FSH-stimulated cAMP Production Research Articles

P-678 Cabergoline has inhibitory effects on FSH-induced cell differentiation and estrogen secretion in a primary culture system of rat ovarian granulosa cells

Abstract Study question How does cabergoline have effects on cell differentiation and hormone secretion induced by FSH stimulation in ovarian granulosa cells? Summary answer Cabergoline suppresses LH receptor expression and Estradiol (E2) secretion in primary cultures of rat ovarian granulosa cells by inhibiting cAMP production induced by FSH stimulation. What is known already Cabergoline is a dopamine receptor 2 agonist that has been used to treat hyperprolactinemia and, more recently, to prevent ovarian hyperstimulation syndrome. Although it has been previously reported that cabergoline suppresses E2 and Progesterone secretion induced by FSH stimulation in a primary culture system of rat ovarian granulosa cells, but the effect on LH receptor expression has not been investigated. It has also been reported that the activation of dopamine receptor 2 suppresses VEGF secretion and reduces the incidence of early-onset OHSS by inhibiting the VEGF/VEGFR2 pathway in research using human luteinized granulosa cells. Study design, size, duration We used a primary culture system of ovarian granulosa cells obtained from immature female rats treated with diethylstilbestrol to examine the effect of cabergoline addition on the changes in ovarian granulosa cells induced by FSH stimulation. Participants/materials, setting, methods FSH and cabergoline were added alone or co-added to a primary culture system of rat ovarian granulosa cells, and the changes induced by them were measured and compared. (1) mRNA expression of Lhcgr, Cyp19a1, and Fshr by RT-qPCR (2) E2 concentration in the culture medium (3) Measurement of hCG binding on the cell surface (4) cAMP concentration Main results and the role of chance Co-addition of cabergoline suppressed each of the changes induced by FSH stimulation as follows. (1) The mRNA expression levels of Lhcgr, Cyp19a1, and Fshr decreased in a concentration-dependent manner (0.01-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, they decreased to 14%, 9%, and 49% of FSH alone, respectively. (2) E2 concentration in the culture media also decreased in a concentration-dependent manner (1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 49% of FSH alone. (3) The amount of hCG binding on the cell surface also decreased in a concentration-dependent manner (0.1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 7% of FSH alone. (4) FSH-stimulated cAMP production decreased in a concentration-dependent manner (0.01-1µM) of co-added cabergoline compared to FSH alone. In the 1µM of co-added cabergoline group, it decreased to 50% of FSH alone. These results suggest that cabergoline suppresses LH receptor and aromatase expression and E2 production by inhibiting cAMP production induced by FSH stimulation. Limitations, reasons for caution Since this is an in vitro experiment using a rat primary culture system, the results of this research cannot be considered directly applicable to humans. Wider implications of the findings Our findings imply that cabergoline administration during the follicular phase may inhibit ovarian granulosa cell differentiation and estrogen secretion. In women taking relatively high doses of cabergoline, attention may need to be paid regarding the effects of cabergoline on follicular development and estrogen secretion. Trial registration number not applicable

Ethanol potentiates follicle-stimulating hormone action in ovarian granulosa cells.

There are many previous reports on the effects of ethanol on physiological function, including reports of elevated blood estrogen levels in women who drank alcohol. However, the mechanism of ethanol's effects on ovarian functions, such as follicle development and hormone secretion, has not been fully clarified. Therefore, in this study, we investigated the impacts of ethanol on these phenomena and their mechanisms using a primary culture system of rat ovarian granulosa cells (GCs). In the present experiment, groups were created in which follicle-stimulating hormone (FSH) or ethanol was added alone or FSH and ethanol were co-added, and mRNA and protein expression in each group was measured for luteinizing hormone receptor (LHR) and sex steroid hormone synthase, as well as for estradiol (E2) production, cAMP production, and FSH receptor (FSHR) internalization rate. The addition of FSH induced mRNA expression of LHR and aromatase, which led to membrane LHR expression and E2 production. The coexistence of ethanol enhanced all these responses. The action of FSH is exerted via cAMP, and the co-addition of ethanol enhanced this cAMP production. Ethanol alone did not induce cAMP production. The enhancing effect of ethanol was also observed for cAMP induced by cholera toxin. Ethanol had no significant effect on the internalization rate of FSHR. In conclusion, ethanol increased FSH-stimulated cAMP production by increasing the activity of adenylyl cyclase, which enhanced FSH actions in rat GCs. Alcohol is an exacerbating factor in several female hormone-related diseases, and the mechanism of ethanol-induced increase in estrogen secretion revealed in this study may be involved in the pathogenesis of these diseases.

