Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance

The impact of vitrification procedures on in vitro matured (IVM) ovine oocytes mRNA content and ability to undergo successful fertilization, cleavage and embronic development was assessed. Vitrified-warmed (n=113) and control (n=140) IVM oocytes were in vitro fertilized and cultured up to blastocyst stage under standard conditions. Vitrified oocytes showed lower cleavage rate (47% vs. 75%, P<0.001) and development to blastocyst stage (17% vs. 57%, P<0.001) than controls. In addition, the timings of the first cleavage and blastocysts production were significantly delayed in the vitrifiedwarmed group (P<0.001 in both cases). In parallel, we analyzed by reverse transcriptase real-time PCR the relative abundance of b-actin, H2A.Z histone, Poli A Polimerase (PAP), Heat Shock Protein 90b (HSP90b), P34cdc2, Cyclin b, Na/K-ATPase and Type I cadherin (E-Cad) transcripts in single IVM controls (n=24) and vitrified-warmed oocytes (n=40). Results were normalized against the exogenous rabbit a-globin mRNA standard and the b-actin housekeeping gene and similarly described a lower abundance of most mRNAs in oocytes subjected to vitrification procedures. When normalized against the exogenous standard mRNA, all transcripts except for b-actin and H2A.Z showed a significantly different abundance in the two classes of oocytes. The same results were obtained after normalization against the internal standard, except for HSP90b and E-Cad transcripts, whose lower abundance in vitrified-warmed oocytes resulted prominent, but not significant (P=0.083 and P=0.068, respectively). The oocyte lower transcripts abundance following vitrification might be an early indicator of poor quality in good correlation with the developmental data to blastocyst stage.