Follicle-stimulating Hormone Signaling Research Articles

Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells

Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G(o/i)-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.

Follicle-stimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus

Japanese flounder ( Paralichthys olivaceus), a teleost fish that has XX (female)/XY (male) sex determination system, exhibits temperature-dependent sex determination. We have previously shown that high water temperature or an aromatase inhibitor treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase ( cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that high water temperature treatment suppresses specifically mRNA expression of the forkhead transcription factor gene foxl2, and follicle-stimulating hormone receptor ( fshr) in gonads during early sex differentiation. Moreover, transient transfection assay shows that the flounder Foxl2 and cAMP analog can activate the cyp19a1 gene transcription in vitro. These results strongly suggest that FSH signaling and Foxl2 are involved in the transcriptional regulation of cyp19a1 gene during gonadal sex differentiation in Japanese flounder with temperature-dependent sex determination.

Transcriptional regulation of the FSH receptor: New perspectives

The cell-surface receptor for the gonadotropin follicle-stimulating hormone (FSH) is expressed exclusively on Sertoli cells of the testis and granulosa cells of the ovary. FSH signal transduction through its receptor (Fshr) is critical for the timing and maintenance of normal gametogenesis in the mammalian gonad. In the 13 years since the gene encoding Fshr was first cloned, the mechanisms controlling its transcription have been extensively examined, but a clear understanding of what drives its unique cell-specificity remains elusive. Current knowledge of basal Fshr transcription highlights the role of an E-box in the proximal promoter which is bound by the basic helix-loop-helix transcription factors upstream stimulatory factor 1 (Usf1) and Usf2. Recent studies utilizing knockout mice and chromatin immunoprecipitation validated the importance of Usf to Fshr transcription and demonstrated a sexually dimorphic requirement for the Usf proteins to maintain normal Fshr expression. Studies have also shown that the promoter region itself is insufficient for appropriate Fshr expression in transgenic mice, indicating Fshr transcription depends on regulatory elements that lie outside of the promoter. Identification of such elements has been propelled by recent availability of genome sequence data, which facilitated studies using comparative genomics, DNase I hypersensitivity mapping, and transgenic analysis with large fragments of DNA. This review will focus on the current understanding of transcriptional regulatory processes that control expression of rat Fshr, including recent advances from our laboratory.

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires β-catenin

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for beta-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by beta-catenin. Additionally, beta-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of beta-catenin. The stimulatory effect of beta-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that beta-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for beta-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.

Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells1

Bone morphogenetic proteins (BMPs) play critical roles in folliculogenesis by modulating the actions of follicle-stimulating hormone (FSH) in the ovary. However, the effects of FSH on the BMP system remain unknown. Here, we have investigated the effects of FSH on BMP signaling using the human granulosa-like tumor cell line KGN. KGN cells express BMP type I and type II receptors and the BMP signaling molecules SMADs. FSH administration upregulated BMP type IA (BMPR1A) and IB (BMPR1B) receptors, activin type II receptor (ACVR2), and BMP type II receptor (BMPR2). FSH also augmented SMAD1 and SMAD5 expression, and conversely, FSH suppressed the expression of the inhibitory SMADs, SMAD6 and SMAD7. Bioassays revealed that FSH enhances BMP-induced SMAD1/5/8 phosphorylation and cellular DNA synthesis induced by BMP6 and BMP7. Since overexpression of BMPR1A and BMPR1B, but not SMADs, significantly enhanced the BMP responses, these type I receptors were revealed to be limiting factors for BMP signaling in KGN cells. BMPs significantly suppressed progesterone synthesis induced by forskolin and dibutyryl-cAMP (BtcAMP) but had no effect on estradiol induced by the same factors. KGN cAMP levels induced by forskolin were not altered by BMPs, suggesting that BMPs regulate steroidogenesis at a level downstream of cAMP synthesis in KGN cells. In this regard, BMPs specifically reduced the STAR transcription, whereas the levels of CYP11A, HSD3B2, and CYP19 stimulated by forskolin as well as BtcAMP were not altered. Collectively, the two major factors, FSH-cAMP pathway and BMP system, are reciprocally and functionally linked. Given that BMPs downregulate FSH receptors in KGN cells, this interaction may contribute to fine-tuning of the mutual sensitivity toward BMP ligands and FSH.

