Differentially expressed miRNAs in follicular cells and cell-secreted vesicles into the bovine ovarian follicular environment

During folliculogenesis the crosstalk between oocytes and follicular cells is crucial to induce cell proliferation and differentiation, for the proper oocyte development and for the acquisition of competence. Extracellular vesicles (EVs) such as exosomes and microvesicles were identified within the follicular fluid and suggested as mediators of intercellular communication. The EVs can transfer bioactive molecules such as lipids, proteins, mRNA and miRNAs. The objective of this study was to analyze differential expression of miRNAs between granulosa cells (GCs) and cumulus-oocyte-complex (COCs) as well as between its respective cell-secreted vesicles. GCs and COCs were collected from 3-6 mm follicles and cultured with medium DMEM supplemented with bovine seric albumine, ITS liquid media supplement (100x), fungizone, peniciline, MEM non-essential amino acids solution (100x) and androstenedione. After 48h the culture media of GCs and COCs was supplemented with FSH (1ng/mL). This treatment aims to mimic the existing FSH stimulation during the phase of follicular growth. Cells and culture medium were collected 48 h after the FSH treatment and stored at -80 ̊C. The culture medium EVs were isolated by ultracentrifugation and total RNA isolation were performed using Trizol® reagent (Invitrogen). Reverse transcription was carried out using the miScript PCR System (Qiagen). Real-time PCR analysis with custom plates of 351 bovine miRNAs was performed, in triplicate, in cells (GCs and COCS) and in its respective EVs (GC-EVs and COC-EVs). Expression levels were calculated using the 2-∆Ct method and data were tested by ANOVA and compared by Tukey’s test at 5%. Bioinformatics analysis of miRNAs present in follicular cells and respective EVs isolated from culture media identified putative regulated pathways in GC and COCs and both EVs. Initially, we identified sixteen miRNAs present only in COCs and eight only in granulosa cells. GCs and COCs had in common 302 miRNAs whose ten were increased (P < 0.05) in COCs: let7a-3p, miR-128, miR-15b, miR-196b, miR-342, miR-411a, miR-497, miR-502b, miR-542-5p, miR-592; and one was increased (P < 0.05) in GCs: miR-454. Functional enrichment indicated regulation of pathways as oocyte meiosis by miRNAs increased in COCs, and signaling pathways such as mTOR, ErbB, TGF-beta by the miRNA increased in GCs. Thirteen miRNAs were identified only in GC-EVs and 30 in COC-EVs. Twenty-four miRNAs were common between GC-EVs and GCEVs, two of which (miR-127 and miR-433) were higher (P < 0.05) in GC-EV, regulating oocyte maturation mediated by progesterone, adherens junctions and cell cycle; and two (miR-631 and miR-323) were higher (P < 0.05) in COCEVs, regulating pathways such as gap junctions and ErbB signaling. The results suggest that miRNAs present in the follicular environment are part of a complex communication network and cellular interactions that regulate and modulate cellular pathways associated with oocyte and follicular development.