Ultrastructure of rabbit ova recovered from ovarian follicles and inseminated in vitro.

Rabbit ova were obtained from presumably preovulatory ovarian follicles of gonadotropin-treated does and inseminated with uterine sperm in vitro. Ova were serially sectioned for light and electron microscopy 0, 1 1/2, 3, 6, and 9 hours after in vitro insemination. Control ova were maintained in culture for 25 hours and examined by light microscopy for cleavage. Little difference in fertilization rate (i.e., ova fertilized/ova inseminated) was observed when criteria were based on ultrastructural evidence (56.5%) and on light microscopic evidence 25 hours post-insemination provided by cleavage of control ova (54.8%). Therefore, observations of cleavage (as in controls here) can be an accurate criterion for fertilization in the rabbit, at least under the in vitro conditions used in this study. After insemination of ova recovered from preovulatory follicles, the developmental sequence was as follows: after 1 1/2 hours, sperm penetration into the vitellus; after 3 hours, male pronuclear formation and second polar body extrusion; after 6 hours, male and female pronuclear enlargement; and after 9 hours, pronuclei in apposition. This sequence compares favorably with the normal temporal sequence of events already well known for fertilization of ovulated ova in the rabbit. Penetrating sperm cells within the matrix of the zona pellucida and the supplementary sperm cells lying in the perivitelline space had undergone the acrosome reaction. Changes associated with the acrosome reaction took place before the penetration of the zona pellucida. The presence of a number of supplementary sperm, usually five to six per ovum, within the perivitelline space of the already activated ovum indicated that the block to polyspermy in these experiments operated mainly at the level of the vitelline membrane, as is the case in the normal fertilization process. The vitelline membrane block to polyspermy occurred rapidly after penetration of the fertilizing sperm into the vitellus and was associated with cortical granule breakdown. Ultrastructural details of rabbit ova recovered from ovarian follicles and inseminated in vitro revealed no distinguishable characteristics when compared with reported observations of ovulated rabbit ova undergoing fertilization in vivo.