【作者】 石运芝；

【导师】 王树迎；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2014， 博士

【摘要】 济宁青山羊是我国一个优秀地方品种，具有性成熟早和繁殖率高的特点。从激素和受体表达水平等角度研究青山羊性发育过程，对于阐明济宁青山羊性成熟早的机理和培育新的高繁殖力山羊品种或品系等具有重要意义。本研究以雌性济宁青山羊为研究对象，应用放射免疫、组织化学、免疫组织化学、透射电镜、实时荧光定量PCR和WesternBlot等方法，从激素水平、细胞水平、亚细胞水平和分子水平等层面系统研究从出生到性成熟山羊血液中雌孕激素的波动、雌孕激素受体在下丘脑-垂体-卵巢轴以及雌孕激素受体在生殖器官发育过程中的增龄性分布特点及变化规律。研究结果显示，在雌性济宁青山羊生后发育阶段：①下丘脑与生殖有关的核团均有ER和PR阳性细胞分布，各核团ERα、ERβ和PR阳性细胞随日龄增长而数量增多，且同一日龄同一核团内ERβ阳性着色明显强于ERα（p<0.05）。PR免疫反应阳性神经元的分布和变化规律与ERβ基本一致。ERα、ERβ和PR阳性细胞在下丘脑中分布的增龄性变化与济宁青山羊性发育和性早熟的特点密切相关；②腺垂体嗜色细胞随着年龄增长而数量增多，体积增大；ERα主要分布于嗜色细胞的细胞核，ERβ和PR主要分布于部分嗜色细胞的细胞质；初情期（D60），垂体嗜色细胞进入形态学快速生长期，ERα和ERβ的表达明显上调。PR阳性细胞AOD的峰值出现在D120，此时青山羊已达性成熟，PR的高表达与垂体的功能状态同步；③卵巢具有发育快和成熟早的特点，初生母羔的卵巢内即有生长卵泡，ERα、ERβ和PR的合成及其mRNA的表达均较高。出生至30日龄，为卵巢形态上的快速生长发育期，重量和体积均显著增加，内、外皮质区的分带消失。60～90日龄，为卵巢机能上的发育完善时期。60日龄已排卵，出现黄体或白体，进入初情期，ERα、ERβ和PR及其mRNA的表达明显上调；90日龄为性成熟期，成熟卵泡最大直径可达6.18mm；除ERα表达下调外，ERβ和PR及其mRNA的表达均维持在高水平状态。初情期之前，卵巢雌激素的优势受体为ERα，性成熟后则为ERβ；④不同日龄子宫内均有ERα、ERβ和PR及其mRNA的表达。ERα、ERβ和PR蛋白免疫阳性产物定位于子宫腔上皮细胞、腺上皮细胞、基质细胞、血管内皮细胞及平滑肌细胞的核中。ERα和ERβ表达从60日龄明显增多，90日龄最多。出生以后，PR出现两个高峰点是120日龄和180日龄。ER和PR及其mRNA在出生后不同发育阶段子宫内的分布及表达状况，为山羊的多胎性提供良好的子宫内环境；⑤输卵管壶腹部和伞部上皮内存在ERα、ERβ、PR、FSHR和LHR，并且这些受体在输卵管上皮发育过程中的表达具有时空特异性。性成熟前（D0～D60），山羊下丘脑-垂体-卵巢轴尚未发育成熟，输卵管壶腹部和伞部上皮中ERα、ERβ、PR、FSHR和LHR的表达少，阳性细胞积分光密度值低，性成熟（D90～D180）后，输卵管上皮中ERα、ERβ、PR、FSHR和LHR的表达升高，激素受体与配体结合进而调节输卵管上皮细胞的分化发育，使壶腹部和伞部上皮中纤毛细胞和分泌细胞的数量增多，上皮细胞含有的糖原或粘液增多，上皮功能更加活跃。D150开始在壶腹部和伞部上皮的游离缘出现“腔排”现象，“腔排”物为全浆分泌的分泌细胞。更多还原

