Tissue & Cell 35 (2003) 121–132
Ultrastructure of the testis in Synbranchus marmoratus
(Teleostei, Synbranchidae): the germinal compartment
F.L. Lo Nostro a,∗ , H. Grier b , F.J. Meijide a , G.A. Guerrero a
a
Laboratorio de Embriologı́a Animal, Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pab. II, 4to piso, Buenos Aires C1428EHA, Argentina
b Stock Enhancement Research Facility, Florida Marine Research Institute, 14495 Harllee Road, Palmetto, FL 34221-9620, USA
Received 19 August 2002; received in revised form 27 December 2002; accepted 27 December 2002
Abstract
Synbranchus marmoratus, is a protogynic diandric species in which two types of males, primary and secondary, are found. In both
types, the germinal compartment in the testes is of the unrestricted lobular type, but in secondary (sex reversed females) males the lobules
develop within the former ovarian lamellae. In the present study, the germinal compartment was examined in both types of males using light
microscopy as well as scanning and transmission electron microscopy. Germinal compartment is limited by a basement membrane and
contains Sertoli and germ cells. During maturation, processes of Sertoli cells form the borders of spermatocysts containing isogenic germ
cells. Characteristically, type A and type B spermatogonia have a single nucleolus and grouped mitochondria associated with dense bodies
or nuage. Type B spermatogonia, spermatocytes and spermatids are joined by cytoplasmatic bridges and are confined within spermatocysts.
Secondary spermatocytes are difficult to find, indicating that this stage is of short duration. Biflagellated spermatozoa have a rounded head,
no acrosome, and possess a midpiece consisting of two basal bodies, each of which produces a flagellum with a typical 9 + 2 microtubular
composition. No associations occur between sperm and Sertoli cells. There were no differences between spermatogenesis in primary and
secondary males in this protogynic, diandric fish.
© 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Synbranchidae; Synbranchus marmoratus; Germinal compartment; Sex reversal; Spermatogenesis; Biflagellated sperm
1. Introduction
Spermatogenesis in fish exhibits characteristic features
(Billard, 1969, 1970a,b, 1984; Grier, 1975a, 1981; Pecio
and Rafinski, 1999; Quagio-Grassiotto and Carvalho, 1999).
Type B spermatogonia (SPGB), clustered within spermatocysts, have a decreased nuclear size and an increased
nuclear chromatin density compared to type A spermatogonia (SPGA), which are individually surrounded by Sertoli
cells (Billard, 1969, 1984). Nuclear chromatin structure in
meiotic SPCI is characterized by the presence of condensing chromatin ending with the appearance of synaptonemal
complexes during pachytene of the first meiotic division
(reviewed by Westergaard and von Wettstein, 1972).
The ultrastructure of teleost spermiogenesis has been reviewed by Mattei (1969, 1970), Yasuzumi (1971) and Billard
(1986). In particular, spermiogenesis has been well documented in Poecilia reticulata (Billard, 1970a), P. latipinna
∗ Corresponding author. Tel.: +54-11-4576-3348;
fax: +54-11-4576-3384.
E-mail address: fabi@bg.fcen.uba.ar (F.L. Lo Nostro).
(Grier, 1973, 1975b), Elomorpha (Mattei and Mattei, 1974),
Gambusia affinis (Grier, 1975a), Lepadogaster lepadogaster
(Mattei and Mattei, 1978a,b), Liza aurata (Bruslé, 1981),
Salmo gairdneri (Billard, 1983), Lepomis macrochirus
(Sprando et al., 1988), Oreochromis niloticus (Lou and
Takahashi, 1989), Mimagoniates barberi (Pecio and
Rafinski, 1999), Plagioscion squamosissimus (Gusmão
et al., 1999), and Sorubim lima (Quagio-Grassiotto and
Carvalho, 2000).
The structure of fish spermatozoa (SPZ) is extremely diverse. This can be partly explained by their diversity of reproductive modes. Most externally fertilizing teleosts have a
simple type of spermatozoon, called aquasperm (Jamieson,
1991). The aquasperm has a round or ovoid head and a short
neck region, or midpiece, containing few mitochondria. A
few species possess biflagellated sperm (Jamieson, 1991;
Mattei, 1991).
In fish, as in all other vertebrates, there are two compartments within the testis: the germinal compartment and the
interstitial compartment, being separated from each other by
a basement membrane. Both compartments have the same
distribution of cells as do their mammalian counterparts.
0040-8166/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-8166(03)00011-9
F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
The gonadal germinal epithelium has been recently redefined and conforms to definitions of an epithelium presented
in histology textbooks. According to the “unifying concept”
(Grier, 2000; Grier and Lo Nostro, 2000), the definitions
apply for both the sexes and across chordate taxa. As such,
the testicular germinal epithelium consists of Sertoli and
germ cells that border an anastomosing tubular or lobular
lumen and are supported by a basement membrane. The
germinal epithelium rests upon a basement membrane that
separates it from the interstitial tissue. In the testicular
germinal epithelium of teleost fish, sperm mature within
spermatocysts, synchronous clones of maturing germ cells
surrounded by processes of Sertoli cells (Callard, 1991;
Grier, 1993).
S. marmoratus is a protogynic, diandric species. Primary
males develop directly as males while secondary males arise
from the sex reversion of females (Lo Nostro and Guerrero,
1996; Lo Nostro, 2000). Both types of males, primary and
secondary, have unrestricted, lobular testes (Lo Nostro and
Guerrero, 1996), spermatogonia being distributed along the
lobules (Lo Nostro et al., 2003) rather than confined to their
distal termini as occurs in the atherinomorphs (restricted
testes) (Grier, 1993). As in another synbranchid species of
fish, Monopterus albus (Chang and Phillips, 1967; Liem,
1963; Nagahama, 1983), sex reversal is a natural, life history
process. It has been recently shown that S. marmoratus testes
possess a mechanism for the preservation of a permanent
germinal epithelium between breeding seasons (Lo Nostro
et al., 2003). However, the ultrastructure of spermatogenesis
in this genus has not been described.
The present paper describes for the first time in a fresh
water sequential hermaphroditic fish, the germinal compartment of the testis during spermatogenesis, both at light and
electron microscopic levels.
123
togynous fish were not reverting their sex. A total of 11
primary males and 39 secondary males were obtained.
2.2. Light microscopy (LM)
Testes were fixed in Bouin’s fluid or 0.1 M phosphatebuffered (pH 7.4) 5% formalin and embedded in paraffin.
Tissue sections were cut at 7 ␮m and stained with hematoxylin–eosin or periodic acid Schiff’s. Nuclei were measured microscopically, expressing values as mean±standard
deviation (X ± S.D.). Micrographs were taken with a
Nikon-Microphot FX microscope.
2.3. Transmission electron microscopy (TEM)
The 2 mm thick sections were fixed in 0.1 M phosphatebuffered (pH 7.2) 2.5% glutaraldehyde for 24 h at 4 ◦ C and
postfixed in 0.1 M phosphate-buffered 1% osmium tetroxide
for 1 h at room temperature. Samples where then dehydrated
and embedded in a mixture of Epon 812 resin and Araldite.
Semithin sections, stained with toluidine blue, were used
for orientation. Ultrathin sections were stained with uranyl
acetate and lead citrate and examined in a Zeiss EM-109
electron microscope.
2.4. Scanning electron microscopy (SEM)
Milt from reproductive males was collected in Eppendorf
tubes, subsequently fixed in 0.1 M phosphate-buffered (pH
7.2) 5% glutaraldehyde for 1 h (adapted from Lahnsteiner
and Patzner, 1991), and centrifuged at 800 rpm for 15 min at
4 ◦ C. The pellet was washed twice in 0.1 M phosphate buffer
(pH 7.2) with 10% saccharose for 30 min, dehydrated in a
graded ethanol series at 500 rpm for 7 min and transferred to
acetone. One drop (0.1 ml) of each sample was critical point
dried, coated with gold, and examined in the microscope.
