Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus).

Accurate embryonic gene activation (EGA) is essential for the embryo's developmental potency and reflects the quality of in vitro produced embryos. In order to describe the dynamic and temporal patterns of EGA in the cat, the mRNA expression of developmental important genes (DNA methyltransferases 1 and 3A, DNMT1 and DNMT3A; gap junction protein alpha 1, GJA1; transcription factor Octamer 4, OCT4; insulin-like growth factors 1 and 2 receptors, IGF1R and IGF2R) was examined by RT-PCR techniques in preimplantation embryos obtained after in vitro maturation and fertilization. Furthermore, influences of intracytoplasmic sperm cell injection and sperm cryopreservation on the relative mRNA abundance in day 4 to 5 morulae were analyzed. Total RNA was obtained from immature and matured oocytes, 2-cell, 4-cell, 8- to 16-cell embryos, morulae and blastocysts. RNA was transcribed into single stranded cDNA by reverse transcriptase. After amplification, a non-felid standard RNA was used for semi-quantitative analysis. Our results showed an increase in transcript abundance from the matured oocyte to 2-cell embryo for all examined genes except for IGF2R, indicating that, in vitro, the embryonic genome is activated shortly after fertilization. However, the activation pattern varied markedly between the different genes. We also found different patterns of mRNA expression for the examined genes in morulae produced either by IVF or ICSI, and using fresh or cryopreserved sperm. Due to high variations within the single groups of compared morulae we were able to observe only a tendency towards higher relative mRNA expression in embryos derived by in vitro fertilization with fresh sperm in comparison to all other groups.