【Abstract】 As a first-class strain in China, the Jining Gray Goat is characterised by early sexualmaturity and high progenitive rate. To study the sexual development of the Jining Gray Goatby investigating its hormone level and receptor expression is of great significance to suchefforts as to elucidate the mechanism for its early sexual maturity and to breed new strains orlines of goats with high progenitive capacity. In this study, the Jining Gray Goat wasinvestigated by means of radioimmunological, histochemical and immunohistochemicalmethods, transmission electron microscope, reverse transcription-polymerase chain reaction(RT-PCR) and Western Blot. The fluctuations of estrogen and progesterone in the blood,age-related distribution changes of estrogen receptors and progesterone receptors in thehypothalamus-hypophysis-ovary axis and in the reproductive organs of the goat from beingborn to sexual maturity were systematically studied on the level of hormones, cells,subcellular fractions and molecules. The study showed after the female goat was born:①ERand PR positive cells were present in reproduction-associated nuclei in the hypothalumus, thenumber of the cells increasing with the goat’s age and positive staining of ERβ being muchstronger than that of ERα（p<0.05） in the same nuclei at the same stage. The age-relateddistribution changes of ERα，ERβ and PR positive cells in the hypothalumus were closelylinked to the goat’s early sexual development and maturity. The distribution and the changerule of PR positive neurons and and that of ERβ are basically identical;②The chromophilcells in the adenohypophysis grew in number and volume with age; ERα was mostly presentin the nuclei of chromophil cells, ERβ and PR in cytoplasm; at puberty (D60), chromophilcells of the pituitary began a growth spurt morphologically with an remarkable increase of theexpression of ERα and ERβ; AOD peak of PR positive cells appeared at D120, when the goatwas sexually mature, and the high expression of PR appeared with the changes of pituitaryfunctions；③The ovary of the goat typically develops fast and matures at an early stage. Thenewborn ewe has growing follicles and high expression of ERα, ERβ, PR and mRNA in itsovary. From0to30days of age, rapid morphological growth was seen, weight and volumeincreased significantly; the boundary between the inner and outer cortex disappeared. Ovarianfunction improved remarkably when the kids were60-90days old. When60days old, the animals began to ovulate, corpus luteum or corpus albicans appeared and the expression ofERα, ERβ and PR and their mRNA significantly increased, which indicated the animalsreached puberty. On day90, the animals manifested sexual maturity, with the biggest maturefollicle6.18mm in diameter; the expression of ERβ and PR protein and their mRNA wasmaintained at a high level, with decreased expression of ERα. Before puberty, the expressionof ovarian ERα (prepubertal dominant receptor) and it’s mRNA was significantly higher thanthat of ERβ (dominant receptor after sexual maturity);④The expression of ERα mRNA, ERβmRNA and PR mRNA was present in the uterus of the goat of different ages. The proteinimmune positive products of ERα, ERβ and PR were located in the nuclei of epithelial cells ofthe uterine cavity, glandular epithelial cells, stromal cells, vascular endothelial cellsand smooth muscle cells. The expression of ERα and ERβ became increasingly strong fromD60, and became strongest at D90. Two peaks of PR appeared separately at D120and D180.The distribution and expression of ER, PR and their mRNA in the uterus of the goat atdifferent postnatal development stages suggested that the goat’s prolificacy was linked to itsexcellent intrauterine condition;⑤ERα, ERβ, PR, FSHR and LHR were present in theepithelium of fallopian tubal ampullae and fimbria tube and these receptors hastime-and-space specific expression in the epithelium of oviduct. Before sexual maturity(D0-D60), the goat’s hypothalamus-pituitary-ovary axis was not mature, there was weakexpression of ERα, ERβ, PR, FSHR and LHR in the epithelium of fallopian tubal ampullaeand fimbria tube, and the intergral optical density of positive cells was low; After sexualmaturity (D90-D180), the expression of ERα, ERβ, PR, FSHR and LHR in the epithelium ofoviduct increased, hormone receptors bound to ligands regulated the differentiation anddevelopment of the epithelium, which increased the number of ciliated cells and secretorycells in the epithelium of fallopian tubal ampullae and fimbria tube; therefore the epithelialcells had more glycogen and mucus and the epithelium functioned more actively. Besides,“cavity discharge” phenomenon was found in the goat aged D150and those older at the freeedge of the epithelium of fallopian tubal ampullae and fimbria tube and the products of“cavity discharge” were holocrine secretory cells.更多还原