Gonadal development and sex differentiation in the cichlid fish

JOURNAL OF MORPHOLOGY 264:191–210 (2005)
Gonadal Development and Sex Differentiation in the
Cichlid Fish Cichlasoma dimerus (Teleostei,
Perciformes): A Light- and Electron-Microscopic Study
Fernando J. Meijide,* Fabiana L. Lo Nostro, and Graciela A. Guerrero
Laboratorio de Embriologı́a Animal, Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria, C1428EHA,
Buenos Aires, Argentina
ABSTRACT Although the overall pattern and timing of
gonadal sex differentiation have been established in a
considerable number of teleosts, the ultrastructure of
early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile
stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels.
This freshwater species adapts easily to captivity and
spawns with high frequency during 8 months of the year,
providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 ⫾ 1°C and a 12:12 hour photoperiod. Gonadal
development was documented from 14 –100 days postfertilization, covering the period of histologically discernible
sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a
differentiated gonochorist, in which an indifferent gonad
develops directly into a testis or ovary. On day 14, the
gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost
fishes. The earliest signs of differentiation, detected from
day 42 onward, include the onset of meiotic activity in
newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity
is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In
contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100,
the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this
time of testicular development is spermatocyte II. J. Morphol. 264:191–210, 2005. © 2005 Wiley-Liss, Inc.
Timmermans, 1985; Hamaguchi, 1992; Foyle, 1993;
Patiño and Takashima, 1995; Patiño et al., 1996;
Strüssmann et al., 1996; Grandi and Colombo, 1997;
Lin et al., 1997; Nakamura et al., 1998; Devlin and
Nagahama, 2002), detailed histological descriptions
of early gonadal development and of the period of
sex differentiation are limited to a few gonochoristic
species.
Careful histological observations of the process of
morphogenesis of the gonads are of primary importance for a precise understanding of the mechanisms
of gonadal sex differentiation (Nakamura et al.,
1998) and could help to develop efficient methods of
directing sexual development in aquaculture species, since they can provide a guide for determining
the hormone-sensitive period in cases of sex manipulation by exogenous steroid treatment (see Hunter
and Donaldson, 1983; Foyle, 1993; Strüssmann et
al., 1996).
The South American cichlid fish Cichlasoma dimerus, a perciform teleost (Nelson, 1994), is common
in quiet shallow waters in the Paraguay and most of
the Paraná river basins (Kullander, 1983). This
freshwater species adapts easily to captivity and
shows notable reproductive features such as a high
spawning frequency (about every 30 days during 8
months of the year, under laboratory conditions) and
acceptable survival rates, providing an appropriate
model for developmental studies. Cichlasoma dimerus lives in pairs that defend a territory. The
females lay their eggs on a cleaned substrate and
KEY WORDS: gonadal development; sex differentiation;
histology; ultrastructure; Cichlidae; Teleostei
Contract grant sponsor: University of Buenos Aires; Contract grant
number: EX 157; Contract grant sponsor: CONICET; Contract grant
number: PIP 4558.
Although a number of articles and comprehensive
reviews on early gonadogenesis and differentiation
of germ cells in teleost fishes are available (Satoh
and Egami, 1972; Satoh, 1974; Bruslé and Bruslé,
1978; Mezhnin, 1978; Yoshikawa and Oguri, 1978;
Ryazantseva and Sakum, 1980; Timmermans and
Taverne, 1983; Colombo et al., 1984; Parmentier and
© 2005 WILEY-LISS, INC.
*Correspondence to: Lic. Fernando J. Meijide, Laboratorio de Embriologı́a Animal, Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Pabellón II, Ciudad Universitaria, C1428EHA, Buenos
Aires, Argentina. E-mail: [email protected]
Published online 23 March 2005 in
Wiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/jmor.10329
192
F.J. MEIJIDE ET AL.
one or more pits are dug on the bottom, to which the
offspring are transferred by mouth after they hatch.
Both parents fan the eggs and the young fry, and
guard their young when these, in a school, become
free-swimming (Staeck and Linke, 1995). Meijide
and Guerrero (2000) described the embryonic and
larval development of this substrate-brooding species under laboratory conditions.
According to Yamamoto (1969), two patterns of
gonadal development exist among teleosts: differentiated gonochorists, in which an undifferentiated
gonad develops directly into a testis or ovary, and
undifferentiated gonochorists, in which the indifferent gonad first develops into an ovary-like gonad
and then about one-half of the individuals become
males and the other half females. Spontaneous intersexes among gonochorists occur mostly in the
undifferentiated species, while in the differentiated
ones the occurrence of intersex is rarely or never
seen (Yamamoto, 1969).
The aim of the present study was to describe the
gonadal development of Cichlasoma dimerus, both
at the light- and electron-microscopic levels, with
special emphasis on the period of gonadal sex differentiation.
MATERIALS AND METHODS
Adult Cichlasoma dimerus used in this study were captured in
Esteros del Riachuelo, Corrientes, Argentina (27° 25⬘ S, 58° 15⬘
W) by local fishermen and transferred to the laboratory. Single
pairs were kept in 45-L aquaria, at 26.5 ⫾ 1°C and a 12:12 h
photoperiod. Laboratory aquaria were well aerated and provided
with a layer of gravel and smooth stones for egg deposition on the
bottom. Fishes were normally fed with pelleted commercial food
and Tubifex worms.
Eggs and larvae were obtained from several spawnings of six
pairs. On the 14th day after spawning, each lot of offspring was
isolated in a 20-L aquarium, where their development was followed until the juvenile stage was reached. A part of the brood
was sampled periodically from day 14 to day 100 postfertilization
(6 ⫾ 0.5 mm to 22 ⫾ 3 mm TL), covering the period of histologically discernible sex differentiation. Temperature and photoperiod conditions were the same as those of the adults. Fry were
initially fed with freshly hatched nauplii of Artemia salina and
later fed finely ground, dried flake food.
Gonadal tissues, either within the abdominal cavity or removed
from fishes immediately after sacrifice, were processed according
to standard techniques for light- and electron-microscopy. For
light microscopy, samples were fixed in Bouin’s liquid, gradually
dehydrated, and embedded in paraffin. Sections, 8 ␮m thick, were
stained with hematoxylin-eosin. The slides were examined and
photographed with a Nikon Microphot FX. For transmission electron microscopy (TEM), samples were fixed in 3% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.4) for 4 – 8 h. The tissue was then
rinsed in 0.1 M phosphate buffer and postfixed in 1% osmium
tetroxide in 0.1 M phosphate buffer (pH 7.4) for 2 h. Tissue was
subsequently rinsed in distilled water, dehydrated in a graded
alcohol series and acetone, and embedded in Spurr resin. Ultrathin sections were made with a Sorvall MT2-B ultramicrotome,
stained with aqueous uranyl acetate and lead citrate, and examined with a Zeiss EM 109 T. Semithin sections, stained with
Toluidine blue, were used for orientation.
RESULTS
With respect to gonadal sex differentiation,
Cichlasoma dimerus is classified as a differentiated
gonochorist, in which ovaries and testes develop directly from undifferentiated gonadal tissue.
Undifferentiated Gonads
On day 14 postfertilization (day 12 posthatching),
paired gonadal primordia are present in the posterior region of the abdominal cavity, immediately
below the kidney, and are suspended from the dorsal
peritoneal wall by short mesenteries (Fig. 1A,B).
Parasagittal sections show a cord-like gonadal tissue in which germ cells form discontinuous clusters,
interspersed with segments formed solely by somatic cells (Fig. 1B). Cross sections through germ
cell clusters reveal that the gonadal primordium
consists of large, round to oval germ cells surrounded by enveloping somatic cells (Fig. 1C). Sexually undifferentiated germ cells contain large, central euchromatic nuclei with a prominent
eccentrically located nucleoli (Fig. 1A,D). The cytoplasm contains cup-shaped mitochondria with tubular cristae (Fig. 1C,E,F), abundant smooth endoplasmic reticulum (Fig. 1C,E), and electron-dense
substance or nuage, a germ cell-specific organelle
(Fig. 1D). Occasional mitotic activity occurs in these
cells (Fig. 1C). Somatic cells are of various shapes.
They have heterochromatic nuclei and relatively little cytoplasm that contains few organelles. Two
types of somatic cells are present: coelomic epithelial
cells, located at the periphery of the gonadal anlage,
and supporting cells, derived from the former and
located in an interior position, where they become
associated with germ cells. The epithelial basement
membrane is present between these two cell types
(Fig. 1C). Neighboring epithelial cells are connected
by desmosomes and tight junctions (Fig. 1G). Support cells tend to envelope germ cells with their
cytoplasmic processes, so that only in rare cases do
germ cells contact each other (Fig. 1C). Occasionally,
germ cells may directly contact the epithelial basement membrane.
By day 25, gonads have become suspended from
the ventral edge of the forming swim bladder by
short mesenteries (Fig. 2A). Germ cell mitotic figures are now more frequent in the undifferentiated
gonad (Fig. 2B,C). Concomitant with mitotic divisions, the number of germ cells is augmented. As a
result, the gonads increase in size and several germ
cells (not one or two as in earlier stages) appear in
cross sections (Fig. 2B). In parasagittal sections,
germ cells acquire continuity and the gonads approach each other at the caudal tip as they converge
towards the urogenital papilla (Fig. 2C). Germ cells
possess large, round to oval nuclei with one, or
rarely two, nucleoli (Fig. 2C,D). Granular and fibrillar components of nucleoli are evident in some germ
Fig. 1. Gonadal development in Cichlasoma dimerus on day 14 postfertilization (6 ⫾ 0.5 mm TL). A,B: LM. C–G: TEM. A: Cross
section of gonadal primordia suspended from the dorsal peritoneal wall by short mesenteries at both sides of the dorsal mesentery.
Scale bar ⫽ 10 ␮m. B: Sagittal section of the gonadal anlage below the kidney. Note the discontinuity of germ cell clusters, which
confers on the gonadal primordium the appearance of a string of beads. Scale bar ⫽ 20 ␮m. C: Cross section of one of the gonadal
anlagen showing germ cells and somatic (epithelial and support) cells. Note that germ cells are wrapped by the cytoplasm of support
cells. A basement membrane supports epithelial cells. Scale bar ⫽ 2 ␮m. D: Primordial germ cell. A large nucleolus is present within
the nucleoplasm. Scale bar ⫽ 2 ␮m. E: Germ cell organelles at higher magnification. Note the presence of a vacuole within the matrix
of some mitochondria. Scale bar ⫽ 0.5 ␮m. F: Detail of a cup-shaped mitochondrion, that appears ring-shaped since only the lip of the
cup has been sectioned. Its cristae are distinctly tubular. Scale bar ⫽ 0.2 ␮m. G: Connections between adjacent epithelial cells. Scale
bar ⫽ 0.2 ␮m. arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; cm, cup-shaped mitochondria; cp, cytoplasmic
process of support cell; d, desmosome; E, epithelial cell; G, germ cell; K, kidney; M, gonadal mesentery; m, mitotic figure; n, nuage; nu,
nucleolus; SU, support cell; tj, tight junction.
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F.J. MEIJIDE ET AL.
Fig. 2. Gonadal development in Cichlasoma dimerus 25 days after fertilization (8 ⫾ 0.7 mm TL). A–C: LM. D,E: TEM. A: Cross
section of the abdominal cavity showing the indifferent gonads below the caudal tip of the swim bladder. Scale bar ⫽ 50 ␮m. B: Gonadal
primordia at higher magnification. Scale bar ⫽ 10 ␮m. C: Sagittal section of the gonads. Note the continuity of germ cells along the
gonad. Scale bar ⫽ 20 ␮m. D: Undifferentiated germ cell showing two nucleoli within the nucleoplasm. Note the presence of the
diplosome near the nucleus. Scale bar ⫽ 1 ␮m. E: Peripheral area of the gonad. Scale bar ⫽ 1 ␮m. arrow, smooth endoplasmic
reticulum; di, diplosome; E, epithelial cell; G, germ cell; K, kidney; M, gonadal mesentery; m, mitotic figure; ml, mitochondria with
lamellar cristae; n, nuage; pf, pars fibrosa; pg, pars granulosa; pv, pinocytotic vesicles; sb, swim bladder; SU, support cell.
cells. Within the cytoplasm, spherical or elongated
mitochondria with lamellar cristae are present in
association with nuage. Centrioles are found in
pairs, called diplosomes, next to the nucleus (Fig.
2D). Epithelial cells show pinocytotic activity (Fig.
2E).
Gonads remain undifferentiated by day 38 (Fig.
3A). Many spherical or oval mitochondria are located around the nuclei of germ cells (Fig. 3B). Nuage are prominent and tend to be located near the
nucleus in association with mitochondria (Fig.
3A,B). Smooth endoplasmic reticulum and annulate
lamellae, forming amphitheater-like structures, are
conspicuous components of the cytoplasm (Fig.
3B,C). Multilamellar bodies are also present. The
flattened, peripheral epithelial cells surrounding the
gonad are connected by tight junctions and desmosomes (Fig. 3D). The inner support cells contain
triangular to elongated nuclei. Cytoplasmic extensions of these cells incompletely invest germ cells
that have recently divided but have not completed
cytokinesis (Fig. 3A,E). Desmosomes occasionally
occur between neighboring support cells. Between
epithelial and support cells, a thin space correspond-
Fig. 3. Gonadal development in Cichlasoma dimerus on day 38
postfertilization (12.5 ⫾ 1 mm TL). TEM. A: Cross section of the
undifferentiated gonad. Separation of the germinal and interstitial
compartments of the gonad is evident. The germinal compartment
contains germ cells and support cells that are derived from epithelial
cells. The interstitial compartment is a thin peripheral space that
contains collagen fibrils and very few interstitial cells. Scale bar ⫽ 3
␮m. B: Indifferent germ cell. Scale bar ⫽ 1 ␮m. C: Area from
B, showing annulate lamellae disposed as an amphitheater. The
outer pores appear in side view while the inner pores are primarily
en face. Scale bar ⫽ 0.3 ␮m. D: Somatic (epithelial and support) cells
at higher magnification. Scale bar ⫽ 0.5 ␮m. E: Area from A,
showing the sinuous cytoplasmic process of a support cell. Note the
presence of four plasma membranes. The inner membranes define a
support cell whose process is ⬃0.05 ␮m across. The outer membranes are those of adjacent germ cells resulting from a recent
mitotic division. Germ cells contact each other at the point marked
with opposite arrowheads. Note also the presence of a “double”
basement membrane (arrowheads) corresponding to folds of the
epithelial basement membrane. The outer basement membrane
supports peripheral epithelial cells, while the inner basement membrane subtends internal support cells that are associated with germ
cells. This basement membrane separates the germinal and interstitial compartments of the gonad. Scale bar ⫽ 0.5 ␮m. al, annulate
lamellae; arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; asterisk, collagen fibrils; cp, cytoplasmic process of
support cell; E, epithelial cell; G, germ cell; I, interstitial cell; ml,
mitochondria with lamellar cristae; n, nuage; nu, nucleolus; pc, pore
complex; SU, support cell; tj, tight junction.
