An Ovarian Component Involved In Subfertility Of The Nsmf Ko Mouse.

Genetic approaches in humans with gonadotropin releasing hormone (GnRH) deficiency causing normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS) have been important to better understand normal reproduction. NSMF (NMDA receptor synaptonuclear signaling & neuronal migration factor), formerly known as NELF (nasal embryonic LHRH factor), gene mutations have been identified in humans with either nHH/KS. However, the phenotype of the Nsmf knockout (KO) mouse is less severe than the human. The Nsmf KO females have reduced numbers of GnRH neurons and delay in vaginal opening, but normal puberty and subfertility. We previously showed Kiss1 mRNA expression was increased in the hypothlami of KO animals and that pituitary gonadotropin responses were not different in wild type (WT) vs Nsmf KO mouse. Our objective in this study was to identify cell types that express Nsmf in the ovary and determine if the subfertility in the female Nsmf KO mouse has a gonadal component. NSMF protein cellular localization was determined in the WT mouse ovary. Kiss1 and Kiss1r mRNA expression was characterized in the KO vs WT 8 week old mice, and ovarian responses to gonadotropins were studied in 3 week old Nsmf KO mice in the diestrus phase. Heterozygous Nsmf mice were bred to homozygosity. Ovaries from KO vs WT mice were sectioned and prepared for immunohistochemistry (IHC) using a monoclonal anti-NSMF antibody. RNA extracted from ovaries of KO and WT animals were subjected to RT-qPCR for Kiss1 and Kiss1r expression. The ΔΔCt, cycle of threshold, method was used to calculate relative gene expression of Nsmf KO vs control using Gapdh expression for normalization. To determine the ovarian response to gonadotropins, WT and KO mice 3 weeks of age were superovulated using PMSG and hCG. Mice were sacrificed and oocytes were removed from the oviducts and counted. Differences were analyzed using the Mann-Whitney U test. 8-week old mice also had serum gonadotropins before and after ovariectomy. Our preliminary findings demonstrate Nsmf mRNA expression in the ovary, and IHC studies and serum gonadotropins after ovariectomy are ongoing. Kiss1r expression is unchanged in Nsmf hypothalamus and ovary, but Kiss1 was upregulated in the hypothalamus and the ovary. Preliminary data suggests that oocyte numbers were modestly decreased in the KO (∼18/ovary) vs WT (30/ovary), but maturity has not been assessed yet. A hypothalamic component appears to be involved in the subfertility of the Nsmf KO mouse, as demonstrated by a decreased number of GnRH neurons as well as our finding of increased Kiss1 expression in the hypothalami of Nsmf KO mice, which we hypothesize is a compensatory increase secondary to deficient NSMF. Therefore we sought to characterize Kiss1 and Kiss1r expression in the ovary. Kiss1r expression was unchanged, but there was a significant increase in Kiss1, which is known to be expressed in granulosa cells in mice. We also demonstrated Nsmf expression in the WT ovary. The reduced number of oocytes in the Nsmf KO mouse supports an ovarian role for NSMF in the subfertility of the Nsmf KO mouse.