Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation.

Resistance ovarian syndrome is a heterogeneous disorder inherited as a Mendelian recessive trait and characterized by infertility, primary amenorrhea, normal karyotype and elevated serum FSH and LH levels. An inactivating mutation, C566T, in FSH receptor gene (FSHR) has been identified initially in Finland. We investigated if an adenovirus expressing a normal copy of human FSHR (Ad-hFSHR) has the ability to: (i) transfect granulosa cell lines, (ii) render the transfected cell lines responsive to FSH stimulation and (iii) transcomplement the malfunctioning form of human FSHR gene with C566T mutation. COS-7, JC-410, JC-410-P450-scc-luc and JC-410-StAR-luc cell lines were infected by Ad-hFSHR followed by treatment with FSH. Functional activity of the Ad-hFSHR was tested by measuring cyclic adenosine monophosphate (cAMP) or luciferase activity in response to FSH stimulation, and showed 2-4.6-fold increases in Ad-hFSHR transfected cells compared with untransfected or Ad-LacZ transfected cells, indicating that Ad-hFSHR is functionally active and expressing hFSHR. Generation of cAMP in cells expressing only mutated hFSHR-T566 showed minimal increase after FSH stimulation. Co-transfection of Ad-hFSHR in these cells carrying the malfunction form of human FSHR caused significant increases of 2.2-7.4-fold in FSH dependent cAMP generation (P = 0.0007). We concluded that adenovirus expressing a normal human FSHR can compensate the inactivating human FSHR-C566T mutation and restore FSH responsiveness.

Ovarian and placental production of progesterone and oestradiol during pregnancy in reindeer.

We obtained uterine and peripheral venous plasma, and samples of luteal and placental tissues from 2- to 7-year-old, Eurasian mountain reindeer (Rangifer tarandus tarandus) from a free-living, semi-domesticated herd in northern Norway in November 1995, and February and March 1996. In November, ovarian venous blood was also collected from four animals. Plasma samples were assayed for progesterone and oestradiol. The tissue samples were examined by light and electron microscopy, steroid dehydrogenase histochemistry, and northern blot analysis for RNAs for 3beta-hydroxy-steroid dehydrogenase (3beta-HSD) and P450 (side chain cleavage (scc)). Peripheral blood was taken from non-pregnant females in the same herd on the same dates. Peripheral progesterone concentrations in pregnant reindeer (3.4 +/- 0.5 ng/ml, n = 8) clearly exceeded those in non-pregnant animals (0.40 +/- 0.14 ng/ml; P < 0.0004 , n = 10) but oestradiol levels were only marginally higher in pregnant (6.0 +/- 0.7 pg/ml) than in non-pregnant (4.8 +/- 0.5 pg/ml; P = 0.35) reindeer at the stages examined. In pregnant animals, peripheral progesterone and oestradiol concentrations rose slightly between November and March but the differences did not reach significance (progesterone, P = 0.083; oestradiol, P = 0.061). In November, progesterone concentrations in the ovarian vein (79 +/- 15 ng/ml) greatly exceeded (P < 0.03) those in the uterine vein ( 10 +/- 4 ng/ml) which in turn exceeded the levels in the peripheral blood (2.8 +/- 0.4 ng/ml; P < 0.29). Oestradiol concentrations were slightly but significantly (P < 0.05) higher in the ovarian (20 +/- 3 pg/ml) than the uterine vein (13 +/- 1 pg/ml) and, in turn, greater (P < 0.03) than in peripheral blood (4.6 +/- 0.4 pg/ml). All samples of luteal tissue consisted exclusively of normal fully-differentiated cells and stained intensely for 3beta-HSD. Isolated groups of placental cells also stained strongly for 3beta-HSD. RNA for P450 (scc) and 3beta-HSD was abundant in all corpora lutea and lower concentrations of P450 (scc) were present in the placenta. 3beta-HSD RNA in the placenta was below the limit of detection. We conclude that the corpus luteum remains an important source of progesterone throughout pregnancy in reindeer but that the placenta is also steroidogenic.

