Regulation of bovine oocyte-specific transcripts during in vitro oocyte maturation and after maternal-embryonic transition analyzed using a transcriptomic approach.
Oocyte/embryo genomics in mammals faces specific challenges due to limited biological material, to the comparison of models with different total RNA contents, and to expression of a specific set of genes often absent from commercially available microarrays. Here, we report experimental validation of a RNA amplification protocol for bovine oocytes and blastocysts. Using real-time PCR, we have confirmed that the profile of both abundant and scarce polyadenylated transcripts was conserved after RNA amplification. Next, amplified probes generated from immature oocytes, in vitro matured oocytes, and in vitro produced hatched blastocysts were hybridized onto a macroarray that included oocyte-specific genes. Following an original approach, we have compared two normalization procedures, based on the median signal or an exogenous standard. We have evidenced the expected difference in sets of differential genes depending on the normalization procedure. Using a 1.5-fold threshold, no transcript was found to be upregulated when data were normalized to an exogenous standard, which reflects the absence of transcription during in vitro oocyte maturation. In blastocysts, the majority of oocyte-preferentially expressed genes were not activated, as previously observed in mouse. Finally, microarray data were validated by real-time PCR on a random subset of genes. Our study sheds new light on and complements previous transcriptomic analyses of bovine oocyte to embryo transition using commercial platforms.
Thelie
Aurore
A
INRA UMR Physiologie de la Reproduction et des Comportements, CNRS UMR, Université de Tours, Haras Nationaux, Nouzilly, France.
Papillier
Pascal
P
Perreau
Christine
C
Uzbekova
Svetlana
S
Hennequet-Antier
Christelle
C
Dalbies-Tran
Rozenn
R