Cultured Porcine Granulosa Cells Research Articles

Inter-relationships between nonapeptide hormones and cyclic nucleotides within cultured porcine granulosa cells

The reciprocal control of nonapeptide hormone (oxytocin, vasopressin) and cyclic nucleotide (cAMP, cGMP) release by porcine granulosa cells was studied. In particular, the influence of vasopressin and oxytocin treatment (10-10 000 ng/ml) on basal and LH-induced cAMP and cGMP output, as well as the effects of dibutyryl cAMP (dbcAMP; cAMP analogue) and forskolin (a stimulator of cAMP formation; 0.1-1000 ng/ml) on vasopressin and oxytocin secretion by cultured porcine granulosa cells were examined. It was observed that the addition of arginine-8-vasopressin or oxytocin stimulated both cAMP and cGMP output from granulosa cells. Moreover, both vasopressin and oxytocin also increased LH-stimulated cAMP and cGMP release. On the other hand, both dbcAMP and forskolin decreased vasopressin secretion. Oxytocin release was stimulated under the influence of dbcAMP. The same stimulating effect occurred with forskolin given at a low dose (1 ng/ml), whilst higher doses of forskolin (10 or 1000 ng/ml) were inhibitory. The present observations demonstrate the reciprocal influence of nonapeptide hormones and cyclic nucleotides in porcine ovarian cells. Oxytocin and vasopressin, like LH, exert their action on the ovary via the activation of cAMP- and cGMP-dependent intracellular mechanisms. cAMP in turn inhibits vasopressin release through a negative feedback mechanism. On the other hand, a reciprocal stimulation of oxytocin and cAMP output in granulosa cells is suggested. Thus, cyclic nucleotides can be both regulators of nonapeptide hormone secretion and mediators of their action within porcine ovaries.

Effects of transforming growth factor- β1 and activin-A on in vitro porcine granulosa cell steroidogenesis

The mammalian ovarian cycle is a strictly regulated process that is dependent on the intimate interactions among the 3 cell types in the follicle — theca, granulosa, and oocyte. The cycle has been shown to be controlled by gonadotropins as well as locally produced peptide factors. In this study, an in vitro culture system was used to study the roles of 2 locally produced ovarian peptide factors, transforming growth factor- β 1 (TGF- β 1) and activin-A, on porcine granulosa cell steroidogenesis. Gonadotropin stimulated cultured porcine granulosa cells (from medium-sized follicles) were pretreated with 100 ng/ml follicle-stimulating hormone (FSH) for 48 h and then treated with 1 ng/ml TGF- β 1, 100 ng/ml activin-A, TGF- β 1 plus activin-A, or received no treatment (control) for 48 h, From our previous studies, the concentrations of the 2 growth factors were determined to produce maximal antisteroidogenic effects in porcine granulosa cells. Progesterone (P 4) production, estradiol-17β (E 2) production, and aromatase activity for gonadotropin-stimulated porcine granulosa cells treated with TGF- β 1, activin-A, and TGF- β 1 plus activin-A were significantly (P < 0.05) reduced fromthat of the control. The same procedures were conducted on basal steroidogenesis studies in which no pretreatment with FSH was performed. Both P 4 and E 2 production and aromatase activity for porcine granulosa cells treated with TGF- β 1, activin-A and TGF- β 1 plus activin-A were significantly (P < 0.05) inhibited compared with the control. Our results indicate that both TGF- β 1 and activin-A can inhibit FSH-stimulated and basal steroidogeneses in porcine granulosa cells and, thus, may act as local atretic factors during follicular development. When the 2 growth factors were given in combination at concentrations that would produce maximal steroidogenic inhibition, they were not able to produce a synergistic effect. These results are consistent with the current theory that TGF- β 1 and activin-A may act via the same messenger system, a serine-threonine kinase.

The direct effect of prolactin (PRL) on follicle stimulating hormone (FSH) receptor binding and progesterone (P) production in cultured porcine granulosa cells.

The direct effect of prolactin (PRL) on follicle stimulating hormone (FSH) receptor binding and progesterone (P) production in cultured porcine granulosa cells.

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.

3β-hydroxy-5-ene Steroid dehydrogenase gene expression regulation in porcine granulosa cells

The objective of this study was to investigate the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) on FSH- and LH-induced 3β-HSD-gene expression in cultured porcine granulosa cells. FSH and LH induced a dose dependent increase in the accumulation of 3β-HSD mRNA, measured by Northern blot. A 1.6- to 1.8-fold increase (p<0.01) was observed with 10 ng/mL of FSH or LH. Maximal levels of 2.5- to 2.9-fold increases, relative to control, were reached at 30 and 100 ng/mL of the gonadotropins. When granulosa cells were treated with PMA (100 nM) just before the addition of FSH, the 3β-HSD rnRNA levels induced by 10 or 30 ng/mL of FSH were inhibited or partially inhibited, respectively. PMA did not inhibit elevated levels of 3β-HSD mRNA induced by FSH at concentrations of 100, 300, and 1000 ng/mL. Alternatively, PMA added just before LH, inhibited LH-stimulated 3β-HSD mRNA levels at all doses of LH tested (10, 30, 100, 300, and 1000 ng/mL). The protein kinase A-stimulators, dibutyryl-cAMP (cAMP) (0.5 mM) and forskolin (10 nM), also elevated the 3β-HSD-gene transcription, 3.5- and 4.0-fold respectively. PMA prevented the stimulation of the 3β-HSD-gene transcription when it was added just before cAMP or forskolin. We concluded that stimulation of PKC by PMA appears to have inhibited the gonadotropin-induced increase in 3β-HSD mRNA levels by preventing cAMP-activated 3β-HSD-gene transcription. The data also suggest that the effect of PMA appears to be more specific for regulation of LH-stimulated intracellular signals than those of FSH. This effect may indicate a site of differential regulation of FSH and LH on the stimulation of 3β-HSD-gene transcription.

Transforming growth factor β receptor expression in cultured porcine granulosa cells

By using primary cultures of porcine granulosa cells as a model, TGFβ receptors have been identified and characterized through three different approaches including cross-linking experiments, Western- and Northern-blotting analysis. In cross-linking experiments, labeled TGFβ was shown to bind to four different molecular species of 300, 168, 72 and 58 kDa. The 300-kDa species may correspond to β-glycan, while the 72- and 58-kDa correspond to TGFβ type II and I receptors, respectively. The presence of these receptors was further demonstrated by Western-blotting analysis using specific polyclonal antibodies. Finally, both the expression of β-glycan, type II and type I mRNA, was confirmed through Northern-blotting analysis as shown by the presence of 6.4, 4.6 and 5.8 kb mRNA, respectively. Additionally, we detected another TGFβ binding protein of 168 kDa which remains to be identified. Together, our present data indicate that the regulatory action of TGFβ on cultured granulosa cells previously reported in several laboratories may be mediated through the receptors identified here.

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.

Growth hormone amplifies insulin-like growth factor I induced progesterone accumulation and P450scc mRNA expression

The interaction of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the acquisition of progesterone biosynthetic capacity were examined in cultured porcine granulosa cells. Basal progesterone production was not affected ( P > 0.05) by GH treatment. However, concurrent treatment with GH produced a 4.1-fold increase (539 versus 2214 ng/culture) in the IGF-I-stimulated accumulation of progesterone. GH potentiated IGF-I induced progesterone production in a dose and time dependent manner, with a time requirement of 48 h or less. The amplified effect of GH was not attributable to changes in cellular protein, DNA content, cell number, plating efficiency or cell viability. Moreover, Northern blot analyses revealed that GH enhanced IGF-I induced expression of the gene encoding cytochrome P450 side chain cleavage. These observations provide further evidence to support the role of GH in the regulation of ovarian steroidogenesis.

