Smeat-seq transcriptomic profiling unveils key regulatory events during Maternal-to-Zygotic transition and supports High-Efficiency culture system development in Tibetan sheep embryos.

Zygotic gene activation plays a pivotal role in early embryonic development by determining embryonic potential. Cumulus-oocyte complexes collected through the egg-cutting method underwent 24-hour in vitro culture at 38.5 °C under 5% CO₂ and saturated humidity. Following in vitro fertilization (IVF), embryos cultured in IVCs showed developmental rates of 82.33% (2-cell), 76.09% (4-cell), 63.34% (8-cell), 45.61% (morula), and 24.71% (blastocyst). Smart-seq transcriptome sequencing of maternal-zygotic gene transformation at the 2-cell, 4-cell, and 8-cell stages revealed pronounced expression differences at the 4-cell stage, with the greatest number of differentially expressed genes occurring between the 2-cell and 4-cell groups. The substantial upregulation of numerous genes suggests extensive transcriptional activation at the 4-cell stage to facilitate critical developmental processes, whereas most downregulated genes were maternally derived. Ribosome biogenesis in eukaryotes emerged as the most upregulated pathway, contrasting with downregulation of the MAPK signaling pathway. KEGG analysis demonstrated that ribosome biosynthesis factors primarily governed gene transcription, modification, and splicing, while MAPK signaling attenuation reflected reduced pathway activity. This sequencing analysis of early Tibetan sheep embryos, building upon optimized in vitro culture conditions, yields important data for improving embryo production and supporting assisted reproductive technologies in this species.