The Novel Research Of Human Sperm Associated Antigen4-like (SPAG4L)

Dyszoospermia and other correlative diseases is an important part of male infertility. Many genes participate in the controls of spermatogenesis, sperm maturity, sperm movement and other processes. About 2000 different genes been reported have taken part in the every procese of male reproduction:testis growth, germ cells differentiation, meiosis, spermatogenesis and so on. Many genes have been studied absolutely, but to the others, there are still many genes which can not been studied throughly. What are the details of the function of them? How do they cooperate with each other to control the different stage of human procreation? So it is very important to do the research of human genes involved in the spermatogenesis.It is reported that some members of SUN gene family may cooperate with the human spermatogenesis. Teacher Xiao Wei Xing has coloned a new sperm gene located in the chromosome 20q 11.21 region, named SPAG4L (TSARG4), from the gene SPAG4 in early research. In july 2001 he submit the full sequence of SPAG4L to GenBanK (GeneBanK Access number:AF401350) and coloned another gene named spag41 (SRG4) (GeneBanK Access number:AY307077) which is a mouse homologous gene of SPAG4L from mouse ESTs. The result of multiple orgnizations revealed that spag41 expressed specifically in mouse testicle by the means of RT-PCR and Northern blot. The result of RT-PCR revealed that spag4l express very low in 2 weeks after mouse's birth. From the third week Spag4l expressed more and more and to it's peak in the period of 4-5 weeks, indicating that spag4l may play important role in the spermatogenesis progress. In situ hybridization, spag4l express obviously in the normal control sperm, mostly in spermatocyte and round spermatids. But in normal sperm and enorchia negative control, No obvious signals were detected, indicating that Spag41 may in charge of the development of mouse spermatogenesis.But to the human homologous gene SPAG4L, there are still many spaces needed to be explored. In this research, we study the expression specility of SPAG4L, the accurate subcellular localization and the key gene region which respond for the subcellular location?the changes of spag41 proteins in the process of mouse meiosis.Chapter One:The bioinformation analyzation of SPAG41 and the expression specialityObjective:We try to clear the expression specility of SPAG4L and to confirm which gene family it should belong to by means of homology analysis with the known SUN-domain proteins. Methods:Comparative homology analysis of full length of SPAG4L with the known SUN proteins, we kown which protein family it belong to and speculate the original function of it. By means of Northern bolt?RT-PCR and In situ hybridization, we clear the expression characteristic of SPAG4L.Result:SPAG4L is a new member of SUN family and may originate from the same ancestor with SUN3, SUN4 (SPAG4). SPAG4L only expressed in human testis, but in other tissues no expression of SPAG4L been detected. SPAG4L expressed in mature male testis, mostly been observed in spermatogonium and spermatocyte, but not in mature sperm, neither in fetus testisConclusion:SPAG4L is a new member of SUN family and expressed specially in human testis, correlating with the mature degree of testis. SPAG4L expressed in mature male testis, mostly been observed in spermatogonium and spermatocyte but not in mature sperm, neither in fetus testis Chapter Two:Prokaryotic expression and purification of SPAG4LObjective:Get the protein of SPAG4L through prokaryotic expression and purified it.Methods:We amplify the 126-379bp of SPAG4L by means of RT-PCR from freash human testis. Then we subclone the fragment to pUCm-T vector and PQE-30 expressing vector. After transforming to Bacterium coli JM109, we extract the protein and examine it by means of Western blot. At last we purify the protein through NI-NTA magnectic agarose beads.Result:We amplify the coding sequence of SPAG4L by the means of RT-PCR and the fragment inserted is about 253bp. The product dealed with restriction enzyme then subclone to expressing vector PQE30. The recominated vector PQE30-SPAG4L been transformed to Bacterium coli JM109 and then successfully expressed out the fusion protein. The fusion protein been purifyied by means of NI-NTA magnectic agarose beads and then confirmed by means of Western Blot.Conclusion:We successfully constructed the prokaryotic expressing vector of SPAG4L and get the fusion protein. Then the purified product which been confirmed by Western Blot can be used into next research. Chapter Three:The subcellular localization of SPAG4L and the expression features in meiosisObjective:Try to clear the accurate subcellular localization of SPAG4L and confirm the key region which decide the correct position. We observed the changing feature of spag4l, the mouse homologous of human SPAG4L, in the development of mouse meiosis and speculate the meaning of it's function.Methods:The accurate subcellular localization of SPAG4L was confirmed by immunofluorescence. To clear the key region which decide the subcellar position we construct a few mutants and infected the constructed EGFP-N1-SPAG4L vectors to Hela cells. We observed the changing features of spag4l, the mouse homologous, in mouse sperm meiosis.RESULT:SPAG4L localized in nuclear envelope and endoplasmic reticulum. The transmember and coiled-coil region but not the SUN region in it's C-terminal are the necessary region for it's accurate position. SPAG4L may involved in the migration and position of nuclear envelope and endoplasmic reticulum in the process of meiosis, indicating that it's importance in spermatogenesis.Conclusion:SPAG4L is a protein of nuclear envelope and endoplasmic reticulum. The transmember and coiled-coil region is the key region for the accurate position. SPAG4L may be a new breakout for us next research of male infernity.