| Objective:Targeted knockout of the rat Trib3 gene was performed to analyze the role of this gene in spermatogenesis in male rats.Methods:(1)CRISPR/Cas9 targeted knockout of Trib3 gene in Wistar rats was performed.The deletion of exon 3 of Trib3 gene in knockout rats was detected and identified by PCR and gene sequencing.They were fed and propagated.The rat genotype was identified by PCR and agarose gel electrophoresis.(2)Preparation of experimental samples: Testicular tissues were obtained from three male rats of different litters at five time points: 8,15,25,31,and 65 for homozygous Trib3-/-,heterozygous Trib3 +/-,and wild-type Trib3 +/+,respectively,and total protein and total mRNA were extracted from testicular tissues,and mRNA was reverse transcribed into c DNA,and testicular tissues were fixed in 10% formaldehyde solution.(3)The transcript expression levels of CDH1 gene,SCP3 gene,and TNP2 gene mRNA at each time point were detected by real-time PCR(qPCR);(4)The expression levels of CDH1 protein,SCP3 protein,and TNP2 protein at each time point were detected by Western blot;(5)The morphological changes of seminiferous tubules in the testis were observed by hematoxylin-eosin staining(HE staining).(6)Immunohistochemical staining(IHC)was used to observe the localization of CDH1 protein,SCP3 protein,and TNP2 protein expression.(7)Mating experiment was used to detect the effect of Trib3 knockout on the fertility of female rats,and adult homozygous female rats,heterozygous female rats,and wild-type female rats were taken;they were mated with homozygous male rats,respectively,and the conception rate of dams,the number of fetuses,and the number of natural births were recorded.(8)A rat testicular cell co-culture system was established to culture homozygous,heterozygous,and wild-type rat testicular cells in vitro to observe the growth and development of spermatogenic cells at all levels in rats of the three genotypes.Results:(1)Trib3 +/-was successfully prepared from positive F0 generation rats;F1 generation rats were obtained from F0 generation self-crossing,and F2 generation rats were obtained from F1 generation self-crossing.Homozygous rats were selected by verifying F2 generation genotype using PCR and agarose gel electrophoresis.(2)Compared with wild-type,the expression of CDH1 mRNA was increased in homozygous 8,15,and 65(P <0.05);the expression of CDH1 mRNA was increased in heterozygous 15,25,and 31(P <0.05);the expression of SCP3 mRNA was increased in homozygous 15 and 25(P < 0.05);the expression of SCP3 mRNA was increased in heterozygous 15 and 25(P < 0.05);the expression of TNP2 mRNA was decreased in homozygous 65(P < 0.05);and the expression of TNP2 mRNA was decreased in heterozygous 65(P < 0.05).(3)Compared with wild-type,the expression of CDH1 protein was decreased in homozygous 25(P < 0.05);the expression of CDH1 protein was increased in heterozygous 15,25,and 31(P < 0.05);the expression of SCP3 protein was increased in homozygous 15(P < 0.05);the expression of SCP3 protein was increased in heterozygous 15(P < 0.05);the expression of TNP2 protein was decreased in homozygous 65(P < 0.05);and the expression of TNP2 protein was decreased in heterozygous 65(P < 0.05).(4)HE staining showed that spermatogonial stem cells were attached to the basement membrane of seminiferous tubules in testicular sections of the three genotypes 8;spermatocytes were observed in testicular sections of both homozygous and heterozygous types at 15 hours,while spermatocytes were not formed in testicular sections of wild-type type,and homozygous and heterozygous seminiferous tubules developed more maturely;a large number of spermatocytes were observed in testicular sections of the three genotypes at 25 hours;a large number of spermatocytes were observed in testicular sections of the three genotypes at 31 hours;and spermatocytes had developed and matured in testicular sections of the three genotypes at 65 hours,spermatocytes were observed close to the testis,and Sertoli cells and Leydig cells could also be observed at the same time.(5)Immunohistochemical staining to observe CDH1 protein expression: testicular sections of rats with the three genotypes of 8 and 65 showed brownish-yellow positive results in spermatogonial stem cells near the basement membrane of seminiferous tubules;SCP3protein expression was detected by immunohistochemical staining: spermatocytes were darker and brownish-yellow on the 15 th day of life in homozygous and heterozygous rats;while testicular tissues were almost not visualized on the 15 th day in wild-type rats,and darker and brownish-yellow near the basement membrane of seminiferous tubules were observed in rats with the three genotypes on the 65 th day;TNP2 protein expression was detected by immunohistochemical staining: round sperm showed brownish-yellow positive results in testicular sections of rats with the three genotypes,TNP2 protein expression: homozygous <heterozygous < wild-type.(6)the number of dissected fetuses and natural litter size in each group were compared,and there was a significant difference in the number of embryos in each group(F = 6.113,P < 0.05);there was a significant difference in natural litter size among the three groups(F = 8.784,P < 0.05).(7)Rat testicular tissue cells were cultured in vitro,and observation showed that homozygous rat testicular cells observed that Sertoli cell growth was relatively scattered,there was no spermatogenic cell accumulation,no obvious cell mass was formed,and spermatogenic cells existed singly;heterozygous rat testicular cells Sertoli cells were relatively scattered,spermatogenic cell proliferation was also more obvious,and spermatogenic cells formed fewer cell clusters;wild-type rat testicular cells had a higher number,Sertoli cells basically climbed the surface of the culture dish,cell proliferation was obvious,and most spermatogenic cells attached to Sertoli cells had formed cell clusters,presenting a shape.Conclusion:(1)Trib3 knockout rats have no embryonic lethality,rats can produce normal gametes,and can be fed and expanded normally,and the offspring rats born can be stably inherited.(2)knockout of the Trib3 gene resulted in a premature meiotic phase in the testes of male rats,and it was speculated that this gene may be involved in the meiotic process of rat spermatogonial stem cells.(3)knockout of the Trib3 gene resulted in a decrease in the number of round sperms in the testes of adult male rats,and it was speculated that this gene may be involved in the process of spermiogenesis.(4)knockout of the Trib3 gene may promote reproductive performance in rats. |
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