Analysis of sperm cytoplasmic mRNA content with special reference to the storage temperature and its possible impact on early embryonic development

Aims: The current study aims to investigate five mRNA necessary for chromatin decompaction and demetilation and mRNA degradation in Oryctolagus cuniculus sperm cytoplasm. The investigation was implemented on three groups, one fresh control and two stored at different temperatures such as 4°C and 15°C to investigate also an impact of storage temperature on possible mRNA self decay. The results would be beneficial for understanding of maternal – paternal genes interplay during the very first hours of the embryonic development and the impact of paternal genes on the basic tasks implementation just before the embryonic genome activation (EGA). This knowledge would help to improve ART success rates by development better medium, storage and ICSI techniques for the cases with unexplained infertility. Materials and Methods: Sperm for the current project was collected once or twice a week (during 7 weeks) using artificial vagina from adult male rabbits known to be fertile. After dilution, the samples were divided into two equal groups. The first group of the samples was stored at 4°C and the second one was stored at 15°C for 72 hours. At the end of this period, a freshly taken ejaculate after examinations was also added as a control group (without previous storage). The mRNA was isolated, cDNA synthesized and qRT-PCR performed. For the gene expression comparison among the groups the ΔCT method was used as a normalization method, and the Independent Samples one-sided T Test was used to decide on the significance of the intergroups differences with the 0.05 level of significance. Statistic analyses were performed using SPSS (Statistical Packag for Social Sciences) for Windows 22. xiv Results: The study has confirmed that sperm cytoplasm indeed contains paternally produced mRNA, and this study could determine five gene transcripts in Oryctolagus Cuniculus sperm cytoplasm: CNOT1, CNOT8, TET3, NPM2 and XRN1. Moreover, CNOT8 and CNOT1 were present the most, XRN1 and NPM2 were present the least. According to the Independent Samples T Test, TET3 transcripts' sensitive to low storage temperatures was claimed with 87% of statistical power at 0.95 significance. Conclusion: The current study has met it's research goals succesfully. It has added valuable research data to the field of research in sperm cytoplasmic content and to the whole field of embryology. This research contribution has also added to the field of knowledge on Oryctolagus cuniculus species.