【作者】 梁鸿雁；

【导师】 周虚；

【作者基本信息】 吉林大学 ， 基础兽医学， 2009， 博士

【摘要】 本研究系统评述了促卵泡素受体(FSHR)和促黄体素受体(LH/CGR)以及能量对繁殖性能影响的研究进展,探讨了初情期前母猪子宫、输卵管中FSHR和LH/CGR的分布与活性,研究了不同日粮能量水平对子宫、输卵管FSHR和LH/CGR表达的影响。研究发现,初情期前母猪子宫、输卵管中存在功能性的FSHR和LH/CGR,FSHR存在于子宫内膜上皮细胞和输卵管粘膜上皮细胞,LH/CGR存在于子宫内膜上皮细胞、子宫肌层血管平滑肌细胞和输卵管粘膜上皮细胞。促性腺激素可能直接调节子宫、输卵管上相应受体表达,也可能通过卵巢上分泌的性腺激素间接调节。不同日粮能量水平能够影响初情期前母猪外周血清FSH、LH、生长激素(GH)、瘦素、胰岛素(INS)及胰岛素样生长因子-I(IGF-I)浓度。高能量水平可提高血清FSH、LH、瘦素、GH、INS及IGF-Ⅰ的浓度;低能量水平则降低血清中这些激素的浓度;不同日粮能量水平影响初情期前母猪子宫、输卵管FSHR和LH/CGR的表达。高能量水平具有促进作用,低能量水平则起抑制作用。母猪发育阶段和发情周期不同,子宫、输卵管中FSHR和LH/CGR表达量存在差异。卵泡期子宫中FSHR和黄体期子宫中LH/CGR的表达量均高于初情期前子宫,输卵管中FSHR和LH/CGR的表达量均在卵泡期最高。能量可能通过血清INS、IGF-Ⅰ、瘦素、GH等代谢媒介引起垂体促性腺激素分泌的变化,影响FSHR和LH/CGR的表达。更多还原

【Abstract】 Two months old crossbred (Landrace×Large White) gilts were used to investigate: 1) the expression and location of the functional FSHR and LH/CGR in both uterus and oviducts. 2) the effects of dietary energy on hormones release in prepubertal gilts 3) the effects of dietary energy on the expression of gonadotropins receptors in extragonadal prepubertal gilts. The present study was to clarify the preliminary molecular biology mechanism of the regulating role of extragonadal gonadotropins in the extragonadal tissues , and to establish theoretical basis for feeding of prepubertal gilts as well.Firstly, we determined the localization of FHSR and LH/CGR in the uteri and oviducts of prepubertal gilts with immunohistochemical method. The uterus, oviducts and ovaries were collected from five prepubertal gilts. the ovaries were used as the positive control, and the negtive control was non-specific IgG instead of specific antibodies. The results showed that LH/CGR and FSHR were present in the uteri and oviducts of prepubertal gilts. In the uteri, FSHR was expressed in the endometrial epithelial cells, but not in the myometrium and perimetrium. LH/CGR was present in the endometrial epithelial cells and the myometrial vascular smooth muscle. However, there was no LH/CGR in the perimetrium. FSHR and LH/CGR were all expressed in the oviductal mucosal epithelium, but not in the myosalpinx and the serosa. The resluts confirmed that the ovaries was not the only target organ of gonadotropins receptors, and FSHR and LH/CGR were also located in the prepubertal extragonadal organs such as uterus and oviducts.Secondly, we investigated the expression of functional FHSR and LH/CGR in the prepubertal uterus and oviducts with radioactive immunoassay, and Western blotting. Fifteen growth-matched crossbred prepubertal gilts were randomly allocated to three groups after one week reared in the same room. One group animals (n=5) were performed ovariectomy. Two days later, the fasting vein blood was collected from all three groups. Then we carried out treatments according to the experimental design. The groupⅠgilts were injected with physiological saline (5 ml per gilt); the groupⅡgilts were injected, i.m., with eCG (300IU per gilt); the groupⅢgilts (ovariectomizd) were injected with eCG (300IU per gilt). Twenty four hours after eCG treatment, vein blood was collected from all three groups for serum preparation, uteri and oviducts were also removed for detection. The results showed that the concentrations of serum FSH and LH were significantly increased after eCG injection(p<0.01)in groupⅡand groupⅢ. The ovary excision removed the major source of in vivo estrogen synthesis and significantly decreased the concentration of serum estradiol compared with that of intact gilts(p<0.01). After eCG injection, the concentration of serum estradiol in the ovariectomized gilts was significantly lower than that of saline injection pig (p<0.01). Regardless of ovaries removed or not, eCG injection could promote the expression of FSHR and LH/CGR in the uterus and oviducts. The FSHR expression level of uterus and oviducts in intact gilts was significantly higher than that of ovariectomized gilts. The similar consequence was found in oviducal LH/CGR(p<0.05). These results showed that the FSHR and LH/CGR in prepubertal uterus and oviducts were active and functional, and the expression of LH/CGR and FSHR were regulated by FSH, LH, and the estrogen.Finally, we observed the effects of dietary energy on the hormones release and the expression of FSHR and LH/CGR on prepubertal gilts with radioactive immunoassay, fluorescence quantitative-PCR and Western blotting. Fifteen gilts were randomly allocated to three groups, the groupⅠfed with the moderate energy level (100%of NRC energy requirements), the groupⅡfed with the low energy level (90% of NRC energy requirements), the groupⅢfed with the diet of high energy level (100%of NRC energy requirements), for 12d. Then the fasting vein blood was collected from all three groups for serum preparation on d1, d5, d9, and d12.Then uterus and oviducts were collected at the slaughter on d12 The results showed that dietary energy could affect the concentrations of peripheral serum FSH, LH, GH, leptin, Ins, and IGF-Ⅰin prepuberty gilts. High energy level significantly enhanced the concentrations of serum FSH, LH, leptin, GH, insulin and IGF-Ⅰ, whereas the change of GH concentration was not significantly(p<0.01). Low energy level significantly reduced the concentrations of serum FSH, LH, GH, leptin, Ins and IGF-Ⅰ. Dietary energy level affected the expression of FSHR and LH/CGR in prepubertal gilts. High energy intake significantly enhanced the expresioon of uterine and oviducal FSHR and LH/CGR, whereas insufficient energy intake restricted FSHR and LH/CGR expression. In the uterus, the expression level of FSHR mRNA was the highest in follicular phase and LH/CGR mRNA expression level was the highest in luteal phase. In the oviducts, The expression level of FSHR mRNA and LH/CGR mRNA were all the highest in follicular phase .Overall, our results indicated that there were functional FSHR and LH/CGR in uterus and oviducts of prepubertal gilts, and confirmed for the first time that FSHR was present in the endometrium epithelial cells. FSH, LH and estrogens regulated the expression of FSHR and LHR in the uterus and oviducts. Gonadotropins could regulate the uterine and oviductal function not only indirectly through gonadal hormone secreted by ovaries. but also directly through gonadotrophins receptors in the uterus and oviducts. These results imply that dietary energy level could influence the expression of FSHR and LH/CGR in uterus and oviducts which might be mediated by insulin, IGF-Ⅰ, leptin and GH, These results provided the hypothesis that gonadotropins might play direct roles on the extragonadal organs and consummate the mechanism of gonadotropins in animal reproduction. Furthermore, we explored the effect and mechanism of energy on reproductive development of prepubertal gilts from aspects of reproductive endocrinology, gene and protein expression, which might provide a theoretical basis for feeding of the prepubertal gilts.更多还原