Abstract:
In vitro maturation (IVM) is the first and the most important step for IVF technique because the success of in vitro embryos production highly depends on oocyte quality. In the present study it was aimed to identify the optimal time of maturation of cattle oocytes, in which IVF techniques are very important and frequently applied. One of the indicators of IVF failure is the apoptosis, occurring in the participating cells on the whole cycle of development. Therefore is essential to identify highly accurate the apoptotic moment. In this respect, the expression of two genes: Bax and Bcl2 was assessed in three stages of maturation of oocytes: time 0, 24h and 48h of cultivation on maturation culture media. These genes have been previously shown to play an essential role in apoptotic process of the cells. The genes expression was evaluated by Quantitative Revers-Transcription PCR, using SYBR Green reagents, having as target the total ARN isolated from the complex oocytes - cumulus cells. The value of genes expression was normalized using as control the Actin gene biomarker. Obtained data were interpreted using 2ΔΔC(T) method and the statistical analysis was performed using ANOVA algorithms. In the determinations, it was found that the expression of the two : Bax and Bcl2 genes are somewhat antagonist, so that at 24 hours of maturation time are both over-expressed comparing to the 0 moment. By comparison, Bcl2 expression is low, which indicates that the apoptotic process has not started and that the stress of in vitro cultivation has not reached the maximum level. Bax gene expression indicates the existence of high stress factors, but not the entry of a cell into apoptosis process. Therefore, it can be considered that the optimum time interval for maturation in culture media is 24h. At 48h the Bax gene overexpression compared to the Bcl2 gene expression indicates the entry of cells into apoptotic process, knowing that a very high level of the Bax gene may have an inhibitory effect on Bcl2 gene experssion.