2. Materials and methods
3. Results
2.1. Animals
A total of 100 fish were captured throughout the year from
flooded habitats in Corrientes Province, Argentina (27◦ 30 S;
58◦ 00 W). Fish were anesthetized with benzocaine (0.1 g/l),
sacrificed by decapitation, and the testes were quickly removed. They were processed for light microscopy in order
to determine the sex as they do not show external sexual
dimorphism and it was necessary to ascertain that these pro-
Testes are located along the mid-dorsal side of the body
cavity and occupy approximately two-thirds of the total
body length. Anatomical differences between primary and
secondary males are evident. The testes of primary males
consist of paired organs joined medially by connective
tissue where ducts are located. In cross-section, testes are
heart-shaped in immature individuals and kidney-shaped
in mature fishes (Fig. 1A). The testes of secondary males
䉳
Fig. 1. Light microscope photograph. (A) Cross-section of primary male testes. (B) Cross-section of secondary male testes. D, dorsal; ed, efferent ducts;
V, ventral. (A) 40×; (B) 40×. Fig. 2. (A) Light microscope photograph. Testicular lobules cross-section. (B) TEM micrograph. SPGA individually
surrounded by Sertoli cell processes. Note the nucleolus with both fibrillar and granular component. bm, basement membrane; cf: collagen fibers; it:
interstitial tissue; L: lobule; m: mitochondria; mb: multilamellate body; n: nucleolus; N: nucleus; nu: nuage; ps: Sertoli cell process; rer: rough endoplasmic
reticulum; S: Sertoli cell. (A) 600×; (B) 3000×. Fig. 3. (A) Light microscope photograph. Testes cross-section showing spermatogonia during mitosis.
(B) TEM micrograph. Two SPGB within a spermatocyst formed by more than one Sertoli cell. bm: basement membrane; cf: collagen fibers; G: Golgi
apparatus; it: interstitial tissue; L: lobule; ly: lysosome; m: mitochondria; n: nucleolus; N: nucleus; nu: nuage; S: Sertoli cell; SPGB: spermatogonia B;
Sy: spermatocyst. (A) 600×; (B) 3000×.
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F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
develop within the single ovary of a female during sex
reversal, a primary morphological criterion used to distinguish primary from secondary males. In this case, efferent
ducts are located dorsally and in lateral supports (Fig. 1B).
The germinal epithelium is located within lobules. The
lobules terminate blindly at the testis periphery and are limited by a basement membrane (Fig. 2A). Lobules contain
two cell types, the somatic Sertoli cells and the germ cells.
Spermatogenesis occurs entirely within spermatocysts, containing isogenic germ cells, whose borders are formed by
multiple Sertoli cells. In both primary and secondary males,
the characteristics of the germinal compartment are similar.
Spermatogenesis itself is a developmental process that consists of three steps: mitotic proliferation followed by meiosis
and spermiogenesis.
4. Germ cells
4.1. Type A and type B spermatogonia represent the
mitotic or proliferative phase of spermatogenesis
SPGA are present throughout the year. These cells are
oval-shaped, and stain lightly with hematoxylin–eosin. They
are the largest germ cells in the testis. They possess a hyaline
cytoplasm and a single prominent nucleus (9.1 ± 0.55 ␮m in
diameter), which is slightly oval or spherical and contains
a distinctive nucleolus. Chromatin is frequently observed in
different degrees of condensation according to their mitotic
stage (Fig. 2A).
SPGA are individually surrounded by Sertoli cell processes and display a very low electron density and regular
outlines in electron micrographs (Fig. 2B). Since the nucleus is voluminous and eccentric in position, there is a
higher concentration of mitochondria in the cytoplasm at
one side of the cell (Fig. 2B). Mitochondria are either round
or elongate with only a slight presence of lamellar cristae
in a clear matrix. Dense osmiophilic material or nuage, is
polymorphic and either free in the cytoplasm or associated
with mitochondria. While annulate lamellae have not been
observed, a prominent Golgi apparatus was detected. Many
polyribosomes are scattered throughout the cytoplasm and
multilamellate bodies are also seen. A conspicuous nucleolus shows both fibrillar and granular components (Fig. 2B).
SPGB are characterized by a decrease in cell size compared to SPGA. The nucleus is elongated and central in location. Its longer axis measures 9.66 ± 0.47 ␮m. Chromatin
aggregation differs according to the cell cycle phase observed (Fig. 3A). After hematoxylin–eosin staining, SPGB
present a hyaline cytoplasm.