GONADAL DEVELOPMENT IN C. DIMERUS
Figure 3
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F.J. MEIJIDE ET AL.
ing to the interstitial compartment of the developing
gonad becomes evident. This space contains a few
interstitial cells and collagen fibrils, and is delimited
by an inconspicuous basement membrane (Fig.
3A,E). Germ cells directly contact the basement
membrane in the areas where support cell processes
do not completely surround them (Fig. 3E).
Ovarian Differentiation
Ovarian differentiation precedes testicular differentiation, as is usual in teleosts. By day 42, many
oogonia have entered meiotic prophase, becoming
primary growth oocytes, which in addition to moderate oogonial mitotic proliferation and somatic
growth results in an enlargement of the presumptive ovary. For the first time, blood capillaries occur
in the dorsal region of the gonad (Fig. 4A). Oogonia
contain a prominent euchromatic nucleus with a
large nucleolus, mitochondria with lamellar cristae,
conspicuous nuage, and abundant smooth endoplasmic reticulum (Fig. 4B). In meiotic oocytes, the nucleus becomes more electron-lucent than that of oogonia and the number of mitochondria increases,
although they seem to be smaller. Nuage and
smooth reticulum are less conspicuous (Fig. 4C).
Synaptonemal complexes are evident within nuclei
of oocytes at the pachytene stage of the first meiotic
prophase (Fig. 4C,D). Support or prefollicle cells contain triangular nuclei that are less chromatic than
those of the other somatic cell types (Fig. 4B,E,F).
Cytoplasmic processes of prefollicle cells enclose oogonia or early oocytes, marking the onset of folliculogenesis (Fig. 4B). Epithelial cells possess an elongated nucleus (Fig. 4B,E). They are joined by tight
junctions and desmosomes (Fig. 4E), and display
conspicuous pinocytotic activity (Fig. 4F,G). A third
type of somatic cell, the prethecal (interstitial) cell,
is observed lying between epithelial and prefollicle
cells. These cells, as well as the collagen fibrils that
surround them, are derived from the stromal connective tissue and not from the coelomic epithelium.
Therefore, they lie in a tissue compartment delimited by an inconspicuous basement membrane, i.e.,
the interstitial or stromal compartment of the ovary,
separating them from the forming follicles (Fig.
4B,E,H). A few mitochondria with tubular cristae
occur in all types of somatic cells (Fig. 4F,G,H).
Active germ cell mitosis and meiosis are observed
by day 50. Likewise, somatic reorganization of the
presumptive ovary begins. Gonads present a triangular or kidney-shape when observed in cross sections (Fig. 5A,B). Clusters of somatic cells are noticeable along the dorsal and ventral edges of the
ovary (Fig. 5B). By day 58, somatic cells from these
areas proliferate and form appendix-like structures
that initially protrude from the gonad (Fig. 5C).
Subsequently, they face each other and finally fuse
to form the ovarian lumen (Fig. 5D). The ovarian
cavity is completely formed by day 65 and numerous
basophilic oocytes at the diplotene stage of meiosis
are present by this time of development. The cytoplasm of these cells becomes highly basophilic and
thin chromosome threads that form a random reticulum occur within the nucleoplasm (Fig. 5D).
On day 80, the ovary consists mainly of follicles
that contain characteristically large, perinucleolar
oocytes with a basophilic cytoplasm (Fig. 6A). These
cells are arrested in the diplotene stage of the first
meiotic prophase and are characterized by the presence of multiple round nucleoli in the peripheral
nucleoplasm (Fig. 6A,B). Masses of an electrondense substance, or nuage, are observed outside the
nuclear envelope, associated with mitochondria
(Fig. 6B,E). As folliculogenesis progresses, the typical ovarian follicle organization exists, i.e., each
perinucleolar oocyte becomes surrounded by a continuous layer of follicle cells that flatten out over its
surface and are separated from the stroma by a
basement membrane (Fig. 6C). Interstitial connective tissue is detected in the periphery of the ovary,
and in the angular interstices between three or more
follicles in the inner region of the gonad (Fig. 6A).
Thecal cells derived from this stromal tissue start to
surround the follicles (Fig. 6C), although a complete
layer of thecal cells is observed only around the more
advanced follicles. Younger oocytes in the pachytene
stage of meiosis are found along the luminal edge,
bordering the ovarian lumen (Fig. 6A,D). Nuage
forming aggregations with mitochondria, smooth endoplasmic reticulum, and Golgi complexes occur in
their cytoplasm. Prefollicle cells located between
pachytene oocytes are joined by desmosomal junctions (Fig. 6E).
By day 100 the ovary contains many developing
follicles, as well as younger oocytes at early stages of
meiotic prophase and only a few oogonia (Fig. 7A,B).
Oogonia (Fig. 7C) and oocytes in early meiotic
prophase (Fig. 7B) reside in the luminal edge of the
developing ovarian lamellae, forming, along with
the epithelial and prefollicle cells, the so-called “germinal epithelium.” Follicles, derived from the germinal epithelium, are composed of a perinucleolar
oocyte and surrounding follicle cells (Fig. 7B). Segregation of the fibrillar and granular components of
nucleoli becomes evident in perinucleolar oocytes.
Nuage are observed outside the nuclear envelope
(Fig. 7D). A well-developed Golgi apparatus and
many mitochondria occur in the cytoplasm (Fig.
7D,E). Formation of microvilli, by folding of the oolemma, has begun at the periphery of these cells.
Microvilli extend from the surface of the oocyte toward the overlying follicle cells (Fig. 7C,F). Two or
three squamous follicle cells (derived from epithelial
cells) surround each perinucleolar oocyte. Thecal
cells (derived from stromal or interstitial connective
tissue cells) form a thin continuous layer around
follicle cells (Fig. 7B,C). Both follicle and thecal cells
have little cytoplasm, containing only a few organelles. The area between follicles, in which thecal
Fig. 4. Onset of ovarian differentiation in Cichlasoma dimerus 42 days after fertilization (15 ⫾ 1.2 mm TL). A: LM. B–H: TEM.
A: Cross section of differentiating ovaries. Note the presence of many early meiotic oocytes with thick strands of condensed chromatin
inside the nucleus. Scale bar ⫽ 30 ␮m. B: Oogonium at the onset of folliculogenesis is encompassed by cytoplasmic processes of a
prefollicle cell. Scale bar ⫽ 2 ␮m. C: Primary growth oocyte in early pachytene stage of the first meiotic prophase. Scale bar ⫽ 3 ␮m.
D: Longitudinal section of a synaptonemal complex inside the nucleus of a pachytene oocyte. The synaptonemal complex appears as
a tripartite structure formed by a thin central component and two thick lateral components that insert onto the nuclear envelope. Scale
bar ⫽ 0.5 ␮m. E: Spatial relations of somatic cells, collagen fibrils, and the basement membrane. Scale bar ⫽ 2 ␮m. F: Detail of a
prefollicle cell associated with pachytene oocytes. Scale bar ⫽ 1 ␮m. G: Area from F, showing the cytoplasm of an epithelial cell
exhibiting mitochondria with tubular cristae and pinocytotic vesicles. Scale bar ⫽ 0.5 ␮m. H: Prethecal cell surrounded by collagen
fibrils. Scale bar ⫽ 1 ␮m. arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; asterisk, collagen fibrils in stromal
compartment; c, blood capillary; cc, central component; cp, cytoplasmic process of prefollicle cell; E, epithelial cell; lc, lateral
component; M, mesovary; mb, multilamellar body; ml, mitochondria with lamellar cristae; mt, mitochondria with tubular cristae; n,
nuage; ne, nuclear envelope; nu, nucleolus; O, oogonium; PF, prefollicle cell; PO, pachytene oocyte; PT, prethecal cell; pv, pinocytotic
vesicles; sc, synaptonemal complex; tj, tight junction.
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F.J. MEIJIDE ET AL.
Fig. 5. Somatic reorganization and formation of the ovarian cavity in Cichlasoma dimerus between days 50 and 65 postfertilization
(15 ⫾ 1.3 mm to 17.5 ⫾ 1.5 mm TL). LM. A: Cross section of differentiating ovaries showing active germ cell mitosis and meiosis (day
50). Scale bar ⫽ 20 ␮m. B: Ovaries showing clusters of somatic cells (double arrow) from which outgrowths will originate (day 50). Scale
bar ⫽ 20 ␮m. C: Ovaries with appendix-like expansion of somatic cell outgrowths (double arrowhead) (day 58). Scale bar ⫽ 100 ␮m.
D: Fusion of outgrowths and formation of the ovarian lumen (day 65). Scale bar ⫽ 50 ␮m. dd, digestive duct; DO, diplotene oocyte; M,
mesovary; m, mitotic figure; O, oogonium; ol, ovarian lumen; PO, pachytene oocyte.
cells, blood capillaries, and other interstitial elements reside, is known as the stromal extravascular
space (Fig. 7B). A prominent basement membrane
separates follicle from thecal or epithelial cells (Fig.
7C,D,F). A single basement membrane is frequently
present at the point where a follicle is attached to
the germinal epithelium, i.e., the follicle cells of the
follicle and the epithelial cells of the germinal epithelium both share a common basement membrane
(Fig. 7D,F). Pinocytotic activity still occurs in epithelial cells (Fig. 7F).
Testicular Differentiation
In contrast to ovarian development, signs of histological differentiation are not observed in the presumptive testis until day 72.
Undifferentiated gonads on day 65 of development
are recognizable as presumptive testes. They retain
the original pear-like shape of indifferent gonads
and are much smaller than ovaries of the same
developmental stage (Fig. 8A, cf. Fig. 5D). The overall appearance of spermatogonia is similar to that of
undifferentiated germ cells at 38 days. The most
characteristic features are the presence of a prominent nucleus with one or two nucleoli, a conspicuous
smooth endoplasmic reticulum, and abundant mitochondria that may be associated with nuage (Fig.
8B,D). Annulate lamellae and multilamellar bodies
are also present (Fig. 8E,F). Support or presumptive
Sertoli cells have triangular to elongated nuclei and
invest spermatogonia with cytoplasmic extensions
(Fig. 8B,D). Desmosomes and tight junctions join
neighboring Sertoli cells. Epithelial cells show pino-
GONADAL DEVELOPMENT IN C. DIMERUS
199
Fig. 6. Ovarian development in Cichlasoma dimerus 80 days after fertilization (19 ⫾ 2 mm TL). A: LM. B–E: TEM. A: Cross section
of the ovary. Scale bar ⫽ 50 ␮m. B: Detail of an early perinucleolar oocyte. Scale bar ⫽ 3 ␮m. C: Area of contact between adjacent
follicles. Note the presence of a basement membrane enclosing each follicle. A thecal cell invests one of the follicles. Scale bar ⫽ 1 ␮m.
D: Nucleus of a pachytene oocyte. Scale bar ⫽ 1 ␮m. E: Prefollicle cell between two adjacent pachytene oocytes. Scale bar ⫽ 1 ␮m.
arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; c, blood capillary; d, desmosome; DO, diplotene oocyte; E,
epithelial cell; F, follicle cell; ga, Golgi apparatus; I, interstitial or stromal tissue; M, mesovary; ml, mitochondria with lamellar cristae;
n, nuage; ne, nuclear envelope; nu, nucleolus; ol, ovarian lumen; pc, pore complex; PF, prefollicle cell, PO, pachytene oocyte; sc,
synaptonemal complex; T, thecal cell.
cytotic activity and are connected by tight junctions
(Fig. 8F). A thin basement membrane, as well as
collagen fibrils, occur between Sertoli and epithelial
cells (Fig. 8D,F). The mesorchium is composed of a
double layer of epithelial cells that show many pinocytotic vesicles. Many collagen fibrils lie between
epithelial cells (Fig. 8B,C).
By day 72, mitotic proliferation of spermatogonia
is occurring and blood vessels become evident in the
dorsal region of the testis. Some spermatogonia A
undergo mitotic divisions with incomplete cytokine-
sis, becoming spermatogonia B (Fig. 9A). The onset
of meiosis is soon noticed in the gonads of the larger
specimens (Fig. 9B). Likewise, a central space that
becomes recognizable as the efferent duct anlage is
distinguished in some sections of the testes (Fig.
9C).
On day 80, meiotic activity is marked so that
spermatocytes at early stages of meiotic prophase
become numerous within the differentiating testis
(Fig. 10A). Isogenic spermatocytes, together with
Sertoli cells, form spermatocysts. Within each cyst,
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F.J. MEIJIDE ET AL.
Figure 7
GONADAL DEVELOPMENT IN C. DIMERUS
germ cells are at the same stage of meiosis. Cytoplasmic processes of Sertoli cells form the walls of
spermatocysts. Spermatocytes I at pachytene stage
show synaptonemal complexes inside the nucleus
(Fig. 10B). The cytoplasm contains mitochondria
with lamellar cristae, annulate lamellae, and
smooth endoplasmic reticulum. Swirls of smooth reticulum are detected as well (Fig. 10B,C). A Golgi
apparatus is also present. Sertoli cells are joined
laterally by desmosomes and tight junctions (Fig.
10D).
By day 100, the unrestricted lobular organization
of the testis can be distinguished. This type of testis
is subdivided into lobules, separated from each other
by interstitial tissue (Fig. 11A,B). Inside the lobules,
an epithelioid arrangement of Sertoli and germ cells
occurs. Sertoli cells are supported by a basement
membrane that limits the testicular lobules and separates the germinal and interstitial compartments
of the testis (Fig. 11B). Germ cells are surrounded by
cytoplasmic extensions of Sertoli cells, forming cysts
(Fig. 11A,D), and never contact the basement membrane. Sertoli cells have pleomorphic nuclei and
small, round mitochondria with poorly developed
cristae. Desmosomes and tight junctions join these
cells laterally. Tight junctions established between
adjacent Sertoli cells form the so-called “Sertoli cell
barrier” (Fig. 11B). Spermatogonia A are isolated
cells, individually surrounded by the cytoplasm of a
Sertoli cell (Fig. 11C). Spermatogenesis occurs
Fig. 7. Ovarian development in Cichlasoma dimerus on day
100 postfertilization (22 ⫾ 3 mm TL). A: LM. B–F: TEM. A: Cross
section of the ovary. Scale bar ⫽ 100 ␮m. B: Ovarian germinal
epithelium, formed by the association of epithelial and derived
prefollicle cells with oogonia and early meiotic oocytes. An adjacent follicle that originated from the germinal epithelium is
present. The follicle is composed of the diplotene oocyte and
surrounding follicle cells. Scale bar ⫽ 5 ␮m. C: Detail of an
oogonium and adjacent follicle. Scale bar ⫽ 1 ␮m. D: Area from a
diplotene (perinucleolar) oocyte. Note how the pars fibrosa encloses the pars granulosa within nucleoli. Scale bar ⫽ 5 ␮m.
