MIWI蛋白在小鼠睾丸发育过程中的研究

发布时间：2018-01-25 04:18

本文关键词： 睾丸发育精子发生 MIWI蛋白 Chromatoid Body 免疫电镜　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 前言生物通过生殖实现亲代与后代个体之间生命的延续。在雄性哺乳动物的生殖过程中,精子发生是个高度协调的过程。精子发生被分为三个阶段：第一阶段是生殖干细胞起始的有丝分裂过程,其通过有丝分裂转变为精原细胞最终成为精母细胞。第二阶段是精母细胞通过减数分裂转变为精子细胞,导致了单倍体圆形精子细胞的产生。第三个阶段是圆形精子细胞经过形态转变而成为精子。目前研究认为,成熟的精子不再有转录发生,因此与精子发生有关的基因在精子发生的早期阶段即开始转录。但这些转录产物并未立即翻译形成蛋白产物,而是呈抑制状态达数天,直到精子发生需要时才进行翻译。在由精原细胞转变为成熟精子的漫长过程中有很多RNA和蛋白小分子物质的参与,是一个精确和可调控的过程。本实验中所涉及的MIWI蛋白便是最近发现的一个重要的作用于该过程的小分子物质。 MIWI蛋白是Argonaute蛋白家族的重要成员之一。Argonaute蛋白家族是RNA诱导沉默复合体(RISC)的核心元件,在RNA干扰(RNAi)中发挥重要作用。当小RNA与具有同源序列的靶mRNA或靶DNA结合后,Argonaute家族蛋白识别部分或者全部的核酸靶标,借以来调节mRNA的稳定性、蛋白的合成和基因组的结构。Argonaute蛋白主要包括Ago和PIWI两个亚家族。其中的PIWI亚家族主要有3个成员,分别是MIWI, MILI和MIWI2,是生殖系细胞中的特异性蛋白,与精子的发生有非常密切的关系,敲除Miwi, Mili或Miwi2基因,都会造成小鼠精子产生明显缺陷,表现为雄性不育。此亚家族与生殖细胞特异性事件相关,并且特异性分布于生殖细胞中,其表达与细胞的生长发育调节相关联。小鼠中的MIWI蛋白是这个亚家族的代表。正是由于生殖细胞表达集中的特异性,尤其是哺乳动物睾丸表达的特异性,对PIWI亚家族蛋白功能的研究主要于精子发生。目前的研究表明,在哺乳动物的生殖细胞中,PIWI亚家族蛋白通过与piRNA (piwi-interacting RNA)的结合形成piRNA复合物(piRC)来调控基因沉默途径。通过对MIWI蛋白的分析研究,有利于我们进一步了解生物配子发生的分子调控及其机理,对理解此蛋白的作用机制也有重要意义,还可以为不育症找到新的靶点,从而为临床治疗开拓思路及提供理论依据。实验材料各年龄段的SPF级C57BL／6J小鼠睾丸组织；常规免疫组化试剂盒、MIWI抗体、羊抗兔二抗、锇酸、2.5%戊二醛、4%多聚甲醛、梯度酒精(70%,80%,90%,95%,100%)、二甲苯、石蜡、苏木素、中性树胶方法 1、提取各年龄段小鼠睾丸组织蛋白,进行免疫印迹实验,检测是否存在MIWI蛋白的表达； 2、制作小鼠睾丸组织石蜡切片,进行免疫组化实验,明确MIWI蛋白在细胞水平的表达定位； 3、进一步采用免疫电镜技术,检测MIWI蛋白在亚细胞水平的表达定位。结果 1.、Western结果显示了MIWI蛋白在小鼠发育的胚胎的各组织中无表达,而是存在于成年小鼠睾丸组织中,并在小鼠发育至6周、8周检测到MIWI蛋白的表达。 2、免疫组化结果显示MIWI蛋白在小鼠睾丸发育的精母细胞与精子细胞时期表达。 3、亚细胞定位显示MIWI蛋白存在于紧贴核膜处,表明其在核质运输中可能起到重要作用,是Chromtoid Body的重要组成成分。讨论 MIWI蛋白在成熟小鼠睾丸中特异性表达。通过本实验研究,MIWI蛋白只在成年小鼠的睾丸中表达,在胚胎期以及出生后4周内小鼠的各组织中均无表达。MIWI蛋白的定位显示了生殖细胞中特异结构的存在。Chromatoid body(CB,拟染色体)是一个生殖细胞特异性的胞质结构。本实验中,电镜结果显示CB是由细丝分支形成的不规则网络,进而结合而成的紧密团块。在很多物种中,细胞中如果存在CB结构,表明该细胞为生殖细胞。目前已知有多种蛋白定位于CB中。在精子细胞中,CB以平行或是垂直的方式沿核膜快速的移动,而且有实验证明CB移动过程中从核中收集基因产物,参与核质间RNA的运输。对于CB这种生殖细胞特异结构的形成和功能的分子机制仍然需要进一步研究。本实验采用免疫电镜技术在亚细胞水平定位MIWI蛋白。本实验中采用了免疫电镜技术在精母细胞中定位MIWI蛋白的表达,可见MIWI蛋白在精母细胞后期聚合,并存在于核膜周围。免疫染色是免疫电镜技术中极为重要的一个步骤,本实验采用的是包埋前染色。免疫电镜实验过程中,即要考虑到保存较好的超微结构,又要能很好地体现免疫阳性反应。包埋前染色后的样本制作有一定的难度,但易于获得良好的免疫反应。
[Abstract]:Preface
The biological mother and offspring between individuals through the continuation of life. In the process of male reproductive reproduction in mammals, spermatogenesis is a highly coordinated process. Spermatogenesis is divided into three stages: initial germline stem cell mitosis in the first stage, through mitosis into spermatogonia eventually become spermatocytes. Second stage spermatocytes by meiosis into sperm cells, resulting in haploid round spermatids. The third stage is the round spermatids transfers into SPERMATOA. The present study suggests that mature sperm has no transcription to occur, and therefore spermatogenesis related genes in the early stage of sperm there is transcription. But these transcripts did not immediately translate into proteins, but were inhibited for several days, until the spermatogenesis only when needed In the translation. By spermatogonial cells into a lot of RNA and small molecular proteins participate in the long process of mature sperm, is an accurate and controllable process involved in this experiment. The MIWI protein is an important role in the recently discovered small molecules in the process.
MIWI protein is an important protein family member of the Argonaute family is the.Argonaute protein RNA induced silencing complex (RISC) of the core components in RNA interference (RNAi) plays an important role. When the small RNA with homologous sequence targeted mRNA or DNA binding, Argonaute family protein identification of some or all of the nucleic acid targets. By the regulation of mRNA stability, structure and synthesis of.Argonaute genome proteins including Ago and PIWI two subfamily. PIWI subfamily include 3 members, namely MIWI, MILI and MIWI2, is a germ cell specific protein, has close relationship with spermatogenesis knockdown of Miwi, Mili, or Miwi2 gene will cause the mouse sperm produced obvious defects, for male sterility. This subfamily and germ cell specific events, and specific distribution in germ cells, the Expression and cell growth regulation associated MIWI protein in mice. Representatives of this subfamily. It is because of germ cell specific expression of focus, especially the specific expression of the mammalian testis, the research of PIWI subfamily function mainly in spermatogenesis.
