JOURNAL OF MORPHOLOGY 264:149 –160 (2005)
Ultrastructure of Oogenesis in the Bluefin Tuna,
Thunnus thynnus
Francisco J. Abascal and Antonio Medina*
Departamento de Biologı́a, Facultad de Ciencias del Mar y Ambientales, Avda. República Saharaui, s/n, E-11510
Puerto Real, Cádiz, Spain
ABSTRACT Ovarian ultrastructure of the Atlantic bluefin tuna (Thunnus thynnus) was investigated during the
reproductive season with the aim of improving our understanding of the reproductive biology in this species. The
bluefin, like the other tunas, has an asynchronous mode of
ovarian development; therefore, all developmental stages
of the oocyte can be found in mature ovaries. The process
of oocyte development can be divided into five distinct
stages (formation of oocytes from oogonia, primary
growth, lipid stage, vitellogenesis, and maturation). Although histological and ultrastructural features of most
these stages are similar among all studied teleosts, the
transitional period between primary growth and vitellogenesis exhibits interspecific morphological differences
that depend on the egg physiology. Although the most
remarkable feature of this stage in many teleosts is the
occurrence of cortical alveoli, in the bluefin tuna, as is
common in marine fishes, the predominant cytoplasmic
inclusions are lipid droplets. Nests of early meiotic oocytes
derive from the germinal epithelium that borders the
ovarian lumen. Each oocyte in the nest becomes surrounded by extensions of prefollicle cells derived from
somatic epithelial cells and these form the follicle that is
located in the stromal tissue. The primary growth stage is
characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth commences with the accumulation of lipid droplets in the
oocyte cytoplasm (lipid stage), which is then followed by
massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage the
lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a
homogeneous mass of fluid yolk in mature eggs. J. Morphol. 264:149 –160, 2005. © 2005 Wiley-Liss, Inc.
KEY WORDS: Thunnus thynnus; oogenesis; ultrastructure; histology; vitellogenesis
Tunas show asynchronous oocyte development
and are considered multiple spawners. Like the
other two species of bluefin tunas (Thunnus maccoyii and T. orientalis), T. thynnus exhibits a migratory and spatiotemporally confined reproductive
pattern, since spawning is restricted to a short period of time and to specific grounds (Schaefer, 2001).
An accurate knowledge of reproductive parameters,
such as sexual maturation, fecundity, and spawning, is essential for population dynamics and stock
management studies in tunas. Histological exami© 2005 WILEY-LISS, INC.
nation of ovarian and testicular tissues is used to
assess the reproductive cycle of teleost fishes (Grier
and Taylor, 1998; Taylor et al., 1998; BrownPeterson et al., 2002; Lo Nostro et al., 2003), including tunas (Hunter et al., 1986; Schaefer, 1996, 1998,
2001), since it provides the most precise picture of
the reproductive state in both females and males.
However, despite the economic importance of T.
thynnus, histological studies on its reproductive biology have been scarce until very recently (Baglin,
1982; Susca et al., 2001; Medina et al., 2002;
Sarasquete et al., 2002; Corriero et al., 2003; Abascal et al., 2002, 2004). Most of these studies are
based on light microscopy, which has considerable
limitations in resolving the entire suite of gametogenic processes, such as folliculogenesis or vitellogenesis, for which only electron microscopy provides
adequate resolution and detail (Grier, 2000).
In the developing ovary of fishes, following oogonial proliferation, early meiotic oocytes become surrounded by prefollicle cells and form the ovarian
follicles. Subsequently, early oocytes enter primary
oocyte growth, which is marked by intense RNA
synthesis and formation of the vitelline envelope,
and then are arrested in diplotene of prophase I.
During vitellogenesis the diplotene oocytes take up
vitellogenin, a protein synthesized by the liver
which is processed into yolk globules. Eventually,
under appropriate hormonal stimuli, the oocytes undergo final maturation, are ovulated, and become
fertilizable eggs (deVlaming, 1983; Tyler and
Sumpter, 1996). Ultrastructural aspects of oogenesis have been documented in several teleost species
(Selman and Wallace, 1982, 1983, 1986; Selman et
Contract grant sponsor: the Spanish government; Contract grant
number: 1FD1997-0880-C05-04; Contract grant sponsor: European
Union; Contract grant number: Q5RS-2002-01355.
*Correspondence to: Antonio Medina, Departamento de Biologı́a,
Facultad de Ciencias del Mar y Ambientales, Avda. República
Saharaui, s/n, E-11510 Puerto Real, Cádiz, Spain.
E-mail: [email protected]
Published online 9 March 2005 in
Wiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/jmor.10325
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F.J. ABASCAL AND A. MEDINA
al., 1986, 1988, 1993; Wallace and Selman, 1990),
including some perciforms (Matsuyama et al., 1991;
Nakamura et al., 1993; Grau et al., 1996; Grier,
2000). However, electron microscope observations
have not as yet been applied to the study of oocyte
development in tunas. The continuous process of egg
formation in Brachydanio rerio was classified by
Selman et al. (1993) into a series of five stages which
can be readily identified on the basis of morphological features that also reflect physiological and biochemical events. This is, therefore, a functionally
consistent classification that can be generally applied to oogenesis in teleosts.
