Carbon disulfide induces embryo loss by perturbing the expression of the mTOR signalling pathway in uterine tissue in mice

Abstract

Understanding of the mechanism of embryo loss is critical for successful pregnancy considering an increase in the incidence of infertility. In this study, we focus on the effect of alterations in the expression of the AKT/AMPK/mTOR signalling pathway in mouse uterine tissue after embryo loss induced by harmful environmental exposure to carbon disulfide (CS2). CS2 is a material used in certain production processes, and women are sometimes exposed to it in occupational settings. We created an animal model of gestating mice exposed to CS2 on gestation days 3 (GD3), 4 (GD4), 5 (GD5) and 6 (GD6) with various corresponding endpoints after the exposure. The uterine tissue was collected according to the endpoint time series to detect the expression levels of mTOR, p-mTOR, pAKT, and pAMPK using western blot, RT-PCR, immunohistochemistry staining, and ELISA. Dietary supplementation with N-carbamoyl glutamic acid (NCG) was used to verify the effect of the mTOR signalling pathway on embryo loss caused by CS2. We detected down-regulation of the levels of the mTOR and p-mTOR proteins; the levels of these two proteins were decreased by 49.35% and 51.44% at the GD5 endpoint after GD4 exposure and by 38.55% and 59.51% after GD3 exposure, respectively. The change in the expression level of mTOR mRNA was consistent with the protein expression, and the mRNA level at the GD5 endpoint was decreased by 55.0% after GD4 exposure (P < 0.05). Additionally, protein expression levels of pAKT were decreased by 49.05%, and the levels of pAMPK were increased by 25.51% at the GD5 endpoint after GD4 exposure (P < 0.05). A similar trend was observed for pAKT and pAMPK at the GD4 endpoint after GD3 exposure, at the GD6 endpoint after GD5 exposure, and at the GD7 endpoint after GD6 exposure (P < 0.05). Supplementation with NCG contributed to recovery from the effects of CS2 by increasing the protein expression levels of mTOR and pAKT by 47.54% and 63.79% (P < 0.05), respectively, while the pAMPK protein level was decreased by 37.15% (P < 0.05) at the GD5 endpoint after GD4 exposure. It should be noted that the number of implanted embryos was significantly increased after supplementation with NCG. Our results indicate that down-regulation of mTOR at the time of implantation is regulated by pAKT and pAMPK, that may be an important factor for embryo loss induced by CS2.