Effects of progestins on progesterone synthesis in a stable porcine granulosa cell line: control of transcriptional activity of the cytochrome p450 side-chain cleavage gene.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450(scc)) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450(scc) mRNA levels after 3-6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 microM increased the transcription rate of P450(scc) transiently expressed in JC-410 cells after 18 h of incubation; 30 microM had no effect, and 100 microM suppressed transcription. Levonorgestrel did not affect transcription of the P450(scc) gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450(scc) gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

The diverse mechanism of action of dichlorodiphenyldichloroethylene (DDE) and methoxychlor in ovarian cells in vitro.

Dichlorodiphenyldichloroethylene (DDE), the most stable metabolite of the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT), and the DDT analog methoxychlor can have adverse effects on reproduction. These chemicals have been identified as having estrogenic activity. The aim of the current study was to examine the effects of dichlorodiphenyldichloroethylene (DDE), methoxychlor, and estradiol-17 beta on steroidogenesis and FSH responsiveness in ovarian cells in vitro. Experiments were performed on a primary culture of porcine granulosa cells and a culture of Chinese hamster ovary (CHO) cells, the latter stably transfected with the FSH receptor (CHO-FSH-R). DDE (10 microM) and estradiol-17 beta (0.1 microM) but not methoxychlor (10 microM), increased proliferation of the granulosa cells. DDE (100 and 10 microM, respectively) decreased FSH-stimulated cAMP synthesis in the granulosa and CHO-FSH-R cells. DDE also decreased progesterone synthesis in the granulosa cells. Methoxychlor (10 microM) inhibited progesterone synthesis in the granulosa cells, but it did not affect the generation of cAMP in either type of cells studied. However, methoxychlor inhibited estradiol-17 beta-stimulated progesterone synthesis in the granulosa cells. We conclude that DDE primarily inhibited the generation of cAMP, while methoxychlor suppressed progesterone synthesis through a mechanism distal to cAMP generation. The present results indicate that DDE and methoxychlor are not limited to a mimicking of the endocrine effects of estradiol-17 beta in cultured ovarian cells. Therefore, a non-estrogenic component of the endocrine disrupting activities of DDE and methoxychlor must be considered in evaluating their reproductive toxicity.

Inhibition of basal and stimulated progesterone synthesis by dichlorodiphenyldichloroethylene and methoxychlor in a stable pig granulosa cell line.

The effects of the insecticide dichlorodiphenyldichloroethylene (DDE) and methoxychlor in a stable pig granulosa cell line, JC-410, were investigated. The studies of DDE and methoxychlor were conducted in combination with studies of cholera toxin, the protein kinase A activator that stimulates cAMP and progesterone synthesis and gene expression of P450 cholesterol side chain cleavage (P450scc), which converts cholesterol to pregnenolone. Administration of DDE at 3000 and 10 000 ng ml (-1) was found to decrease progesterone synthesis 0.49- and 0.25-fold, respectively, and to block the stimulatory effect of 100 ng cholera toxin ml (-1), after 24 h incubation. At 1-100 ng ml (-1), methoxychlor did not affect progesterone synthesis after 48 h incubation. However, 1000 ng methoxychlor ml (-1) decreased progesterone synthesis 0.32-fold, and both 100 and 1000 ng methoxychlor ml (-1) blocked the stimulatory effect of cholera toxin. At 3000 and 10 000 ng ml(-1), DDE decreased cAMP synthesis 0.66-and 0.36-fold, respectively. At 300, 3000 and 10 000 ng ml (-1), DDE also decreased cholera toxin-stimulated cAMP synthesis 0.84-, 0.68-, and 0.52-fold, respectively. Administration of 1-100 ng methoxychlor ml (-1) did not affect basal or cholera toxin-stimulated cAMP synthesis. Cholera toxin increased P450scc mRNA 1.4-fold after 24 h incubation, while 3000 and 10 000 ng DDE ml (-1) led to 0.39- and 0.18-fold reductions, respectively. The stimulatory effect of cholera toxin on P450scc mRNA was blocked by 3000 and 10 000 ng DDE ml(-1). Cholera toxin increased P450scc mRNA 3.48-fold after 48 h incubation, while 100 and 1000 ng methoxychlor ml (-1) increased P450scc mRNA 1.79- and 3.0-fold, respectively, and further increased the stimulatory effect of cholera toxin 6.47- and 5.44-fold, respectively. The results of the present study indicate that DDE inhibits granulosa cell steroidogenesis by affecting cAMP production and P450scc gene expression. However, methoxychlor appears to inhibit steroidogenesis by a mechanism occurring before the conversion of cholesterol into pregnenolone.

