Performing Department
Animal and Avian Sciences
Non Technical Summary
One of the main reasons for economic loss in cattle is embryonic mortality. While most research has focused on understanding the mechanisms behind early embryonic mortality before day 20 of gestation, recent reports show that significant loss also occurs during early placental development after day 20 of gestation. The process of placental development is complex, and errors in placental development can lead to high levels of reproductive failure, including pregnancy loss and suboptimal fetal development. Therefore, studying the early placenta can help develop strategies to improve reproductive performance in domestic species. However, this requires a thorough understanding of the molecular, cellular, histological, and metabolic events that underlie placental development. The long-term goals of this research are to understand how the placenta develops, how the placenta interacts with the uterus, and what cells and what molecules are involved in the process of placental development. The aim of this application is to characterize placental cell types in the maternal endometrium during early stages of development in cattle, using cutting-edge techniques. This research is the next step towards achieving our long-term goal.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Embryonic mortality is one of the primary contributors to economic loss in cattle. Most efforts have been directed toward understanding mechanisms associated with early embryonic mortality prior to day 20 of gestation. However, recent reports indicate that substantial embryonic losses also occur during early placental development after conceptus elongation. The long-term goals of this research are to 1) elucidate the cellular, histological and metabolic processes taking place in the developing bovine placenta from Days 20 to 50 of gestation; and 2) use this fundamental knowledge to develop management strategies to decrease reproductive loss. Specific objectives of this proposal are to: 1) perform multi-transcriptomic analyses to characterize novel invasive trophoblast lineages in the caruncular endometrium during early placentomal development; and 2) perform confocal, Airyscan super-resolution, and multiphoton microscopic analyses to examine the microanatomy of the uterine-placental interface during the early stages of placentation. Gaining knowledge in this area will lead to a better understanding of the causes of and potential strategies to prevent embryonic loss, which is a major economic problem for the beef and dairy industries. Eventual efforts will focus on improving fertility to increase the sustainability of animal production and ensure that this aspect of American agriculture remains competitive.
Project Methods
For Object 1, we will perform multi-transcriptomic analyses to characterize novel invasive trophoblast lineages in the caruncular endometrium during early placentomal development. Estrus will be synchronized in crossbred beef (Hereford x Angus) heifers using previously described protocols and heifers bred using semen from a single sire. A sufficient number of heifers will be bred to account for 50% pregnancy rates and pregnancy confirmed with ultrasound on Day 30. Pregnant heifers (n=8/Day) will be hysterectomized on Days 20, 30, 42, and 50 of pregnancy. At hysterectomy, uteri will be trimmed free of the cervix and oviduct and opened along the mesometrial border. We will take care to remove the attached chorioallantoic tissue because we are focused on invasive trophoblasts not trophoblasts within the chorion and collect only the top portion (~3 mm thick) of the caruncular endometrium. In advance of scRNA-seq data analyses portions of the freshly collected caruncular tissues will be minced into small pieces with scissors and digested with an enzyme cocktail Accutase (ThermoFisher Scientific). Following digestion, the cells will be filtered through a 40 μm cell strainer to dissociate cell clumps into single cells, and 5% FBS will be added to stop enzymatic digestion. The cell suspension will be centrifuged at 300 g for 5 min. Cells will be washed once and resuspended in PBS/bovine serum albumin (BSA). In advance of LCM coupled with bulk RNA-seq and spatial transcriptomics analyses intact uterine-placental interface will be frozen in OCT and stored at −80°C.For Objective 2, we will perform confocal, Airyscan super-resolution, and multiphoton microscopic analyses to examine the microanatomy of the uterine-placental interface during the early stages of placentation. Some portions of intact uterine-placental interface obtained from Days 20, 30, 42, and 50 for Objective 1 will be fixed in 4% paraformaldehyde for H&E and Masson's trichrome staining and immunohistochemistry, or fixed in 2%formaldehydeand 2.5 %glutaraldehydefor electron microscopic (EM) examination. Immunofluorescence staining for proteins that are known markers for TGCs (PAGs) and invasive trophoblast cells (SHMT2), uterine LE cells (cytokeratin and E-cadherin) as well as newly identified markers for trophoblast cells identified from Objective 1 will be performed. Confocal and multiphoton microscopy images will be captured using a Zeiss 780 confocal microscope with 6 laser lines and a Coherent Ultra pulsed femtosecond laser and an Airyscan super-resolution detector.