The equine embryo remains mobile and without physical attachment to the endometrium for a prolonged period in early pregnancy. Embryo-maternal signalling is critical during this time but is poorly understood, while a definitive mechanism of maternal recognition of pregnancy remains elusive. In this study we used an endometrial organoid system to examine the maternal response to embryo secretions by analysing the endometrial phosphoproteome following exposure to embryo-conditioned medium.
Mare endometrial organoids were derived from isolated endometrial glands from uteri obtained immediately post-mortem. Embryos were recovered by uterine lavage from Standardbred mares at day 8-9 post-ovulation and cultured in protein-free medium for 12 h to generate embryo-conditioned medium. Organoids were primed with estradiol and progesterone and co-incubated for 30 min with embryo-conditioned (n=9) or control (n=3) media (diluted 1:100). Organoids were processed to remove media and secretome components. Proteins were extracted from cell pellets with urea-based extraction buffer, digested with trypsin, TMT labelled and enriched for phosphopeptides. Phosphopeptides were detected via LC-MS/MS.
We detected a total of 4,218 phosphorylation sites mapping to 795 protein IDs. Abundances of 27 phosphopeptides were altered between control media-exposed and embryo-conditioned media-exposed organoids (16 increased and 11 decreased). Notable findings include altered phosphorylation of PGRMC1 (progesterone receptor membrane component 1) and MGLL (monoglyceride lipase). PGRMC1 forms part of a non-classical system of progesterone action, with changes in PGRMC1 phosphorylation suggesting an alternative route of progesterone receptor activation is involved in equine embryo-maternal signalling. MGLL is involved in prostaglandin synthesis and may be important for the inhibition of prostaglandin synthesis during maternal recognition of pregnancy.
In summary, this study describes the immediate response of mare endometrial organoids to compounds released by the embryo and identifies novel pathways likely to be implicated in signalling between the embryo and maternal system in the horse.