In the present study, folliculogenesis and oocyte
development in the northern Atlantic bluefin tuna
are described ultrastructurally. A deeper knowledge
of the cellular events culminating in the production
of mature eggs might help improve our understanding of the annual reproductive cycle in this and other
tuna species.
MATERIALS AND METHODS
Adult female bluefin tuna, Thunnus thynnus (Linnaeus, 1758),
were caught by trap in the Strait of Gibraltar (SW Spain) and by
purse seine in the spawning grounds around the Balearic Islands,
during the fishing seasons of 1999 and 2000.
For light microscopy (LM), 0.5 cm-thick cross sections, comprising the whole ovarian wall, were fixed for 48 –96 h in 4%
phosphate-buffered formaldehyde (10% commercial formalin). After dehydration in a series of ascending concentrations of ethanol,
the tissue samples were embedded in either paraffin or plastic
medium (2-hydroxyethyl-methacrylate). The 3-␮m thick plastic
sections were stained with Toluidine blue, while 6-␮m paraffin
sections were stained with hematoxylin-eosin. LM micrographs
were taken on a Leitz DMR BE microscope. For transmission
electron microscopy (TEM), small fragments of ovary were fixed
for 3– 4 h at 4°C in 2.5% glutaraldehyde buffered in sodium
cacodylate 0.1 M, pH 7.2. After washing in cacodylate buffer for
1 h the samples were postfixed for 1 h at 4°C in cacodylatebuffered osmium tetroxide. Following several short washes in
buffer, they were dehydrated through ascending concentrations
of acetone and finally embedded in epoxy resin (Epon 812). Thin
sections (⬃80 nm thick) picked up on copper grids were stained
with uranyl acetate and lead citrate and examined in a JEOL
JEM 1200 EX TEM.
RESULTS
The ovaries of Thunnus thynnus are paired, elongate organs, attached to the abdominal roof by the
dorsal mesentery. Posteriorly, the ovaries extend
into short oviducts that open into the cloaca. The
ovarian tissue forms longitudinal lamellae that extend into the reduced central lumen. A squamous
epithelium, the so-called germinal epithelium
(Grier, 2000), lines these lamellae at the luminal
surface. The pattern of ovarian development is asynchronous (Baglin, 1982; Susca et al., 2001; Medina
et al., 2002; Corriero et al., 2003); therefore, during
the breeding season all stages of oocyte development
can be found in the gonad (Fig. 1A). The complete
process of oogenesis was divided into five stages:
formation of early oocytes from oogonia and follicu-
logenesis, primary oocyte growth, secondary oocyte
growth (comprising lipid stage and vitellogenesis),
and maturation.
Early Oocytes and Folliculogenesis (Oocyte
Diameter: ⬃10 –20 ␮m)
Oogonial proliferation was not observed in adult
bluefin tuna ovaries, and oogonia (seldom identifiable) were not easily distinguished from early meiotic oocytes. As in another perciform, Centropomus
undecimalis, oogonia appear to have a more irregular nucleus than the oocytes, whose nuclei are spherical (Grier, 2000). Oogonia and oocytes, prior to initiation of the primary growth stage, are usually
found beneath the squamous epithelial layer bordering the ovarian lamellae, and are usually grouped in
nests where development seems to be synchronous
(Fig. 1B). Both oogonia and early oocytes have a high
nucleus– cytoplasm ratio, and the nucleus has a single, central nucleolus. The modest amount of cytoplasm contains many ribosomes, round mitochondria with few cristae, some Golgi complexes, and a
poorly developed endoplasmic reticulum (Fig. 1C–
F). The nuclear envelope exhibits numerous pores
through which an electron-dense, granular material
is exported from the nucleus to the cytoplasm and
forms the “nuages” (Fig. 1D) that frequently appear
associated with mitochondria (Fig. 1C,E). Prefollicle
cells, derived from the epithelial somatic cells, surround the germ cell nests and send thin processes
between adjacent early meiotic oocytes that separate them from each other (Fig. 1E). This event
takes place at the zygotene-pachytene stage (Fig. 1E
inset). The prefollicle cells surrounding oocyte nests
show intricate interdigitations and are joined by
desmosomes (Fig. 1F). Eventually, they lose contact
with the ovarian lumen, sink under the germinal
epithelium, and constitute a follicular epithelium
around the oocytes. At this stage desmosomes between adjacent follicle cells are rarely found.
