Evidence for a novel thioredoxin-like catalytic property of gonadotropic hormones.

It has been proposed that dithiol-disulfide interchange and oxidation-reduction reactions may play a role in hormone-induced receptor activation. Inspection of the sequences of the gonadotropic hormones revealed a homologous tetrapeptide (Cys-Gly-Pro-Cys) between the beta subunit of lutropin (LH) and the active site of thioredoxin (TD). The beta subunit of follitropin (FSH) has a similar sequence (Cys-Gly-Lys-Cys). Thioredoxin is a ubiquitous protein serving as an electron donor for ribonucleotide reductase, but it also exhibits disulfide isomerase activity. The catalytic activity of TD was assayed by its ability to reactivate reduced and denatured ribonuclease. In this assay, the purified ovine FSH and bovine LH preparations tested were approximately 60 and approximately 300 times, respectively, as active as TD on a molar basis. This heretofore unsuspected catalytic property of FSH and LH may be important in understanding their mechanism of receptor activation and signal transduction.

Properties of particulate and detergent-solubilized adenylate cyclase of rat testis. Effects of follitropin stimulation.

Basal, fluoride and follitropin stimulated activities of adenylate cyclase have been studied in the testes of immature rats. The enzyme was maximally activated (about twice the basal activity) by low concentrations of follitropin, with an apparent Km of about 9 . 10(-10) M. Both Mg2+ and Mn2+ activate the enzyme but the apparent Ka for Mg2+ is about 10 times that for Mn2+. However, the apparent Km values for MgATP2- and MnATP2- are nearly the same (1.4 . 10(-4) M) and the cation activation of the enzyme is mainly through an increase in V. Ca2+ inhibited all expressions of testicular adenylate cyclase activity. Follitropin and fluoride stimulated adenylate cyclase activity at all Mg2+ concentrations but did not significantly affect the apparent Ka for Mg2+ or the Km for the substrate (MgATP2-). The stimulatory effect of the hormone or fluoride is mainly through an increase in V. Testicular adenylate cyclase could be solubilized with Triton X-100 or Lubrol-PX. The detergent-solubilized enzyme exhibited Km for substrate and Ka values for divalent cations similar to those of the membrane-bound enzyme and remained responsive to stimulation by fluoride. The stimulatory effect of follitropin, however, was lost. Responsiveness to follitropin was also lost by membrane-bound adenylate cyclase after treatment with phospholipase, although the fluoride effect was unchanged. These results reflect the essential role of lipids in the regulation of the follitropin-specific response.

Bovine luteinizing hormone. Circular dichroism and thermal difference spectra.

The circular dichroism of bovine luteinizing hormone in the far ultraviolet (200-240 nm) and near ultraviolet (240-320 nm) is reported as a function of pH, reduction and denaturation. The significance of side-chain ellipticity in the native and fully randomised hormone is critically examined, using denatured and reduced ribonuclease and insulin as bases for comparison. Disulphide groups make no measurable contribution to the side-chain ellipticity. Judged by the spectra of the subunits both isolated and recombined, those treatments which promote subunit disociation without causing chain unfolding, reversibly decrease the ellipticity in the near ultra-violet with minimal effect in the far ultraviolet. Thermal perturbation difference spectroscopy of bovine luteinizing hormone and its subunits shows that, in common with the ovine and porcine hormones, there are two tyrosine residues located at the interface between the subunits and inaccessible to water. Only two or three of the five water-accessible tyrosine residues are reactive to N-acetylimidazole.

Structure--function relationships of the glycoprotein hormones and their receptors.

The primary structures of the glycoprotein hormones follitropin (FSH), lutropin (LH), human choriogonadotropin (hCG) and thyrotropin (TSH) have been determined, hCG has been crystallized and initial diffraction data obtained. Studies with synthetic peptides have provided information on regions involved in receptor interaction and signal transduction. The receptors for the glycoprotein hormones have been prepared by gene cloning methods and their primary structures deduced. As Leo Reichert and colleagues discuss here, although cAMP is involved in glycoprotein hormone signal transduction, recent evidence also implicates other second messengers, especially Ca2+ and may include both the phosphatidylinositol pathway and activation of Ca2+ channels.

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes.

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.

Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein.

In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.

Modulation of follicle-stimulating hormone-sensitive rat testicular adenylate cyclase activity by guanyl nucleotides.

We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 microM). The Km for hormone activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hRSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity.

Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase.

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride.

Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-[2-3H] inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total [3H]inositol phosphates [[3H]inositol mono-, di-, and triphosphates (IP, IP2, and IP3)] in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%. These results suggest that phospholipase-C activity in Sertoli cells is modulated by a pertussis toxin-sensitive G-protein(s), but does not appear to be affected by FSH.

The effects of aprotinin on follicle-stimulating hormone binding and signal transduction in bovine calf testis.

Aprotinin (bovine pancreatic trypsin inhibitor), a serine protease inhibitor, caused a dose-dependent inhibition of [125I]human FSH ([ 125I]hFSH) binding to 1) an FSH receptor-enriched light membrane fraction prepared from bovine calf testes homogenates, 2) Triton X-100-solubilized FSH receptor, and 3) proteoliposomes containing incorporated FSH receptor-G-protein-adenylate cyclase (AC) complexes. Equilibrium binding studies with the solubilized receptor indicated that the effect of aprotinin on [125I]hFSH binding was due to a decrease in the Ka of the receptor, with no change in FSH-binding capacity. The rate of association of [125I]hFSH with its receptor was reduced by 50% in the presence of aprotinin, but no effect on dissociation of FSH-receptor complexes was evident. Aprotinin, at a concentration (250 microM) that inhibited binding of [125I]hFSH to the membrane receptor by 25%, completely inhibited basal, fluoride-stimulated and FSH-stimulated AC activity. However, aprotinin, at a concentration (50 microM) that had little effect on [125I]hFSH binding, markedly enhanced basal AC activity (3.4-fold) to the level of fluoride and FSH stimulation. Aprotinin did not inhibit [3H]5'-guanylylimidodiphosphate binding to FSH receptor-enriched membranes, suggesting that its effects on the affinity of the receptor for FSH and on AC activation were not mediated through an interaction with FSH receptor-associated G-protein. No serine protease activity could be detected in any of the receptor or hormone preparations used in this study. The ability of aprotinin to inhibit binding of [125I]hFSH to the Triton X-100-solubilized receptor and to the soluble receptor incorporated into proteoliposomes as well as to the FSH receptor-enriched membrane fraction, all of which are free of serine protease activity, is consistent with the notion that aprotinin may directly interact with the FSH receptor to sterically hinder binding of FSH. Furthermore, the apparent direct effect of aprotinin on basal as well as FSH-stimulated AC activity suggests its general usefulness in studies on the mechanism of signal transduction for ligands thought to operate via the cAMP second messenger system.

Guanine triphosphate-binding site regulation by follicle-stimulating hormone and guanine diphosphate in membranes from immature rat Sertoli cells.

GTP binding to Sertoli cell membranes has been investigated using [3H]5'-guanylyl-beta gamma-imidodiphosphate [[3H]Gpp(NH)p], a nonhydrolyzable analog of GTP. Binding of [3H]Gpp(NH)p Gpp(NH)p to membranes prepared from Sertoli cells in serum-free culture was proportional to membrane protein concentration in the range of 5-50 micrograms. Competitive displacement studies using adenine (ATP, ADP, and AMP) and guanine nucleotides [GTP, GDP, GMP, and Gpp(NH)p] indicated that only GTP, its analog Gpp(NH)p, and GDP were effective ligands. The relative potencies were Gpp(NH)p much greater than GTP greater than GDP, as characterized by ED50 values of 0.8, 2.5, and 4 microM, respectively. Competitive inhibition by GTP, however, was similar to that by Gpp(NH)p in the presence of a nucleoside triphosphate-regenerating system, suggesting the involvement of an active GTPase. Equilibrium binding studies indicated a single high affinity site for GTP with a Ka of 3.3 +/- 0.2 X 10(7) M-1. This value was supported by other studies in which an association rate constant of 1.8 X 10(6) M-1 min-1 and a dissociation rate constant of 2.4 X 10(-2) min-1 were estimated. Maximal binding of [3H]Gpp(NH)p to Sertoli cell membranes ranged from 30-55 pmol/mg protein. FSH enhanced [3H]Gpp(NH)p binding by about 50% (P less than 0.05), reflecting an increase in the number of available binding sites rather than an effect on Ka. When GDP was preincubated with membranes in the absence of FSH, the number of available binding sites for [3H]Gpp(NH)p was decreased. This reduction in available binding sites by pretreatment with GDP could be reversed by adding FSH during the equilibrium binding analysis. These studies have demonstrated specific high affinity binding of Gpp(NH)p to Sertoli cell membranes with an affinity comparable to that required for activation of FSH-sensitive adenylate cyclase. Furthermore, a potent GTPase activity associated with the Sertoli cell membrane is responsible for rapid hydrolysis of GTP to GDP and may participate in inactivation of GTP-dependent adenylate cyclase activity. The role of FSH in the regulation of nucleoside binding appears to be in facilitating exchange of GTP for GDP by enhancing the release of bound GDP.

