Apoptosis related gene transcripts in buffalo cumulus oocytes complex in relation to its morphology and in vitro maturation conditions
Abstract
The present study was carried out on Cumulus Oocyte Complex (COCs) aspirate from buffalo ovarian follicles obtained from slaughter house and investigated for the presence of Bcl-2 and Bax gene transcript in poor and good quality COCs graded mainly on the basis of cumulus cells layers enclosing oocyte and was further related with outcome of in vitro fertilization (IVF) rates in these groups. The overall recovery rate of COCs per ovary was 0.84 with 1667 COCs recovered from 1965 ovaries. After their grading the average recovery rate of grade-I COCs was 0.38, lower as compare to grade-II with 0.48. The grade-I COCs had homogenous ooplasm with hazy zona pellucida, visible only as a dark structure enclosing ooplasm and oocyte completely enclosed inside several layers of cumulus cells. In poor grade COCs, the ooplasm was heterogeneous, and oocyte was completely or partially denuded with clearly visible zona pellucida and
the cumulus layers were already expanded and detached from oocyte while handling. Good quality COCs showed maximum maturation percent 79.48 and minimum 68.57 with expanded cumulus cell layers and single polar body formation after in vitro maturation (IVM). In poor quality COCs this percentage varied between 8.69 and 4.83 percent in addition there was nuclear chromatin condensation, ooplasm shrinkage and increase in perivitelline space. Maturation was observed only in partially denuded COCs under grade-II COCs. Percent maturation was very less in poor quality COCs as compare with good quality. The percent cleavage rate after IVF under good quality COCs was found to vary between 64.44 percent and 55.31 with overall average of 61.57 %. In poor quality oocytes cleavage percentage ranged between 5.55 and 2.77 with overall 4.91 percent. This result clearly indicates that the cleavage rate in good quality COCs is much higher as compared to poor quality COCs graded on the basis of their morphology. Furthermore, zygote obtained from grade-I COCs developed beyond two celled stage in contrast from grade-II COCs were unable to develop beyond two celled stage and degenerated gradually. The blastomeres in such incompetent embryos were also not identical in size. The fertilized COCs showed formation of secondary polar body and unfertilized in both grade were found to degenerate gradually. For the first time our study showed the presence of Bcl-2 and Bax transcripts in COCs of buffaloes, which was detectable in COCs of both good and poor grade. No significant difference was observed for transcript level of Bcl-2 in grade-I COCs before and after maturation but the transcripts for Bax was up-regulated after maturation. Under grade-II COCs transcript level for Bax was relatively more as compare Bcl-2 before maturation but after maturation no transcripts were evident for both genes but β-actin, positive control was amplified. The genomic DNA isolated from COCs after IVM showed the presence of high molecular weight DNA along with diffuse pattern in gel indicating presence of low molecular weight fragmented nucleic acid, possibly generated due to action of caspase activated DNAse, but fragmentation of DNA giving ladder like pattern was not clearly evident which is hallmark for apoptosis. Such changes were not present in the DNA isolated from immature COCs in which only DNA of high molecular weight was evident possibly due to less number of apoptotic cells at the start of IVM