ELECTRON MICROSCOPE EVALUATION OF RABBIT EGGS EXPOSED TO SPERMATOZOA TREATED WITH CAPACITATING AGENTS

Abstract

The effects of bovine follicular fluid (BFF), human follicular fluid (HFF), uterine fluid, McCoy''s modified medium, .beta.-amylase, trypsin and chymotrypsin on rabbit spermatozoa were investigated in vitro. The treated spermatozoa were then exposed to eggs in vitro. Cleaved cells were examined by EM for the presence of cortical granules. There was no visible change in untreated ova or in ova incubated without spermatozoa in McCoy''s medium, HFF, BFF or medium containing enzymes. They did not develop pronuclei or cleave. Similar results were obtained when ova were exposed to spermatozoa incubated in fertilization medium or in Tyrode''s solution. Eggs that were placed with spermatozoa capacitated in vivo developed to the 2-, 4- and 8-cell stages and showed no cortical granules under EM. Cleavage occurred in ova exposed to spermatozoa preatreated with enzymes or body fluids, except for .beta.-amylase and BFF. Spermatozoa placed in preovulatory uterine fluid did not induce cleavage but 60% of ova cleaved when incubated with spermatozoa treated with uterine fluid from a primed doe. The physiological action of postovulatory uterine fluid on spermatozoa was therefore significantly different from that of preovulatory fluid. When examined by light microscopy, eggs exposed to treated spermatozoa appeared to develop normally. Polar bodies were extruded and the cleaved cells were indistinguishable from control ova. EM showed that these ova retain large numbers of cortical granules. The granules were not randomly distributed but were present in some segments of the cortical cytoplasm and absent in others. The homogeneity of the population of cortical granules is questionable since only some disappear. Mitochondria, yolk bodies, the Golgi complex, nuclei and nucleoli could not be distinguished from those of fertilized ova. These results suggest that ova were activated rather than fertilized, implying that spermatozoa are converted to activating agents by incubation in test media. Alternatively, if these ova are fertilized, the effect of the spermatozoa on the ovum is not identical to those produced by spermatozoa capacitated in vivo. Experiments to establish that these ova are fertilized are in progress.