Cytological events associated with in vitro aged and fertilized rabbit eggs

The morphogenetic events associated with rabbit eggs aged in vitro for 12 to 50 hours prior to mixing with sperm have been examined by light and electron microscopy. After 12 hours in culture, morphological alterations of the meiotic spindle and the cortex of unfertilized eggs were evident. By 24 to 50 hours in culture, unfertilized eggs contained subnuclei, structures which formed when individual and/or groups of meiotic chromosomes dispersed and becmae invested by a double-laminated structure reminiscent of a nuclear envelope. Although most eggs obtained 11.5 to 12 hours after induced ovulation and in vitro fertilized displayed morphogenetic patterns similar to those described for in vivo fertilized ova, some (10%) contained three pronuclei. Many eggs obtained 13 to 15 hours after induced ovulation and subsequently mixed with sperm in vitro appeared to undergo processes of fertilization typical of in vivo fertilized eggs, however, approximately 30% contained subnuclei in association with the male pronucleus. Few eggs (15%) aged 12 hours prior to in vitro fertilization displayed patterns of pronuclear development and association typical of fertilized unaged ova. Subnuclei developed in many of the fertilized ova. Supernumerary sperm nuclei, which did not develop into male pronuclei, were observed in some zygotes. Cleavage of eggs aged 12 hours prior to fertilization was abnormal or retarded. After 24 hours in culture approximately 16% of the eggs fertilized. Seventy percent of the fertilized eggs failed to support the development of a male or female pronucleus.