338 EFFECT OF OXYGEN CONCENTRATION ON NUCLEAR MATURATION, CUMULUS EXPANSION AND GLUTATHIONE LEVEL IN PORCINE OOCYTES DURING IN VITRO MATURATION

Abstract

Contrary to experimental animals, it is well known that pig oocytes show low developmental competence after IVM, fertilization and culture despite of the attempts to improve the IVM technology. One of the reasons causing such low developmental ability of porcine oocytes seems to be the culture condition, especially the gas phase for IVM because there is a large differences in oxygen tension between in vitro and in vivo conditions. Indeed, our preliminary study revealed that oocytes matured in vivo had larger perivitelline space than oocytes matured in vitro, which could be affected by in vitro culture condition. The present study was conducted to evaluate the effect of oxygen tensions on nuclear maturation, cumulus expansion and glutathione synthesis of porcine oocytes during IVM. COCs at the germinal vesicle stage were collected from ovaries of prepubertal gilts and cultured in modified NCSU37 either under 2, 5, or 20% O₂ for 44 h (Group 2%, Group 5%, and Group 20%, respectively). Five percent CO₂ was used for all groups. After culture, the cumulus expansion was morphologically evaluated by classification to three grades (Grade 1: excellent [the length of expanded cumulus cells was longer than the diameter of the oocyte], Grade 2: good [the length of expanded cumulus cells was less than the diameter of oocyte], Grade 3: poor [oocyte having partial or single layer of expanded cumulus cells]). All experiments in this study were replicated more than 5 times. Data were analyzed by ANOVA and then shown as mean ± SD%. The rate of Grade 1 in Group 2% (16.8 ± 8.3%, 32/189) was significantly lower than those in Group 5% (68.2 ± 11.2%, 149/228) and Group 20% (78.6 ± 6.9%, 162/201) (P < 0.05). As for rates of Grade 2 and Grade 3, there were no significant differences between the groups. After evaluation of cumulus expansion, cumulus cells were removed and oocytes were stained by aceto-orcein for evaluation of nuclear maturation. The rates of metaphase II-stage oocytes were 41.0 ± 12.4% (86/210), 47.6 ± 20.5% (119/263) and 47.7 ± 12.9% (100/199) in Group 2%, Group 5%, and Group 20%, respectively. There were no significant differences among the groups. In order to clarify the effect of oxygen concentration on cytoplasmic maturation, COCs were cultured for 44 h and glutathione level of the oocytes was measured by 5,5′-dithio-bis-2-nitro-benzonic acid-glutathione disulfide reductase recycling method. Regardless the oxygen concentration, glutathione level was increased from the start of culture (6.2 ± 3.9 pmol/oocyte). But there were no significant differences in the glutathione level among groups. These results suggest that oxygen concentration during IVM could affect cumulus expansion but not nuclear maturation and cytoplasmic glutathione level in pig oocytes.

This work was supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.