Caffeine and oocyte vitrification: Sheep as an animal model

Abstract

Oocyte cryopreservation is valuable way of preserving the female germ line. Vitrification of immature ovine oocytes decreased the levels of both maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in metaphase II (MII) oocytes after IVM. Our aims were 1) to evaluate the effects of vitrification of ovine GV-oocytes on spindle assembly, MPF/MAP kinases activities, and preimplantation development following IVM and IVF, 2) to elucidate the impact of caffeine supplementation during IVM on the quality and development of vitrified/warmed ovine GV-oocytes. Cumulus-oocyte complexes (COCs) from mature ewes were divided into vitrified, toxicity and control groups. Oocytes from each group were matured in vitro for 18 h in caffeine free IVM medium and denuded oocytes were incubated in maturation medium supplemented with 10 mM (+) or without (-) caffeine for another 6 h. At 24 h.p.m., oocytes were evaluated for spindle configuration, MPF/MAP kinases activities or fertilized and cultured in vitro for 7 days. Caffeine supplementation did not significantly affect the percentages of oocytes with normal spindle assembly in all the groups. Caffeine supplementation during IVM did not increase the activities of both kinases in vitrified groups. Cleavage and blastocyst development were significantly lower in vitrified groups than in control. Caffeine supplementation during the last 6 h of IVM did not significantly improve the cleavage and blastocyst rates in vitrified group. In conclusion, caffeine treatment during in vitro maturation has no positive impact on the quality and development of vitrified/warmed ovine GV-oocytes after IVM/IVF and embryo culture.

Keywords: Caffeine; GV; MPF/MAPK; Oocytes; Ovine; Vitrification.

Fig. 1

Representative images of spindle (green) morphology and chromatin (blue) alignment in ovine oocytes following immunostaining at 24 h.p.m. (A–C) show normal morphology (symmetrical barrel shape meiotic spindle with chromatin aligned regularly along the equatorial plane of spindle), while (D–F) illustrates abnormal morphology in vitrified, toxicity and control oocytes, respectively. Scale bar = 50 µm.

Fig. 2

Effects of caffeine supplementation during IVM on maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities of ovine oocytes vitrified at the germinal vesicle stage. Oocytes from control, toxicity and vitrified groups were incubated in IVM medium either supplemented with + or without − caffeine (10 mM) at 18–24 h.p.m., at 24 h.p.m., MPF and MAPK activities were detected in oocytes with an in vitro double-kinase by phosphorylation of histone H1 and myelin basic protein (MBP), respectively. A) Relative kinase activities (cpm/mm²). B) Phospho-image of PAGE, ³²P radioactivity representing kinase activities. Ten oocytes were analyzed in each group and three replicates were repeated for each experimental group. Data are presented as means ± SEM.

Fig. 3

Effects of caffeine supplementation during IVM of ovine oocytes vitrified at the germinal vesicle stage on mean cell numbers of blastocysts produced after IVF and embryo culture. Mean cell numbers per blastocyst were determined by staining of day 7 blastocysts with Hoechst 33342 (10 µg/mL). Data are presented as means ± SEM. Bars with different letters denote significant differences at (P ≤ 0.05).