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The role of calcium ions in fertilization of mouse and rat eggs in vitro.

In attempts to define suitable conditions for the in-vitro fertilization of mammalian eggs, it has been shown that the osmolality of the medium affects the fertilization rate of mouse and hamster eggs (Miyamoto & Chang, 1973b), and the pH values of the medium affect the fertilization rate of hamster eggs (Bavister, 1969; Miyamoto et al., 1974), mouse eggs (Iwamatsu & Chang, 1971; Miyamoto et al., 1974) and rat eggs (Miyamoto et al., 1974). Iwamatsu & Chang (1971) showed that calcium ions affect the capacitation of spermatozoa and fertilization of mouse eggs in vitro in the presence of bovine follicular fluid. It has recently been shown that guinea-pig spermatozoa fail to fertilize eggs in calcium-free media (Yanagimachi & Usui, 1974). The achievement of fertilization in vitro by epididymal spermatozoa of mice (Toyoda et al., 1971) and rats (Toyoda & Chang, 1974) has made it possible to define suitable conditions for in-vitro fertilization of these eggs in a defined physico-chemical environment. The present study was designed to determine if calcium ions have a particular role to play in the fertilization in vitro of mouse and rat eggs in a chemically defined medium. Mature female DDN mice and immature female Wistar rats were induced to superovulate and their eggs were recovered according to the procedures reported elsewhere (Miyamoto & Chang, 1973a; Toyoda & Chang, 1974). Eggs in the cumulus clot were washed three times in 1 ml of a test medium in order to remove the oviducal fluid. The medium used in the present experiment was essentially the same as that used by Toyoda & Chang (1974), except that the concentration of CaCl2 was varied. In some cases, the cumulus egg clot was placed in a watch glass containing 0-5 ml of 0-003% hyaluronidase in a test medium. After the removal of the cumulus cells, the hyaluronidase-treated eggs were washed three times. A mouse sperm suspension was prepared by mincing the cauda epididymidis of a mature male DDN mouse in a watch glass containing 2 ml test medium. A rat sperm suspension was prepared by placing spermatozoa obtained from the cauda epididymidis of a mature male Wistar rat in 0-5 ml test medium covered with paraffin oil. About 0-02 ml of fresh sperm suspension was added to 0-5 ml test medium containing eggs under paraffin oil in a plastic dish. In some cases,