260 THE EFFECTS OF L-ARGININE ON CUMULUS CELL INTEGRITY AND MEIOTIC RESUMPTION DURING IN VITRO MATURATION OF BOVINE OOCYTES IN THE PRESENCE OF OVARIAN FOLLICLE HEMI-SECTIONS

Abstract

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 < 0.05) in cumulus–oocyte complexes supplemented with at least 4 mM l-arginine [39.4 ± 9.8, 34.4 ± 18.1, 33.2 ± 0, 66.7 ± 24.1, 64.3 ± 15.9, 60.6 ± 12.8, 51.9 ± 19.3 (means ± SD) of CC with intact membrane for control with HS, without HS, and 2.5, 4, 5, 6, and 10 mM l-arginine, respectively], but a higher concentration (50 mM) of l-arginine had the opposite effect (20.3%). Similarly, concentrations of 4, 5, and 6 mM l-arginine increased the proportion of oocytes that reached intermediate stages (metaphase I, telophase I) of nuclear maturation when compared with the control with HS (P < 0.05; 72.6 ± 12.5, 74.1 ± 9.5, 67.2 ± 19.3 v. 48.0 ± 11.7) and caused a trend for an increase in the proportion of oocytes at the metaphase-II stage [3.4 ± 4, 7.7 ± 10.9, 14.1 ± 14.4, 19.4 ± 13.4, 22.1 ± 28.2, 6.9 ± 6, 9 ± 0 (means ± SD) for the control with HS and 2.5, 4, 5, 6, 10, and 50 mM l-arginine respectively]. In conclusion, l-arginine improved the integrity of CC and may play a role in the nuclear oocyte maturation process in a dose-response manner. Further investigations are necessary to clarify the role of NO in these cellular processes. Supported by FAPERJ.