The zona-free hamster oocyte penetration test: a simple procedure using a DNA fluorescent stain to detect the male pronucleus formation.

In order to increase the value of the zona-free hamster oocyte penetration test, a comparatively simple and fast method using the fluorochrome Hoechst 33342 was developed. Human spermatozoa were washed and incubated 1 hr medium BWW for capacitation. Hamster oocytes were stripped of cumulus oophorus and zona pellucida with hyaluronidase and trypsin, washed and used immediately. Thirty oocytes were placed in a drop of BWW containing 3,5.10(6)/ml of human spermatozoa under mineral oil. The sperm-oocyte preparation was incubated for 3 hr at 37 degrees C, during the last 15 min of incubation, the fluorochrome Hoechst 33342 (H) was added and incubation was allowed to proceed until the incubation time was over. Observations showed that the female pronucleus, eccentrically placed, gives a bright green-bluish fluorescence whereas chromatin of sperm heads shows different stages of decondensation and also a bright fluorescence. This inexpensive method has given consistent results in a large number of cases and provides an additional new approach to the "penetration test" as a proof of the capacity to form a "male pronucleus".