Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice

Abstract

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild-type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and β and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle-stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised-oestradiol valerate-treated mice, we evaluated ex vivo hypothalamic gonadotrophin-releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss-10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised-oestradiol silastic capsule-treated mice (proestrous-like environment), we evaluated morning and evening kisspeptin neurone activation (c-Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERβ was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss-10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs.In WTs, pulsatile Kiss-10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss-10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c-Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described. K E Y W O R D SGABAB1KO mice, GnRH pulsatility, kisspeptin, oestrogen receptors, ovulation

New methods to investigate the GnRH pulse generator

“…Application of baclofen, a GABA B receptor agonist, was also shown to suppress kiss1 evoked increases in intracellular calcium levels which are known to be dependent on voltage-gated calcium channels, indicating that the dominant GABA B receptor may act to counterbalance the excitatory drive provided by KNDy neurons. This idea is consistent with existing evidence of increased GnRH pulse frequency in mice with a global knockout of the GABA B receptor ( Catalano et al 2010 , Di Giorgio et al 2019 ). Interestingly, at the GnRH soma, the hyperpolarisation effects exerted by GABA B receptor activation were abolished by kiss1 administration whereas co-application of baclofen after kiss1 administration did not completely reverse the kiss1-induced depolarisation of GnRH cell bodies ( Zhang et al 2009 ).…”

GABAergic input through GABAB receptors is necessary during a perinatal window to shape gene expression of factors critical to reproduction such as Kiss1

“…Efficiencies of 2 Ϯ 0.1 were considered optimal. Quantitative measurements of Kiss1r, Tac2, Pdyn, Th, Esr1, Esr2, Pgr, Gnrh1, Cyp19A1, Gad1, Gabbr1, Gabbr2, and Ppib2 cDNA were performed by qPCR in a total volume of 10 L, as previously described (24), whereas Kiss1 qPCR was performed in a total volume of 30 L. Amplification was carried out in StepOne real-time PCR systems (Applied Biosystems). Each sample was analyzed in duplicate along with nontemplate controls to monitor possible contamination.…”

Robust GABAergic Regulation of the GnRH Neuron Distal Dendron

“…We show here that GABA B receptor activation completely and acutely abolishes kisspeptin activation of the dendron. Interestingly, mice with a global knockout of the GABA B receptor exhibit increased GnRH pulse frequency and, in response to a constant kisspeptin stimulus, GnRH pulses have an increased mass per pulse in these mice ( 10 , 26 ). Thus, it is possible that GABAergic inputs to the distal dendron provide a mechanism for the regulation of pulse generation at the level of the dendron itself.…”