Serum exosome profile as related to early pregnancy status in the mare
Date
2011
Authors
Hergenreder, Joanna R., author
Bruemmer, Jason E., advisor
Bouma, Gerrit J., committee member
Veeramachaneni, D. N. Rao, committee member
Clay, Colin McKeown, committee member
Han, Hyungchul, committee member
Journal Title
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Volume Title
Abstract
During early pregnancy in the mare the conceptus and mare must communicate in order to establish and maintain pregnancy. This coordinate communication is most pronounced between days 12 and 16 post-ovulation, at which time the conceptus is highly mobile throughout the uterus preventing endometrial prostaglandin F2α release and subsequent luteolysis. The mechanism behind successful establishment and maintenance of pregnancy in the mare is currently unknown. Recently, cell-secreted vesicles, called exosomes, were detected in high amounts in serum of pregnant women. Exosomes are 50-100 nm vesicles containing bioactive materials such as mRNA, miRNA, and protein. Exosomes can mediate immune-responses through membrane protein interaction and delivery of bioactive products into cells. Interestingly, exosomes have been described in various body fluids, including urine, breast milk, and serum. We hypothesized that exosomes are present in serum in the mare and that their relative amount differs with pregnancy status. To test this hypothesis, we determined the presence and relative amount of exosomes in serum of pregnant and non-pregnant mares. Serum samples were obtained from mares in a cross-over design, with each mare serving as both a pregnant treatment and non-mated control (n=3/day). Blood samples were obtained by jugular venipuncture on days 12, 14, 16, and 18 post-ovulation. Serum was removed, snap frozen, and stored at -80°C. Exosome isolation, for flow-cytometry and transmission electron microscopy (TEM), was performed using ExoQuick (System Biosciences, Inc.), a precipitation solution designed to isolate exosomes from fluids. After exosome isolation, samples were analyzed using flow cytometry with 100 nm sized beads as an internal control and a counting bead standard for relative amount determination. Flow cytometry analysis revealed the presence of exosomes in serum of both pregnant and non-pregnant mares in variable amounts. Furthermore, analysis revealed the presence of two distinct size populations, one of smaller exosomes (< 100 nm) previously undescribed, which were more abundant in mare serum from day 12 of pregnancy, and the second of the expected 100 nm size at each day examined. TEM analysis validated the results from the flow cytometry as each population, determined by size and granularity, was visually characterized. Along with the 100 nm and slightly smaller sized vesicles, TEM also revealed the presence of vesicles slightly larger than 100 nm, with small amounts of vesicles ∼200 nm in size, indicating the presence of exosomes as well as microvesicles. Therefore, we conclude that exosomes are present in mare serum and further characterization of such populations can provide clues about the intercellular mode of communication in early pregnancy.
Description
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Subject
microvesicles
intercellular communication
mare
early pregnancy
exosomes