Histological characterization of oocyte developmental stages of suruvi Steindachneridion scriptum kept in captivity

Stages of oocyte development were described for suruvi females (Steindachneridion scriptum) kept in captivity. Oocytes of 21 mature or maturing females and ovaries of 12 immature females were fixed in Karnovsky solution for 4 and 12 hours, respectively. After, they were processed for routine histology, embedded in paraffin and stained with hematoxylin-eosin. Slides were examined under a light microscope and the oocytes were measured using the LAS EZ software, using the average between the largest and the smallest diameter of each oocyte for representation. There were six types of oocytes: Chromatin nucleolus: found in immature females, with average diameter of 9.5 ± 4.46 μm; Perinucleolar: present in all developmental stages with average diameter of 67.6 ± 15.61 μm; Cortical alveolar: measuring 175.6 ± 56.58 μm; Vitellogenic: average diameter of 797.65 ± 136.10 μm; Mature: with average diameter of 1184.98 ± 171.13 μm and Atretic, which were found in mature or maturing females of suruvi.


Introduction
Most fish species native to Brazil with potential for cultivation migrates towards the source of the river in the breeding season (Paulino, Miliorini, Murgas, Lima & Felizardo, 2011).This migratory behavior is restricted in fish ponds, preventing these fish reach the physiological preparation for reproduction (Pereira et al., 2009).The animals do not receive certain external stimuli and lose endocrine response appropriate for inducing final gonadal maturation.Thus, the ovaries develop only partially (complete vitellogenesis) and enters the 'dormant stage' (Andrade & Yasui, 2003).
During the 'dormant stage', there is reduced ovarian activity and adjustments to the environment for the release of oocytes when environmental conditions are more favorable to the development and survival of the offspring (Zaniboni Filho & Weingartner, 2007).This stage can persist for several months before spawning and if the changes in the environment are not sufficient to cause the final maturation and release of oocytes, there will be follicular atresia.Some factors are known for inducing atresia, such as stress caused by critical environmental factors (e.g.oxygen, temperature, etc.) or by inadequate management during the dormancy period (Murgas, Drumond, Pereira & Felizardo, 2009;Zaniboni Filho & Weingartner, 2007).
The successful captive breeding requires the selection of mature fish showing higher chances to respond positively to induction of final maturation and spawning, comprising the selection of individuals that completed vitellogenesis and with ovaries in the 'dormant stage'.In this way, the identification of the oogenesis stage is critical to the proper selection of mature animals.
Suruvi (Steindachneridion scriptum) of the order Siluriformes and family Pimelodidae, is found in the Uruguay and upper Paraná river basins (Garavello, 2005), and is one of the species listed in the Red Book of endangered species in Brazil, showing important characteristics for inclusion in the South American fish farming.Studies have demonstrated great difficulty in selecting mature suruvi females, because they do not have significant external signs indicating their gonadal development stage.
Histological studies provide knowledge and more detailed observation of the oogenesis process, assisting in the identification of reproductive mechanisms in fish, which is key to understanding the life cycle, the establishment of guidelines to work with spawning induction in fish farming and control standards to fisheries exploitation (Zaniboni-Filho & Resende, 1988).
In this context, this study provides the first histological characterization of the oocyte development of S. scriptum females kept in captivity, with observations in different months of the year.In this way, it will be possible to obtain samples of all oocyte developmental stages, enabling their description so as to they can serve as reference for future work on the reproduction of this species.

Material and methods
The suruvi females analyzed in this study are derived from the first filial generation (F1) of wild broodstock caught in the upper Uruguay river and from the same spawning with induced reproduction held in September 2009 by the Laboratory of Freshwater Fish Biology and Culture (LAPAD/UFSC) (Authorization 016/2011 -NUPESC/IBAMA/SC).All specimens are individually identified with internal marks PIT-tag (Passive Integrated Transponder).
To obtain oocytes at all maturity stages, females stocked in two different rearing systems were evaluated in different months of the year.Immature females kept in the laboratory (n = 12) had weight and length (mean ± standard deviation) of 302.63 ± 76.53 g and 31.71± 2.59 cm, respectively.Maturing and mature females kept in ponds (n = 21) had weight and length of 1921.21 ± 47379 g and 54.14 ± 7.13 cm, respectively.
Immature females were killed for the removal of the ovaries in July 2013.In mature or maturing females, oocytes were taken through ovarian biopsy performed between September and December 2013, using a plastic cannula 10.The decision for biopsy to obtain these samples was to avoid the need to sacrifice these animals, which would compromise the stock of breeders of this species in LAPAD.Both ovaries and oocytes were fixed in modified Karnovsky's solution (2% Paraformaldehyde + 2% Glutaraldehyde + 0.1 M phosphate buffer -pH 7.4) and kept for 12 and 4h, respectively (Karnovsky, 1965).After, they were transferred to 70% alcohol.The material was subjected to histological dehydration in different alcohol solutions, clearing and paraffin embedding.Sections were cut in a Minot microtome to a thickness of 4 and 2 μm for gonads and oocytes, respectively, and stained with hematoxylin-eosin and mounted with Erv-Mount.
Slides were photographed under a light microscope (Leica ICC50 HD) and oocytes were measured using the LAS EZ software, using the average between the largest and the smallest diameter of each oocyte for representation (Zaniboni-Filho & Resende, 1988).The developmental stages of the oocytes were described using as suggested by Grier, Uribe-Aranzábel and Patiño (2009).

