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PubMed2002

Differentiation of chicken gonad as an endocrine organ: expression of LH receptor, FSH receptor, cytochrome P450c17 and aromatase genes.

The gonad is an endocrine organ secreting sex hormones and also a target of pituitary gonadotrophins. The expression of mRNAs encoding LH receptor (LHR), FSH receptor (FSHR), P450c17 and P450aromatase in the developing gonads of embryos between day 4 and day 6 of incubation was determined using a RT-PCR to elucidate the chicken gonad as a target organ of gonadotrophins. Although expression of mRNAs encoding LHR, FSHR and P450c17 was detected at day 4 of incubation in both sexes, mRNA encoding P450aromatase appeared at day 6 in female embryos only, indicating that mRNAs encoding gonadotrophin receptors can be identified before sexual differentiation. Quantitative PCR analysis revealed that expression of mRNA encoding LHR and FSHR remained low in male gonads from day 4 to day 6 of incubation, whereas they increased on day 6 in female gonads. The sexual dimorphism in the expression of mRNAs encoding LHR and FSHR was confirmed in the sexually differentiated gonads of embryos at day 12 of incubation (LHR in ovary ratio LHR in testis = 7 ratio 1; FSHR in ovary ratio FSHR in testis = 9 ratio 1).

Animals↗
PubMed1993

Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells.

Activin, a dimer of beta-subunits of inhibin, has been found to induce FSH receptor on cultured rat granulosa cells. The effect of activin on FSH receptor messenger RNA (mRNA) levels has not been elucidated. To study the effect of activin on FSH receptor mRNA levels, we used a specific complementary RNA probe to evaluate changes in FSH receptor transcripts in cultured rat granulosa cells. Granulosa cells obtained from immature diethylstilbestrol-treated rats contained two predominant FSH receptor mRNA transcripts (5.5 and 2.4 kilobases). Compared to the control, the treatment of granulosa cells with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum of about a 4-fold increase at 24 h. FSH receptor mRNA markedly decreased after 48 h and maintained a level comparable to that found in the control. The FSH receptor expression was also increased by activin. Scatchard analysis of the binding of rat FSH to granulosa cells showed that the increase in FSH binding after activin treatment was due to an increase in the receptor number and not the affinity of binding. Treatment of granulosa cells for 24 h with activin (20-300 ng/ml) increased FSH receptor mRNA in a dose-dependent manner to a maximum of about a 4-fold increase at a concentration of 100-300 ng/ml. We analyzed rat type II activin receptor mRNA transcripts in cultured rat granulosa cells with a specific complementary RNA probe to study the action of activin on granulosa cells. Granulosa cells contained two predominant rat type II activin receptor mRNA transcripts (6.0 and 3.0 kilobases). Furthermore, we measured intracellular cAMP production by activin to examine the mechanism by which activin acts on granulosa cells. In result, activin alone did not increase intracellular cAMP accumulation. In conclusion, this study demonstrates that the effect of activin A on the induction of FSH receptor expression is associated with a change in FSH receptor mRNA levels, suggesting that modulation of follicle development occurs.

Activins↗
PubMed1992

A synthetic peptide corresponding to amino acids 9-30 of the extracellular domain of the follitropin (FSH) receptor specifically binds FSH.

