Formation of male pronuclei in partitioned human oocytes

Ninety-five metaphase II human oocytes, aged in vitro for either one day or for two days, and five fresh immature oocytes with no visible germinal vesicle nucleus were partitioned into small cytoplasts after removal of the zone pellucida and exposure to cytochalasin B. Seventy-one metaphase II and four immature oocytes were used as intact zone-free controls. The cytoplasts derived from each partitioned oocyte and all the zone-free whole oocytes were exposed to normal or subfertile donor sperm and later assessed for signs of male pronucleus development. A total of 711 fragments (an average of 8 fragments per partitioned egg) with a mean diameter of about 50 μm were produced from the 100 partitioned oocytes. When exposed to normal sperm, 76% of the 1-day old metaphase II fragments, 67% of the 2-day-old metaphase II fragments, and 83% of the immature oocyte fragments were fertilized. The mean number of decondensing nuclei per partitioned oocyte was 11.9 for the 1-day- old metaphase II oocytes, 6.4 for the 2-day old metaphase II oocytes, and 13 for the immature oocytes. The mean number of decondensing nuclei per a whole zone-free oocyte was 5.6 for the 1-day-old metaphase II oocytes (p < 0.05), 6.7 for the 2-day-old metaphase II oocytes (NS), and 13 for the immature oocytes. When exposed to subfertile sperm, 54% of the 1-day old metaphase II fragments and 28% of the 2-day-old metaphase II fragments were fertilized. The mean number of decondensing nuclei per partitioned oocyte was 8.8 for the 1-day-old metaphase II oocytes and 2.7 for the 2-day-old metaphase II oocytes. The mean number of decondensing nuclei per a whole zone free oocyte was 4.5 for the 1-day-old metaphase II oocytes (p < 0.05) and 2.4 for the 2- day-old metaphase II oocytes (NS). Over 50% of the penetrated fragments contained a single decondensing sperm; the number of decondensations within each was related to the fragment diameter. Fragments containing a sing le sperm head were more prevalent when their diameter was 40-50 μm for normal sperm and 50-60 μm for subfertile sperm. The present study demonstrates that when human oocytes are partitioned into several small membrane-bound fragments, each fragment retains the ability to fuse with sperm and to support apparently normal sperm head decondensation and pronucleus formation.