【作者】 黄丽波；

【导师】 王树迎；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2013， 博士

【摘要】 促性腺激素(GTH)、促性腺激素释放激素(GnRH)、生长激素(GH)及Ghrelin在哺乳动物的生殖调控中起着关键作用，其生理调控作用主要是通过与相应的受体FSHR、LHR、GnRHR、GHR结合来介导完成。因此，研究这些激素和受体在生后发育过程中垂体、卵巢内的表达与分布情况，是理解其在生殖发育调控中如何影响垂体-卵巢轴作用机理的基础。本试验以D0-D180日龄的雌性济宁青山羊为研究对象，采用免疫组织化学、RT-PCR和Western-blot方法研究从出生至性成熟(D0-D180)过程中垂体-卵巢轴中这些激素和受体表达定位以及mRNA表达规律，结果表明：D0-D180日龄血清中雌激素(E2)含量差异显著，孕激素变化趋势与E2相近，含量均以D0为多，D30显著下降，在D90和D180分别出现两个峰值，P是D120骤然上升。FSH在D0、D90有所升高；LH变化趋势基本同E2，在D90出现峰值。P的峰值出现是在LH之后，符合LH在促进排卵后，刺激P分泌的生理规律。而E2、FSH、LH含量在D90时处于高峰，可能是济宁青山羊发情早的原因之一。垂体内FSH、LH、Ghrelin、GnRH、GnRHR、GH、GHR、AR阳性细胞及其mRNA在D0-D180日龄山羊腺垂体内均有分布和表达，阳性细胞呈区域状分布，随着日龄增长阳性区域也增大。垂体细胞内GH、Ghrelin与LH分布特点和mRNA变化趋势相同，以D60显著高于其他月龄(p<0.05)，其次是D30、D90，提示在D30-90阶段垂体细胞通过大量合成分泌这些激素，调控垂体自身及卵巢等器官的功能活动，这可能是济宁青山羊性成熟早的物质基础。垂体和卵巢GnRHR、GHR、AR mRNA表达量在D60-D180间变化不大，提示受体含量在生后发育至性成熟阶段是相对恒定的，真正影响功能发挥的是取决于其相应激素含量和分布位置。在垂体和卵巢内有Ghrelin、GnRH免疫阳性细胞和mRNA表达，可能是源于垂体和卵巢对Ghrelin、GnRH的“需求”，并通过自身细胞以自分泌或旁分泌的方式来产生。AR阳性细胞的存在说明垂体和卵巢细胞也接受雄激素的调控，AR在垂体是D60阶段高表达，与绵羊相同；而AR在卵巢随着卵泡发育，在基质细胞和颗粒细胞内表达量增多。FSHR、LHR、Ghrelin、GHR阳性细胞在各月龄山羊的卵巢内分布位置因日龄不同而呈现较大差异。特别是在刚出生的D0阶段存在明显差异。在刚出生时集中分布在不规则原始卵泡的卵母细胞内，但随月龄增长，是在少数次级卵泡上存在，D30-180阶段常在1-2个生长卵泡上分布。当次级卵泡发生闭锁，则可在卵泡壁上见有环形排列的Ghrelin、GHR阳性细胞均匀分布。卵巢内动脉管壁的平滑肌上呈GHR、FSHR、LHR、GnRHR、GnRH阳性，而Ghrelin在动脉管壁周围的结缔组织呈强阳性。几种物质的mRNA表达量与组化结果变化趋势基本相近，结论：垂体内的FSH、LH、GH、Ghrelin、GnRH、GnRHR在生后发育过程中阳性细胞分布及mRNA表达量与生长阶段密切相关，几种激素可能协同调控垂体自身及卵巢等器官的功能活动。FSHR、LHR、Ghrelin、GnRHR、GHR在卵巢内分布的差异性说明其对于卵泡发育过程中调控随机体生长而异，其细胞内的mRNA是否表达相应的蛋白与卵泡发育相关联。垂体和卵巢还可以通过自身细胞以自分泌或旁分泌的方式产生Ghrelin、GnRH。更多还原