OR08-4 A Novel Mutation in the Follicle-Stimulating Hormone Receptor (Ile423Thr) in a Woman with Normal Pubertal Development and Primary Amenorrhea: In Vivo, In Vitro, and In Silico Studies

Women with inactivating mutations in the FSHR may exhibit an array of phenotypes, from lack of pubertal development, primary amenorrhea, and complete insensitivity to FSH, to secondary amenorrhea and premature ovarian failure. In contrast to the LHCGR, only 19 inactivating mutations in the FSHR have been described to date. We report herein the case of a 22 year old women who presented with normal pubertal development but primary amenorrhea, associated with elevated serum FSH and LH levels (27-30 IU/L), low serum estradiol (E2) (21-40 pg/ml) and normal serum AMH (2.2-2.5 ng/ml) levels. After administration of recombinant FSH (total dose 1425 IU/9 days) serum E2 levels rose from 20 to 65 pg/ml with a parallel increase in number (from 5 to 17 follicles) and size (from 3 to 10 mm diameter) of follicles, and ovarian volumes (from 4 to 10 cm3). Sequencing of the FSHR revealed a new homozygous mutation in codon 423, where ATC was changed to ACC, generating the Ile423Thr substitution at the junction of the transmembrane domain (TM) 2 and the exo-loop (EL) 1 of the FSHR. The mother and two half-brothers were heterozygous for the same mutation. The molecular physiopathogenesis of the novel Ile423Thr mutation was analyzed by a series of in silico and in vitro biochemical studies in HEK293 cells transiently expressing either the wild-type (Wt) FSHR or the Ile423Thr mutant cloned into the pSG5 vector, and the results were compared with those obtained in cells transfected with the expression-deficient Asp408Tyr mutant, which leads to a more severe form of hypergonadotropic hypogonadism (1). Compared with the Wt FSHR, the plasma membrane expression of the mature (~80 kDa) form of the Ile423Thr mutant was slightly reduced, while that of the Asp408Tyr mutant was remarkably low, as disclosed by immunoblotting. A dissociation in the responses to FSH-stimulated signaling between the mutant receptors was found: compared with the Wt FSHR, FSH-stimulated cAMP production and ERK phosphorylation were reduced by 67% and 27% in the Ile423Thr mutant and by 52% and 43% in the Asp408Tyr FSHR, respectively. In silico molecular dynamics simulations of the FSHRs in an explicit membrane environment unveiled important differences in the behavior of both mutants vs the Wt receptor, particularly in the interhelical interactions of Thr423 with TM7 and EL2, and Tyr408 with TMs 5 and 7. Concurrently, these data suggest that conformational differences during the inactive (particularly during trafficking of the receptor proteins from the endoplasmic reticulum to the plasma membrane) and active (agonist-occupied receptor) states, account for the distinct expression levels and differential signaling exhibited by the Ile423Thr and Asp408Tyr mutant FSHRs. (1) Bramble MS, et al. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing. Hum. Reprod. 2016; 31;905-914.

Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and β in the rat granulosa cell in vitro.

The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.