Primary Oocyte Growth (Oocyte Diameter:
⬃20 –120 ␮m)
As in other fishes, this stage starts once the oocyte
enters into arrested meiotic diplotene (Wallace and
Selman, 1990; Grier, 2000). The cytoplasm of primary growth-phase oocytes is highly granular due to
an abundance of ribosomes. Nucleoli are first scattered throughout the nucleoplasm and afterwards
migrate toward the nuclear envelope (Fig. 2A). The
volume of the oocyte increases as a result of proliferation of membranous organelles and accumulation
of ribonucleoproteins in the cytoplasm (Fig. 2A). As
a consequence, the nucleus– cytoplasm ratio decreases considerably (Fig. 1B). Mitochondria of variable shape, Golgi complexes, and nuage bodies are
usually found close to the nucleus (Fig. 2A,B).
OOGENESIS IN BLUEFIN TUNA
151
Fig. 1. Oogenesis in Thunnus thynnus. A: Section of an ovary with follicles at all developmental stages. LM. B: Section of a nest of
early oocytes (ON) located beneath the epithelium bordering the ovarian lumen (L). LM. C–F: Early meiotic oocytes. TEM. Gc, Golgi
complex; m, mitochondria; MNO, migratory nucleus oocyte (maturation stage); N, nucleus; n, nuages; nu, nucleolus; p, nuclear pores;
PFC, prefollicle cells; PGO, primary growth oocyte; POF, post-ovulatory follicle; sc, synaptonemal complex in pachytene oocyte (see
inset in E for further detail); VO, vitellogenic oocyte. Arrows, extensions of prefollicle cells between adjacent pachytene oocytes;
arrowheads, desmosomes between prefollicle cells.
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F.J. ABASCAL AND A. MEDINA
Fig. 2. A–F: Primary growth oocytes of Thunnus thynnus. TEM. bl, basal lamina; FC, follicle cell; Gc, Golgi complex; ld,
lipid droplet; m, mitochondria; mv, microvilli; N, nucleus; n, nuage; nu, nucleolus; O, cytoplasm of oocyte; TC, thecal cell; ve,
vitelline envelope.
OOGENESIS IN BLUEFIN TUNA
During this stage, membrane processes begin to
extend from the surface of both the oocyte and (to a
much lesser extent) the follicle cells. The vitelline
envelope (also referred to as zona radiata or chorion
in the relevant literature) begins to be deposited as
a homogeneous, moderately electron-dense material
(Fig. 2C,D). Shortly thereafter, an inner, more
electron-dense layer of the vitelline envelope differentiates (Fig. 2E,F). Golgi bodies, some profiles of
rough endoplasmic reticulum (RER), and smooth
vesicles are present (Fig. 2E). Occasionally, small
lipid droplets become apparent in the ooplasm at the
end of this stage (Fig. 2F).
A continuous follicular layer (the granulosa)
formed by flattened cells encompasses the oocyte
(Fig. 2D–F). The follicle cells are elongated, with
free ribosomes, mitochondria, small vesicles, and
cisternae of the RER. A distinct, thick basal lamina
separates this cell layer from the overlying thecal
cells (Fig. 2D–F). The inner thecal layer is vascularized and contains small capillaries, whereas the
outer thecal cells form a rather thin squamous layer.
Lipid Stage (Oocyte Diameter: ⬃120–250 ␮m)
The beginning of this stage is marked by the appearance of lipid droplets that are clearly discernible
in light microscopy (Fig. 3A). Lipid synthesis continues throughout this period and extends into the
vitellogenic stage, leading to an increase in lipid
stores that eventually become distributed randomly
in the ooplasm (Fig. 3A,B). These lipid droplets are
homogeneous, moderately electron-dense, and display a smooth, nonmembrane-bound surface (Fig.
3B,C). The nuclear envelope has numerous pores
and the nucleoplasm contains a finely granular chromatin. As in the preceding stage, small amounts of
granular material are apposed to the nuclear envelope at the ooplasmic side. Large parts of nucleolar
material lie beneath the nuclear envelope in the
peripheral nucleoplasm (Fig. 3D).
At this stage the weakly basophilic cytoplasm becomes more vesicular and contains free ribosomes,
mitochondria, and some dictyosomes that are more
common at the periphery of the oocyte (Fig. 3E). The
vitelline envelope has thickened slightly in comparison to the preceding stage, and the follicle cells have
become higher (Fig. 3E). In close association with
cisternae of the RER, cortical alveolus-like vesicles
are occasionally seen. These cytoplasmic inclusions
are relatively scarce and have an electron-lucent,
finely granular content (Fig. 3F).
Vitellogenesis (Oocyte Diameter:
⬃250 –500 ␮m)
This stage is characterized by substantial growth
of the oocyte, which is primarily accounted for
by uptake of exogenous material into the ooplasm.
During vitellogenesis, the oocytes accumulate
153
membrane-bound yolk platelets that increase in size
as oocyte development progresses (Fig. 3A). Vitellogenic oocytes have abundant finger-like projections
that are embedded in the layered vitelline envelope
(Fig. 4A–E). These well-developed oocyte cytoplasmic microvilli traverse the thickened vitelline envelope through pore canals and then intertwine to
form bundles beneath the follicle cell layer (Fig.
4A,C,D). Although some of these microvillar bundles
penetrate deeply into the intercellular spaces between follicle cells, they never reach the basal lamina at the opposite side of the follicular epithelium
(Fig. 4A,C–E).
The height of the follicular epithelium increases
during vitellogenesis; the follicle cells become
thicker and contain round mitochondria and a moderately developed RER (Fig. 4A,E). Golgi bodies and
cytoplasmic vesicles are relatively scarce. The borders between follicle cells appear clearly marked
because of the high electron-density of the extracellular substance (Fig. 4A,C–E). This dense substance
also pervades through the bundles of microvilli, thus
outlining the microvillar profiles under and between
the follicle cells (Fig. 4A,D,E). These observations
suggest that the follicular epithelium is rather permeable to substances from the surrounding thecal
layers via intercellular spaces. The basal lamina
that externally invests the follicle is thick and fibrous, and displays a parallel orientation of the collagen fibers in relation to the follicular surface (Fig.
4A,C,D). Two layers of flattened thecal cells envelope the follicle around the basal lamina. Capillaries
are commonly found in the inner thecal layer.
Aside from the presence of microvilli, another evidence of the absorptive capacity of vitellogenic oocytes is the appearance of numerous pits and spherical vesicles (⬃100 nm in diameter) in the peripheral
cytoplasm immediately beneath the plasma membrane. They enclose an electron-dense material that
resembles in appearance the intercellular substance
found between follicle cells (Fig. 4A,B,D–G). At high
magnification these pits and vesicles were found to
be clathrin-coated (Fig. 4G), which is indicative of
selective transport of specific ligands. Single or multiple coated vesicles are frequently observed to bud
from deep tubular membrane invaginations, thus
suggesting intensive uptake of exogenous substances (Fig. 4D–G). The cortical region of the
ooplasm is rich in free ribosomes but lacks membranous organelles (Fig. 4A,B,F). Under this region the
most conspicuous structures are mitochondria with
tubular cristae and also electron-lucent vesicles that
are somewhat larger than the endocytotic vesicles
(Fig. 4A). It is not known whether these vesicles
belong to the endoplasmic reticulum or they play a
role in the plasma membrane recycling system. Fusion of these vesicles with the plasmalemma is not
frequently observed.
Shortly after the extracellular material is internalized, the endocytotic vesicles lose their clathrin
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F.J. ABASCAL AND A. MEDINA
Fig. 3. Lipid stage oocytes of Thunnus thynnus. A: Ovary showing lipid stage and vitellogenic oocytes. LM. B–F: Details of lipid
stage oocytes. TEM. Arrowheads, traffic of granular material through nuclear pores; bl, basal lamina; cav, cortical alveolus-like vesicle;
FC, follicle cell; ld, lipid droplet; LSO, lipid stage oocyte; mv, microvilli; N, nucleus; nu, nucleolus; PGO, primary growth oocyte; rer,
rough endoplasmic reticulum; TC, thecal cell; ve, vitelline envelope; VO, vitellogenic oocyte.
Fig. 4. Vitellogenic oocytes of Thunnus thynnus (A–G). TEM. bl, basal lamina; e, uncoated endocytotic vesicles (endosomes); FC,
follicle cells; is, intercellular space; m, mitochondria; mv, microvilli; N, nucleus; ny, nascent yolk platelets; O, oocyte; T, thecal cell
layers; TE, theca externa; TI, theca interna; ve, vitelline envelope; y, yolk platelet. Arrows, gathering of oocyte microvilli into bundles;
arrowheads, coated endocytotic pits and vesicles (for further detail, see G, which is an enlargement of the area indicated in E); double
arrowheads, multiple budding of endocytotic vesicles from long tubules connecting with the plasma membrane.
156
F.J. ABASCAL AND A. MEDINA
Fig. 5. Vitellogenic oocytes of
Thunnus thynnus. TEM. A–C:
Details of yolk platelets featuring the dense central core (c),
which shows a crystalline arrangement (see inset in C for
further detail), and the peripheral granular matrix (p); cav,
cortical alveolus-like vesicle; ld,
lipid droplet; y, yolk platelet.
coat and appear to coalesce with others to form
larger vesicles (endosomes and nascent yolk platelets) (Fig. 4E,F). As a result of this process, the
ooplasm of vitellogenic oocytes stores yolk platelets
whose size increases centripetally (Figs. 4B, 5A).
Large lipid droplets, which continue to accumulate
during the vitellogenic stage, alternate with yolk
platelets at the central ooplasm (Fig. 5B). The yolk
platelets are membrane-bound inclusions that consist of a central, electron-dense core embedded in a
more electron-lucent granular matrix (Fig. 5A–C).
