A synthetic peptide corresponding to amino acid residues 90 to 95 of human follicle-stimulating hormone beta-subunit delays the onset of puberty in female Swiss Webster mice.

We have recently reported that a synthetic peptide corresponding to amino acid residues 81-95, a receptor-binding region of the human FSH-beta-subunit, and a subdomain within this region, hFSH-beta-(90-95) (DSTDCT), prolonged vaginal estrus when administered intraperitoneally (ip) to normally cycling Swiss Webster mice. These results were similar to those we reported for a synthetic peptide corresponding to hFSH-beta-(34-37) [TRDL, a subdomain within receptor-binding region hFSH-beta-(33-53)] in the same model system. TRDL also accelerated the onset of puberty in immature mice. We now report the effects of hFSH-beta-(90-95) in prepubertal female mice. In two separate experiments, a single ip injection of 200 microg/g body weight (BW) hFSH-beta-(90-95) in phosphate buffered saline (PBS, vehicle) administered to mice on day 28 delayed first vaginal estrus by 3 days in 50% (4/8) and 62.5% (6/8) when compared to mice given vehicle alone on day 28. Vaginal opening was also delayed in mice receiving hFSH-beta-(90-95) when compared to mice injected with vehicle alone. Serum estradiol levels of vehicle-injected control mice in first vaginal estrus were three-fold higher than in mice treated with hFSH-beta-(90-95), whose vaginal smears showed no evidence of first estrus. No significant differences in ovarian or uterine weights, or serum progesterone levels, were observed between vehicle-injected control mice achieving first vaginal estrus and mice receiving hFSH-beta-(90-95) in whom first estrus was delayed. The contrasting effects on the onset of puberty of hFSH-beta-(90-95) (delay) and hFSH-beta-(34-37) (acceleration) may reflect synthetic peptide binding to different domains of the FSH receptor, resulting in variable post-binding effects. These results are consistent with our earlier study suggesting that FSH-beta-subunit-related synthetic peptides can induce significant in vivo effects on the onset of puberty in female mice.

Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone beta-subunit receptor-binding domain 81-95 [hFSH-beta-(81-95)] modulate the in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.

In vivo effects of human follicle-stimulating hormone-related synthetic peptide hFSH-beta-(81-95) and its subdomain hFSH-beta-(90-95) on the mouse estrous cycle.

We have previously reported that a synthetic peptide corresponding to amino acid residues 81-95 of the human (h) FSH-beta subunit inhibited binding of [125I]hFSH to bovine calf testis membranes and stimulated estradiol biosynthesis in primary cultures of rat Sertoli cells. We have now obtained several lines of evidence demonstrating in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle. 1) A single i.p. injection of 200 micrograms/g BW hFSH-beta-(81-95) significantly (p < 0.001) prolonged vaginal estrus in comparison to that in vehicle-injected control mice. 2) Vaginal smears taken at estrus from mice given hFSH-beta-(81-95) were characterized by the complete absence of epithelial casts, a hallmark of spontaneous ovulation in mice. 3) Mice receiving hFSH-beta-(81-95) had significantly (p < 0.001) lower serum estradiol at proestrus and serum progesterone at diestrus than vehicle-injected control mice. 4) The proestrous effects of estrogen on uterine ballooning and weight gain, clearly evident in vehicle-injected control mice, were not observed in mice treated with hFSH-beta-(81-95). A synthetic peptide corresponding to the carboxy-terminal region of hFSH-beta-(81-95), hFSH-beta-(90-95), inhibited binding of [125I]hFSH to bovine calf testis membranes, antagonized FSH-stimulated estradiol biosynthesis by primary cultures of rat Sertoli cells, and prolonged vaginal estrus in normally cycling mice. A synthetic peptide corresponding to the amino-terminal domain, hFSH-beta-(81-86), was inactive in vitro and had no effect on the mouse estrous cycle. The results of the present study provide additional evidence for in vivo effects of FSH-related synthetic peptides.

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.

Cultured human granulosa cells secrete a follicle stimulating hormone receptor-binding inhibitor.

