Involvement of obestatin,cyclin-dependent kinase and protein kinase C in control of feline ovarian cell viability and hormones release

The aim of our in vitro study was to understand the role of obestatin, cyclin-dependent kinase (CDK) and protein kinase C (PKC) in the control of basic feline ovarian cell functions (viability, ovarian hormones release), as well as the role of protein kinases in mediating the effect of obestatin on these processes. For this purpose, we analyzed the effect of obestatin (0, 10 and 100 ng/mL) alone or in combination with CDK blocker olomoucine (100 ng/mL) or PKC blocker calphostin-c (100 ng/mL) on cultured feline ovarian fragments or granulosa cells. The release of progesterone (P4), testosterone (T) and estradiol (E2) by isolated ovarian follicular fragments were evaluated by ELISA. Granulosa cell viability was analysed using the Trypan blue exclusion test. It was observed that the addition of obestatin alone significantly increased the granulosa cell viability (at dose 100 ng/mL), promoted the release of P4 (at all doses added) and IGF-I (at dose 100 ng/mL) but decreased T (at all doses added). E2 output was below the detection limit in all groups. The addition of either olomoucine or calphostin-c reduced cell viability, P4, T and IGF-I release. Both olomoucine and caplhostin-c inverted the stimulatory effect of obestatin on granulosa cell viability and were able to prevent stimulatory action of obestatin on ovarian cell viability and on hormone and growth factor release and change it to an inhibitory action. These observations show that obestatin can directly regulate (mostly promote) basal feline ovarian cell functions (hormone release and viability). The inhibitory action of CDK and PKC blockers on these functions suggests, that both CDK and PKC can be promoters of ovarian cell viability and steroidogenesis in cats. Furthermore, the ability of both CDK and PKC to prevent olomoucine action demonstrates that obestatin action on the feline ovary could be mediated by these kinases.     

Kisspeptin as autocrine/paracrine regulator of human ovarian cell functions: Possible interrelationships with FSH and its receptor

The present study aims to examine the role of kisspeptin (KP), FSH, and its receptor (FSHR), and their interrelationships in the control of basic human ovarian granulosa cells functions. We investigated: (1) the ability of granulosa cells to produce KP and FSHR, (2) the role of KP in the control of ovarian functions, and (3) the ability of KP to affect FSHR and to modify the FSH action on ovarian functions. The effects of KP alone (0, 10 and 100 ng/mL); or of KP (10 and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured human granulosa cells were assessed. Viability, markers of proliferation (PCNA and cyclin B1) and apoptosis (bax and caspase 3), as well as accumulation of KP, FSHR, and steroid hormones, IGF-I, oxytocin (OT), and prostaglandin E2 (PGE2) release were analyzed by the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. KP given at a low dose (10 ng/mL) stimulated viability, proliferation, inhibited apoptosis, promoted the release of progesterone (P4), estradiol (E2), IGF-I, OT, and PGE2, the accumulation of FSHR, but not testosterone (T) release. KP given at a high dose (100 ng/mL) had the opposite, inhibitory effect. FSH stimulated cell viability, proliferation and inhibited apoptosis, promoted P4, T, E2, IGF-I, and OT, but not PGE2 release. Furthermore, KP at a low dose promoted the stimulatory effect of FSH on viability, proliferation, P4, E2, and OT release, promoted its inhibitory action on apoptosis, but did not modify its action on T, IGF-I, and PGE2 output. KP at a high dose prevented and inverted FSH action. These results suggest an intra-ovarian production and a functional interrelationship between KP and FSH/FSHR in direct regulation of basic ovarian cell functions (viability, proliferation, apoptosis, and hormones release). The capability of KP to stimulate FSHR, the ability of FSH to promote ovarian functions, as well as the similarity of KP (10 ng/mL) and FSH action on granulosa cells’ viability, proliferation, apoptosis, steroid hormones, IGF-I, OT, and PGE2 release, suggest that FSH influence these cells could be mediated by KP. Moreover, the capability of KP (100 ng/mL) to decrease FSHR accumulation, basal and FSH-induced ovarian parameters, suggest that KP can suppress some ovarian granulosa cell functions via down-regulation of FSHR. These observations propose the existence of the FSH-KP axis up-regulating human ovarian cell functions.     