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells.

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.

Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor. Role in hormone signaling and cell proliferation.

Follicle-stimulating hormone (FSH) regulated growth and function of the ovarian follicle was previously thought to be mediated solely through activation of G(s)-coupled receptors. In this study, we show for the first time that this function is predominantly mediated through the alternatively spliced and novel growth factor type 1 receptor (oFSH-R3) that is also present in the ovary. Immortalized granulosa cells lacking endogenous FSH receptors, when transfected with either oFSH-R3 cDNA (JC-R3) or the G(s)-coupled oFSH-R1 (JC-R1), expressed the corresponding glycosylated receptor. In JC-R3 or JC-R1 cells labeled with bromodeoxyuridine or [(3)H]thymidine, FSH stimulated the cells to progress through S-phase and divide. The growth promoting effect of recombinant FSH in JC-R3 cells was preceded by the rapid activation of ERK1 and ERK2. This effect was hormone-specific and transient. In JC-R3 cells inhibitors like calphostin C, PD98059, Ag 18, or calcium chelators EGTA or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM inhibited both mitogen-activated protein kinase activation and bromodeoxyuridine incorporation. FSH induced phosphorylation of the FSH-R3 receptor was blocked by pretreating cells with calphostin C. There was no cAMP induction by FSH in JC-R3 cells. The cAMP independent growth promoting effect of FSH is mediated by activation of Ca(2+) and mitogen-activated protein kinase-dependent pathways. Thus, alternative splicing of a G-protein coupled receptor creates the expression of a novel receptor motif that can mediate a widely recognized function of the glycoprotein hormone.

Ovarian cancer gene therapy: repeated treatment with thymidine kinase in an adenovirus vector and ganciclovir improves survival in a novel immunocompetent murine model.

OBJECTIVE: Our purpose was to assess the effect of multiple injections of the system of herpes simplex virus thymidine kinase in an adenovirus vector and ganciclovir on survival in a murine model of human epithelial ovarian cancer. STUDY DESIGN: In this work we tested the ability of the system of thymidine kinase delivered by an adenovirus vector and ganciclovir to treat ovarian cancer in a novel murine model for epithelial ovarian cancer, SaskMouse. SaskMouse was developed by injecting LM-1 cells, a murine epithelial ovarian cancer cell line, intraperitoneally into a syngeneic C57BL/6N x C3H/He mouse strain. The cells developed into multiple cancer implants on different abdominal organs, leading to ascites and rapid death. The model has an intact immune system, as evidenced by the inability of different human cancer cells to develop into cancers when injected into the mice intraperitoneally. RESULTS: The system of thymidine kinase delivered by an adenovirus vector and ganciclovir was applied to SaskMouse. Mice were either untreated (group 1), treated with one intraperitoneal injection of adenovirus- thymidine kinase at 250 plaque-forming units/cell (group 2), or treated with two intraperitoneal injections of adenovirus-thymidine kinase at 250 plaque-forming units/cell on days 0 and 23 (group 3). Survivals were 23 +/- 2, 27 +/- 2, and 35 +/- 4 days, respectively (P <.05). Antiadenoviral antibodies were assayed both in the serum and in the peritoneal fluid of treated mice. Despite high antibody titers in serum, there were no detectable antibodies in the peritoneal fluid. CONCLUSION: Our data suggest that multiple intraperitoneal injections of the combination of thymidine kinase delivered by an adenovirus vector and ganciclovir are effective in prolonging survival in the presence of ovarian cancer. There are potential implications for other abdominal malignancies.

