International Journal of Chemical Studies
Vol. 5, Issue 5 (2017)
Correlation between Clusterin expression and lipid peroxidation status of Caprine semen
Author(s): M Vengatachalam, K Loganathasamy, V Leela, J Ramesh, K Vijayarani and Bhaskaran Ravi Latha
Abstract: The present investigation has been undertaken to study the transcription pattern of clusterin (CLU) gene in buck semen and to assess in vitro sperm characteristics of buck semen which are screened for CLU gene. Fresh semen samples were collected from 12 bucks maintained in the organized goat farms by artificial vagina. Normalization of initial concentration of spermatozoa was carried out in all buck semen samples before proceeding for RNA isolation. So, initial concentration of each sample was made equal. The spermatozoa were isolated from buck semen samples by swim- up protocol using sperm TALP. Total RNA from the buck spermatozoa were extracted and first strand cDNA was synthesized from 1μg total RNA by using commercial kits. Confirmation of CLU and GAPDH gene was performed by conventional PCR with product size 302 and 227 bp, respectively. The eluted cDNA was sequenced commercially and was subjected to BLAST analysis. Sequencing of the cloned CLU gene from buck semen showed 98% identity to the known caprine sequence (EMBL Accession NO: XM_018052776.1).Absolute quantification of CLU gene transcripts in semen samples from 12 bulls was performed by plotting standard curve. Variations in levels of CLU gene transcripts (2500-22546000 copies) were found among 12 different buck semen samples. Relative normalized gene expression was analyzed between housekeeping gene (GAPDH) and target gene (CLU) for all the 12 buck semen samples. Expression of GAPDH did not show significant difference. But, expression of CLU was up regulated in buck number 2, 4, 7, 8, 10 and 12 and there was no change in expression of CLU in buck number 1,3,5,6,9 and 11, when compared to threshold regulation of GAPDH. LPO status of semen samples was assessed by estimation of MDA concentration. Variations in MDA (1.12-1.96 μmol/ml) concentration from 12 different buck semen samples were highly (positively) correlated (p<0.01) with expression levels of CLU gene transcripts in spermatozoa. In vitro sperm characteristics were also studied from 12 buck semen samples. Variations in sperm characteristics such as sperm motility (60.0 - 80.0%), sperm viability (72.0 - 93.0%), sperm morphology (73.0 - 91.0%), plasma membrane integrity (50.0 - 82.0%), acrosome integrity (81.0 - 93.0%), DNA integrity (82.0 - 93.0%) and MMP (46.0 - 74.0%) were found among buck semen samples. All in vitro sperm characteristics were highly (negatively) correlated (p<0.01) with expression levels of CLU gene transcripts in spermatozoa. From this study, it is evident that ejaculated buck semen has variations in transcription pattern of CLU gene in spermatozoa among bucks and expression levels of CLU transcripts have positive correlation with malondialdehyde (MDA) concentrations of buck semen samples and negative correlation with in vitro sperm characteristics.
Pages: 1997-2000 | 537 Views 11 Downloads
How to cite this article:
M Vengatachalam, K Loganathasamy, V Leela, J Ramesh, K Vijayarani, Bhaskaran Ravi Latha. Correlation between Clusterin expression and lipid peroxidation status of Caprine semen. Int J Chem Stud 2017;5(5):1997-2000.