Temporal Dynamics of Extracellular Vesicle-Associated miRNAs at the Maternal-Embryo Interface During Porcine Conceptus Elongation

Benjamin T. Vyzourek¹, Jeremy R. Miles², Alexandria P. Snider², Lea A. Rempel², Angela K. Pannier^1†

¹Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
²Livestock Bio-Systems Research Unit, U.S. Meat Animal Research Center, Clay Center, Nebraska, United States of America

^†e-mail: apannier2@unl.edu [corresponding author(s) are marked with the dagger(s)^† in the author list]

Abstract Text:

With the goal of advancing reproductive efficiency in pigs, this study investigates the role of extracellular vesicle (EV)-mediated miRNA signaling in maternal-embryo communication during porcine conceptus elongation. Benjamin T. Vyzourek and Angela K. Pannier affiliated with the Biological Systems Engineering department at the University of Nebraska-Lincoln, collaborate with Jeremy R. Miles, Alexandria P. Snider, and Lea A. Rempel from the U.S. Meat Animal Research Center to explore gestational-stage-specific EV miRNA dynamics and their implications for embryonic development and implantation success.

Prior to implantation, developing pig embryos undergo a rapid transition elongating from spherical (~1mm) to filamentous ( >100mm) morphology within a 3-day period. Conceptus elongation significantly influences reproductive efficiency of the pig, as molecular crosstalk between maternal and embryonic tissues during this critical phase directly impacts maintenance of pregnancy and embryonic survival. Extracellular vesicles (EVs) have emerged as key mediators of communication that facilitate essential signaling for successful embryogenesis. Notably, EV-mediated microRNA (miRNA) transfer could play a crucial role in post-transcriptional gene regulation within both embryonic and maternal tissues, contributing to processes including the initiation and completion of conceptus elongation. To investigate the dynamics of EV-associated miRNAs during elongation, gilts were bred and harvested at gestational days 8, 9, 10, 11, or 12. Reproductive tracts were collected, and each uterine horn was flushed with RPMI-1640 medium. EVs were isolated from the uterine flushings via ultracentrifugation, followed by nano-flow cytometry analysis with subsequent miRNA extractions and quantifications. Following incubation with a lipid membrane dye, nano-flow cytometry revealed that lipid-positive EVs accounted for an average of 91.18 ± 1.54% (day 8), 98.1 ± 0.167% (day 9), 96.6 ± 0.634% (day 10), 92.3 ± 3.05% (day 11), and 89.8 ± 2.03% (day 12) of the total particle population. Corresponding lipid-positive particle concentrations peaked on day 8 before trending downward on gestational days 9, 10, and 11, followed by a slight recovery and increase on day 12. Similarly, recovered miRNA concentrations averaged 5.83 ± 2.17, 3.18 ± 0.392, 2.64 ± 0.523, 0.973 ± 0.151, and 2.41 ± 0.763 ng/µl across gestational days 8 to 12.  In conclusion, these findings indicate a gestational-day dependent effect on EV secretion and associated miRNA cargo, with potential implications for conceptus elongation and implantation. The observed elevations in EV and miRNA levels on day 8 may support the initiation of conceptus elongation, whereas those on day 12 could contribute to uterine preparation for implantation. Subsequent RNA sequencing analyses will further elucidate these relationships. Overall, this study further supports the potential role of EV-mediated miRNA signaling in coordinating key reproductive events required for implantation success.

This research was funded through the Agriculture and Food Research Initiative (AFRI) awarded by the National Institute of Food and Agriculture (NIFA): USDA-AFRI 2021-67015-34416. USDA is an equal opportunity provider and employer.

 
Key words: Pig, Pregnancy, Uterus, Embryo, Extracellular Vesicles, miRNA.