Functional haplotypes of ARID4A affect promoter activity and semen quality of bulls.

The AT-rich interaction domain 4 A (ARID4A) has an important role in regulating Sertoli cell function and male fertility. Its molecular mechanisms, however, remain largely unknown. In this study, two single nucleotide polymorphisms (SNPs) (g.53 G > T, ss 1966531596, and g.826 G > A, rs 210809648) were identified in the promoter region of ARID4A in 215 Chinese Holstein bulls using polymerase chain reaction (PCR)-restriction fragment length polymorphism and created restriction site-PCR. Results revealed that bulls with g.53 G > T-GG and g.826 G > A-G G genotype exhibited higher sperm deformity rate than those with g.53 G > T-TT and g.826 G > A-AA genotype (P <  0.01). Furthermore, three haplotypes (H1 (GG), H3 (TG), H4 (TA)) and six haplotype combinations (H1H1, H1H3, H1H4, H3H3, H3H4, H4H4) were obtained. The bulls with H4H4 exhibited lower sperm deformity rate than those with H1H1 and H1H3 (P <  0.05). In addition, results of bioinformatics analysis revealed that ARID4A has two promoters and that two SNPs of ARID4A are located in transcription factor binding sites. Compared with g.53 G > T-G and g.826 G > A-G allele, there was a greater fluorescence intensity in g.53 G > T-T and g.826 G > A-A allele by transient transfection in MLTC-1 cells and the luciferase report assay. qRT-PCR indicated the ARID4A expression was greater in bull spermatozoa with H4H4 haplotype combination than those with H1H1 haplotype combination (P < 0.05). Results of the present study indicate that g.53 G > T and g.826 G > A are functional mutations that are involved in regulation of ARID4A gene expression by affecting promoter activity and thus semen quality of Chinese Holstein bulls.