| Objective: In this paper,we discussed the distribution of Nesfatin-1/NUCB2 in the hypothalamus-pituitary-ovarian axis,and analyzed the expression of Nesfatin-1/NUCB2 mRNA in rat hypothalamus during puberty development and the relation of reproductive hormone genes.We investigated the effect of exogenous Nesfatin-1 on puberty onset,gonadotropin,gonadal hormone secretion and gene expression.The time and space expression of Nesfatin-1/NUCB2 and its mRNA,the mechanism and effect of Nesfatin-1on puberty onset were explored,which will be provided as the scientific theoretical basis for further researching for Nesfatin-1/NUCB2 in regulation of the reproductive system.Methods:(1)Hypothalamus,pituitary,ovary and uterus were separated from female adult SD rats,and then fixed with 4% paraformaldehyde,made into paraffin sections to observe the distribution of Nesfatin-1 by immunohistochemistry.(2)The expression of Nesfatin-1/NUCB2 mRNA,Tac2 mRNA,GnRH mRNA were determined by real-time fluorescent quantitative PCR(qRT-PCR)in the hypothalamus of rats during puberty development,and analysed the correlation between the genes.(3)The rats were treated with intraperitoneal injection using 2% sodium pentobarbital to anesthesia,and then were fixed in a stereotaxic apparatus.Female rats at prepuberty and adult were treated with 50 pmol Nesfatin-1 by intracerebroventricular injection respectively.Blood samples were collected at 15 minutes and 60 minutes,respectively.The concentrations of LH and FSH in the serum were determined with enzyme-linked immunosorbent(ELISA).(4)Famale rats at prepuberty were administrated with Nesfatin-1 by intracerebroventricular injection and continued to feed until the day of vaginal opening.After decapitation,the blood,hypothalamus,pituitary and right ovary were rapidly collected.The expression of LH,FSH,GnRH and its receptor and ER? m RNA were determined with qRT-PCR.Left ovaries were collected for wet weight,routine paraffin section and HE staining to observe histological structure.The concentration of estrogen in the serum was assayed by ELISA.Results:(1)Nesfatin-1/NUCB2 were mainly distributed in the arcuate nucleus,supraoptic nucleus,paraventricular nucleus,lateral hypothalamic area,anterior pituitary,ovocyte,corpus luteum,interstitial cells,granulosa cells,connective tissue of chorion,myometrium,endometrium gland and mesothelium.(2)Nesfatin-1/NUCB2 mRNA was expressed in the hypothalamus of rat during puberty development and changed with thedevelopment period.The expression of Nesfatin-1/NUCB2 mRNA were positively correlated with Tac2 mRNA(r=0.940,P<0.001),and the highest expression at prepuberty was found compared to other periods(P<0.01).(3)Serum LH level was enhanced at 15 and 60 minutes after intracerebroventricular injection of Nesfatin-1during the prepuberty,LH level was more significantly(P<0.05)at 15 min compared to60 min,but FSH level was not obvious,enhanced at 15 min(P<0.05)and it was unable to modify FSH level at 60 min(P>0.05).Nesfatin-1 could not affect the secretion of gonadotropin in adult rats(P>0.05).(4)Nesfatin-1 could significantly advance vaginal opening,enhance ovarian weight and serum estrogen secretion(P<0.05).The result of HE staining showed that large ovarian corpus luteum was observed in Nesfatin-1 group.Intracerebroventricular injection of Nesfatin-1 significantly increased the expression of GnRH?GnRHR?LH??LHR?FSH??ER? mRNA(P<0.05),but the expression of FSHR mRNA was not significant(P>0.05).Conclusion:(1)Nesfatin-1/NUCB2 is widely distributed in rat hypothalamus,pituitary ovary and uterus.(2)Nesfatin-1/NUCB2 mRNA is changed with the development period and the highest expression is found in the prepuberty,Nesfatin-1/NUCB2 mRNA is positively correlated with Tac2 m RNA.(3)Nesfatin-1 play a role in the gonad axis at the central level and enhance the secretion of FSH/LH in pituitary of rat during prepuberty not adult.(4)Nesfatin-1 accelerate the puberty onset and increase the gene expression of GnRH?GnRHR?LH??LHR?FSH??ER?. |
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