Ultrastructure of the germ cells in the testis of matrinxã,Brycon

Tissue & Cell, 1999 31 (6) 540–544
© 1999 Harcourt Publishers Ltd
Article no. tice.1999.0064
Tissue&Cell
Ultrastructure of the germ cells in the
testis of matrinxã, Brycon cephalus
(Teleostei, Characidae)
E. Romagosa1, M. Y. Narahara1, M. I. Borella2, S. F. Parreira2,
N. Fenerich-Verani3
Abstract. This study describes at ultrastructural level the germ cells in the testis of matrinxã (Brycon cephalus)
raised in captivity. The specimens ‘matrinxã’ were maintained in four breeding tanks of 200 m2, at the Aquaculture
Research Center at Vale do Ribeira–CEPAR, from Fishery Institute, in Pariquera-Açu City, São Paulo, Brazil. The
samples were collected from March 1994 to February 1996. The testis has been classified as tubular unrestricted
spermatogonial type, in which four stages of germ cells can be distinguished as follows: spermatogonia, spermatocytes (primary and secondary); spermatids and spermatozoa. © 1999 Harcourt Publishers Ltd.
Keywords: ultrastructure, testis, spermatogenic cells, Brycon cephalus, matrinxã
Introduction
Materials and methods
Matrinxã is a native species of economical importance in
the Amazon Basin (Brazil). It is a Brycon genus and a
Characidae family teleost fish. The fattening of this species
has been developed by pisciculturists, and the results have
been available to anyone interested in this area (Scorvo
Filho et al., 1998). Brycon cephalus is a very popular fish
for sport fishing, because it fights hard when hooked, which
is very attractive for the anglers, bringing more people to the
practice of this activity (Romagosa, 1998).
There are no records of this species in the literature. This
paper describes at ultrastructural level the germ cells of
matrinxã, Brycon cephalus, raised in captivity at the Ribeira
Valley Region.
Twenty-one male specimens of matrinxã, Brycon cephalus,
weighing 1.5–2.0 kg were used. This study was carried out
from March 1994 to February 1996 at the ‘Centro de
Pesquisa em Aquicultura do Vale do Ribeira’ (CEPAR –
Ribeira Valley Aquaculture Research Center), which
belongs to the Fishery Institute in Pariquera-Açu, São
Paulo, Brazil (24°43¢ S and 47° 53¢W). The fish were raised
in four breeding tanks of 200 m2, with an initial density of
1.0 fish/m2 in each tank, and were fed twice a day with a
balanced diet containing 28% crude protein.
The fish were anesthetized with Benzocaine (ethylaminobenzoate) and quickly decapitated. Every 2 months
the data on total length, and body and testes weights of each
fish were recorded. The testes weights varied from 0.001 to
1.53 g. In summer they were exposed to 18 h of light daily,
in an average temperature of 17°C. The average rainfall was
269.7 mm in the summer and 19.8 mm in the winter.
The left testis were rapidly removed and sliced into
2–3 mm cross-sectional slabs from middle region. Small
blocks were fixed in 3% glutaraldehyde in 0.1 M phosphate
1
Centro de Pesquisa em Reprodução e Larvicultura, Instituto de Pesca,
São Paulo 2Instituto de Ciências Biomédicas, USP, São Paulo
3
Departamento de Hidrobiologia, UFSCar, São Paulo, Brasil
Received 2 March 1999
Accepted 29 June 1999
Correspondence to: E. Romagosa, Centro de Pesquisa em Reprodução e
Larvicultura, Instituto de Pesca, Av. Francisco Matarazzo, 455, São Paulo,
05031-900 Brazil. Tel.: + 11 3871 7566; E-mail: [email protected]
One part of a PhD thesis submitted at UFSCar (Universidade Federal de
São Carlos, São Paulo, Brazil) – recipient of CNPq fellowship.
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FISH GERM CELL ULTRASTRUCTURE
Fig. 1 Spermatogonia: mitochondria (long arrow); annulate lamellae (short arrow) and desmossomes (d). X 1350.
Fig. 2 Primary spermatocytes (C1); spermatids (T); spermatozoa (Z) and cystic cells (★). X 1440.
