128 NEURONATIN TRANSCRIPT ANALYSES AS MARKER TO CONFIRM SUCCESSFUL FERTILIZATION IN BOVINE BLASTOCYSTS

Abstract

In humans, intracytoplasmic sperm injection (ICSI) has become widely applied to overcome infertility. In addition to its clinical usefulness, ICSI is also a valuable research tool for studying fundamental aspects of gamete interaction during fertilization. Oocyte activation is a prerequisite for decondensation of the sperm head and formation of the male pronucleus regardless of whether the spermatozoon enters the oocyte by injection or by fertilization. In several species, the injection procedure itself is sufficient to activate the oocyte. However, the bovine oocyte is rarely activated by the mechanical stimulation of injection alone. Therefore, additional stimuli are required during ICSI, which may also initiate parthenogenetic development. Because blastocyst origin, either via IVF or after parthenogenetic activation, could not be distinguished at the morphological level via microscopic evaluation, the paternal genome needs to be determined, employing a molecular approach. Therefore, the aim of this study was to validate the expression of neuronatin (NNAT) mRNA, a paternally expressed gene, via RT-PCR, to verify whether bovine blastocysts were generated by fertilization or parthenogenesis. Bovine cumulus-oocyte-complexes were recovered from abattoir-derived ovaries by the slicing method. After 24 h of maturation, fertilization was achieved using a standard protocol. Presumptive zygotes were cultured in SOFaa for a period of 8 days. To generate parthenogenetic blastocysts, oocytes were activated, employing the following protocol. After 24 h of maturation, the oocytes were exposed to 5 µM ionomycin for 5 min and were then incubated in 1.9 mM 6-dimethylaminopurine for 4 h. Afterwards, treated oocytes were cultured in SOFaa for 8 days. Blastocysts from both experimental groups (IVF-derived and parthenogenetic) were stored at −80° until analysis. RT-PCR was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–510) for 6 embryos stemming from the IVF-group and 3 embryos out of the chemical-activated group. The NNAT mRNA was expressed in 5 blastocysts stemming from the IVF group; one blastocyst did not show NNAT mRNA expression, suggesting that it was derived from parthenogenesis. Conversely, NNAT mRNA could not be detected in all blastocysts derived from chemical activation, indicating that only the maternal genome was present. These results clearly demonstrate that NNAT mRNA expression is a reliable method to prove the paternal participation in early bovine embryo development.