APPLICATIONS OF SCANNING ELECTRON MICROSCOPY ASSOCIATED WITH
CRYOFRACTURE FOR ANALYSIS OF FISH OVARIES
Abstract
Scanning Electron Microscopy (SEM) allows a precise identification of
the surface of the structures. When associated with the cryofracture
technique, it allows a more detailed analysis, since the samples have
their internal structures exposed. Thus, the protocol and results of SEM
associated with cryofracture in Astyanax lacustris ovaries are
presented here in order to characterize the external and internal
structure of the oocytes. A. lacustris ovaries were collected,
prefixed in 4% paraformaldehyde, fixed in osmium tetroxide (2% OsO
4) at 4 ºC and kept overnight in phosphate-buffered
saline. The samples were washed, immersed in DMSO at different
concentrations, followed by their fracture in 50% DMSO cooled by liquid
N 2 and then immersed in 50% DMSO until returning to
room temperature. The fractured samples were taken for maceration in 1%
osmium, in a shaker for 15 hours. Afterwards, the samples were
post-fixed in 2% OsO 4 for 2 hours at 4 ºC, dehydrated
in an increasing ethanol series and taken to the critical point
apparatus Balzers CPD-030 using liquid CO 2. This was
followed by the assembly of the blocks and the metallization of the
sample with gold ions, with the analyzes carried out in a Scanning
Electron Microscope Jeol JSM-7401F 5V. It was found that the
cryofracture technique provided images with greater internal detail of
the oocytes. Some specific characteristics of fish oogenesis could only
be observed using this new technique, with emphasis on the oocyte
envelope, the micropyle and the zona pellucida, as well as the
connective membrane associated with the ovarian structures.