Effects Of Trib3 Gene Knockout On Spermatogenesis In Male Rats

Objective The purpose of this study was to explore the role of Trib3 gene in reproduction by studying the expression of Trib3 gene in testis of wild-type rats at different stages.Methods(1)Expression of Trib3 gene in the spermatogenesis of wild-type rats: Twenty-one male wild-type Wistar rats of 2-65 days old were selected from three different nests at 21 time points,and the testis tissue was extracted and the m RNA transfer level of TRIB3 gene at each time point was detected by real-time q PCR The expression level of TRIB3 was detected by blot,and the expression and location of TRIB3 were observed by immunohistochemistry.(2)Effect of Trib3 gene knockout on reproductive function of male rats:The adult male homozygous(-/-),heterozygous(+ /-),wild-type(+ / +)Wistar rats were selected to mate with the wild female rats,and the pregnancy rate,the number of fetal rats and the number of natural production were analyzed.The testis tissue differences of the offspring of the three genotypes were observed by HE staining,RT-PCR product sequencing confirmed homozygous and heterozygous mutations;total testicular protein was extracted,and the expression of TRIB3 protein was detected by Western blot after gene knockout;he staining and immunohistochemical staining were used to compare the morphological structure of testis and the expression of TRIB3 protein;real time The expression of TRIB3 gene and each marker gene were observed by q PCR,and the spermatogenesis was speculated.The activity index and concentration of epididymal sperm were extracted by computer-aided analysis.Result(1)The expression of TRIB3 m RNA was found in testis of wild-type rats from 2 to 65 days old,which was higher at 2-12 days old and reached the peak at 4 days old;there was no significant difference between 14-16 days old and 2-12 days old(P > 0.05);the expression of TRIB3 m RNA was significantly decreased at 20-35 days old and 14-16 days old(P < 0.05);the expression of TRIB3 m RNA was significantly increased at 40-65 days old and 20-35 days old(P < 0.05).The expression of TRIB3 protein was found in 2-65 days old and higher in 2-12 days old;the expression of TRIB3 decreased significantly in 14-16 days old and 2-12 days old(P < 0.05);there was no significant difference between 20-35 days old and 14-16 days old(P > 0.05);the expression of TRIB3 increased significantly in 40-65 days old and 20-35 days old(P < 0.05).Immunohistochemical staining showed that the cytoplasm of spermatogonial stem cells and some testicular interstitium of 5-day-old testis showed positive results,and the connective tissue fibers of testicular interstitium of 12-day-old,18-day-old,29 day old and 40 day old showed positive results.(2)Alignment with wild-type bases,homozygous and heterozygous TRIB3 genes Exon3 was absent;the expression of TRIB3 protein was positive in all genotypes of rats;the differences of embryo number and natural production number in all groups were statistically significant(P < 0.05),and the testis tissue structure of the offspring of the three genotypes of 18 day old rats was complete,no significant difference was found;the tissue morphology of testis pill of adult rats was compared,no significant difference was found among the genotypes,and the expression of TRIB3 protein in each group was at The m RNA expression of CDH1,SYCP3 and TP2 were not significantly different among the three genotypes(P > 0.05).The expression of pure TRIB3 m RNA was not detected,and the expression of heterozygous TRIB3 m RNA was lower than that of wild-type TRIB3 m RNA;The sperm concentration of homozygous and heterozygous rats was significantly lower than that of wild type rats(P < 0.05),and there was no significant difference in sperm motility index between each group(P > 0.05).Conclusion(1)The gene may be involved in the proliferation of rat spermatogonial stem cells.(2)After the TRIB3 gene was knocked out,the rats were able to produce sperm and copulate offspring.The testis structure of the offspring copulated by the gene knockout rats and wild female rats was not significantly abnormal compared with that of wild-type offspring rats.(3)Knockout of TRIB3 gene will lead to the decrease of sperm number in rats without affecting the sperm viability index,which may be one of the key genes involved in sperm production.(4)TRIB3 gene may play a role in testicular stroma.