The peripheral matrix of the yolk platelets is of
variable thickness. Sometimes it is lost in areas
where the outer membrane is apposed directly to the
central core (Fig. 5B). High-magnification micro-
graphs show a crystalline arrangement of the central core material with a periodicity of ⬃4.5 nm (Fig.
5C, inset). Electron-lucent patches of variable size
are seen in some yolk platelets (Fig. 5A).
Maturation (Oocyte Diameter: ⬃500–1,000 ␮m)
The primary event of oocyte maturation is the
resumption of meiosis. The commencement of this
stage is marked by migration of the nucleus toward the animal pole of the oocyte (Fig. 6A), which
is followed by breakdown of the nuclear envelope.
Concomitant with the nuclear migration, the lipid
droplets begin to fuse into larger lipid inclusions
OOGENESIS IN BLUEFIN TUNA
157
Fig. 6. Thunnus thynnus oocytes at maturation. A,B: LM
micrographs. C–E: TEM micrographs showing consecutive
stages of yolk proteolysis (1– 4)
(C,D) and the cortical ooplasm
(E). Arrows, coalescence of yolk
platelets; arrowheads, proteolytic yolk platelets; FC, follicle
cells; ld, lipid droplet; mv, microvilli; N, nucleus; O, oocyte;
ve, vitelline envelope (chorion);
y, yolk platelets.
that occupy the central portion of the oocyte,
where they eventually form a very large central
lipid droplet (Fig. 6A,B). Throughout this stage
the yolk platelets coalesce as they undergo internal disorganization, probably due to hydrolysis of
their contents. First, the pale areas in the central
core increase in number and size (Fig. 6B), and
then the core materials disaggregate into irregular patches that remain embedded in a highly
electron-lucent matrix (Fig. 6C,D). The processes
of proteolysis and coalescence of yolk globules lead
to hydration of the oocyte with the formation of a
continuous mass of fluid yolk that occupies the
entire volume of the oocyte.
The layered vitelline envelope reaches ⬃6 ␮m in
thickness and the follicle cells become more vacuolated. Following ovulation the follicular epithelium
shrinks and folds, giving rise to postovulatory follicles.
DISCUSSION
The terminology and features used to differentiate
successive stages of oocyte formation and matura-
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F.J. ABASCAL AND A. MEDINA
tion vary according to authors and species. In this
study the follicle development in Thunnus thynnus
was divided into five distinct stages on the basis of
histological and ultrastructural observations. This
staging reflects morphological as well as physiological and biochemical events, and is compatible with
other classifications commonly used in teleosts (deVlaming, 1983; Nagahama, 1983; Selman and Wallace, 1986; Wallace and Selman, 1990; Selman et al.,
1993; Tyler and Sumpter, 1996). We herein use the
term “lipid stage” to refer to the transitional stage
between primary growth and vitellogenesis. However, we avoid the term “cortical alveolus stage”
commonly used in teleosts because, as in other marine species, cortical alveoli are scarce in, if not
absent from, bluefin tuna oocytes. Lipid stage oocytes are characterized by the presence of cytoplasmic lipid droplets that can be distinctly identified on
the light microscope (Susca et al., 2001; Medina et
al., 2002; Corriero et al., 2003). Although in most
teleosts studied thus far cortical alveoli have been
reported to become apparent before lipid droplets
and yolk granules, there are exceptions (West,
1990). Within the perciforms, Mayer et al. (1988)
found cortical alveoli to appear after both lipid droplets and yolk granules in the sea bass, while in the
red sea bream (Matsuyama et al., 1991) and the
amberjack (Grau et al., 1996) they were observed
shortly after the commencement of lipid droplet formation.
The present ultrastructural observations on bluefin tuna oogenesis do not differ significantly from
those reported previously for other teleost fishes
(Selman and Wallace, 1986; Wallace and Selman,
1990; Selman et al., 1993; Grier, 2000). It is generally accepted that teleost oocytes arise within the
germinal epithelium bordering the ovarian lumen
(Wallace and Selman, 1990; West, 1990; Grier,
2000). The presence of early meiotic oocytes within
the ovarian luminal epithelium suggests the germinal role of this layer in bluefin tuna, although, as
reported in previous studies (Selman and Wallace,
1986; Wallace and Selman, 1990; Selman et al.,
1993), oogonial proliferation was not observed. Oogonial mitotic divisions are difficult to detect in
adult teleost ovaries during the breeding season,
since the overwhelming majority of germ cells are
more advanced in development. Early meiotic oocytes appeared grouped in nests and, as in other
teleost species (Selman et al., 1993; Grier, 2000),
their development seems to be synchronous even
though the germ cells within the same cluster soon
become isolated from each other by prefollicle cells.
