Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
(N/A)
Non Technical Summary
Successful maintenance of pregnancy in mammals depends on the so-called 'maternal recognition of pregnancy' (MRP). During MRP, the developing embryo sends a biochemical message to its mother to ensure that she makes the physiological changes necessary for continued support of pregnancy. The most important of these changes is extending the lifespan of the corpus luteum (CL), a temporary ovarian organ that produces progesterone, a hormone essential for pregnancy maintenance. Although known in many species, the MRP signal has not been discovered in horses. This is important because approximately 25% of early pregnancy losses in horses have been reported to involve loss of CL function. This project will investigate the hypothesis that the early horse embryo releases the hormone chorionic gonadotropin (the beta subunit: CG-beta) which, in turn, ensures CL maintenance. The studies will examine whether the early horse embryo produces CG-beta, and whether that CG-beta is able to extend CL lifespan. Specifically, the project will examine when CG-beta is first produced by the embryo, and whether it is secreted into the uterus and enters the mare's blood in appreciable amounts. Finally, we will examine whether CG-beta is able to prevent the demise of the CL at the end of a normal estrous cycle. The results of this study will help us better understand how the embryo communicates with its mother during early pregnancy, and whether CG-beta plays a critical role in pregnancy maintenance. If CG-beta transpires to be an important embryonic signal it could become a valuable basis for designing treatments to reduce the risk of early pregnancy loss, and for developing tests for (the health of) early pregnancy. Ultimately, knowing the identity of the MRP signal should help us reduce the incidence of early pregnancy loss, and thereby reduce financial losses to the horse breeding industry.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The objectives of the current proposal are to: 1) characterize the initial expression, peak abundance, and expression pattern of CGB in equine conceptus within 10-14 days after ovulation, 2) identify CGB peptide in conceptus' secretome embryo-conditioned culture media (in vitro) using deep proteome sequencing, 3) identify CGB peptide in maternal blood using deep proteome sequencing, and 4) investigate the outcomes of intravenous administration of eCG in non-pregnant mares on luteal and endometrial functions.
Project Methods
All animal and experimental protocols that will be used in the current study will follow the Institutional Animal Care and Use Committee of the University of Kentucky (protocol number 2024-4479). Mixed breed mares with varying ages (n=40) and a body condition score (BCS) between 5-6 will be selected from the UK research herd (Maine Chance Farm). For all proposed experiments, mares will be evaluated to ensure that they are clinically healthy and pass a breeding soundness exam (BSE). Mares will undergo transrectal ultrasonography every other day to determine the stage of the estrous cycle. If a CL is detected, mares will receive a luteolytic dose of PGF2α (0.01 mg/kg) to begin a new estrous cycle (return to estrus). When a pre-ovulatory follicle (≥35 mm) is detected in the absence of a CL and the presence of uterine edema, mares will receive a dose of 2500 IU of hCG intravenously for ovulation induction and the mare will be artificially inseminated with 500 million progressively motile sperm, previously collected from a confirmed fertile stallion(s). If ovulation does not occur within 48 hours after insemination, an additional artificial insemination will be performed. Mares will be subjected to transcervical uterine flushing to recover embryos on specific days after ovulation (within the MRP time window) as described below for each experiment. Briefly, the mare will be restrained in stocks and mares displaying discomfort will be lightly sedated, if necessary, with xylazine (0.2-0.5 mg/kg IV), have their tail wrapped, and their vulva and perineum cleansed. The operator (PI), wearing a sterile obstetrical sleeve will introduce a silicon Foley catheter or modified endotracheal tube (based on the gestational age of the conceptus) transcervically, and the conceptus (embryo and membranes) will be recovered by lavage using warm and sterile Dulbecco's phosphate buffered saline (DPBS). The recovered embryo will be transferred to the lab (<5 min) for specific procedures aligned with the research objectives described below. After the embryo collection, PGF2α will be administered to the mares in to begin a new estrous cycle. The previous procedures will be repeated until we achieve the required number of conceptuses/experiments as described below for each experiment.Experiment 1: CGB expression in equine conceptus during MRP time window. In this experiment, conceptuses will be recovered on days 10, 11, 12, 13, and 14 after ovulation (n=8/time point; a total of 40 embryos). The conceptuses will be examined and manipulated under a stereo microscope. The recovered conceptus will be rinsed three times with DPBS. The capsule will be ruptured/collapsed using a microblade and the blastocoel fluid will be retrieved and frozen at -80 °C until protein extraction and western blot (WB) analysis. After the blastocoel fluid is removed, five conceptuses (embryo and membrane) per time point will be transferred into separate cryovials, snap-frozen in liquid nitrogen, and stored at -80°C until protein extraction for WB analysis. The remaining three conceptuses per time point will be fixed with 4% formaldehyde for 24 hours and then embedded in paraffin for later IHC analysis.Experiment 2: Deep proteomic analysis of the equine conceptus secretome. In this experiment, conceptuses will be recovered on days 9, 10, 11, 12, and 13 after ovulation (n=5/time point; a total of 25 embryos). The conceptuses will be incubated separately for 24 hours (+1 day) in an embryo culture medium. After incubation, the conceptuses will be removed, and the conceptus-conditioned media will be utilized for proteomic analysis.Experiment 3: Deep proteomic analysis of the equine plasma from pregnant and non-pregnant mares. Blood samples will be collected from mares assigned for day 14 conceptus recovery (experiment 1). The samples will be collected daily from day 8 until day 14 post-ovulation in heparinized blood tubes. In this experiment, we are collecting samples two days before the MRP window to provide a better insight into the blood profile changes. In this experiment, pregnancy diagnosis will be done on days 12-14 after ovulation using transrectal ultrasonography and based on that mares will be classified into pregnant and non-pregnant groups (at least 8 mares per group). Plasma will be harvested and stored at -20°C until deep proteome sequencing. For experiments 2 and 3, untargeted, bottom-up, LC-MS/MS and LGC will be utilized to enhance detection of low-abundant proteins and peptides at the Mass Spectrometry and Proteomics Core Facility, UK.Experiment 4: Effect of eCG (CGB) on luteal and endometrial functions in non-pregnant mares. Non-pregnant mares will receive eCG (Folligon; Intervet, The Netherlands) via i.m. injections on day 9 (4 IU/kg), day 10 (10 IU/kg), and day 11-14 (16 IU/kg) after ovulation (eCG group; n=8) or a similar volume of saline solution (control group; n=8). The mares will be examined daily until the next ovulation is detected in both groups, this will include an assessment of CL volume and blood flow using B-Mode and Color Doppler ultrasonography, respectively. Moreover, blood samples will be collected from the jugular vein daily to analyze blood P4 and eCG and 13, 14- dihydro-15-keto PGF2α (also known as, prostaglandin F metabolite; PGFM) using commercially available kits. In addition, endometrial biopsies will be collected on day 14 after ovulation using Jackson uterine biopsy forceps. The biopsy will be removed from the instrument with a sterile needle (NIPRO Medical Corporation) and transferred into RNAlater (Thermo Fisher Scientific), kept at 4°C overnight, and then kept at −80°C until RNA isolation for RNA-seq.