SPERMATOGENESIS IN THE YELLOWTAIL TETRA Astyanax altiparanae : A HISTOLOGICAL ANALYSES WITH EMPHASIS TO SPERMATOGONIAL AND SPERMATID TYPES *

This study aimed to detailed description of the characteristics of the different germ cell types found in Astyanax altiparanae during spermatogenesis. In this purpose, testes from 25 adult male’s specimens of A. altiparanae were sampled and submitted to the usual techniques for light microscopy. Based on nuclear shape, chromatin condensation, nucleoli quantity and cell size were identified four spermatogonial types: type A undifferentiated (Aund.*); type A undifferentiated (Aund.); type A differentiated (Adif.); and type B spermatogonia. Spermatocytes were observed in different phases of meiosis (leptotene/zygotene/pachytene and diplotene), metaphase I and II and secondary spermatocytes, being distinguish mainly by their chromosomal organization inside the nucleus. Were also identified three different types of spermatids, which were named as initial, intermediate and final, which can be differentiated by increase in nuclear condensation and spacing among cells inside the cysts, possibly by flagella arise and reduction in nuclear diameter . Thus, this study contribute to a better understand of spermatogenesis in this and other fish species.


INTRODUCTION
Teleost represent the most diversify and abundant group among vertebrates, being formed for about 96% from all fish species.The success of this group is assigned to a series of adaptation, reflecting the huge diversity of morphology, life cycle pattern, but mainly in the reproduction type, characterized by varied reproductive strategy such as spawning rhythm, fecundity, maturity age, annual reproductive cycle, etc. (ALEXANDRINO et al., 1985;VAZZOLER, 1996;REIS et al., 2003;NÓBREGA et al., 2006;GRIER and ARANZÁBAL, 2009).
The main testicular function is to keep spermatogenesis, one complex and extremely organized process, in which one initial diploid cells, named spermatogonia, undergo proliferation and differentiation to produce spermatozoa, which are haploid cells (GRIER et al., 1980;NÓBREGA et al., 2009;SCHULZ et al., 2010).
Spermatogenesis in fish and other anaminote develop inside spermatocysts, which are formed by germ cells in synchronic development surrounded by cytoplasmic process of Sertoli cells, which are responsible for germ cells mechanical and nutritional.Didactically, spermatogenesis may be divided in three phases: spermatogonial or proliferative phase in which spermatogonia undergo successive mitotic divisions.This phase is specie-specific; spermatocitary or meiotic phase, in which the genetic material is duplicated, recombined and segregated; and spermiogenic phase, characterized by morphological and physiological changes of spermatids, with variation according the species, to originate the spermatozoa (GRIER et al., 1980;RUSSELL et al., 1990;VERÍSSIMO-SILVEIRA et al., 2006;NÓBREGA et al., 2009;SCHULZ et al., 2010;SIQUEIRA-SILVA et al.;2012).Despite the advantages provided by histological studies in attempt to better understand fish spermatogenesis, few data considering the basic aspect of this process and both a detailed morphological description and behavior from germ cells during this process are available (JAMIESON, 1991;LEAL et al., 2009).
Thus, the aim of the present study was the morphological description of germ cells characteristics in Astyanax altiparanae differentiating them according to the subdivisions in spermatogonial, spermatocytary and spermiogenic lineage during spermatogenic cycle.Popularly known as yellowtail tetra, this is a small fish (reaching a maximum of 20 cm), distributed in High Paraná, Paranapanema, Tibagi and Iguaçu basin (Brazil) in both lotic and lentic environments, being a substantial part of bigger fish diet, besides to be appreciated by human (GARUTTI and BRITISKI, 2000;ORSI et al., 2002;DE BEM, 2009).The choice of species was motivated by its good livestock characteristics, such as ease of handling, small size, early sexual maturity at 4 months of age, and intertidal spawning, which enable it as an appropriate model for investigating the biology of teleost and for the development of distinct studies in laboratory conditions (CASTILHO-ALMEIDA, 2007;YASUI et al., 2014).

Animals
Twenty-five sexually mature male specimens of A. altiparanae of standard size ranging from 12 to 15 cm were selected from excavated tanks at Hydrobiology Engineer Souza Dias Station (CESP).Sexually mature males are easily distinguished from females by presenting hooks in anal fin.
Experimental procedures were carried out in strict accordance with the guide for care and use of laboratory animals of the University Estadual Paulista (UNESP).The research ethics committee of UNESP approved the protocols (Permit Nº: 006/2012/CEUA).All surgical procedures were performed under anesthesia with 0.5 g of Bol.Inst.Pesca, São Paulo, 41(esp.):697 -705, 2015 benzocaine in 5 mL of absolute ethanol, and all efforts were made to minimize fish suffering.Fish were identified and a group of voucher individuals was deposited in the collection of fishes of DZSJRP Ichthyology Laboratory, Department of Zoology and Botanic IBILCE/UNESP under the register number DZSJRP008999.

