PLCz functional haplotypes modulating promoter transcriptional activity are associated with semen quality traits in Chinese Holstein bulls

PLoS One. 2013;8(3):e58795. doi: 10.1371/journal.pone.0058795. Epub 2013 Mar 15.

Abstract

The sperm-specific phospholipase C zeta (PLCz) is a candidate sperm-borne oocyte-activating factor that triggers a characteristic series of physiological stimuli via cytoplasmic Ca(2+) oscillations during fertilization. The molecular mechanisms involved in the regulation of PLCz gene expression remain largely unknown. To explore the genetic variations in the 5'-flanking region of the PLCz gene and their common haplotypes in Chinese Holstein bulls, as well as to determine whether these variations affect bovine semen quality traits and transcriptional activity, DNA samples were collected from Chinese Holstein bulls and sequenced for the identification of genetic variants in the 5'-flanking region of PLCz. Two genetic variants were identified, and their haplotypic profiles were constructed. The two novel genetic variations (g. -456 G>A and g. +65 T>C) were genotyped in 424 normal Chinese Holstein bulls. Bioinformatics analysis revealed that both loci are in transcription factor binding sites of the core promoter region. The association studies revealed that the two genetic variations and their haplotype combinations significantly affected semen quality traits. Using serially truncated constructs of the bovine PLCz promoters and the luciferase reporter, we found that a 726 bp (-641 nt to +112 nt) fragment constitutes the core promoter region. Furthermore, four haplotypes, H1H1 (GTGT), H2H2 (GCGC), H3H3 (ATAT), and H4H4 (ACAC), were significantly associated with semen quality traits and successfully transfected into MLTC-1 cell lines. The luciferase reporter assay showed that the different haplotypes exhibited distinct promoter activities. Maximal promoter activity was demonstrated by the H2H2 haplotypes, as compared with the other haplotypes. To the best of our knowledge, this study is the first report on genetic variants and their respective haplotypes in the 5'-flanking region of PLCz gene that can influence the semen quality of Chinese Holstein bulls as well as contribute to the transcriptional activity of the PLCz promoter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Flanking Region
  • Animals
  • Base Sequence
  • Cattle
  • Cell Line
  • Gene Expression
  • Genetic Variation
  • Genotyping Techniques
  • Haplotypes*
  • Male
  • Molecular Sequence Data
  • Phosphoinositide Phospholipase C / genetics*
  • Promoter Regions, Genetic*
  • Quantitative Trait, Heritable*
  • Reproduction / genetics
  • Semen Analysis*
  • Transcription, Genetic*
  • Transcriptional Activation

Substances

  • Phosphoinositide Phospholipase C

Grants and funding

This work was partially supported by grants the National Science and Technology Pillar Program during the 12th Five-Year Plan of China (Grant no. 2011BAD19B04), the Modern Agro-industry Technology Research System (Grant no. CARS-37), State Major Project of Transgenic (2011ZX08007-004), the Well-bred Program of Shandong Province (Grant no. 2012LZ10-06), and the Key Scientific and Technological Project of Shandong Province (Grant no. 2009GG20002033). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.