Molecular Characterization And Expression Of Meiotic Recombination Pathway-related Genes Spata22,Rad51 And DMC1 In The Testis Of Cattle-yak,Yellow Cattle And Yak

Cattle-yak is a first-generation(F1)hybrid of the yak and yellow cattle,has a tall body,and the overall body structure is symmetrical.The production capacity of milk and meat,and the working ability of cattle-yak is better than that of yak,but the F1 generation male is sterile.The male hybrid sterility restricts gene flow hence posing a challenge to the improvement of the yellow yak through hybridization.It is of great significance to study the mechanism of male sterility in the yak.Therefore,identifying and determining the expression of genes and key regulatory factors during germ cell development,especially the gene recombination pathway,is fundamental in understanding the hybrid male sterility in the cattle-yak.The Spata22,Rad51 and DMC1 genes and their interacting partners play crucial roles in prophase I of meiosis.Their proteins accumulate at sites of double-strand breaks and assist in double-strand break repair and promote meiotic recombination.At present,the expression,distribution and functional characteristics of Spata22,Rad51 and DMC1 in the cattleyak have not been explored.Therefore,the purpose of this work is to clone and analyze the gene sequences of Spata22,Rad51 and DMC1,and also detect the location and expression of upstream regulatory factors in the testis of the sexually matured and immature yak,cattle and cattle-yak as well as do a comparative analysis of testicular morphological parameters of their testes.This would help reveal the relevant mechanism of sterility in the cattle-yak.To achieve this aim,a three-part experiment was conducted using gene cloning,RT-PCR,Western blotting,immunohi stochemi stry,immunofluorescence,TUNEL labelling and histomorphological analysis were used.The main results are summarized as follows:1.Histo-morphology analysis showed that there was no difference in the composition and structure of the reproductive epithelium between cattle and yak.The germ cells,Sertoli cells,and stromal cells were normal in the different developmental stages.In contrast,there are spermatogonia and very few primary spermatocytes in the seminiferous tubules of cattle-yak,but no secondary spermatocytes,spermatids and spermatozoa in the late developmental stage are observed.Further observation showed that the nuclei of some of the germ cells are highly condensed,a typical feature of cells undergoing degeneration and apoptosis.There were lots of immature spermatogenic cells and mature spermatids in the seminiferous tubules of the yellow cattle and yak compared to the cattle-yak.Of the cells found in the matured cattle-yak,45.6%were spermatogonia,33.1%were spermatocytes and 21.3%were Sertoli cells.In immature cattle-yak,38.7%,36.2%,and 25.1%were spermatogonia,spermatocytes,and Sertoli cells respectively.This suggests that the number of germ cells decreases with increasing age through germ cell death by the activation of the apoptotic pathway.2.The morphological comparisons of the seminiferous tubules of the different species and age groups revealed no significant difference in seminiferous tubule diameter,tubule cross-sectional area and cross-sectional perimeter.However,there was a noticeably widened seminiferous tubule lumen area and lumen perimeter,and the thickness of the lamina epithelia was remarkably decreased.3.The coding region(CDS)of the cattle-yak Spata22 and Rad51 genes were cloned and sequenced successfully.No mutation sites were found for Rad51,but 13 mutations were found in the Spata22 sequence compared to the yellow cattle sequence.Phylogenetic analysis showed that the cattle-yak was in the same class as other members of Bovidae such as the wild yak and buffalo.4.The expression of Spata22,Rad5,DMC1 and their co-localizing partners and upstream regulators(MEIOB,BRME1,BRCA2,and HSF2BP)in the sexually matured and immature cattle-yak,yak,and yellow cattle were determined using qRT-PCR.The relative mRNA expression of these genes was significantly down-regulated in the cattle-yak compared with the matured and immature yellow cattle and yak.The tissue-specific expression of these genes was determined by finding their expression in the gonads(testes and ovaries)and some somatic tissues(kidney,liver,spleen,lungs,and heart).The results showed that Spata22,MEIOB,HSF2BP,BRME1 and DMC1 were only expressed in testes and ovaries.However,Rad51 and BRCA2 were not tissue-specific and were expressed in all the tissues examined.In addition,the expression of Spata22,MEIOB,Rad51,and DMC1 were higher in the testis than in the ovary.5.The expression and localization of Spata22,Rad51,and DMC1 proteins were done using Western blot,immunohistochemistry(IHC),and immunofluorescence(IF).Western blot results showed that the expression level of Spata22,Rad51 and DMC1 was higher in yak and yellow cattle,the results were consistent with the trend of the mRNA expression.Immunohistochemical(IHC)and immunofluorescence(IF)results showed that Spata22,Rad51,and DMC1 were mainly expressed in the spermatogonia,primary spermatocytes,and secondary spermatocyte,but not in round spermatids and spermatozoa.This is consistent with previous studies of intracellular depletion of these proteins at late pachytene in spermatocytes.The positive reaction intensity of Spata22,Rad51 and DMC1 in yak and cattle testis was higher than that in cattle-yak testis.6.To determine the stage of meiotic arrest in the cattle-yak,we determined the presence and expression levels of histone HIST1H1T(H1t)and HoxA4(midpachynema makers)and Cdc25c(a late-pachynema maker)in the testes tissue.The results showed that the mRNA expression of the pachytene markers was not detected in the testis of the cattle-yak,which proved that the meiotic arrest occurred at or before the pachytene stage.7.The degree of germ cell apoptosis and the relative mRNA expression of apoptosis-related genes were determined using TUNEL labelling and qRT-PCR.TUNEL staining was strongly positive in seminiferous tubules,which were mainly distributed in the basal and middle parts of spermatogenic epithelium,indicating that spermatogonia,primary and secondary spermatocytes were apoptotic.Also,a significantly higher number of apoptotic-positive cells were detected in the cattle-yak compared to the yellow cattle and yak.This indicates that a large number of germ cells in the testis of the yak cattle are dying by apoptosis.A disorder in the expression of the recombination pathway genes causes the death of primary spermatocytes by p53-dependent apoptosis.There was higher mRNA expression of p53 and Bax in the cattle-yak.Also,we found significantly lower levels of Bcl-2 in the testicular tissues of yellow cattle and yak compared to the cattle-yak.This suggests that either a process activating apoptosis is potentiated or a process inhibiting apoptosis is compromised in the germ cells of the cattle-yak.Conclusion:① The coding region(CDS)sequences of Spata22 and Rad51 were cloned successfully.No mutation sites were found in the Rad51 sequence,but 13 mutations were found in the Spata22 sequence② During the development of the cattle-yak testis,there was a lot of germ cell apoptosis,which led to the enlargement of the area and perimeter of the seminiferous tubules.The thickness of the seminiferous epithelium decreased significantly,which was unfavourable for spermatogenesis.Finally,no spermatogenic cells except spermatocytes were found in seminal tubules.③Due to the low expression of Spata22,Rad51,and DMC1 and related regulatory factors during meiosis of spermatogenesis,the spermatocyte meiosis was inhibited,which made the spermatogenesis process stop at the first meiotic metaphase and pachytene stage.