E: Detail of the Golgi apparatus found in perinucleolar oocytes.
Note how stacked membrane cisternae form a circular structure.
A condensing vacuole (immature secretory granule) with
electron-dense content is observed at the trans face. Scale bar ⫽
0.5 ␮m. F: Formation of microvilli in the periphery of perinucleolar oocytes. Note that the epithelial cell of the germinal epithelium and the follicle cell of an adjacent follicle attached to the
germinal epithelium both share a common basement membrane,
i.e., the basement membrane that supports the germinal epithelium is “one” with that of the follicle at the point of attachment. In
the lower part of the micrograph (around asterisk), the basement
membrane appears to split; one branch subtends the germinal
epithelium and the other branch extends over the surface of the
follicle cell. Scale bar ⫽ 0.5 ␮m. arrow, smooth endoplasmic
reticulum; arrowhead, basement membrane; asterisk, collagen
fibrils; cv, condensing vacuole; DO, diplotene oocyte; E, epithelial
cell of germinal epithelium; es, extravascular space; F, follicle
cell; ga, Golgi apparatus; mi, microvilli; ml, mitochondria with
lamellar cristae; n, nuage; ne, nuclear envelope; nu, nucleolus; O,
oogonium; ol, ovarian lumen; PF, prefollicle cell; pf, pars fibrosa;
pg, pars granulosa; PO, pachytene oocyte; pv, pinocytotic vesicles;
T, thecal cell.
201
within the cysts. Inside each cyst, germ cells at a
similar stage of development are joined by cytoplasmic connections that are sometimes difficult to detect (Fig. 11D). Mitochondria with lamellar cristae,
nuage, and annulate lamellae are present in the
cytoplasm of spermatocytes I (Fig. 11D,E). The latest stage of spermatogenesis detected at this time of
testicular development is spermatocyte II. Compared to spermatocytes I, spermatocytes II have decreased cell and nuclear sizes (Fig. 11A). Clumps of
chromatin are located in the central and peripheral
nucleoplasm. Cell limits of spermatocytes II are difficult to observe and the cytoplasm has few organelles (Fig. 11F). The interstitial or stromal tissue
is barely differentiated. The most common cell type
identified is a fibroblast-like cell. Its nucleus usually
is elongated and quite irregular in shape (Fig. 11G).
Myoid cells are recognized by the presence of microfilaments running parallel to the long axis of the cell
and tend to be surrounded by collagen fibrils (Fig.
11H). Leydig cells cannot be identified ultrastructurally. Blood capillaries, granulocytes, and collagen
fibrils are conspicuous in the periphery of the testis
(Fig. 11D,I).
Figure 12 shows the time scale of the most important events and features during gonadal development and sex differentiation of Cichlasoma dimerus.
DISCUSSION
In teleost fishes, primordial germ cells arise at
extragonadal locations and migrate to the genital
ridges (gonadal anlagen) during specific developmental phases (Braat et al., 1999). The genital ridge
forms as a longitudinal thickening of mesoderm that
protrudes into the coelomic cavity ventral to the
developing kidney and lateral to the dorsal mesentery (Devlin and Nagahama, 2002). Once within the
genital ridge, germ cells increase in number due to
mitotic proliferation (Patiño and Takashima, 1995;
Braat et al., 1999). In several teleost species such as
the medaka (Oryzias latipes), intensive mitotic activity in the germ line is initiated upon arrival of the
germ cells at the gonadal anlagen (Satoh and Egami,
1972; Hamaguchi, 1982). In contrast, the proliferative activity of germ cells is preceded by a period of
quiescence in other species (Timmermans and Taverne, 1983; Parmentier and Timmermans, 1985; Lin
et al., 1997). The initial gonadal primordium (day 14
postfertilization) of Cichlasoma dimerus increases
in length without substantial proliferation of germ
cells. As a result, germ cell clusters become cord-like
and discontinuous, and are interspersed with gonadal segments formed solely by somatic cells (Fig.
1B). The presence of germ cells in cord-like, discontinuous clusters intercalated with germ cell-free
spaces during the early gonadal development has
also been reported in other teleosts (Mezhnin, 1978;
Ryazantseva and Sakum, 1980; Parmentier and
Timmermans, 1985; Strüssmann et al., 1996; Lin et
Fig. 8. Testicular development in Cichlasoma dimerus 65 days after fertilization (17.5 ⫾ 1.5 mm TL). A: LM. B–F: TEM. A: Cross
section of indifferent gonads (presumptive testes). Scale bar ⫽ 50 ␮m. B: Cross section of the testis at higher magnification. Scale bar ⫽
3 ␮m. C: Detail of the mesorchium shown in B. Scale bar ⫽ 1 ␮m. D: Spermatogonium showing two nucleoli within the nucleoplasm
and accompanying Sertoli cells. Scale bar ⫽ 2 ␮m. E: Detail of annulate lamellae located next to the nuclear envelope. Note how pore
complexes of annulate lamellae appear much like those of the nuclear envelope. Scale bar ⫽ 0.5 ␮m. F: Epithelial cells. Scale bar ⫽
1 ␮m. al, annulate lamellae; arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; asterisk, collagen fibrils; cp,
cytoplasmic process; dd, digestive duct; E, epithelial cell; M, mesorchium; mb, multilamellar body; ml, mitochondria with lamellar
cristae; n, nuage; ne, nuclear envelope; nu, nucleolus; pv, pinocytotic vesicles; S, spermatogonium; SE, Sertoli cell; tj, tight junction.
GONADAL DEVELOPMENT IN C. DIMERUS
al., 1997). By day 25, primordial gonads enlarge
mainly by mitotic proliferation of germ cells and
become suspended from the ventral edge of the swim
bladder by short mesenteries (Fig. 2A). In longitudinal sections, germ cells acquire continuity, i.e.,
germ cell-free segments are no longer present (Fig.
2C).
Until day 40, the gonad shows a relatively simple
pattern of cellular associations and no signs of somatic or germ cell sex differentiation can be recognized. As in other teleosts, germ cells of Cichlasoma
dimerus prior to gonadal sex differentiation are
characterized by their relatively large size and a
conspicuous nucleus with finely granular chromatin
and one or two prominent nucleoli. At the ultrastructural level, germ cells can be distinguished by
the presence of extensive smooth endoplasmic reticulum, mitochondria, nuage, and annulate lamellae
(Figs. 1C,D, 2D, 3A,B). Interestingly, the shape of
mitochondria in undifferentiated germ cells seems
to vary during early stages of gonadal development.
