211 Inhibition of protein palmitoylation during in vitro maturation accelerated oocyte meiotic maturation but decreased blastocyst rate in bovines

Protein palmitoylation (PP) is a reversible post-translational modification by linkage of palmitic acid to some proteins. PP mediates protein association to membranes and is involved in subcellular protein traffic, interactions, and molecular signalling, thus regulating protein localisation and activity. In bovine oocyte and cumulus cells (CC), expression of enzymes, regulating PP, varied during oocyte maturation. The objective was to evaluate a role of PP for oocyte maturation and competence. Bovine cumulus-oocyte complexes were matured in 500 μL drops of TCM199 containing 3 mg mL⁻¹ BSA, 0.5 mM pyruvate, and 100 ng mL⁻¹ EGF in the absence (control), or in the presence of 2-bromopalmitate (2BP), an inhibitor of PP. After 4 h and 16 h in vitro maturation (IVM), the oocytes were stripped, and meiotic stages were analysed using Lamin A/C and/or DAPI nuclear staining. CC viability was checked using Trypan blue. Control and 2BP-treated oocytes after 24 h IVM underwent IVF/in vitro culture (IVC) procedures using BO-IVF and BO-IVC mediums (IVF Bioscience). Oocyte maturation rate was determined during post-IVF stripping by counting the oocytes with polar bodies. Cleavage and blastocyst rates were checked at Days 3 and 7 post-IVF. Blastocyst cell number was determined by DAPI. Data were analysed by ANOVA following Tukey test. Viability of CC was not affected by 2BP after 16 h IVM (Table 1). However, oocyte maturation rate (% of Telophase and Metaphase-II) increased dose dependently in 2BP-treated groups compared to control (P < 0.0001). Moreover, 2BP (50 µM) led to a lower rate of immature oocytes with Lamin A/C positive germinal vesicle at 4 h IVM, compared to control: 74.4% and 95.7%, respectively (Chi2, P < 0.005). Although oocyte maturation rate was similar in all the groups (Table 1), cleavage rate was significantly lower in 50 µM 2BP group. Blastocyst rate was significantly decreased in 50 µM 2BP groups as compared to control. Blastocysts in control had more cells than in 2BP groups (P < 0.05). In conclusion, inhibition of PP during IVM significantly decreased blastocyst rate and quality. Although 2BP accelerated meiotic resumption and maturation, oocyte developmental competence was affected. PP is thus important for oocyte meiotic progression in bovine.

Table 1. Viability of cumulus cells and oocyte maturation rate after 16 h IVM and embryo development and quality

This research was supported by the Russian Science Foundation (project No 19-16-00115-Π).