168 Melatonin slightly alleviates the effect of heat shock on nuclear maturation of the bovine oocyte

Abstract

Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means±s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7v. 55%, respectively; chi-squared, P<0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32v. 13.7%, respectively; chi-squared, P<0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8±5.32v. 33.9±4.08 pixels intensity, respectively; P<0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0±4.78; P<0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.