Altered GnRH response to Kiss-10/estradiol and ovulation impairment in GABAB1KO female mice.

DI GIORGIO N; BIZZOZZERO MARIANNE; BOURGUIGNON NS; TABARES FLORENCIA; LIBERTUN, C; LUX LANTOS V

Orlando

Congreso; Kisspeptin 2017: Brain and Beyond (3rd World Conference); 2017

Kisspeptin is expressed in the AVPV in the anterior hypothalamus (POA-AH), involved in the positive E2 feedback in females, and in the ARC, in the medial basal hypothalamus (MBH), involved in the negative E2/T feedback in both sexes. Other neuropeptides and neurotransmitters are also involved in the regulation of reproduction such as GABA. We have shown that female mice lacking functional GABAB receptors, GABAB1KO (KO), showed impaired reproduction with altered cyclicy, reduced pregnancies and altered GnRH physiology. We demonstrated that a high percentage of Kiss1 neurons express GABAB receptors in hypothalamic and extra-hypothalamic nuclei. While Kiss1 expression was very significantly increased in the amygdala intriguingly, its expression in AVPV or ARC was unaltered in KO mice. This suggested that Kiss1 mRNA expression in these nuclei was not involved in the above-described reproductive alterations. Here we evaluated in WT and KO females the ovulation index and the GnRH response to kisspeptin and/or E2 in ex vivo incubated the hypothalami..After two regular cycles and a clear proestrus, WT and KO females were sacrificed in first estrus, serum samples were collected to measure FSH (RIA) and ovaries were pressed between two slides and the number of ova in the ampulla were counted. We performed GnRH pulsatility studies in whole hypothalami (HT). Adult WT and KO females were ovariectomized (OVX), treated with E2 valerate (VAL, 10ug/kg) and sacrificed 7 days post-ovariectomy. Sera were collected and LH, FSH and E2 were determined (RIA). HT were incubated in Krebs-Ringer buffer with one pulse (15 min) of Kiss-10/hour (10-7M) in absence or presence of E2 (10-8M) or E2 alone. Medium was collected at 9-min intervals, replacing it each time with fresh medium, for 8 h. GnRH in the media was measured by RIA. In a second set of brains of OVX-VAL females, POA-AH and MBH were immediately dissected after sacrifice and stored to determine Kiss1r expression (qPCR).Approximately 80 % of WT females ovulated on the morning of estrus, significantly higher than KO mice (14%). In agreement, first estrus FSH levels in KO females were significantly lower than in WTs.OVX+VAL females had lower LH compared to OVX and higher E2 than estrous females, without differences between genotypes. FSH levels did not vary between OVX and OVX+VAL females. Additionally, OVX+VAL KO females had increased HMB Kiss1r expression; .in POA-AH, Kiss1r levels were similar between genotypes. GnRH. pulse frequency was increased by the combination of E2 and Kiss-10 only in WT HT, while Kiss-10 or E2 alone had no effect. Conversely, in KO HT, E2 increased the number of GnRH pulses and this was reversed by the combination of E2-Kiss-10. We conclude that the low fertility rate in KO females may be due to a decreased response in terms of GnRH pulses in the presence of Kiss-10 and E2 even in the presence of increased Kiss1r that impacts ovulation and estrus FSH levels. Research Support: CONICET-ANPCYT-Fundación René Barón- Fundación Williams (VLL); ANPCYT-UBA (CL). Key words: Kisspeptin, GABA, GnRH, estradiol