Fshr Transcript Research Articles

B-Vitamin and Homocysteine Status Determines Ovarian Response to Gonadotropin Treatment in Sheep1

Maternal B-vitamin status and homocysteinemia can affect fertility and pregnancy establishment, although the direct effects on ovarian follicle and oocyte development are not known. We report on the effects of restricting the supply of vitamin B(12) and methionine from the diet of mature female sheep on ovarian folliculogenesis following follicle-stimulating hormone (FSH) stimulation. The study was split into three batches and involved 76 animals. Surprisingly, the number of growing, estrogen-active antral follicles following FSH treatment was enhanced (P = 0.005) following this dietary intervention. This increase occurred even in the presence of modest live-weight loss (batch 1 only) and depressed plasma insulin concentrations, suggesting a breakdown in the regulation of follicular responsiveness to FSH. This dietary intervention also increased plasma homocysteine concentrations. Physiological concentrations of homocysteine increased granulosa cell proliferation (P < 0.001), estradiol production (P = 0.05), and FSHR transcript expression (P = 0.017) during culture. Transcript levels for growth differentiation factor 9 and bone morphogenetic protein 15 in oocytes from treated ewes were increased (P < 0.05) in the first two batches. Furthermore, regression of BMP receptor 2 (BMPR2) transcript expression and diet on follicle number revealed a significant interaction (P = 0.01); BMPR2 transcript expression was associated with follicle number only in vitamin B(12)/methionine-restricted animals. Because FSHR transcript expression also was positively (P = 0.007) related to follicle number, the effects of diet may have arisen through enhanced FSH and BMP signaling. Although this remains to be confirmed, the data support an intraovarian impact of vitamin B(12)/methionine-deficient diets.

Dynamics of avian ovarian follicle development: Cellular mechanisms of granulosa cell differentiation

In vertebrate species that ovulate one or a limited number of ovarian follicles per reproductive cycle, the cellular processes by which follicle selection (cyclic recruitment) is mediated and final differentiation is initiated remain largely unknown. In the hen ovary, the selection of a single follicle into the preovulatory hierarchy on an approximate daily basis occurs from a small cohort of prehierarchal follicles measuring approximately 6- to 8-mm in diameter. Given that the granulosa layer undergoes a dramatic alteration in phenotype subsequent to follicle selection, of particular interest are the cell signaling and associated transcriptional mechanisms that regulate this transition. Recent studies suggest that granulosa cells from prehierarchal follicles are normally maintained in an undifferentiated state by inhibitory MAP kinase (MAPK) signaling mediated by epidermal growth factor receptor ligands (EGFRLs). One of the earliest markers for differentiating granulosa cells is elevated expression of FSH receptor ( fshr) mRNA and enhanced FSH-induced cyclic adenosine monophosphate (cAMP) production. EGFRL/MAPK signaling is proposed to inhibit fshr transcription via its ability to induce Inhibitor of differentiation/DNA binding (Id) protein isoforms, Id1, Id3 and Id4. Subsequent to follicle selection, cAMP-induced Id2 expression is considered both sufficient and necessary for fshr transcription. Two working models are proposed which predict that enhanced FSHR expression and the progression of granulosa cell differentiation occurs as a result of a decline in MAPK signaling from within granulosa cells (internal model for differentiation) and/or elevated cAMP signaling promoted by an endocrine, neuroendocrine or neuronal factor (external model).

In Vivo Regulation of Follicle-Stimulating Hormone Receptor by the Transcription Factors Upstream Stimulatory Factor 1 and Upstream Stimulatory Factor 2 Is Cell Specific

Pituitary FSH promotes pubertal timing and normal gametogenesis by binding its receptor (FSHR) located on Sertoli and granulosa cells of the testis and ovary, respectively. Studies on Fshr transcription provide substantial evidence that upstream stimulatory factor (USF) 1 and USF2, basic helix-loop-helix leucine zipper proteins, regulate Fshr through an E-box within its promoter. However, despite the strong in vitro support for USF1 and USF2 in Fshr regulation, there is currently no in vivo corroborating evidence. In the present study, chromatin immunoprecipitation demonstrated specific binding of USF1 and USF2 to the Fshr promoter in both Sertoli and granulosa cells, in vivo. Control cells lacking Fshr expression showed no USF-Fshr promoter binding, thus correlating USF-promoter binding to gene activity. Evaluation of Fshr expression in Usf1 and Usf2 null mice further explored USF's role in Fshr transcription. Loss of either gene significantly reduced ovarian Fshr levels, whereas testis levels were unaltered. Chromatin immunoprecipitation analysis of USF-Fshr promoter binding in Usf-null mice indicated differences in the composition of promoter-bound USF dimers in granulosa and Sertoli cells. Promoter-bound USF dimer levels declined in granulosa cells from both null mice, despite increased USF2 levels in Usf1-null ovaries. However, compensatory increases in promoter-bound USF homodimers were evident in Usf-null Sertoli cells. In summary, this study provides the first in vivo evidence that USF1 and USF2 bind the Fshr promoter and revealed differences between Sertoli and granulosa cells in compensatory responses to USF loss and the USF dimeric composition required for Fshr transcription.

Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica

Eel species have a striking life cycle with a blockade of puberty until the oceanic migration. We report the first molecular data on eel gonadotropin receptors. The partial sequences cloned covered two-third of the open reading frame and included most of the extracellular and transmembrane domains. Phylogenetic analysis partitioned the two eel gonadotropin receptors into the two teleost FSHR and LHR clusters, respectively. Real-time quantitative RT-PCR was used to quantify the expression of eel gonadotropins and their receptors. Similar levels of pituitary FSH-β and LH-β transcripts were found in the immature previtellogenic female eels. In contrast, ovarian FSHR mRNA level was at 100- to 185-fold higher than that of LHR. This revealed that FSHR rather LHR would mediate gonadotropin stimulation of the early stages of ovarian growth. Chronic treatment with fish pituitary homogenates, applied to induce eel sexual maturation, stimulated pituitary LH-β but suppressed FSH-β transcripts. In the ovaries, both FSHR and LHR mRNA were significantly increased in experimentally matured eels. Treatments with sexual steroids showed a stimulatory effect of estradiol-17β (E 2) on pituitary LH-β mRNA levels, while FSH-β transcripts were suppressed by E 2 or testosterone (T). In contrast, neither E 2 nor T-treatment had any significant effect on ovarian FSHR nor LHR transcripts. This suggests that steroid feedbacks may be responsible for the opposite regulation of pituitary gonadotropins in experimentally matured eels, but are not involved in the regulation of gonadotropin receptors. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors in the eel. They provide new foundation for basic and applied research on eel reproduction.

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.

Silencing ofFshrOccurs through a Conserved, Hypersensitive Site in the First Intron

Expression of the FSH receptor (Fshr) is restricted to testicular Sertoli cells and ovarian granulosa cells, thereby limiting the direct targets of FSH action to these somatic cells of the gonads. Earlier studies indicate that transcription of Fshr in the gonads requires elements outside the gene's immediate 5' flanking sequence. To help uncover candidate regulatory sequences, comparative genomics and deoxyribonuclease I hypersensitivity mapping were employed. A total of 156 evolutionarily conserved sequences were found, and partial deoxyribonuclease I hypersensitivity mapping across 45 kb of 5' flanking sequence and the first intron identified four hypersensitive sites, DHS1-4. Notably, DHS1 and DHS2 localized to conserved sites in the promoter region and exon 1 and correlated with the active state of the gene. DHS3 also corresponded to a conserved site (site 7) but was more pronounced in nonexpressing myoid cells, suggesting a role in gene silencing. Transient transfection analysis of DHS3 confirmed its role in gene silencing, a function that was promoter, cell type, and position dependent. Protein-DNA binding studies on DHS3 revealed that octamer transcription factor 1 (OCT-1) and GATA-4 bound site 7, in vitro, and transient transfection analysis showed that their binding sites were required for silencing activity. Furthermore, chromatin immunoprecipitation revealed that OCT-1 bound to site 7 in the endogenous gene, but only in myoid cells. In contrast, GATA-1 bound site 7 predominantly in Sertoli cells, suggesting that it attenuates silencer activity. The findings reveal that OCT-1 binds within DHS3 to silence Fshr transcription and implicate members of the GATA family in the modulation of this activity.

Cyclic and maturation-dependent regulation of follicle-stimulating hormone receptor and luteinizing hormone receptor messenger ribonucleic acid expression in the porcine ovary.

We sought to characterize the ovarian expression of mRNAs for the receptors for FSH (FSHr) and LH (LHr) at the different stages of the estrous cycle of the pig and to localize the receptor mRNAs to individual cell types in follicles and corpora lutea as a function of their developmental status. Northern blot analyses indicated that multiple FSHr and LHr mRNA transcripts occur on Days 0, 4, 7, 12, and 16 of the estrous cycle. In situ hybridization analyses revealed considerable cyclic variation in expression among small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr mRNA expression was strongly positive and was spatially restricted to granulosa cells. FSHr mRNA expression was strongly positive in granulosa cells of medium follicles but had declined in granulosa cells of mature follicles on Day 0. LHr expression in small follicles, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4, was strongly positive in small follicles on Day 7 and in medium follicles on Days 12 and 16, and had declined in large mature follicles on Day 0. LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mRNA was expressed weakly in the theca cells, and FSHr mRNA expression in granulosa cells was absent. In the corpus luteum, FSHr mRNA was not expressed, but LHr mRNA was expressed in some cells on Days 4 and 7, was expressed maximally on Day 12, and had declined partially by Day 16. These studies indicate that there is maturation-dependent regulation of FSHr and LHr expression in the adult porcine ovary. FSHr mRNA content decreases in parallel with FSHr protein content as follicles increase in size.