354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION

Abstract

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10^-7, 10^-5, 10^-4, and 10^-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 µg mL^-1 FSH, 5.0 µg mL^-1 LH, 0.2 mM pyruvate, and 10 mg mL^-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 µg mL^-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 × 10⁶ sperm cells mL^-1 in TALP-IVF medium supplemented with 2 µM penicillamine, 1 µM hypotaurine, 250 µM epinephrine, and 20 µg mL^-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL^-1 gentamicin). All cultures were at 38.5°C under 5% CO₂ in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 ± 3.7%; P < 0.05), whereas the groups cultured with l-NAME had lower MII rates (78–82%; P < 0.05), irrespective of concentration (P > 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P > 0.05). Blastocyst rates (34.0 ± 7.2% to 41.5 ± 4.8%; P > 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 ± 4.7; P < 0.05) than in the treated groups (2.7 ± 4.8 to 4.4 ± 6.4; P > 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.