Carbohydrate moiety of follitropin receptor is not required for high affinity hormone-binding or for functional coupling between receptor and guanine nucleotide-binding protein in bovine calf testis membranes.

Previously, we have extensively studied FSH-receptor interactions using bovine calf testis membranes, and demonstrated that the high-affinity FSH binding to receptors and coupling of FSH receptors with guanine nucleotide-binding protein (Gs protein) in a GTP-sensitive state are important initial events in FSH action. In this study, using the same plasma membrane system, we examined the glycoprotein nature of the FSH receptor and determined the contribution of carbohydrate moieties to these functions of the FSH receptor. Our approach involved enzymic deglycosylation of FSH receptors present in calf testis plasma membranes and then removal of incompletely deglycosylated FSH receptors by lectin affinity chromatography. Following treatment of testis membranes with peptide N-glycosidase, the receptor, as identified by ligand-blot analysis, had a higher electrophoretic mobility indicating a decrease in M(r) from 240-200K. Treatment of testis membranes with neuraminidase caused a reduction (to approximately 225K) in the size of the receptor consistent with desialylation. However, digestion with O-glycosidase (endo-alpha-N-acetylgalactosaminidase) did not affect the mobility of the FSH receptor. These results suggest that bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains, a finding which is consistent with recent predictions that N-linked glycosylation, but not O-linked glycosylation sites are present in cloned FSH receptor from rat testis. Moreover, calf testis membranes after treatment with peptide N-glycosidase F, were solubilized with Triton X-100 under optimum conditions that preserve the physical and functional coupling of FSH receptors with guanine nucleotide-binding protein, and then subjected to lectin affinity chromatography. Scatchard analysis indicated that intact and deglycosylated FSH receptors bound 125I-human FSH with similar affinities. In the presence of GTP, the binding of 125I-human FSH to intact and deglycosylated receptors decreased similarly and in a noncompetitive manner. Treatment of testis membranes with NAD plus cholera toxin, but not NAD plus pertussis toxin, eliminated the GTP effect on FSH binding to enzymic deglycosylated as well as intact receptors, suggesting that the guanine nucleotide binding protein mediating GTP regulation of FSH binding in these membranes is probably Gs protein. Our results suggest that the bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains consistent with recently predicted N-linked glycosylation sites of cloned FSH receptor of rat testis. The bovine testis FSH receptor does not require N-linked carbohydrate for high-affinity hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by proteoliposomes and cultured rat sertoli cells: evidence for involvement of voltage-activated and voltage-independent calcium channels.

We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-[3-thiotriphosphate]) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

In vivo effects of follicle-stimulating hormone-related synthetic peptides on the mouse estrous cycle.

We have previously shown that a synthetic peptide amide corresponding to residues 34-37 (TRDL) of the human (h) FSH beta-subunit inhibited binding of [125I]hFSH to bovine calf testis membrane receptors and antagonized FSH-stimulated estradiol biosynthesis in primary cultures of rat Sertoli cells. These in vitro effects would have additional significance if they could be confirmed in an in vivo model system. We have obtained several lines of evidence supporting in vivo effects of TRDL on the mouse estrous cycle. 1) A single i.p. injection of 200 microg/g BW TRDL induced persistent vaginal estrus, characterized by the complete absence of epithelial casts in 87% of the mice treated, as determined by vaginal cytology. 2) A synthetic peptide representing a larger receptor-binding domain of the hFSH beta-subunit, hFSHbeta-(33-53), that contains the TRDL sequence had a similar effect, but hFSHbeta-(38-53) lacking the TRDL sequence, did not. 3) A series of unrelated synthetic peptides, tested at a comparable dose (200 microg/g BW), were also without effect, as was a D-amino acid analog of TRDL, TR(D)DL. 4) Serum estradiol levels at proestrus in TRDL-treated mice were significantly lower than those in vehicle-injected control mice. 5) The effect of estrogen on uterine ballooning and weight gain, seen in all vehicle-injected control mice at proestrus, did not occur in 97% of the mice treated with TRDL. 6) The ovaries of TRDL-treated mice taken during persistent vaginal estrus contained a greater number of large hemorrhagic preovulatory follicles and fewer corpora lutea than those in ovaries taken at estrus from vehicle-injected control mice. Taken together, these results indicate disruption of the normal mouse estrous cycle by the TRDL peptide and represent the first demonstration of in vivo effects of gonadotropin-related synthetic peptides on reproductive processes.

Functional properties of polyclonal antibodies raised against the N-terminus region (residues 9-30) of the follicle-stimulating hormone (FSH) receptor: significance of this receptor region in FSH recognition and signal transduction.

In this study, we raised polyclonal antibodies in rabbits against a synthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Binding of [125I]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) peptide antibody. Preimmune serum had no effect. Lineweaver-Burke plot analysis of [125I]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibody effectively competed with FSH at a hormone-binding site on the receptor. Also, antireceptor peptide antibody, but not preimmune serum, inhibited the ability of FSH to stimulate the conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forskolin (which are known to act through the Gs-protein and catalytic unit of adenylate cyclase, respectively) was not inhibited by antireceptor peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane receptor. No fluorescent staining of receptor was observed when cells were incubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized membrane preparations from rat Sertoli cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and then subjected to Western blot analysis, antireceptor peptide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occupied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), as shown by indirect immunofluorescence staining of rat Sertoli cells and the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raised against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagonist, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of granulosa cell lactate production by follicle-stimulating hormone and androgen.

Lactate accumulation in granulosa cell cultures (prepared from estrogen-pretreated immature rat ovaries) increased with human FSH (hFSH) concentration in the culture medium. In 48-h cultures, maximal stimulation (approximately 25%) occurred in the presence of more than or equal to 100 ng hFSH/ml. Human CG (hCG) (3-1000 ng/ml) had no effect. Testosterone and 5 alpha-dihydrotestosterone (10(-8)-10(-6) M) did not affect basal lactate accumulation but they enhanced (dose dependent) the response to hFSH: lactate levels after 48 h of treatment with 10(-7) M testosterone plus 100 ng/ml hFSH were 100% higher than those in untreated control cultures. Lactate was refractory to estradiol and progesterone (10(-8)-10(-6) M) even in the presence of hFSH. Progesterone accumulation showed a qualitatively similar pattern of response to the gonadotropins and sex steroids. As expected, the progesterone response to hFSH (100 ng/ml) plus testosterone (10(-7) M) was progressively suppressed in the presence of 10(-7)-10(-5) M nonsteroidal antiandrogen (SCH16423). Lactate accumulation was also reduced. However, maximal inhibition did not exceed 18% in the presence of SCH16423 at 10(-6) or 10(-5) M as compared with the 80% inhibition of progesterone accumulation observed at 10(-5) M. In the absence of androgenic steroid, the lactate response to hFSH was increased approximately 30% by the high dose of SCH16423. A corresponding synandrogenic action of the drug on FSH-sensitive progesterone accumulation was not observed. These results are evidence that carbohydrate metabolism in differentiating granulosa cells is subject to direct and specific control by FSH and androgenic steroid.

Polyclonal antibodies against follitropin (FSH) receptor interfere with hormone binding, but mimic the effects of FSH.

We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.

A new role for follicle-stimulating hormone in the regulation of calcium flux in Sertoli cells: inhibition of Na+/Ca++ exchange.

Elucidation of mechanisms regulating intracellular calcium levels in steroidogenic tissues is important for understanding control of cellular function. We have previously described FSH receptor-mediated flux of 45Ca++ into cultured rat Sertoli cells and receptor-enriched proteoliposomes via voltage-sensitive and voltage-independent calcium channels. In the present study, we report heretofore unrecognized inhibitory effects of FSH on Na+/Ca++ exchange in these two systems. An outwardly directed Na+ gradient, developed by preincubating Sertoli cell monolayers in buffer made hypertonic with NaCl, resulted in uptake of 45Ca++ that was unaffected by calcium channel blocking agents, ruthenium red or methoxyverapamil, but was enhanced by ouabain, a specific inhibitor of Na+/K(+)-ATPase. Sodium-dependent 45Ca++ flux into Sertoli cells was inhibited in a concentration-related manner by increased extracellular Na+ (up to 135 mM). FSH consistently and reproducibly (28.9 +/- 3.8%, 10 separate assays) reduced sodium-dependent 45Ca++ influx in the absence or presence of ouabain. A lesser effect on Na+/Ca++ exchange was seen when Li+ replaced Na+ in the preincubation buffer, and a marked reduction occurred when Sertoli cells were incubated in buffer containing KCl, presumably due to membrane depolarization. FSH-sensitive Na+/45Ca++ exchange was also observed when using FSH receptor-enriched proteoliposomes. Our earlier calcium channel studies indicated that FSH affects Ca++ entry into Sertoli cells via a receptor-mediated process. The results reported here demonstrate that the interaction of FSH with its receptor is associated with changes in Na+/Ca++ exchange as well, and suggest that this activity may also be involved in regulating intracellular free Ca++ levels in the Sertoli cell.