Dual actions of phorbol ester on cytochrome P450 cholesterol side-chain cleavage messenger ribonucleic acid accumulation in porcine granulosa cells.

In earlier studies in cultures of porcine granulosa cells prepared from small antral follicles, steroidogenesis-related loci were inhibited by treatment for 48 h with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC). In the present investigation, cells were incubated in serum-free medium for 48 h, with various agents present during the last 2-24 h. With TPA at 30 ng/ml, the FSH-stimulated cAMP accumulation was markedly enhanced at all time points. FSH increased the concentration of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA throughout the 24-h incubation. At 4 and 8 h, TPA increased the accumulation of P450scc mRNA, having an additive effect with FSH. However, at 24 h, TPA markedly suppressed the FSH-induced increased in P450scc mRNA. Pretreatment of cells with FSH did not shorten the time required for TPA to become inhibitory. The stimulatory effect of 8-bromo-cAMP on P450scc mRNA also was augmented by TPA at 4 h, but significant inhibition was not observed at 24 h. The concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA, intended to be used for correction of gel loading, was stably increased by both cAMP and TPA. These effects of TPA suggest multiple actions of PKC(s) on the regulation of P450scc expression and other endpoints in ovarian granulosa cells.

Effects of metabolic hormones and growth factors on forskolin and dibutyryl adenosine 3′,5′-cyclic monophosphate induced steroidogenic responses by porcine granulosa cells in vitro

The present experiments were conducted to examine the influence of metabolic hormones and growth factors on progesterone and estradiol secretion by porcine granulosa cells in culture and to determine the possible site(s) of action of these agents on steroidogenesis. Porcine granulosa cells, obtained from medium-sized (4–6 mm) nonatretic follicles of prepubertal gilts slaughtered at a commercial abattoir, were cultured at a density of 2 × 106 viable cells well−1 in serum-free Eagle's Minumum Essential Medium for 72 h with 0, 10 or 100 ng mL−1 of GH, insulin or IGF-I or IGF-II. At 72 h, each treatment group was divided into three subgroups, one subgroup remained untreated (basal) while either 25 μM FK or 3 mM dbcAMP was added to the other two subgroups. Additionally, androstenedione (10−7 M) was provided to all cultures as a substrate for estradiol biosynthesis. Media were collected at 96 h for determination of progesterone and estradiol content. Both IGF-I and IGF-II increased (P &lt; 0.01) basal progesterone and estradiol secretion and enhanced the FK- and dbcAMP-induced stimulation of progesterone and estradiol secretion (P &lt; 0.01). Insulin, at 100 ng mL−1, had a modest but significant positive effect on both FK- and dbcAMP-stimulated steroidogenic responses. In contrast, GH inhibited basal, FK- and dbcAMP-induced progesterone and estradiol production. Based on the stimulatory effects of both IGFs above those seen with dbcAMP alone and the inhibitory effects of GH in the face of dbcAMP stimulation, we conclude that their effects are likely mediated by non-cAMP dependent pathways. Key words: Steroidogenesis, granulosa cells, growth hormone, IGF, insulin, pigs

Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells.

We have examined porcine granulosa cells (pGCs) for the presence of immunodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) on the activation of these kinases. Cell lysates prepared from untreated monolayer cultures of pGCs were subjected to Western immunoblotting analysis using monoclonal antibodies to ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mol wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatment of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 min resulted in electrophoretic mobility shifts of ERK1 and ERK2 suggesting hyperphosphorylation. Immunoprecipitation with an antiphosphotyrosine antibody (PY20), followed by Western analysis using pan-ERK, revealed a marked concentration-dependent increase in tyrosine phosphorylation of ERK2 in response to EGF treatment. The mobility shift and tyrosine phosphorylation of ERK2 was observed as early as 1 min after treatment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assays revealed significant MBP kinase activity associated with ERK1 and ERK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Although ERK1 displayed a moderate mobility shift in response to EGF, tyrosine phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kinase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the ERK54 and ERK87 was not effected regardless of EGF concentration or duration of treatment. These data demonstrate for the first time that pGCs contain immunodetectable MAP kinases. EGF, in a concentration- and time-dependent manner, increases tyrosine phosphorylation and MBP kinase activity (i.e. activation) of ERK2, and to a lesser degree ERK1, suggesting that the activation of MAP kinase may mediate the mitogenic action of EGF in pGCs.

Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells

We have examined porcine granulosa cells (pGCs) for the presence of immunodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) on the activation of these kinases. Cell lysates prepared from untreated monolayer cultures of pGCs were subjected to Western immunoblotting analysis using monoclonal antibodies to ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mol wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatment of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 min resulted in electrophoretic mobility shifts of ERK1 and ERK2 suggesting hyperphosphorylation. Immunoprecipitation with an antiphosphotyrosine antibody (PY20), followed by Western analysis using pan-ERK, revealed a marked concentration-dependent increase in tyrosine phosphorylation of ERK2 in response to EGF treatment. The mobility shift and tyrosine phosphorylation of ERK2 was observed as early as 1 min after treatment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assays revealed significant MBP kinase activity associated with ERK1 and ERK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Although ERK1 displayed a moderate mobility shift in response to EGF, tyrosine phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kinase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the ERK54 and ERK87 was not effected regardless of EGF concentration or duration of treatment. These data demonstrate for the first time that pGCs contain immunodetectable MAP kinases. EGF, in a concentration- and time-dependent manner, increases tyrosine phosphorylation and MBP kinase activity (i.e. activation) of ERK2, and to a lesser degree ERK1, suggesting that the activation of MAP kinase may mediate the mitogenic action of EGF in pGCs.

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

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

The inhibitory action of taxol on granulosa cell steroidogenesis is reversible.

Taxol is a novel anticancer agent extracted from the bark of Pacific yew trees. The drug has been approved by the FDA for the treatment of advanced ovarian cancer and is in clinical trials for other malignancies, including breast cancer. The goals of this study were to determine whether taxol adversely and irreversibly affects ovarian granulosa cell steroidogenesis. Cultured porcine granulosa cells were treated with taxol (0.12-12 microM) or vehicle (0.01-1% ethanol) in the absence or presence of 10(-9) M hCG in a time- and dose-response study. Morphological changes were recorded every 2 h, and media were collected for the measurement of progesterone (P4) and 17 beta-estradiol. Taxol suppressed both basal P4 and 17 beta-estradiol production and hCG-stimulated P4 production in a time- and dose-dependent manner and drastically changed cell shape by causing disorganization of microtubule bundles and other subcellular organelles. hCG partially reversed the steroid inhibition induced by taxol. These changes are not attributed to ethanol used as the vehicle, because ethanol at higher concentrations than that present in taxol did not suppress P4 production. When taxol was removed from the culture, P4 production returned to control levels. The results of this study show that taxol causes a significant, but reversible, inhibition of granulosa cell steroidogenesis. This inhibitory effect can be partially overcome by co-treatment with hCG.

Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1.

Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doublet), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBP-4. These results were compared to those in cultures of immature granulosa cells from 1-3-mm follicles, in which 22- and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22- and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperation between LH-RH and LH in the direct action on the ovary: LH-RH stimulation of LH/hCG receptors, basal and LH-induced cAMP and cGMP release by porocine granulosa cells in vitro

LH-Rh effects on LH/hCG receptors content, on basal and LH-stimulated cAMP and cGMP release by cultured porcine granulosa cells, were investigated by radioreceptor analysis and radioimmunoassay. It was found that LH-RH additions (10, 100, 1000 or 10,000 ng/ml medium) increased the number of LH/hCG binding sites in granulosa cells. LH or hCG (0.1, 1. 10 or 100 ng/ml) increased both cAMP and cGMP secretion by the cell culture. LH-RH alone (0.1, 1, 10, 100 or 1000 ng/ml) had the same stimulating effect on both cyclic nucleotides. The addition of LH-RH (100 ng/ml) to the medium supplemented with LH (10, 100, 1000, 10,000 or 100,000 ng/ml) enhanced LH-stimulated cAMP and cGMP output form the culture. The present observations suggest tha LH-RH can act as synergist of LH and of hCG increasing a number of LH/hCG receptors and stimulating basal and LH-induced cyclic nucleotide release by ovarian cells.

