Transcriptome analysis of buffalo bull spermatozoa for identification of fertility associated gene(s)
Abstract
Keeping in view the importance of sperm RNA and its correlation with fertility, the proposed study was
carried out to determine the gene expression between high fertile and sub-fertile buffalo bull spermatozoa and
identify the genes potentially associated with bull fertility through transcriptome analysis. These fertilityassociated
genes were further validated with qRT-PCR and their possible role in fertilization through in-vitro
fertilization.
Ten bulls were classified on the basis of conception rates (CR), where bulls having CR <40% and 50%
were considered as sub- and high-fertile bulls, respectively. Total motility, rapid motility, average path velocity,
curvilinear velocity, amplitude of lateral head, beat cross frequency, straightness, linearity and intactness of plasma
membrane of sperm for high-fertile bulls were significantly higher than sub-fertile bulls. Semen samples from
these bulls were utilized for sperm enrichment using three different density gradient methods viz., Percoll,
BoviPure and Iodixanol. Purified sperm pellets were subjected to RNA isolation using conventional and kit
methods with and without TRIZOL. Maximum yield of RNA was achieved when the semen sample was purified
using BoviPure as density gradient followed by isolation of RNA by RNAqueous + heated TRIZOL method.
Further, AKAP4, CRISP2, Fertilin- â and PLCZ1 transcripts showed significantly higher expression in high- fertile
than sub-fertile groups. Transcriptome analysis was done by Next generation sequencing and a total of 33715 and
21788 transcripts were expressed in sub- and high-fertile groups, respectively. When these transcripts were
annotated with the available sequence of buffalo, 1248 and 802 transcripts were uniquely expressed in the sub- and
high-fertile groups, respectively and 2527 transcripts were identified as common in both the groups. When in silico
translated these transcripts, 258 proteins were observed to be commonly present in both the groups. Apart from
these, 6 and 18 proteins were specifically present in the high and sub-fertile group, respectively.
The expression of AKAP4, CRISP2, Fertilin- â and PLCZ1 was evaluated in IVF derived embryos. All
the four transcripts differentially expressed only in sperm and embryo up to cleaved and blastocyst stage, but these
genes didn‟t express in immature and mature oocytes. Thus, the present study provides the first evidence of
differential expression of AKAP4, CRISP2, Fertilin â and PLCZ1 genes in high and sub-fertile bulls and their
transmission to embryo indicating the role of sperm RNA in fertility and embryo development.