INTRODUCTION
The use of scanning electron microscopy (SEM) allows precise
identification of the surface of structures through the generation of
three-dimensional images.1 This technique is based on
a beam of electrons, produced by an electron source gun that is
generated by a filament or crystal that focuses on a region of the
sample. Then, this beam is transmitted to a cathode screen, and the
generated image is the result of an interaction that occurs between
beam-sample.2 However, to perform the technique,
several aspects must be considered, such as: electrical conductivity,
hardness, and porosity of the sample to be analyzed.3For example, samples that have low conductivity need to undergo a
metallic coating, usually a gold film is used. 3
SEM analyses are used to reveal microstructures, showing the size,
structure, and shape of particulate or fractured surfaces, such as:
polymers, proteins, seeds, inorganic, and organic
compounds.1,4-5 In this regard, the association of the
cryofracture technique adds to the characteristics that can be observed
under the microscope. This protocol allows for an analysis with more
detailed observations, since the samples have their internal structures
exposed, promoting a finer resolution of structures, and using the depth
of field created by SEM.6
As evidenced by Riehl7 in a study with teleosts, the
pattern found on the surface of the oocyte can be used as one of the
criteria for identification and differentiation of species. Macchiarelli
et al.5 also report that this technique allows the
morphofunctional classification by understanding the vascular plexuses.
It is clear, then, that the analysis of oocyte structures involves the
use of high precision techniques, as proposed in this study. It is worth
mentioning that in teleosts, as in other vertebrates, the oocyte
development in a mature egg is a regulated process under the influence
of environmental and endocrine factors.8 Thus, the
clearer the understanding of the morphological characteristics of the
oocytes, the easier and more complete the understanding of the dynamics
of oogenesis will be.9 The understanding of cellular
growth dynamics is used also to describe the reproductive cycle,
allowing the recognition of the reproduction period and the
morphological modifications that occur.10
SEM can therefore be used as a complementary tool to light microscopy,
as it provides more details.4 However, until now, no
studies have been found that used SEM associated with cryofracture for
the description and comparison of fish oocytes, so it is not possible to
estimate its real application in these models. Thus, the protocol and
results of SEM associated with cryofracture in Astyanax lacustrisovaries are presented here in order to characterize the external and
internal structure of the oocytes.