Variation Of PLCz And Its Associations With Sperm Quality Traits In Chinese Holstein Cattle

Phospholipase C-zeta(PLCz) is one of five subtypes of the Phospholipase C (PLC) which is a key enzyme in phosphatidyl inositol turnover during signal transduction. PLCz expressed in the testicular tissue specially, which can provoke and trigger the Ca2+oscillations and oocyte activation as well as the important roles in acrosomal reaction, fertilization and the succedent embryonic development. Study on the PLCz gene about the molecular genetic characteristics and the correlation to the sperm quality traits provides theoretical foundation on the marker-assisted selection of the bull in high prolificacy, as it plays an important role during sperm-egg fusion in mammals.1. Analysis of PLCz gene promoter transcriptional activityThe objectives of this study were to clone and characterize the expression and regulation of bovine PLCz in sperm through the analysis of single nucleotide polymorphisms (SNPs) using DNA sequencing methods and PCR-restriction fragment length polymorphism (PCR-RFLP) to study their association with sperm quality and fertility of 424 normal Chinese Holstein Bulls from three bull stations (including Beijing Dairy Center, Shanghai Bright Dairy Center, Shandong OX Bull Station).To explore genetic variations in the 5’-flanking region of PLCz gene as well as their frequent haplotypes in the Chinese Holstein Bulls and determine whether such variations have an impact on bovine sperm quality traits and transcriptional activity, DNA samples were collected from Chinese Holstein Bulls and sequenced for identifying genetic variants in the 5’-flanking region of PLCz. Two genetic variants were identified and haplotype profiles were constructed for the genetic variants. The two novel genetic variations (g.-456 G>A and g.+65 T>C) were genotyped in 424 normal Chinese Holstein Bulls, which are both located in the core promoter region and in the transcription factor binding sites through the bioinformatic analysis. The association results showed that the two genetic variations and their haplotype combinations have significant impact on the sperm quality traits.Using serially truncated bovine PLCz promoter luciferase constructs, we found that a 726 bp (-641 nt to +112 nt) fragment constitutes the core promoter region. In addition, four haplotypes H1H1 (GTGT), H2H2 (GCGC), H3H3 (ATAT) and H4H4 (ACAC), which have significant association with the sperm quality traits, were transfected into MLTC-1 cell lines. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. Maximal activity was shown for H2H2 haplotypes compared with the others’ promotor activity. The results of this study demonstrate, for the first time, that genetic variants and their haplotypes in the 5’-flanking region of PLCz gene can impact on the Chinese Holstein Bulls sperm quality traits and contribute to the transcriptional activity of the PLCz promoter.2. Identification and characterization of PLCz gene novel splice variantWe characterized a novel transcript referencing PLCz sequence in the testis tissues of both adult and fetal Chinese Holstein bulls by using reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis. The novel splice variant (PLCz-svl) was derived from the PLCz complete transcripts (PLCz-complete) via alternative splicing (AS) that the pattern was alternative 3’splice sites. Quantitative real-time PCR analysis demonstrated that the expression levels of the PLCz gene complete transcript was not only significantly different from the novel splice variant (PLCz-svl) in the same bovine testis tissues but also different from adult versus fetal cows. Moreover, PLCz protein sequencing analysis showed that PLCz-svl made the 453th-457th amino acids deletion, leading to the transcription termination stopped in advance and the protein structure was different with the original PLCz protein, eventually. In summary, we concluded that the novel splice variant may suggest some potential novel roles in the expression and the function of bovine PLCz.3. Polymorphism of PLCz gene and its correlation with sperm quality traitMolecular cloning of the bovine PLCz yielded a 53160 bp sequence which transcripted into a 2176 bp codons and encoded a number of 634 amino acid. We detected eight novel SNPs, g.27529 T>A and g.27597 T>C located in the seventh intron, g.47969 G>A, g.48020 T>C, g.48079 G>A, g.48127 G>T and g.48384 G>T in the twelveth intron, thus the SNP g.27768 G>C in the eighth exon was identified as a non-synonymous mutation [GCC(Ala)>CCC (Pro)] at 337th amino acid. Significantly, linkage disequilibrium analysis showed that g.27529 T>A, g. 27597 T>C and g.27768 G>C (SNP1); g.47969 G>A, g.48020 T>C, g.48079 G>A, g.48127 G>T and g.48384 G>T (SNP2) were completely linked. Correlation analysis with the sperm quality traits showed that the SNP2 marker had a respective effect on the percentages of viable sperm (p<0.05). Whereas, the SNP1 marker had no significantly relationship with the sperm production traits (P>0.05).In addition, The association analysis between nine combined haplotypes and bull reproductive traits showed that the bulls with the combined haplotype TTAB was significantly higher with ejaculate volume (P<0.05), sperm density(P<0.01) and frozen semen motility (P<0.05) than the other haplotype combinations; as well as the TTAB haplotype with initial sperm motility was higher than that with the TTBB haplotype combination(P<0.05); but the TTAB haplotype with deformity rate was lower than that with the CTAB haplotype combination(P<0.05). The result indicated that the TTAB combined haplotype may be regard as a new molecular marker associated with sperm quality traits which in marker-assisted selection of Chinese Holstein Bulls breeding programs.