We previously reported that human follicular fluid contained a protein that inhibits binding of 125I-human FSH to its membrane receptor (FSH-BI) and demonstrates FSH-like agonist activity in vitro. The cellular origin of FSH-BI was unknown, although ovarian granulosa cells seemed a likely source. To address this question, human granulosa cells were collected from patients during routine in-vitro fertilization (IVF) or gamete intra-Fallopian transfer (GIFT) procedures. Cells from 98 patients were cultured and then examined for their ability to secrete FSH receptor-binding inhibitory activity into the culture medium. The function of the cultured cells was confirmed by their ability to respond to added FSH with conversion of exogenous androstenedione to oestradiol. Radioreceptor assays were performed individually on cell culture medium obtained from granulosa cell cultures from these 98 patients. Cultured granulosa cells, under basal conditions (in the absence of FSH stimulation), secreted significant FSH-BI activity into the culture medium. In order to accumulate enough material for further study, this culture medium was pooled and lyophilized. The lyophilized medium retained FSH-BI activity, and also demonstrated FSH agonist activity by stimulating oestradiol synthesis in cultured rat Sertoli cells. A fraction showing a single component after purification by polyacrylamide gel electrophoresis had an estimated molecular weight of 25000, and inhibited 125I-human FSH binding to receptor by 50% at 2.5 microg/ml. The results indicate that human granulosa cells secrete a protein with FSH-like activity having potential significance as a local regulator of FSH action in the ovary.

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.

D-amino acid substitution of residues 32 to 46 of the glycoprotein hormone common alpha-subunit: development of a synthetic glycoprotein hormone antagonist.

We have used single- or double-point D-amino acid substitutions to study the structure-function relationships involving residues 32 to 46 of the glycoprotein hormone common alpha-subunit (GPHa) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. D-Amino acid substitution analogs of GPHa(32-46) were synthesized and tested in both FSH and hCG radioligand receptor assays using bovine calf testis membranes as receptor source. Correct orientation of the amino acid side chains was generally of paramount importance for peptide interaction with receptor and bioactivity. Most substitutions with corresponding D-amino acids did not enhance the potency of native GPHa(32-46). A significant increment in peptide potency, however, was observed by inversion of configuration at positions Ser-34 and Thr-39 with D-amino acid isoforms. Based on the relative potency of each peptide analog. [D-Ser-34, D-Thr-39]GPHa(32-46) was approximately twofold more potent than native peptide GPHa(32-46) in both FSH and hCG radioligand receptor assays. [D-Ser-34, D-Thr-39]GPHa(32-46) also markedly inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. The present study is unique in that it represents the first report of utilizing D-amino acid substitution to develop more potent peptide analogs related to the glycoprotein hormone common alpha-subunit region 32-46. Our results offer hope for the development of more potent and stabile peptide antagonists of possible usefulness in fertility regulation and control.

Synthetic peptides corresponding to residues 551 to 555 and 650 to 653 of the rat testicular follicle-stimulating hormone (FSH) receptor are sufficient for post-receptor modulation of Sertoli cell responsiveness to FSH stimulation.

We have recently demonstrated that synthetic peptides corresponding to the third cytoplasmic (3i) loop (residues 533 to 555) and a region in the carboxy-terminal cytoplasmic tail (residues 645 to 653) of the rat testicular follicle-stimulating hormone receptor (FSHR) affected signal transduction in rat testis membranes and cultured rat Sertoli cells. In order to define more precisely the peptide domains involved, we synthesized truncated peptide amides corresponding to FSHR residues 551-555 (KIAKR) and 650-653 (RKSH), respectively. These two regions were chosen since they contained a minimal structural motif present in G protein activator regions of several other G protein-coupled receptors (i.e., B-X-X-B-B or B-B-X-B, B representing a basic amino acid). Neither peptide inhibited binding of FSH to testis membrane receptors. Each peptide significantly reduced FSH-stimulated estradiol biosynthesis by intact cultured rat Sertoli cells. The same results were obtained with streptolysin O-permeabilized Sertoli cells. No effect was noted on forskolin-induced steroidogenesis, indicating that the peptide effects were not due to interaction with adenylyl cyclase. Each peptide amide, however, induced concentration-dependent increases in guanine nucleotide exchange in rat testis membranes. Our results indicate that interaction of FSH receptor with its associated G protein may involve relatively restricted peptide sequences, and include residues 551-555 (KIAKR) in the third cytoplasmic loop, and residues 650-653 (RKSH) in the carboxy-terminal cytoplasmic tail of the FSH receptor.

