Cloning And Sequencing Of Yak KDM1A And KDM4B And Their Expression In Yaks And Cattle-yaks Testes

The hybrid of cattle(Bos taurus)with yaks(Bos grunniens)is called the cattle–yak.All female cattle-yaks are fertile,but male cattle-yaks from F1 to F3 has no normal reproductive capacity and cannot produce sperm.To date,there is no clear conclusion on the mechanism for cattle-yak male sterility.Lysine histone demethylase(KDMs)may affect histone methylation levels suggests that they might play important roles during spermatogenesis.The objective of this study was to explore the possible role of KDM1 A and KDM4 B for male sterility of yak hybrids.The experimental materials were sexually mature adult yaks(n=10)and F1 cattle–yaks(n=10).The epididymes of sexually mature yaks and cattle–yaks were made into paraffin sections,and the histological structure of the epididymis was observed under a microscope after staining.Total RNA was extracted from the testes of yaks and cattle–yaks.The coding sequences(CDS)of KDM1 A and KDM4 B,were cloned by RT-PCR.The levels of KDM1 A and KDM4 B in the testes of yaks and cattle–yaks were detected using real-time PCR and Western blotting for m RNA and protein,respectively.The histone methylation modifications of H3K36me3 and H3K27me3 were compared between testes of yaks and cattle–yaks using ELISA.The main results of this study are follows:(1)Histological structure of yak and cattle–yaks epididymis were observed under microscope,A large amount of sperm can be found in the epididymal ducts of the yaks.However,in the epididymis sections of cattle–yaks,no sperm was observed.(2)Sequencing results of KDM1 A of yak testis showed two variants similar to cattle.KDM1 A variant 1 has a CDS of 2622 bp,encoding 873 amino acids,while variant 2 has a CDS of 2562 bp,encoding 853 amino acids.Compared to variant 1,variant 2 is missing a 60 bp fragment while the rest of the sequence is identical.Compared with cattle two variants,yak KDM1 A two variants have five nucleotide differences in the CDS but identical amino acid sequence.The CDS of KDM1 A gene variant 1 shares 93.29%～99.81% sequence similarity with six animal species,namely,cattle(99.81%),wild yak(99.50%),sheep(97.75%),pig(93.77%),dog(93.29%),and horse(93.60%).The CDS of the KDM4 B gene was 3351 bp in length,coding 1116 amino acids.Compared with cattle,yak KDM4 B lacks a segment of 25 amino acids in the middle of the sequence.Compared to cattle,yak KDM4 B has 28 nucleotide differences and results in 9 amino acid differences.The KDM4 B gene sequence shares 87.74%～99.07% identity with six animal species,namely,cattle(99.07%),wild yak(97.94%),sheep(96.84%),pig(90.95%),dog(88.46%),and horse(87.74%).These results suggest that the yak KDM1 A and KDM4 B genes are basically conserved.(3)The m RNA levels of KDM1 A and KDM4 B in the testes of yaks and cattle–yaks were assayed using real-time PCR.The results showed that the relative m RNA levels of the KDM1 A and KDM4 B genes in the testes of cattle-yaks were reduced significantly than those in yaks(P<0.01).The expressions of KDM1 A and KDM4 B proteins in testes were tested by Western blotting.The results showed the expressions of the KDM1 A and KDM4 B proteins in cattle–yaks were also reduced significantly than those in yaks(P<0.01).(4)The ELISA results showed that the level of H3K36me3 was significantly decreased in the testes of cattle–yaks compared with yaks(P<0.05),but not for H3K27me3.The above results indicate that cattle–yak spermatogenesis may be affected by the levels m RNA and protein of KDM1 A and the trimethylation of histone H3K36.The results of this study provide new data for further epigenetic interpretation of the molecular mechanism for cattle–yaks male sterility.