Fine structure of sheep fertilization in vitro.

The fertilization events of the sheep oocyte have been investigated by electron microscopy. Thirty-two oocytes recovered from the oviducts of superovulated ewes were fertilized in vitro by a procedure that has given rise to full embryonic development (Crozet et al.: Gamete Res 16:159-170, 1987) and were analyzed at different stages of fertilization. Sperm incorporation in the ooplasm occurred as early as 2 h postinsemination (PI). One hour later, a fertilization cone containing very few cytoplasmic organelles developed above the decondensing sperm chromatin. It was a transitory structure which disappeared when the male pronucleus (PN) was formed. The second meiotic division of the oocyte resumed soon after sperm incorporation; abstriction of the second polar body occurred by 3-4 h PI; the polar body constriction was associated with microfilaments. Both the male and female PN were formed at 5 h PI and were surrounded by nuclear envolopes containing pore complexes. The female PN was characterized by patches of condensed chromatin; the male PN was closely associated with the sperm neck and proximal midpiece. Thereafter the PN migrated toward the center of the egg, where the spindle of the first cleavage division was observed at 21 h PI. Dispermic ova analyzed at 2-4 h PI contained 2 decondensing sperm heads and 2 fertilization cones which have reached the same stage, thus suggesting that a few sperm may penetrate quite simultaneously. Multipronucleate eggs were characterized by disturbance of the PN migration.