Table 1.
Means and standard errors (SE) of sperm quality traits in 307 Chinese Holstein bulls.
Table 2.
Primer information used for cloning the bovine HIBADH gene promoter region.
Fig 1.
HIBADH expressed in different bull tissues.
(A) The transcripts of HIBADH were detected by RT-PCR in lung, spleen, heart, testis, epididymis, and ejaculate semen. GAPDH was used as the positive control. (B) Western blot analysis of HIBADH in adult bull tissues and ejaculate (with β-actin as the control).
Fig 2.
Immunolocalization of HIBADH in bull seminiferous epithelium and epididymis.
(A, B, C, and D) Localization of HIBADH in adult bull testis, caput epididymis, corpus epididymis, and cauda epididymis, respectively. The brown area indicates the expressed protein, whereas the blue area is the nuclear DNA signal (negative control). These cells were photographed using an inverted microscope (OLYMPUS) at 40 × 10. Images were obtained on a 50 μm scale plate.
Fig 3.
HIBADH is expressed and localized in bull spermatozoa.
The arrow heads indicate that HIBADH protein was mainly localized at sperm neck-piece and mid-piece, and to a lesser extent, in the sperm head. (A) Negative control. (B) Nuclear DNA signals (blue). (C) HIBADH protein signals (green). (D) Merging of HIBADH protein (green) and nuclear DNA signals (blue). Image was obtained using an inverted microscope (OLYMPUS) at 40 × 10. Images were obtained on a 5 μm scale plate.
Fig 4.
Structure of HIBADH, location of the identified SNP (g.-165 T>C), and PCR-RFLP patterns.
Patterns for g.-165 T>C genotypes TT, TC, and CC. M: Marker. Digestion with Sma I of the amplified HIBADH gene g.-165 T>C locus produced fragments of the following sizes: 967 bp for genotype TT; 967, 710, and 257 bp for genotype TC; and 710 and 257 bp for genotype CC.
Fig 5.
The core promoter region sequence of HIBADH from the -703 bp to +175 bp of Chinese Holstein bulls.
The green-highlighted TSS is marked as A with +1 (the nucleotide sequence numbered +1 is the first A of the TSS). The numbering of nucleotides is relative to the ATG. The boxed sequences represent the putative transcription factor binding sites. The nucleotides highlighted in blue represent the previously reported SNP (g.-165 T>C, rs 133569227).
Table 3.
Genotypic frequencies, allelic frequencies, and genetic diversity (PIC, He, Ne, and x2) of the bull HIBADH gene at position g.-165 T>C.
Table 4.
Least square means and SE for semen quality traits of different genotypes in the HIBADH gene of 307 Chinese Holstein bulls.
Fig 6.
Scheme of the 5′-flanking region of the HIBADH gene and the identification of the core promoter region.
The red arrow represents the previously reported SNP (g.-165 T>C, rs 133569227). Fragments pGL3-1548 (-1,548 bp to +792), pGL3-1013 (-1,013 bp to +792), pGL3-703 (-703 bp to +792), pGL3-703→+175 (-703 bp to +175), and pGL3+175 (+175 bp to +792) were amplified by PCR to produce the reporter constructs. Each fragment with wild-type alleles was cloned into the pGL3-Basic vector and transfected into MLTC-1 cells. Relative luciferase activity of a series of truncated constructs in the HIBADH 5x-flanking region was measured by dual luciferase assays of the MLTC-1 cells. For each construct, plasmid DNA extracted from 6 to 9 colonies was used. Results are presented as the average fold of firefly luciferase activity vs. the Renilla control vector (mean x S.D., n = 6 to 9). The asterisk indicates P < 0.01 vs. the pGL3 basic control.
Fig 7.
The effect of the SNP g.-165 T>C on HIBADH transcriptional activity in MLTC-1 cells.
The 5′-flanking region of the HIBADH gene containing T or C loci (designated as pGL3-T and pGL3-C) from the HIBADH core promoter (-703 bp to +175 bp) was transiently transfected into MLTC-1 cells. The empty vector (pGL3) provided the lowest level of luciferase activity among all the loci constructs. The pGL3-T genotype showed 58% higher transcriptional activity than the genotype pGL3-C. For each construct, individual plasmid DNA extracted from 6 to 9 colonies was used. Results are presented as the average fold change of firefly luciferase activity vs. the Renilla control vector (mean ± S.D., n = 6 to 9) (Black star indicates P < 0.05).
Fig 8.
The promoter methylation profile of the HIBADH in the high-motility group (Bulls 1, 2, and 3) and the low-motility group (bulls 4, 5, and 6).
We amplified a 175 bp fragment with 17 CpG sites in the core promoter region of each bull. Each fragment was cloned into a pEASY-T3 vector and transformed into E. coli DH5α cells for clone sequencing. Sixty selected clones (10 clones per bull) and a total of 1,020 CpG sites were analyzed. No significant difference in the frequency of DNA methylation of the HIBADH promoter between the high-performance (5.88%, n = 30 clones) and low-performance bulls (6.66%, n = 30 clones) (P > 0.05) was found, and all clones were hypomethylated (<50% of CpG sites on a given methylated strand). The 7th CpG site showed higher methylation level in the high-motility group (73.33%, 22/30) than in the low-motility group (23.33%, 7/30) (P < 0.01). Black circle represents Methylated CpG, Blank circle represents Unmethylated CpG.
Table 5.
Initial sperm motility parameters and promoter methylation levels of the six selected adult bulls.