2 stimulated steroidogenesis in cultured rat granulosa cells.

Progesterone biosynthesis and metabolization to 20 alpha-hydroxyprogesterone was stimulated in granulosa cells cultured in the presence of 20 ng/ml of follicle stimulating hormone (FSH) or increasing concentrations of PGE2 (10(-9)-10(-7)M). Concurrent treatment with the synthetic progestin R5020 (10(-6) M) enhanced the FSH or PGE2 stimulated progesterone and 20 alpha-hydroxyprogesterone accumulation in culture media, as well as delta 5-3 beta-hydroxysteroid dehydrogenase activity in granulosa cell homogenates. These findings may represent another example of an autocrine control mechanism in which the steroidogenic product of the granulosa cell exerts an ultra-short loop regulation of its own production.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up‐regulated cyclin gene expression

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin‐dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H₇ or activator PMA (phorbol 12‐myristate 13‐acetate) for 24 h in serum‐free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1–10 μg/ml) significantly increased the number of GCs in SYF in a dose‐dependent manner, and this stimulatory effect was inhibited by H₇, but enhanced by PMA. Meanwhile, the PCNA‐LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 μg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H₇. Furthermore, GS up‐regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H₇ attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC‐involved up‐regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.     

Effects of follicle-stimulating hormone and 17β-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

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 (F₁, mature), third largest (F₃, 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 F₁ 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 (F₃) granulosa cells than by differentiated (F₁) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F₁) and differentiating (F_5–7) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F₃) 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 (F₁) 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 (F₁) 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 (F₃) 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.     

Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.     

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochemical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-I). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H(89) and PKC inhibitor H(7), respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5)M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8)M) also increased PGC colony number (P<0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H(89) or H(7). Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.     

Follicle-stimulating hormone and estradiol regulate antrum-like reorganization of granulosa cells in rat preantral follicle cultures

Formation of a fluid-filled antrum results from the actions of FSH and estrogen on preantral ovarian follicles in most mammalian species. To investigate the novel proposal that hormone-regulated cell-cell interactions mediate antrum formation, we isolated preantral follicles from infant (10- or 11-day-old) Wistar rats and cultured them in a substratum-adherent manner in Minimum Essential Medium supplemented with 2 mM hypoxanthine, 3 mg/ml bovine serum albumin, 5 micrograms/ml insulin, 5 micrograms/ml transferrin, and 5 ng/ml selenium. Similar cultures were previously shown to support oocyte growth and acquisition of meiotic competence. In the absence of FSH, follicles attached to the plastic surface and granulosa cells spread-out uniformly around granulosa cell-enclosed oocytes. FSH treatment caused certain follicles to show an increase between culture days 3 and 7 in appearance of conspicuous antrum-like reorganization of the granulosa cells, but without forming a completely enclosed fluid-filled cavity. This response was biphasic over 10-500 ng/ml FSH, with an optimal concentration of 50 ng/ml resulting in a mean of 37.8 +/- 4.7% of follicles showing antrum-like reorganization for 3 similar experiments. Estradiol-17 beta alone at 10(-10)-10(-8) M was without effect on this response, but at 10(-10) and 10(-9) M, it significantly augmented the action of an optimal concentration of FSH by about 2-fold in 4 experiments. In these experiments, the effect of 10(-8) M estradiol was not significantly different from FSH alone, indicating that the response to estradiol was also biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandin on luteinizing hormone-stimulated proliferation of theca externa cells from chicken prehierarchical follicles

The effect of prostaglandin (PG) on proliferation of chicken theca externa cells from prehierarchical small yellow follicles (SYF) was evaluated and involved signaling pathways as well as mRNA expression of cAMP response element binding protein (CREB1), cyclins (CCND1 and CCNE1) and cyclin-dependent kinases (CDKs) were investigated. Results showed that PGE₁ (1–100 ng/ml) manifested a similar proliferating effect as LH on theca externa cells, and this stimulating effect was restrained by the prostaglandin receptor antagonist SC19220 at 10⁻⁷ to 10⁻⁵ M. Moreover, prostaglandin synthase inhibitor indomethacin (10⁻⁷ to 10⁻⁵ M) suppressed LH-induced increase in the cell number. In addition, PGE₁-stimulated cell proliferation was also predominantly hindered by H₈₉ (PKA inhibitor) instead of H₇ (PKC inhibitor). Meanwhile, BrdU incorporation experiment displayed similar changes with the cell number. Furthermore, H₈₉, SC19220 and indomethacin abolished the PGE₁-stimulated increase in the expression of CREB1, CCND1/CDK6 and CCNE1/CDK2 mRNAs, indicating that cAMP/PKA/CREB1 signaling cascade was involved in PGE₁-stimulated DNA synthesis. In conclusion, PG could promote proliferation of theca externa cells from prehierarchical follicles through changes in cyclin D1/CDK6, cyclin E1/CDK2 and CREB1 mRNA expression via cAMP/PKA and CREB1 signaling cascade. These results suggest that PG may promote development of chicken prehierarchical follicles and is related to dominant follicle selection in laying hens.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Leptin affects proliferation-, apoptosis- and protein kinase A-related peptides in human ovarian granulosa cells

The aim of our in vitro studies was to understand the role of leptin in controlling proliferation, apoptosis, and protein kinase A (PKA) in human ovarian cells. We analyzed the in vitro effects of leptin (0, 1, 10 or 100 ng/ml) on the accumulation of proliferation-related peptides (PCNA, cyclin B1), apoptosis-associated peptide (Bax) and the intracellular signaling molecule PKA in cultured human granulosa cells using immunocytochemistry and Western immunoblotting. It was observed that leptin stimulated in a dose-dependent manner the accumulation of PCNA (at doses 1-100 ng/ml), cyclin B1 (at doses 10 or 100 ng/ml), Bax (at doses 10 or 100 ng/ml) and PKA (at doses 1-100 ng/ml) in cultured human ovarian cells. These observations suggest the ability of leptin to control directly human ovarian cell functions: proliferation, apoptosis, and intracellular messenger PKA.     

