Principal Investigator: Mariana Diel de Amorim

Co-PI: Bettina Wagner

DESCRIPTION (provided by applicant): 

It is estimated that about 17.3% of all pregnancies in horses are lost by day 15 post-ovulation, with some of those failures related to the mare’s ability to recognize its pregnancy. The ability to understand events around the time of maternal recognition of pregnancy (MRP) will help develop targeted new therapies that will reduce these losses. The equine embryonic factor responsible for the MRP is unknown. Recent research has demonstrated that the swine trophoblast has a duplicated interleukin 1 beta (IL1B) gene, IL1B2, that is unique to the embryonic tissue and is essential for pregnancy establishment. The investigation of this form of IL-1β has not been studied in the equine embryo. Therefore, our broad objective is to characterize the expression and protein levels of IL1B2 in the equine conceptus and investigate the function of IL1B in in vitro primary endometrial cell culture. The research will be accomplished by two steps: 1) Fertile mares (n = 10) will be bred to a single stallion, and serum, equine embryos, and endometrial biopsy samples will be collected on days (D) 11, 13 and 16 post-ovulation; 2) endometrial biopsies will be taken for primary endometrial cell cultures to be treated with: a) high and low dose of IL-1β recombinant protein with and without progesterone (P4) and/or estradiol (E2) supplementation; b) high and low dose of IL-1β2 recombinant protein with and without P4 and/or E2 supplementation; c) high and low dose of IL-1 receptor antagonist with and without P4 and/or E2 supplementation; d) oxytocin; e) DMSO as a control; and f) P4 and/or E2 as a control for the supplemented wells. Specific aims include: 1) to compare gene expression levels of IL1B, IL1B2, IL1 receptors, and prostaglandin-related genes of the equine conceptus, endometrium (D11, 13 and 16), and cell culture RNA; 2) to investigate protein levels of C-C motif chemokine ligands (CCL) CCL2, CCL3, CCL5, and CCL11, along with TNF-α, IFN-γ, IFN-α, IL-1β, IL-4, IL-10 and IL-17A in the conceptus, endometrium, and cell culture supernatant using an established and validated fluorescent-bead based multiplex assay in collaboration with the Wagner Lab; 3) to compare the levels of prostaglandin (PG) (PGE2 and PGF2α) in the cell culture supernatant. We anticipate that IL1B2 will be expressed, IL-1β2 protein levels will be high in all ages of conceptuses, and that IL-1β recombinant protein supplemented with estradiol will increase prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and prostaglandin E2, so PGE2 can provide luteal support during the critical period of MRP in the mare. This research will provide more insight into the molecular regulation of endometrial and embryonic communication and provides the basis for future therapies to help maintain corpus luteum function and reduce embryonic losses, which is desirable by the horse’s industry that seeks therapies to keep mares out of heat.