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

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

FSH and testosterone signaling in Sertoli cells

Testosterone and follicle-stimulating hormone (FSH) are required to obtain full reproductive potential. In the testis, somatic Sertoli cells transduce signals from testosterone and FSH into the production of factors that are required by germ cells as they mature into spermatozoa. Recent advances in identifying new signaling pathways that are regulated by FSH and testosterone have allowed for refinement in the understanding of the independent, overlapping and synergistic actions of these hormones. In this review, we discuss the signaling pathways that are regulated by FSH and testosterone as well as the resulting metabolic and gene expression changes that occur as related to Sertoli cell proliferation, differentiation and the support of spermatogenesis.

Essential Role of the Oocyte in Estrogen Amplification of Follicle-Stimulating Hormone Signaling in Granulosa Cells

The establishment of dominant ovarian follicles that are capable of ovulating fertilizable oocytes is a fundamental determinant of female fertility. This process is governed by pituitary gonadotropins as well as local ovarian factors. Within the follicle, estrogen acts in an autocrine/paracrine manner to enhance FSH action in the granulosa cells. These effects include the augmentation of P450aromatase expression and estradiol production. This feed-forward effect of estrogen is believed to play a key role in follicle dominance. Here we found the essential role of the oocyte in this physiological process using primary cultures of rat granulosa cells. In the presence, but not absence, of oocytes, estrogen amplified FSH-stimulated increases in mRNA expression of P450aromatase, FSH receptor, LH receptor, and inhibin alpha-, betaA-, and betaB-subunits as well as cAMP production. Thus, oocytes mediate the estrogen enhancement of FSH action in the granulosa cells. In comparison with FSH, cotreatment with estrogen and oocytes failed to amplify the stimulatory effects of forskolin or 8-bromoadenosine-cAMP on granulosa cell responses including P450aromatase mRNA expression and cAMP production, indicating that estrogen/oocytes amplify FSH action at a site upstream of adenylate cyclase. These findings support the novel conclusion that communication between the oocyte and granulosa cells plays a crucial role in mediating estrogen action during FSH-dependent folliculogenesis.

Activin and Follicle-Stimulating Hormone Signaling Are Required for Long-Term Culture of Functionally Differentiated Primary Granulosa Cells from the Chicken Ovary1

Follicle-stimulating hormone, activin A, and transforming growth factor (TGF) alpha are important regulators of chicken granulosa cell (cGC) function. Hence, we aimed to test whether these growth factors are useful for establishing a suitable in vitro cell culture model system of primary cGC. Although cGC are easily isolated from distinct follicular stages, a long-term cGC culture system for in vitro studies has been unavailable. Here, we report a novel, long-term cell culture system that allows for cGC proliferation in vitro while maintaining the epithelial phenotype that granulosa cells exhibit in vivo. The cGC rapidly lose their epithelial morphology and acquire a mesenchymal or fibroblastoid phenotype when cultured in the absence of activin A. This process is strongly enhanced by TGFalpha, a well-known granulosa cell mitogen. However, FSH stimulates cGC proliferation without enhancing morphological changes and dedifferentiation. Interestingly, a combination of both activin A and FSH stimulates cGC proliferation and supports maintenance of differentiated epithelial morphology. Furthermore, activin A and FSH synergistically induce granulosa cell-specific differentiation markers such as inhibin alpha and chicken zona pellucida protein C, suggesting that cultured cGC resemble functionally differentiated granulosa cells. Our data demonstrate that activin signaling is necessary to sustain a morphologically differentiated phenotype of cGC in vitro. The results also suggest a pivotal importance of activin signaling for granulosa cell function in vivo.

Endocrine changes associated with the growth of pre-vitellogenic oocytes in coho salmon, Oncorhynchus kisutch

It has been hypothesized that in salmonids there are two periods when growth can alter the decision to mature; thus, for a fall spawning species, an ‘initiation’ period in the fall is followed by a ‘permissive’ period for continuing maturation in the spring. Research in mammalian and avian models have shown that follicle-stimulating hormone (FSH), steroidogenic acute regulatory protein (StAR) and 17β-estradiol are key components controlling oocyte growth. In the present study we sampled two-year-old female coho salmon during the proposed fall ‘initiation’ period. Histological analyses of the ovaries indicated that from August to December (11–15 months prior to spawning) oocytes developed from the late perinucleolar to mid-cortical alveoli stage. The accumulation of cortical alveoli during this period was accompanied by significant increases in GSI, pituitary and plasma FSH, and plasma 17β-estradiol. In addition, small but significant increases in transcript levels for FSH receptor and StAR occurred during the fall. Such changes in FSH signaling are consistent with a proposed role for FSH stimulation of oocyte growth via steroid production during the fall initiation period.