SPGB are grouped within spermatocysts, bordered by
Sertoli cell processes (Fig. 3A and B). Although is difficult to detect, TEM reveals the presence of intercellular
bridges between neighboring SPGB. These spermatogonia also possess polyribosomes, multilamellate bodies and
SER. Lysosomes and a prominent Golgi apparatus are also
observed (Fig. 3B). Their mitochondria are similar to those
observed in SPGA, but fewer. Nuage may be associated with
mitochondria, but not always. There is a single, spherical
nucleolus with granular and fibrillar components (Fig. 3B).
4.2. Meiosis
Primary spermatocytes (SPCI) are oval cells as visualized
with light microscopy. Their cytoplasm is hyaline, and it is
hard to distinguish the cellular limits. The nucleus is elongated and its longer axis increases (11.7 ± 0.60 ␮m). The
presence of different degrees of condensation in the chromatin of SPCI belonging to different spermatocysts reveals
that these cells are in distinct stages of meiotic prophase
(Fig. 4A). A single, homogeneous nucleolus persists until
pachytene. Several intercellular bridges remain (Fig. 4B).
The centrioles are found near the nucleus; the cytoplasm
also contains an abundance of multilamellate bodies, inconspicuous SER and a Golgi complex (Fig. 4B). Mitochondria are smaller than in SPGB. They are rounded, having
lamellar cristae and an electron-dense matrix. They tend to
be associated with nuage, while the isolated nuage becomes
scarce (inset of Fig. 4B). During pachytene, synaptonemal
complexes are evident (Fig. 4B).
Secondary spermatocytes (SPCII) are rarely observed in a
testis section. Compared to SPCI, there is a decrease in cell
and nuclear size. They possess a voluminous, spherical nucleus, 6.1 ± 0.4 ␮m in diameter, with condensed chromatin
that stains strongly with hematoxylin (Fig. 5A). Their cytoplasm is reduced, rendering it poorly visible. Under TEM,
the cytoplasmatic limits are easily seen only if there are
intercellular spaces. The nucleus is regularly outlined and
clumped or mottled due to its dense chromatin (Fig. 4C).
The cytoplasm has few organelles. Mitochondria are small,
spherical; possess lamellar cristae and an electron-dense matrix. Polyribosomes, SER, Golgi apparatus and multilamellate bodies are also observed. Centrioles are found close to
the nucleus. Intercellular bridges join SPCII (Fig. 4C).
Spermatids undergo a shape remodeling and a size reduction during spermiogenesis. Their nuclei become smaller
as the chromatin condenses (Fig. 5A). Early spermatids,
with spherical nuclei (5.68 ± 0.42 ␮m in diameter), appear nearly contiguous. The nucleus is characterized by
the presence of dense, irregular chromatin strands. Evident
cytoplasmatic bridges whose limits have an electron-dense
material interconnect SPD and variable intercellular spaces
are observed (Fig. 5B). The topographical location, distribution and structure of organelles in spermatids is initially
similar to that observed in secondary spermatocytes, including the presence of nuage that is associated with scattered,
spherical mitochondria (Fig. 5B).
4.3. Spermiogenesis
The size of SPD decreases during spermiogenesis.
Therefore, intercellular spaces become larger within the
F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
125
Fig. 4. (A) Light microscope photograph. Testes cross-section showing spermatocysts with different stages of spermatogenesis. Note spermatozoa within
the lobule lumen (arrow). (B) and (C) TEM micrographs. (B) SPCI in pachytene stage. Inset: mitochondria and nuage at higher magnification. (C)
SPCII. c: centriole; G: Golgi apparatus; ib: intercellular bridges; it: interstitial tissue; Ll: lobule lumen; m: mitochondria; mb: multilamellate body; n:
nucleolus; nu: nuage; ps: Sertoli cells process; S: Sertoli cell; sc: synaptonemal complex; ser: smooth endoplasmic reticulum; SPCI: spermatocyte I;
SPG: spermatogonia; SPZ: spermatozoa; sy: spermatocysts. (A) 600×; (B) 3000×; inset 4800×; (C) 4400×.
