Anti-Follicle Stimulating Hormone monoclonal antibody, clone A1039

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (AD-CYAP1) also stimulates all three gonadotropin subunits. When cells were stimulatedwith both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression. Summary Sentence KP10 increased all three gonadotropin subunit promoters with an increasing expression of ADCYAP1 type I receptor in pituitary gonadotroph LbetaT2 cells overexpressing kisspeptin receptor.

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after G alpha(s) knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and G alpha(s) knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on G alpha(s). In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs.