119 ENDOMETRIAL CO-CULTURE MODELS FOR THE IN VITRO INVESTIGATION OF EARLY EMBRYO–MATERNAL CROSSTALK IN CATTLE

Abstract

Cellular and molecular changes in the endometrium during the preimplantation period are orchestrated by a complex network of molecules. The endometrium consists mainly of epithelial (EC) and stromal cells (SC) which produce a variety of active factors, such as prostaglandins (PG), in response to hormones and embryonic signals. However, it is unknown whether and how EC and SC influence each other in their reaction to these endo- and paracrine stimuli. To address this question, we measured the PG secretion of separately cultured EC and SC, and of EC+SC co-cultures in an insert system (0.2 µm membrane pore size) allowing exchange of secreted factors, but no direct contact between EC and SC. Bovine endometrial cells were isolated from cow uterus by a combination of mechanical and enzymatic procedures on Day 8 of the estrous cycle (ovarian morphology). Isolated EC and SC (90% purity) were cultured in DMEM/F12 containing 10% fetal bovine serum at 37°C in an atmosphere of 5% CO₂ in air. PGE₂ and PGF_2α concentrations in media from separately cultured EC and SC or co-cultured EC+SC were measured by specific ELISAs after 24 h of stimulation with (i) 100 nm oxytocin (OXT); (ii) 100 ng mL^–1 interferon tau (IFNT); or (iii) the respective solvent as non-stimulated control. EC responded to OXT stimulation with a decreased PGE₂/PGF_2α ratio, although the difference was not significant (PGE₂/PGF_2α ratio non-stimulated: 2.4 ± 0.9; PGE₂/PGF_2α ratio stimulated: 1.5 ± 0.8; P > 0.05 v. non-stimulated control, evaluated by t-test). No differences in the ratios of PGE₂/PGF_2α release into the medium were observed when comparing OXT-treated SC with non-treated SC. Incubating co-cultures of EC and SC with OXT resulted in a statistically significant, 2.5-fold decrease in the PGE₂/PGF_2α ratio as compared to non-stimulated cells (0.8 ± 0.2 v. 2.0 ± 0.7; P < 0.05; t-test). In contrast, IFNT stimulation of EC and SC co-cultures shifted PG secretion toward the pregnancy protective PGE₂ (PGE₂/PGF_2α ratio non-stimulated: 0.99 ± 0.44; PGE₂/PGF_2α ratio stimulated: 2.36 ± 0.96; P < 0.05 v. non-stimulated control, by t-test). Conclusions: Our findings indicate that interactions between EC and SC regulate the responsiveness of the co-culture system to OXT and the pregnancy recognition molecule IFNT. We propose that co-culture of both cell types should be used for studying mechanisms of early embryo–maternal communication.