spermatocyst. Mitochondria possess lamellar cristae with
an electron-dense matrix and still are associated with nuages (Fig. 5C). There is a well-developed Golgi apparatus,
as well as free ribosomes and polyribosomes (Fig. 5C
and D). Transformation of spermatids into mature spermatozoa (spermiogenesis) involves a complete reorganization
of the nucleus, a loss of the cytoplasm as a “residual body,”
and the development of two flagella (Fig. 5E and G). As
spermiogenesis progresses, chromatin condensation continues, transforming from granular to homogeneous (compare
Fig. 5F and G). The centrioles are initially located near
the nuclear membrane (Fig. 5B). A flagellum forms from
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F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
Fig. 5. (A) Light microscope photograph. Testes cross-section showing spermatocysts with different maturation stages. Note spermatozoa within
spermatocysts (arrow), not associated with Sertoli cells. (B)–(G) TEM micrographs. (B) Early spermatids connected by cytoplasmic bridges. Note the
intercellular spaces. (C) Cytoplasmatic bridges between spermatids in advanced stage of spermiogenesis. Note the presence of nuage during this stage.
(D) Detail of a prominent Golgi apparatus within a spermatid. (E) Basal bodies and two flagella. Note that mitochondria approach the posterior part of the
nucleus. (F) Spermatids in final stage of spermiogenesis. Desmosomes are observed between Sertoli cells of neighboring spermatocysts. (G) Spermatozoa
are free within lobule. bb: basal body; d: desmosomes; f: flagellum; G: Golgi apparatus; ib: intercellular bridges; it: interstitial tissue; Ll: lobule lumen;
m: mitochondria; nu: nuage; p: polyribosomes; ps: Sertoli cell process; rb: residual body; S: Sertoli cell; ser: smooth endoplasmic reticulum; SPCII:
spermatocyte II; SPD: spermatids; SPZ: spermatozoa; sy: spermatocysts. (A) 600×; (B) 4400×; (C) 12,000×; (D) 20,000×; (E) 20,000×; (F) 4400×;
(G) 3000×.
F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
each centriole prior to the mitochondrial arrangement in
the region that will become the midpiece (Fig. 5C and E).
Due to this arrangement, the SPD acquires polarity. Desmosomes are observed between Sertoli cells of neighboring
spermatocysts (Fig. 5F).
SPZ are the smallest germ cells, having a nuclear diameter
of only 2.5 ± 0.49 ␮m and appearing as small spheres due to
the high chromatin condensation (Figs. 4A and 5A). SEM
confirms this as SPZ from a milt sample show a spherical
head and a mitochondrial collar (Fig. 6B). During mid and
late maturation class, SPZ are released from spermatocysts
and fill the lumina of lobules (Figs. 4A and 5A).
127
SPZ are revealed to be biflagellate cells with a rounded
head (Fig. 6A) lacking an acrosome (Fig. 6C). Biflagellated
spermatozoa utilize both centrioles as basal bodies for the
development of each flagellum. Fig. 6C clearly shows two
flagella arising from the same cell. The midpiece is short, and
contains two parallel basal bodies each composed of nine
triplets (Fig. 6C, D and E). The basal bodies are surrounded
by a ring of various mitochondria (Fig. 6E). The flagella are
approximately 50 ␮m in length. Each axoneme has a typical
9 + 2 arrangement of microtubules, nine pairs of peripheral
microtubules and one central pair (inset of Fig. 6E). The nine
peripheral microtubules of both flagella are continuous with
Fig. 6. (A) Light microscope photograph. Milt sample, note the biflagellated sperm. (B) SEM micrograph. Spermatozoa from a milt sample, note the
mitochondrial collar. (C), (D), (E) TEM micrographs. (C) Spermatozoa with rounded head and two flagella arising from a same cell. (D) Detail of basal
bodies of biflagellated sperm. (E) Cross-section of basal bodies (triplets) and mitochondrial collar. Inset: flagella in cross-section. Note the typical 9 + 2
arrangement of microtubules. bb: basal body; cm: cytoplasmic membrane; f: flagellum; m: mitochondria; mc: mitochondrial collar; mi: microtubules. (A)
200×; (B) 10,000×; (C) 14,500×; (D) 50,000×; (E) 20,000×; inset 20,000×.
F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
those of the basal bodies, and the central pair ends at a short
distance behind them. No cytoplasmic channel is observed
between the flagellum and the cytoplasm (Fig. 6C and D).