In the common snook, Centropomus undecimalis,
desmosomes and tight junctions are lost once the
epithelial cells associate with leptotene oocytes to
become prefollicle cells (Grier, 2000). Conspicuous
desmosomes, however, keep the prefollicle cells
tightly joined during zygotene and pachytene in the
bluefin tuna, but as the oocyte becomes surrounded
by follicle cells and sinks into the ovarian stroma
these junctions are no longer observed. A characteristic of early oocytes is the presence of electrondense nuages close to the nuclear envelope. Nuages
continue to be present throughout primary growth
until the lipid stage, in contrast to the condition in
the common snook (Grier, 2000), where the nuages
disappear prior to the completion of folliculogenesis.
According to Nagahama (1983), Wallace and Selman (1990), West (1990), Tyler and Sumpter (1996),
Grier (2000), and Guimarães and Quagio-Grassiotto
(2001) the primary growth stage is characterized by
an intense transcriptional activity. The occurrence
of multiple nucleoli at the periphery of the nucleus,
as well as numerous perinuclear nuages, appears to
indicate an intense transport of ribonucleoproteins
from the nucleus to the cytoplasm. Aggregations of
organelles, mainly mitochondria in association with
nuages, are seen close to the nucleus. At the end of
this stage the vitelline envelope begins to form, penetrated by short microvilli.
During the following secondary growth phase the
oocyte accumulates nutrients for the development of
the future embryo. The first inclusions to become
visible in the ooplasm of bluefin tuna oocytes are
small lipid droplets that gradually increase in number and size throughout the lipid stage and also
during most of the remaining secondary growth. As
commonly observed in teleosts, particularly the marine species (deVlaming, 1983), the lipid droplets
begin to coalesce centripetally into larger lipid inclusions at the beginning of the maturation stage, eventually forming a single central oil droplet. The lipid
content of the bluefin tuna ovary was found to increase significantly throughout secondary growth,
neutral lipids (primarily triacylglycerol and steryl/
wax ester classes) predominating over polar lipids
(Mourente et al., 2002). These data appear to indicate that the lipids accumulated into the egg oil
droplet during secondary growth mostly consist of
neutral lipids.
In many teleosts, the precursors of the cortical
alveoli appear when the follicle diameter is ⬃200
␮m (deVlaming, 1983; Selman et al., 1986, 1988;
Tyler and Sumpter, 1996; Guimarães and QuagioGrassiotto, 2002). Membrane-bound vesicles resembling the cortical alveoli of other teleosts appeared to
originate in association with RER cisternae in bluefin tuna oocytes. The extremely low frequency of
these structures makes it unlikely that they play a
significant role during the cortical reaction at fertilization, as occurs in other species (deVlaming, 1983;
Selman et al., 1986, 1988; Tyler and Sumpter, 1996).
A massive incorporation of exogenous substances
appears to be mainly responsible for the enormous
enlargement that the oocyte undergoes during secondary growth. The presence of a well-developed
microvillar border, and the formation of numerous
pits and vesicles at the oocyte surface, provide ample
evidence of the great capacity of bluefin tuna vitel-
OOGENESIS IN BLUEFIN TUNA
logenic oocytes to incorporate extracellular substances. The pathway of yolk incorporation into the
ooplasm has been revealed in Cyprinodon variegatus
(Selman and Wallace, 1982) and Fundulus heteroclitus (Selman and Wallace, 1983) using electrondense tracers. Circulating macromolecules were
shown to pass between the endothelial cells of the
thecal capillaries, then between the follicle cells, and
finally through the pore canals of the vitelline envelope (Selman and Wallace, 1982, 1983, 1986). In
Thunnus thynnus the intercellular spaces of the follicular epithelium are filled with an electron-dense
substance that is very similar in appearance to that
found in the endocytotic pits and vesicles of the
oocyte. Bundles of microvilli from the oocyte accumulate in the subfollicular space, and some of them
penetrate deeply into the intercellular spaces between follicle cells, a phenomenon that also has been
observed in C. variegatus (Selman and Wallace,
1982) and in F. heteroclitus (Selman and Wallace,
1983, 1986). The dense extracellular substance diffuses between the microvilli located in the interfollicular and subfollicular spaces; hence, the long microvillar processes convey the exogenous substances
to the oocyte surface by capillarity. Once this material has reached the oocyte surface, it appears to
enter the ooplasm by endocytosis. Since clathrincoated pits are major ports of entry of macromolecules into the cell (Smythe, 2003), the abundance of
coated pits and vesicles in the cortical cytoplasm of
teleost oocytes (Selman and Wallace, 1982, 1983,
1986; Wallace and Selman, 1990; Selman et al.,
1993) points to a selective uptake of the yolk precursor, vitellogenin, which would then be translocated
to the nascent yolk platelets. The sequence of events
leading to formation of the mature yolk platelets can
be inferred from ultrastructural observations of the
cortical ooplasm. Endosomes apparently resulting
from coalescence of endocytotic vesicles fuse with
one another or with preexistent yolk vesicles in the
cortical ooplasm (Selman and Wallace, 1982, 1983),
thus contributing to the formation of larger yolk
platelets that reside in the central region of the
oocyte. The whole process is quite fast, since in C.
variegatus circulating horseradish peroxidase is incorporated into peripheral yolk platelets within 20
min after intraperitoneal injection of the tracer (Selman and Wallace, 1982). In calf chromaffin cells,
Artalejo et al. (2002) calculated that the duration of
clathrin-coated vesicle-based endocytosis is ⬃10
min. Thus, although the volume of the clathrincoated vesicles of bluefin tuna oocytes is rather
small (⬃5 ⫻ 10– 4 ␮m3), their great number and the
supposedly fast completion of the endocytotic process suggest a high rate of vitellogenin uptake so as
to be capable to maintain the production of a batch
of mature oocytes every 1.2 days, which is the average interspawning interval estimated for T. thynnus
during the breeding season (Medina et al., 2002).
