Date of Award:

5-2024

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Heloisa M. Rutigliano

Committee

Heloisa M. Rutigliano

Committee

S. Clay Isom

Committee

Irina Polejaeva

Abstract

During pregnancy, the maternal immune system must be altered to protect the partially non-self fetus from attack. Our previous studies show an inflammatory response in the uterus of cows carrying somatic cell nuclear transfer (SCNT), commonly known as cloned, pregnancies due to abnormalities in proteins expressed by placental trophoblast cells. Between 30 and 90 days the rate of pregnancy loss is 50-100% for SCNT compared to 2-10% for artificial insemination (AI) pregnancies. Abnormal communication between the maternal and fetal systems during placentation is a major cause of this loss. The trafficking of extracellular vesicles (EVs), membrane-bound cargo carriers, potentially represents a form of fetal-maternal crosstalk. The aims of this study were to determine the role of trophoblast-derived EVs in bovine pregnancies established by AI and SCNT. We hypothesized that maternal immune cells treated with EVs from SCNT pregnancies will stimulate a more pro-inflammatory immune response than cells treated with AI EVs.

Cattle pregnancies were established by AI or SCNT (n = 6/group) and collected at 42±3 days, a time of major embryonic loss in cattle. Placental tissue was cultured in EV-free medium for 21 days. EVs were collected from trophoblast supernatant by size exclusion chromatography, a method of separating particles by size. Peripheral blood mononuclear cells (PBMCs) were collected from day 35-70 AI pregnant cows and isolated by density gradient centrifugation. PBMC populations were sorted for CD4+ helper T cells, CD8+ cytotoxic T cells, and CD14+ macrophages/monocytes and cultured with EVs for 24 hours. Reverse transcription quantitative polymerase chain reactions using primers for pro- and anti-inflammatory genes were performed on the collected cells using the Fluidigm BioMark system to assess relative gene expression. Experimental data were analyzed as a randomized block design using SAS® University Edition Version 3.8, where block was cow and experimental unit was the cell culture well. The interaction between treatment and block was investigated.

Our data reveal changes in the relative gene expression level in maternal immune cell populations between AI EV-treated and SCNT EV-treated cells. Rather than inducing either a solely anti-inflammatory or pro-inflammatory immune response, our findings suggests that EVs from both healthy and abortion-prone pregnancies may mediate communication between the fetal and maternal systems to establish balance between the expression of anti- and pro-inflammatory genes during early gestation.

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