Occurrence and Ultrastructural Characterization of "nuage" during Oogenesis and Early Spermatogenesis of Piaractus Mesopotamicus Holmberg, 1887 (teleostei)

We investigated the occurrence and ultrastructurally characterized electrondense nuclear material (nuage) released from the nucleus during oogenesis and early spermatogenesis of Piaractus mesopotamicus, a fish from Pantanal Matogrossense (Brazil) having a seasonal reproductive cycle. The female germ cells presented two instances of nuclear material extrusion: in the oogonia and in the oocyte in the perinucleolar phase. In males, material with similar morphology and behavior occurred in the spematogonia. In all cases, this material was associated to mitochondria. The possible function of this material is discussed. RESUMO Ocorrência e caracterização ultra-estrutural de " nuage " durante a ovogênese e o início da espermatogênese de Piractus mesopotamicus Holmberg, 1887 (Teleostei) Foi investigada a ocorrência e caracterizada ultra-estruturalmente a eliminação de material nuclear eletrondenso (nuage) para o citoplasma durante a ovogênese e durante os estágios iniciais da espermatogênese de Piaractus mesopotamicus, um peixe do Pantanal Matogrossense de ciclo reprodutivo sazonal. Constataram-se nas células germinativas femininas dois momentos de eliminação desse material, na ovogônia e no ovócito em fase perinucleolar. Nas células masculinas, material com morfologia e comportamento muito semelhante foi encontrado na espermatogônia. Em todos os casos, o material associou-se a mitocôndrias. A possível função desse material foi discutida.

The aim of the present investigation is to characterize the presence of nuage during oogenesis and early spermatogenesis of P. mesopotamicus, a fish from Pantanal Matogrossense (Brazil) having a seasonal reproductive cycle, in order to contribute to the knowledge of the behavior of the nucleuscytoplasm exchanges during gametogenesis.

MATERIAL AND METHODS
In Pantanal Matogrossense (State of Mato Grosso do Sul, Brazil), female and male specimens of Piaractus mesopotamicus (Teleostei) were collected in feeding and breeding sites at the Aquidauana and Miranda rivers during the months of April and September.Ovary and testis fragments were fixed in 2.5% gluraldehyde in 0.1 M sodium cacodylate buffer, and then post-fixed in 1% osmium tetroxide in the same buffer.After a rinse in the buffer, the material was dehydrated in a standard acetone series (30% to 100%) and included in Epon 812 resin.Semi-thin sections were analyzed under a light microscope after being stained with methylene blue and azure II, as well as with toluidine blue.Ultrathin sections were contrasted with uranile acetate and lead citrate and then examined and photographed under a Zeiss EM9S2 transmission electron microscope (TEM).