These organelles are initially cup-shaped and possess tubular cristae. In addition, some mitochondria
appear vacuolated (Fig. 1E,F). Subsequently, mitochondria change to a round or elongated shape with
typical lamellar cristae (Fig. 3B,D). This modification in the structure of mitochondria may reflect a
process of maturation of these organelles during
early gonadal development. The electron-dense material referred to as nuage may be the most definitive marker of early germ cells in a wide range of
organisms (Eddy, 1975). It appears as discrete,
electron-dense cytoplasmic inclusions that tend to
be associated with mitochondria (Eddy, 1975) or
with annulate lamellae (Kessel, 1983). This material, also referred to as “germinal dense bodies,”
“nucleolus like bodies,” and “intermitochondrial cement,” has been observed consistently, not only in
primordial germ cells but also in oogonia, oocytes,
spermatogonia, spermatocytes, and even spermatids
of fishes (Satoh, 1974; Bruslé and Bruslé, 1978;
Hamaguchi, 1982, 1992; Azevedo, 1984; Billard,
1984; Gevers et al., 1992; Flores and Burns, 1993;
Grandi and Colombo, 1997; Quagio-Grassiotto and
Carvalho, 1999; Grier, 2000; Guimaraes and
Quagio-Grassiotto, 2001; Lo Nostro et al., 2003;
Ravaglia and Maggese, 2003). Nuage appears to be
synthesized in the nucleus (Azevedo, 1984) and contains ribonucleoproteins (Toury et al., 1977) that
may represent ribosomal components that are subsequently assembled in the cytoplasm (Flores and
Burns, 1993). In C. dimerus, nuage is observed close
to the nuclear envelope (Figs. 1D, 4E, 11E) and/or in
association with mitochondria (Figs. 2D, 6E, 8D) or
with annulate lamellae (Fig. 8E). Annulate lamellae, an infrequently found membranous complex,
appear as stacked cisternae with pore complexes
similar to those of the nuclear envelope. Stacks of
lamellae commonly appear in parallel array (Fig.
8E) and sometimes may have a concentric arrange-
203
Fig. 9. Testicular development in Cichlasoma dimerus on day
72 postfertilization (18 ⫾ 1.7 mm TL). LM. A: Cross section of a
testis showing spermatogonial mitotic activity. B: Cross section of
a testis with meiotic figures. C: Testis showing efferent duct
anlage. Scale bars ⫽ 20 ␮m. c, blood capillary; E, epithelial cell;
ed, efferent duct; M, mesorchium; m, mitotic figure; PS,
pachytene spermatocyte; SA, spermatogonium A; SB, spermatogonium B; SE, Sertoli cell; ZS, zygotene spermatocyte.
ment (Fig. 3C). The function of this organelle is not
clear, and how it is formed is likewise disputed.
Lamellae are sometimes continuous with the rough
endoplasmic reticulum, and this has led to the notion that they are derived from this organelle (Wallace and Selman, 1990). On the other hand, the
structural similarities of the nuclear envelope and
annulate lamellae, together with their frequent juxtaposition, has also led to the conclusion that they
arise from the nuclear envelope and represent a
storage form of nuclear membrane (Kessel, 1968,
1983). Annulate lamellae have been observed in rapidly dividing cells and germ cells (Satoh, 1974; Bozzola and Russell, 1992). Contrary to the observations of Billard (1984), who reported the presence of
annulate lamellae only in spermatogonia A of adult
Poecilia reticulata, in the present study this organelle was detected both in spermatogonia (Fig.
8E) and pachytene spermatocytes (Figs. 10B, 11E) of
C. dimerus. The organelle designated as annulate
lamellae by Flores and Burns (1993) in spermatogonia of Xiphophorus maculatus and oogonia of X.
nigrensis may actually correspond to smooth endoplasmic reticulum, since pore complexes characteristic of annulate lamellae were not clearly observed.
Fig. 10. Testicular development in Cichlasoma dimerus 80 days after fertilization (19 ⫾ 2 mm TL). A: LM. B–D: TEM. A: Cross
section of the testis. Scale bar ⫽ 10 ␮m. B: Cyst of spermatocytes at pachytene stage. Note the cytoplasmic continuity between
spermatocytes belonging to the same cyst. Spermatocysts are separated by cytoplasmic extensions of Sertoli cells. Scale bar ⫽ 2 ␮m.
C: Spermatocyte cytoplasm, showing mitochondria with lamellar cristae and swirls of smooth reticulum. Scale bar ⫽ 1 ␮m. D: Sertoli
cells. Scale bar ⫽ 0.5 ␮m. al, annulate lamellae; arrow, smooth endoplasmic reticulum; arrowhead, basement membrane; asterisk,
collagen fibrils; c, blood capillary; cp, cytoplasmic process; d, desmosome; E, epithelial cell; er, smooth endoplasmic reticulum swirl; I,
interstitial cell; M, mesorchium; ml, mitochondria with lamellar cristae; ne, nuclear envelope; pc, pore complex; pf, pars fibrosa; pg,
pars granulosa; PS, pachytene spermatocyte; SA, spermatogonium A; sc, synaptonemal complex; SE, Sertoli cell; SP, spermatocyst; tj,
tight junction.
GONADAL DEVELOPMENT IN C. DIMERUS
205
Fig. 11. Testicular development in Cichlasoma dimerus on day 100 postfertilization (22 ⫾ 3 mm TL). A: LM. B–I: TEM. A: Sagittal
section of a testis showing the unrestricted lobular organization. Scale bar ⫽ 20 ␮m. B: Interstitial tissue between adjacent lobules.
A basement membrane underlies Sertoli cells from the different lobules. Tight junctions that form the Sertoli cell barrier occur at the
apical ends of Sertoli cells. Scale bar ⫽ 0.5 ␮m. C: Spermatogonia A. Note that they are separated by thin cytoplasmic processes of
Sertoli cells. Scale bar ⫽ 3 ␮m. D: Cyst of pachytene spermatocytes I. The nucleus appears in only one of the two cells shown. Note
the presence of a granulocyte next to a blood capillary in the periphery of the gonad. Scale bar ⫽ 2 ␮m. E: Nuclear envelope and
adjacent organelles from a spermatocyte I. Scale bar ⫽ 0.5 ␮m. F: Cyst of spermatocytes II. Scale bar ⫽ 3 ␮m. G: Interstitial cell
(fibroblast). Scale bar ⫽ 1 ␮m. H: Myoid cell. Note the presence of cross-sectioned microfilaments (double asterisk) within the
cytoplasm. Scale bar ⫽ 0.5 ␮m. I: Granulocyte. Note the voluminous dense granules within the cytoplasm. Scale bar ⫽ 1 ␮m. al,
annulate lamellae; arrowhead, basement membrane; asterisk, collagen fibrils; c, blood capillary; ce, centriole; cp, cytoplasmic process;
d, desmosome; FI, fibroblast; GR, granulocyte; I, interstitial tissue; L, lobule; ml, mitochondria with lamellar cristae; mt, mitochondria
with tubular cristae; n, nuage; ne, nuclear envelope; MY, myoid cell; PS, pachytene spermatocyte; SA, spermatogonium A; sc,
synaptonemal complex; SE, Sertoli cell; SP, spermatocyst; SI, spermatocyte I; SII, spermatocyte II; tj, tight junction.
206
F.J. MEIJIDE ET AL.
Fig. 12. Time scale of the most important events and features of Cichlasoma dimerus gonadogenesis at 26.5°C. The onset of germ
cell meiotic activity, a key sign of gonadal differentiation, takes place 1 month earlier in females than in males. GC, germ cells; FG!,
folliculogenesis; SG!, spermatogenesis; SII, spermatocyte II.
