Expression Profiling Of Testis-Specific Genes During Testicular Development In Mice

ObjectiveInfertility is a kind of disease which is caused by multiple factors that among which the male factor account for 50%of all the causes.Male infertility could be divided into two types clinically including the type that causes have been known clearly and the type that causes are unknown which is usually known as primary infertility. Primary infertility takes a 75%in clinical diagnosis that all patients have normal results in clinical examination.It has been concerned now that primary infertility is caused by genetic factors.There is no clinical method to examine primary infertility on the level of genetic.If we know which genes have defect in the developing testis we could adopt appropriate treatment to relative genes.So screening and finding genes inducing male infertility could be the foundation of gene therapy.Find the abnormal expressed gene and to institute a genetic diagnosis of male infertility could guide the therapy and have great sense.Materials and MethodsMaterialsTestes from thirteen groups of different ages C57 BL/6J mice(6d,8d,10d,12d, 14d,16d,18d,20d,28d,56d,16w,24w,30w).Methods1,RNA PreparationTotal RNA was isolated from each testis using Trizol Reagent according to the instructions and measures the concentration.2,Reverse transcription polymerase chain reaction(RT-PCR)Total RNA isolated from developing testes was prepared for reverse transcription using a RT kit.Twoμg total RNA was used to synthesis cDNAs.β-actin was amplified in each developing testes to judge if the cDNAs are available and adjuest the concentration of the cDNAs by the densitometric analysis of bands.3,PCRIn previous job,we screening about 500 testis-specialized expressed gene in mice using silico subtraction and among which 339 genes has homology with human.We amplify those 339 genes in developing testes of mice of thirteen groups.4,PAS-hematoxylin StainingTestes were dissected,fixed in the paraform and embedded in paraffin.Sections(5 lm) were prepared and stained with PAS and hematoxylin.ResultThe expression of these 339 testis-specific genes during testicular development could be divided into 4 types as below:1,Genes express in every developmental stage of testis and have uniform expression brightness(5 in total).2,Genes express in every developmental stage of testis,however,have different expression brightness:①the expression amount decrease progressively during the testis development.(2 in total );②the expression amount increase progressively during the testis development.(44 in total).3,Genes do not express in every developmental stage of testis but have uniform expression brightness(60 in total).4,Genes do not express in every developmental stage of testis and have different expression brightness:①the expression amount increase progressively from a early stage(138 in total );②the expression amount decrease progressively from a early stage(4 in total ).Conclusion1,We identify the testis-specialized gene exclusively expressed in the developing testis of mice.2,All these testis-specialized gene have been amplified in the developing mice testes successfully and summarize the difference of expression,as well as the expression table.DiscussionIt has great sense in studying the spermatogenesis and the spermateliosis,as well as male fertility to identify the expression model of these genes.Several genes had been researched already,such as Speml,Catsper3,Catsper4 and so on.It is clear that knowing when and where those genes express in the developing testis has great sense for future study of each gene.What's more,these genes could be classified and then made into cDNA chip.We can extract RNA from patients' sperm and then hybridize with the cDNA chip.Making an analysis and comparison with the clinical symptom,we can find the abnormal gene and make a genetic diagnostic criterion.