Dichlorodiphenyldichloroethylene potentiates the effect of protein kinase A pathway activators on progesterone synthesis in cultured porcine granulosa cells.

The insecticide dichlorodiphenyltrichloroethane (DDT) and its major metabolite p,p'-dichlorodiphenyldichloroethylene (DDE) have been implicated as endocrine-modulating chemicals. The DDT metabolite p, p'-DDE has been found contaminating human tissues and follicular fluid because of dietary exposure. We investigated the effects of DDE on progesterone synthesis in a stable porcine granulosa cell line, JC-410, and in primary cultures of porcine granulosa cells. Progesterone synthesis was not affected by 0.1-100 ng/ml DDE in the JC-410 cells. However, 10 ng/ml DDE increased 8-bromo-cAMP (8-Br-cAMP)-stimulated progesterone synthesis 0.4-fold (P < 0.05) over the levels observed with 1 mM 8-Br-cAMP alone. The effect of cholera toxin (CT) on progesterone synthesis was increased 0.7-fold (P < 0.05) by 10 ng/ml DDE over the value observed with 30 ng/ml CT alone. In primary cultures of porcine granulosa cells, 10 ng/ml DDE potentiated CT-stimulated progesterone synthesis 1.2-fold over the value observed with CT alone. In the JC-410 cells, 1 and 10 ng/ml DDE increased CT-stimulated cytochrome P450-cholesterol side-chain cleavage (P450(scc)) mRNA levels 0.3- and 0.4-fold, respectively, over the values obtained with CT alone. Neither basal nor CT-stimulated cAMP levels were changed by DDE. We conclude that DDE affects granulosa cell response to protein kinase A activators by altering the expression of the P450(scc) gene.

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins.

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3beta-hydroxy-5-ene steroid dehydrogenase (3beta-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17beta (estradiol), androstenedione, 5alpha-dihydrotestosterone, levonorgestrel, and 5-cholesten-3beta, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5alpha-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5alpha-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3beta-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3beta-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.

Regulation of steroidogenesis in jc-410, a stable cell line of porcine granulosa origin.

We investigated the regulation of steroidogenesis in a cell line of porcine granulosa origin, JC-410. Cells responded to the protein kinase-A activators, cholera toxin and forskolin, with increased accumulation of intracellular cAMP. Histochemically, cells were shown to contain 3beta-HSD, the enzyme which converts pregnenolone to progesterone. The JC-410 cells produced progesterone and responded to the protein kinase-A activators with an increase in progesterone synthesis. Progesterone levels were also increased by 25-hydroxycholesterol, pregnenolone, estradiol and androstenedione. Follicle-stimulating hormone and luteinizing hormone had no effect on cAMP or progesterone accumulation. Androstenedione was required for the synthesis of estradiol by JC-410 cells. Steady-state levels of mRNA for the steroidogenic enzymes 3beta-HSD and P450scc were increased by treatment with cholera toxin, whereas P450arom was not changed. These cells express the steroidogenic enzymes genes in a similar fashion to primary cultures of porcine granulosa cells. The JC-410 cells may represent a valuable model to study second messenger regulation and the molecular mechanisms involved in steroidogenesis in granulosa cells.

Establishment of a stable steroidogenic porcine granulosa cell line.

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.

FSH bioactivity in commercial preparations of gonadotropins.