The flagellar axis is perpendicular to the base of the nucleus
(Fig. 6C and D). SPZ are not inserted within the Sertoli cell
cytoplasm (Figs. 4A, 5A and 5G).
5. Sertoli cells
Sertoli cells have pleomorphic nuclei, and in general
are located at the periphery of the spermatocyst (Figs. 2A,
3A, 3B, 4A and 5A). When evident, nucleoli are eccentric (Fig. 7B). More than a single Sertoli cell comprises
the spermatocyst wall (Fig. 3B). They are supported by a
basement membrane that limits the testicular lobules, separating them from the interstitial compartment (Fig. 7A), and
adopting the shape of the available space (Figs. 2A, 3A,
4A and 7B). Sertoli cell cytoplasm is difficult to distinguish
and does not stain differentially from that of germ cells as is
observed using light microscopy. With the increased resolution afforded by electron microscopy, adjacent Sertoli cells,
from the same or different spermatocysts, interdigitate in a
complex manner and form the borders of the spermatocysts
(Figs. 3B and 7A). Desmosomes and tight junctions join
Sertoli cells laterally (Fig. 7A and B). Their cytoplasm is
rich in intermediate filaments. Ribosomes grouped forming polyribosomes, rough (RER) (Fig. 2B) and smooth
endoplasmic reticulum are poorly developed. Mitochondria possess electron-dense granules and few cristae, and a
Golgi apparatus is prominent (Fig. 7B). Conspicuous pores
appear in the nuclear membrane (Fig. 7A and B). Many
pinocytotic vesicles occur along the basal and lateral surfaces of the cell. Sertoli cells phagocyte residual bodies
and degenerating cells. These phagocytosed cells appear as
large vacuoles within the cytoplasm of Sertoli cell, along
with prominent lysosomes (Fig. 7B).
6. Efferent ducts
In both type of males, the efferent ducts possess a simple epithelium. Their cells vary from squamous to cubical
according to reproductive condition (Fig. 8A). Nuclei are
oval and contain one nucleolus. Mitochondria, numerous
microfilaments, polyribosomes and pinocytotic vesicles are
noticed within the cytoplasm (Fig. 8B and C). Junctions between these epithelial cells consist of desmosomes and tight
129
junctions near the lumen (Fig. 8B and C). Myoid cells and
collagen fibers are located below the basement membrane
upon which the duct epithelium rests (Fig. 8B).
7. Discussion and conclusions
Since S. marmoratus is a diandric species, both primary
and secondary males were collected and examined in this
study. The testis observed in secondary males differs from
that of primary males in that the testicular tissue resides
within the former ovarian lamellae, which project into a lumen, the former ovarian lumen (Lo Nostro and Guerrero,
1996). However, no differences were observed in the gross
morphology of the germinal compartments and spermatogenesis between the two types of males. Therefore, the different stages of spermatogenesis were characterized using
either type of male.
Sertoli cell processes completely envelop SPGA, isolating
them from contact with both the basement membrane and
the lobule lumen. This isolation persists throughout spermatogenesis even when isogenic clones of germ cells develop synchronously within spermatocysts, until the release
of sperm into the lobule lumen (spermiation). Intercellular
bridges represent an incomplete type of cell division that has
only been observed in germ cells committed to form sperm,
i.e. germ cells enclosed within spermatocysts (Clérot, 1971).
Beginning with SPGB through the time when spermatids
cast off their excess of cytoplasm as residual bodies, intercellular bridges connect the germ cells. As in SPGB, the
presence of these bridges has often been reported between
spermatocytes whether they occur in cysts (Clérot, 1971;
Russo and Pisanó, 1973; Billard, 1984) or in mammals
where spermatocysts do not occur (Dym and Fawcett, 1971).
Billard showed that one cell may have several cytoplasmic
bridges in the guppy, as described in the present study for S.
marmoratus. This would suggest that the cell retains these
links with sister-cells over several divisions. Thus, all the
cells in a spermatocyst may remain in contact with each
other by these bridges, as proposed by Clérot (1971). It is
believed that the exchange of molecules between germ cells
via their intercellular bridges is responsible for synchronous
development (Gilbert, 2000).