Results and discussion
Histological observations of germ cells in suruvi, at the level of light microscopy, allowed the characterization of six types of oocytes.
The number of oocyte developmental stages may vary according to species and the criteria used by the authors.For S. scriptum, the oogenesis process was divided into five stages, considering the oogonia as STAGE I. Some authors consider the oogonia as the initial cells in the oogenesis process (Fávaro, Frehse, Oliveira & Júnior, 2005;Santos et al., 2006), however, other authors do not include oogonia as a developmental stage (Gonçalves, Bazzoli & Brito, 2006;Núñez & Duponchelle, 2009).
The Chromatin nucleolus oocytes (STAGE I) (Figure 1) were the smallest oocytes, appearing grouped in 'clusters of germ cells' (CG) inserted in ovigerous lamellae in vascularized areas, with average diameter (± SD) of 9.5 ± 4.46 μm (Figure 5).The cytoplasm of these cells is thin and the nucleus is large and rounded, with little affinity for dyes, usually with a single central and slightly basophilic nucleolus.In turn, Perinucleolar oocytes (STAGE II) appeared generally separated from the 'cluster', probably due to their increased volume (Figure 1).The cytoplasm is more basophilic than in the previous stage, with a gradual decrease in this characteristic according to the increase in cell size.
There was a reduction in the nuclear-cytoplasmic ratio.The average diameter (± SD) of these oocytes was 67.6 ± 15.61 μm (Figure 5).The nucleus of smaller diameter oocytes has one or two spherical and intensely basophilic nucleoli (Figure 1B), which become larger and more numerous in accordance with increase in cell size, gradually migrating to the nuclear periphery.These oocytes showed many different shapes, round, triangular, rectangular or oval.In the stage II, the oocytes had highly basophilic cytoplasm and nucleoli, associated with intense protein synthesis (Takahashi, Gointein & Nakaghi, 2008).Oocytes at this stage were found in slides made from tissues of females in all maturity stages, unlike the Chromatin nucleolus oocytes, which were observed only in immature females.The occurrence of oocytes stage II in all maturity stages was expected, because they represent oocytes of the 'reserve stock', which will give rise to cell populations that will begin vitellogenesis in the next breeding season (Ganeco, Nakaghi, Urbinati, Dumont-Neto & Vasques, 2001).Figure 1A and B illustrate samples taken in July.All 12 females evaluated in this period presented primarily oocytes at stages I and II, with few exceptions given the appearance of a small number of oocytes at the beginning of the Cortical alveolar stage.Thus, the oocytes that represent the subsequent stages in the other figures were all obtained from ovarian biopsy.
In Cortical alveolar oocytes (STAGE III), cortical alveoli appeared arranged in the periphery of the cytoplasm, with translucent granules advancing into the cell with the development of the oocyte (Figure 2), as observed for Brycon orbignyanus (Ganeco et al., 2001), Cathorops spixii (Fávaro et al., 2005) and Brycon orthotaenia (Gonçalves et al., 2006) and unlike that found for Katsuwonus pelamis (Grande, Murua, Zudaire & Korta, 2012) and Hypostomus strigaticeps (Takahashi et al., 2008), where the appearance of these structures began around the nucleus and was displaced to the cell periphery according to the oocyte development.The nucleus and cytoplasm increased compared to the previous stage; however, the cytoplasm increased at a greater extent.Oocytes presented an average diameter (± SD) of 175.6 ± 56.58 μm (Figure 5).
The cytoplasm in the perinuclear region continues basophilic.The nucleus is weakly stained with multiple nucleoli occupying the periphery.There is the appearance of a continuous acidophilic layer, the vitelline membrane, which is surrounded by a layer of follicular cells.The appearance of the vitelline membrane and follicular cell layer assists the identification of this developmental stage, as reported for Hypostomus strigaticeps (Takahashi et al., 2008).
Vitellogenic oocytes (STAGE IV) showed average diameter (± SD) of 797.65 ± 136.10 μm (Figure 5).This stage was observed at the onset of deposition of yolk granules from the periphery of the cytoplasm, which become pinkish by eosin (Figure 3).The nucleus keeps the characteristics of the previous stage.The vitelline membrane becomes thicker and follicular cells grow and become more elongated and evident (Figure 3B).Oocytes of this stage were characterized by the presence of highly acidophilic yolk granules; the vitelline membrane and the follicle cells become more evident.The oocytes more developed of this stage will evolve to complete vitellogenesis and will be released in the next spawning (Núñez & Duponchelle, 2009).In Mature oocytes (STAGE V), basophilia disappears almost completely (Figure 4).The nucleus kept its characteristics with very small nucleoli, lost its spherical shape and shrunk.The vitelline membrane was radially arranged around the oocyte (zona radiata) (Figure 4B).Apparently, they are more mature cells observed in the germ line, occurring only in mature ovaries.Oocytes have an average diameter of 1184.98 ± 171.13 μm (Figure 5).
In oocytes of Stage V, the cytoplasm was filled with acidophilus yolk granules, similar to that observed for various freshwater fish (Gonçalves et al., 2006;Núñez & Duponchelle, 2009;Zaniboni-Filho & Resende, 1988).At this stage, freshwater fish oocytes are ready to be released from the follicle (Brown-Peterson, Wyanski, Saborido-Rey, Macewicz & Lowerre-Barbieri, 2011).When vitellogenesis is completed, ovarian activity starts to decrease until the environmental conditions are favorable for the development of eggs and survival of the offspring, when ovulation and spawning occur (Zaniboni Filho & Weingartner, 2007).Atretic oocytes were not classified as a developmental stage, being considered structures derived from the oogenesis process (Figure 6).These oocytes exhibited the disruption of the vitelline membrane and the yolk granules lost their individuality, turning into an acidophilic mass (Figure 6B).The nucleus was no longer visible.Atretic follicles are formed by oocytes that started the process of oocyte development, and may have completed it or not, but are reabsorbed before release.The follicular development stops and begins the degenerative processes that are most often seen in vitellogenic oocytes (Miranda, Bazzoli, Rizzo & Sato, 1999).These atretic follicles were found in oocyte samples from mature or maturing females.The presence of follicular atresia is usually observed in ovarian vertebrates, both in natural environment and under captive conditions (Saidapur, 1978), nevertheless, some factors may increase the frequency of follicular atresia and may even adversely affect fecundity (Sharma & Bhat, 2014).