Inhibition of rat granulosa cell differentiation by overexpression of Gαq

Activation of FSH and LH receptors in undifferentiated granulosa cells (i.e., no prior exposure to FSH) results in comparable induction of progesterone production, but activation of the LH receptor is less effective than FSH in inducing aromatase and the native LH receptor. Because the LH receptor can also activate the Galphaq signaling pathway, we investigated whether activation of this pathway could be responsible for these differences. Overexpression of Galphaq inhibited FSH induction of both the estradiol and progesterone biosynthetic pathways as well as mRNA levels for cholesterol side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and P450aromatase (aromatase). This suppression was associated with a reduction (P < 0.05) in FSH-stimulated cAMP production. Lower cAMP levels were not due to reduced FSH receptor (FSHr) mRNA levels or reduced levels of Galphas. Phosphodiesterase (PDE) activity and regulator of G-protein signaling 2 (RGS2) mRNA levels were significantly (P < 0.05) increased by Galphaq, both of which could account for diminished cAMP levels. We conclude that Galphaq signaling pathway inhibits both estradiol and progesterone production comparably and thus activation of this pathway does not seem to account for differences between FSH and LH in the regulation of aromatase and the LH receptor.

Microtubule-independent and Protein Kinase A-mediated Function of Kinesin KIF17b Controls the Intracellular Transport of Activator of CREM in Testis (ACT)

Kinesins are motor proteins that transport their cargos along microtubules in an ATP-dependent manner. The testis-specific kinesin KIF17b was shown to directly regulate cAMP-response element modulator (CREM)-dependent transcription by determining the subcellular localization of the activator of CREM in testis (ACT), the testis-specific coactivator of CREM in postmeiotic male germ cells. CREM is a crucial transcriptional regulator of many important genes required for spermatid maturation, as demonstrated by the complete block of sperm development at the first steps of spermiogenesis in crem-null mice. To better understand the complex regulation of postmeiotic germ cell differentiation, we further characterized the ACT-KIF17b interaction, the function of KIF17b, and the signaling pathways governing its action. In this study, we demonstrated that the abilities of KIF17b to shuttle between the nuclear and the cytoplasmic compartments and to transport ACT are neither dependent on its motor domain nor on microtubules, thus revealing a novel microtubule-independent function for kinesins. We also showed that the cyclic AMP-dependent protein kinase A mediates the phosphorylation of KIF17b, and this modification is important for its subcellular localization. These results indicate that cyclic AMP signaling controls CREM-mediated transcription in male germ cells through modification of KIF17b function.

Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure.

Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 33.1 years (range 15-57). Ovarian failure had been diagnosed at a median age of 29 years (range 15-39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10-30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production.

Genetic and functional analyses of polymorphisms in the human FSH receptor gene.

To determine the influence of FSH receptor variants Thr307-Asn680 (TN) and Ala307-Ser680 (AS) on ovarian function, we investigated the frequency of these gene polymorphisms by using restriction fragment length polymorphism analysis and observed their effects on clinical manifestations. In a population of 522 Japanese women, the overall frequency of TN/TN (NN), TN/AS (NS), and AS/AS (SS) was 41.0, 46.9 and 12.1% respectively. In polycystic ovary patients, the NS population was significantly larger when compared with the spontaneously ovulating group (66.7 versus 43.5%, P < 0.05). In the SS group, a significantly higher (46%) basal level of serum FSH was observed as compared with that in the NS group (P < 0.05). A higher dose of the exogenous gonadotrophin was required to achieve ovulation induction in the SS group as compared with the NS group (P < 0.05). At the time of hCG administration, estradiol levels per oocyte retrieved for IVF in the SS group were significantly lower as compared with the levels in the NS and NN groups (P < 0.05). There were no significant differences in FSH-stimulated cAMP production and PI turnover as well as ligand-binding affinity between the two receptor isoforms when overexpressed in transfected 293T cells. These results suggest that although FSH receptor polymorphisms have no discernible effect on FSH receptor function in vitro, there are associations between the genotype and some aspects of patient status.