Sertoli cell proliferation and FSH signalling in African catfish, Clarias gariepinus

Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cyst-forming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Mullerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values).

Mathematical model of FSH-induced cAMP production in ovarian follicles.

During the terminal part of their development, ovarian follicles become totally dependent on gonadotropin supply to pursue their growth and maturation. Both gonadotropins, follicle-stimulating hormone (FSH) and luteining hormone (LH), operate mainly through stimulatory G protein-coupled receptors, their signal being transduced by the activation of the enzyme adenylyl cyclase and the production of second-messenger cAMP. In this paper, we develop a mathematical model of the dynamics of the coupling between FSH receptor stimulation and cAMP synthesis. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in the different states of FSH receptors (free, bound, phosphorylated, and internalized), coupling efficiency (activated adenylyl cyclase), and cAMP response. Classical analysis shows that, in the case of constant FSH signal input, the system converges to a unique, stable equilibrium state, whose properties are here investigated. The system also appears to be robust to nonconstant input. Particular attention is given to the influence of biologically relevant parameters on cAMP dynamics.

Deletion of follicle-stimulating hormone (FSH) receptor residues encoded by exon one decreases FSH binding and signaling in the rat.

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (DeltarFSHR) cDNAs were expressed in insect cells. The large deletion DeltarFSHRS9-N30 and a smaller deletion, DeltarFSHRS9-S18, did not bind (125)I-hFSH. However, DeltarFSHRK19-R29 and DeltarFSHRD300-F315 bound (125)I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants DeltarFSHR S9-N30 or DeltarFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although (125)I-hFSH binding to DeltarFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas DeltarFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to DeltarFSHR.

Alterations in sperm characteristics of follicle-stimulating hormone (FSH)-immunized men are similar to those of FSH-deprived infertile male bonnet monkeys.

The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle-stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)-induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields alphat values, which is another index of chromatin packaging. Higher alphat values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA-FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross-reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate.

Screening Male Intracytoplasmic Sperm Injection Candidates for Mutations of the Follicle Stimulating Hormone Receptor Gene

Follicle stimulating hormone (FSH) is considered to be essential for spermatogenesis. Therefore, genetic abnormalities of FSH signalling on testicular Sertoli cells would be expected to affect sperm production negatively in males. Inactivating FSH receptor mutations have been reported earlier in both males and females. All affected males had elevated FSH serum concentrations and abnormal sperm parameters. We postulated that inactivating FSH receptor mutations might be a cause of oligozoospermia or azoospermia and reviewed the clinical data of 151 male intracytoplasmic sperm injection (ICSI) candidates with special attention to FSH serum concentrations. The exclusion criteria for mutation screening of the FSH receptor gene were: a history of operative sterilization or testicular malignancy, congenital abnormality other than cryptorchidism, and a chromosomal aberration or a Y-chromosome microdeletion. The inclusion criteria were: male (ICSI candidate) with azoospermia or oligoasthenoteratozoospermia (OAT) and elevated FSH serum concentrations. In total, 23 males with OAT and five males with azoospermia were tested for mutations of the coding sequences and the intron-exon boundaries of the FSH receptor gene by polymerase chain reaction (PCR) followed by single strand conformation polymorphism analysis (SSCP). Neutral polymorphisms were readily detected using this technique in both probands and controls. None of the 28 selected patients showed a pathogenic FSH receptor mutation. Mutations in the FSH receptor gene are not a common cause of infertility in ICSI candidates.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R -/- mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R -/- mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Mechanisms of desensitization of follicle-stimulating hormone (FSH) action in a murine granulosa cell line stably transfected with the human FSH receptor complementary deoxyribonucleic acid

