57 The effect of prematuration culture using C-type natriuretic peptide and extracellular vesicles recovered from follicles at different stages of development on oocyte chromatin compaction and embryonic development in bovine species

Extracellular vesicles (EVs) are nanoparticles secreted by cells and play an important role in communicating with neighbouring or distant cells. In the ovarian follicle, EVs mediate communication between the oocyte and follicular cells, coordinating follicular growth and the acquisition of oocyte competence and maturation. However, as the size and concentration of EVs secreted into the follicular fluid, as well as their content, change during antral follicle growth and development, it is possible that EVs retrieved from different follicle categories differentially modulate oocyte development and capacities. The aim of this study was to evaluate the acquisition of meiotic and developmental competence in bovine oocytes cultured in an in vitro prematuration system (PM) with C-type natriuretic peptide (NPPC) and EVs recovered from follicles at different stages of development. The cumulus–oocyte complexes (COCs; n = 499) were PM during 8 h in TCM-199 with 0.3% bovine serum albumin (BSA), 1 × 10⁻⁴ UI/mL r-hFSH, 100 nM NPPC and 10% vol./vol. EVs (<200 nm) according to the treatments: PreDev (EVs recovered from follicles at predeviation time); DevF1 (largest follicle at the time of deviation); PostDev (postdeviation); and PreOv (preovulatory). Oocytes from the control group were PM without EV supplementation. Meiosis progression in oocytes was evaluated (1% Hoechst 33342) after PM and immature oocytes at germinal vesicle stage (GV) were classified according to the chromatin compaction (from least (GV0) to most (GV3) compacted). Other sample of PM COCs were cultured for 22 h in IVM medium (TCM-199 with bicarbonate, 0.5 mg/mL FSH, 100 IU/mL hCG, and 10% fetal calf serum), fertilized and cultured to the blastocyst stage. All data were analysed by ANOVA followed by Tukey’s test (P < 0.05). At the end of PM, few oocytes exhibited chromatin compaction in the GV0 configuration (0% to 3.8 ± 2.0%; P > 0.05) and most were homogeneously distributed between GV1, GV2 (31.2 ± 6.4% to 42.5 ± 6.4%; P > 0.05) and GV3 (27.5 ± 7.2% to 48.1 ± 7.2%; P > 0.05). The percentage of structures in GV1 differed between the PostDev and PreOv (7.7 ± 4.1% vs 27.4 ± 4.1%; P < 0.05) groups, but both were similar (P > 0.05) to the other groups (control: 20.0 ± 4.1%; PreDev: 20.6 ± 4.1%; and DevF1: 23.7 ± 4.7%). No oocyte reached metaphase II stage after PM. There was no difference between groups regarding cleavage rates (75.0 ± 7.3% to 82.1 ± 4.8%; P > 0.05) and embryonic development to the blastocyst stage (38.3 ± 4.7% to 48.1 ± 7.9%; P > 0.05). However, the total number of blastocyst cells was higher in the PostDev group compared to control (151.0 ± 6.6 vs 116.6 ± 9.9; P < 0.05), and both were similar (P > 0.05) to the other groups (PreDev: 122.7 ± 9.8; DevF1: 126.6 ± 10.6; and PreOv: 133.2 ± 8.8). Our results indicate that the supplementation of PM culture medium with EVs retrieved from different categories of follicles differentially modulates bovine oocyte chromatin compaction and the quality of the resulting embryos, suggesting that EVs signalling during the acquisition of oocyte competences is distinct according to the stage of antral follicle development.

This research was supported by CAPES and FAPESP (#19/11174–6).