Penetration of human eggs by human spermatozoa in vitro.

Abstract

The timing of sperm penetration through the zona peliucida, and fertilization of human eggs had received little attention in the literature. Edwards et al. (1969) inseminated eggs matured in vitro with ejaculated spermatozoa which had been washed and found that spermatozoa did not penetrate the anna pellucida earlier than 7 h after insemination. A similar time interval between insemination and anna penetration by spermatozoa has been reported by others (Bavister et al., 1969; Overstreet and Hembree, 1976). This time interval has been considered to represent the time required for sperm capacitation, acrosome reaction and passage of the reacted spermatozoa through the zona pellucida (Austin, 1969; Austin et al., 1973). We report here that under our experimental conditions, sperm penetration into human eggs can occur much faster than previously reported. MATERIALS AND METHODS Immature oocytes were collected at laparoscopy by flushing ovarian follicles with Dulbecco’s phosphate buffered saline supplemented with 556 mM glucose and 0.1% bovine serum albumin. The recovery rate and criteria used for selection of viable oocytes were as reported by Lopata and coworkers (1974). The selected oocytes were thoroughly rinsed with the same buffer and transferred into 0.2 ml of a culture medium under liquid paraffin in a plastic petri dish. The culture medium was Ham’s F-b supplemented with 1 mM sodium pyruvate, 10 mM sodium lactate, penicillin G (100 units/mi) and 15% heat-inactivated human serum. The oocytes were incubated for 2 days at 37#{176}C under 5% CO2 in air. When examined at the end of incubation, the follicle cells that were originally surrounding each oocyte were found as a compact cell mass attached to an almost naked oocyte. The oocytes were mechanically freed from the follicle cell mass and then washed twice with BWW medium (Biggers et al., 1971), supplemented with 15% heat-inactivated human serum and placed in 0.2 ml of fresh, BWW medium (serum-supplemented) under paraffin oil in a petri dish. Freshly ejaculated spermatozoa were washed 3 times with BWW medium by centrifugation (1,000 rpm for 10 mm each) and added to the droplet containing the oocytes. The final concentration of spermatozoa in the droplet was about 5 X io spermatozoa/mI. The dish was incubated at 37#{176}C Accepted January 20, 1978. Received November 10, 1977. under 5% CO2 in air. Two oocytes, selected at random, were removed from the dish at 2, 4 or 7 h after insemination (total number of oocytes studied = 6) and were fixed in 5% glutaraldehyde, postfixed with 2% osmium tetroxide, dehydrated in graded ethanol and embedded in an epon-araldite mixture. The oocytes were studied using both light and transmission electron microscopy for signs of zona penetration and fertilization.