PLCz Functional Haplotypes Modulating Promoter Transcriptional Activity Are Associated with Semen Quality Traits in Chinese Holstein Bulls

Qing Pan,Jinming Huang,Yan Zhang,Jifeng Zhong,Qiuling Li,Mei Liu,Qin Gao,Changfa Wang,Zhihua Ju,Chao Qi,Lei Zhou,Lingling Wang,Claire Wade

doi:10.1371/journal.pone.0058795

Qing Pan, Jinming Huang + Show 11 more

Open Access

https://doi.org/10.1371/journal.pone.0058795

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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 Ca2+ 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.

Highlights

Fertility is one of the most economically important production traits in cattle improvement
Prediction of the promoter region of phospholipase C zeta (PLCz) gene The bioinformatics analysis predictions indicated that the core promoter region of the PLCz gene is located in the 2461 bp to +100 bp region
The bovine PLCz gene is composed of 14 exons and encodes a protein with 634 amino acids; PLCz is a key protein during sperm-egg fusion in mammals [35]

Summary

Introduction

Fertility is one of the most economically important production traits in cattle improvement. Fertility is greatly affected by the genetic make-up and the environment. Artificial insemination (AI) is known as one of the most valuable techniques for the genetic improvement of semen quality traits in dairy cattle herds [1]. The quantity and quality of semen from bull (including the ejaculate volume, sperm density, initial sperm motility, frozen semen motility, and the sperm deformity rate) are affected by heredity, breeding conditions, the physiological status of the parents, temperature, and other factors. Several reports have described the use of candidate genes for MAS to influence semen quality and fertility in boars [3,4,5], goats [6], and bulls [7,8,9,10,11,12]

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