187 9-cis RETINOIC ACID IMPROVES MATURATION RATE AND ALTERS GENE EXPRESSION OF IN VITRO-MATURED OOCYTES IN EGYPTIAN BUFFALO

Abstract

Retinoic acid, a metabolite of vitamin A, regulates oocyte maturation through multiple mechanisms, including gene expression modulation or preventing oxidative stress. Effects of retinoic acid during oocyte maturation have been reported in several species; however, there have been no studies illustrating these effects in buffalo. Therefore, the objective of this study was to investigate the influence of 9-cis retinoic acid (9-cisRA), an active metabolite of vitamin A, on maturation rate and gene expression during in vitro maturation of buffalo oocytes. Cumulus-oocyte complexes (n = 360) were aspirated from surface follicles of Buffalo ovaries collected from local abattoirs and transported to the laboratory in physiological saline (0.9% NaCl) containing antibiotics (100 µg mL⁻¹ of streptomycin sulfate and 100 IU mL⁻¹ of penicillin) and maintained at 30°C. Grade A cumulus-oocyte complexes (evenly granulated cytoplasm and surrounded by multiple layers of cumulus cells) were randomly divided into 4 groups (90 oocytes/group) and allocated in TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 50 μg mL⁻¹ of gentamycin, and 10 μg mL⁻¹ of FSH and contained 0 (control), 5, 50, or 200 nM of 9-cisRA for maturation. After 24 h, maturation rate was calculated as a percentage based on polar body extrusion. In addition, gene expression patterns were analysed for antioxidant related genes (SOD1, CAT, GPX4, HOMX1, and PRDX1) and oocyte quality-related genes (GDF9 and BMP15) using quantitative real-time PCR with GAPDH as a housekeeping gene. Fold changes (FC) were calculated using ΔΔCt method (FC ≥2; P < 0.05). The results showed that maturation rate (based on the extrusion of polar body) was significantly higher in 5 nM 9-cisRA oocyte group (49.4 ± 2.1%) compared with the control group (35 ± 1.8%); in contrast, the 200 nM 9-cisRA oocyte group showed the lowest maturation rate (27.2 ± 2.7%). However, the 50 nM 9-cisRA oocyte group showed no significant differences (31.2 ± 3.8%) compared with control group .Oocytes treated with 5 and 50 nM 9-cisRA during in vitro maturation showed significant up-regulation of SOD1 (3.4 and 3.08 FC), CAT (2.7 and 1.8 FC), and HOMX1 (4.5 and 4 FC), and significant down-regulation of BMP15 (−3.7 and −3.6 FC), respectively, compared with the control group. Moreover, GPX4, PRDX1, and GDF9 genes were highly expressed in the 50 nM compared with the control group (13.2, 10.4, and 1.8 FC, respectively). In contrast, the 200 nM 9-cisRA group showed significant down-regulation of CAT (−60.3 FC), GDF9 (−2.5 FC), and BMP15 (−9.7 FC) compared with the control group. In conclusion, these results suggested that a low concentration of 9-cisRA (5 nM) in maturation media can improves maturation rate of buffalo oocytes and up-regulates the expression of oxidative stress response-related genes.