Structural determinants in the second intracellular loop of the human follicle-stimulating hormone receptor are involved in G s protein activation

In the present study, we analyzed the structural determinants present in the second intracellular loop (IL-2) of the human follicle-stimulating hormone (FSH) receptor (R) involved in G s protein-mediated signal transduction. Human embryonic kidney 293 (HEK-293) cells, stably expressing wild-type (Wt) human FSHR (HEK-293 (+)), were transiently transfected with plasmids containing cDNAs encoding the entire IL-2 or several IL-2 sequences mutated in R467 (a residue located at the center of the conserved ERW motif in the glycoprotein hormone receptors), T470 (a potential site for phosphorylation by protein kinase-A and -C) or L477 (a residue conserved in all glycoprotein hormone receptors). Expression of the IL-2 Wt in HEK-293 (+) cells reduced the maximum FSH-stimulated cAMP production significantly by ∼40%; similar results were observed with the R467A and R467K IL-2 mutants. The IL-2(R467H), IL-2(T470A), the triple R467A/T470A/L477A IL-2 mutant and the IL-2 of the oxytocin receptor (G q/11-coupled) had no effects on Wt FSHR-mediated intracellular signaling whereas the L477A mutation provoked a higher (∼55%) inhibition of FSH-stimulated cAMP than the free, Wt IL-2. These results suggested a specific role of IL-2 residues in FSHR function. Site directed mutagenesis of the FSHR and the expression of resulting mutants in HEK-293 cells were performed in order to corroborate the effects of these substitutions. Expression of FSHR(R467H), FSHR(R467A) and FSHR(T470A) failed to mediate ligand-provoked G s protein activation, whereas the R467K mutant behaved as the Wt receptor. Interestingly, the expression of L477A, L477D and L477P FSHR mutants conferred elevated basal cAMP levels to HEK-293 cells. This study indicates that the IL-2 of the human FSHR possesses amino acid residues that are important for both coupling the receptor to the G s protein (R467 and T470) and maintaining the receptor molecule in an inactive conformation (L477). It appears that this particular intracellular domain may act as a conformational switch to produce the activation of G proteins as has been reported for the IL-2 of other G protein-coupled receptors.

Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III.

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of these receptors, it has been suggested that either the hFSHR as a whole, or the i3/TM VI region of the hFSHR, is less susceptible to mutation-induced constitutive activation. However, as shown herein, substitution of a highly conserved leucine residue in transmembrane III (TM III) of the hFSHR (Leu 111.18) with arginine causes a 5-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Interestingly, this mutant is unresponsive to further hormonal stimulation. Substitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP production by the hFSHR. Because Leu 111.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human beta2-adrenergic receptor (hbeta2-AR). Substitution of L124 in the hbeta2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our data support a model whereby Leu 111.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu 111.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.

Stable transfection of the rat follicle-stimulating hormone receptor complementary DNA into an immortalized murine Sertoli cell line

A plasmid expressing the rat FSH receptor (R) cDNA under the Simian virus (SV) 40 promoter/enhancer was stably transfected into a mouse Sertoli cell (SC) line (MSC-1) established from transgenic mice carrying a fusion gene of the human anti-Müllerian hormone (AMH) promoter sequences linked to the SV40 T-antigen gene ( Peschon et al., 1992). The original cell line has numerous SC characteristics, but it was reported not to express the inhibin- α and follicle-stimulating hormone (FSH)R genes. The new FSHR expressing cell line possessed ≈2000 per cell with equilibrium association constant ( K a) of 1.5×10 9 l/mol. In Northern blots, an FSHR mRNA species of 2.6 kb was found. The cells responded to recombinant human FSH (recFSH) and pertussis toxin (PT) with stimulated cAMP production. Moreover, PT enhanced the FSH-stimulated cAMP production in these cells, indicating the presence of a functional Gi protein. 12- O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the FSH-stimulated cAMP production of the cells, which effect was similar to that observed previously upon protein kinase C (PKC) activation in rat seminiferous tubules in vitro. Hence, the FSHR signalling, and its modulatory pathways, were intact in the FSHR expressing MSC-1 cell line. RT-PCR with inhibin- α specific oligonucleotide primers, followed by Southern hybridization, indicated that, unlike previously shown, the original and the FSHR expressing MSC-1 cells do express the inhibin α gene. FSH stimulation of the cells decreased their proliferation and, unexpectedly, the inhibin- α mRNA levels. The cells have functional features both from neonatal and mature SC. A feature of the former cells is the lack of FSH-stimulated up-regulation of inhibin- α expression; in fact FSH decreased this message. The antiproliferative, and apparently differentiating, effect of FSH on these cells resembled mature SC functions. Since adult SC do not proliferate in vitro, the new FSHR expressing and proliferating cell line provides a useful in vitro model for studying some facets of SC functions, though keeping in mind that these transformed cells do not behave identically with adult SC in vivo. The constitutive expression of FSHR in these cells allows the study of posttranscriptional events in the FSHR regulation.

