Sperm penetration through zona pellucida and perivitelline space in the hamster

Sperm penetration through zona pellucida was observed in tubal fluid containing eggs and spermatozoa, which had been recovered under a layer of paraffin oil previously insufflated with a mixture of 5% CO2 and 95% air and warmed to 38°C before each experiment. A Nikon F 35 mm camera and a Bolex Rex‐5 16 mm movie camera, synchronized with Xenon‐gas flash epuipment, were used with a microscope for recording this fast‐occurring phenomenon. In golden hamsters, 284, 306, 312 and 293 tubal eggs were recovered in paraffin oil after 4, 4.5, 5 and 5.5 hours, respectively, following delayed mating; 1, 11, 13 and 8 eggs were observed to be either attached or approached by spermatozoa, of which 1, 9, 10 and 1 eggs respectively were subsequently penetrated by the spermatozoa. The change in the CO2 tension of the paraffin oil had no apparent effect on the incidence of in vitro fertilization. Nevertheless, either the containment of eggs and spermatozoa in the small space between a cover slip and glass slide, or the dilution of tubal fluid with the liquid medium of 0.1 ml hamster serum and 0.4 ml CDM 199 was detrimental to the in vitro environment of fertilization. As in a previous investigation, most spermatozoa penetrated obliquely through the zona pellucida, but very few spermatozoa penetrated directly through it. The duration of time for spermatozoa to traverse the zona pellucida varied from about 4 to 22 minutes. Although almost all tubal eggs were completely penetrated as a result of fertilization in the oviduct of golden hamsters, yet a rather high percentage, 36%, of spermatozoa penetrated incompletely through the zona pellucida in vitro. Following sperm penetration through the zona pellucida, the vibration of ooplasm was initially induced by the impact of the two gametes, and then the rotation of ooplasm was subsequently caused by the migration of spermatozoa in the perivitelline space. Nearly 40 hamster eggs were observed and photographed in vitro during the process of fertilization, the largest number of mammalian fertilizations yet reported.