As deduced on the basis of cloning experiments, the putative extracellular domain of pituitary glycoprotein hormone (lutropin (LH), thyrotropin (TSH), and FSH) receptors (rec) is sufficiently large to suggest involvement in hormone binding. Comparison of the amino acid sequences of the extracellular domains of the glycoprotein hormone receptors indicates that the FSH receptor has a peptide sequence in the external domain close to the amino terminus (residues 9-30) which has no sequence homology to receptors for LH or TSH. To examine whether this region is involved in FSH-receptor interaction, we studied the hormone-binding properties of a corresponding synthetic peptide in several systems. (1) Binding of 125I-hFSH to receptor-containing bovine testis membranes was inhibited by preincubation with FSH rec-(9-30) peptide amide in a concentration-dependent manner. (2) 125I-labeled rec-(9-30) peptide amide bound to ovine, bovine, or human FSH preparations, and the binding was inhibited by solubilized bovine FSH receptor. 125I-labeled rec-(9-30) peptide amide, however, did not bind to LH or TSH. (3) 125I-hFSH bound to unlabeled rec-(9-30) peptide amide, and the binding was inhibited by excess unlabeled FSH, but not by LH or TSH. (4) Scatchard analysis indicated that the FSH rec-(9-30) peptide amide contained a single class of FSH binding sites with a Ka = 1.1 x 10(6) M-1. (5) The binding of 125I-labeled rec-(9-30) peptide amide to hFSH, bFSH or oFSH was effectively inhibited by rabbit polyclonal antibodies raised against rec-(9-30) peptide amide but not by preimmune rabbit serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

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.

Adenylyl Cyclases↗
PubMed1993

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)

Amino Acid Sequence↗
PubMed1995

Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function.

The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated form of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated receptor is glycosylated, through the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that of the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Therefore, further experiments, done in the context of the full-length receptor, were performed to determine the actual sites of glycosylation in the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collectively disrupt the potential sites for N-linked glycosylation. Western blot analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 276 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydrate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH binding activity is not observed with nonglycosylated mutant receptors where both sites have been collectively disrupted. Similarly, when cells expressing the wild type FSHR were treated with tunicamycin to prevent N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed from wild type receptors. Our results demonstrate that while N-linked carbohydrates on the FSH receptor are not required directly for the binding of hormone, a carbohydrate at either Asn174 or Asn276 is required for the efficient folding of the nascent receptor protein into a conformation that allows high affinity binding of hormone.

Amidohydrolases↗
PubMed1994

Follicle-stimulating hormone (FSH) receptors and FSH-responsive adenosine 3',5'-cyclic monophosphate production in porcine granulosa cells decline with follicular growth.

Comparison of binding of 125I-porcine FSH to porcine granulosa cells from small and large ovarian follicles indicated that binding is dependent on both time and temperature. At 37 C, binding of ligand is more than four times higher in cells from small follicles than in cells from large follicles within 30 minutes of the start of incubation. Binding to cells from both small and large follicles is more stable for a longer period of time at 20 C than at 37 C. Equilibrium saturation binding analysis of 125I-pFSH binding indicated that binding is saturable and of high affinity. Granulosa cells from small and large follicles have similar affinities for 125I-pFSH. However, granulosa cells from small follicles have 4.7-times as many FSH receptors as granulosa cells from large follicles. The sensitivity and responsiveness of the adenylyl cyclase system to FSH were assessed by incubating cells from small and large follicles with increasing concentrations of FSH in a defined medium containing isobutylmethylxanthine (1 mM), an inhibitor of phosphodiesterase. Granulosa cells from small follicles are more sensitive to FSH and have a greater cAMP response to FSH than granulosa cells from large follicles. Thus, both active FSH receptors and FSH-responsive adenylyl cyclase activity decline during follicular growth.

1-Methyl-3-isobutylxanthine↗
PubMed2005

Association of human follitropin (FSH) receptor with splicing variant of human lutropin/choriogonadotropin receptor negatively controls the expression of human FSH receptor.

A splice variant of human lutropin (LH)/choriogonadotropin (CG)-receptor [hLHR(exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a normal menstrual cycle. Supported by a detergent-soluble binding assay and a receptor biotinylation experiment, the receptor binding assay shows hLHR(exon 9) is neither expressed at the cell surface nor has the capability of binding to hCG. In addition, hLHR(exon 9) was confirmed in the endoplasmic reticulum (ER) by endoglycosidase H treatment. A coimmunoprecipitation experiment clearly showed that hLHR(exon 9) and constitutively inactivate mutant-LHRs, which stay in the ER, form an association with the human follitropin (FSH)-receptor (hFSHR). This suggests that in the presence of mutant-LHR, hFSHR, which is trapped in the ER and associated with hLHR(exon 9), is unable to come up to the plasma membrane. This phenomenon is specific among gonadotropin receptors because human TSH receptor failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the hFSHR receptor protein level within the cells, which impaired cAMP production. To elucidate the mechanism underlying the decrease in hFSHR protein by this receptor complex, we performed a Percoll fractionation experiment, which indicated that the receptor complex drove hFSHR to the lysosome instead of the plasma membrane. These results reveal a novel mechanism of FSHR expression regulation.