Contact-dependent cell interactions determine hormone responsiveness and desensitization in rat granulosa cells.

The maintenance of associations between granulosa cells (GCs) is necessary for FSH-stimulated induction of LH receptors. In cultures in which these associations have been disrupted, FSH fails to induce LH receptors. As FSH exerts its action in GCs via cAMP, we have examined if the aggregation state of GCs plays a role in modulating FSH-stimulated cAMP production. GCs were obtained from the ovaries of diethylstilbestrol-primed immature rats. Cells were prepared as aggregate or dispersed populations by isolating GCs in either the presence or absence of Ca2+. Nonviable cells were removed by a brief exposure to trypsin. We have shown previously that trypsin treatment in the absence of Ca2+ removes a class of cell adhesion molecules, termed cadherins, from the plasma membranes of GCs. Hence, the dispersed GCs are incapable of reaggregating. Dispersed or aggregate GC preparations were incubated with different doses of human FSH (0-1 microg) for 0-60 min in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Incubations were terminated, and the cAMP accumulated was measured using a specific RIA. As desensitization to hormonal stimuli is a characteristic property of many G protein-coupled response systems, cAMP production of cell aggregates and dispersed cells in response to a repeated stimulation with FSH was assessed. Our results indicate that aggregate GCs have a significantly attenuated cAMP response to all doses of FSH compared with dispersed GC preparations. Changing cell densities did not alter the nature of these responses, indicating that nonspecific cell interactions were not responsible for this difference. The number of FSH receptors and their affinity were unaltered in the two cell preparations. Cholera toxin- and forskolin-stimulated cAMP production were similar in the two preparations, demonstrating that the changes in responsiveness did not arise from alterations in G protein activation or adenylate cyclase activity. Only the aggregate GCs could be desensitized. The dispersed cells displayed undiminished cAMP responsiveness to a second FSH stimulation. Finally, culture of the GC preparations with cholera toxin induced LH receptors in GC aggregates only. LH receptor induction in dispersed cell cultures required the addition of estradiol. These results indicate that contact-dependent cell interactions can modulate GC cAMP production in response to FSH. cAMP responses, however, were not the sole determinant of cell differentiation, as assessed by LH receptor induction. We speculate that cell-cell interactions within the follicular epithelium are important determinants for cell differentiation leading to follicle selection for ovulation or atresia.

Contact-Dependent Cell Interactions Determine Hormone Responsiveness and Desensitization in Rat Granulosa Cells

