Seasonal variations of extracellular vesicle-coupled microRNAs in equine follicular fluid
Ovarian follicular fluid (FF) comprises a dynamic milieu whose composition alters under seasonal variation, influencing follicle development and oocyte developmental competence. Various cell subtypes (e.g. granulosa, theca, and cumulus) produce cell-secreted nanoparticles, coined extracellular vesicles (EVs), which contain bioactive regulatory molecules such as microRNAs (miRNAs) known to mediate intrafollicular communication through various forms of signaling and information transfer. Markedly, the hallmark feature of EVs transferring cellular knowledge propagates their appealing nature as direct indicators of cellular status, albeit homeostasis, dysfunction, or disease. Photoperiod alterations influencing the regulatory content of FF-EVs as downstream modifiers remain largely unknown, particularly in seasonal breeding animals. Here, we aimed to unpack the impacts of seasonal variation on the miRNA expression profiles of FF-EVs in the mare. Equine pre-ovulatory follicles were monitored and aspirated using ultrasound-guided transvaginal techniques to obtain follicular fluids during non-breeding (spring anovulatory, SAN) and breeding seasons (spring ovulatory, SOV; summer, SUM; and fall ovulatory, FOV). 97 miRNAs were found to be differentially expressed between groups. Pairwise comparisons revealed specific clusters, including six miRNAs involved in the spring transition (miR-149-200b-206-221-328-615) and many others dysregulated during that of the summer (miR-143-192451- 302b-100, and let 7c), the peak time point for breeding. Bioinformatic analyses led to unveiling significant enrichments in various biological functions, such as transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Ultimately, the miRNA content of seasonally divergent FF-EVs poses as potentially relevant indicators of intrafollicular mechanisms directing seasonal transitions and their functional regulatory roles in the pre-ovulatory stage, governing subsequent ovulation. Overall design: Seventeen Quarter horse mares weighing 400 to 600 kg, spanning the ages 8 to 14 years old were used for in vivo collections of FF samples during the SAN, SOV, SUM, and FOV seasons. During the SAN season, ovarian follicles =6 mm in diameter were ablated under ultrasound guidance. During the SOV, SUM, and FOV seasons, dominant follicles (= 28 mm in diameter) were monitored daily via ultrasonography until ovulation, and thereafter,  follicle ablations were performed on days 10–12 post-ovulation to induce new follicular waves. Daily ultrasonographic tracking of healthy/growing follicles was performed until follicles reached the pre-ovulatory diameter size (30-35 mm). After a minimum of three consecutive days of tracking, selected pre-ovulatory follicles (n = 6-12) were assigned to experimental groups, consisting of SAN (March, the transition season, when dominant anovulatory follicles develop after the deep anestrus season, before the spring ovulatory season), SOV (April and May, the beginning of the ovulatory season, with regular cyclic activity), SUM (June to July; Ovulatory season) and FOV (September, the final period of the ovulatory season), before the mares approached the receding phase (fall anovulatory) and inactive phase (winter anestrous). Only animals showing continuous ultrasonographic follicle growth for at least three consecutive days, the presence of uterine edema (estrus-like), and the absence of a corpus luteum at the time of FF collection were used.