Sampling and analyses
Testes were dissected, cuted into transverse and longitudinal sections, fixed in 4% paraformaldehyde with 2% glutaraldehyde solution in Sorensen phosphate buffer, pH 7.4, for a minimum period of 24 hours.Then, the samples were dehydrated in ethanolic solutions at increasing concentrations and were impregnated in glycol methacrylate (Technovit 7100/historesin; HeraeusKulzer, Wehrheim, Germany).Finally, the samples were sectioned to a thickness of 3.0 μm using a microtome (LEICA RM 2145; Leica Instruments GmbH, Heidelberg Nussloch, Germany) equipped with a glass blade and stained with Haematoxylin and Eosin.Photoprocessing and histological analyses were completed with a Zeiss optical microscopy equipped with an AXIOCAM-MRc5 camera (Carl Zeiss Microimaging GmbH, Göttingen, Germany).Measure from cell nuclei diameter was performed using Motic Images Plus 2.0 program.One hundred nuclei were measured for each spermatogonial type and spermatozoon and 4500 nuclei for each spermatid type.

RESULTS
Spermatogenesis in A. altiparanae was subdivided according the stage of germ cells inside germinal epithelium.Thus, morphological characteristics of germ cells allowed identification of four spermatogonial types and three different types of spermatids, besides spermatocytary stage commonly found during reproductive cycle in fish.

Spermatogonial types in mitotic or proliferative phases
Spermatogonial types were differentiated according to nuclear shape, chromatin condensation, nucleoli quantity and cell size, being divided in: -Type A undifferentiated spermatogonia (Aund.*):these cells are isolated and dispersed throughout the germinal epithelium (Figure 1).Their nucleus show an irregular and elongated shape, being a little basophilic and heterochromatic; the average diameter was 7.67 ± 0.63 µm.More than one wellevidenced nucleolus is observed (Figure 2A).-Type A undifferentiated spermatogonia (Aund.):are also isolated and dispersed throughout the germinal epithelium, however, are smaller than the previous one.These cells show an elongated and more basophilic nucleus with average diameter of the 6.12 ± 1.16 µm.In this spermatogonial type nucleoli are not well evidenced (Figure 2B).-Type B spermatogonia (SpgB): these cells are also grouped in cysts and the last germ cells to undergo mitotic divisions during spermatogenesis.Their nucleus is rounder, showing an increase in the amount of spread heterochromatic material.Nuclear average diameter of this spermatogonial type was 4.83 ± 0.54 µm and the nucleoli are little evidenced (Figure 2D).Meiotic phase, which is also named spermatocytary, starts after differentiation of spermatogonia B in primary spermatocyte.

Meiotic or spermatocytary phases
In this phase, spermatocytes were found in different phases from meiosis and were mainly distinguished by their nuclear characteristics, such as size, condensation and chromosome arrangement: -Leptotene/Zygotene (L/Z), pachytene and diplotene primary spermatocytes and secondary spermatocyte: Leptotene/Zygotene spermatocytes show rounded nucleus with apparently clearest chromatin and some heterochromatic points that are subjacent to the nuclear envelope (Figure 3A); Cells in pachytene (P) are apparently smaller than the previous one, show a denser nucleus with more condensed chromosomes (Figure 3B); Spermatocyte in diplotene phase (D) are found in the same cyst with that ones in metaphases I (M).In this cell phase, chromosome reach their maximum nuclear condensation (Figure 3C).
At the end of first meiotic division, secondary spermatocytes (Sc2) are found.In A. altiparanae they were characterized by their round nucleus and dense chromatin (Figure 3D).

Spermiogenesis and sperm generation
After secondary spermatocyte undergo the second meiotic division, spermatids are originated giving start to spermiogenic phase, also called differentiation phase, which in this species can be observed in three different stages of development, according to the increase in nuclear compression and spacing among them inside the cysts, probably due to the appearance of flagellum and elimination of cytoplasmic reside.The spermatid types in this species were named initial, intermediate and final spermatids.  the cysts.Its nucleus is very condensed and surrounded by a narrow strip of cytoplasm.Nucleus average diameter of this spermatid type is 1.31 ± 0.19 µm (Figure 4C).
At the end of spermatogenesis the sperm are formed.It is smaller than final spermatids with nuclear average diameter 1.02 ± 0.16 µm.These cells are found only into tubular lumen (Figure 4D).