Alternative Splicing↗
PubMed2005

Fish FSH receptors bind LH: how to make the human FSH receptor to be more fishy?

In mammals, the interactions between glycoprotein hormones and their cognate receptors are highly specific; unintended cross-reactivity under normal physiological conditions has not been observed. The interactions between fish gonadotropins and their receptors, on the other hand, appeared to be less discriminatory. For example, the catfish follicle-stimulating hormone (FSH) receptor was highly responsive to both catfish luteinizing hormone (LH) and catfish FSH. Similarly, the FSH receptor of coho salmon bound both salmon FSH and LH. In contrast, LH receptors of both species were found to be rather specific for their cognate LH. This paper intends to summarize the current situation with special emphasis to our comparative structure-function studies that aim at elucidating the molecular basis of ligand selectivity (in mammals) and ligand promiscuity (in fish).

Animals↗
PubMed1999

Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms.

In a search for pathophysiological causes of idiopathic male infertility we investigated the occurrence of mutations of the FSH receptor in 48 men with this disorder. The entire FSH receptor gene was analyzed by single stranded conformation polymorphism analysis (SSCP). A heterozygous point mutation without functional consequences, exchanging Val to Ala in codon 341, was found in one patient. SSCP analysis led to the identification of 2 polymorphisms in exon 10 associated in 2 discrete FSH receptor allelic variants, i.e. Thr307-Asn680 and Ala307-Ser680. The frequency and distribution of the two allelic variants was further analyzed in 86 proven fathers and 75 infertile men by SSCP (codon 307) and restriction fragment length polymorphism (codon 680). The 2 receptor isoforms showed similar Mendelian distribution in proven fathers and in infertile men. Serum FSH, inhibin B, and combined testicular volume did not differ between subjects with different receptor isoforms. Binding studies in transiently transfected COS-7 cells showed similar binding affinity for the two receptor variants. Moreover, the Ala307-Ser680 and the Thr307-Asn680 FSH receptors responded in vitro to FSH with comparable cAMP production. These data suggest that different isoforms of the FSH receptor with similar functional properties exist in normal and infertile men. We conclude that mutations of the FSH receptor or the FSH receptor genotype do not play a pathogenic role in male idiopathic infertility. The possibility that different FSH isoforms might interact differently with the 2 receptor variants remains to be investigated.

Animals↗
PubMed2001

Functional importance of bovine myometrial and vascular LH receptors and cervical FSH receptors.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Adenylyl Cyclases↗
PubMed1993

Developmental changes of testicular activin and FSH receptor mRNA and plasma FSH and inhibin levels in the rat.

To investigate the changes in the FSH receptor and the activin receptor during sexual maturation in rat testes, we examined the mRNA levels of the receptors by Northern blot analysis. With a full length rat activin receptor cRNA probe in this study, Northern blot analysis revealed two activin receptor mRNAs (6Kb and 3Kb) in testes. The large messenger (6Kb) was low on day 7, and gradually increased by day 35. On the other hand, the small messenger (3Kb) was low on day 7, began to increase on day 21, and had increased dramatically by day 35. Therefore, compared with our previous data, developmental changes in the mRNA of the activin receptor of male and female gonads showed certain differences. The expression of 3Kb activin receptor mRNA in rat testis may be relevant to the stimulated spermatogenesis in this period. 2.4Kb and 5.5Kb FSH receptor mRNA were revealed on day 7 and decreased to 80% on day 14 and remained at the same level. The Scatchard plots of FSH binding data showed that the binding affinities of testicular FSH were constant at each stage of development. Changes in the mRNA level in FSH receptor were followed by changes in the concentration of FSH receptor in testis. The rise in the plasma FSH level was concomitant with the decrease in the plasma inhibin level by day 14.

