Early embryogenesis is highlighted by the emergence of several embryonic end extraembryonic lineages. One such lineage is the primitive endoderm, which will eventually give rise to the yolk sac. Once believed to be a vestigial structure, the yolk sac is now believed to play a more prominent role in embryogenesis as it provides nutrients to the preimplantation embryo. The endoderm may also interact with the trophectoderm lineage, as they develop in close contact within the embryo. The efficiency of developing primitive endoderm in vitro is considerably low, leading to a lapse in our understanding of its development and function in cattle and other ruminants. The goal of the first study was to establish a protocol for developing primitive endoderm cultures and characterizing these cells. Bovine embryos were produced in vitro, and primitive endoderm outgrowths were created with fibroblast growth factor 2 (FGF2) supplementation. These cells can be produced in culture with 80.3 5.6% efficiency. Furthermore, outgrowths can be maintained in culture for 6-8 weeks before reaching a quiescent state. A true bovine primitive endoderm cell line does not currently exist, however, these cells hold potential in improving the current understanding of early lineage specification in cattle.
A second set of studies was performed to examine the effects of maternal obesity on the preimplantation conceptus and endometrium. Exposure to maternal obesity in utero affects offspring development at the postnatal, adolescent, and adult stages of development; however, its impacts on early embryogenesis are not well studied. This work utilized an obese ewe model. Once the obese phenotype was established, ewes were bred. Conceptus and endometrial tissue were collected at D 14 of pregnancy, and samples were processed for RNA-sequencing analysis. There were no differences in pregnancy rate, ovulation rate, or pregnancies/ovulation between obese and lean animals. At an RPKM threshold of 0.2, fold-change 2, and FDR 0.05, 669 and 21 differentially expressed genes (DEGs) were identified between obese- and lean-derived endometrial samples and conceptus samples, respectively. Likewise, 137 DEGs were identified between male and female conceptuses. The PANTHER GO-Slim Biological Process system identified several biological processes affected by obesity in both the endometrium and conceptus tissue. GO terms do not currently exist for "placenta" and "trophoblast", so a literature search was conducted to identify DEGs involved in implantation and placentation. This revealed 125 placentation DEGs in the endometrium, and 4 DEGs in conceptuses between obese and lean groups. A follow-up study was conducted to examine the abundance of transcripts with regulatory roles in embryogenesis. Conceptuses exhibited differential expression of DNA methyltransferase 1 (DNMT1) based on obesity exposure, fibroblast growth factor receptor 2 (FGFR2) in a sex*obesity interaction, and peroxisome proliferator-activated receptor gamma (PPARG) and prostaglandin-endoperoxide synthase 2 (PTGS2) in a sex-specific manner. Collectively these results identify the preimplantation period as a susceptible time to maternal obesity in both conceptus and endometrial tissue.
Together, these studies aim to provide a better understanding of the events controlling early embryogenesis, and insight into the implication of insults during this time. These findings will prove to be beneficial in establishing the link between maternal health, endometrial function, and subsequent offspring outcomes, with the hope of promoting a more viable embryo and thus healthier offspring. / Ph. D. / Early embryogenesis in cattle is afflicted with high embryonic loss, costing the dairy and beef industries a fair portion of their profits. The mechanisms behind these losses are not well understood, however, cellular miscommunications during lineage specification are likely to blame. Of particular interest is the endoderm lineage, which gives rise to the embryonic yolk sac. Initially believed to be a transient structure, we now believe the yolk sac is indispensable in embryogenesis as it provides nutrients to the preimplantation embryo. Our current understanding of primitive endoderm and the resulting yolk sac in cattle is severely lacking because few primitive endoderm in vitro models exist. A portion of the following work is focused on developing a protocol for producing primitive endoderm cell lines in vitro. This work improved the rate of producing primitive endoderm cells in vitro and characterized those cells. These cells will be used as a tool to better understand the mechanisms involved in early embryogenesis. Furthermore, they may help identify targets for manipulating early development to lessen the high rate of embryonic loss in cattle.
The stage of early embryogenesis may also be particularly susceptible to intrauterine stressors, such as maternal obesity, because of the lineage segregation events occurring at this time. Insults to the earliest lineages can have lasting developmental effects, as these cell types will give rise to the embryo proper, yolk sac, and placenta. The effects of maternal obesity have been extensively studied in the postnatal, adolescent, and adult stages of development, however, insights into the effects on early embryogenesis are missing. The final studies of the following work are focused on the effects of maternal obesity on the preimplantation ovine conceptus and endometrium. This work utilized RNA-sequencing technology as well a qRT-PCR to identify differential gene and transcript expression in conceptus and endometrial samples collected from lean and obese ewes. Following analysis, we identified several crucial biological processes affected by maternal obesity. Of particular interest were those involved in implantation and placentation, indicating developmental programming events during early embryogenesis may be at fault for the abnormal offspring outcomes observed in previous studies. This work highlights the susceptibility of the preimplantation conceptus to maternal obesity and identifies the endometrium as a mediator between maternal nutrition and conceptus development. Additionally, this work identifies alterations in genes involved in placentation in both the conceptus and endometrium, indicating developmental programing events have occurred.
As a whole, this work developed a new tool for examining early embryogenesis and the specification events that occur during that time. It also examines the embryonic impacts of maternal obesity during that critical window of development. These findings will prove to be invaluable in factors involved in early embryo development and function in ruminant species.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/94375 |
| Date | 13 April 2018 |
| Creators | McCoski, Sarah R. |
| Contributors | Animal and Poultry Sciences, Ealy, Alan D., Lee, Kiho, Rhoads, Michelle, Eyestone, Willard H. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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