鸡胚性腺发育的组织形态学研究

发布时间：2018-10-20 15:14

【摘要】：由于鸡的性别决定及鸡胚性腺发育的分子机理不清楚,所以鸡胚性腺发育过程及其分子调控成为重要的研究方向。目前关于鸡胚性腺的研究已经深入到各个方面,大部分研究集中在性腺发育的分子调控机理,然而关于鸡胚胎期性腺基础形态系统的研究则相对匮乏,仅有极少数零散的研究报道;若没有正常时基本形态作为参照标准,很难判断实验操作效果,因此系统的研究正常状态下性腺的基本形态显得尤为重要。有鉴于此,本研究以0.5天为间隔,采集整个孵化期不同日龄的鸡胚性腺,对其进行性别鉴定分组后,借助石蜡切片和冰冻切片,利用HE染色观察不同倍数物镜下性腺组织的基本形态,了解雌雄性腺基本的结构组成以及二者间的差异;利用PAS染色、SSEA-1免疫组化和c-Kit免疫组化定性追踪生殖细胞的变化过程;利用油红O染色、FOXD1免疫组化和SOX9免疫组化研究支持细胞的生成时间和形态变化。通过定性地分析雌雄性腺中几种主要细胞和性腺结构,以期能深入、系统地从基本组织形态水平丰富整个胚胎期性腺的状态及其变化趋势,同时寻找雌雄性腺组织中主要细胞类型鉴定方法,并拟清几个主要细胞分化或形成的时间,从而全方位的丰富鸡性腺发育的基础学,为今后对鸡胚性腺的研究提供基础对照。主要研究结果如下:(1)HE染色低倍镜下观察发现,雄性鸡胚性腺,分左右两侧,且两侧均发育,但左侧稍大。而雌性鸡胚性腺虽也分为左右两侧,但雌性仅左侧发育,呈不对称发育。(2)HE染色高倍镜下观察发现,雄性性腺无皮、髓之分,含有大量管状结构,在胚胎期第8天清晰可见;雌性有明显的皮、髓质,且髓质紧靠肾的中段。在胚胎期第5.5天已清晰可见皮质部分。髓质部分含有大量空泡状结构,胚胎期第8天可见。(3)PAS染色与SSEA-1免疫组化结果发现原始生殖细胞系在雄性胚胎期性腺的第12.5天左右完全分化;在雌性胚胎期性腺的第11天左右完全分化。(4)油红O染色、SOX9免疫组化以及FOXD1免疫组化结果发现:睾丸支持细胞在胚胎期第6.5天左右开始生成。初期细胞核大,为圆形,而后分裂为多个,体积有所减小。(5)本研究获得了6.5-19天的HE染色、油红O染色、FOXD1免疫组化的图谱,为今后对鸡胚性腺的深入研究奠定了良好的形态学基础。
[Abstract]:Because the sex determination of chicken and the molecular mechanism of chicken embryo gonad development are not clear, the process of chicken embryo gonad development and its molecular regulation become an important research direction. At present, the study of chicken embryonic gonads has been deeply studied, most of which focus on the molecular regulation mechanism of gonad development, but the basic morphological system of chicken embryonic gonad is relatively scarce. There are only a few scattered research reports; it is difficult to judge the effect of experimental operation without normal basic morphology, so it is very important to systematically study the basic morphology of gonads in normal state. In this study, the chicken embryo gonads of different days of incubation period were collected at intervals of 0.5 days. After sex identification, paraffin sections and frozen sections were used. HE staining was used to observe the basic morphology of gonad under different objective lens, to understand the basic structure and the difference between male and female gonads, to trace the change of germ cells qualitatively by PAS staining, SSEA-1 immunohistochemistry and c-Kit immunohistochemistry. The formation time and morphology of Sertoli cells were studied by oil red O staining, FOXD1 immunohistochemistry and SOX9 immunohistochemistry. By qualitatively analyzing several main cell and gonad structures in the female and male gonads, we hope to enrich the state and tendency of the gonads throughout the embryonic period from the basic tissue morphology level in depth and systematically. At the same time, the identification method of the main cell types in the female and male gonad tissues was found, and the time of differentiation or formation of several main cells was determined, so as to enrich the basic science of chicken gonad development in all directions, and to provide the basic control for the future study of the chicken embryo gonads. The main results were as follows: (1) under low power HE staining, the gonads of male chicken embryos were divided into left and right sides and developed on both sides, but the left side was slightly larger. The female embryo gonads were also divided into left and right sides, but the female developed only on the left side and developed asymmetrically. (2) the male gonads were found to have no skin, medullary division, and a large number of tubular structures, which could be seen on the 8th day of embryo. Females have distinct skin, medulla, and medulla close to the middle of the kidney. The cortex was clearly visible on day 5.5 of embryo. The medulla contains a large number of vacuole structures, which can be seen on the 8th day of embryonic phase. (3) PAS staining and SSEA-1 immunohistochemical results showed that the primordial germ cell line was fully differentiated on the 12th day of the male embryonic gonad. (4) Oil red O staining, SOX9 immunohistochemical and FOXD1 immunohistochemical results showed that testicular Sertoli cells began to form around the 65th day of embryo. At the beginning, the nucleus was large, round, and then divided into several. (5) in this study, 6.5-19 days of HE staining, oil red O staining, FOXD1 immunohistochemical patterns were obtained. It provides a good morphological basis for the further study of chicken embryo gonads.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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