RESULTS AND DISCUSSION
In the testes and ovaries of this species of fish, as in others, oogonia and spermatogonia are found alone, or in groups of two or three in the follicle wall (Dadzie, 1969;Shrestha & Khanna, 1978;Grier, 1981;Romagosa, 1991;Romagosa et al., 1993Romagosa et al., , 2000)).In our study, these germ cells usually were large with the nucleus occupying a considerable part of their volume, presenting in the cytoplasm many spherical mitochondria and very few endoplasmic reticulum profiles (Figs.1A and 2A, B).The nuclear chromatin presented completely dispersed in such a way that the generally spherical and very electrondense nucleolus became very conspicuous (Figs.1A and 2A).Various electrondense granules, intensely stained by toluidine blue, were found attached to the cytoplasmatic side of the nuclear envelope of these cells (Figs. 1A and 2A, B).This material, showing the same morphology and topology of previously described nuage, which occurs in several species (Kessel, 1968;Kato, 1968;Eddy, 1975;Clérot, 1976Clérot, , 1979;;Cruz-Landim & Cruz-Höfling, 1979;Bruslé, 1980;Dohmen, 1985;Cruz-Höfling & Cruz-Landim, 1990;Reunov et al., 2000).In the spermatogonia, the nuclear envelope frequently presented a pronounced depression, where the nuage lodged (Fig. 2B).When this material detached from the nuclear envelope, it formed dense granules that associated with groups of mitochondria in oogonia (Fig. 1A) as well as in spermatogonia (Fig. 2B).This association has been observed in other species of fishes (Grier, 1981;Bruslé, 1980;Silva, 1987).The material transferred from the nucleus was similar in both sexes in morphology, staining qualities, and association with mitochondria.The mitochondria were poor in cristae and presented diverse sizes (Figs.1A and 2B).
As this material stained, it showed morphology and electrondensity characteristics similar to the nucleolar material (Figs.1A and 2A).We suggest that it is constituted by ribonucleoproteins.According to Clérot (1979), the association with mitochondria might mean a role in this organelle replication, since these germ cells increase the mitochondria number in the cytoplasm.Nevertheless, although it is known that mitochondria import proteins from cytoplasm (Alberts et al., 1989), nothing is known about nucleic acid import during their replication.
According to Reunov et al. (2000), during spermatogenesis of the sea urchin Anthocidaris crassispina, modified mitochondria associates with nuclear nuage and release some of their content into the cytoplasm, suggesting that the germ plasm is supplemented by the mitochondrial genome.
Therefore, the nuage-mitochondria association seems to have an important role in oocyte development, but it is unlikely to have the same in spermatogonia since their cytoplasm is lost during spermatogenesis, and even spermatogonial mitochondrial derivates are left out of the oocyte in fertilization.
During spermatogenesis the nucleus is expected to have few opportunities for gene expression.Therefore, it is possible that the material extruded is long-lasting RNA to be used during gamete maturation.This may result from the transcription of genes responsible for the synthesis of cell division regulators, since these spermatogonia intensely divide mitotically before starting meiosis, and the meiotic division occurs without an interphase.If no new RNA is produced after the spermatogonia proliferation starts, the longlasting RNA, entering the cytoplasm in the spermatogonia, may even have some function during spermiogenesis.
In females a new elimination of nuclear material took place during the primary growth stage of the oocyte (Fig. 1B).At a certain stage of this process, nuage dispersed in an area surrounded by mitochondria (Fig. 1C).In some follicles this material was very electrondense, and after mitochondrial association became thread-like (Fig. 3A), occupying a great extension of the cytoplasm (Cruz-Landim & Cruz-Höfling, 1979).In a more advanced dispersion stage, many annulated lamellae appeared in the cytoplasm and could be seen associated with this electrondense material.
The extrusion of material from the micronucleoli to the cytoplasm produced within the nuclear matrix vacuoles visible under light (Fig. 3B) and by transmission electron microscopy (Fig. 3C).After a while, the vacuoles seemed to collapse, leaving only very electrondense membranous residues (Figs.1C and 3D).
Therefore, material transferred later, in the oocyte, is thought to constitute germ plasm, which according to Eddy (1975) is constituted mainly of ribonucleoproteins with a ribosomic function, but more recent data point to the presence of long-lasting mRNA in its composition (Alberts et al., 1989).
In P. mesopotamicus it is seen, in initial phases of oocyte I maturation, being produced in specific chromosome regions and accumulating in the micronucleoli, before passing throughout the nuclear pore complexes to the cytoplasm.
In conclusion, whereas the female germ cells presented two moments of nuclear material extrusion (in the oogonia and in the perinucleolar phase of the oocyte), in males the extrusion was present only in spermatogonia.In all cases the material is probably constituted of ribonucleoproteins from ribosomes, or mRNA.It is possible that the nuclear material reaching the cytoplasm in oogonia and in spermatogonia are similar and supply information about the final destiny and function of these cells, while material transferred during oocyte perinucleolar phase enter into the germ plasm constitution.

Fig. 1 -
Fig. 1 -A.Transmission electron micrograph (TEM) of an oogonium, showing the large nucleus (n) with a prominent nucleolus (nu).Notice electrondense material (arrows) in the cytoplasmic side of the nuclear envelope, which may be associated with spherical mitochondria (m).ser, smooth endoplasmic reticule; Bars = 2 µm.B. Detail of the nuclear region of a perinucleolar oocyte.Notice in the nucleus (n) a micronucleolus (mn) and nuage (arrow´s head) associated with the cytoplasmic face of the nuclear envelope and the nucleolar material going towards the nuclear envelope.cy, oocyte cytoplasm; Bars = 3 µm.C. Perinuclear region of an oocyte´s cytoplasm, showing nuage (arrowhead) associated with mitochondria (m).Notice in the nucleus (n) lamellar structures (la).mn = micronucleolus; Bars = 3 µm.