Research On Genetic Polymorphism And Point Mutation In Male Sterility

BackgroundApproximately 15%-20% of couples are infertile with males and females half and half.15% of male infertility is due to azoospermia,most often NOA(1% male).Non-obstructive azoospermia may be due to cryptorchidism,Klinefelter syndrome,and chemotherapy or radiotherapy.15%-30% of non-obstructive azoospermia men have aneuploidy or Y chromosome deletion.However,a large percentage of infertile men are diagnosed as idiopathic,reflecting the lack of understanding basic mechanisms of regulating spermatogenesis and sperm function.Therefore,approaches that are more reliable and noninvasive are needed for the diagnosis of NOA.Identified the genetic factors of male infertility will be able to provide valuable insight into the etiology of idiopathic infertility,will contribute to the development of targeted therapies.Single nucleotide polymorphism(SNP)is one of the most common human heritable variation,and mainly refers to a DNA sequence polymorphisms in the genomic level by a single nucleotide mutation,resulting in the diversity of chromosomal genome between species including humans.SNPs in the human genome are widespread,averaging 500 to 1000 base pairs have one,and in total up to 3 million or more.SNPs may be distributed to non-coding gene segment or encoding gene segments,and SNPs which is not in protein coding regions may still affect gene splicing,transcription-binding and degradation of messenger RNA.SNP frequency in the population is greater than 1%,while the mutation frequency in the population is less than 1%.Point mutation is a type of mutation,in the genetic material DNA or RNA,making a single base nucleotide substitution into another nucleotide.Typically also including the insert or delete of a single base pair.Mutation is a single base change,if in the coding region may affect the encoded amino acid thus affecting protein function.Population frequency of mutation is very low.There are many types of mutations,and mutations fate undecided.A mutation may be fixed in the population,and may be disappear due to selection or random drift.Here we through SNP screening and mutation model research,analysis the two DNA genetic markers,and hope to contribute to the clinical diagnosis and treatment.Methods and ResultsIn the previous study,we identified three risk loci associated with NOA.One,rs7099208,is located within the last intron of FAM160B1 at 10q25.3.In this study,we analysed expression Quantitative Trait Loci(eQTL)of FAM160B1,ABLIM1 and TRUB1,the three genes surrounding rs7099208.The expression level of FAM160B1 was reduced for the homozygous alternate genotype(GG)of rs7099208,but not for the homozygous reference or heterozygous genotypes.The expression levels of ABLIM1 or TRUB1 were unaffected by the rs7099208 genotype.Using immunohistochemistry,we showed that FAM160B1 is predominantly expressed in human testes,where it was found in spermatocytes and round spermatids.We examined testes from 17 patients with NOA and five with obstructive azoospermia(OA)and found that expression of FAM160B1 was reduced,or undetectable,in NOA patients,but not in OA cases or normal men.We determined that FAM160B1 was expressed in germ cells in mouse testes.Using a mouse germ cell line(GC2)as a model,knockdown of FAM160B1 resulted in a dramatic reduction in cell number,reduced cell viability,and ultimately cell death.We concluded that rs7099208 is associated with NOA via a reduction in the expression of FAM160B1.N-glycosylation disorders are an important cause of the disease,and recently the molecular mechanism of the relationship has begun to slowly opened.DPAGT1 gene encodes N-acetylglucosamine-1-phosphate transferase,and catalytic the first step of LLO in the synthesis of the precursor.DPAGT1 mutant could lead to a CDG syndrome type I with N-glycosylated proteins disorder.In our study,we found when 166 amino acids of DPAGT1 protein coding regions aspartic acid(Asp)was mutated to glycine(Gly),the male mice were completely infertile.DPAGT1 mutant germ cell was blocked in the round spermatid stage,and belong development arrest of NOA.Histology showed DPAGT1 mutant male mice testes lumen were smaller,within multinucleated giant cells and vacuole formation.The electron microscope results revealed in DPAGT1 mutant testes round spermatids showed multiple dispersed acrosome vesicle and elongated sperm nuclei highly concentrated,suggested defects in sperm deformation process.By proteomics analysis between DPAGT1 mutant and widetype testes found that there was a class of glycoprotein protein with significantly reduced glycosylation and not change protein levels.These glycoprotein protein included SUN3,SUN4,Lamin,SUN5,SPACA1 and ZPBP1,which were important in sperm deformation.Immunofluorescence showed that,in DPAGT1 mutant mice these glycoproteins occurs disorder due to lack of sugar chain,which was one of the causes of abnormal sperm deformation.In summary,we hypothesized DPAGT1 mutant affected a group of protein glycosylation which plays an important role in the structure of the sperm head and the deformation process,resulting in spermatogenesis arrest and male sterility.