196 The association of dibutyryl cAMP and sildenafil on meiotic arrest of bovine oocytes

For in vitro embryo production, the cumulus–oocyte complex (COC) is aspirated from the ovary. Upon removal from the follicle, the oocyte resumes meiosis spontaneously, which occurs asynchronously to cytoplasmic maturation. This study aimed to evaluate different modulators of cyclic nucleotides to arrest meiosis in bovine oocytes for prematuration culture. COCs aspirated from abattoir ovaries were prematured in TCM-199 with 0.4% BSA, 0.2 mM sodium pyruvate, and 50 µg mL⁻¹ gentamicin (25 COCs/group; five replicates), supplemented with modulators dibutyryl cAMP (dbcAMP, a cAMP analog; 1 mM or 10 mM ), sildenafil (S, a cGMP degradation inhibitor; 100 µM), or both: 1 mM dbcAMP (A1), 10 mM dbcAMP (A10), sildenafil (S), A1+S (A1S), and A10+S (A10S). The control group (C) was cultured without modulators. After 9-h prematuration culture (38.5°C, 5% CO₂ in air), oocytes were denuded, fixed, stained with Hoechst 33342 (10 µg mL⁻¹, 20 min), and subjected to immunofluorescence with anti-lamin mouse monoclonal antibody (1:1000 in PBS, overnight) and goat anti-mouse Alexa Fluor 488 secondary antibody (1:1000 in PBS, 1 h; nuclear envelope staining). Images were captured using a ×63 objective (Leica DMi8 inverted microscope and Thunder Imaging System). To assess meiosis arrest, oocytes were classified based on chromatin configuration and nuclear envelope presence into germinal vesicle (GV) stages 1, 2, and 3, and GV breakdown (GVBD; meiosis resumption). GVBD and GV stages rates were analyzed by one-way ANOVA and Tukey’s test (JMP software, SAS Institute). Significant differences were considered when P < 0.05. For meiosis resumption (P = 0.0005), C and A1 showed the highest GVBD proportions and were similar to each other (51.6% and 46.2%, respectively), indicating that the lower dbcAMP concentration did not maintain immaturity. A10 and S (~30% GVBD) did not differ from C, suggesting that even a higher dbcAMP concentration or S individually was not efficient. In contrast, the combination of dbcAMP at both concentrations and S (A1S and A10S) successfully maintained meiosis arrest with the lowest GVBD rates (11.1% and 17.7%, respectively). Considering GV stage distribution, C had the most oocytes resuming meiosis (51.6% GVBD), with no differences in the distribution among GV1, GV2, and GV3 (4.9%, 13.0%, and 24.5%, respectively; P = 0.0001). A1 also showed higher GVBD rates (46.2%) and a similar distribution among GV1, GV2, and GV3 stages (9.5%, 9.4%, and 21.9%, respectively; P = 0.0001). Although A10 showed the majority of oocytes in GV3 (48.3%), it exhibited similarly high GVBD (30.3%; P = 0.0001), but lower proportions of GV2 (6.3%) and GV1 (6.9%). S had similar GVBD (30.7%), GV2 (20.4%), and GV3 rates (32.4%), and a lower rate of oocytes in GV1 (6.8%; P = 0.0484). A1S and A10S showed a higher proportion of oocytes in GV3 (P = 0.0001 for both; 39.3% and 43.2%, respectively), and increased earlier GV stages (GV1, 14.3% and 17.8%; GV2, 22.2 and 10.3%, respectively). Results indicate a synergistic effect of modulating cGMP (sildenafil) and cAMP (dbcAMP) in effectively sustaining meiosis arrest and increasing earlier GV stages, possibly reducing spontaneous maturation during IVM. Further studies will assess complete maturation over 24 h with modulators.

Financial support was provided by FAPESP; PHS 2023/16602-1; grant no. 2021/09886-8.