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.