132 Activation of Toll-like receptor 2 in early bovine embryos promotes their developmental competence in vitro

In vitro-produced (IVP) embryo technology holds significant potential for enhancing reproductive efficiency in cattle. However, despite advancements, the overall efficiency and viability of IVP embryos still lag behind those of in vivo embryos. Consequently, improving the developmental potential and quality of IVP embryos remains a critical objective in assisted reproductive technology. Toll-like receptor 2 (TLR2), a conserved innate immune receptor, is highly expressed in the female reproductive tract. Recently, we demonstrated that TLR2 is expressed in bovine sperm and its activation increases calcium influx and hyperactivation, which improves the fertilization capability of sperm and subsequent embryo development (Ma et al. 2022 Front. Cell Dev. Biol.10, 810961). However, the precise role of the TLR2 system in early bovine embryos remains poorly understood. In this study, we adapted an in vitro culture model to investigate the potential impact of TLR2 activation on the developmental competence of IVP bovine embryos. Initially, we examined TLR2 expression using immunofluorescence in different stages of early bovine embryos. Subsequently, embryos at Day 1.5 were exposed to a TLR2 agonist (control [C], 0 ng mL⁻¹; treatment [T], 100 ng mL⁻¹) and cultured continuously until Day 7. Following this treatment, the blastocyst formation rate was analyzed. Furthermore, embryo development speed was tracked by capturing images every 6 h using a time-lapse imaging system. Additionally, the impact of TLR2 activation on calcium influx (measured with Fluo-4 a.m.), autophagy (using the Cyto-ID kit, Enzo Life Sciences), and quality (evaluated through cathepsin B activity and apoptosis-TUNEL) in embryos were analyzed. Statistical analysis was performed as a t-test using GraphPad Prism 5, and data were presented as mean ± s.e.m.; reported differences were considered significant at P ≤ 0.05. The immunofluorescence analysis revealed robust expression of TLR2 at all stages of early embryo development. Notably, TLR2 activation led to a significant increase in the blastocyst rate on Day 7 (35.0 ± 3.4 vs. 49.8 ± 4.5%, n = 5; total embryos: C, 297; T, 294) in IVP embryos. The time-lapse analysis demonstrated that TLR2-activated embryos exhibited accelerated growth compared with control embryos. Furthermore, TLR2 activation in early embryos significantly elevated cytosolic calcium levels (1.0 ± 0.03 vs. 1.2 ± 0.04 relative fluorescence intensity [RFI], n = 5; C, 93; T, 110) and induced autophagy (1.0 ± 0.01 vs. 1.1 ± 0.01 RFI, n = 5; C, 96; T, 111) in Day 7 embryos. Additionally, TLR2 activation suppressed cathepsin B activity (1.0 ± 0.1 vs. 0.7 ± 0.1 RFI, n = 3; C, 49; T, 56) and reduced apoptotic cell index (10.1 ± 0.7% vs. 7.1 ± 0.4%, n = 5; C, 66; T, 86), suggesting an improvement in the quality of IVP embryos. Collectively, these findings suggest that the preimplantation embryo possesses a well-developed TLR2 system that regulates cytosolic calcium signaling, autophagy, and apoptosis levels, ultimately contributing to enhanced developmental competence. This promising approach holds the potential for producing high-quality embryos and improving the efficiency of in vitro production of bovine embryos.