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring 3H-H 2O released from [1β- 3H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 μM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.

Follicle-stimulating hormone (FSH) increases FSH receptor messenger ribonucleic acid while decreasing FSH binding in cultured porcine granulosa cells.

The goal of the present studies was to compare direct effects of porcine FSH (pFSH) on [125I]pFSH-binding sites with effects of pFSH on FSH receptor mRNA in cultured porcine granulosa cells. Cells from immature follicles were cultured on laminin-coated plates in serum-free medium for up to 6 days in the absence or presence of pFSH (1-100 ng/ml) or cholera toxin (0.04-400 ng/ml), which activates adenylyl cyclase independently of the FSH receptor. RRA indicated that [125I] pFSH binding to cells cultured without stimulator increased more than 10-fold with time in culture. Addition of pFSH to cultures resulted in a dose-dependent decrease in binding, assessed after removal of bound pFSH. Equilibrium saturation binding analysis indicated that pFSH (10 ng/ml) caused a 39% decrease in binding sites in cells cultured for 6 days. At the same time, pFSH increased progesterone production 9.5-fold. Cholera toxin (4 ng/ml) increased [125I]pFSH binding 110% and progesterone production 8.9-fold. Northern hybridization analysis of cultured granulosa cell mRNA using a porcine FSH receptor cDNA revealed three transcripts for the FSH receptor [2.2, 3.5, and 4.2 kilobases (kb)], with the major transcript being 4.2 kb in length. Addition of either pFSH (10 ng/ml) or cholera toxin (10 ng/ml) to cultures of granulosa cells increased the intensity of pFSH receptor transcripts compared with control values, with the 4.2-kb message remaining predominant. Hybridization with a porcine LH receptor cDNA revealed different transcripts (2.4, 4.0, 4.7, 7.0, and 11.0 kb), with the major transcript being 4.7 kb in length. Addition of either pFSH or cholera toxin to the cultures increased the intensity of all LH receptor transcripts; however, cholera toxin was more effective than pFSH. pFSH and cholera toxin increased the intensity of each species to different extents, although the 4.7-kb transcript remained predominant. These results indicate that exposure to FSH in culture results in down-regulation of the FSH receptor. Down-regulation is accompanied by increased FSH receptor mRNA levels, suggesting that FSH enhances FSH receptor synthesis.

Identification of a Molecular Species in Porcine Ovarian Luteal Glutathione S-Transferase and Its Hormonal Regulation by Pituitary Gonadotropins

Glutathione S-transferase (GST) was purified to a homogenous state from the cytosol fraction of porcine corpora lutea. The present study showed that the final enzyme preparation consisted of a single molecular species of hepatic GSTA1-1, which has been identified from its enzymatic properties, amino acid composition, N-terminal amino acid sequences, and immunological reactivity. Furthermore, it is suggested that ovarian GST is involved in steroidogenesis, especially in the step of progesterone synthesis, from the following results. (1) The final enzyme preparation had a significant Δ 5-3-ketosteroid isomerase activity, which acts in steroid synthesis. (2) GST activity in the cultured porcine granulosa cells was remarkably increased in the luteinizing process, which was induced by addition of pituitary gonadotropins such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the culture system. (3) Changes of GST activity induced by FSH and LH were closely associated with progesterone production. (4) GST was detected within luteinizing and theca interna cells, but not in granulosa cells, by immunohistochemical staining of ovary tissues using anti-ovarian luteal-GST antibodies.