159
Incorporation of exogenous materials by pinocytosis continues to occur until the early maturation
stage in oocytes of Fundulus heteroclitus, but it
stops abruptly at the time of germinal vesicle breakdown (Selman and Wallace, 1983). Likewise, morphologic evidence of endocytosis was not observed in
maturational follicles of Thunnus thynnus.
As in other teleost species (Kjesbu and Kryvi,
1989; Selman et al., 1993), mature yolk globules of
Thunnus thynnus have a main proteic body embedded in a superficial matrix. As oocyte development
proceeds through maturation, the yolk platelets lose
their regular inner structure. Apart from nuclear
transformations (germinal vesicle migration and
breakdown), the most striking event that occurs
during the maturation stage in many marine teleosts is proteolysis and fusion of yolk platelets,
which culminate in the formation of a continuous
mass of fluid yolk. Hydration during final maturation causes a considerable increase in volume and
appears to be important in the production of pelagic
(buoyant) eggs (deVlaming, 1983; Tyler and
Sumpter, 1996).
ACKNOWLEDGMENTS
We thank Almadraba Cabo Plata, S.A., and Ginés
J. Méndez Alcalá (G. Méndez España, S.L.) for cooperation in the sampling. The invaluable assistance of Dr. César Megina, Agustı́n Santos and the
staff of the Electron Microscopy Unit (University of
Cádiz) is greatly appreciated. Prof. Harry Grier
kindly solved some doubts about early oogenesis.
The authors thank two anonymous reviewers for
helpful comments and recommendations.
LITERATURE CITED
Abascal FJ, Medina A, Megina C, Calzada A. 2002. Ultrastructure of Thunnus thynnus and Euthynnus alletteratus spermatozoa. J Fish Biol 60:147–153.
Abascal FJ, Megina C, Medina A. 2004. Testicular development
in migrant and spawning bluefin tuna (Thunnus thynnus (L.))
from the eastern Atlantic and Mediterranean. Fish Bull 102:
407– 417.
Artalejo CR, Elhamdani A, Palfrey HC. 2002. Sustained stimulation shifts the mechanism of endocytosis from dynamin-1dependent rapid endocytosis to clathrin- and dynamin-2mediated slow endocytosis in chromaffin cells. Proc Natl Acad
Sci U S A 99:6358 – 6363.
Baglin RE Jr. 1982. Reproductive biology of western Atlantic
bluefin tuna. Fish Bull 80:121–134.
Brown-Peterson NJ, Grier HJ, Overstreet RM. 2002. Annual
changes in germinal epithelium determine male reproductive
classes of the cobia. J Fish Biol 60:178 –202.
Corriero A, Desantis S, Deflorio M, Acone F, Bridges CR, de la
Serna JM, Megalofonou P, De Metrio G. 2003. Histological
investigation on the ovarian cycle of the bluefin tuna in the
western and central Mediterranean. J Fish Biol 63:108 –119.
deVlaming V. 1983. Oocyte developmental patterns and hormonal involvements among teleosts. In: Rankin JC, Pitcher TJ,
Duggan RT, editors. Control processes in fish physiology. London: Croomhelm. p 176 –199.
160
F.J. ABASCAL AND A. MEDINA
Grau A, Crespo S, Riera F, Pou S, Sarasquete MC. 1996. Oogenesis in the amberjack Seriola dumerili, histochemical and ultrastructural study of oocyte development. Sci Mar 60:391– 406.
Grier H. 2000. Ovarian germinal epithelium and folliculogenesis
in the common snook, Centropomus undecimalis (Teleostei:
Centropomidae). J Morphol 243:265–281.
Grier HJ, Taylor RG. 1998. Testicular maturation and regression
in the common snook. J Fish Biol 53:521–542.
Guimarães ACD, Quagio-Grassiotto I. 2001. Ultrastructural aspects of oogenesis and oocyte primary growth in Serrasalmus
spilopleura (Teleostei, Characiformes, Characidae). Tissue Cell
33:241–248.
Guimarães ACD, Quagio-Grassiotto I. 2002. The ultrastructural
aspects of vitellogenesis or oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Serrasalminae). J
Submicrosc Cytol Pathol 34:199 –206.
