Sperm TLR2/1 activation modulates the paternal RNA cargo and enhances bovine embryonic development

In vitro fertilization (IVF) and in vitro embryo production (IVP) are crucial technologies for accelerating genetic gain in cattle. However, IVP efficiency remains suboptimal, as in vitro-derived embryos frequently exhibit reduced developmental competence compared to their in vivo counterparts. Previous studies indicate that activation of the sperm Toll-like receptor 2/1 (TLR2/1) complex via Pam3CSK4 (PAM3) enhances sperm-oocyte interactions and improves cleavage and blastocyst rates; however, the underlying molecular mechanisms remain unexplored. In this study, frozen-thawed active sperm from three high-fertility Holstein bulls were allocated to control or PAM3-treated groups before IVF. Both sperm and the resulting day 7 blastocysts were subjected to RNA sequencing (RNA-seq) to investigate whether TLR2/1 activation modulates the paternal RNA cargo and if these alterations persist in the embryonic transcriptome. PAM3 treatment significantly improved developmental outcomes, specifically cleavage and blastocyst rates, although the magnitude of the effect varied among bulls. Transcriptomic analysis revealed distinct bull-specific RNA profiles and demonstrated that PAM3 treatment induced selective remodeling of sperm transcript abundance, involving both coding and non-coding RNA fractions. Conversely, day 7 embryos derived from PAM3-treated sperm did not exhibit global transcriptomic divergence from controls. This suggests that the molecular consequences of sperm TLR2/1 activation may be restricted to earlier developmental stages or regulated through specific pathways rather than broad transcriptomic remodeling at the blastocyst stage. In conclusion, these findings suggest that TLR2/1 signaling modulates paternal RNA cargo, potentially contributing to improved fertilization and bovine IVP efficiency by facilitating sperm penetration of the oocyte.