mTOR signalling pathway as a key-modulator in placenta development: cell functionality and gene expression of trophoblast adaptive response during the early stage of pregnancy in sheep

During the early stage of placentation in sheep (15-23 Days), normal conceptus development is affected by trophoblast cells (oTCs) functionality whose dysregulation leads to early pregnancy loss, characterised by inadequate nourishment and oxygen supply. In this crucial developing period, oTCs metabolism is mostly supported by endometrial histotrophic factors, including FGF2, involved in cell differentiation and function through the modulation of specific cellular mechanisms. Among the others, mTOR is known as cellular “nutrient sensor”, but its downstream regulation in oTCs is still poorly understood. Therefore, our hypothesis was that oTCs adapt to overcome poor-growth factors in the uterine environment through mTOR signalling pathway modulation. The main aim was to set up an in vitro culture system from early sheep placenta in order to study trophoblast adaptive response in suboptimal environments occurring in impaired pregnancy. Firstly, primary oTCs from 21-days old sheep placenta collected at the slaughterhouse were characterised by cell morphology, immunofluorescence and PCR to detect specific trophoblast markers. Then, oTCs primary cells and oTr cell line were subjected to different treatment (50 ng/ml FGF2, 100 nM rapamycin – mTOR inhibitor – for 24h) to study their effects on cell functionality, gene and protein expression profile. oTCs showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, such as bi- and multinucleated syncytium-plaques, expressing peculiar trophoblast markers and progesterone release in culture medium. Cell functionality tests relieved similar results in both cell systems. FGF2 promoted cell proliferation and migration in normal culture conditions, whereas mTOR-inhibition induced a significant decrease. Invasion activity wasn’t influenced by FGF2 when mTOR signalling pathway was activated. On the contrary, when it was inhibited, cell invasiveness was affected by FGF2. mTOR-inhibition leaded to a significant low-cell motility both in presence and absence of FGF2, but its supplementation seemed to restored oTCs activity even when mTOR was prevented, as confirmed by mTOR phosphorylation even in presence of the inhibitor. Interestingly, mTOR-inhibition influenced endocrine trophoblast marker regulation, indeed oPL expression wasn’t affected by FGF2 supplementation as observed in the control, while IFN-τ was drastically reduced. Present findings support that FGF2 acts to modulate oTCs behaviour by regulating mTOR signalling pathway. The study provides new insight regarding how mTOR-inhibition influences trophoblast migration and invasion activity, essential for conceptus development. Moreover, mTOR involves in the expression of hormonal trophoblast markers, suggesting that it plays a crucial role in the early placenta growth and foetal-maternal crosstalk.