Oxygen levels regulating embryonic genome activation

Involuntary infertility affects some 18% of the world's population. Assisted reproductive technologies (ART), including in vitro fertilization (IVF), are for many the only hope on their journey to have children. However, the success rate of IVF is still 33-42%, and one of the main bottlenecks in ART is that 45-50% of the fertilized oocytes stall in development and do not reach the blastocyst stage of embryo development. The stalling of in vitro embryos mostly happens around day 3 of development, which coincides with the embryonic genome activation (EGA) event in humans. Due to ethical concerns about using human embryos for research and the scarcity of the material, studying EGA in humans is challenging. Therefore, in this thesis, we tackled this problem by establishing bovine as a model for human embryogenesis for higher similarity than, e.g., mouse. We achieved this by developing a method to perform RNA sequencing using single cells/embryos with limited numbers of samples. STRT-N seq is a 5' end sequencing method, making it possible to distinguish between degrading maternal transcripts and novel embryonic transcripts (Paper I). Next, we applied this method to identify previously predicted but unconfirmed paired-like homeobox (PRDL) genes in bovine that are similar to those involved in human EGA. We provided evidence of the PRDL genes in bovine and cloned them from the specific embryo development stages, showing their involvement in bovine EGA. This confirms that bovine models are indeed useful for studying human embryogenesis, as many PRDL genes are absent from mice, rendering them unsuitable for such studies (Paper II). We then applied this knowledge to understand the impact that oxygen levels have on in vitro embryo development, especially on EGA and metabolism. We found that the highest blastocyst formation rate happened in hypoxic culture conditions that induced timely EGA and energy metabolism genes. On the contrary, normoxia slowed down maternal transcript degradation, which is necessary for the embryo to achieve totipotency and reprogramming. In addition, these embryos had several key metabolic pathways downregulated, making them less likely to be able to respond to the environment, and this might potentially be the reason why the embryos grown in normoxia have a lower success rate in IVF. Finally, we tested the hypothesis of a sequential hypoxia culture system by switching to ultrahypoxia after day 3 of development and showed that these embryos had a similar transcriptomic profile to hypoxia embryos, but a significantly lower number reached the blastocyst embryo stage (Paper III). Overall, this thesis introduced a new RNA sequencing technology applicable to various embryology studies and offered insights into EGA in bovine embryos. It also showed the impact of IVF culture conditions on EGA and the metabolism of in vitro embryos to improve embryo culturing protocols for ART.

List of scientific papers

This thesis is based on the following original articles:

I. Optimized single-cell RNA sequencing protocol to study early genome activation in mammalian preimplantation development. Nina Boskovic, Gamze Yazgeldi, Sini Ezer, Mari H. Tervaniemi, Jose Inzunza, Spyridon Panagiotis Deligiannis, Barış Yaşar, Tiina Skoog, Kaarel Krjutškov, Shintaro Katayama, Juha Kere. STAR Protocols. 2023 Volume 4, Issue 3, 102357.
https://doi.org/10.1016/j.xpro.2023.102357

II. Molecular cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos. Barış Yaşar, Nina Boskovic, Marilin Ivask, Jere Weltner, Eeva-Mari Jouhilahti, Piibe Vill, Tiina Skoog, Ülle Jaakma, Juha Kere, Thomas R. Bürglin, Shintaro Katayama, Tõnis Org, Ants Kurg. BMC Genomics 25, 1048 (2024). https://doi.org/10.1186/s12864-024-10969-w

III. Oxygen level alters energy metabolism in bovine preimplantation embryos. Nina Boskovic, Marilin Ivask, Gamze Yazgeldi Gunaydin, Barış Yaşar, Shintaro Katayama, Andres Salumets, Tõnis Org, Ants Kurg, Karolina Lundin, Timo Tuuri, Carsten O. Daub, Juha Kere. Scientific Reports 15, 11327 (2025)
https://doi.org/10.1038/s41598-025-95990-z