DISCUSSION
Morphological characteristics of spermatogenesis in teleost assist the comprehension of testicular functions, contributing to understand the numerous tactic and reproductive strategies developed by this group (VAZZOLER, 1996;NÓBREGA et al., 2009).Spermatogenic study in A. altiparanae enabled histological characterization of four spermatogonial and three spermatid types.
According to VILELA et al. (2003), there is a difference among spermatogonial types in fish, varying according both the nuclear chromatin arrangement and germ cell size, which is speciesspecific and can be genetically defined (ANDO et al., 2000).Differently from observations in A. altiparanae, five types of spermatogonia were described in zebrafish (Danio rerio) and they were classified as type-A undifferentiated (Aund.*),type-A undifferentiated (Aund.),type-A differentiated (Adif.)and initial and final type-B spermatogonia (LEAL et al., 2009).However, in Nile tilapia (Oreochromis niloticus), only three types of spermatogonia were differentiated, being named type-Aor immature spermatogonia, type-A or mature differentiated spermatogonia and type-B spermatogonia (VILELA, et al., 2003;SCHULZ et al., 2005) and for the South American species, Gymnotus carapo, only two types of spermatogonia were observed, defined as type-A spermatogonia (SPGA) and type-B spermatogonia (SPGB) (VERGÍLIO et al., 2012).

Spermatogonia
are fundamental to spermatogenesis, since they can develop distinct functions.Aund.* spermatogonia, also named isolated spermatogonia, for example, is the biggest germ cell inside the epithelium, being frequently named as stem cell, due to its renewal potential in each reproductive cycle and for giving origin to differentiated daughter cells, which will contribute to tissue functions.Spermatogonial cells show fundamental role in the transmission of inherited characters, since spermatogonia compromised with sperm generation, named type-A undifferentiated spermatogonia, carry all parental genetic material (MORENA et al., 1996;De ROOIJ and RUSSELL, 2000;NÓBREGA et al., 2010;SCHULZ et al., 2010;VERGÍLIO et al., 2012).Type-A differentiated spermatogonia are originated from previous ones.It show cytoplasmic bridges among them, due to incomplete cytokinesis during mitosis process (LEAL et al., 2009;SIQUEIRA-SILVA et al., 2012), and can be found in cysts with two to eight germ cells according to the species (SCHULZ et al., 2010).
After mitotic divisions, secondary or type-B spermatogonia are originated.They are the last spermatogonial generations and can form cysts with 16 or more cells according to the species, as in D. rerio, whose total number of spermatogonia B per cyst was about 208 cells (LEAL et al., 2009;SCHULZ et al., 2010).In A. altiparanae only one type of spermatogonia B was identified.However, in D. rerio these cells were divided between initial and final spermatogonia B (LEAL et al., 2009), corroborating De ROOIJ andRUSSELL (2000) idea who admit that spermatogonial terminology is confuse, due to the complexity of spermatogonial kinetics.Type-B spermatogonia differentiated in primary spermatocytes, giving origin to spermatocytary phases in which meiosis occur (GRIER et al., 1980;SILVA, 1987;VERÍSSIMO-SILVEIRA et al., 2006;NÓBREGA et al., 2008;SCHULZ et al., 2010).
In this phase, spermatocytes can be distinguished by their size and chromosome condensation, but mainly by "meiotic figure" or arrangement of the chromosomes into the nucleus (LEAL et al., 2009).Morphological characteristics of A. altiparanae spermatocytes are similar to observations already described to other teleost, as for the species G. carapo (VERGÍLIO et al., 2012) highlighting that this phase might be highly preserved in this group.
After secondary spermatocytes finish the second meiotic division, the spermatids are originated.These cells are committed to undergo events of cell differentiation, characterized by two main morphological phenomena: intracellular Bol.Inst.Pesca, São Paulo, 41(esp.):697 -705, 2015 movements and structural changes.Together, both of these phenomena are named spermiogenesis, which can also vary among species (ROOSE-RUNGE, 1962;BRUSLÉ, 1981;BILLARD, 1984;SILVA, 1987;LO NOSTRO et al., 2003).As can be notice by difference in spermatid types, since other Neotropical fish such as Cyphocharax modestus, Potamorhina altamazonica and Steindachnerina insculpta presented only two types of spermatids, named early and late spermatid (QUAGIO-GRASSIOTTO et al., 2003).However, LEAL et al. (2009) also estimated three spermatid types to D. rerio species, which separated from the studied species around 153 million years ago.Thus, the number of spermatids does not seem to follow a set pattern among Teleostei fish.

CONCLUSION
Considering these aspects, it can be concluded that this study provides important tools for better understanding of spermatogenesis in teleosts.Moreover, understanding of the morphological events of spermatogenesis in this species, enable it as an appropriate model for the development of methodologies such as germ cell transplantation involving other Neotropical fish and Characiform species.
Bol. Inst.Pesca, São Paulo, 41(esp.):697 -705, 2015 -Type A differentiated spermatogonia (Adif.):differently from the previous ones, type A differentiated spermatogonia are grouped in two or more cells in a cyst.Their nucleus is round/oval, presenting little condensed chromatin and only one evidenced nucleolus.The nucleus average diameter of this spermatogonia type was 4.55 ± 2.16 µm (Figure2C).