The effect of obestatin on porcine ovarian granulosa cells

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P(4)), testosterone (T) and estradiol (E(2)) was measured by EIA. Addition of obestatin (1-100ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100ng/ml, obestatin promoted the secretion of P(4), but not T or E(2). Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.     

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Puncturevine (Tribulus terrestris L.) affects the proliferation,apoptosis, and ghrelin response of ovarian cells

The objective of our study was to examine the direct effects of the medicinal plant Tribulus terrestris L. (puncturevine) on the basic functions of ovarian cells, including their proliferation, apoptosis, and response to the physiological hormonal stimulator ghrelin. In the first series of experiments, porcine ovarian granulosa cells were cultured with or without puncturevine extracts at concentrations of 0, 1, 10, or 100 μg/ml. In the second series of experiments, these cells were cultured with ghrelin at concentrations of 0, 1, 10, or 100 ng/ml, either alone or in combination with puncturevine (10 μg/ml). The expression levels of the proliferation marker PCNA and the apoptosis marker bax were analyzed via quantitative immunocytochemical methods. Puncturevine was found to stimulate the accumulation of both proliferation and apoptotic markers. Additionally, ghrelin alone could promote the proliferation and apoptosis of ovarian cells. The presence of puncturevine reversed ghrelin-stimulated apoptosis and instead induced apoptotic inhibition. However, puncturevine did not modify the proliferation-inducing effect of ghrelin. These observations demonstrated that (1) puncturevine directly promotes cell proliferation and apoptosis, turnover, of ovarian cells; (2) ghrelin is involved in the regulation of ovarian cell apoptosis and proliferation, consistent with existing evidence; (3) puncturevine antagonizes and even reverses the effects of the hormonal regulator, ghrelin, on ovarian cell apoptosis, but not proliferation; and (4) puncturevine affects not only the basic functions of ovarian cells but also their responses to upstream hormonal regulators.     

Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

In vitro assessment of iron effect on porcine ovarian granulosa cells: secretory activity, markers of proliferation and apoptosis

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.     

Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells

The effect of insulin and insulin-like growth factor-1 (IGF-1) on progesterone secretion by porcine granulosa cells and their modulatory effect on follicle-stimulating hormone (FSH)-induced responses were examined. For comparative purposes, growth hormone (GH), previously shown to stimulate IGF-1 secretion, was also included. Granulosa cells from ovarian follicles (3 to 5 mm) were cultured in multiwell plates for the first 48 hours, either in the presence or absence of 1% fetal bovine serum (FBS). Following plating, all cultures were maintained in serum-free media. The addition of only insulin, but not IGF-1 or GH, enhanced progesterone secretion under both culture conditions. When low-density lipoprotein was provided as steroid substrate, a stimulatory effect of insulin on progesterone accumulation was observed with a minimum dose of 10 ng/ml. Granulosa cells cultured in serum-free media from the time of plating secreted less progesterone and were less responsive to FSH compared with cultures plated with 1% FBS. Only insulin, but not IGF-1, enhanced FSH responses to threefold in cells cultured with 1% FBS. However, when cells were cultured in serum-free media from the time of plating, both insulin and IGF-1, but not GH, potentiated the responses to FSH, but insulin was more potent than IGF-1. Insulin-like growth-factor-1 binding studies with granulosa cells indicate the presence of specific high-affinity binding sites (Kd 3.96 nM). A dose of 100 ng/ml of insulin had negligible cross-reactivity with IGF-1 receptors.     

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.     

Effects of angiogenin on granulosa and theca cell function in cattle

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

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 (1-5mm) and/or large (8-22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens (Gallus domesticus)

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0-100ng/ml) and/or LH (0-100ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10ng/ml (p<0.01). Testosterone, up to 10ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1ng/ml (p<0.001). However, at supraphysiological concentrations of LH (10 and 100ng/ml) there was no further increase in progesterone production caused by testosterone (p>0.05). Finally, the addition of 2-hydroxyflutamide (0-1000mug/ml) to hen granulosa cells cultured with 10ng/ml of testosterone reduced progesterone production in a dose response manner (p<0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.     

