320 EFFECT OF OXYGEN TENSION AND FOLLICLE CELLS DURING IN VITRO CULTURE OF PORCINE OOCYTES IN FOLLICULAR FLUID ON THEIR MATURATION AND FERTILIZATION

Abstract

If we could use porcine follicular fluid (pFF) as a solo in vitro maturation (IVM) medium, the preparation of complicated medium wouldn't be necessary. In this study, we investigated the effects on nuclear maturation and subsequent IVF of oxygen tension and follicle cells (FC) during IVM of porcine oocytes in pFF using static (S) and rotating (R) systems. In Experiment 1, all of the cumulus–oocyte complexes (COCs) in pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the pFF after filtration through 212 µm mesh, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. The COCs were transferred to 2 mL (in a 35-mm petri dish) or 3.5 mL (in a 15-mL test tube) of MpFF with or without FC (5.2 × 10⁶ cells mL⁻¹) added and were cultured for 48 h at 38.5°C in 5% CO₂ and 20% O₂ (in air) or 5% O₂ using the S or R culture systems (5O₂/FC−/S, 5O₂/FC+/S, 20O₂/FC−/S, 20O₂/FC+/S, 5O₂/FC−/R, 5O₂/FC+/R, 20O₂/FC−/R, and 20O₂/FC+/R groups). The oxygen tension had no effect on nuclear maturation except for the FC−/R groups. In the FC−/R groups, cumulus cells were detached from oocytes and their maturation rates were so low that it was difficult to discuss the effect of oxygen tension. When cultured with FC, oocytes were surrounded with expanded cumulus cells and matured to metaphase II (MII) at higher rates in 5O₂/FC+/R (67.7%) and 20O₂/FC+/R (65.9%) than those in 5O₂/FC+/S (17.5%) and 20O₂/FC+/S (35.7%) (chi-squared test; P < 0.05). In contrast, when cultured without FC, oocytes matured to MII at higher rates in 5O₂/FC−/S (78.3%) and 20O₂/FC−/S (76.5%) than those in 5O₂/FC−/R (32.7%) and 20O₂/FC−/R (11.4%) (P < 0.05). In Experiment 2, oocytes were cultured in 5O₂/FC−/S, 20O₂/FC−/S, 5O₂/FC+/R, or 20O₂/FC+/R; fertilized in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and fixed 10 h after IVF. Oocytes cultured in 20O₂/FC+/R showed a significantly higher sperm penetration rate (95.9%) than those cultured in 5O₂/FC−/S and 20O₂/FC−/S (88.0% and 88.4%, respectively), but were similar to those cultured in 5O₂/FC+/R (90.9%) (P < 0.05). Oocytes cultured in 5O₂/FC+/R formed a male pronucleus at a higher rate (77.1%) than those cultured in 5O₂/FC−/S, 20O₂/FC−/S, and 20O₂/FC+/R (52.8, 51.6, and 63.3%, respectively) (P < 0.05). These results indicate that oxygen tension had no effect on IVM and IVF, except for a higher male pronucleus formation rate in 5O₂/FC+/R than in 20O₂/FC+/R, for porcine oocytes cultured in pFF, and that the addition of FC to pFF in the rotating culture system promoted nuclear maturation and a high male pronucleus formation rate after IVF, especially under 5% O₂. In contrast, when the static culture system was used for IVM, the addition of FC was found to be detrimental to oocyte maturation in pFF.