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.     

The promoter of murine follicle-stimulating hormone receptor: functional characterization and regulation by transcription factor steroidogenic factor 1

The promoter of the FSH receptor (R) gene has been cloned from several species. Although some of its regulatory elements have been identified, its function still remains poorly characterized. Using transient transfections of luciferase reporter constructs, driven by various fragments of the murine (m) FSHR promoter, we identified a cell-specific promoter region. This domain is located in the distal part of the mFSHR promoter, -1,110 to -1,548 bp upstream of the translation initiation site, and it contains two steroidogenic factor 1 (SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and protein closely correlated in various steroidogenic cell lines with activity of the transfected mFSHR promoter/luciferase reporter construct carrying the distal activator domain. A dose-dependent increase in FSHR promoter activity was shown in nonsteroidogenic HEK 293 cells transiently transfected with SF-1 cDNA. SF-1 was found to bind to a nonconsensus 5'-CAAGGACT-3' SLBS-3 motif in the distal part of the promoter; formation of the SF-1/SLBS-3 complex could be reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a significant loss of SF-1-mediated mFSHR promoter activity. The second SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In conclusion, we have identified a regulatory domain in the mFSHR promoter participating in the cell-specific regulation of FSHR expression. We demonstrated for the first time that the mFSHR promoter possesses functional SF-1 binding sites and thus belongs to the group of SF-1-regulated genes. These findings provide further evidence for the key role of SF-1 in the regulation of genes involved in gonadal differentiation and endocrine functions.     

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.     

New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype.

Premature ovarian failure occurs in almost 1% of women under age 40. Molecular alterations of the FSH receptor (FSHR) have recently been described. A first homozygous mutation of the FSHR was identified in Finland. More recently, we described two new mutations of the FSHR in a woman presenting a partial FSH-resistance syndrome (patient 1). We now report new molecular alterations of the FSHR in another woman (patient 2) who presented at the age of 19 with primary amenorrhea contrasting with normal pubertal development. She had high plasma FSH, and numerous ovarian follicles up to 3 mm in size were evidenced by ultrasonography. Histological and immunohistochemical examination of ovarian biopsies revealed the presence of a normal follicular development up to the antral stage and disruption at further stages. DNA sequencing showed two heterozygous mutations: Asp224Val in the extracellular domain and Leu601Val in the third extracellular loop of FSHR. Cells transfected with expression vectors encoding the wild type or the mutated Leu601Val receptors bound hormone with similar affinity, whereas binding was barely detectable with the Asp224Val mutant. Confocal microscopy showed the latter to have an impaired targeting to the cell membrane. This was confirmed by its accumulation as a mannose-rich precursor. Adenylate cyclase stimulation by FSH of the Leu601Val mutant receptor showed a 12+/-3% residual activity, whereas in patient 1 a 24+/-4% residual activity was detected for the Arg573Cys mutant receptor. These results are in keeping with the fact that estradiol and inhibin B levels were higher in patient 1 and that stimulation with recombinant FSH did not increase follicular size, estradiol, or inhibin B levels in patient 2 in contrast to what was observed for patient 1. Thus, differences in the residual activity of mutated FSHR led to differences in the clinical, biological, and histological phenotypes of the patient.     

Maintenance of sexual immaturity in male mice and bucks by immunization against N-terminal peptides of the follicle-stimulating hormone receptor

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.     

Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids.

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.