小鼠胚胎雌雄原始生殖细胞发育的比较研究

发布时间：2018-05-14 11:04

本文选题：原始生殖细胞 + 增殖率　；参考：《南方医科大学》2010年硕士论文

【摘要】： C57BL/6J小鼠是标准的近交系小鼠,其胚胎期生殖发育的过程与人的有许多相同之处,而且遗传背景清楚,便于研究,是胚胎生殖发育研究较理想的生物模式材料。胚胎组织切片标本的制作与观察是研究生殖发育的一个必不可少的重要手段,为形态学研究与免疫荧光技术等研究提供技术平台。目前小鼠胚胎的生殖发育仍有很多难题未被解决,已有的资料大多是通过体外分离培养研究所得,很多体内的发育问题仍困惑着科学家,需要进行更多的研究来解答。C57BL/6J小鼠妊娠期19-21天,在胚胎第11天至13.5天之间生殖嵴与原始生殖细胞的发育变化明显可见。8.5天-10.5天时原始生殖细胞向生殖嵴移动并增殖。11.5天时多半进入生殖嵴,失去其运动能力,并大量增殖。13.5天之后原始生殖细胞在生殖嵴内的增殖完成。接着雌性生殖细胞即开始进入减数分裂,在15.5天大多数进入第一次减数分裂并最终停留在前期的双线期。而雄性生殖细胞则处于静止状态,出生后5天再开始有丝分裂。目前国内外关于小鼠生殖嵴胚胎发育形态学研究的全面报道较少,而且很少有研究关注不同性别之间胚胎期生殖细胞发育的区别。本研究以C57BL/6J小鼠胚胎生殖嵴为实验对象,采用石蜡切片苏木精-伊红染色、免疫荧光组织化学法以及蛋白印迹分析等方法,研究C57BL/6J小鼠在11天至13.5天之间生殖嵴发育的形态学改变以及雌性和雄性之间原始生殖细胞在数量与增殖率的区别,初步探讨正常C57BL/6J小鼠雌性与雄性原始生殖细胞发育的区别,为生殖胚胎发育的分子水平调控的深入研究提供了依据。一、鼠整胚组织切片标本的制作与观察分别取不同天数的小鼠胚胎(11天,11.5天,12天,12.5天,13天,13.5天),每个胚胎取少量组织PC R法鉴定胚胎性别。整胚组织采用4%多聚甲醛固定液固定,接着从低浓度乙醇开始梯度脱水,二甲苯透明,浸蜡后包埋,5um连续切片。石蜡块切片后做HE染色。选择含有生殖嵴的切片,光学显微镜下观察11天至13.5天胚胎生殖嵴的组织形态学变化以及性别分化特征。组织切片观察发现,小鼠胚胎的细胞与组织的形态被很好的展现出来,我们能很轻松的观察到从11天至13.5天生殖嵴形态结构上的改变、胚胎性别分化的特征以及原始生殖细胞的形态。结果发现,小鼠胚胎11-13.5天生殖嵴的发育迅速。11天的鼠胚生殖嵴已经形成,且其中可见迁移来的原始生殖细胞。原始生殖细胞体积大,核大且呈圆形或卵圆形。11.5天鼠胚,生殖嵴变厚变长。12天的鼠胚生殖嵴性别尚未分化。12.5天的鼠胚开始出现性别分化的迹象。13天的鼠胚性别明显分化,雄性的原始性索开始分化为实心原始生精小管。13.5天的鼠胚,雄性原始生精小管结构明显,雌性皮质索孤立细胞团形成原始卵泡。生殖嵴石蜡切片的制作,可以为生殖嵴免疫荧光研究提供材料,为更进一步研究小鼠胚胎生殖发育以及原始生殖细胞的发育提供了实验依据。二、生殖嵴中原始生殖细胞的免疫荧光组织化学检测雌雄性生殖细胞特异性表达小鼠脉管同系物(Mouse vasa homolog, Mvh)。采用免疫荧光双染的方法用Mvh抗体标记原始生殖细胞,用增殖细胞核抗原(Proliferating Cell Nuclear Antigen, PCNA)标记处于增殖中的细胞。从每个胚胎的连续切片中间隔选取3-4张切片进行免疫荧光双染,拍照后截取相同大小的面积,计数Mvh阳性与Mvh与PCNA共阳性的细胞数,即原始生殖细胞数与正处于增殖中的原始生殖细胞数。根据后者与前者的比值计算出原始生殖细胞的增殖率。结果发现,11、天生殖嵴面积小,未参与计数。对于原始生殖细胞的数量与增殖率,11.5天-12.5天的雌性和雄性胚胎之间都没有显著差别(P0.01),但其数量在雄性13天(135.83±4.57)和13.5天(152.23±5.54)显著多于雌性13天(97.08±4.72)和13.5天(121.22±9.27)(P=0.000),增殖率在13天雄性(20.23±0.34%)较雌性(16.95±0.35%)高(P=0.000),13.5天没有显著区别(P0.01)。雌性和雄性胚胎原始生殖细胞的增殖率都在13天最高,数量在13.5天最多。三、蛋白印迹法检测生殖嵴中Mvh和PCNA蛋白的表达取13天、13.5天胚胎的生殖嵴,提取蛋白做蛋白印迹检测,分析比较雌性与雄性Mvh与PCNA蛋白的表达。结果发现：以β-tubulin为内参,Mvh蛋白的表达在13天与13.5天雄性显著高于雌性,PCNA表达在13天雄性也高于雌性,13.5天没有显著区别。说明13天、13.5天雄性原始生殖细胞数量较雌性多,13天时雄性生殖嵴内的细胞增殖率较雌性高,与免疫荧光结果大致相同。本实验结果表明C57BL/6小鼠雌性和雄性胚胎的原始生殖细胞发育是有显著区别的。在原始生殖细胞进入生殖嵴且性别分化明显之后、停止增殖之前,即主要在胚胎期13天雄性的原始生殖细胞较雌性增殖迅速,且数量上显著高于雌性,这为更进一步研究小鼠胚胎发育过程中原始生殖细胞的基因表达变化以及某些基因敲除后对不同性别小鼠生殖的影响提供了实验依据。
[Abstract]:In this study , the reproductive development of C57BL / 6J mice was studied by means of morphological study and immunofluorescence technique .

