EVs-miRNAs and polyamines regulate embryo dormancy and implantation

Abstract

Embryonic diapause is a reproductive strategy widely employed in animals to ensure the birth of offspring in a favorable environment. Understanding diapause is an important platform for studying implantation. Implantation failure is a significant cause of infertility affecting 15% couples at reproductive age. The ovarian hormones, estrogen (E2) and progesterone (P4) coordinately regulate the implantation process. MicroRNAs (miRNAs) can be packed into nanoparticles termed extracellular vesicles (EVs) and transmitted to distant recipient cells modulating their biological activities. Polyamines are small aliphatic chains with two or more primary amino acids and are ubiquitously expressed in all species. Inhibition of polyamines biosynthesis by DMFO induces embryo dormancy in vivo. In this thesis, it is hypothesized that E2 and P4 control embryo dormancy and implantation via regulating the expression of EVs-miRNAs and polyamines. The hypothesis was tested with the delayed implantation mouse model. In this model, diapause was induced by ovariectomy in morning of Day 4 of pregnancy and followed by daily P4 injection. The dormant blastocysts were reactivated by a single E2 injection. EVs isolated from uterine luminal fluid (ULF) of delayed implantation mice (Dor-EVs) induced embryonic diapause ex vivo; the treated blastocysts maintained their morphology for over 10 days in culture and produce viable pups after embryo transfer. MiRNAs sequencing identified a set of miRNAs that have a high expression in Dor-EVs. Among them, let-7 was selected for study. Let-7a, let-7g and let-7g in endometrial cells derived EVs induced embryo dormancy ex vivo. Bioinformatic analysis and experimental validation showed that let-7 directly targeted c-Myc and Rictor (a main component of mTORC2 signaling). The expression of both molecules was downregulated in dormant embryos. Inhibition of c-Myc decreased mTORC1 signaling significantly but did not influence mTORC2 signaling. Therefore, let-7 induced embryo dormancy via inhibition of c-Myc, mTORC1 and mTORC2 signaling pathways. Depletion of endogenous polyamines was necessary for embryo dormancy. DMFO treatment inhibits activity of ODC1, the rate limiting enzyme in polyamines biosynthesis. Exogenous polyamines supplementation partially reversed the stimulatory effect of Dor-EVs on diapause. The expression and biosynthesis of endometrial polyamines increased during reactivation. Exogenous polyamines activated dormant embryos via mTOR signaling pathway. E2 and P4 control embryo behaviors via regulating the status of endometrial cells both in delayed implantation mice model and ovariectomized mice. P4 upregulated the expression and secretion of let-7a, which induced embryonic diapause. While, E2 increased the concentration of polyamines, which facilitated the reactivation of dormant embryos. In conclusion, P4 and E2 control embryo implantation and diapause by regulating the expression of miRNAs and polyamines. This study provides a new insight on regulation of implantation and may help establishing potential biomarkers for successful implantation. (436 words)