FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR ED ), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR ED . In addition to the known hormone-binding site, FSHR ED provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.F SH is a gonadotropin that stimulates steroidogenesis and gametogenesis in the gonads. Secreted by the anterior pituitary gland, FSH regulates the menstrual cycle and ovarian follicular maturation in women and supports sperm production in men. FSH acts by binding to the FSH receptor (FSHR) on the granulosa cell surface in ovaries and the Sertoli cell surface in testes. The stimulated receptor leads to the dissociation of α-and βγ-subunits of G protein heterotrimer inside the cell. The α-subunit activates adenylyl cyclase, resulting in an increase of cAMP levels, and ultimately leads to the increased steroid production that is necessary for follicular growth and ovulation in women. The free βγ dimers recruit G protein-coupled receptor (GPCR) kinases to the receptor, which, in turn, lead to the recruitment of β-arrestin to the receptor (1). FSH is used clinically for controlled ovarian stimulation in women treated with assisted reproductive technologies and also for the treatment of anovulatory infertility in women and hypogonadotropic hypogonadism in men. The central role of FSH in human reproduction makes its receptor a unique pharmaceutical target in the field of fertility regulation (2-4).The glycoprotein hormone (GPH) family has four members: FSH; two other pituitary hormones, luteinizing hormone and thyroid-stimulating hormone (TSH); and one placental hormone, chorionic gonadotropin. The four members are homologous in sequence, structure, and function. Each member is a heterodimer composed of a common α-subunit and a hormone-specific β-subunit. The crystal structures of FSH and human CG (hCG) revealed that both α-and β-subunits adopt similar folds of cystineknot architecture (5-7). The assembled α-and β-heterodimers bind to their respective receptors with high affinity and hormone specificity, resulting in similar signaling pathways but distinct biological responses.

ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein beta, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

Background: A carbohydrate of follicle-stimulating hormone (FSH) has been proposed to sterically block other FSH molecules from binding to the putative receptor (FSHR) trimer.Results: FSH increases its receptor binding by 3-fold when the steric hindrance is removed.Conclusion: FSHR forms a functional trimer.Significance: This knowledge may improve designs of therapeutic drugs targeting FSHR.

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.