During the past 2 decades, commercial preparations of FSH have been extensively used to superovulate cattle. The problems that have been encountered in superovulation of cattle include high variability in the ovulation rate and subsequent yield of viable embryos. The lack of predictability in superovulatory trials has been attributed to difficulties in standardizing the potency of commercial FSH preparations. Traditionally, FSH potency has been tested in bioassays that utilize specific responses in whole animals or primary cell cultures. Whole animal bioassays lack sensitivity, while primary cell culture bioassays, which use fresh cells, have inherent variability within each preparation. An FSH bioassay that employed a stable chimeric cell line expressing the human FSH-R was used to provide an accurate measurement of FSH bioactivity. The hormonal potency of 2 commercial preparations of FSH used to superovulate cattle was determined using FSH immuno- and bioassays. Commercial FSH preparations differed in potency. One commercial product, prepared in 4 different years, showed no difference in the immunoactive levels of FSH. In the same product stored under identical conditions, FSH bioactivity varied from year to year. There was variability in FSH bioactivity both between and within commercial products. The lack of correlation between bioactivity and immunoactivity of commercial FSH preparations may explain, in part, the variability observed in superovulation of cattle.

Growth hormone as an amplifier of insulin-like growth factor-I action: potentiated oestradiol accumulation.

The interaction between GH and IGF-I in modulating oestradiol biosynthetic capacity was examined in cultured porcine granulosa cells. Granulosa cells (2 x 10(6) viable cells/culture) were initially cultured for 96 h (treatment period) in an androstenedione-free medium in the absence or presence of IGF-I (0 or 50 ng/ml), with or without GH at 100 ng/ml. At the conclusion of this period, culture media were discarded and the cells were washed twice with the androstenedione-free medium and reincubated for an additional 24 h (test interval) in an androstenedione (10(-7) M)-supplemented medium for oestradiol accumulation. GH alone induced no stimulation (P > 0.05) of basal oestradiol accumulation. In contrast, concurrent treatment with IGF-I produced a 4.3-fold increase (26 vs 112 ng/ 2 x 10(6) cells per 24 h, P < 0.001) in oestradiol accumulation. GH amplified IGF-I-induced oestradiol production in a dose (minimal dose requirement of 0.3 ng/ml)- and time (minimal time requirement of 24-48 h)-dependent manner. Studies on the site(s) of action indicated that GH exerts its amplifying effects on IGF-I-induced oestradiol production both proximal and distal to cAMP generation. As the specificity study and the inhibitory study indicated, GH amplification of IGF-I-induced oestradiol production is a process involving gene transcription and/or translation and the synergism is not solely specific to IGF-I as IGF-II-induced oestradiol production was also amplified in the presence of GH.

3ß-hydroxy-5-ene Steroid dehydrogenase gene expression regulation in porcine granulosa cells : Differential effect of FSH and LH on gene transcription.

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 3ß-HSD-gene expression in cultured porcine granulosa cells. FSH and LH induced a dose dependent increase in the accumulation of 3ß-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 3ß-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 3ß-HSD mRNA induced by FSH at concentrations of 100, 300, and 1000 ng/mL. Alternatively, PMA added just before LH, inhibited LH-stimulated 3ß-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 3ß-HSD-gene transcription, 3.5- and 4.0-fold respectively. PMA prevented the stimulation of the 3ß-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 3ß-HSD mRNA levels by preventing cAMP-activated 3ß-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 3ß-HSD-gene transcription.

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.

Gonadotropin-releasing hormone stimulates glycoprotein hormone alpha-subunit messenger ribonucleic acid (mRNA) levels in alpha T3 cells by increasing transcription and mRNA stability.

Pulsatile GnRH stimulates gonadotropin secretion, whereas continuous exposure to GnRH causes pituitary desensitization and suppressed levels of LH and FSH. At the level of gene expression, continuous GnRH also causes partial or complete suppression of the LH beta and FSH beta genes, but expression of the alpha-subunit gene is stimulated without evidence of desensitization. In this report, we examined the transcriptional and posttranscriptional mechanisms by which GnRH controls alpha-gene expression using the gonadotrope-derived alpha T3 cell line. Continuous GnRH caused a 4- to 5-fold accumulation of alpha mRNA over 72 h, without evidence of a decline. In contrast, measurements of alpha-gene transcription, either by nuclear run-on assays or using a stably integrated alpha LUC reporter gene, revealed that GnRH caused a transient increase in alpha-promoter activity, followed by a decline after 4-6 h. The prolonged accumulation of alpha mRNA at a time when transcriptional activity had abated was accounted for by independent effects of GnRH on alpha mRNA stability. After prior treatment with GnRH, its removal either by washout or using a GnRH receptor antagonist caused an abrupt decline in steady state alpha mRNA levels (t1/2, < 2 h). Readdition of GnRH prevented the decay in alpha mRNA, and experiments using the transcriptional inhibitor actinomycin-D confirmed that this effect of GnRH did not require transcription. Consistent with these results, pulse-chase analyses of mRNA stability demonstrated that GnRH increased the alpha mRNA half-life 6.7-fold, from 1.2 h in the absence of GnRH to 8.0 h in the presence of GnRH. We conclude that GnRH induces a transient burst of alpha-gene transcription that is accompanied by marked induction of mRNA stability.

