DISCUSSION
Electron microscopy techniques are highly disseminated and used in the
most diverse areas of science, precisely because they promote the
analysis of very small details and structures.2 Among
them, Scanning Electron Microscopy (SEM) allows the analysis of surfaces
and has been used in the study of fish, mainly in reproductive
characteristics, for a long time.4,7,14 However, its
use has been restricted to the study of the oocyte surface, highlighting
adhesive structures or the micropyle, as well as the shape of
spermatozoa.7,15-18
By associating the cryofracture technique with SEM, other possibilities
can be approached, such as the study of the relief of other oocyte
structures, such as the cells of the oocyte envelope, the structuring of
the zona pellucida, the yolk vesicles, among others. These aspects are
described in this study for the first time and bring details different
from what is possible to analyze in light microscopy.
Some challenges can be present in this technique, such as its cost and
the specialized training required for its operation.19Boyde and Wood20 also describe that wet tissues
experience less distortion compared to those subjected to the
dehydration and drying process, so this type of tissue needs careful
handling. This care in handling the samples is even more important when
one considers that certain intracellular structure, such as microtubules
and intermediate filaments, cannot be fully revealed only by fracturing
methods, as they are involved in the cytoplasmic matrix. So, it depends
on the use of osmium, which detaches this cytoplasmic matrix. But it is
noted that membranous structures remain
well-preserved.21 Finally, according to Pathan et al.22, it has been demonstrated that this technique
requires a meticulous sample preparation, or it can result in artifacts
or modifications that may alter the original characteristics of the
sample.
First, one can consider the situation that electronic microscopy
techniques, in general, promote better conservation of the samples that
are analyzed, both due to the size of the samples and because of the
applied fixative solutions (which have greater power of
fixation).23-24 In this sense, it is expected that
these techniques present less distortions of what is analyzed and what
actually exists.24 This can be seen in this study when
observing the structure of theca cells, which here are columnar. When
analyzed under light microscopy, it is common to describe these cells as
squamous or cubic. This difference could be explained by the
manipulation of the samples for light microscopy, which can generate a
greater modification of the structures to the point where they can be
analyzed.
Another situation regarding these distortions can be observed in the
micropyle. This structure is often described as an entry funnel for
sperm.15,17,25-28 However, the SEM associated with
cryofracture shows that this structure is more irregular and has large
chamfers associated with the plasma membrane and the zona pellucida.
These descriptions differ from what exists in the literature, possibly
because micropyle descriptions are made from SEM images where only a
portion of this structure that is visible on the outside of the oocyte
is analyzed.
Still analyzing the peripheral region of the oocyte, one observes its
association with the follicular cells through the zona pellucida. This
structure is described as a projection region of the cells involved, one
towards the other, establishing closer communications between
them.29-30 Thus, the zona pellucida promotes a
facilitated exchange of factors, nutrients, and many other signals that
are regulated by follicular cells to promote oocyte
development.30 In this sense, the presence of pores in
this region is highlighted here as possible ways to also facilitate the
bidirectional distribution of substances (follicular cells–oocyte).
These structures are observed in several other works, mainly those that
highlight the ultrastructure of the micropyle, as it is possible to
observe all these characteristics in the same
images.15,17,25-28
Other characteristics were also observed, such as squamous follicular
cells and several yolk vesicles. These structures have a format and
distribution like that observed in other microscopic techniques and in
the descriptions of several species of fish. Thus, these characteristics
can be consulted in more detail in other
studies.10,29-34
Finally, the distribution and appearance of the connective membrane that
surrounds and structures the ovarian tissue is highlighted. In light
microscopy this membrane is observed as a very thin structure and with
little detail regarding how it is associated with the cells of the
germinal epithelium.31-32,35 When observed on SEM,
still associated with cryofracture, it is possible to observe in more
detail the reach of this membrane. Thus, it is observed that the
connective membrane has a leaf aspect, covering all the structures of
the ovary and distributing the blood vessels. It is known that the
vasculature of the ovary depends on the stage of maturation of the
organ, 34-35 so it is believed that this structuring
of the connective membrane may undergo changes throughout the
reproductive period, requiring its study at other times of development
of the ovary of fish.
Once the specific characteristics of fish have been discussed, it is
emphasized here that this study was based on other studies in mammals,
since there is no information with this technique for fish. Among the
characteristics compared, Makabe et al.36 showed in
their study the essential relationship between germ and somatic cells of
mammals and humans, in addition to highlighting the specialization of
the membrane, such as the adhesion zone, desmosomes and gap junctions,
which result in the zona pellucida. The same can be seen when analyzing
the results presented here, where the relationship established between
the oocyte, the follicular cells and the theca cells is observed,
guaranteeing the adequate development of the follicle until its
maturation. As for the zona pellucida, it was observed here that it is
full of pores that are open and without plugs on its surface,
reaffirming the patterns found by Ganeco and Nakaghi15for the Characidae family.
In the same way that the SEM technique added to cryofracture promotes
differentiated responses for analyzes in mammals, some specific
characteristics of fish could only be observed in more detail using this
new technique. It is noteworthy, in this sense, that with the technique
of cryofracture associated with maceration, it is possible to observe
the interior of vitellogenic oocytes more easily, with emphasis on the
characteristics of their surroundings, such as the oocyte envelope, the
micropyle and the zona pellucida, as well as of the connective membrane
associated with ovarian structures.