Gonadotropin stimulated protein phosphorylation in porcine granulosa cells

Treatment of granulosa cells with luteinizing hormone (LH) or follicle-stimulating hormone (FSH) stimulated the phosphorylation of a 58,000 molecular weight protein found in the 900 x g pellet. The phosphorylation of this protein was rapid, being significant at 1 min. LH treatment for 30 min induced greater phosphorylation of this protein than did FSH. LH and FSH also appeared to stimulate the phosphorylation of different 900 x g pellet proteins. Since both are known to utilize cAMP as a second messenger, the finding of these unique gonadotropin-induced phosphorylations may point to an additional regulatory mechanism.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Receptor-mediated uptake of lipoproteins by cultured porcine granulosa cells

Receptor-mediated uptake of low density lipoprotein (LDL) has been shown to provide a major source of cholesterol for a variety of cell types, particularly steroidogenic cells. In this study, the functional significance of lipoproteins in porcine ovarian granulosa cells and their mechanism of uptake by the cell was examined. Porcine LDL and high density lipoprotein (HDL) were isolated using a KBr density gradient, and the purity of both lipoproteins was confirmed by single corresponding bands on agarose gel stained for lipid and protein. Purified LDL and HDL were radioiodinated and labelled with colloidal gold for binding and tracer studies respectively. Both lipoproteins bind to cell surface and are internalized within 30 min at 37 degrees C. The cultured granulosa cells possess more HDL binding sites than LDL binding sites and are more responsive in progesterone production when supplemented with HDL. These results suggest that granulosa cells may preferentially utilize HDL over LDL as a source of cholesterol for steroidogenesis.     

Insulin mitigates apoptosis of porcine follicular granulosa cells by downregulating BimEL

Follicular growth or atresia is governed by the survival and apoptosis of granulosa cells. Increasing evidence shows that follicle growth is influenced by energy intake, which is positively related to insulin levels. However, the function of insulin in granulosa cell survival is poorly understood. This study focused on the effects of insulin on porcine medium follicle granulosa cell survival. In the present study, we showed that insulin markedly mitigated the apoptosis of porcine granulosa cells following serum starvation. Moreover, insulin activated the PI3K/Akt pathway to downregulate bim mRNA expression and simultaneously promoted the phosphorylation of BimEL through activating ERK 1/2, both of which reduced the level of BimEL. The results demonstrate that insulin protected the granulosa cells against apoptosis by reducing levels of the pro-apoptotic protein BimEL. However, the concentration of insulin (1 μg/ml) was relatively high. High levels of insulin partly combined with the IGF-1 receptor to play its roles in granulosa cells. This experiment provides new insight into the role of insulin in granulosa cells and sheds light on nutrition-reproduction interactions.     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Effects of Fusarium mycotoxins on steroid production by porcine granulosa cells

Fusarium mycotoxins, such as trichothecenes and zearalenone, are common grain and foodstuffs contaminants. Some of these like deoxynivalenol (DON) can negatively impact pregnancy success in swine, but evidence for direct ovarian effects of DON, zearalenone, and its major metabolite, alpha-zearalenol (ZEA) is meager. To evaluate the effects of two mycotoxins, DON and ZEA on porcine granulosa cell(s) (GC) proliferation, steroidogenesis and gene expression, pig GC from small follicles (1-5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing control (no mycotoxins) or mycotoxins (at various doses/combinations). Both DON and ZEA had biphasic effects on IGF-I-induced estradiol production, increasing estradiol production at smaller doses and inhibiting at larger doses. ZEA at 3,000 ng/mL (9.37 microM) increased IGF-I-induced progesterone production and at 30 ng/mL (0.0937 microM) and 300 ng/mL (0.937 microM) were without effect, but these doses of ZEA increased FSH-induced progesterone production. ZEA at 3,000 ng/mL inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. DON inhibited progesterone production at 100 ng/mL (0.337 microM) and 1,000 ng/mL (3.37 microM) but at 10 ng/mL (0.0337 microM) was without effect. DON at 1,000 ng/mL (but not at 10 ng/mL) completely inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. The concomitant treatment of ZEA had little effect on the dose response to DON. DON increased IGF-I-induced cell numbers at 10 and 100 ng/mL and inhibited cell numbers at 1,000 ng/mL, whereas ZEA had no effect on GC numbers. Only a combined treatment of DON and ZEA increased serum-induced cell proliferation. In conclusion, mycotoxins have direct dose-dependent effects on GC proliferation, steroidogenesis and gene expression. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine.     

Estrogen stimulation of progesterone synthesis by porcine granulosa cells in culture

Estrogen stimulates synthesis of progesterone by porcine granulosa cells in tissue culture. This enhancement is inhibited by cycloheximide, suggesting that protein synthesis is required for the effect. Estrogen is synergistic with hCG in increasing progesterone synthesis. Sequential treatment with hCG followed by estradiol causes maximal stimulation of progesterone production.     

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells.

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-terminal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.     

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20 alpha-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20 alpha-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was x 2 and x 1.6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.     

Steroid hormones modulate prolactin binding by cultured porcine granulosa cells.

We have studied the effects of the gonadal steroids — testosterone, 17β-estradiol, progesterone, and 5α-dihydrotestosterone on the prolactin-binding activity of porcine granulosa cells maintained in monolayer culture. Testosterone, estradiol, and progesterone all significantly enhanced prolactin binding (55%, 107%, and 112% above control, respectively). In contrast, the non-aromatizable androgen, 5α-dihydrotestosterone, caused an insignificant suppression of prolactin binding. The anti-androgen, cyproterone acetate, did not influence prolactin binding when used alone, and did not inhibit the effects of testosterone. These data suggest that the stimulatory effects of testosterone may require aromatization to estradiol.     

Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro

To understand the role of protein kinase A (PKA) in the control of ovarian secretory activity, we examined effects of stimulators (db-cAMP, 6-Phe-cAMP, Sp-cDBIMPS) or inhibitors (Rp-cAMPS, KT5720) of PKA on the release of insulin-like growth factor I (IGF-I), progesterone (P) and estradiol (E) by cultured porcine granulosa cells using RIA. All the PKA stimulators db-cAMP (10-10000 ng/ml), 6-Phe-cAMP (10-10000 pmol) or Sp-cDBIMPS (1-10000 pmol) increased IGF-I almost at all doses tested. P release was stimulated by db-cAMP (at doses 100-10000 ng/ml), Sp-cDBIMPS (at 10-1000 pmol) and 6-Phe-cAMP (at 1000 and 10000 pmol). The release of E was stimulated by Sp-cDBIMPS (1-100 pmol), db-cAMP (1000 and 10000 ng/ml) and 6-Phe-cAMP (1000 and 10000 pmol). Since Sp-cDBIMPS, which activates preferentially PKA isozyme type II, showed stimulating effects at doses lower than those of 6-Phe-cAMP, a preferential activator of both, type I and II of PKA, it is assumed that PKA type II is more important for the control of ovarian steroidogenesis than type I. A PKA inhibitor Rp-cAMPS inhibited release of IGF-I (10000 pmol), P (1000 pmol) and E (1000 and 10000 pmol), whereas Rp-cAMPS, at doses higher than 1000 pmol, tended to reverse this inhibitory effect. Other PKA inhibitor KT5720 suppressed P (at 10-1000 ng/ml), but not IGF-I or E release.The stimulation of growth factor and sex steroid release by PKA activators, and suppression of the secretion some of these substances by PKA inhibitors may indicate the implication of PKA (probably site B) in up- and down-regulation of ovarian IGF-I and steroid release.     

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.     

Cessation of transition-phase follicle growth in the guinea pig by follicle-regulatory protein

The granulosa cell produces a protein inhibitor of aromatase activity (follicle-regulatory protein: FRP), which recently was purified to homogeneity. To determine the possible involvement of FRP in follicular maturation, we examined the size distribution of follicles and their morphological patterns as well as serum steroid levels after the systemic administration of FRP and/or gonadotropin to guinea pigs, which have 5-6 days between luteolysis and ovulation in a 16-day cycle. FRP was partially purified from porcine follicular fluid by ammonium sulfate precipitation (0-35%), Dye Matrex Orange A Chromatography, dialysis, and lyophylization. To investigate the effect of pregnant mare's serum (PMS) during the periovulatory period in follicular development, adult guinea pigs underwent unilateral ovariectomy on Days 10, 12, and 14 of the estrous cycle (N = 6 each). Guinea pigs were injected twice daily with vehicle or PMS (5 IU) and 2 days thereafter the remaining ovaries were removed. Another group of guinea pigs received, in addition, intraperitoneal injections of FRP (1 mg) each morning from Day 8 of estrus until they were killed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 micron), and stained with Azan for comparative study via light microscopy. All follicles greater than 400 micron were classified by size, and the atretic pattern was determined by mural granulosa cell pyknosis and antral sloughing. The distribution of follicular size was not affected by hemicastration at Day 10, although the percentage of total atretic follicles decreased. In the PMS-treated group, there was a significant decrease in the number of viable follicles (700-899 micron) after hemicastration. Also pronounced in follicles of this size was the lack of mid-atretic follicles. After injections of FRP for 3 or 5 days, the overall number of follicles was almost doubled as compared to the number found in the normal ovary. Additionally, there was a significant increase in the percentage of follicles that were recently atretic, although the total percentage of atretic follicles was unchanged. After hemicastration at Day 10 followed by FRP treatment for 2 days, the total percentage of atretic follicles in the remaining ovary decreased to 18% compared with 35% in the normal ovary, 46% in the hemicastrated plus PMS-treated group, and 38% in the hemicastrated and PMS- and FRP-treated group (all p less than 0.01). Treating the hemicastrated animal with PMS increased the percentage of atretic follicles in all groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.     

The glycosaminoglycan chondroitin-4-sulfate alters progesterone secretion by porcine granulosa cells

Porcine granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) antral follicles were cultured in monolayer for 2 to 3 days with 0 to 3 mg of chondroitin-4-sulfate (C-4-S)/ml in the presence or absence of 0.5 microgram follicle-stimulating hormone (NIH-FSH-S13)/ml. Testosterone (1.4 microgram/ml) was added to some cultures as substrate for estrogen synthesis. Progesterone and estrogen secreted into the media were measured by radioimmunoassay. Concentrations of C-4-S similar to concentrations of chondroitin sulfates (CS) reported for small antral or atretic follicles inhibited both basal and FSH-stimulated progesterone secretion. Progesterone secretion was not inhibited by C-4-S when pregnenolone was added to the media. Thus 3 beta-hydroxysteroid dehydrogenase activity was not inhibited by C-4-S. Estrogen secretion was also not inhibited by even the highest concentration of C-4-S tested. Testosterone did not influence C-4-S inhibition of progesterone secretion. Granulosa cells from medium-sized follicles were more sensitive to C-4-S than cells from small follicles. Granulosa cells from large follicles were completely resistant to C-4-S inhibition of progesterone secretion. These observations suggest that C-4-S may play a role in altering gonadotrophin-stimulated and basal progesterone secretion in follicles during differentiation of granulosa cells.