Pharmacological mTOR pathway activation does not enhance trophectoderm differentiation in bovine embryos.

The formation of the blastocyst is a highly orchestrated process involving different cellular and molecular aspects. In mice, the mTOR pathway influences the Hippo pathway, leading to expression of trophectoderm (TE)-related genes. However, these mechanisms are not fully characterized in bovine embryos, and biological pathways are not always conserved across species. We hypothesized that mTOR pathway positively influences the TE differentiation in bovine embryos. This study aimed to evaluate the effects of the mTOR agonist MHY1485 (MHY) on TE formation in in vitro-produced bovine embryos. Embryos were treated at 96 h post insemination (hpi) with 2 µM MHY or DMSO (vehicle) until 144 hpi or 192hpi. At 144 hpi, morulae were collected for gene expression analysis of CDX2, GATA3, OCT4, SOX2, TFAP2C and YAP1. No significant differences in mRNA expression were observed between the Control, DMSO or MHY groups. At 192 hpi, embryos were fixed for confocal microscopy to assess the TE marker GATA3 and the Hippo-related transcription factor YAP1. While total cell and inner cell mass counts remained unaffected, TE cell numbers were significantly reduced in the DMSO group compared to both the Control and MHY groups. Furthermore, nuclear YAP1 intensity was lower in the DMSO group compared to the MHY group, in which YAP1 was lower than the Control. In conclusion, pharmacological activation of the mTOR pathway did not positively influence TE differentiation in bovine embryos; however, it effectively rescued the unexpected negative effects induced by the vehicle (DMSO) on TE cell number and YAP1 signaling.