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

RESUMO

In order to increase the value of the zona-free hamster oocyte penetration test, a comparetively simple and fast method using the fluorochrome Hoechst 33342 was developed. human spermatozoa were washed and incubated 1 hr mediumBWW for capacitation. hamster oocytes were stripped of cumulus oophorus and zona pellucida with hyaluronidase and trypson, washed and used immediately. Thirthy oocytes were placed in a drop of BWW containing 3,5.10(6)/ml of human spermazoa 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 <> as a proof of the capacity to form a <>