108 EFFECT OF CAFFEINE TREATMENT ON MPF AND MAPK ACTIVITY AND PARTHENOGENETIC ACTIVATION OF VITRIFIED MII OVINE OOCYTES

Abstract

Cryopreservation of oocytes is still an open problem because of their structural sensitivity to the cooling and freezing process. Meiotic spindle disorganization and chromosomal aberrations are frequently observed, possibly due to the alteration of molecules involved in the meiotic cell cycle regulation and spindle formation such as maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). In this study we treated MII ovine oocytes with caffeine before vitrification and we evaluated the effect on (1) MPF and MAPK activity and (2) oocyte spontaneous or induced parthenogenetic activation. Oocytes from slaughterhouse sheep ovaries were in vitro-matured for 21 h in TCM-199 + 10% FCS + FSH, LH + cysteamine, and then incubated for 2 h with (+) or without (-) caffeine (20 mM; Sigma-Aldrich, Milan, Italy). Thereafter, MII oocytes were vitrified by equilibration with 10% ethylene glycol (EG) + 10% DMSO (30 s), exposure to 20% EG + 20% DMSO + 0.5 M sucrose (20 s), loading onto the Cryotop, and plunging into liquid nitrogen. After warming, groups of (+) and (-) oocytes were used for MPF and MAPK assays or (A) cultured in TCM-199 + 10% FCS for 24 h to evaluate spontaneous parthenogenetic activation, or (B) activated with ionomycin (5 �M, 5 min; Sigma) and cultured as in A. Fresh (+) and (-) oocytes were processed as above as controls. All oocytes were stained with glycerol-Hoechst 33342 to evaluate chromatin configuration. After vitrification, similar rates of spontaneous parthenogenetic activation between (+) and (-) oocytes (12/38, 31.6% vs. 16/38, 42.1%, respectively) were observed. However, the number of oocytes with a regular chromatin configuration in the MII plate was significantly higher (P < 0.01; chi-square test) in vitrified (+) compared to (-) oocytes (22/38, 57.9% vs. 4/38, 10.5%, respectively). After incubation with ionomycin, a significantly (P < 0.05) lower proportion of activated oocytes was recorded in the (+) compared to the (-) group (19/49, 38.8% vs. 32/53, 60.4%, respectively). In the control oocytes, the treatment with caffeine significantly (P < 0.01) decreased the activation rate after ionomycin stimulation compared to that of non-caffeine-treated control oocytes (6/51, 11.8% vs. 28/46, 60.9%, respectively), but no difference in spontaneous parthenogenetic activation was found between (+) and (-) groups (6/38, 15.8% vs. 6/46, 13.0%, respectively). Analysis of MPF and MAPK activities showed that caffeine treatment significantly increased (P < 0.05; ANOVA) the levels of the 2 kinases of vitrified MII oocytes, and these values are comparable to those of MII fresh oocytes. These results show that caffeine can increase MPF and MAPK activity in vitrified ovine oocytes and thus may contribute toward maintaining a regular chromatin organization in the MII plate and toward the decrease in parthenogenetic iomomycin-induced activation. Experiments are in progress to test the efficiency of caffeine treatment to improve the development competence of vitrified MII ovine oocytes.