Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology.

Abstract: The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.