305 ROYAL JELLY TREATMENT DURING OOCYTE MATURATION IMPROVES IN VITRO MEIOTIC COMPETENCE OF GOAT OOCYTES BY INFLUENCING INTRACELLULAR GLUTATHIONE SYNTHESIS AND APOPTOSIS GENE EXPRESSION

Abstract

Royal jelly (RJ) is a secretion product from the cephalic glands of nurse bees that has extraordinary properties and remarkable health effects. Over the years, antioxidative and antiapoptotic properties of RJ have been experimentally investigated. Here we hypothesised that supplementary RJ in in vitro maturation (IVM) medium would (i) improve cumulus expansion (ii) oocyte nuclear maturation, (iii) glutathione (GSH) content, and (iv) mitochondrial activity, and (v) also affect the mRNA abundance of the (Bax, Bcl-2, and p53) transcripts involved in oocyte apoptosis. To test these hypotheses, goat ovaries were collected from a local abattoir and transported to the laboratory. Cumulus-oocyte complexes (COC) with multilayered compact cumulus investment and evenly granulated cytoplasm were selected and randomly allocated to the experiments. To evaluate the effects of RJ on meiotic competence after maturation in vitro, IVM medium was supplemented with concentration of 0.0 (RJ-0), 2.5 (RJ-2.5), 5.0 (RJ-5), and 10.0 (RJ-10) mg mL^–1 of RJ. After IVM, oocytes of each group were evaluated for cumulus expansion (visual assessment), stage of nuclear maturation (Hoechst staining), intracellular level of GSH (Cell Tracker blue staining), mitochondrial activity (MitoTracker Deep Red staining), and relative expression of Bax, Bcl-2, and p53 genes (qRT-PCR assay). Differences were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P < 0.05 to be significant. Supplementation of the maturation media with RJ did not appear to affect cumulus expansion (P > 0.05). Our results revealed that maturation rate was higher (88.0%) in the RJ-10 group when compared with the RJ-2.5 (71.5%) and control (RJ-0) groups (60.0%; P < 0.05), but similar with the RJ-5 group (81%; P > 0.05). A higher (P < 0.05) GSH content was detected when comparisons were made between each concentration of RJ-treated (i.e. RJ-2.5, RJ-5, and RJ-10) oocytes and the control (RJ-0) oocytes; however the differences were not significant when RJ groups were compared. No difference (P > 0.05) was observed among RJ-treated and untreated oocytes regarding their mitochondrial activity after IVM. Based on these results, the concentration of 10 mg mL^–1 (RJ-10) was selected for evaluation of Bax, Bcl-2, and p53 transcripts abundance. Our results revealed that the expression of Bax mRNA was decreased (P < 0.05) in RJ-10 group when compared with control (RJ-0) group. Furthermore, there was an increased (P < 0.05) expression of Bcl-2 transcripts in RJ-10 group when compared to the control (RJ-0) group. The p53 transcript also tended to be higher in RJ-10 group than in the control (RJ-0) group, although this difference was not statistically significant (P > 0.05). In conclusion, results of this study showed that adding RJ to maturation medium at optimum concentration increased the nuclear maturation and GSH synthesis, but not activity of the mitochondria; this improvement was associated with expression of apoptosis-related genes in goat oocytes.