One of the most common and prominent histological features marking the beginning of gonadal differentiation in teleosts is the appearance of meiotic
figures in germ cells of the presumptive ovary. This
feature was observed in Cichlasoma dimerus 42
days after fertilization (Fig. 4A). The initiation of
meiotic activity has frequently been reported as the
first recognizable sign of ovarian differentiation (Satoh and Egami, 1972; Nakamura and Takahashi,
1973; Yoshikawa and Oguri, 1978; Takashima et al.,
1980; Colombo et al., 1984; Piferrer and Donaldson,
1989; Nakamura and Nagahama, 1993; Patiño and
Takashima, 1995; Nakamura et al., 1998). The onset
of ovarian meiosis is soon accompanied by an increase in germ cell mitosis (Fig. 5A). However, in
ovaries of some species increased germ cell mitosis
may actually precede meiosis (Lebrun et al., 1982;
Hamaguchi, 1982; Strüssmann et al., 1996). Moreover, Hamaguchi (1982) reported that germ cells in
female medaka Oryzias latipes proliferate more actively than those in males, and indicated that this
difference in the rate of germ cell mitosis is the first
morphological sign of sex differentiation in this species. Our findings also show a higher mitotic activity
in the female lineage similar to that observed by
Hamaguchi, although it becomes evident a few days
after the onset of meiotic activity. In C. dimerus,
histological sex differentiation of female germ cells
coincides with the appearance of blood capillaries
(Fig. 4A) and occurs 1 week before somatic cell clusters are detected in the gonad (Fig. 5B). In the channel catfish (Ictalurus punctatus), ovarian somatic
differentiation (appearance of small tissue outgrowths) precedes the onset of meiosis by only 3
days (Patiño et al., 1996). Likewise, germ cell and
somatic differentiations begin almost simultaneously in the tilapias Oreochromis mossambicus
and Tilapia zilli (Nakamura and Takahashi, 1973;
Yoshikawa and Oguri, 1978). Although relatively
rare, there are some other cichlid species, e.g., O.
aureus and O. niloticus, in which the somatic organization of the ovary may start well before meiosis
can first be detected (Eckstein and Spira, 1965; Nakamura and Nagahama, 1985).
In most teleosts the ovarian lumen is characteristically formed by the fusion of outgrowths developed
from somatic cell clusters at the periphery of the
gonad that enclose part of the coelom and form a
lumen that is lined internally by the former coelomic
epithelium (Fig. 5C,D). This state is referred to as
the cystovarian condition (Hoar, 1969) or ovary of
the cystovarian type (Dodd, 1977). In Cichlasoma
dimerus, most oocytes are in the early diplotene
stage of meiotic prophase by the time the ovarian
cavity is completely formed (Fig. 5D).
The transition from oogonium (Fig. 4B) to oocyte
(Fig. 4C) is characterized by a distinctive change in
the nucleus that is associated with the onset of meiosis. Most importantly, the conspicuous nucleolus
disappears as synaptonemal complexes organize at
the pachytene stage. Subsequently, as early meiotic
oocytes (Fig. 4A) progress through the primary
growth phase of oogenesis and reach the perinucleolar stage (Fig. 6A), the nucleo-cytoplasmic ratio decreases and the cytoplasm becomes highly basophilic due to the accumulation of cytoplasmic
mRNA. The appearance of multiple nucleoli in the
peripheral region of the oocyte nucleus, or germinal
vesicle (Fig. 6B), indicates an intense transcription
of ribosomal RNA (Selman and Wallace, 1989).
With respect to the origin of somatic cells within
the ovary of Cichlasoma dimerus, it seems clear that
follicle cells originate from epithelial cells which in
turn are derived from the coelomic epithelium, as
proposed by other authors (see Tokarz, 1978; Wallace and Selman, 1990; Hamaguchi, 1992; Grier,
2000), while thecal cells are derived from the connective tissue lying beneath the epithelial basement
GONADAL DEVELOPMENT IN C. DIMERUS
membrane, i.e., the subepithelial connective tissue
that gives rise to the ovarian stroma. Nagahama
(1983) and Begovac and Wallace (1988) indicated
that stromal connective tissue elements become organized to form the thecal layer around salmonid
and syngnathan follicles, respectively. Likewise, it
has been established that somatic cells from the
ovarian stroma of the common snook Centropomus
undecimalis (Grier, 2000) and the swamp eel Synbranchus marmoratus (Ravaglia and Maggese,
2002) become distributed around follicle cells and
form the theca. The collagen-filled spaces between
prefollicle and epithelial cells (Fig. 4B,E,H) correspond to the same interstitial compartment in which
prethecal cells are observed. It is delimited by a
basement membrane and has direct connections
with the pericapillary spaces (Abraham et al., 1984).
In the later stages of ovarian development, this compartment is referred to as the extravascular space
(Fig. 7B), as proposed by Grier (2000) and adapted
from Abraham et al. (1980). Thus, prethecal cells,
thecal cells, blood vessels, and other interstitial cells
all reside within the stromal extravascular space.
The source of the ovarian follicles is the germinal
epithelium, which consists of the epithelial and derived prefollicle cells that surround either oogonia or
oocytes (Fig. 7B). The germinal epithelium borders
the ovarian lumen and is supported by a basement
membrane that separates it from the underlying
stromal compartment of the ovary (Fig. 7B,C). According to Grier (2000), thecal cells are derived from
a different ovarian compartment than is the follicle
and should not be considered part of it. The follicle is
therefore defined by its origin from the germinal
epithelium and is composed of the oocyte and surrounding follicle cells. A single basement membrane
is frequently observed at the point of attachment of
ovarian follicles to the germinal epithelium, i.e., epithelial cells from the germinal epithelium and follicle cells from attached follicles share a common
basement membrane at the point of attachment
(Fig. 7D,F). This feature was also observed in the
ovary of common snook, C. undecimalis (Grier,
2000).
The appearance of microvilli due to folding of the
oolema in the periphery of perinucleolar oocytes on
day 100 (Fig. 7C,F) indicates that the initiation of
vitelline envelope formation is about to take place.
Moreover, condensing vacuoles at the trans face of
the Golgi apparatus (Fig. 7E), considered immature
secretory granules (Bozzola and Russell, 1992),
might contain the electron-dense material that
would form the vitelline envelope when released by
exocytosis and deposited around microvilli.
As is typical among teleosts, the testes of Cichlasoma dimerus remain histologically indifferent
longer than the ovaries. The precocious differentiation of female germ cells (day 42, Fig. 4A) contrasts
with the late appearance of the first meiotic
prophase feature in spermatocytes (day 72, Fig. 9B).
207
Thus, using meiosis of germ cells as a criterion,
gonadal differentiation becomes apparent 1 month
earlier in females than in males (Fig. 12). The onset
of meiosis in the testis occurs concomitantly with an
increase in germ cell mitosis, some of these mitotic
divisions giving rise to spermatogonia B (Fig. 9A).
However, it should be noticed that there is still a
lack of definitive criteria for the detection of the very
first discrete signs of testicular differentiation. According to Nakamura et al. (1998), the formation of
efferent duct anlagen, coincident with the appearance of the first blood capillaries, are reliable criteria by which testicular differentiation can be recognized, even though germ cells remain at the gonial
stage. Taking into account this sex-specific characteristic of somatic tissue, ovarian and testicular differentiation seems to start at the same time in certain tilapia species (Nakamura and Takahashi,
1973; Nakamura and Nagahama, 1985, 1989). In C.
dimerus, sex differentiation of the testicular soma,
i.e., appearance of blood capillaries and the efferent
duct anlage, occurs concomitantly with or shortly
before the onset of male germ cell meiosis (day 72,
Fig. 9). Therefore, we consider that changes in the
germ cells should be used as the main clues in deciding gonadal sex. Nevertheless, in those species in
which changes in male germ cells occur at a considerably later period of testicular development, the
behavior of somatic cells may provide valid criteria
for determining testicular differentiation at an earlier phase of development.