The maintenance of associations between granulosa cells (GCs) is necessary for FSH-stimulated induction of LH receptors. In cultures in which these associations have been disrupted, FSH fails to induce LH receptors. As FSH exerts its action in GCs via cAMP, we have examined if the aggregation state of GCs plays a role in modulating FSH-stimulated cAMP production. GCs were obtained from the ovaries of diethylstilbestrol-primed immature rats. Cells were prepared as aggregate or dispersed populations by isolating GCs in either the presence or absence of Ca2+. Nonviable cells were removed by a brief exposure to trypsin. We have shown previously that trypsin treatment in the absence of Ca2+ removes a class of cell adhesion molecules, termed cadherins, from the plasma membranes of GCs. Hence, the dispersed GCs are incapable of reaggregating. Dispersed or aggregate GC preparations were incubated with different doses of human FSH (0–1 μg) for 0–60 min in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Incubations were terminated, and the cAMP accumulated was measured using a specific RIA. As desensitization to hormonal stimuli is a characteristic property of many G protein-coupled response systems, cAMP production of cell aggregates and dispersed cells in response to a repeated stimulation with FSH was assessed. Our results indicate that aggregate GCs have a significantly attenuated cAMP response to all doses of FSH compared with dispersed GC preparations. Changing cell densities did not alter the nature of these responses, indicating that nonspecific cell interactions were not responsible for this difference. The number of FSH receptors and their affinity were unaltered in the two cell preparations. Cholera toxin- and forskolin-stimulated cAMP production were similar in the two preparations, demonstrating that the changes in responsiveness did not arise from alterations in G protein activation or adenylate cyclase activity. Only the aggregate GCs could be desensitized. The dispersed cells displayed undiminished cAMP responsiveness to a second FSH stimulation. Finally, culture of the GC preparations with cholera toxin induced LH receptors in GC aggregates only. LH receptor induction in dispersed cell cultures required the addition of estradiol. These results indicate that contact-dependent cell interactions can modulate GC cAMP production in response to FSH. cAMP responses, however, were not the sole determinant of cell differentiation, as assessed by LH receptor induction. We speculate that cell-cell interactions within the follicular epithelium are important determinants for cell differentiation leading to follicle selection for ovulation or atresia.

A mutation in the follicle-stimulating hormone receptor occurs frequently in human ovarian sex cord tumors.

A subset of ovarian tumors, referred to as sex cord-stromal tumors, produce endocrine manifestations due to the secretion of estrogens or androgens. Because gonadotropins induce the growth, differentiation, and function of the steroid-producing cells of the ovary, we hypothesized that mutations in the FSH receptor (FSH-R) might occur in this group of tumors. Ovarian sex cord tumors (n = 13), small cell carcinomas of the ovary (n = 3), and control DNA specimens (n = 116) were screened for mutations in the transmembrane domains of the FSH-R. A heterozygous T-->C mutation was found at nucleotide 1777 that converts codon 591 from phenylalanine to serine (F591S). This sixth transmembrane domain mutation was found in 9 of 13 (69%) sex cord tumors and 2 of 3 ovarian small cell carcinomas, but it was not present in control specimens, including 5 normal ovaries, 5 nonsex cord ovarian tumors, 16 thyroid tumors, or 90 specimens of peripheral blood leukocyte DNA, suggesting that this nucleotide change is not a polymorphism. The functional effects of identified mutations were assessed by expression of the wild-type or the F591S mutant FSH-R in COS-7 cells. The F591S mutation eliminated FSH-stimulated cAMP production, and a similar effect was observed when this mutation was introduced into the homologous location of the LH receptor. The high prevalence of the F591S mutation in the FSH-R suggests that it plays a role in the development of ovarian sex cord tumors.

Low levels of follicle-stimulating hormone receptor-activation inhibitors in serum and follicular fluid from normal controls and anovulatory patients with or without polycystic ovary syndrome.