The so-called nuage, a germ cell specific organelle, is
either free or associated with mitochondria (Kalt, 1973;
Hamaguchi, 1993). It is typically found only in SPGA,
as in the medaka, Oryzias latipes (Hamaguchi, 1993). In
the guppy, P. reticulata, nuage has been associated with
䉳
Fig. 7. Sertoli cells. TEM micrographs (A) and (B). (A) Cytoplasmatic processes of Sertoli cells interdigitating in a complex manner. Junctions between
them are evident. (B) Sertoli cells containing a phagocyted cell that appear as a large vacuole within the cytoplasm. bm, basement membrane; d,
desmosomes; G: Golgi apparatus; it: interstitial tissue; m: mitochondria; n: nucleolus; N: nucleus; np: nuclear pores; ps: Sertoli cell process; SPGA:
spermatogonia A; t: tight junctions; v: vacuole. (A) and (B) 7000×. Fig. 8. Efferent ducts. (A) Light microscope photograph. Cross-section of efferent ducts
from a mature male. (B) and (C) TEM micrographs. (B) Junctions between epithelial cells. (C) Desmosomes and tight junctions at higher magnification.
bm: basement membrane; cf: collagen fibers; d: desmosome; ec: epithelial cells; ed: efferent duct; m: mitochondria; if: intermediate filaments; my: myoid
cell; p: polyribosomes; pv: pinocytotic vesicle; rb: residual body; SPZ: spermatozoa; t: tight junctions. (A) 200×; (B) 12,000×; (C) 30,000×.
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F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
mitochondria in SPGA, SPGB and metaphase spermatocytes (SPC) during the first meiotic division, and it may
have resulted from a cytoplasm–nuclear exchange (Billard,
1984). In S. marmoratus, nuage and the aggregates that it
forms with mitochondria occur mainly in spermatogonia but
the presence of nuages is observed up to the SPD stage. In
the SPD of the common snook Centropomus undecimalis,
nuages have also been observed (Grier, unpublished data).
The precise role of nuage is not known (Hamaguchi, 1993),
but its presence is presumably associated with synthetic
activities of these cell types.
Another organelle that is characteristic of germ cells are
annulate lamellae, which have been described in spermatogonia of Pimephales notatus (Schjeide et al., 1972), S. gairdnieri (Van den Hurk et al., 1982), and P. reticulata (Billard,
1984), and in primary spermatocytes of Lycodontis afer
(Mattei et al., 1967). Contrarily, annulate lamellae have not
been found in S. marmoratus in any spermatogenic stage.
In S. marmoratus, the larger nuclear diameter is registered
in the SPCI stage. This is coincident with observations performed in L. aurata by Bruslé and Bruslé (1978). SPCII are
scarce and difficult to observe, probably due to their short
lifetime. These cells would divide rapidly to become SPD.
Contrarily, SPCII are numerous in M. barberi (Pecio and
Rafinski, 1999).
The presence of a prominent Golgi apparatus and polyribosomes in the SPD is presumably associated with a high
level of synthetic activity contrarily to oogenesis, in which
the most important synthetic activity is detected in the
prophase I.
A primitive type of spermatozoon, named aquasperm
(Jamieson, 1991), is observed in S. marmoratus. The sperm
nucleus is rounded or spherical, and lacks an acrosome,
which coincides with the presence of a micropyle in the
oocyte (Ravaglia, 2000). A variant of the anacrosomal
teleostean aquasperm is the biflagellated sperm, as observed
in S. marmoratus. Biflagellated sperm have been observed
in only a few other teleosts such as the Siluriformes:
Ictaluridae, Ictalurus punctatus (Poirier and Nicholson,
1982), Malapteruridae, Malapterurus sp. (Mattei, 1988),
and Pimelodidae Rhamdia sapo (Jamieson, 1991). Biflagellated sperm also occur in the Perciformes: Apogonidae
Paranocheilus sp. (Mattei and Mattei, 1984), Gobioesociformes: L. lepadogaster (Mattei and Mattei, 1978a,b),
Batrachoidiformes: Porichthys notatus (Stanley, 1965), Opsanus tau (Casas et al., 1981), and in the Myctophiformes:
Lampanyctus sp. (Mattei and Mattei, 1976).
