Identification Of Mutational Genes Of Male Infertility In MIJ Rat

Objective: Couples of10%~20%are in trouble of infertility, half ofwhich are caused by male infertility, named male infertility. Development ofmale reproductive system is regulated by many gene, insufficiency of functionof whichever genes result in abnormal or infertility of male reproductivesystem, however, the molecular mechanisms of those genes are still not clear.In the present, the major research techniques of pathogenic mechanismtake advantage of model of experimental animals, inartificial experimentalanimals are similar to diseases of humans in occurrence and development, andhave significant value to study of human diseases. MIJ Rat is spontaneousinbred line rat with male sterile, which was breeded in Department ofLaboratory Animal Science, Hebei Medical University, characterized by delayof orchiocatabasis, cryptorchidism, hypoevolutism of organs of generation(such as testicle, epididymis, penis, deferent duct, prostate and seminalvesicles), oligospermia and dyszoospermia and so on. These characters aresimilar to cryptorchidism, small testopathy and oligospermia of male infertility.MIJ rats have same genotype and inherited character, and easy to obtain theidentical results, is better experimental animal model for male infertility inhuman. Here we analyzed the differential expression between MIJ rat andnormal rat using gene microarray and screened out the significant differentialexpression genes involving in regulation and development of reproductionsystem, so then provided the basic scientific basis for mechanism of humanmale infertility.Methods:1SamplingFive pairs of testicles were collected from4weeks MIJ rat and normal rat,respectively.10left testicles were analyzed using gene microarray, and10right testicles were detected by Real-time PCR. 2mRNA ExtractionTotal RNA was extracted from testicles of MIJ infertility and normal Rat,respectively with Trizol method. RNAs were further purified withQiagen-RNeasy Kit. Agarose gel electrophresis was used to analyze thequality of total RNA.3Labeling and HybridizationcDNA probes were reversely transcribed from mRNA with the directtranscription method. Cy5-dUTP and Cy3-dUTP were used to label mRNAprobes from testicles of MIJ infertility and normal Rat. After the degenerationof hybridization solution of mixed probes and microarray, hybridizationsoluteon was dropped onto the sampling area of microarray, covered with acover slip, palced into the hybridization cabin, sealed with parafilm, and thenhybridization in42℃hybridization oven for16-hour.4Analysis of gene microarrayThe microarray was scanned with confocal fluorescence scanner and theraw data was gaided. The significant up-regulation and down-regulation geneswere identified between MIJ and normal rat with threshold: P<0.05, FoldChange (FC)>2.0or <0.5. The data were further interrogated with GeneOntology (GO) and KEGG Pathway Analysis. We screen candidate genesinvolving in male infertility according to previous reports in the literature.5RT-PCR verification of differentially expressed genes about infertility.RNAs of MIJ infertility and normal rat’s testis tissue were extracted toundergo reverse transcription. The differential expression genes was detectedby Real-time PCR, results of gene microarray were verified.Results:1Agarose gel electrophoresis showed clear bands of28s and18s rRNA,suggesting satisfactory purification and integrality of RNA.2Analysis of gene microarray revealed that328differential expressiongenes were found,248of which were down-regulation genes,80wereup-regulation genes. Analysis of differential expression genes with GO andKEGG Pathway showed that the genes involving in development of reproduction system had a role in bioprocess, celelular component and signalpath, such as development of male genitals and gonad, spermatid apoptosis,fertilization, acrosomal vesicle, G-protein coupled receptor signal transductionpathway. Next, we screened out nine down-regulation genes，such as Asb1，Klhl10，Pitx2，Insl3，Zpbp，Zmynd15，Pvrl3，Acr and Plcd4，and oneup-regulation gene Igfbp3in view of previous studies and results of genemicroarray to validate the differential expression by real time RT-PCR.3The results from real time RT-PCR showed that expression levels ofAsb1，Klhl10，Pitx2，Insl3，Zpbp，Zmynd15，Pvrl3，Acr and Plcd4of MIJinfertility rat had a marked down-regulation compared with those genes ofnormal rat. While expression of Igfbp3gene was significantly up regulatedcompared with normal rat. These results were in line with gene microarray.Conclusions:1Analysis of gene microarray showed that expression of genes of MIJinfertility rat had a significant differences compared with normal rat. Theseresults prompted us that pathological process of male reproduction system wasregulated by multiple proteins and metabolic pathway.2The results from real time RT-PCR showed that expression levels ofAsb1、Klhl10、Pitx2、Insl3、Zpbp、Zmynd15、Pvrl3、Acr、Plcd4、Igfbp3involving in infertility were coincident with results of gene microarray, andvalidated the reliability of gene microarray.3In the present study, we screened out the genes, which had a criticalrole in development of male reproductive system, and may associate withdefect of MIJ infertility rat reproduction system. These findings gave usscientific basis for pathogenic mechanism and clinical treatment of maleinfertility.