| The time of puberty onset is directly associated with sexual maturation and reproductive performance in the animal, but its mechanism remains unclear. Studies have shown that GnRH secreted by the GnRH neuron is a decisive factor in puberty onset in the developmental regulation of animal puberty. It is generally believed that the hypothalamus GnRH neurons is sensitive to negative feedback effect of gonadal steroids at prepubertal animal, therefore after birth to puberty onset, GnRH secretion of the hypothalamus has been inhibited because of the effect of gonadal steroid hormones, thus GnRH release pulse is low amplitude, low frequency pulse. The puberty onset of animal dates from relieving the mechanism of the hypothalamic gonadotropin inhibition, but there is no receptor of gonadal hormone on GnRH neurons, then, there should be other gonadal hormone-sensitive neurons which mediate gonadal hormones in the regulation of GnRH. Recent studies have found that neurokinin B and Kisspeptin neurons are expressed receptor of gonadal hormone, both can promote to release of GnRH and puberty onset. Therefore, exploring the relationship between NKB, Kisspeptin, GnRH and receptors of gonadal hormone is particularly important.The most researches of NKB, Kisspeptin and GnRH are mostly focused on different species of the central nervous system and its relationship at puberty, the distribution in the reproductive axis and the research about the relationships among NKB, Kisspeptin, GnRH and gonadal hormone remains unclear. In this experiment, subcutaneous implantation, ovariectomized and other treatment methods were used, the distribution of NKB, Kisspeptin and GnRH in the reproductive axis of female SD rats was investigatedwith trinal immunofluorescence assays. Image-pro plus 6.0 software was used to measure the average fluorescence intensity and the number of immunoreactive cells in the reproductive axis. Real-time PCR, enzyme immunoassay, radioimmunoassay and other analysis methods were used to detect the variation of NKB, Kisspeptin wand GnRH when the level of gonadal hormone changed in the body. It will provide a scientific basis for further study about the role and mechanism of NKB, Kisspeptin and GnRH on reproductive function in female rats.The results showed that:(1)the days of vulva opening in the rats of group E2, E2+P4, OVX+E2, OVX+E2+NKB and OVX+E2+P4+NKB were more significantly earlier than group Oil (P<0.01), but the difference of vulva opening time among the five groups was not significant (P>0.05). (2) NKB mainly distributed in the arcuate nucleus, ventromedial nucleus and paraventricular nucleus, adenohypophysis, and ovarian luteal cells, stromal cells, the theca cells and granulosa cells of the secondary follicle in all rats. The fluorescence intensity expression in different position exists difference (P<0.05). â‘ The immunofluorescence intensity on the arcuate nucleus was significantly higher than the ventromedial nucles and paraventricular nucleus (P<0.05). â‘¡The number of NKB-positive neurons in the arcuate nucleus of rat with group ovariectomized were significantly higher than group E2 and E2+P4 (P<0.05). NKB-immunoreactive cells in the rat periventricular nucleus is less (P<0.05), and only distributed in group OVX+E2+P4+NKB, OVX+E2+P4 and E2+P4. â‘¢The fluorescence signal of NKB on the hypothalamus and pituitary in group Oil rats was significantly stronger than group E2, E2+P4, OVX+E2 and OVX+E2+P4 (P<0.05).â‘£The average fluorescence intensity of the ovarian luteal cells, ovarian follicle and granulosa cells was significantly higher than the stromal cells (P<0.05). (3) GnRH was mainly distributed in the rat median eminence, arcuate nucleus and paraventricular nucleus, and the fluorescence signal of median eminence is strongest. â‘ The positive cells of GnRH in the hypothalamic less than ovariectomized rats in the group E2 and E2+P4 (P<0.05). â‘¡The mean fluorescence intensity of GnRH in the median eminence of the rat was significantly higher than the arcuate nucleus and preoptic area (P<0.05), and in the same nucleus, the mean fluorescence intensity of GnRH in group E2 and E2+P4 were also higher. (4) Kisspeptin mainly distributed in the arcuate nucleus, paraventricular nucleus, preoptic area and the periventricular nucleus of the rat hypothalamic. â‘ The number of Kisspeptin positive cells was the highest in the arcuate nucleus, followed by the paraventricular nucleus and preoptic area, less in the periventricular nucleus (P<0.05). The number of Kisspeptin positive cells in the paraventricular nucleus increased in the implanted rats. (5)The expressions of the gene of Tac-2, GnRH and Kiss-1 in group OVX+E2, OVX+E2+P4, OVX+Oil, OVX+Oil+NKB, OVX+E2+NKB and OVX+E2+P4+NKB, were significantly higher than in group E2, E2+P4 and Oil (P<0.05). The level of ERa gene expessed in group OVX+E2+NKB, OVX+E2+P4+NKB and OVX+Oil+NKB was higher than in group E2, E2+P4 and Oil (P<0.05). ERa Gene expression was positively correlated with Tac-2 and GnRH gene but negatively correlated wiht Kiss-1 gene. (6)The concentration of NKB in the serum of the group Oil was significantly higher than group OVX+Oil and OVX+E2 (P<0.05). The difference of the concentration of NKB among group E2+P4, OVX+E2+P4+NKB and OVX+E2+NKB was no significant (P>0.05). The concentration of NKB in the serum of the group E2 and OVX+Oil+NKB was significantly low (P<0.05). (7) The concentration of E2 in the cycling blood at group E2+P4 and E2 was significantly higher than the other groups (P<0.05), the level of P4 of the blood were highest in group E2+P4, E2, OVX+E2+P4 and Oil rats (P<0.05). (8)The correlation coefficient between NKB and E2 was -0.81423, was highly negative correlation. The correlation coefficient between NKB and P4 was-0.478593, showed moderate negative correlation.In conclude:(1) Estradiol and NKB can induce the early time of puberty onset in rats. (2) NKB mainly distributed in the hypothalamic arcuate nucleus, ventromedial nucleus and paraventricular nucleus, adenohypophysis, and ovarian luteal cells, granulosa cells, the theca cells and stromal cells in rats. Kisspeptin mainly distributed in the arcuate nucleus, ventromedial nucleus and preoptic area in rats. GnRH mainly located in the median eminence, arcuate nucleus and preoptic area in rats. NKB and Kisspeptin expressed higher in the arcuate nucleus which GnRH distributed, suggesting that there was a regulation relationship among NKB, Kisspeptin and GnRH, and the arcuate nucleus was the key position in the regulation of reproduction between NKB and Kisspeptin. (3)Gonadal hormones inhibit the NKB, GnRH and Kisspeptin protein and gene expression in the hypothalamus of rat. (4)The hypothalamic NKB neurons mediated regulation of ERa on reproduction. (5)NKB widely distributed in the ovary, it may be involved in the regulation of ovarian function. |
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