LOW-VOLTAGE SCANNING ELECTRON-MICROSCOPY OF MAMMALIAN FERTILIZATION IN-VITRO - PREPARATION OF OOCYTES

To optimize specimen-processing protocols for investigations of fertil ization in mammals, with high-resolution, low-voltage scanning electro n microscopy (LVSEM), bovine oocytes matured in vitro were prepared by either aldehyde fixation, conductive staining, or high-pressure cryoi mmobilization with freeze-substitution. Samples prepared by these diff erent preparative techniques were coated with either platinum or gold and imaged with an LVSEM operating at 1.5 keV. Additionally, aldehyde- fixed oocytes, sputter-coated with gold, were imaged with a convention al scanning electron microscope (cSEM). The results show that bovine o ocytes prepared by aldehyde fixation or cryoimmobilized by high-pressu re freezing produced superior images of the zona pellucida (ZP) compar ed with those from the conductive stained samples. A comparison of LVS EM and cSEM images suggests that the gold-coating employed in cSEM pre paration obscures the ZP detail seen with the LVSEM, but not with the cSEM. Application of these procedures provide not only a new view of t he ZP but may be useful in elucidating the molecular mechanism of game te interactions during the fertilization process.