Prolactin modulates steroidogenesis by rat granulosa cells: I. Effects on progesterone

Either testosterone or follicle-stimulating hormone (FSH) stimulates progesterone secretion by granulosa cells from rats but the combination of the two hormones increases progesterone production in a synergistic manner. We have investigated the effects of graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with testosterone (0.5 microM), FSH (300 ng/ml), or FSH + testosterone on progesterone secretion by granulosa cells at two stages of differentiation. Relatively undifferentiated granulosa cells from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats were cultured in defined (serum-free) medium for 3 days. More highly differentiated granulosa cells were obtained on the morning of proestrus from the preovulatory follicles of 30-day-old rats induced to undergo an estrous cycle by injection with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% fetal bovine serum. Prolactin alone did not enhance the negligible secretion of progesterone by cells from HPX rats, but increased progesterone secretion by cells from proestrous rats. Prolactin significantly enhanced the stimulatory effects of testosterone or FSH alone on cells from both HPX and proestrous rats. When cultures containing both FSH + testosterone were treated with prolactin, progesterone secretion by cells from proestrous rats was significantly enhanced, whereas secretion by cells from HPX rats was significantly depressed. Therefore when cells from HPX rats were cultured with both FSH and testosterone, the direction of the effect of prolactin was reversed from that observed with prolactin + FSH or testosterone alone, and from that observed when cells from proestrous rats were cultured with prolactin + FSH + testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of ghrelin and some intracellular mechanisms in controlling the secretory activity of chicken ovarian cells

The general aim of these in-vitro experiments was to determine whether ghrelin controls the secretory activity of chicken ovarian cells and whether its action is mediated by TK-, MAPK-, CDK- or PKA-dependent intracellular mechanisms. We postulated that particular protein kinases could be considered as mediators of ghrelin action (a) if they are controlled by ghrelin, and (b) if blockers of these kinases modify the action of ghrelin. In our in-vitro experiments we investigated whether ghrelin altered the accumulation of TK, MAPK, CDK and PKA in chicken ovarian cells and whether ghrelin, with or without blockers of MAPK, CDK and PKA, affected the secretion of progesterone (P4), testosterone (T), estradiol (E2) or arginine-vasotocin (AVT). In the first series of experiments, the influence of a ghrelin 1-18 analogue (1, 10 or 100 ng/mL) was studied on the expression of TK, MAPK and PKA in cultured chicken ovarian granulosa cells. The percentage of cells containing TK/phosphotyrosine MAPK/ERK1, 2 and PKA was determined using immunocytochemistry. Ghrelin increased the expression of both TK and MAPK. The low concentration of ghrelin (1 ng/mL) increased the accumulation of PKA in ovarian cells whilst the high concentration (100 ng/mL) decreased it. The 10 ng/mL concentration had no effect. In the second series of experiments, the effects of the ghrelin analogue combined with an MAPK blocker (PD98059; 100 ng/mL), a CDK blocker (olomoucine; 1 microg/mL), or a PKA blocker (KT5720; 100 ng/mL), were tested for their effects on the secretion of hormones by cultured fragments of chicken ovarian follicular wall. P4, T, E2 and AVT secretions were measured using RIA and EIA. Ghrelin increased T and decreased E2, but did not affect P4 or AVT secretion. The PKA blocker promoted P4 secretion and suppressed E2 and AVT, but did not affect T secretion. It prevented or even reversed the effect of ghrelin on T and E2, but did not modify its effect on AVT secretion. The MAPK blocker enhanced P4 and T and reduced AVT, but did not affect E2 secretion. It was able to prevent or reverse the effect of ghrelin on T and E, and it induced a stimulatory effect of ghrelin on AVT secretion. The CDK blocker reduced the secretion of AVT, but had no effect on steroid hormone secretion. It induced the stimulatory influence of ghrelin on the secretion of P4 and AVT, but did not modify the effect of ghrelin on other hormones. These observations clearly demonstrate that ghrelin is a potent regulator of the secretory activity of ovarian cells and of TK, MAPK and PKA. Furthermore, they suggest that MAPK-, CDK- and PKA-dependent intracellular mechanisms are involved in the control of ovarian secretion and that they mediate the effects of ghrelin on these processes.