541
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ROMAGOSA ET AL.
buffer (pH 7.2), during 18 h, at 4°C, and postfixed in 1.0%
osmium tetroxide, dehydrated, and embedded in Spurr resin
(Spurr, 1969), also at 4°C, for 2 h, at the Departamento de
Histologia e Embriologia do Instituto de Biomédicas da
USP, São Paulo, where they were processed. The fragments
were cut at approximately 0.5 mm and stained with 1.0%
toluidine blue in satured boric acid. Finally, 60 nm ultrathin
sections were obtained and constrasted with 2.0% uranyl
acetate and lead citrate for 5 min, and examined in a JEOL
100 CX-II microscope.
Results and discussion
The testes of Brycon cephalus are elongate paired organs,
ventrally disposed along the gaseous bladder, to which they
are attached by a mesorchium. They become narrow at their
caudal extremity where they join themselves, before
opening at the genital pappila (Romagosa, 1998).
There are two compartments in the testis of matrinxã: (1)
the seminiferous, containing cystic (Sertoli) cells and germ
cells: spermatogonia, spermatocytes, spermatids and spermatozoa, which are found inside the tubules. The cystic
cells are in the intratubular compartment, forming the wall
of the cysts, and are always associated to the germ cells, but
with very distinct morphological features; (2) the interstitial
compartment, outside the tubules, contains elements from
the connective tissue as fibrocytes, collagenous fibers,
myoid cells, and blood vessels. The basal membrane and the
insterstitial cells or Leydig are also present.
The testis of matrinxã is composed of a large number of
seminiferous tubules with different diameters, containing
spherical or elongated cysts inside. The spermatogenic cells
of each cyst are in the same development stage. According
to Grier et al. (1980), the fishes have two different testicular
types. The testis of matrinxã belongs to tubular unrestricted
spermatogonial type. The macroscopic anatomy and the
histology of the testis of Brycon cephalus were described by
Romagosa (1998). It is similar to most oviparous freshwater
teleosts in South America (Andrade & Godinho, 1983;
Zaniboni Filho et al., 1988; Romagosa et al., 1993).
Seminiferous compartments
Germ cells
The germ cells are found in the testis of Brycon cephalus in
four stages of development:
Stage I: spermatogonia
The spermatogonia are the largest cells of the testis. The
nucleus is spherical, the chromatin is granular and
uniformly distributed. The proeminent nucleolus is spherical and electrondense. The cytoplasm contains spherical
mitochondria with few cristae, free ribosomes, agranular
and granular endoplasmic reticulum, and a well developed
system of double membranes perforated by a series of pores
or ‘annuli’, with eight to ten cisternae, called annulate
lamelae (Fig. 1). Electrondense structures in the cytoplasm
are the ‘mitochondrial cementum’ when they are close to
mitochondria or mitochondria cluster. These structures were
also observed by Andrade and Godinho (1983) and Silveira
et al. (1990). Primary spermatogonium divides mitotically
to give rise to secondary spermatogonia, which are initially
very similar to their mother-cell, except for their nuclei,
which is slightly smaller. The primary and secondary spermatogonia appear during the reproductive cycle, and can be
observed outside the cysts. A similar observation was made
for other teleost fishes as the freshwater Piaractus
mesopotamicus (Romagosa et al., 1993).
Desmossome-like junctions were seen between spermatogenic cells and the cyst wall (Fig. 1), as described by
Russell (1977). The presence of desmossome-like junctions
in South American freshwater fish is not described
frequently. The peculiar characteristics of these structures in
the testis of non-mammalian vertebrates and mammals
suggest that they are highly conserved during evolution
(Sprando & Russell, 1987).
Stage II: primary and secondary spermatocytes
Primary spermatocytes originate from late secondary spermatogonia and are seen in various phases of the meioticprophase. They show spheric central nucleus, with a regular
shape, occupying a large part of the cell (Fig. 2). The nucleolus is not visible, and the chromossomic masses frequently
found are called synaptonemic complex. The presence of
synaptonemic complexes in the spermatocytes of Piaractus
mesopotamicus was also described by Romagosa (1991). It
is possible to visualize cytoplasmatic bridges connecting the
spermatocytes in the studied species. These same structures
were described by Andrade and Godinho (1983) and
Romagosa (1991).
