The Study Of MIWI Protein During Mouse Testis Development

BackgroundOrganisms perpetuate their lives through reproduction from one generation to another. During the reproduction of male mammalian, spermatogenesis is a highly coordinated process. Spermatogenesis can be divided into three stages:The first stage is mitosis which initiates from germ stem cell,spermatogonial through this transfer into spermatocyte. The second stage is that spermatocytes transfer into spermatids, resulting in haploid round spermatisds. The third stage is spermiogenesis that round spermatids transfers into spermatoa. Recent studies have shown that mature sperm did not have transcription activities, so the genes which is related to spermatogenesis transcribe at the early stage of spermatogenesis. These transcriptional products were not translated into proteins immediately, while were inhibited for several days, until they were needed in the process of spermatogenesis. There are many RNA and small molecular proteins involving the process of spermatogonia transferring into mature spermatoa. And it is a accurate and controllable process. MIWI protein which involved in our research, is a small molecular which plays an important role in this process.MIWI protein is one of important family members which belonged to Argonaute. Argonaute protein family is the key element of RNA induced silencing complex (RISC), and functions in RNA interference (RNAi). As small RNA bind with targeted mRNA or DNA which possess homologous, Argonaute family proteins recognize partial or complete nucleic acid target, using to adjust the stability of mRNA, protein synthesis and the structure of genome. Argonaute protein can be divided into two subfamily, Ago and PIWI. PIWI subfamily include three members:MIWI MILI and MIWI2. They are germ cell-specific protein and are close to spermatogenesis. Knocking out miwi, mili or miwi2 genes would result in sperm defects and male infertility. PIWI subfamily is related to germ cell-specific events, and they distribute in germ cells exclusively, especially in mammalian testis. The study of PIWI subfamily function focuses on spermatogenesis.Recent studies indicate that, in mammalian germ cell, PIWI subfamily protein forms piRNA complex (piRC) combining together with piRNA to regulate gene silencing pathway. According to the analysis of MIWI protein, it contributes to further understand the molecular regulation and mechanism of gamotogenesis, and to understand the mechanism of this protein, to find the new target of infertility, so as to open for new ways for clinic treatment and provide theory basis.MaterialsSPF C57BL/6J mice testis, IHC kit, MIWI antibody, osmic acid,2.5% Glutaral, 4% paraformaldehyde, gradient alcohol, xylene, paraffin, hematoxylin,netual gumMethods1. Proteins were extracted from each stage of mice testis tissues, performing western blot to determine the expression of MIWI.2. Mice testis were made into paraffin section,and the localization of MIWI proteins were detected by IHC techniques.3. Adopting Immuno-EM technique to determine the localization of MIWI protein on subcellular level.Resultsl.The results of western blotting indicated that MIWI protein is not expressed during the development of mice embryonic tissues,while it exists in the testis of adults mice. And the protein can be detected during 6 and 8 weeks.2.The results of Immunohistochemistry indicate that MIWI protein localized in spermatocytes and spermatids during mice testis development.3.Subcellular localization display that MIWI protein is to be in close proximity with nuclear envelope,indicating that MIWI proteins are likely to play an important role in the transportation between the nuclear and cytoplasm, and are a kind of important components in CBs.DiscussionMIWI protein was Exclusively expressed in the testis of adult mice.According to our sthdy,we demonstrate that MIWI protein was expressed exclusively in the testis of adult mice,while it is not expressed in any other tissues of mice which is at embryonic stage and within postnatal 4 weeks. The localization of MIWI protein exhibited a kind of specific structure in germ cells. Chromatoid body is a germ cell-specific cytoplasmic structure. In our study, the results of immunoelectron microscopy display that The chromatoid body is a finely filamentous, lobulated irregular network, and forming into a compact mass.In many species,if CB structure is existed,the cell is considered as germ cell. Recently we know that there are several components in CB. In spermatids, CBs move quickly around the nuclear envelope in a parallel or vertical manner. There are several experiments show that CBs collect gene products when they move, and participate in the transportation of RNA between nuclear and cytoplasm. The molecular mechanism of formation and function of CB which is a kind of germ cell-specific structure needs further study. We perform immunoelectron microscope method to localize MIWI protein in subcellular level. The result shows that MIWI protein get together in the later stage of spermatids,and exist around nuclear envelope. Immuno staining is a key step during the whole immunoelectron microscope methods, and we adopt preembedding staining method. In the process of immunoelectron microscope, we need not only consider the preservation of ultrastructure, but also highlight the positive reaction. Although it is hard to perform the preembedding staining, the result is much better than post-embedded staining.