Localization And Function Of SLXL1, SLX2, XLR5C And Mitochondria In Germ Cells

Spermatogenesis is a complex differentiation process essential for all the animal species with sexual reproduction. And this process can be divided into three stages:mitosis in spermatogonia, meiosis in spermatocytes, and spermiogenesis. On the molecule level, spermatogenesis is a process that a series of specific-genes are programmed express and transcript. However, mammalian spermatogenesis is nowadays still poorly understood at the molecular level. Three novel genes (including Slxll, Slx2 and Xlr5c) were found, however, there localization and function is not defined. In this study, the localization and function of these genes (proteins) in the tissue or cells (including spermatogenesis and meiosis) were identified by using cell biology technology, development biology technology, molecule biology technology, RNAi technology and transgenic technology. This study can help us to understand the mechanisms of spermatogenesis and meiosis in the testis. In addition, it can help us to further study the mechanisms underlying infertility, develop a male pill that protects against pregnancy, explore the pathogen of sterility and provide a basis for effective treatment.Adenosine triphosphate (ATP) generated by mitochondria is the main resource of energy for mammalian cells. At present, it is the hot points for the mitochondria distribution, structure and function in cell biology. However, the localization, distribution and function of mitochondria in buffalo germ cells were unknown. In this study, the localization and function of mitochondria in buffalo oocytes maturation, fertilization and preimplantation embryos development were investigated. This study can help us to understand the mechanism of buffalo oocyte maturation. And the research on mitochondria translocation in buffalo oocyte and embryo might be used as a marker to estimate the potential oocyte and embryo and might be helpful to understand reproductive physiology and assisted reproductive technology in human.Therefore, a series experiments assorted in 5 parts were carried out in this study.Partâ… , SLXL1, a novel testis-specific gene, was cloned, expressed and identified. And its encoding protein might be an acrosomal associated protein and was involved in fertilization.The results showed that:(1) On mRNA level, Slxll mRNA was only detected in the testis during multiple tissues and different stages of spermatogenesis in the testis by RT-PCR. (2) SLXL1 antigens were designed and its plasmid was expressed in E.coli and then a polyclonal antibody of SLXL1 was obtained. (3) On protein level, Slxll encodes a protein of 18 kDa that is specifically expressed in the testis during multiple tissues and different stages of spermatogenesis in the testis by western blotting. (4) SLXL1 was exclusively expressed in the acrosome of post-meiotic germ cells (including round spermatozoa, elongation spermatozoa, spermatozoa in epididymis and mature spermatozoa) by immunohistochemistry techniques. And the trend of SLXL1 expression was associated with the formation of acrosome. (5) SLXL1 interacts with DKKL1, which is another acrosome-associated protein and co-localized in the acrosome. (6) Using a neutralizing antiserum from the rabbit, we also demonstrated the in vitro fertilization rate was significantly reduced by the SLXL1 antibody. To the best of our knowledge, SLXL1 was an acrosome associated protein and is involved in fertilization.Partâ…¡, SLX2, a novel gene, was cloned, expressed and identified. And its encoding protein might be a meiosis-associate protein and play an important role in the formation of meiosis, homologous recombination, double strand repair and sex chromosomes inactivation.The results showed that:(1) On mRNA level, Slx2 mRNA was only detected in the testis and ovarian during multiple tissues and different stages of testis and ovarian by RT-PCR. (2) SLX2 antigens were designed and its plasmid was expressed in E.coli and then a polyclonal antibody of SLX2 was obtained. (3) On protein level, SLX2 is specifically expressed in the testis and ovarian during multiple tissues by western blotting. (4) SLX2 and yH2AX co-localized in the XY body during meiosis by immunohistochemistry techniques. SLX2 distribution is so similar to yH2AX which is a protein marker of double strand repair and sex chromosomes inactivation in meiosis. (5) SLX2 as a novel interacting protein interacts with SYCE2 and histone acetyltransferase KAT5 (also known as TIP60) through yeast two-hybrid screening and Co-IP. yH2AX interacts with KAT5/TIP60. These results indicated that SLX2 along with SYCE2 may associate with the homologous recombination and synaptonemal complex assembly. These results indicated that SLX2 along with SYCE2 may associate with the homologous recombination and synaptonemal complex assembly. In conclusion, SLX2 interaction with SYCE2, KAT5/TIP60 and yH2AX in the meiosis might take part in the homologous recombination, double strand repair and sex chromosomes inactivation.Partâ…¢, Xlr5c, a novel gene, was cloned, expressed and identified. And its encoding protein might be take part in the first meiosis in the spermatocyteâ… .The results showed that:(1) On mRNA level, Xlr5c mRNA was only detected in the testis and ovarian during multiple tissues and different stages of testis and ovarian by RT-PCR. (2) XLR5C antigens were designed and its plasmid was expressed in E.coli and then a polyclonal antibody of XLR5C was obtained. (3) On protein level, Xlr5c encodes a protein that is specifically expressed in the testis and ovarian during multiple tissues and different stages of testis by western blotting. XLR5C might be a time-related gene and involved in the early stage of spermatogenesis. (4) The expression of XLR5C in the testis was localized in the nuclear of male germ cells, particularly in spermatogenis by immunohistochemistry. In addition, XLR5C was detected in the cytoplasm of oocytes. In conclusion, XLR5C may play a role in the biological process of spermatogenesis and oocyte meiosis.Partâ…£, This study was research on the localization of X chromosome in germ cells. And the localization of X chromosome was a method of sex identification of spermatozoa and early embryo in bovine and buffalo.The results showed that:(1) The X chromosome probe was prepared by PCR and microisolate technique. And the probe was specifically for the bovine metaphase chromosomes. (2) The bovine chromosome probe was used for identification of sorted spermatozoa and blastomere biopsy in bovine and buffalo. The results indicated that FISH was an efficient method for sexing identification of sorted spermatozoa and early embryo in bovine and buffalo.Partâ…¤, This study was research on the function and distribution of active mitochondria in buffalo oocytes maturation, fertilization and preimplantation embryos development.The results showed that:(1) The distribution of active mitochondria was found in oocytes which cultured before and after maturation in vitro. The active mitochondria in oocytes were transferred from outer cytoplasm to inner cytoplasm and chromatin along with the oocytes maturation in vitro. (2) The active mitochondria aggregated around the two pronuclei in fertilized eggs. (3)The active mitochondria were dramatically changed in the different stage of preimplantation embryos. The results indicated that the apoptosis percentage of ovarian granulose cells might associate with those follicular sizes. In addition, the active mitochondrial distribution was dramatically changed and might play an important role in buffalo oocytes maturation and preimplantation embryos development.