Zinc supplementation influences 8-cell stage global transcriptional activity and improves preimplantation embryo development in cattle

One of the unsolved ques&ons in mammalian early embryogenesis relates to the morphofunc&onal and molecular processes that drive transcrip&onal reac&va&on of the embryonic genome aKer fer&liza&on. This gap of knowledge limits the ability to recapitulate this transi&on in vitro and further improve the efficiency of in vitro embryo produc&on. In this study, we tested the hypothesis that zinc, emerging as a key component in several conserved processes regula&ng female germ cell growth, fer&lity, and pregnancy, is a cri&cal determinant of transcrip&onal regula&on in bovine early embryogenesis. To this end, immature oocytes collected from abaQoir-derived ovaries were subjected to standard in vitro matura&on and fer&liza&on, and then cul&vated in control condi&ons, which do not include the supplementa&on of trace elements (control) or in the presence of two concentra&ons of zinc (0.15 or 1.5 µg/ml) in the form of zinc sulfate. Zinc concentra&ons were chosen based on the content in the follicular fluid and the female genital tract. In the first set of experiments, the global transcrip&onal ac&vity was directly assessed by uridine incorpora&on with a commercial Imaging Kit in embryos cultured in the absence (control) or in the presence zinc aKer 64 hours of culture when bovine embryos typically reach the 8-cell stage. Whole mount samples were imaged using an EclipseE600 fluorescence microscope equipped with a Sight DS-U3 camera (Nikon) at 40X magnifica&on. For each embryo, mul&ple images were acquired at different focal planes, using the nuclear staining with DAPI as a reference. Only embryos whose nuclei corresponded to the number of blastomeres observed under bright field condi&ons were considered, while embryos showing mul&nucleated or anucleated blastomeres were discarded. A total of 478 blastomeres (of 66 embryos at the 5-8 cell stage) and 312 blastomeres (of 28 embryos at the > 8 cell stage) were included in the analysis. The blastomeres’ fluorescence intensity was calculated with ImageJ aKer background subtrac&on. Data analysis showed that transcrip&onal ac&vity levels of the blastomeres did not significantly differ between the control and the treatment with a higher zinc concentra&on. On the contrary, there was a significant increase in levels of transcrip&onal ac&vity when low concentra&ons of zinc were used (Two-way ANOVA followed by Tukey’s mul&ple comparison test, p<0.05). Subsequently, we tested the effect of supplementa&on of the two doses of zinc on the ability to reach the blastocyst stage of development aKer 8 days of culture. A total of 584 oocytes in 5 biological replicates were included in the study. Data analysis showed that the low concentra&on promotes a higher blastocyst rate when compared to the higher one (Kruskal-Wallis followed by Dunn’s mul&ple comparison test, p<0.05), while the control group showed intermediate blastocyst rates. In conclusion, our studies indicate that zinc supplementa&on has a dose-dependent effect on the ability of the embryo to develop in vitro. The increase in developmental competence obtained in the presence of low concentra&ons of zinc appears to be correlated to a s&mula&on of the global transcrip&on process of the embryo. Further studies are necessary to iden&fy which molecular mechanisms govern this process. Funded by RL PSR2014-2020 No.202102146691 (R-INNOVA) and PSR2022 Piano di Sostegno alla Ricerca: Linea 2 – Azione A - Molecular and structural responses to stressors in different cells and &ssue models.