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Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17β-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control −ve). The addition of 5.0-mM L-arg increased (P 0.05) at 22 hours. All concentrations of L-arg tested increased (P 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control −ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control −ve and treatment with 5-mM L-arg progressively decreased (P

It has been shown that phosphatidylinositol 3 kinase (PI3K) participates in oocyte maturation by regulating the activity of important reactions related to the resumption of meiosis and energy metabolism. Changes in the enzyme activity caused by in vitro conditions may impair important events of oocyte maturation and consequently the production of blastocysts. The study aimed to verify the effect of the addition of wortmannin (a specific inhibitor of PI3K) to the in vitro maturation medium on nuclear and cytoplasmic maturation of bovine oocyte, as well as on the production of blastocysts. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with FCS and 0 (control) or 20 nM of wortmannin and then fertilized and cultured in vitro in the absence of inhibitor. Twenty-two hours after in vitro maturation the determination of PI3K activity of oocytes by Western blot was performed using anti-PI3K subunit P85 antibody/peroxidase. The activity quantification was performed by densitometry of the bands using the Gel Perfect software (Bozzo and Retamal 1991 Arch. Biol. Med. Exp. 63, 510). To check the effect of treatment on energy metabolism, glucose and glycogen concentration of oocytes was quantified by Glucox 500 test (Doles Reagentes e Equipamentos Para Laboratórios Ltd., Goiânia, Brazil). The oocyte viability determination was performed by double labelling with propidium iodide and calcein AM. To determine the nuclear maturation, the oocytes were stained with 2% acetic orcein, being considered matured those with chromosomes in metaphase plate. To assess the meiotic spindle organisation, the oocytes were labelled with anti-α tubulin-FITC and propidium iodide. The distribution of actin filaments and mitochondria was determined by rhodamine-phalloidin labelling. The distribution of cortical granules was observed by labelling the oocytes with Lens culinaris–fluorescein isothiocyanate (FITC). The cleavage and blastocyst rates were determined at 48 and 168 h post-fertilization, respectively, both calculated on the number of oocytes placed to mature. The treatment means were compared by t-test (SAS Institute Inc., Cary, NC, USA, 2003). Wortmannin produced a reduction around 40% of PI3K activity; however, the levels of glucose and glycogen were not altered. No changes were found in the viability of in vitro matured oocytes or in nuclear maturation rate (P ≥ 0.05). Treatment did not promote any change in the organisation of meiotic spindle, distribution of actin filaments, and positioning of mitochondria. However, oocytes treated with wortmannin showed a significant increase (P ≤ 0.05) in the migration of cortical granules compared with controls (87.4% ± 11.4 and 72.8 ± 11.8%, respectively). The cleavage rate was not influenced by treatment, but the blastocyst rate was higher when oocytes were matured in presence of wortmannin (34.2 ± 6.4% v. 20.0 ± 5.0%, respectively; P ≤ 0.05). The results indicate that partial inhibition of PI3K activity in bovine oocytes treated with wortmannin during in vitro maturation did not affect nuclear maturation, but improved the migration of cortical granules, which seems to have contributed to the increased the blastocyst rate.

Nuclear maturation of the oocytes, characterised by a resumption of meiosis, occurs at the end of the growing phase in preovulatory follicles, after the peak of gonadotropins. The mechanisms and substances involved, by which gonadotropins lead to the final nuclear maturation, are still unknown. The withdrawal of immature oocytes from the follicular environment is sufficient to trigger the spontaneous resumption of meiosis, hindering the study of these events in the in vitro model. However, the co-culture of immature oocytes with hemi-sections (HS) of follicular walls holds the meiosis arrest and is therefore an alternative system for evaluating the role of certain substances in the process of oocyte maturation. Nitric oxide is an important signalling molecule. There is evidence that NO is involved in the mechanisms that hold the meiotic arrest, and also in the events that lead to the resumption of nuclear maturation. Through the action of the NO synthase enzyme, l-arginine releases NO as a by-product. Thus, l-arginine may be considered a natural NO donor. This study tested the possible involvement of NO in the oocyte maturation process in in vitro conditions mimicking the in vivo environment. Different concentrations of l-arginine (2.5, 4, 5, 6, 10, and 50 mM) were added in the culture medium (200 µL of TCM-199/albumin). Groups of 20 cumulus–oocyte complexes were matured for 22 h at 38.5°C with 8 HS from the follicular wall. In addition, two controls (with and without HS) were processed. Membrane integrity of cumulus cells (CC) was evaluated, as well as the nuclear maturation of the oocytes at 22 h of culture, by double staining with Hoechst 33342 (10 µg mL–1) and propidium iodide (10 µg mL–1) and staining with 1% orcein, respectively. The results were assessed through ANOVA, and means were compared by t-test. There was an increase in the proportion of CC with intact membranes (P