58 Effects of inhibition of endocytosis in cumulus cells viability, maturation rates, and embryo development of bovine oocytes

Endocytosis is a natural process in which cells take substances into their interior through invagination of the plasma membrane. Dynasore is a pharmacological inhibitor of dynamin-dependent protein that prevents endocytosis in several cell types. Thus, we aimed to evaluate the effect of a dynamin inhibitor in cumulus cell viability, oocyte IVM, and parthenogenetic embryo development. Three primary culture cells were establishede, and dynasore was used as the endocytosis inhibitor. The cells were cultured in a 25-cm² flask with DMEM-F12 with 10% of FCS until the second passage. At this time, the cells were trypsinized and divided into five groups with 2 × 10⁵ cells each: the control (1% of DMSO) and experimental groups with 20 μM, 40 μM, 80 μM, and 160 μM of Dynasore diluted in DMSO and then in DMEM-F12. The treatment was performed for 30 min at 38.5°C and 5% of CO₂. Cells were then centrifuged at 200g for 5 min and washed two times with DMEM-F12.The cells were stained with Hoechst 33842 and propidium iodide for 15 min at room temperature to evaluate live versus dead cell rates by flow cytometry (CytoFLEX®, Beckman Coulter Life Sciences). For evaluation of the effects of dynasore on maturation rates and embryo development, bovine ovaries were collected from slaughterhouse in three different routines. The selected oocytes were treated for 30 min before the IVM, with the same concentrations of dynasore as previously mentioned. Oocytes were washed in IVM medium (TCM-199 with 10% of FCS, 0.5 μg mL⁻¹ LH, 50 μg mL⁻¹ FSH, 0.2 mM pyruvate, and 50 μg mL⁻¹ gentamicin) and kept 19 h in IVM medium. Then, the oocytes were denuded with trypsin and the first polar body (1PB) extrusion was evaluated. At 26 h of maturation, the oocytes with 1PB were chemically activated with ionomycin (5 μM) for 5 min and with 6-DMAP (2 mM) for 3 h and were cultured in SOF for 7 days. All analyses were performed in triplicates and used the program Prisma 9.0. ANOVA was applied to evaluate cumulus cell viability and the Chi-squared test for maturation and embryo development rates. The significance level was 5% for all analyses performed. Regarding the percentage of dead cells, only the group 160 μM (42.4%) differed significantly (P < 0.05) from the control group (22.3%); the 20 μM (25.0%), 40 μM (34.8%), and 80 μM (39.3%) groups did not differ from each other (P > 0.05) or from the previous groups. Regarding maturation rate, the 160 μM group showed the highest rate (70.9%; n = 78/110) and differed significantly (P < 0.05) from the control group (56.0%; n = 61/109) and 20 μM (49.5%; n = 53/107). However, the control and 20 μM groups did not differ from each other (P > 0.05). The 40 μM group (68.0%; n = 70/103) differed significantly (P < 0.05) from the 20 μM group, while the 80 μM group (62.6%; n = 67/107) showed a trend (P = 0.0538) in relation to the 20 μM group. The other groups (40 μM, 80 μM, and 160 μM) did not differ (P > 0.05) from each other. No difference (P > 0.05) was observed between all groups in relation to cleavage and embryo development: control group (82.0; 47.5%), 20 μM (83.0; 52.8%), 40 μM (83.3; 58.3%), 80 μM (83.6; 47.8%), and 160 μM (84.6; 55.1%), respectively. In summary, the highest dose of dynasore increased the number of dead cumulus cells and increased IVM (1PB) without affecting embryonic development. That allows us to use dynasore in future experiments to study the effects of the endocytosis inhibition in cumulus cells.

Financial support was provided by FAPESP (process numbers 2021/06645-0; 2023/01511-0; 2023/09646-2).