The Mutation Of USP26 Gene Related With Spermatogenesis Patients

In recent years,Infertility has become a worldwide problem, male infertility proportion is as high as 50% in many causes of infertility.In male infertility,the incidence of sperm is rising,it has reach up to 15%-20%.The reason of it is so complex that it brings many difficulties in clinical diagnosis and treatment of complex factors. As the clinical application of advanced technology,a part of reasons which lead to male infertility have been confirmed,such as,hereditary disease,autosomal or sex chromosome abnormalities;testicular pathological changes by itself:like testicular trauma、testicular inflammation、testicular torsion and vascular lesions;endocrine disease、pituitary function、pituitary tumor、adrenal function hyperfunction or low、hyperthyroidism or a low,all of these reasons can affect sperm production and cause no sperm. But at present there are still some cases unable to ascertain the exact pathological mechanism.The reasons of male infertility are mainly including: chromosome abnormalities、lack of Y chromosome was born of pure genes and mutations and the X chromosome of pure gene mutation. Ubiquitin-specific protease 26 gene is located on chromosome X and the gene is associated with male infertility. Ubiquitin proteasome belongs to the deubiquitinating enzyme(DUB) family,this enzyme can produce antagonism in the binding of ubiquitin and protein, and can reduce the abnormal degradation of protein.However, This effect of the gene mutation inhibits the enzyme to some extend. Ubiquitin-specific protease 26 gene encodes a protein consisting of 913 amino acids, the gene is located on Xq26.2, consists of 2794 bases, only a single exon. This gene belongs to ubiquitination enzymes family and morever belongs to one of the numerous candidate genes who are associating with male sterility. The gene specific expression in the testis,its common mutation types are insertional mutagenesis and point mutation. The research associated with this gene about its physiological function is still very little. In this experiment, through comparing the expression differences of USP26 gene polymorphism between patients with azoospermia and normal men, through the seqman software to analysis and compare of the sequencing results,we can determine the method and site of mutation what locates in 259-1206 and length of 947 bp gene fragment of ubiquitin-specific protease 26 gene.This study can help us to understand and reveal the mechanism of the occurrence of azoospermia.waht’s more,this study may important for diagnosis and treatment of azoospermia.ObjectiveThrough the seqman software to analysis and compare of the sequencing results,the first object is to determine the method and site of mutation what locates in 259-1206 and length of 947 bp gene fragment of ubiquitin-specific protease 26 gene;the second object is to determine the relationship between the polymorphisms of ubiquitin-specific protease 26 gene and the man with azoospermia.Materials and Methods 1. Materials:According to the WHO standard,using microscopy after Spermcentrifuged,no sperm was found can be diagnosised withazoospermia.Azoospermia can be divided into obstructive and non obstructive.Excluded due to absence of the vas deferens or insemintion duct obstruction leadto obstructive azoospermia,and chromosomal abnormalities,Y chromosomemicrodeletion etc.We selected 50 cases of non obstructive azoospermia patientsto the Third Affiliated Hospital of Zhengzhou University Hospital ReproductiveCenter as case group,At the same time,50 male with normal fertility ascontrols.Excluded for male infertility over the implementation of operationtreatment or have children by assisted reproductive technology.In the earlymorning fasting acquisition case group and the control group of upper limbveinblood 3ml,the collected specimen displacement EP tube and stored in-20 DEGC to be measured. 2. Methods: In strict accordance with the kit method used to extract the case groupand the control group of peripheral blood DNA, PCR in peripheral blood ofpatients with amplified genomic gainubiquitin specific protease 26gene fragments, seqman software through the analysis and comparison of thesequencing results, determine the ubiquitin specific protease 26 genemutation in259-1206, length 947 bp gene fragment and sites in the way. 3. Statistical analysis:Analysis of the sequencing result with seqman software,Data were analyzed by SPSS 17 statistical software,X2 test was usesed toanalysis the difference between the case group and control group.Bilateral inspection level of alpha = 0.05,P<0.05 had statistical significance.Results 1. The selected case group and control group, no chromosomal abnormalities or andY chromosome micro defects. 2. The Results of gene sequencing analysis showed that :In 259-1206 genes beingmeasured,12 of 50 spermatogenesis patients exhibited changes in the Usp26gene,these changes including compound mutations of 363-364 insert ACA and inthe site of 496, T replace C, no changs were found in 460 and 1044. No otherchanges were found in the remaining patients and normal fertile controls. 3. The end of the data which had been measured, 12 of 50 spermatogenesis patientsexhibited changes in the Usp26 gene, these changes including compoundmutations of 363-364 insert ACA and in the site of 496, T replace C, at thesame time with the change of the amino acid sequence of mutation. the rate ofMutation is 24%, X2=13.636,P=0.000(<0.005). 4. No other changes were found in the remaining patients and normal fertilecontrols(P=1>0.05),has no statistical significance.Conclusion 1. The mutations of USP26 gene might be related with spermatogenesis patients,thegene of USP26 may lead to the semen quality serious decline or in some way itmay be regulation the process of sperm, at last,it Leading to the male sperm. 2. Ubiquitin-specific protease 26 can specific express in multiple phases of thesperm and testicular tissue,it is an important part of ubiquitin-protease,thisubiquitin-specific protease can make the protein degradation,it also can make theUbiquitin precursor been activated, Participate in the degradation of protein andfunctional, Then in the process of spermatogenesis plays a very important role.