The aim of this study was to evaluate morphologic and biochemistry alterations caused by the addition of sodium nitroprusside (SNP), a NO donor, on bovine oocyte maturation in vitro. Bovine ovaries were collected at a local abattoir. COC were cultured in TCM-199 with 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, and antibiotics. Analysis of variance was conducted and the means were compared by t-test at a level of 5%. Experimental design: (1) evaluation of the oocyte plasma membrane viability and integrity using Annexin V/propidium iodide (PI) and Hoechst 33342/PI staining, respectively; (2) microtubule and microfilament organization, and migration of cortical granules by immunofluorescence; (3) oocyte glutathione content and concentration of NO3-/NO2-using the method of Griess (Ricart-Jane D et al. 2002 Nitric Oxide 6, 178-185) and (4) embryo development. In Experiment 1, the addition of 1 mM SNP caused cellular death in the majority of the oocytes [100%, AnnexinV/PI (+) and 80.7% Hoescht/IP (+)] differing from the control group and the 0.01 mM SNP (P < 0.05). In Experiment 2, the microtubule staining was observed in the cytoplasm in both control group and 0.01 mM SNP; however, the group treated with 1 mM of SNP exhibited clear defects in spindle and chromatin arrangements (P < 0.05). No alterations in microfilaments disposition was observed in the control group and 0.01 mM SNP. However, after the addition of 1 mM, the microfilaments arranged into clusters, and not below of the cortex. Oocytes treated with 1 mM SNP (68.2%) showed total cortical granule migration to the periphery of the ooplasm and were similar to the control group (72.2%) (P > 0.05). Nevertheless, in the group treated with 0.01 mM SNP the total cortical granule migration was greater (86.8%, P < 0.05). In Experiment 3, the glutathione content of oocytes cultured in the presence of 1 mM SNP was lower (4.4p mol) when compared to the control group (5.4p mol) and 0.01 mM SNP (5.5 pmol) (P > 0.05). The concentration of NO in the medium were similar to both control group (6.0 ± 3.0 μM) and treated with 0.01 mM SNP (15.8 ± 1.9 μM), however, the treatment with 1 mM SNP increased 10 times (59.9 ± 12.0 μM; P < 0.05) this concentration. In Experiment 4, cleavage rates and embryo development were similar for groups control and 0.01 mM SNP (P > 0.05). Even so, in the group treated with 0.01 mM there was a greater blastocyst cell number when compared to the control group (256.8 ± 52.5 and 196.9 ± 54.0, respectively-P < 0.05). These results indicate that: (1) the addition of 0.01 mM SNP increased the quality of the oocyte maturation, leading to a higher percentage of cortical granules migration and blastocyst cell numbers, in a different pathway from that of glutathione; (2) the addition of 1 mM of SNP caused a cytotoxic effect, leading to cellular death with loss of viability and integrity of plasma membrane, absence of nuclear maturation/organization of cytoskeleton and reduction of the glutathione content, although with no intervention in the migration of cortical granules.
Effects of inhibiting nitric oxide synthase on cumulus expansion and nuclear maturation of sheep oocytes
