116 In Vitro Transcriptomic Response of Bovine Oviduct Epithelial Cells to Direct or Indirect Embryo Contact

Abstract

We observed that in vitro transcriptomic response of bovine oviduct epithelial cells (BOEC) to the early embryo could be the result of a contact-dependent signalling effect or interactions with embryo secretions. In order to determine this, BOEC were co-cultured directly with embryos or indirectly with embryo-conditioned media (CM); BOEC from the isthmus of oviducts at early luteal phase were cultured with TCM-199+10% fetal calf serum (FCS) in 4-well plates in 5% CO₂ in air at 38.5°C for 6 days until confluence. In vitro 2- and 8-cell embryos as well as their CM were produced in parallel. A day before co-culture, BOEC medium was replaced with SOF+10% FCS. Groups for 2- and 8-cell embryos were established: BOEC in direct contact with embryos; BOEC in the same well as embryos but not in indirect contact; BOEC with embryo CM; and BOEC without embryos, as a control. Polyester mesh was used to maintain embryos position on top of the cells. After 48 h of co-culture, BOEC were recovered for gene expression analysis (4 replicates). The relative abundance of candidate genes previously shown to be affected by the presence of embryo in vivo (Maillo et al. 2015 Biol Reprod. 92, 144) [SMAD6 (BMP signalling pathway); ROCK1, ROCK2 (cytokinesis); SOCS3 (inflammatory response); PRELP (extracellular matrix)] or in vitro (Schmaltz-Panneau et al. 2014 Anim. Reprod. Sci. 149, 103-106) [GPX4, NFE2L2 (oxidative stress); SCN9A (sodium ion binding); EPSTI1 (tissue remodelling); IGFBP3 (insulin-like growth factor binding); TDGF1 (BMP signalling pathway); AGR3 (regulation of ciliary beating)] was assessed by RT-qPCR. H2A.Z and ACTG1 were used as housekeeping genes. Statistical analysis was assessed by ANOVA. The BOEC responded to the presence of 2-cell embryos only when in direct contact by significantly decreasing abundance of NFE2L2. Both direct and indirect embryo contact or culture with CM significantly decreased GPX4, ROCK2, and SCN9A transcripts compared with control. The presence of 2-cell embryos irrespective of being in direct or indirect contact reduced the expression of SMAD6 compared with the control and CM groups. In the case of CM, expression of IGFBP3 was enhanced compared with the control but was similar to the presence of the 2-cell embryos. In the presence of 8-cell embryos, direct contact with BOEC significantly down-regulated the expression for GPX4 and SOCS3, whereas expression of SCN9A was up-regulated. The opposite was observed when compared with control. The presence of 8-cell embryos down-regulated the expression of SMAD6 and ROCK2 compared with the CM group, whereas direct or indirect contact with BOEC or culture with CM down-regulated the expression of PRELP compared to control. In conclusion, these results provide evidence for a differential affect on the transcriptome of BOEC in vitro depending on embryo stage. These changes may be related either with direct embryo contact or embryo secretions released into the media.

Research supported by Spanish MINECO-AGL2015-70140-R; AGL2015-66145-R; OECD-Co-operative Programme TAD/CRP JA00092482.