Papel inibitório dos neurônios KNDy na regulação estrogênica da secreção de gonadotrofinas

Abstract

Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin-B, and dynorphin (KNDy) play a crucial role in the control of gonadotropin-releasing hormone (GnRH) neurons and, thus, on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. According to the KNDy hypothesis, neurokinin-B stimulates kisspeptin while dynorphin signaling inhibits kisspeptin, resulting in the generation of GnRH/LH pulses. However, the neuroendocrine mechanisms involved in the estrogenic regulation of the KNDy neuron to control LH and FSH secretion are not fully understood. Therefore, our first objective was to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the role of this neuronal population in the control of pulsatile LH and basal FSH secretion under different estrogenic conditions. We then characterized the expression of the kappa (Oprk1)-, mu (Oprm1)-, and delta (Oprd1)-opioid receptors in kisspeptin neurons of the ARC (Kiss1ARC) and rostral periventricular area of the third ventricle (Kiss1RP3V), and in GnRH (Gnrh1) neurons of female mice and rats under different estrogenic conditions. To achieve the first objective, adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) or blank saporin (Control). In order to accomplish the second objective, female mice and rats with low (diestrus) and high (proestrus) endogenous estradiol (E2) levels were perfused and brains were analyzed by RNAscope in situ hybridization. Our results shows that diestrous NK3-SAP rats with 74% loss of KNDy neurons displayed increased frequency, amplitude, and baseline levels of LH pulses and higher basal FSH secretion. After ovariectomy, parameters of LH and FSH secretion were increased but did not differ between NK3-SAP and Control ovariectomized (OVX) rats. In a second cohort of NK3-SAP rats with 64% KNDy neuron loss, OVX rats treated with E2 (OVX+E2) displayed reduced LH pulsatile secretion and, in this condition, the NK3-SAP lesion again increased LH pulse frequency but not pulse amplitude or baseline levels. Thus, the partial loss of KNDy neurons facilitates LH and FSH secretion in the presence of E2, suggesting that the dynorphin signaling might be responsible for the inhibitory role of KNDy neurons on the gonadotrophin output. Regarding the opioid receptors, in both mice and rats, there was a high expression of Oprk1 in Kiss1ARC neurons, which was reduced on proestrus compared to diestrus. On the other hand, Oprd1 expression in Kiss1ARC neurons was unchanged, while the Oprm1 expression in Kiss1ARC neurons was differently modulated by estrous cycle depending on the species. The Kiss1RP3V neurons displayed a high degree of co-expression of Oprd1, Oprm1, and Oprk1, unchanged across the cycle. All opioid receptors were found in Gnrh1 neurons, and the co-expression of Oprk1 was higher on proestrus than on diestrus. In conclusion, the submaximal loss of KNDy neurons accelerates and amplifies LH pulses and FSH secretion. This inhibitory effect of KNDy neurons depends on the presence of E2 and is possibly mediated by dynorphin. Accordingly, Oprk1, Oprm1, and Oprd1 are abundantly expressed in the GnRH network. Thus, Oprk1 expression Kiss1ARC and GnRH neurons is modulated by E2 and possibly involved in the mechanism of negative-feedback to restrain gonadotrophin secretion. Keywords: kndy; rp3v; gnrh; fsh; lh; opioid peptide receptors; estrous cycle