A synthetic peptide corresponding to glycoprotein hormone alpha subunit residues 32-46 inhibits gonadotropin binding to receptor.

A synthetic peptide strategy was used to study structure-function relationships between residues 32 to 46 of the glycoprotein hormone alpha subunit (GPH alpha) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. A peptide amide corresponding to this region [GPH-alpha-(32-46)] inhibited both 125I-hFSH and 125I-hCG binding to their respective calf testis membrane receptors. The concentration at which GPH-alpha-(32-46) peptide amide inhibited FSH binding by 50% (IC50) was 36 microM, and for hCG it was 54 microM. GPH-alpha-(32-46) peptide amide also inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. In order to determine the involvement of individual residues within this region of the glycoprotein hormone alpha subunit in receptor binding inhibitory activity, truncated and alanine-substituted peptide analogs were synthesized and tested in both FSH and hCG radioligand receptor competition assays. Based on the relative potency of each peptide, we conclude that Phe-33, Arg-35, Arg-42, Ser-43 and Lys-44 may be important, and Cys-32 is required, for inhibition of FSH and hCG binding to their respective receptor. Our results demonstrate involvement of the glycoprotein hormone alpha-subunit in receptor binding, identify residues 32 to 46 as a receptor binding domain, and define the relative importance of specific residues within this region of the alpha subunit for hormone-receptor interaction.

Selective effects of charge on G protein activation by FSH-receptor residues 551-555 and 650-653.

Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

Identification of amino acid residues 300-315 of the rat FSH receptor as a hormone binding domain: evidence for its interaction with specific regions of FSH beta-subunit.

We previously reported that residues 9-30 of the extracellular N-terminus domain of the rat FSH receptor, which has no homologous sequence in receptors for related pituitary glycoprotein hormones, represented a specific FSH binding domain. Further examination of its deduced primary structure identified another region, residues 300-315, which was also unique to the FSH receptor. To determine whether this region of the FSH receptor was involved in hormone binding, a synthetic peptide corresponding to residues 300-315 was studied with respect to its ability to bind FSH, as well as a series of nine overlapping synthetic peptides corresponding to the entire primary structure of the hormone specific FSH beta-subunit. 125I-FSH rec-(300-315) peptide bound to immobilized human, ovine and bovine FSH, but not to prolactin or ovalbumin. Of the nine synthetic peptides studied, binding was restricted to FSH beta residues 21-35, and to a much lesser extent (20%) to residues 11-25. All binding was abolished in the presence of excess solubilized FSH receptor. Earlier studies indicated that although FSH binds to FSH rec(9-30) peptide, residues 11-25 or 21-35 of the FSH beta-subunit were not involved. Our results suggest the FSH receptor N-terminus, extracellular residues 300-315, may define a FSH binding site, and that binding of FSH beta-subunit may occur via interactions with FSH beta 21-35 and 11-25.

A decapeptide corresponding to the partial amino acid sequence of a high molecular weight human FSH receptor-binding inhibitor is a specific inhibitor of FSH binding.

We previously reported purification of a protein (approximately equal to 57 kDa) from human follicular fluid having FSH binding inhibitory (FSH-BI) activity. Purified hFSH-BI was cleaved with cyanogen bromide and trypsin. The resulting peptide fragments were separated by HPLC and sequence information for individual fragments was obtained. A ten amino acid sequence of hFSH-BI derived from this procedure was identified, and a corresponding peptide amide (BI-10) was synthesized and utilized for further study. A protein database search revealed no significant identity between this decapeptide and other known proteins. We examined the ability of BI-10 to inhibit binding of 125I-hFSH to FSH-receptor enriched bovine testes membranes utilizing a radioligand receptor assay (RRA). BI-10 inhibited binding of 125I-hFSH to its receptor in a concentration-related manner, with an ED50 of 300 microM. BI-10 had no effect on 125I-hCG binding to receptor even at concentrations up to 1000 microM, suggesting that the effect of BI-10 was specific for the interaction between FSH and its receptor. To assess bioactivity of BI-10, we investigated its effect on FSH-stimulated conversion of androstenedione to estradiol by rat Sertoli cells in primary culture in vitro. Inhibition of FSH-stimulated estradiol synthesis (FSH antagonist activity) was significant at a BI-10 concentration of 1000 microM. BI-10 also significantly inhibited FSH-stimulated cAMP accumulation in primary cultures of Sertoli cells when examined at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells.

