152 Seasonal dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid
J. Feugang A , A. Gad B , G. Ishak C D , S. Gebremedhn B E , M. Gastal C , N. Dlamini A , E. Gastal C and D. Tesfaye BA Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA
B Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, USA
C Animal Science, School of Agricultural Sciences, Southern Illinois University, Carbondale, IL, USA
D Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq
E Genus PLC, DeForest, WI, USA
Reproduction, Fertility and Development 35(2) 204-204 https://doi.org/10.1071/RDv35n2Ab152
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Ovarian follicular fluid (FF) comprises blood plasma exudates and follicle cell secretions. This biofluid is a microenvironment containing various molecules involved in complex interactions with follicular cells to govern oocyte maturation during follicle growth. The presence of extracellular vesicles (EVs) in the FF, containing various biomolecules such as microRNA (miRNA), critically influences multiple biological processes occurring within the follicle. We hypothesised that different factors affect EVs-miRNA content leading to fertility impairments. Here, we investigated the EVs-miRNA expression variations within the equine FF throughout the year. Adult mares (n = 17) were maintained in the pasture, with ad libitum access to water and trace-mineralised salt. During the anovulatory season (spring transitional anovulatory [SAN]), transvaginal ultrasound (US)-guided ablations of all ovarian follicles > 6 mm were executed to induce a new follicular wave; thereafter, daily US scanning of ovarian follicles was performed until a growing follicle reached dominance (30–35 mm), confirmed over three consecutive days of monitoring. During the ovulatory seasons (spring ovulatory [SOV], summer [SUM], and fall ovulatory [FOV]), mares were allowed to cycle normally. 12 days after ovulation, follicles were ablated, and ovaries were daily scanned until a dominant follicle reached 30–35 mm, as aforementioned. FF samples (52) were collected using transvaginal US-guided follicle aspiration. Samples were centrifuged, and supernatants were stored at −80°C until use. Frozen-thawed FF of 3–4 mares were pooled (four pools/season) and aliquots (0.5 mL/pool) were ultracentrifuged (120,000–70 min, −4°C). Pelleted EVs were characterised (Western blot, NTA, and TEM), and miRNA-enriched total RNA was isolated from EVs (Exosomal RNA Isolation kit). Small-RNA library preparation and RNAseq (NextSEqn 500; Illumina) were performed (Novogene Co. Ltd.). Analyses of detected miRNAs were conducted for differential expression (at P < 0.05), target gene prediction, and pathway enrichment. Totals of 231, 231, 238, and 249 miRNAs were detected in SAN, SOV, SUM, and FOV seasons, respectively, corresponding to 283 unique miRNAs across all groups. Numerous miRNAs were common to all groups (201) or specific to SAN (9), SOV (4), SUM (13), and FOV (13) seasons. Among the top 20 highly abundant miRNAs in each group, 29 were unique, and 16 differentially expressed miRNAs (including miR-7, miR-22, miR-192, miR-218, and miR-486-5p) revealed ∼880 specific targets across groups. Target RNA associated with the ovulatory seasons, specifically those affected within the SUM group (517), revealed significant enrichments for transferase, kinase, activator, and serine-threonine protein kinase activities. They were predominantly located within the nucleus and endosome to activate DNA-binding transcription factor activities. In conclusion, the present finding provides greater insight into the seasonal variations of FF EV-miRNA pools affecting the ovarian activity of mares as seasonal breeders.
This research was supported by USDA-ARS (#6066-31000-015-00D).