In patients with normogonadotropic anovulation, either with or without polycystic ovary syndrome (PCOS), factors interfering with FSH action may be involved in arrested follicle development. The aim of this study is to assess whether factors inhibiting FSH receptor activation are elevated in serum or follicular fluid from anovulatory patients, as compared with regularly cycling women. For this purpose, a Chinese hamster ovary cell line, stably transfected with the human FSH receptor, has been applied. FSH-stimulated cAMP secretion in culture medium was measured in the presence of serum or follicular fluid. Chinese hamster ovary cells were stimulated with a fixed concentration of FSH (3 or 6 mIU/mL) to mimic FSH levels in serum or follicular fluid. Samples were added in concentrations ranging from 3-90% vol/vol to approach protein concentrations occurring in serum or follicular fluid. In the presence of 10% vol/vol serum from regularly cycling women (n = 8), FSH-stimulated cAMP production was inhibited to 42 +/- 2% (mean +/- SEM of 2 experiments, each performed in duplicate) of cAMP production in the absence of serum, whereas a similar cAMP level (up to 38 +/- 4% of the serum-free level) was observed at higher concentrations of serum (30-90% vol/vol). The inhibition of FSH-stimulated cAMP production in the presence of serum samples from normogonadotropic anovulatory patients, without (n = 13) or with (n = 16) PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained during the follicular phase in 25 regularly cycling women and follicular fluid samples (n = 25) from 5 PCOS patients were tested in a slightly modified assay system. In the presence of 10 or 30% (vol/vol) follicular fluid, FSH-stimulated cAMP levels were decreased to 68 +/- 2% and 55 +/- 2% (mean +/- SEM of a single experiment in triplicate) of the cAMP levels in the absence of follicular fluid, respectively. There was no correlation between the degree of cAMP inhibition and follicle size, steroid content (androstenedione or estradiol concentrations), or menstrual cycle phase. Furthermore, no differences in inhibition were found, comparing PCOS follicles with size- and steroid content-matched follicles obtained during the normal follicular phase. It is concluded that inhibition of FSH receptor activation by proteins present in serum or follicular fluid is constant (60 and 40%, respectively) and independent from the developmental stage of the follicle, either during the normal follicular phase or in patients with normogonadotropic anovulation. Inhibition of FSH receptor activation may be of limited significance for normal and arrested follicle development.

Application of a CHO cell line transfected with the human FSH receptor for the measurement of specific FSH receptor activation inhibitors in human serum

Effects of FSH on ovarian follicular development can be modulated by factors present in serum or by locally produced factors in follicular fluid. Some of these factors may act directly on the FSH receptor. A Chinese hamster ovary cell line (CHO-F3B4) stably transfected with the human FSH receptor has been used to measure the effects of these modulators on FSH-stimulated adenylate cyclase activity. After incubation of CHO-F3B4 cells with human recombinant FSH (recFSH) for 4 h, cAMP levels were elevated 100-230 times above basal levels (ED50 24.9 mU/ml recFSH). cAMP production was inhibited after the addition of increasing amounts (up to 90% of the incubation volume) of hypogonadotrophic human serum (HS) at a fixed stimulatory dose of 30 mU/ml recFSH. At 10% HS the cAMP response was diminished to approximately 40-60% of the original value, whereas at a concentration of 90% HS the cAMP values were diminished to 30%. Effects of serum components on cell viability could be excluded, since forskolin- and cholera-toxin-stimulated cAMP production were not affected by preincubation of the cells in the presence of HS. The FSH-stimulated oestradiol production in rat Sertoli cells, which has been used frequently for in vitro bioassays of FSH, was almost completely inhibited by the addition of human serum, suggesting that serum has more pronounced effects on events downstream of receptor activation. Various specific FSH binding inhibitors have been demonstrated by radioreceptor assays to be present in serum. In order to assess whether such FSH receptor binding inhibitors would also inhibit receptor activation, the specific conditions used in the radioreceptor assays (buffers of low ionic strength) were also used to measure the effects of serum on FSH receptor activation. Under these conditions (a low-salt buffer, corrected for low osmolarity with 200 mM sucrose), CHO-F3B4 cells responded to FSH stimulation in a similar way to that observed in normal buffers. When CHO-F3B4 cells were incubated in this low-salt buffer with a fixed low dose of FSH (3 mU/ml), the addition of 3-90% (v/v) dialysed HS inhibited the FSH-stimulated cAMP accumulation to a similar extent to that in standard conditions. The observed inhibition of adenylate cyclase activation by the low-molecular-mass fraction (< 10 kDa) of HS could be attributed to the presence of salts in this fraction, since the addition of PBS in similar concentrations displayed an equal degree of inhibition. It is concluded that the inhibitory effects of serum on FSH-stimulated cAMP production in CHO-F3B4 cells are small, compared with the inhibition of aromatase induction in rat Sertoli cells. The strong inhibition of aromatase in rat Sertoli cells may result from the effects of serum acting on the FSH receptors as well as on other pathways not related to the FSH receptor. Therefore, measurement of aromatase in Sertoli cells is not suitable for the detection of inhibitors of FSH receptor activation. The CHO-F3B4 cells are useful for the measurement of whether inhibition of FSH receptor activation occurs in serum or follicular fluid from patients with disturbed follicle development.

Lack of endogenous adenosine receptor activation explains the insensitivity of neonatal rat Leydig cells to treatment with pertussis toxin

We have previously shown that the G i protein-mediated negative modulation of FSH-stimulated cAMP production in neonatal rat Sertoli cells can be blocked by pretreatment with pertussis toxin (PT), an inhibitor of the G i protein function. In contrast, hCG-stimulated testosterone (T) production by neonatal Leydig cells was insensitive to PT action, despite the demonstration of G i protein in these cells by immunohistochemistry. We now tested the hypothesis that the missing G i protein function in neonatal Leydig cells is due to absence of its endogenous activator, adenosine. It was first reconfirmed in dispersed cells of 7-day-old rats that PT preincubation enhanced FSH-stimulated cAMP production (i.e. Sertoli cell function), but had no effect on hCG-stimulated T production (Leydig cell function). The adenosine agonist n-phenylisopropyladenosine (PIA 1 μmol/l), acting through the G-protein, attenuated FSH-stimulated cAMP production, and this effect was abolished when the G i protein was inactivated by preincubation with PT. PIA (1 μmol/l) also inhibited ( P < 0.05−0.01) the basal and hCG-stimulated rates of Leydig cell T production, and the PIA effect was abolished by PT preincubation. In conclusion, the present data show that the apparent absence of a functional G i protein function of neonatal Leydig cells is due to insufficient endogenous levels of the stimulating ligand, adenosine. When the G i protein function is activated by exogenous adenosine agonist (PIA), the enhancement of hCG-stimulated T production by PT can be demonstrated. At the same time, the endogenous level of adenosine is sufficient for activation of the G i protein function in Sertoli cells.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Modulation of gonadotropic and purinergic responsiveness by islet activating protein in sertoli cells from immature rats.

In the present study we have examined the effects of islet activating protein (IAP) on the regulatory effects of FSH, glucagon and (-)N6-(R)-phenyl-isopropyladenosine (PIA), an adenosine A1 receptor agonist, on the formation of cAMP and estradiol-17 beta (E2) in Sertoli cell cultures isolated from immature (19-day-old) rats. FSH (NIH-FSH-S-15) (1.25 micrograms/ml) caused a more than 10-fold stimulation of the level of both cAMP and E2 in the spent media from Sertoli cell cultures during an 18 h incubation. Both responses were reduced by 80% in the presence of PIA (10(-6) M). When the cultures were preincubated for 24 h with increasing concentration of IAP, the inhibitory effects of PIA were counteracted in a concentration-dependent manner. Moreover, preincubation with IAP (> 20 ng/ml) caused a significant stimulation of FSH-stimulated cAMP production even in the absence of PIA. PIA inhibited FSH-stimulated cAMP production in a concentration dependent manner. However, when the cells were preincubated with IAP (100 ng/ml) for 24 h, the inhibitory effects of PIA were completely abolished, and PIA now actually caused a slight stimulation of cAMP production. Both FSH and glucagon stimulated cAMP production in a concentration-dependent manner. Preincubation with IAP (100 ng/ml for 24 h) resulted in an increase in maximal stimulation of cAMP production for both FSH and glucagon. When adenylyl cyclase (AC) activity was measured directly in isolated membrane particles from Sertoli cells cultured in the presence of IAP (100 ng/ml) for 24 h, both basal and FSH-stimulated AC activity were significantly higher than in membrane particles from control cells. These results provide a further characterization of the functional Gi component coupled to the AC complex in cultured rat Sertoli cells, mediating the inhibitory effects of adenosine and possibly other endogenous substances on cAMP production.

Growth hormone-releasing hormone in testicular interstitial and germ cells: potential paracrine modulation of follicle-stimulating hormone action on Sertoli cell function.

GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.