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533-555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533-555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533-555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533-555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533-555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic peptide corresponding to residues 645-653 in the carboxyl terminal cytoplasmic domain of the rat testicular follicle stimulating hormone receptor modulates G protein coupled-receptor signaling in rat testis membranes and in intact cultured rat Sertoli cells.

We examined the involvement of the carboxyl terminal cytoplasmic domain (residues 645-653) of the rat testicular FSH receptor in FSH signal transduction utilizing light membranes prepared from immature rat testes, intact cultured rat Sertoli cells, and a synthetic peptide approach. This region of the FSH receptor was selected because of its structural similarity to receptor-G protein contact sites identified in other G protein-coupled receptors. FSHR- (645-653) peptide amide promoted guanine nucleotide exchange in rat testis membranes, presumably via its interaction with membrane-associated G protein, but did not inhibit FSH binding to testis membrane receptors. When incubated with intact cultured Sertoli cells from immature rat testes, FSHR- (645-653) peptide amide consistently and significantly stimulated basal cAMP and estradiol biosynthesis. The peptide had no effect on forskolin stimulation of cAMP and estradiol, but inhibited FSH stimulation of each. FSH binding to receptor was unaffected by the peptide, these results suggest peptide interaction with receptor-associated G protein. The effects of FSHR- (645-653) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. These results suggest that peptide effects in intact Sertoli cells were related to peptide entry into the cell, presumably during receptor-mediated endocytosis of FSH, or by diffusion. Synthetic peptide amides not satisfying structural criteria for G protein coupling had no effect on either guanine nucleotide exchange or estradiol biosynthesis, even at concentrations significantly higher than used for FSHR- (645-653) peptide amide.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification in human ovarian follicular fluid of proteins that share an epitope region unique to the extracellular domain of the follicle-stimulating hormone receptor.

Recently we identified a unique region, residues 9-30 in the extracellular domain of the FSH receptor, capable of binding FSH but not LH or TSH. We have shown that polyclonal antibodies raised against this region specifically recognized intact FSH receptors present on plasma membranes of cultured rat Sertoli cells. In the present study, plasma membranes from human granulosa-lutein cells were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by Western blot analysis. Antireceptor peptide antibody, but not preimmune serum control, recognized intact human FSH receptors, suggesting that human and rat FSH receptors share this unique N-terminus epitope region. Recent cloning studies have identified, in addition to full length receptor, the presence of FSH receptor-spliced messenger RNA variants, which encode receptor proteins with variable lengths of hydrophilic extracellular domains, but lacking transmembrane domains. Such proteins could theoretically represent secreted forms of the receptor. In this study, we used a polyclonal anti-FSH receptor (residues 9-30) peptide antibody to investigate whether FSH receptor-related soluble proteins might also be present in human ovarian follicular fluid (FF). In an enzyme-linked immunosorbent assay specific for the FSH receptor, antireceptor peptide antibody, but not preimmune serum serving as control, identified significant immunoreactivity in several human ovarian FF samples, suggesting that protein(s) present in FF share a common epitope with the extracellular domain of the FSH receptor. The apparent levels of FSH receptor-related activity in FF samples, expressed relative to the FSH receptor (residues 9-30) peptide, ranged from 31-55 ng/mL. Passing of the samples through 0.22-micron filters or subjecting the samples to high speed centrifugation did not alter the activity profiles of the samples ruling out effects due to contamination with plasma membranes from granulosa cells. When human FF samples were subjected to gel permeation chromatography, at least four distinct protein peaks were resolved, in the molecular mass range between 70-460 kilodaltons, each of which was recognized by the FSH receptor 9-30 peptide antibodies. Our results provide initial evidence for the presence in human ovarian FF of proteins sharing epitope with the extracellular domain of the FSH receptor and presumably derived from the granulosa cell. Since we have previously shown that the epitope region, represented by residues 9-30 in the extracellular domain of the FSH receptor specifically binds FSH, the proteins in human FF sharing this epitope may have functional significance.