The present study shows that in mammalian germ cells, PIWI protein with piRNA (piwi-interacting RNA) with the formation of the piRNA complex (piRC) to regulate gene silencing pathway. According to the analysis of MIWI protein, help us to further understand the molecular regulation and mechanism of biological gametogenesis, and also have important significance to understand the mechanism of this protein, can also find new targets for infertility, so as to provide a theoretical basis for clinical treatment and explore new ideas.
Experimental materials
The SPF level C57BL / 6J mice testis tissues in all age groups, routine immunostaining kit, MIWI antibody, Goat anti rabbit two anti osmium, 2.5% glutaraldehyde, 4% paraformaldehyde, gradient alcohol (70%, 80%, 90%, 95%, 100%), xylene, paraffin, hematoxylin and neutral gum.
Method
1, the mice testis tissue protein was extracted from all ages, and the immunoblotting experiment was carried out to detect the expression of MIWI protein.
2, the paraffin section of the mouse testis tissue was made, and the immuno histochemical experiment was carried out to determine the expression of MIWI protein at the cell level.
3, immuno electron microscopy was used to detect the expression of MIWI protein at subcellular level.
Result
1., Western results showed that MIWI protein was not expressed in the tissues of mouse developing embryos, but existed in adult mouse testis tissues, and the expression of MIWI protein was detected at 8 weeks after developing in mice for 6 weeks.
2, the immunohistochemical results showed that MIWI protein was expressed in spermatocyte and spermatocyte during mouse testicular development.
3, subcellular localization shows that MIWI protein exists at the close to the nuclear membrane, indicating that it may play an important role in the nuclear transport and is an important component of the Chromtoid Body.
discuss
The specific expression of MIWI protein in mature mouse testis. Through this experimental study, MIWI protein was expressed only in the testis of adult mice, during embryonic and postnatal 4 weeks mice in different tissues showed no localization.MIWI protein expression showed the presence of.Chromatoid body specific structure in the germ cells (CB, pseudo chromosome) is a cytoplasmic structure of a germ cell specific. In this experiment, the results of SEM showed that the CB is formed by the filament branch of the irregular network, and then combined into clumps. In many species, if there is a CB cell structure, the cells into germ cells. Now known to have a variety of protein located in CB. In sperm cells, CB in parallel or vertical movement along the membrane quickly, and experiments have proved that CB mobile collected from nuclear gene products involved in nucleocytoplasmic transport. RNA for the CB The molecular mechanism of the formation and function of reproductive cell specific structure still needs further study. The subcellular location of MIWI protein by immune electron microscopy. The expression was used in the experiment of immune electron microscopy localization of MIWI protein in spermatocytes in the visible polymerization of MIWI protein in spermatocyte stage, and exists in the nucleus. Immunostaining is an extremely important step in immune electron microscopy, the experiment uses the pre embedding immunoelectron microscopy staining. In the experimental process, namely to consider the preservation of ultrastructure, but also can well reflect the immunoreactivity. The pre embedding staining after the sample production has a certain degree of difficulty, it is easy to obtain a good immune response.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R321

【参考文献】