Abstract
The oviduct is host to the period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of epigenetic marks as differentiation proceeds. This reprogramming period is vulnerable to changes in environmental conditions, such as compromised maternal health or diet. Likewise, ART related factors,
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such as culture medium composition, light, temperature and oxygen tension, also affect the epigenetic of early developing embryos. In this respect, we demonstrated that conventional IVM and IVF markedly alter the dynamics of DNA (de)methylation and the expression of genes related to DNA (de)methylation in bovine zygotes. We were not able to determine whether the differences are due primarily to IVM or also to the IVF procedure. It is generally believed that, at the zygote stage, control of embryo development depends primarily on maternal mRNA transcripts since embryonic transcription (genome activation) doesn’t begin until between the 4-8 cell stages in bovine in vivo embryos and between the 8-16 cell stages in bovine in vitro embryos. However, a minor embryonic genome activation event has also been described to take place at in vivo the 2 cell stage. We designed and successfully 3D-printed an oviduct-on-a-chip model using a stereo-lithographic technique. Bovine oviduct epithelial cells (BOECs) cultured in the device regained and maintained their ciliated and cuboidal to columnar epithelium for a period of at least 6 weeks, with a mixed population of ciliated and secretory cells comparable to that in the in vivo oviduct epithelium. In this system, the oviduct cells in culture conditioned the apical medium, which in turn supported physiological sperm-oocyte interaction and fertilization, and completely abolished polyspermic fertilization and parthenogenetic activation of oocytes. Since our first oviduct-on-a-chip prototype was found to release components (phthalates and polyethylene-glycol) toxic to developing embryos, we identified a non-toxic material (PDMS) to create an improved oviduct-on-a-chip. BOECs grown in the new oviduct-on-a-chip were responsive to steroid hormone stimulation, mimicking the luteal- and pre-ovulatory phases. Moreover, the improved oviduct-on-a-chip supported not only fertilization, but also embryo development up to the blastocyst stage. The zygotes resulting from on chip (CH) culture were more similar to their in vivo counterparts (VV) than to conventional in vitro (VT) zygotes, in terms of their global DNA methylation level and transcriptome. Interestingly, VV and CH zygotes exhibited lower global DNA methylation levels than VT zygotes, which is presumably a factor of the up-regulation of genes related to DNA demethylation in 80% of the VV and 50% of the CH zygotes (G2). This reduced level of DNA methylation seems to be essential to the minor embryonic genome activation event, since an up-regulation of genes related to transcription and translation initiation was observed in G2 zygotes. Suggesting that standard IVP conditions delay zygote minor transcriptome activation, but that the delay can be partially ameliorated using our oviduct-on-a-chip platform. Finally, our results highlight the importance of using a more in vivo-like environment to study pathways related to normal fertilization and embryo development in vitro.
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