Secondary spermatocytes, which arise from the division
of primary spermatocytes, are present in large numbers
within the cysts. Cysts of secondary spermatocytes are
rarely observed. In the secondary spermatocyte the nucleus
is evident, still large, the chromatin is irregularly distributed
and concentrated in one of the nuclear poles, as a semicircle
(Fig. 3). Those organells observed in the cells of the
previous stage are seen in the cytoplasm (Fig. 3). The
secondary spermatocytes give rise to spermatides.
Stage III: spermatids
The spermatids are seen in different stages of spermiogenesis as indicated by the degree of chromatin condensation
and the presence of flagella. Initially, they contain chromatin in coarse granules, evenly distributed. It is possible to
observe the presence of cytoplasmatic bridges among the
spermatids. With the progress of the spermiogenesis
process, the chromatin condenses into dense clots, and the
cell becomes smaller and more electrondense.
Simultaneously, the migration of the proximal and distal
centrioles occurs: the first is located near the nuclear
membrane, and the other near the tail of the spermatozoa.
The volume of cytoplasm decreases considerably, some
FISH GERM CELL ULTRASTRUCTURE
Fig. 3 Secondary spermatocytes (C2) and spermatids (T). X 985.
Fig. 4 Various spermatozoa (Z). X 2594.
Fig. 5 Spermatozoon: head (H), centrioles proximal (1) and distal (2). X 13 935.
543
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ROMAGOSA ET AL.
mitochondria increase in volume, and encompass the initial
part of the flagellum, forming the mitochondrial sheath, the
future connection piece (Figs 2 & 3).
The process of spermiogenesis involves some alterations
that are similar to those described by Romagosa (1991), for
Piaractus mesopotamicus, also raised in captivity: chromatin condensation, mitochondrial migration, displacement
of centrioles (proximal and distal) and formation of the
flagellum (Figs 2 & 3). The elimination of spermatid cytoplasm must occur as residual bodies. They are formed and
extruded by the spermatid into the spermatocyst lumen in
Lepomis macrochirus (Sprando & Russell, 1988). In
matrinxã, the nuclear membrane should also be investigated, due to the great number of pores accumulated at the
caudal pole after nuclear condensation.
Stage IV: spermatozoa
Brycon cephalus spermatozoa are primitive, according to
Grier (1981), Billard (1983), Cruz Landim and Cruz Höfling
(1986), Mattei (1988) and Romagosa (1991), and their
configuration correspond to the same found in most of the
teleost with external fecundation. It is possible to verify that
the spermatozoa of matrinxã are relatively simple, consisting
of head, neck or connection piece, and tail (Figs 2 & 4).
Spermatozoa are seen in the lumen of the tubule as well as in
the spermatic duct. The head is round, small, with high
eletrondensity, and is covered by plasmalemma (Fig. 5).
The absence of acrosome, observed in the spermatozoa
of matrinxã and other teleost (Romagosa, 1991; Matos et
al., 1993), is justified by the presence of a structure called
micropyle in the ovules of some species of freshwater fish
(Ricardo et al., 1996), which helps the penetration.
The connection piece of the matrinxã spermatozoa is
surrounded by a short mitochondrial necklace with few mitochondria, located in the inferior portion of the piece (Fig. 5).
The long spermatozoa tail of matrinxã represents a typical
flagellum, with 9+2 axonome pattern, typical of primitive
species (Romagosa, 1991; Matos et al., 1993). The initial
portion of the flagellum is surrounded by a mitochondrial
sheath, forming a short connection piece. Cytoplasmatic
projections are evident in the posterior region of the connection piece. The tail of the spermatozoa is essentially
composed by the flagellum, and this is the region where the
cell gets its propulsion. The flagellum has the function of
keeping the efficiency of the beating and movement of the
structure (Baccetti, 1970).
Cystic cells
The cystic cells (Sertoli cells) are the somatic envelope of
the sperm line cells. They are distributed in the periphery of
the cysts or around the spermatogonia. The cystic cells
show an irregular-shaped nucleus, decondensed chromatin
and evident nucleolus; cytoplasm with extensions that inter-
cross and connect through the desmossomes. A similar
structure was observed by Andrade and Godinho (1983) in
Leporinus silvestrii.
ACKNOWLEDGEMENTS
We are grateful to CEPTA/IBAMA, Pirassununga, SP, for
providing the fish utilized in this study, and to Dr Patrícia
Gama for the review of the English manuscript.
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