Activin Receptors↗
PubMed2001

The expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNA in the porcine ovary.

The aim of this study was to visualize the expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNAs in various structures of porcine ovary. Porcine ovaries were frozen in liquid nitrogen, and 8 microm sections were prepared for in situ hybridization. In the small, medium and large antral follicles as well as in early, midluteal and regressing corpora lutea, mRNAs for androgen receptor, P450 aromatase and FSH receptor were detected. In small antral follicles high levels of mRNAs for androgen and FSH receptors were observed, mainly in the granulosa layer, while mRNA expression for P450 aromatase was negligible. As follicles grew, amount of mRNAs for androgen receptor and FSH receptor decreased, and that for P450 aromatase increased. Small amounts of androgen receptor mRNA were also present in corpora lutea at all examined stages. P450 aromatase mRNA was not detected in early and midluteal corpora lutea. However, regressing corpus luteum showed a weak expression of aromatase mRNA.

Animals↗
PubMed2002

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice.

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.

Amino Acid Substitution↗
PubMed1994

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

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

Animals↗
PubMed2003

Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter.

AIM: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. METHODS: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5'-flanking region fragment of its promoter. RESULTS: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. CONCLUSION: A 1.5kb 5'-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

Animals↗
PubMed2001

Follicle-stimulating hormone (FSH) induced internalization of porcine FSH receptor in cultured porcine granulosa cells and Chinese hamster ovary cells transfected with recombinant porcine FSH receptor cDNA.

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

Animals↗
PubMed1997

Ontogeny of FSH receptor messenger ribonucleic acid transcripts in relation to FSH secretion and testicular function in sheep.

The role of alternative splicing of the FSH receptor gene in the generation of FSH receptor proteins and testicular function remains an enigma. To address this issue, this investigation was conducted to determine variations in the expression of alternate FSH receptor mRNA transcripts in relation to changes in FSH release, hormone binding activity and testicular function during pubertal development of ram lambs from two genotypes of sheep (Romanov and a cross between Booroola x DLS) with different sexual precocity. Serum 17 beta-estradiol and testosterone concentrations were used as indices of Sertoli cell and testicular function. The results indicated that increases in Sertoli cell and testicular function normally seen during pubertal development are accompanied by age-dependent reductions in concentration of functional FSH receptors, as determined by binding of iodinated FSH to testicular membrane preparations. During the course of these changes, FSH release was either maintained at a steady level in Romanov lambs or it was gradually reduced in the Booroola x DLS cross, thus indicating that the testis had become more responsive to hormonal signal. This acquisition of heightened sensitivity was also associated with contrasting changes in the level of expression of FSH receptor mRNA transcripts. For both geno-types of sheep, 5 distinct species of mRNA transcripts of approximately 1.1, 1.5, 2.0, 2.5 and 6.5 kb were highly expressed from 11 to 22 weeks of age. Amongst these transcripts, the 1.1 kb molecular species was the most abundant. Specific probing for a previously cloned transcript called 151A1 representing the first 4 exons of the FSH receptor gene revealed a paradoxical increase in the level of expression from 11 weeks up to a maximum at 18-22 weeks of age for both genotypes. Collectively, the results indicated that contrasting changes in the production of specific alternatively spliced mRNA transcripts may mediate changes in FSH receptor expression which apparently accounts for the augmentation in sensitivity and function of the testis during pubertal development. Furthermore, the data provide the first important indication that the novel truncated transcript (151A1), which predictably encodes a soluble protein of either intra- or extracellular fate, could be physiologically relevant.

Animals↗