178 Cellular and extracellular expression of stress response transcription factors in male and female bovine pre-implantation embryos under oxidative stress conditions

Abstract

Pre-implantation embryo development is a critical stage, in which several development and stress-response related transcription factors (TF) are involved. Exposing embryos to environmental insults alter some of these stress response-related TF. However, their expression pattern in male and female embryos and their release via exosomes is still unclear. Here, we aimed to investigate the effect of culture-induced oxidative stress on development and expression pattern of stress-related TF in male and female embryos and in respective spent media coupled with exosomes. For this, bovine male and female zygotes were in vitro produced using sexed semen and cultured under 5% and 20% oxygen in exosome-depleted SOFaa medium (SOF with amino acids). Blastocysts were subjected to total RNA isolation followed by quantitative RT-PCR analysis of the selected TF (Nrf2, KLF4, NOTCH1, SREBF2, E2F1, CAT1, SOD1, and OCT4), as well as protein abundance analysis using immunofluorescence and related phenotypes analysis, including reactive oxygen species (ROS) level and total cell count. Furthermore, the spent embryo culture media were collected for exosomes isolation and expression analysis of candidate TF. The data were statistically analysed using one-way ANOVA followed by multiple pair-wise comparisons using the Tukey post hoc test. Results showed that the blastocyst rates of both male (29.9% v. 34.9%) and female (16.7% v. 26.5%) bovine embryos were significantly lower in 20% than in 5% oxygen level. Female blastocysts subjected to the higher oxygen level showed increased ROS level (37.66 ± 1.70 v. 45.32 ± 2.05 in male and 29.42 ± 1.44 v. 45.51 ± 2.06 in female) and significantly reduced total cell count compared with the male embryo counterpart (136.55 ± 7.8 v. 112.75 ± 2.9 in male and 138.75 ± 2.0 v. 88.25 ± 4.3 in female cultured in 5% and 20% oxygen levels, respectively). Consequently, the expression levels of Nrf2, KLF4, SREBF2, CAT1, SOD1, and OCT4 were significantly increased in male embryos exposed to oxidative stress compared with those cultured under the lower oxygen level. However, NOTCH1 and E2F1 were significantly increased in female embryos exposed to oxidative stress compared with the male counterparts. The mRNA level of SREBF2 was significantly increased in male embryos cultured under both 5% and 20% O₂ compared with female embryos. The protein expression level of Nrf2 and KLF4 was higher in embryos cultured at 20% v. 5% O₂ with greater Nrf2 abundance in male embryos. Consequently, the male embryos produced at 20% O₂ released a higher number of exosomes enriched with Nrf2, SOD1, and NOTCH1 mRNA than the other groups. Interestingly, the exosomal mRNA expression level of E2F1 tended to be higher in female embryos exposed to oxidative stress than their male counterparts. Taken together, the male embryos were more tolerant to oxidative stress than female embryos via the activation Nrf2-mediated oxidative stress response and development related TF. The release of these TF via exosomes could enhance cellular homeostasis maintenance under oxidative stress.