Pituitary Kisspeptin Receptor Knock Out Mice Display Altered Pituitary Function

The hypothalamic‐pituitary‐gonadal (HPG) axis controls the development and maintenance of reproductive function. The HPG axis is comprised of gonadotropin‐releasing hormone (GnRH) neurons in the hypothalamus that regulate the gonadotrophs in the anterior pituitary gland, stimulating the secretion of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the bloodstream. LH and FSH play essential roles in spermatogenesis in males, folliculogenesis and ovulation in females, and steroidogenesis in both sexes. Recently, kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons has been shown by our group and others to play an essential role in HPG axis function. However, whether kisspeptin signaling via the Kiss1r affects reproductive function at the level of pituitary is not yet known. Using Cre/Lox technology, we knocked out the Kiss1r gene specifically in pituitary gonadotropes (PKiRKO) by crossing a αGSUCre mouse with a floxed Kiss1r 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) levels in PKiRKO male mice compared with WT male mice, while stimulated LH and FSH levels were no different between WT and PKiRKO female mice. To directly assess the effects of KISS1 on pituitary gonadotroph function that are mediated via the KISS1R 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. This culture system was then applied to calcium flux assays using the calcium indicator dye Fluo‐1. These calcium flux assays indicated that the WT male pituitaries were more responsive to GnRH (100nm) and kisspeptin (1nm) than PKiRKO males. Interestingly a combination of 1nM kp10 and 30nM GnRH potentiated the calcium response in WT males but not their PKiRKO littermates, suggesting that there is interplay of both KISS1 signaling and GnRH signaling at the level of the pituitary to augment pituitary action. These findings indicate overall that the pituitary Kiss1r may plan an important modulatory role and contribute to normal reproductive function. Support or Funding Information National Institutes of Health T32DK007751, JHU UMD Diabetes Research Center (P30 DK079637), RO1 DK101591 and R01HD068777. The American Physiological Society Porter Developmental and Minority Affairs Committee This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .