Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1-7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10−9M Kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4 hours of treatment in both cell lines. Negative regulation by 17β-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17β-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin-GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1-7 and GN11 cell lines.

The role of IGF-I in the negative regulation of GH expression and release is demonstrated by in vitro and in vivo models; however, the targets and mechanisms of IGF-I remain unclear. We have developed a cell-specific knockout mouse in which the IGF-I receptor was ablated from the somatotroph in order to validate and characterize IGF-I negative regulation; we termed this the somatotroph IGF-I receptor knockout (SIGFRKO) mouse. The SIGFRKO mice demonstrated increased GH gene expression and secretion as well as increased serum IGF-I. Compensatory changes were noted with decreased GHRH and increased somatostatin mRNA expression levels. SIGFRKO mice had normal linear growth, but by 14 wk of age weighed significantly less than controls. Furthermore, metabolic studies revealed SIGFRKO mice had significantly less fat mass and body percent fat. These data support somatotroph IGF-I negative regulation and suggest that hypothalamic feedback limits the extent of GH release. The SIGFRKO mouse is a model delineating the mechanisms of IGF-I regulation in the hypothalamic-pituitary axis and demonstrates compensatory mechanisms that mediate growth and metabolic function in mammals.

Mice lacking estrogen receptor alpha in the pituitary gonadotroph (PitEsr1KO) were generated to determine the physiologic role of pituitary estrogen signaling in the reproductive axis. PitEsr1KO female mice are subfertile or infertile and have elevated levels of serum luteinizing hormone (LH) and LH beta subunit gene expression, reflecting a lack of estrogen negative feedback effect on the gonadotroph. While serum LH values are elevated in PitEsr1KO mice, the degree of elevation is much less than that observed in ESR1-null mice, indicating that the hypothalamus must also have an important role in estrogen negative feedback. PitEsr1KO mice also demonstrate a defect in estrogen positive feedback, as surge LH values and estrous cyclicity are absent in these mice. Although sex steroid feedback in the reproductive axis is thought to involve discrete anatomic regions that mediate either a positive or negative estrogen effect, PitEsr1KO mice demonstrate novel evidence that localizes both estrogen positive feedback and estrogen negative feedback to the gonadotroph, which suggests that they may be mechanistically related.