163 Effect of melatonin and its receptors on bovine oocyte maturation and cumulus cell gene expression after heat shock in vitro: preliminary results

Abstract

The aim of this study was to assess the effect of melatonin (MLT) during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) under normal and heat stress conditions, on oocyte nuclear maturation and expression of genes related to antioxidant, apoptotic, and expansion activities in cumulus cells (CC), and whether effects are mediated by melatonin receptors (MT1, MT2, or both). The COC were submitted to IVM (25-30/group) in TCM-199 supplemented with 3mg mL−1 BSA, 0.5µg mL−1 FSH, 11µg mL−1 sodium pyruvate and 10µg mL−1 gentamicin. Melatonin (10−6 M) was used alone or associated with luzindole (10−6 M; LUZ) or 4-phenyl-2-propionamidotetralin (10−6 M; 4-P-PDOT). Groups were matured under normal conditions for 24h (38.5°C, control, and 38.5°C+MLT) and heat shock (cultured for 14h at 41°C, 41°C+MLT, 41°C+MLT+LUZ, and 41°C+MLT+4-P-PDOT), and then incubated for the remaining 10h at 38.5°C. The CC were collected and evaluated by real-time quantitative PCR for GPX4, SOD1, and SOD2 (antioxidant enzymes); BAX, CASP3, and P53 (apoptosis-related); and HAS1, HAS2, and PTX3 (expansion-related) transcripts. Gene expression data were normalized by the geometric mean of housekeeping genes ACTB, GAPDH, and PPIA. Relative expression levels were calculated by the comparative method of 2−ΔΔCt (Livak and Schmittgen 2001 Methods, 25, 402-408). Maturation rates (metaphase II; MII) were determined by staining oocytes with Hoechst 33342. Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on maturation rate (3 replicates/treatment). Significance level was 5% (GraphPrism software, GraphPad Inc., San Diego, CA, USA). After 24h of IVM, the MII rate decreased (P<0.05) for the 41°C group (30±14.1%, 21/70) compared with the control at 38.5°C (76.1±14.7%, 51/67; P<0.05) and addition of MLT to the 41°C group reversed the decrease (41°C+MLT, 55.3±7.1%, 26/47) and was similar to that of the control (P>0.05). When the heat stress with MLT group was associated with MT inhibitors, MII rates decreased relative to control (P<0.05) and were similar to 41°C without MLT and to each other (41°C+MLT+LUZ, 31.6±8.9%, 24/76; and 41°C+MLT+4-P-PDOT, 30±9.0%, 21/70; P>0.05). Addition of MLT did not differ from control without the hormone (38.5°C+MLT, 90.5±6.7%, 38/42; P>0.05). For gene expression in CC, only the 41°C+MLT+LUZ group increased (P<0.05) transcripts for CASP3 compared with control. There was no difference for antioxidants (GPX4, SOD1, and SOD2), expansion (HAS1, HAS2, and PTX3), or apoptotic (BAX and P53) transcripts for any of the groups (P>0.05). In conclusion, MLT reversed the negative effects on nuclear maturation caused by heat stress conditions during IVM; this effect was probably mediated by the MT2 receptor. However, MLT did not influence the antioxidant enzymes, apoptosis, or expansion-related transcripts in CC, but inhibition of MT1/MT2 receptors increased CASP3 transcripts, suggesting a possible role of the receptors on apoptosis in these cells. Further studies are necessary to improve our knowledge on the role of MLT in heat stress during IVM. The authors acknowledge FAPESP for funding (HF-2016/24884-3; CLVL-2015/20379-0).