In his classification of teleostean sperm, Mattei (1991) did
not include the biflagellated sperm within the type I or II.
Briefly, type I aquasperm typically have a small rounded or
ovoid nucleus and no acrosome. Two centrioles are present
distal to the nucleus. The proximal centriole is often at right
angle to the distal one, which forms the basal body of the
flagellum; one or both of the centrioles may or may not be
located in a basal fossa of the nucleus if, as frequently occurs, a fossa is present. In the type II aquasperm, the rotation
of the flagellar axis in relation to the nucleus does not take
place. The flagellum remains parallel to the base of the nucleus. Although a depression is usually found at this point,
the centrioles remain outside it. However, although S. marmoratus possesses a biflagellated sperm, this could belong
to a “type I” designation regarding the flagella position with
regard to the nucleus and the shape of the head.
Many examinations on the cytology of the teleost testis
described attachment devices between adjacent Sertoli cells.
These ranged from complex interdigitations, to desmosomes and tight junctions. During spermatogenesis, there
is a considerable increase in the volume of spermatocysts
as germ cells divide and undergo maturation. Since Sertoli cells form the walls of the spermatocysts, they must
accommodate these changes in dimension. As spermatocysts enlarge, the complex interdigitations between Sertoli
cells presumably passively unfold to allow the cells to be
stretched. To maintain the integrity of the spermatocyst
wall, however, it is incumbent upon Sertoli cells to form
firmer functional contacts. This is accomplished by the
development of inter-Sertoli desmosomes and tight junctions. Furthermore, the presence of tight junctions has been
shown in some teleost species to result in the formation
of a blood–testis barrier (Pudney, 1993). In amniota testis,
Sertoli–Sertoli tight junctions have to be intermittently dismantled to allow migration of maturing germ cells from
the basal to adluminal compartment. This does not occur in
the teleost testis since all germ cells develop as an isogenic
clone within the spermatocyst lumen (Pudney, 1993).
In S. marmoratus, more than a single Sertoli cell comprises the border of a spermatocyst and they are joined
laterally by desmosomes and tight junctions. They would
probably have a blood–testis barrier function, at least in
mature testes. All of these Sertoli cell characteristics define
the testicular germinal epithelium as they fulfill the criteria
used to define an epithelium (Grier and Lo Nostro, 2000). In
addition to their importance as part of the germinal epithelium, Sertoli cells function as phagocytes, phagocytizing
residual bodies and degenerating germ cells including residual sperm (Grier, 1993). In S. marmoratus testes, Sertoli
cell involvement in all of these processes were observed.
Sertoli cells persist in the lobules after spermiation, and
between reproductive cycles (Lo Nostro et al., 2003).
During the process of spermiation, the hypertrophied
Sertoli cells may be transformed into efferent duct cells in
the atherinomorph teleosts whose testes structure has been
examined (Pandy, 1969; Van den Hurk et al., 1974; Grier
et al., 1980; Grier, 1981), or they may degenerate (Billard,
1986). According to Grier (1993), the efferent ducts in
Poecilidae are a region of cell turnover and also involve
Sertoli cell hypertrophy and transformation into columnar
efferent duct cells. Sertoli cells have been reported to degenerate at the end of spermatogenesis in the mosquito fish,
G. affinis (Melden, 1950), and in the guppy, P. reticulata
(Billard, 1970a). However, the evidence for these reports
requires further investigation. In S. marmoratus, the efferent
F.L. Lo Nostro et al. / Tissue & Cell 35 (2003) 121–132
duct locations was already mentioned by Lo Nostro and
Guerrero (1996). Epithelial cells possess similar cytoplasmatic characteristics to Sertoli cells and the presence of
junctions between epithelial cells, consisting of desmosomes and tight junctions (zonula occludens) near the
lumen, would indicate a possible testis barrier function or
improve the spermatocysts barrier that already exists.
Acknowledgements
The authors would thank I. Farias and L. Zimerman for the
electron microscopy assistance. We are indebted to A. Solari
and J. Burns for their valuable suggestions. F. Antonelli and
I. Quagio-Grassiotto are also acknowledged for the donation
of two figures. This work was supported from the following
grants: UBA (TW41) and CONICET (PIP 0539/98).
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