276 CORTICAL GRANULE MIGRATION AND PRONUCLEAR FORMATION IN BOVINE OOCYTES TREATED WITH BUTYROLACTONE I DURING TRANSPORT

Abstract

Butyrolactone I (BLI) can be used to maintain meiosis blocked during transport of oocytes to the laboratory, aiming to maintain their quality and competence. This work evaluated the effect of BLI during transport of bovine oocytes on the cortical granules (CG) distribution, and fertilization. Meiotic block culture was in basic medium (TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, and 75 mg mL^-1 gentamicin) with 10 μLM BLI (B10) or 100 (AM BLI + 0.3% BSA (B100). During meiotic block, oocytes were cultured in cryogenic vials (15/vial) containing 60 μLL of medium recovered with mineral oil, packed in a portable incubator (Thawing Unit MT 35/42, Minitub) at 38.5°C during the first 5 h, and then transferred to a incubator at 38.5°C and 5% CO₂ in air for the remaining 19 h. Then, they were matured at 38.5°C and 5% CO₂ in basic medium supplemented with 0.6% BSA and hormones (maturation medium, MM) for 20 h. As controls, a group of oocytes was matured in MM during 24 h, without prior meiotic block. Control oocytes were cultured at 38.5°C and 5% CO₂ (control 1, C1) or in the same portable incubator conditions in which the blocked oocytes were cultured (C2). A group of oocytes was fixed immediately after follicular aspiration (C0). At the end of cultures, a group of oocytes (n = 853) were stained with 1 μg mL^-1 Lens culinaris (FITC-LCA) and assessed for the distribution of CG (arrangement in clusters = immature; peripheral = mature). The remaining oocytes (n = 563) were fertilized for 18 h, and zygotes were stained with 10 μg mL^-1 Hoechst 33342 and assessed for sperm penetration and pronuclear formation. Oocytes with 2 pronuclei (2PN) were considered to present normal fertilization, and oocytes with >2PN were considered to be polyspermic. The data were analyzed by ANOVA and Tukey’s test, and correlation between CG positioning and pronuclear formation was analyzed with the Pearson correlation test. After meiotic block, the proportion of immature CG were higher (P < 0.05) in B100 (90.7%) and C0 (88.2%) than in B10 (69.0%). After maturation, mature CG rates in C2 (61.7%) were similar to B10 (52.3%) and B100 (72.9%), but only B100 was similar to C1 (83.6%). Irrespective of the presence or absence of BLI, culture of oocytes in the portable incubator adversely affected the normal pronuclear formation rates, which were higher (P < 0.05) in C1 (78.9%) than in C2 (56.8%), B10 (56.0%), and B100 (51.8%). The incidence of >2PN was higher (P < 0.05) in B10 (5.9%) than in C1, C2, and B100 (0.0, 1.4, and 0.0%, respectively). There was a fine correlation between mature CG and normal pronuclear formation (R = 0.5; P < 0.05). We conclude that (1) CG migration is reversibly blocked by BLI at a concentration of 100 μLM during transport; (2) oocyte transport in the portable incubator, but not BLI itself, adversely affects the normal pronuclear formation; and (3) CG maturation is essential for normal pronuclear formation.

Financial support: FAPESP.