When the going gets tough, the tough get going: Does melatonin drive trophoblast cells' fate in a hypoxic environment during early pregnancy in sheep?

Melatonin plays a crucial role in regulating reproductive physiology in sheep by improving embryo viability, especially under suboptimal conditions such as nutritional stress [1]. Additionally, this molecule exerts antioxidant activity in human placental insufficiency [2]. Melatonin has recently been proposed as a modulator of autophagy; however, the melatonin-mediated autophagy differentially drives cell fate in placental cells derived from physiological or compromised pregnancies [3]. Given its dual action, we hypothesize that melatonin serves as a potential partner for trophoblasts to overcome the early stage of pregnancy, which physiologically occurs under hypoxic conditions. Here, we explore the effect of melatonin on early trophoblast cell behaviour and its potential mitigating effect in CoCl₂-induced hypoxia. Previously characterized ovine primary trophoblast cells (oTCs) were 24h treated with 250 µM melatonin (MEL) with/without 200 µM CoCl₂. Then, we tested proliferation, migration, progesterone release (P4), antioxidant activity (H₂O₂ metabolism), and cell fate modulation (apoptosis, autophagy). Autophagy process was evaluated through the expression of key autophagic proteins, including the mammalian target of rapamycin (mTOR), beclin-1 (BCLN1), and the autophagosome marker (LC3B I-II). First, melatonin receptor expression was observed in both MEL and MEL-CoCl₂ cells, while GLUT1 (hypoxia marker) was increased under CoCl₂ conditions (P<0.01), confirming that oTCs responded to the treatments. Migration and P4 was slightly reduced in MEL cells (P<0.05). CoCl₂ exposure suppressed cell proliferation and migration (P<0.0001); however, MEL-CoCl₂ oTCs functionality was partially restored (P<0.05). Melatonin reduced H₂O₂ levels only in the hypoxia-induced environment (P<0.0001). mTOR phosphorylation decreased (indicated autophagy activation) in both MEL and CoCl₂ oTCs (P<0.01); however, its expression remained unaltered in MEL-CoCl₂ compared to CoCl2 cells. The increase in BCLN1 and the LC3B-II/LC3B-I ratio in CoCl₂ oTCs suggested an increase in autophagic activity (P<0.01), as evidenced by LC3B detection in immunofluorescence staining. Autophagosomes were also observed in MEL oTCs. LC3B was also detected in MEL-CoCl₂ cells, but the LC3B-II/LC3B-I ratio was decreased compared to CoCl2 oTCs. Lastly, the TUNEL assay showed an increased apoptosis in both MEL and CoCl₂ cells compared to control (P<0.001; P<0.0001); however, cell death decreased in MEL-CoCl₂ oTCs (P<0.01). As far as we know, this is the first study proposing an integrative approach to elucidate how melatonin drives cell decisions in a hypoxia-induced environment in the early sheep placenta. Melatonin differentially acts on trophoblast functionality depending on whether conditions are normal or hypoxic. Interestingly, melatonin seems to shift the balance toward autophagy rather than apoptosis, promoting cell survival and adaptation under hypoxic stress. In conclusion, we propose that the conceptus benefits from melatonin supplementation, particularly when it is forced to grow in a suboptimal environment. [1]Vázquez et al. Effect of exogenous melatonin on embryo viability and uterine environment in undernourished ewes, Anim Reprod Sci, 141: 52-61, 2013 [2]Fantasia et al. The role of melatonin in pregnancies complicated by placental insufficiency: A systematic review, Eur J Obstet Gynecol Reprod Biol, 278: 22-28, 2022 [3]Sagrillo-Fagundes et al. Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells, PloS one, 14: e0202458, 2019