Evidence that a calmodulin-like calcium-binding domain of the FSH beta-subunit is involved in FSH-induced calcium uptake by Sertoli cells.

We have previously shown that a synthetic peptide amide corresponding to residues 1-15 of the human FSH beta-subunit (hFSH-beta-(1-15)) possesses structural characteristics and calcium-binding properties similar to the calcium-binding loops of calmodulin (CaM). The calcium-binding property of hFSH-beta-(1-15) correlated well with its ability to stimulate uptake of calcium (as 45Ca2+) by cultured rat Sertoli cells and proteoliposomes enriched with bovine calf testis FSH receptors. A sequence found in the calcium-binding loops of CaM and a number of other calcium-binding proteins can be represented by the motif +-+-+-+-+--+, where + represents a calcium-binding residue and - represents a non-binding residue. A sequence containing a similar motif appears in hFSH-beta-(1-15) between residues 4 and 15: +-++-+---+-+. Using a synthetic peptide strategy, we undertook to determine whether the first three residues of hFSH-beta-(1-15) were required to induce uptake of calcium by cultured rat Sertoli cells and FSH receptor-enriched proteoliposomes, and to assess whether rearrangement of the putative calcium-binding ligands (+) of hFSH-beta-(1-15) to correspond to their linear sequence in CaM would enhance the ability of hFSH-beta-(1-15) to induce calcium uptake in these two model systems.(ABSTRACT TRUNCATED AT 250 WORDS)

A tetrapeptide within a receptor-binding region of human follicle-stimulating hormone beta-subunit, hFSH-beta-(34-37), regulates sodium-calcium exchange in Sertoli cells.

In a previous study, we showed that binding of FSH by cultured rat Sertoli cells significantly inhibited basal levels of Na+/Ca2+ exchange. Similar inhibition was observed when proteoliposomes enriched with bovine calf testis follicle-stimulating hormone (FSH) receptors were stimulated with FSH. In the present study, we screened a series of overlapping synthetic peptide amides, representing the entire primary structure of the beta-subunit of hFSH, for their effects on sodium-dependent calcium uptake (as 45Ca2+) by monolayer cultures of Sertoli cells from immature rats. hFSH-beta-(33-53), previously identified as a receptor binding region of hFSH-beta-subunit, significantly (P < 0.05) inhibited Na+/Ca2+ exchange. A tetrapeptide [TRDL, hFSH-beta-(34-37)] contained within this sequence, was observed to be equally as active as hFSH-beta-(33-53) at 200 microM, suggesting that the regulatory effect of hFSH-beta-(33-53) on sodium-dependent 45Ca2+ influx was due to residues 34-37. hFSH-beta-(81-95) also inhibited Na(+)-dependent calcium influx, although to a lesser extent than hFSH-beta-(33-53) or hFSH-beta-(34-37). Sodium-dependent 45Ca2+ entry into Sertoli cells was enhanced in a concentration-related manner when extracellular sodium was replaced by equimolar concentrations (up to 135 mM) of choline chloride. hFSH-beta-(34-37) significantly reduced basal uptake of 45Ca2+ in choline-containing buffer, but was without effect in buffer containing 135 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Purification of a high molecular weight follicle-stimulating hormone receptor-binding inhibitor from human follicular fluid.

In a previous study we reported the presence in human follicular fluid (hFF) of a FSH receptor-binding inhibitor (hFSH-BI) with FSH agonist activity, which was immunologically similar to FSH but could be distinguished from FSH on the basis of its greater stability in acid. We have now purified hFF-derived hFSH-BI after molecular sieving on Sephracyl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulose followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of dialyzed hFF, based on its ability to inhibit the binding of [125I]hFSH to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cells. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two bands of almost identical mobility, with an estimated mol wt of 57,000, compared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western blot analysis of the SDS-PAGE fraction. The Western blot confirmed the detection of two bands with very similar mobilities and estimated mol wt of 57,000, which were clearly distinguishable from that of immunologically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable amino acid compositions. The amino acid composition of hFSH-BI was clearly distinct from those of hFSH, hLH, hCG, and the alpha-subunit of human inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.