【摘要】：由于雞的性別決定及雞胚性腺發(fā)育的分子機(jī)理不清楚,所以雞胚性腺發(fā)育過(guò)程及其分子調(diào)控成為重要的研究方向。目前關(guān)于雞胚性腺的研究已經(jīng)深入到各個(gè)方面,大部分研究集中在性腺發(fā)育的分子調(diào)控機(jī)理,然而關(guān)于雞胚胎期性腺基礎(chǔ)形態(tài)系統(tǒng)的研究則相對(duì)匱乏,僅有極少數(shù)零散的研究報(bào)道;若沒有正常時(shí)基本形態(tài)作為參照標(biāo)準(zhǔn),很難判斷實(shí)驗(yàn)操作效果,因此系統(tǒng)的研究正常狀態(tài)下性腺的基本形態(tài)顯得尤為重要。有鑒于此,本研究以0.5天為間隔,采集整個(gè)孵化期不同日齡的雞胚性腺,對(duì)其進(jìn)行性別鑒定分組后,借助石蠟切片和冰凍切片,利用HE染色觀察不同倍數(shù)物鏡下性腺組織的基本形態(tài),了解雌雄性腺基本的結(jié)構(gòu)組成以及二者間的差異;利用PAS染色、SSEA-1免疫組化和c-Kit免疫組化定性追蹤生殖細(xì)胞的變化過(guò)程;利用油紅O染色、FOXD1免疫組化和SOX9免疫組化研究支持細(xì)胞的生成時(shí)間和形態(tài)變化。通過(guò)定性地分析雌雄性腺中幾種主要細(xì)胞和性腺結(jié)構(gòu),以期能深入、系統(tǒng)地從基本組織形態(tài)水平豐富整個(gè)胚胎期性腺的狀態(tài)及其變化趨勢(shì),同時(shí)尋找雌雄性腺組織中主要細(xì)胞類型鑒定方法,并擬清幾個(gè)主要細(xì)胞分化或形成的時(shí)間,從而全方位的豐富雞性腺發(fā)育的基礎(chǔ)學(xué),為今后對(duì)雞胚性腺的研究提供基礎(chǔ)對(duì)照。主要研究結(jié)果如下:(1)HE染色低倍鏡下觀察發(fā)現(xiàn),雄性雞胚性腺,分左右兩側(cè),且兩側(cè)均發(fā)育,但左側(cè)稍大。而雌性雞胚性腺雖也分為左右兩側(cè),但雌性僅左側(cè)發(fā)育,呈不對(duì)稱發(fā)育。(2)HE染色高倍鏡下觀察發(fā)現(xiàn),雄性性腺無(wú)皮、髓之分,含有大量管狀結(jié)構(gòu),在胚胎期第8天清晰可見;雌性有明顯的皮、髓質(zhì),且髓質(zhì)緊靠腎的中段。在胚胎期第5.5天已清晰可見皮質(zhì)部分。髓質(zhì)部分含有大量空泡狀結(jié)構(gòu),胚胎期第8天可見。(3)PAS染色與SSEA-1免疫組化結(jié)果發(fā)現(xiàn)原始生殖細(xì)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)油紅O染色、SOX9免疫組化以及FOXD1免疫組化結(jié)果發(fā)現(xiàn):睪丸支持細(xì)胞在胚胎期第6.5天左右開始生成。初期細(xì)胞核大,為圓形,而后分裂為多個(gè),體積有所減小。(5)本研究獲得了6.5-19天的HE染色、油紅O染色、FOXD1免疫組化的圖譜,為今后對(duì)雞胚性腺的深入研究奠定了良好的形態(tài)學(xué)基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S831

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