Gonadotropin-releasing hormone causes transcriptional stimulation followed by desensitization of the glycoprotein hormone alpha promoter in transfected alpha T3 gonadotrope cells.

Pulsatile GnRH regulates the biosynthesis and secretion of gonadotropins. Continuous administration of GnRH is known to desensitize gonadotropin secretion, but its effects on gonadotropin gene expression are less well characterized. We used a cell line of gonadotrope lineage (alpha T3 cells) to examine GnRH regulation of glycoprotein hormone alpha-subunit gene transcription. The alpha-subunit promoter, linked to a luciferase reporter gene (alpha LUC), was stably transfected into alpha T3 cells. Treatment with GnRH stimulated alpha LUC activity 3-fold. Stimulation of alpha LUC by GnRH was transient, with maximal activity after 6 h of treatment, followed by a return to baseline after 24 h. Stimulation of alpha-promoter activity by GnRH was inhibited entirely by a 10-fold molar excess of antide, a GnRH antagonist. Antide partially blocked GnRH stimulation even when added 4 h after GnRH, suggesting that a brief exposure to GnRH is not sufficient for maximal transcriptional stimulation. alpha LUC activity was also stimulated by treatment with 8-bromo-cAMP (3.5-fold), phorbol 12-myristate 13-acetate (TPA; 2.6-fold), or Bay K 8644 (3.3-fold). To assess whether the transient nature of GnRH stimulation was due to transcriptional desensitization, cells were pretreated with GnRH, followed by a second treatment with GnRH, cAMP, TPA, or Bay K. After pretreatment with GnRH, no further stimulation was seen after the addition of GnRH or TPA, but alpha LUC activity was further stimulated after the addition of either cAMP or Bay K. These findings indicate that the pathway for transcriptional activation by GnRH is desensitized and suggest that GnRH also desensitizes TPA-mediated stimulation. Similarly, pretreatment with TPA, but not cAMP or Bay K, prevented subsequent stimulation by GnRH. We conclude that GnRH transiently stimulates alpha gene transcription and that desensitization occurs with continuous exposure to GnRH, probably because of down-regulation of the protein kinase-C pathway.

Alterations in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) mRNA accumulation induced by beta-adrenergic stimulation in cultured porcine granulosa cells.

1. In primary cultures of porcine granulosa cells incubation with isoproterenol (10(-7)-10(-5) M) produced a dose-dependent increase in 3 beta-HSD mRNA, 3 beta-HSD content and progesterone production which ranged from 1.5- to 5-fold. 2. These effects were completely blocked by alprenolol. Terbutaline (10(-6) M) increased 3 beta-HSD mRNA, 3 beta-HSD content and progesterone production (1.5- to 3-fold), an effect which could be prevented by alprenolol. 3. Treatment with dobutamine (10(-6) M) produced no significant change in 3 beta-HSD mRNA accumulation. 4. The results suggest that beta-adrenergic agonists have the capacity to regulate transcription and translation of 3 beta-HSD mRNA in granulosa cells and this effect is mediated by the beta 2 adrenergic receptor.

Protein kinase C, an endogenous regulator of hormone-induced cyclic AMP induction in porcine luteal cells.

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction. The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.