Localization And Energy Negative Balance Abundance Of OB-R_b In The Sheep Hypothalamus-Pituitary-Ovary

This research was study on before puberty in Han sheep,Randomized three groups,they are the low nutrition group,the standard nutrition group and the acute fasting group.We used immunohistochemical medthod and fluorescent quantitation PCR(FQ-PCR)technology, aimed to associate the disposition and localization in Hypothalamus,Pituitary and Ovary of OB-Rb. Explore how to regulate the body's energy balance and reproductive breeding of the gene, provided rationale for the link between nutrition and reproduction.The research was constructed the standard real-time fluorescent PCR and standard curve.Total RNA was isolated from the hypothalamus,pituitary,ovaries of Han sheep by Trizole method. cDNA fragment was generated by reverse transcription-polymerase chain reaction(RT-PCR)and cloned into pMD(?) 18—T Vector, The recombinant plasmid was identified by several kinds of specific methods and confirmed that the cDNA fragment sequence was full consistent with the published sheep OB-Rb gene sequence on Genebank.According to the determined concentration of plasmid fluorescence quantitative reaction was carried out using gradient dilution plasmid,standard curve was made and the plasmid standard was constructed.The disposition and localization of OB-Rb mRNA in hypothalamus,pituitary,ovary were detected by immunohistochemical medthod. The results showed that OB-Rb positive particles were distributed in hypothalamus generally,especially arcuate nucleus,paraventricularis nucleus and supraoptic nucleus; OB-Rb positive particles of pituitary were concentrated into pituitarium anterius cells; and OB-Rb positive particles of ovaries appeared in granular cell;granule cells of all types of follicles,but megagametocyte didn't discover the existence of the positive particles.The change expression abundance of OB-Rb mRNA in hypothalamus,pituitary and ovary detected with FQ-PCR technology in Han sheep at three groups. The results suggested that:the OB-Rb mRNA expression in the hypothalamus,the low nutrition group was significantly lower than the standard group and the acute fasting group (P<0.05), the standard group was significantly lower than the acute fasting group (P<0.05); the OB-Rb mRNA expression in the pituitary, the low nutrition group was significantly lower than the standard group and the acute fasting group (P<0.05),but the standard group and the acute fasting group was not significantly (P>0.05); the OB-Rb mRNA expression in the ovary, the low nutrition group was significantly lower than the standard group and the acute fasting group (P<0.05),but the standard group and the acute fasting group was not significantly (P>0.05)The research explored the distribution and localization of OB-Rb mRNA in the hypothalamus, pituitary,ovaries,determined the express regularity between leptin and OB-Rb mRNA in different nutrition, revealed the relation among several hormones in different nutrition,all of above were good for studying for the link between nutrition and reproduction.