Differential roles of high and low affinity guanosine 5'-triphosphate binding sites in the regulation of follicle-stimulating hormone binding to receptor and signal transduction in bovine calf testis membranes.

We have previously shown that FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein (Gs) in membranes of calf testis. Using N-ethylmaleimide (NEM), forskolin, and cholera toxin as probes, we have investigated the role of low and high affinity GTP-binding sites of stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs) in the activation of adenylate cyclase. When calf testis membranes were exposed to NEM (1 mM), FSH binding to receptors was slightly (30%) decreased, but the receptors showed continued sensitivity to GTP, resulting in a further decrease in [125I]human FSH binding to receptors. Pretreatment of membranes with NEM (up to 20 microM) produced no effect on GTP-binding. A dose-dependent decrease in high affinity GTP-binding sites, however, was observed at higher (greater than 50 microM) NEM. Adenylate cyclase activity was reduced in response to GTP gamma S or NaF concomitant to a decrease in high affinity GTP-binding sites in membranes treated with 50-100 microM NEM, or completely abolished in membranes exposed to 300 microM NEM. Stimulation by forskolin indicated that the significant inhibition of adenylate cyclase activity occurring in membranes exposed to low NEM (50-100 microM) was not due to inactivation of catalytic unit of adenylate cyclase by NEM. Pretreatment of membranes with 100 micrograms/ml cholera toxin and NAD slightly (18%) reduced specific FSH binding but did not affect Gpp(NH)p-binding. However, adenylate cyclase stimulation by GTP plus FSH in these membranes was significantly enhanced. When membranes were treated with higher concentration of cholera toxin (250 micrograms/ml), the adenylate cyclase stimulation by GTP plus FSH was abolished due to uncoupling of FSH receptors from Gs and a significant decrease in high affinity GTP-binding sites. Our results suggest that high affinity GTP-binding sites of Gs coupled to FSH receptors are essential for FSH and guanine nucleotide activation of adenylate cyclase. The low affinity binding sites bind GTP and thereby regulate FSH binding but are not involved in the activation of adenylate cyclase.

A synthetic peptide corresponding to amino acid residues 34 to 37 of human follicle-stimulating hormone beta-subunit accelerates the onset of puberty in male and female mice.

We have previously reported that a synthetic peptide amide corresponding to residues 34-37 (TRDL, threonine, arginine-aspartic acid-leucine) of the beta-subunit of human FSH induced prolonged vaginal estrus in normally cycling female mice (see Ref. 15). These results represented the first demonstration of an in vivo effect of a gonadotropin-related synthetic peptide on reproductive processes. We have extended these studies to examine possible effects of TRDL on the onset of puberty in female mice. In two replicated experiments, vehicle-injected control mice attained first vaginal estrus by day 39. An ip injection of 200 ug TRDL/g BW to 28-day-old prepubertal female mice, however, accelerated the onset of first vaginal estrus by 7 days in 11 of 12 (11/12) (Exp 1) and 7/9 (Exp 2) mice. Serum estradiol levels were significantly (P = 0.017) elevated in TRDL-treated mice, whereas progesterone was unchanged. Uteri of TRDL-treated mice were significantly (P = 0.003) heavier than uteri of vehicle-injected control animals of the same age and body weight. Intraluminal fluid accumulation (ballooning) at proestrus was absent in 20/21 TRDL-treated females, as were oviductal ova and ovarian corpora lutea. These phenomena are characteristic of the first estrous cycles of female mice isolated from males. To obtain further evidence for in vivo effects of TRDL, we assessed the ability of TRDL to accelerate the onset of puberty in male mice. When given as five consecutive daily ip injections of 200 ug/g BW to 28-day-old prepubertal male mice, TRDL significantly increased testis weight, when compared with vehicle-injected control mice of the same age and BW (171.3 +/- 3.8 mg vs. 151.6 +/- 4.3 mg, P = 0.001) and induced a 6.5-fold increase in serum testosterone levels. These studies confirm the previously reported in vivo activity of a synthetic peptide corresponding to human FSH-beta subunit 34-37 (TRDL) in adult female mice and extend its effects to the acceleration of the onset of puberty in immature male and female mice.