103 Evaluating the IFN-t content in extracellular vesicles released by bovine embryos produced in vitro in the blastocyst stage

Embryo-maternal communication is a crucial step to maintain maternal receptivity and subsequent implantation. In cattle, interferon tau (IFN-t) is the main embryonic molecule to induce the primary signal for pregnancy recognition, activating pathways that prevent the lysis of the corpus luteum and induce the remodeling of the endometrium. However, before the peak of IFN-t at Day 15 of pregnancy, the embryo is already releasing signals to prepare the maternal side for pregnancy recognition. As extracellular vesicles (EVs) released during early embryonic development, E-EVs play an important role. They are internalized by endometrial cells, modifying the expression of several classical and nonclassical IFN-t-stimulated genes. There is no evidence of the presence or the absence of IFN-t in EVs released on Days 5–9 (D5–D9) of development by embryos, in consequence it is not clear if the effect of E-EVs is due to the presence of IFN-t or other signaling molecules. In this study, we evaluated the content of protein and mRNA of IFN-t in EVs released by preimplantation embryos during D5–D9 of development. Bovine embryos were produced by IVF and cultured in groups until D5 of development (morula stage). Fifty morulae were selected and cultured individually in SOF medium until D9 (hatched stage). Culture medium from hatched good-quality blastocysts was collected for EV isolation and further procedures. Both the E-EV-enriched fraction from embryonic culture (G1) and the E-EV-depleted fraction of culture medium (G2) were used as experimental groups. Isolated E-EVs were characterized by their morphology, using transmission electron microscopy; size and concentration, using nanoparticle tracking analysis; and presence of surface markers (membrane, CD9; internal, Alix and TSG101; and one negative marker, APOA1). The presence of IFN-t ARNm in G1 and G2 was evaluated by RT-PCR. Original SOF medium (without embryos) was used as negative control, while blastocysts were used as a positive control to confirm the PCR product by the melting curve and size in a 2% agarose gel. All samples were lysed and subjected to reverse transcription. The quality of the cDNA was analyzed by the amplification of ACTB. The presence of IFN-t was evaluated in both groups by Dot Blot, using a rabbit polyclonal antibody against bovine IFNT1. Original SOF medium was used as the negative control, while SOF+IFN-t (50 pg mL⁻¹) was used as the positive control. The detection limit was assessed by serial dilutions of IFN-t, with 0.05 ng mL⁻¹ being the minimum concentration detected by this technique. No statistical analysis was performed because the expected result was presence or absence. Both ACTB and IFN-t mRNAs were detected in all blastocysts with a maximum CT of 20 and 28, respectively. Only the positive control showed a signal in the Dot Blot analysis. Under our experimental conditions, no IFN-t mRNA or protein was detected in G1, G2, or the negative control. The absence of IFN-t mRNA or protein in E-EVs and culture medium of bovine blastocysts suggests that the response of the endometrial cells to E-EVs might be mediated by other molecules independent of IFN-t. It consolidates the existence of redundant mechanisms to ensure early embryo-maternal communication.

This study was funded by the Agencia Nacional de Investigacion y Desarrollo (ANID), projects 1210334 and 21220042.