Similar to observations in other teleosts (Bruslé
and Bruslé, 1978; Billard, 1984), changes from spermatogonia (Fig. 8B,D) to spermatocytes (Fig. 10B)
during testicular development in Cichlasoma dimerus include an increasing nucleo-cytoplasmic ratio, the disappearance of the nucleolus as synaptonemal complexes organize during the pachytene stage
of meiosis, and the reduction in the number of mitochondria and the amount of endoplasmic reticulum. According to Patiño and Takashima (1995),
zygotene spermatocytes can be recognized with light
microscopy by the “bouquet” distribution of the chromosomes, despite the absence of the large nucleolus
typical of zygotene oocytes. Our findings show that
zygotene spermatocytes exhibit both the bouquet
arrangement of chromosomes and a conspicuous nucleolus within the nucleoplasm (Fig. 9B).
Sertoli cells are derived from epithelial cells
(Hamaguchi, 1992; Grier, 1993) and their cytoplasmic processes form the spermatocyst wall. To maintain the integrity of the cyst, it is incumbent upon
Sertoli cells to establish firm junctional contacts.
This is accomplished by the development of interSertoli desmosomes and tight junctions. The presence of tight junctions between Sertoli cells has been
shown in several teleosts to result in the formation
of a blood–testis barrier (Nagahama, 1983; Pudney,
1993). According to Grier (1993), the term “blood–
testis barrier” should be replaced by “Sertoli cell
208
F.J. MEIJIDE ET AL.
barrier” since the barrier does not include the participation of blood vessel endothelial cells or lobule
boundary (myoid) cells. In most teleost species analyzed thus far, tight junctions are usually accompanied by the presence of desmosomes (Pudney, 1993),
as observed during Cichlasoma dimerus testicular
development (Figs. 10D, 11B).
Unlike the restricted lobular testis typical of
atherinomorphs, in which spermatogonia are confined to the distal termini of the lobules which contain an orderly progression of developing spermatocysts (Grier, 1993), the testis of Cichlasoma dimerus
is of the unrestricted lobular type, i.e., spermatogonia and spermatocysts at various stages of development are distributed throughout the length of the
lobules (Meijide, unpubl. obs.), which has been observed in most teleosts (Grier, 1993). By day 100
postfertilization, testicular lobules are quite well developed, spermatocyte II being the latest stage of
spermatogenesis found within them (Fig. 11A,F).
Based on a strict definition of an epithelium, as
indicated in histology textbooks, one criterion is that
epithelia border a body surface, lumen, or tube. In
100-day-old juvenile C. dimerus, solid cords of germ
cells and Sertoli cells are observed within the lobules (Fig. 11A). When most, but not all, of the criteria that define an epithelium apply to a tissue, then
the term “epithelioid” applies (Ross et al., 1995).
Therefore, germ and Sertoli cells in juvenile C. dimerus are organized as an “epithelioid” tissue until
maturation, when a lobular lumen develops. Solid
cords of spermatogonia and Sertoli cells that are also
epithelioid tissue have been reported in adult cobia
(Rachycentron canadum) during the regression and
regressed classes of their reproductive cycle (BrownPeterson et al., 2002). In contrast to the germinal
compartment in the testis of juvenile C. dimerus, the
interstitial tissue appears scarcely differentiated,
most interstitial cells having the appearance of fibroblasts. In this study, steroid-producing cells
(Leydig cells) were not detected ultrastructurally in
the testicular sections analyzed. However, further
inspection of the testes might be necessary since
Leydig cells were detected before the onset of meiosis in other cichlid species (Nakamura and Nagahama, 1989; Nakamura et al., 1998) and in the
medaka Oryzias latipes (Satoh, 1974). Interstitial
steroidogenic cells are usually characterized by having a round nucleus, numerous mitochondria with
tubular cristae, extensive smooth endoplasmic reticulum, and many free ribosomes (Satoh, 1974; Nagahama, 1983; Nakamura and Nagahama, 1989; Patiño and Takashima, 1995; Pudney, 1996; Lo Nostro
et al., 2004). Interestingly, Nicholls and Graham
(1972) found evidence for the origin of Leydig cells
from fibroblast-like connective tissue elements in
the testis of Cichlasoma nigrofasciatum.
The role of the basement membrane in establishing the separation between the germinal compartment and the interstitium or stroma of the gonad
has been adequately addressed in male (Grier,
1993), female (Grier, 2000), and hermaphroditic (Lo
Nostro et al., 2003, 2004) adult fishes. The significance of the basement membrane forming this separation is that it is apparently a constant throughout vertebrate evolution. In this study, it is shown
that separation of tissue compartments by the basement membrane is readily apparent in the undifferentiated gonad, i.e., before sexual differentiation of
the gonads takes place. The germinal compartment
contains germ cells and support cells, the latter being derived from epithelial cells. Within the interstitial compartment, collagen fibrils and a few interstitial cells are initially observed (Fig. 3A,E). During
ovarian differentiation, germ cells (oogonia) transform into oocytes when entering meiosis, while the
encompassing support cells become prefollicle cells.
Within the ovarian stroma, interstitial cells, which
become prethecal cells, are numerous. Collagen
fibrils and the first blood capillaries are observed as
well (Fig. 4). In the testis, support cells become
Sertoli cells, which encompass spermatogonia and
spermatocytes (Figs. 10A,B, 11C,D), while interstitial cells give rise to fibroblasts (Fig. 11G), myoid
cells (Fig. 11H), and presumably Leydig cells. Collagen fibrils, blood capillaries and granulocytes are
also detected within the testicular interstitium (Fig.
11D,I).
Since the onset of meiotic activity is coincident
with the appearance of the first blood vessels in the
gonad (around 42 days in females and 72 days in
males), one might hypothesize that germ cell sex
differentiation in Cichlasoma dimerus is dependent
on the influence of certain endocrine factors
(namely, gonadotropins) that reach the gonad via
the circulatory system. In this context, the possibility that the hypothalamo-pituitary-gonadal axis is
involved in triggering gonadal sex differentiation is
currently under debate (see Fitzpatrick et al., 1993;
Kah et al., 1993; Feist and Schreck, 1996; Baroiller
and Guiguen, 2001; Miranda et al., 2001; Devlin and
Nagahama, 2002; Pandolfi et al., 2002).
During this study it was observed that gonadal
development of Cichlasoma dimerus was both ageand size-dependent. The chronology of gonadal sex
differentiation is variable from one species to the
next, and also within a given species where the
growth rate is an important factor (Bruslé and
Bruslé, 1982). Individuals that grow faster sexually
differentiate earlier. Therefore, different degrees of
gonadal development may be recognized among individuals from a same clutch, even when reared
under the same conditions. In C. dimerus, asynchrony of gonadal development is noticed from 50
days postfertilization onward. Then the development and differentiation of the gonad is more related to body size than to age, as evidenced in other
teleost species (see Colombo et al., 1984; Grandi and
Colombo, 1997).
GONADAL DEVELOPMENT IN C. DIMERUS
ACKNOWLEDGMENTS
The authors thank I. Farı́as for EM sample processing, L. Zimerman for technical assistance in the
TEM, J.P. Vittori for photographic work, and H.
Grier for valuable suggestions.
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