Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.