Conclusion
Samplings conducted in different months of the year enabled the characterization of the five types of oocytes that make up the complete oocyte development cycle of Steindacheniridion scriptum females kept in captivity, namely: Chromatin nucleolus, Perinucleolar, Cortical alveolar, Vitellogenic and Mature.In addition, atretic oocytes were also found in mature or maturing females.

Figure 1 .
Figure 1.Cross section of the ovary of Steindachneridion scriptum at immature stage.A-Ovary with Chromatin nucleolus oocytes (I) and perinucleolar oocytes (II): Chromatin nucleolus oocytes forming clusters of germ cells (CG) inserted in ovigerous lamellae; Perinucleolar oocytes (II), with gradual increase in nucleus size and in number of nucleoli, as they grow; Connective cells (CC); Capillaries (C).B-Detail of Perinucleolar oocytes (II) with varied sizes and shapes, the nucleus displaced to the periphery of the cell; (*) Perinucleolar oocytes with one or two nucleoli.

Figure 5 .
Figure 5. Minimum, maximum and average diameter of oocytes of Steindachneridion scriptum in each developmental stage.STAGE I: Chromatin nucleolus; STAGE II: Perinucleolar; STAGE III: Cortical alveolar; STAGE IV: Vitellogenic; STAGE V: Mature.

Figure 6 .
Figure 6.Atretic oocyte of Steindachneridion scriptum, obtained by ovarian biopsy.A-General aspect of the atretic oocyte, with no visualization of the nucleus.B-Detail of this follicle showing yolk granules (GV) losing their individuality and disruption of the vitelline membrane (arrow).