Hunter JR, Macewicz BJ, Sibert JR. 1986. The spawning frequency of skipjack tuna, Katsuwonus pelamis, from the South
Pacific. Fish Bull 84:895–503.
Kjesbu OS, Kryvi H. 1989. Oogenesis in cod, Gadus morhua L.,
studied by light and electron microscopy. J Fish Biol 34:735–
746.
Lo Nostro F, Grier H, Andreone L, Guerrero GA. 2003. Involvement of the gonadal germinal epithelium during sex reversal
and seasonal testicular cycling in the protogynous swamp eel,
Synbranchus marmoratus Bloch 1795 (Teleostei, Synbranchidae). J Morphol 257:107–126.
Matsuyama M, Nagahama Y, Matsuura S. 1991. Observations on
ovarian ultrastructure in the marine teleost, Pagrus major,
during vitellogenesis and oocyte maturation. Aquaculture 92:
67– 82.
Mayer I, Shackley SE, Ryland JS. 1988. Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. I. An histological and histochemical study of oocyte development. J Fish Biol
33:609 – 622.
Medina A, Abascal FJ, Megina C, Garcı́a A. 2002. Stereological
assessment of the reproductive status of female Atlantic northern bluefin tuna, Thunnus thynnus (L.), during migration to
Mediterranean spawning grounds through the Strait of
Gibraltar. J Fish Biol 60:203–217.
Mourente G, Megina C, Dı́az-Salvago E. 2002. Lipids in female
northern bluefin tuna (Thunnus thynnus thynnus L.) during
sexual maturation. Fish Physiol Biochem 24:351–363.
Nagahama Y. 1983. The functional morphology of teleost gonads.
In: Hoar WS, Randall DJ, Donaldson EM, editors. Fish physiology, IX A. Reproduction, endocrine tissues and hormones.
New York: Academic Press. p 223–276.
Nakamura M, Specker JL, Nagahama Y. 1993. Ultrastuctural
analysis of the developing follicle during early vitellogenesis in
tilapia, Oreochromis niloticus, with special reference to the
steroid-producing cells. Cell Tissue Res 272:33–39.
Sarasquete C, Cárdenas S, González de Canales ML, Pascual E.
2002. Oogenesis in the bluefin tuna, Thunnus thynnus L.: a
histological and histochemical study. Histol Histopathol 17:
775–788.
Schaefer KM. 1996. Spawning time, frequency, and batch fecundity of yellowfin tuna, Thunnus albacares, near Clipperton
Atoll in the eastern Pacific Ocean. Fish Bull 94:98 –112.
Schaefer KM. 1998. Reproductive biology of yellowfin tuna (Thunnus albacares) in the eastern Pacific Ocean. Inter-Amer TropTuna Comm Bull 21:201–272.
Schaefer KM. 2001. Reproductive biology of tunas. In: Block BA,
Stevens ED, editors. Tuna: physiology, ecology and evolution.
London: Academic Press. p 225–270.
Selman K, Wallace RA. 1982. Oocyte growth in the sheepshead
minnow: uptake of exogenous proteins by vitellogenic oocytes.
Tissue Cell 14:555–571.
Selman K, Wallace RA. 1983. Oogenesis in Fundulus heteroclitus.
III. Vitellogenesis. J Exp Zool 226:441– 457.
Selman K, Wallace RA. 1986. Gametogenesis in Fundulus heteroclitus. Am Zool 26:173–192.
Selman K, Wallace RA, Barr V. 1986. Oogenesis in Fundulus
heteroclitus. IV. Yolk vesicle formation. J Exp Zool 239:277–
288.
Selman K, Wallace RA, Barr V. 1988. Oogenesis in Fundulus
heteroclitus. V. The relationship of yolk vesicles and cortical
alveoli. J Exp Zool 246:42–56.
Selman K, Wallace RA, Sarka A, Qi X. 1993. Stages of oocyte
development in the zebrafish, Brachydanio rerio. J Morphol
218:203–224.
Smythe E. 2003. Clathrin-coated vesicle formation: a paradigm
for coated-vesicle formation. Biochem Soc Trans 31:736 –739.
Susca V, Corriero A, Bridges CR, De Metrio G. 2001. Study of the
sexual maturity of female bluefin tuna: purification and characterization of vitellogenin and its use in an enzyme-linked
immunosorbent assay. J Fish Biol 58:815– 831.
Taylor RG, Grier HJ, Whittington JA. 1998. Spawning rhythms
of common snook in Florida. J Fish Biol 53:502–520.
Tyler CR, Sumpter JP. 1996. Oocyte growth and development in
teleosts. Rev Fish Biol Fish 6:287–318.
Wallace RA, Selman K. 1990. Ultrastructural aspects of oogenesis
and oocyte growth in fish and amphibians. J Electron Microsc
Tech 16:175–201.
West G. 1990. Methods of assessing ovarian development in
fishes: a review. Aust J Mar Freshw Res 41:199 –222.