Establishment of an in vitro model to study autophagy during early placenta development in sheep

During the early stage of placentation in sheep (Day 16-23 of pregnancy), normal embryo development depends on trophoblast cell functionality, whose dysregulation results in early pregnancy loss. As immature placentation and limited uterus-conceptus contact, vascularization is still insufficient to provide adequate nourishment. It means the placenta physiologically copes with a suboptimal environment; thus, trophoblast cells adopt adaptive strategies for supporting embryo growth. Autophagy is an intracellular degradation process promoting cell survival in response to stressful conditions. Its regulation passes through the mechanistic target of rapamycin (mTOR), also known as a placental nutrient sensor. So, the hypothesis is that mTOR drives trophoblast adaptive response to the adverse uterine microenvironment changes due to its balancing role between nutrient use and autophagic recycling. Therefore, the present research aims to establish an in vitro model of primary ovine trophoblast cells, and then study the effects on placental functionality under adverse conditions (fibroblast growth factor-2, FGF2 deficiency; starvation, STARV) in mTOR suppressed/activated system (with or without rapamycin supplementation). Here, a new in vitro model from 21-day-old sheep placenta (oTCs) was set up and characterized. oTCs showed typical ruminant trophoblast-cell-like features, including placental morphological properties, progesterone secretion, and trophoblast markers' expression. FGF2 activated mTOR signaling by promoting cell proliferation, migration, and motility, whereas it failed to influence invasiveness. Contrary, an mTOR-inhibited system drastically decreased all trophoblast activities, but FGF2 supplementation restored motility even when mTOR was suppressed. Interestingly, a reduction of endocrine trophoblast marker expression was observed in rapamycin treatment, especially for interferon-tau and epithelial-cadherin. Autophagy activation was confirmed both in rapamycin-treated and low-nutrient conditions, through the detection of specific autophagic markers. However, mTOR activation seems to be severely modified only following rapamycin treatment, while prolonged starvation allowed mTOR reactivation. Nutrient deprivation promoted migrative activity compared to a normal environment. Moreover, the autophagy-activated system did not affect the progesterone release. Nutrient carrier genes' expression revealed how amino acid transporters remain largely undisturbed except for SLC43A2 and SLC38A4 which are downregulated in starved and rapamycin-treated oTCs, respectively. The study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality, indicating its crucial role in placenta growth and fetal- maternal crosstalk. Additionally, the present findings suggest that the placenta adapts to adverse conditions in the early stage of placentation by balancing, in a mTOR-dependent manner, nutrient recycling and transport with relevant effects for placental functional properties, which could potentially impact conceptus development and survival.