mTOR is an essential gate in adapting the functional response of ovine trophoblast cells under stress-inducing environments.

During the early stage of pregnancy in sheep (Day 16-23 of pregnancy) normal embryo development depends on placenta functionality. As immature placentation, vascularization is still insufficient to provide adequate nourishment [1]. 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, such as nutrient and oxygen deprivation. Autophagy is mainly regulated by the mechanistic target of rapamycin (mTOR), which is also known as a placental nutrient sensor [2]. Here, we tested the hypothesis that trophoblast cells may adapt to adverse conditions through an mTOR-dependent regulation of cell survival. Therefore, the main aim was to shed light on how mTOR drives placenta adaptive response to low-nutrient environments. A previously characterized in vitro model of 21-day-old primary sheep trophoblast cells (oTCs) was employed [3]. oTCs were cultured in a normal environment (nutrient-enriched DMEM/F12, CTR), then subjected to 24-h mTOR inhibitor (100nM rapamycin, RAPA) and low-nutrient conditions (MEM, STARV). Gene and protein profiles were explored to assess autophagy modulation from the beginning to the end of the process, including mTOR activation, autophagic markers mRNA expression (ATGs), and autophagosome detection (LC3BI-II). Moreover, cell motility (wound healing assay) and the expression of solute carriersʼ genes (SLCs) on treated-oTCs were studied to evaluate whether mTOR activation/suppression affects trophoblast functionality. Autophagy activation was confirmed both in rapamycin-treated and low-nutrient conditions by LC3BII higher expression compared to the normal environment. However, mTOR activation seems to be severely modified only following rapamycin treatment whereas prolonged starvation allowed mTOR reactivation. Nutrient deprivation promoted trophoblast bi-dimensional movement, on the contrary, mTOR-inhibition reversed cellular functionality. Furthermore, mRNA expression of amino acids transporters remains largely undisturbed except for SLC43A2 and SLC38A4 which are downregulated in starved and rapamycin-treated cells, respectively. Present findings suggest that mTOR inhibition affects placenta adaptation to suboptimal environments. Despite this, sheep trophoblast cells display uncommon responses to pro-autophagic stimuli. A low-nutrient availability stimulates placenta functionality, instead of preventing it. Indeed, we observed that prolonged starvation induces mTOR reactivation in an autophagy-dependent manner. In conclusion, sheep trophoblast cells may adapt to adverse conditions in the early stage of placentation by balancing, in an mTOR-dependent manner, nutrient recycling and transport with relevant effects for in vitro functional properties, which could potentially impact conceptus development and survival. [1] Grazul-Bilska et al. Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in the maternal placenta, Reproduction, 140:165-174, 2010. [2] Wang et al. Chapter 2-mTOR: The Master Regulator of Conceptus Development in Response to Uterine Histotroph During Pregnancy in Ungulates. Molecules to Medicine with mTOR, Academic Press, 2016. [3] Viola et al. Modulatory role of mTOR in trophoblast adaptive response in the early stage of placentation in sheep, Reproduction, 165: 313-324, 2023.