Preparation and observation of tissue sections of mouse whole embryo tissue

The histological changes of embryonic germ cells and the characteristics of sex differentiation were observed in the embryonic germ cells from 11 to 13.5 days . The morphological changes and the sex differentiation of the embryonic germ crista were observed from 11 to 13.5 days . The changes of the morphological structure of the genital ridge from 11 to 13.5 days were observed . The characteristics of the sex differentiation and the morphology of the primordial germ cells were observed .

The results showed that the reproductive ridge of mouse embryo in 11 - 13.5 days was developed rapidly . The reproductive ridge of mouse embryo in 11 days had been formed , and the primordial germ cells migrated were observed .

Immunofluorescence histochemical detection of primordial germ cells in the genital ridge

Male and female germ cells specifically express the mouse vascular homologue ( Mouse vasa sarcoma , Mv h ) . The number of cells with the same size was taken from the continuous section of each embryo , and the number of cells with the same size was taken after taking pictures . The number of cells with the same size was counted after taking pictures . The number of primordial germ cells and the number of primordial germ cells in the proliferating cell were counted . The proliferative rate of primordial germ cells was calculated from the ratio of the latter to the former .

The results showed that there was no significant difference in the number of primordial germ cells ( 135.83 卤 4.57 ) and 13.5 days ( 152.23 卤 5.54 ) than that in females ( 97.08 卤 4.72 ) and 13.5 days ( 125.22 卤 9.27 ) ( P = 0.000 ) , but there was no significant difference between males ( 20.23 卤 0.34 % ) and females ( 16.95 卤 0.35 % ) ( P = 0.000 ) .

3 . Expression of PCNA protein in the genital ridge by Western blotting

The results showed that the expression of PCNA in 13 days and 13.5 days was significantly higher than that in females and 13.5 days . The results showed that the number of male primordial germ cells was higher in 13 days and 13.5 days .

The results showed that the primordial germ cell development of the female and male embryos of C57BL / 6 mice was significantly different . After the primordial germ cells entered the genital ridge and the sex differentiation was obvious , the primordial germ cells were more rapidly and significantly higher than that of the female in 13 days of the embryonic stage , which provided an experimental basis for the further study of the gene expression changes of the primordial germ cells during the mouse embryo development and the influence of certain gene knockout mice on the reproduction of different sex mice .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.1

【共引文献】