Davina Bonte1, Vanessa Thys2, Petra De Sutter2, Annekatrien Boel2, Luc Leybaert3, Björn Heindryckx2. 1. Ghent-Fertility and Stem cell Team (G-FaST), Department of Human Structure and Repair, Ghent University Hospital, C. Heymanslaan 10, Ghent 9000, Belgium. Electronic address: Davina.Bonte@UGent.be. 2. Ghent-Fertility and Stem cell Team (G-FaST), Department of Human Structure and Repair, Ghent University Hospital, C. Heymanslaan 10, Ghent 9000, Belgium. 3. Physiology group, Department of Basic and Applied Medical Sciences, Ghent University, C. Heymanslaagn 10, GhentGhent 9000, Belgium.
Abstract
RESEARCH QUESTION: To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? DESIGN: The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. RESULTS: After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca2+ dynamics in response to SrCl2 or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl2 exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. CONCLUSIONS: Vitrified-warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified-warmed human oocytes is required.
RESEARCH QUESTION: To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? DESIGN: The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. RESULTS: After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca2+ dynamics in response to SrCl2 or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl2 exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. CONCLUSIONS: Vitrified-warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified-warmed human oocytes is required.