Proliferating effects of the flavonoids daidzein and quercetin on cultured chicken primordial germ cells through antioxidant action

Primordial germ cells (PGCs) are undifferentiated pluripotent stem cells, whose proliferation is influenced by many internal and external factors. In the present study, a PGC-somatic cell co-culture model was established to evaluate effects of the flavonoids daidzein (DAI) and quercetin (QUE) on proliferation of PGCs from embryonic chickens. PGCs were isolated from the germinal ridge of 3.5-4day embryos and cultured in 5% fetal calf serum (FCS)-supplemented Medium 199. PGC subculture was carried out on chicken embryonic fibroblast feeder (CEF) or follicular granulosa cell feeder (GCF) layers. The subcultured PGCs were challenged with flavonoids alone or in combination with a reactive oxygen substance (ROS)-producing system on CEF for 48h. The results showed a better supporting effect of CEF than GCF. Flavonoids (1microg/ml) significantly promoted PGC proliferation, which could be markedly inhibited by ROS. The oxidative damage by ROS was further manifest by decreased superoxide dismutase activity and glutathione levels. In addition, activation of protein kinase A (PKA) by forskolin significantly stimulated PGC proliferation, but PKA inhibitor H89 inhibited the proliferating effects induced by DAI and QUE. These results indicated that cultured PGCs respond to exogenous agents on proliferation and that antioxidant flavonoids could restore the intracellular antioxidant system and promote PGC proliferation via their antioxidant action involving the PKA signaling pathway.     

Stage-specific expression of bone morphogenetic protein type I and type II receptor genes: Effects of follicle-stimulating hormone on ovine antral follicles

We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3-5 and >5mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3-5mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10ng/ml) or a combination of 5ng/ml FSH with 1ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5ng/ml compared to control. A further increase in FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). The combination of FSH (5ng/ml) and E2 (1ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells (P<0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.     

Non-steroidal inhibition of granulosa cell aromatase activity in vitro

Treatment of cyclic rats with the substituted triazole R151885 (1,1-di (4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol causes delayed ovulation with suppressed blood oestradiol levels. To determine if R151885 can exert a direct action on ovarian oestrogen biosynthesis, we studied its effect on steroidogenesis in granulosa cell cultures from prepubertal rat ovaries. The cells were incubated for 48 h in medium containing 100 ng human FSH/ml and 10(-7) M testosterone to induce steroidogenic enzymes. When R151885 was also present in the culture medium, there was a marked and concentration-dependent reduction in granulosa cell oestradiol production. Inhibition was half-maximal at approx 3 X 10(-7) M and almost complete at 10(-5) M R151885. Progesterone and 20 alpha-hydroxy-4-pregnen-3-one production were unaffected except by the highest concentration of the substituted triazole (36% inhibition at 10(-5) M). Direct assessment of aromatase activity in the 48-h cultured monolayers (oestradiol formation during a 3-h incubation with 10(-7) M testosterone) was made to determine if the inhibitory effect of R151885 was due to reduced aromatase induction/activation. This was not the case, since cells cultured in the presence of 10(-6) or 10(-5) M R151885 had levels of aromatase up to 60% higher than those cultured in its absence. To determine acute effects of R151885 on testosterone (10(-7) M) aromatization, 3-h incubations were carried out using granulosa cell suspensions with high extant aromatase activity due to stimulation by ovine FSH (100 micrograms sc, twice daily for 2 days) in vivo. The triazole acted as an apparent competitive aromatase inhibitor (apparent Km for testosterone 2.5 X 10(-8) M in the absence of R151885 rising to 4.4 X 10(-8) M in the presence of 10(-7) M R151885). Its potency as an aromatase inhibitor was approximately 10 times greater than that of the naturally occurring steroidal aromatase inhibitor 5 alpha-dihydrotestosterone. Various structurally related substances proved to be even more potent aromatase inhibitors than R151885. The most active were also substituted 4,4'-difluorophenyl derivatives containing an imidazolyl or pyridyl moiety instead of the 1,2,4-triazolyl substituent in R151885. This study has identified a novel series of nonsteroidal substances which have the characteristics of potent and specific inhibitors of testosterone aromatization by rat granulosa cells in vitro.     

Diacylglycerol rather than Ca2+ mediates GnRH inhibition of FSH induced steroidogenesis in ovarian granulosa cells.

Treatment of cultured granulosa cells with PLC or GnRH stimulated the rapid generation of DAG and phosphoinositide turnover. The PKC activators PLC (3 mU/ml) and TPA (10(-7)M) or the decapeptide GnRH (10(-6)M) elicited similar inhibitory responses on FSH or cAMP stimulated granulosa cell steroidogenesis. Mobilization of intracellular Ca2+ with A23187 (10(-8)M) was followed by a slight increase in the steroidogenic activity of cultured granulosa cells, whereas elevation of extracellular K+ (50 mM) largely augmented the steroid biosynthetic activity of the granulosa cells. These results suggest that the inhibitory effect of GnRH on granulosa cell steroidogenesis is mediated by generation of DAG, rather than by increases in intracellular Ca2+ concentrations.