100 Alternatives for pH control during in vitro maturation of bovine cumulus-oocyte complexes impact the expression of cell quality marker genes

The aim of the present study was to evaluate the effect of alternative methods to control the pH of the culture medium during in vitro maturation (IVM) of cumulus-oocytes complexes (COCs) on gene expression in oocytes and cumulus cells. Bovine ovaries were obtained at a local slaughterhouse and subsequently transported to the laboratory. For IVM, the selected COCs were randomly distributed in three groups and cultured in maturation medium for 24 h as follows in eight replicate experiments: (i) chemical reaction (ChR-Group) system (n = 160 COCs), based on the addition of an effervescent tablet, composed of 0.011 g of citric acid and 0.021 g of sodium bicarbonate in a tube with distilled water connected to a second tube containing the maturation medium; (ii) a culture medium based on 20 mM TCM-HEPES (n = 180 COCs), independent of a controlled atmosphere of CO₂; and (iii) as a control group CNT-Group (n = 160 COCs), a conventional incubator system was used with 7% of CO₂, 38.5°C and saturated humidity. After 24 h of IVM, a pool of 20 COCs/group were denuded by mechanically removing cumulus cells through successive pipetting in PBS solution with hyaluronidase. Total RNA from cumulus cells and oocytes was extracted with TRIzol reagent according to the manufacturer’s recommendations. Reverse transcription was performed using a commercial kit (ProtoScript^®). The real-time PCR was performed using the a Luna^® kit. The target genes evaluated in both cumulus cells and oocytes were BCL2 Associated X protein (BAX), indicating proapoptotic processes, bone morphogenetic protein 15 (BMP15), involved in the maturation and quality of oocytes and follicular development, and amphiregulin (AREG) and epiregulin (EREG), related to the expansion of cumulus cells and the resumption of nuclear maturation of oocytes. Analyses were performed in SAS version 9.4 using the MIXED procedure, a P-value lower than 0.05 was considered significant. The pH value of the oocyte maturation medium in the three culture systems fluctuated within the desired range (7.14–7.38). In cumulus cells, BAX expression was significantly higher (P < 0.05) for the TCM-HEPES group (3.30 ± 0.51) compared with the CNT-Group (1.00 ± 0.55). No differences were observed (P > 0.05) in the expression of BAX between the ChR-Group and the CNT-Group. EREG expression was lower in the CNT-Group (1.00 ± 0.40) compared with the ChR-Group (3.20 ± 0.37). For the expression of the BMP15 and AREG genes, no differences were observed between groups (P > 0.05). In oocytes, expression of BAX, AREG, and EREG genes did not show significant differences (P > 0.05) between groups. However, higher BMP15 expression (P < 0.05) was observed in the CNT-Group (1.00 ± 0.18) compared with the TCM-HEPES group (0.27 ± 0.19). The adaptation of a closed system, which uses the CO₂ derived from a chemical reaction, showed promising results, without affecting quality of the structures in culture as it maintained similar gene expression pattern to the conventional system.