【Abstract】 Gonadotrophin (GTH),gonadotropin-releasing hormone (GnRH), growth hormone (GH)and Ghrelin plays a key role in mammalian reproductive regulation, their physiologicalregulatory function mainly are mediated through binding receptors, FSHR and LHR, GnRHR,GHR. Therefore, studying the expression and distribution of these hormones and theirreceptor in pituitary and ovary during postnatal development, is base of understanding how toaffect the pituitary-ovarian axis during their reproductive development regulation.We studied the expression of GTH, GnRH and GH, and their receptors in pituitary-ovarian axis of female Jining goat aged from day0(D0) to day180(D180) at the level ofprotein and mRNA using the methods of IHC, RT-PCR and Western-blot; detected thelocalization and mRNA expression of these hormones and receptors.. The results show that,The content of serum estrogen (E2) had significant differences from D0to D180day.Progesterone (P) had similar changing trend with E2, the level of them were high at D0,dropped significantly at D30, and appeared two peak at the D90and D180respectively, andthe level of P at rised sharply D120.. The level of FSH was also increased on D0and D90;With E2, the change trend of LH was similar with that of E2, appeared peak at D90. The peakof E2, FSH, and LH levels appeared at D90may be one of the reasons of early estrus in jininggrey goats.2. FSH, LH, Ghrelin, GnRH, GnRHR, GH, GHR, AR positive cells and their mRNAwithin the anterior pituitary all showed specific distribution and expression from D0to D180.immunoreactive positive cells showed regional distribution, and positive reactivity increasedwith age. Pituitary GH, Ghrelin and LH distribution in cells and mRNA expression have thesame change trend, with the characteristic of significant peak at the D60(p <0.05), followedby D30, D90, this hints at the stage of D30-90, the pituitary gland can regulate the function ofitself and ovary through a lot of synthetic secretion of the hormones in pituitary cells, it maybe the material basis for the early sexual maturity in jining grey goats.GnRHR, GHR, AR mRNA expression in pituitary gland and ovary had a little ofchanges between the D60and D180, indicating these hormone receptors were relativelyconstant in postnatal and sexual maturity stage, the effect of these hormones on pituitarygland and ovary may depends on the level of themselves.The Ghrelin-ip, GnRH-ip cell andmRNA expression in pituitary and ovary may be the result of "requirements" of pituitary and ovary. The expression of AR-ip cells in pituitary and ovary also indicate that androgen has afunction of regulation for pituitary and ovary.The distribution of FSHR, LHR, Ghrelin and GHR positive cells in ovaries appearedbigger difference with different ages, especially in postbirth. The immunoreactivityconcentrated in irregular primordial follicle oocytes just postborn, but also appeared in aminority of secondary follicle with growth, on phase of D30to D180are often distributed in1-2growing follicles. When secondary follicle appears atresia, Ghrelin, GHR positive cellswere distributed evenly with style of a circular permutation in the follicular wall. GHR, andLHR,FSHR,GHR,GnRHR,GnRH is positive on the smooth muscle of artery walls withinovary, and Ghrelin showed strong positive surrounding connective tissue of artery walls.Amount of mRNA expression of several substances showed similar changing trend with theresults of immnohistochemistry stain.In summary, FSH, LH, GH, Ghrelin, GnRH, GnRHR immunoreactivity cells and theirmRNA expression in anterior pituitary during postnatal was closely related to the growthstage, several kinds of hormones can synergistically regulate the function and activity ofpituitary and ovary. Distribution differences of FSHR,LHR,Ghrelin,GnRHR and GHR inovary illustrate that the regulation for follicular development vary with the body growth,whether the translation of these hormone receptor mRNA associate with folliculardevelopment. Pituitary and ovary can produce Ghrelin, GnRH through the way of autocrine orparacrine.更多还原