Low-voltage scanning electron microscopy of mammalian fertilization in vitro: Preparation of oocytes

To optimize specimen-processing protocols for investigations of fertilization in mammals, with high-resolution, low-voltage scanning electron microscopy (LVSEM), bovine oocytes matured in vitro were prepared by either aldehyde fixation, conductive staining, or high-pressure cryoimmobilization with freeze-substitution. Samples prepared by these different 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 conventional scanning electron microscope (cSEM). The results show that bovine oocytes prepared by aldehyde fixation or cryoimmobilized by high-pressure freezing produced superior images of the zona pellucida (ZP) compared with those from the conductive stained samples. A comparison of LVSEM and cSEM images suggests that the gold-coating employed in cSEM preparation obscures the ZP detail seen with the LVSEM, but not with the cSEM. Application of these procedures provide not only a new view of the ZP but may be useful in elucidating the molecular mechanism of gamete interactions during the fertilization process.