Exploring the Role of Pituitary Kisspeptin Receptor on Gonadotrophic Function In Vivo and In Vitro

Abstract

The hypothalamic‐pituitary‐gonadal (HPG) axis is classically comprised of gonadotropin‐releasing hormone (GnRH) neurons in the hypothalamus that regulate the anterior pituitary gland, stimulating the gonadotrophs to secrete luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the bloodstream. LH and FSH after being synthesized and released by the pituitary gland go on to play essential roles in folliculogenesis, ovulation, and steroidogenesis. Kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons has been shown by our group and others to play an essential role in HPG function. While the gonadotropes of the anterior pituitary express the Kiss1r gene, a physiological role for kisspeptin signaling at the level of the pituitary has not been demonstrated. We knocked out the Kiss1r gene specifically in pituitary gonadotropes by crossing the αGSUCre mouse with the floxed Kiss1r mouse, producing the PKiRKO mouse. Q‐RT‐PCR and immunohistochemistry were used to demonstrate a disruption in pituitary Kiss1r mRNA and KISS1R protein in PKiRKO mice relative to controls. Q‐RT‐PCR demonstrated a reduction in Kiss1r mRNA by 88% and 64% in the pituitary of male and female PKiRKO mice (n=8), respectively, compared with wild type (WT) mice (n=8). Immunostaining for KISS1R protein levels exhibited similar trends for protein knock down as observed for the relative mRNA levels. Our results revealed no difference in the age of puberty between WT and PKiRKO littermates, as assessed by the ages of vaginal opening, and first estrus for female mice, and preputial separation for male mice. Furthermore, we saw no difference in ovarian and testes weight respectively in female and male mice. While there were no differences in basal LH and FSH levels, upon performing a GnRH stimulation test in vivo, we observed a significant attenuation (P<0.05) in stimulated luteinizing hormone (LH) serum of PKiRKO male mice compared with WT male mice, while stimulated LH and FSH levels were no different between WT and PKiRKO female mice. These changes in pituitary sensitivity in males were associated with lower mRNA levels of GnRH receptor and estrogen receptor alpha. To directly study the effects of KISS1 on the pituitary gonadotroph, we sought to develop an in vitro primary culture system. To test the system, a GnRH dose response and time course study was performed on dispersed and adherent pituitary cells harvested from WT male and female mice. Groups of cells were treated with either 5nM, 30nM or 100nM GnRH, for a duration of 30 and 60 minutes. A significant increase in LH levels was observed in both male and female mice pituitary cells when treated with either 30nM or 100nM GnRH for 60 minutes. Female pituitary cells also showed a significant increase in LH secretion at the 30 minute time point. Therefore, a 30nM treatment for 60 minutes is suitable for GnRH stimulation and will be used for future studies. Included in those studies will be; WT and PKiRKO pituitary cells treatment with/without GnRH and with/without different doses of Kp10 (kisspeptin agonist). LH and FSH levels in the media will be measured after a 60 minute incubation. These studies will allow us to assess the direct effects of KISS1 on pituitary gonadotroph function that are mediated via the KISS1R. Overall this work will provide insight into the signaling of kisspeptin and Kiss1r on the gonadotroph neuron and how it contributes to both normal and abnormal reproductive physiology.Support or Funding InformationWe acknowledge support from the following grants 1.) U01HD066432 2.) P30DK079637 3.) R01068777 4.) R01DK101591