The desensitization of follicle-stimulating hormone (FSH)-evoked cAMP synthesis occurs upon continuous or repeated hormonal stimulation, and it involves the hormone-receptor interaction and post-receptor events. These mechanisms were studied in a murine granulosa cell line (KK-1) stably transfected with the human FSH receptor (hFSHR) complementary deoxyribonucleic acid (cDNA) under a powerful viral promoter. Hence, the FSHR transcriptional regulation was eliminated from the experimental model. Stimulation of the cells with recombinant human FSH (rhFSH) or a phorbol ester, 12- O-tetradecanoylphorbol-13 acetate (TPA), resulted in clear desensitization, i.e. subsequent rhFSH-stimulated cAMP formation was 73.4±2.2%, ( P<0.001) and 66.3±3.4%, ( P<0.0001), respectively, of that of cells preincubated in medium. TPA prestimulation evoked also clear inhibition (65–74% of control) of rhFSH or forskolin (a non-specific activator of adenylate cyclase) induced progesterone production. The suppression by TPA preincubation of the rhFSH-induced cAMP synthesis was completely abolished by the protein kinase C (PKC) inhibitor staurosporine (STR). Preincubation with STR exhibited a significant ( P<0.0001) increasing effect on the rhFSH-stimulated cAMP accumulation. The specific involvement of PKC was further evidenced by other inhibitors, all of them exerted significant elevation of cAMP synthesis following rhFSH restimulation. Furthermore, only the PKC β isoform appeared to be constitutively expressed in these cells during desensitization. Prestimulation of the G-protein activity by sodium fluoride (NaF) or cholera toxin (CT), followed by rhFSH challenge, accounted for a decrease in the cAMP-mediated responsiveness, down to 69.4±2.8 or 74.2±1.9% of control ( P<0.001), respectively, indicating that the post-receptor events are critical for desensitization. [ 125l]iodo-rhFSH binding to the cells did not change significantly during desensitization and the different stimulations. In contrast, ≈50% increase ( P<0.001) occurred in the steady-state levels of FSHR mRNA in the cells stimulated with FSH. This was apparently due to prolonged half-time of mRNA, and not to altered transcription, since the FSHR cDNA was driven by a powerful viral promoter. In accordance, the cells transfected with Simian Virus (SV40) promoter-driven luciferase gene did not display alterations in luciferase activity following stimulatory treatments. The effects of the post-receptor stimulations (NaF or CT) on [ 125l]iodo-rhFSH binding were minor (8–12% reduction). Taken together, these data provide evidence that the agonist-responsive hFSHR desensitization appears through a PKC- β isoform-mediated modulation of cAMP production. The desensitization of FSH action involves modifications of functional properties of the existing components of the FSH signal transduction complex, and does not require concomitant suppression of transcription or translation of the FSHR gene.

Screening male intracytoplasmic sperm injection candidates for mutations of the follicle stimulating hormone receptor gene.

Follicle stimulating hormone (FSH) is considered to be essential for spermatogenesis. Therefore, genetic abnormalities of FSH signalling on testicular Sertoli cells would be expected to affect sperm production negatively in males. Inactivating FSH receptor mutations have been reported earlier in both males and females. All affected males had elevated FSH serum concentrations and abnormal sperm parameters. We postulated that inactivating FSH receptor mutations might be a cause of oligozoospermia or azoospermia and reviewed the clinical data of 151 male intracytoplasmic sperm injection (ICSI) candidates with special attention to FSH serum concentrations. The exclusion criteria for mutation screening of the FSH receptor gene were: a history of operative sterilization or testicular malignancy, congenital abnormality other than cryptorchidism, and a chromosomal aberration or a Y-chromosome microdeletion. The inclusion criteria were: male (ICSI candidate) with azoospermia or oligoasthenoteratozoospermia (OAT) and elevated FSH serum concentrations. In total, 23 males with OAT and five males with azoospermia were tested for mutations of the coding sequences and the intron-exon boundaries of the FSH receptor gene by polymerase chain reaction (PCR) followed by single strand conformation polymorphism analysis (SSCP). Neutral polymorphisms were readily detected using this technique in both probands and controls. None of the 28 selected patients showed a pathogenic FSH receptor mutation. Mutations in the FSH receptor gene are not a common cause of infertility in ICSI candidates.

Follicle stimulating hormone and its receptor: future perspectives.

In this report, we review our present effort in the field of molecular reproductive endocrinology: to identify a small molecular weight follicle stimulating hormone (FSH) agonistic molecule. To achieve this goal we require a number of molecular tools. We have cloned and expressed the human gonadotrophin, FSH and the human FSH receptor and developed a reliable high throughput assay. We have also proposed a model to explain FSH receptor activation and from that model, begun to create small molecules predicted to induce FSH signal transduction without binding to the extracellular domain of the membrane protein. In this report, we summarize our efforts to date and discuss our future research efforts in this area.