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The Nature of Exocytosis in the Yolk Trophoblastic Layer of Silver Arowana (Osteoglossum bicirrhosum) Juvenile the Representative of Ancient Teleost Fishes.

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THE ANATOMICAL RECORD 292:1745–1755 (2009)
The Nature of Exocytosis in the Yolk
Trophoblastic Layer of Silver Arowana
(Osteoglossum bicirrhosum) Juvenile, the
Representative of Ancient Teleost Fishes
MARTA JAROSZEWSKA AND KONRAD DABROWSKI*
School of Environment and Natural Resources, The Ohio State University,
Columbus, Ohio
ABSTRACT
We have chosen the silver arowana (Osteoglossum bicirrhosum), a
representative of the most ancient teleost family Osteoglossidae, to address
the question of yolk nutrients utilization. Silver arowana have particularly
large eggs (1–1.5 cm of diameter) and a unique morphology of the yolk. We
present evidence that the yolk cytoplasmic zone (ycz) in the ‘‘yolksac juveniles’’ is a very complex structure involved in sequential processes of yolk
hydrolysis, lipoprotein particles synthesis, their transport, and exocytosis.
Vacuoles filled with yolk granules in different stages of digestion move
from the vitellolysis zone through the ycz to be emptied into the microvillar
interspace in the process of exocytosis. The area of the ycz with the abundance of the mitochondria must play an important role in providing energy
for both the transport of vacuoles and the release of their contents. Therefore, we postulate that the function of yolk syncytial layer (ysl) as the
‘‘early embryonic patterning center’’ transforms in fish larvae or yolksac
juveniles into a predominantly specialized role as the yolk trophoblastic
layer (ytl) involved in yolk nutrients utilization. In addition to discovering
the mechanism of transformation of the ysl function into ytl function, we
suggest that the machinery involved in nutrient mobilization and exocytosis in yolk of arowana yolksac juveniles can be very attractive system for
studies of regulatory processes in almost all secretory pathways in animal
C 2009 Wiley-Liss, Inc.
cells. Anat Rec, 292:1745–1755, 2009. V
Key words: osteoglossidae; yolksac juvenile; yolk trophoblastic
layer; microvillar layer; exocytosis
The fishes are a large and diverse group containing
five subclasses (Holocephali, Elasmobranchii, Cladistia,
Chondrostei, and Neopterygii) divided into 60 orders
(Nelson, 2006). Although the early ontogeny varies
substantially across subclasses and, also, orders, the
development of embryos and larvae or ‘‘yolksac
juveniles’’ (the nomenclature according to Balan, 1999)
in all fishes is based on the use of materials accumulated
in the yolk as the source of nutrients and energy. The
most characteristic feature of the yolk is the syncytial
layer, which is derived from collapsed marginal blastomeres in early cleavage stages and forms a syncytium
between the yolk cell and inner cell mass, the precursor
of the embryo (Betchaku and Trinkaus, 1978; Trinkaus,
1993; Krieger and Fleig, 1999). Researches indicate that
C 2009 WILEY-LISS, INC.
V
Grant sponsor: USAID [Aquaculture Collaborative Research
Support Program (A-CRSP)]; Grant number: LAG-G-00-9690015-00; Grant sponsor: Nicolaus Copernicus University
(NCU); Grant number: 325-B.
*Correspondence to: Konrad Dabrowski, The Ohio State University, School of Environment and Natural Resources, 210
Kottman Hall, 2021 Coffey Rd., Columbus, OH 43210.
E-mail: dabrowski.1@osu.edu
Marta Jaroszewska is currently affiliated with Nicolaus Copernicus University, Institute of Ecology and Environment Protection, Laboratory of Histology and Embryology of Vertebrates,
Gagarina 9, 87-100 Torun, Poland
Received 4 September 2008; Accepted 22 June 2009
DOI 10.1002/ar.20996
Published online 18 September 2009 in Wiley InterScience (www.
interscience.wiley.com).
1746
JAROSZEWSKA AND DABROWSKI
the yolk syncytial layer (ysl) in teleosts serves as the primary motor for blastoderm epiboly (Betchaku and Trinkaus, 1978; Trinkaus, 1984a,b, 1993; Solnica-Krezel and
Driever, 1994). The induction and/or patterning of anterior neural tissue, the body axes, formation of the ventrolateral mesoderm and induction of the nodal-related
genes in the ventrolateral marginal blastomeres during
early embryonic development are also attributed to the
ysl (Ho et al., 1999; Hyodo et al., 1999; Chen and Kimelman, 2000). The ysl function is connected with the morphogenesis of the organizer epithelium (D’Amico and
Cooper, 2001; Cooper and Virta, 2007), the regulation of
cardiac tissue morphogenesis (Sakaguchi et al., 2006),
and the formation of the liver bud (Li et al., 2007).
As the teleost fish embryo and larva mass increases
exponentially, the function of the ysl as the ‘‘early
embryonic patterning center’’ (Ho et al., 1999; Sakaguchi et al., 2006) is transformed into those functions
predominantly involved in yolk digestion, synthesis,
and release of nutrients from the yolk to the growing
body (Krieger and Fleig, 1999). In contrast to other
lower vertebrates, including non-teleost fish, teleosts do
not have a connection between the endoderm of the
presumptive gut and yolk reserves during the whole
embryonic and early larval development (Kunz, 2004).
As it was demonstrated in the larvae of one of
the most evolutionarily advanced group of teleosts,
cichlids (order Perciformes), the ysl is engaged in yolk
mobilization and involved in releasing metabolized
vitellogenic nutrients into the vitellus vasculature
(Fishelson, 1995).
The ysl function of directing cell movement and nourishing do overlap at some developmental stages. Then,
the function of the ysl in initiating cell movement
becomes less distinct after the completion of gastrulation, and its role in yolk utilization becomes more profound. Most studies thus far dealt with the
histochemical aspect of the yolk utilization sequence in
teleost development (Vernier and Sire, 1977a,b; Walzer
and Schönenberger, 1979a,b; Sire et al., 1994). Poupard
et al. (2000) described the process of the utilization of
yolk reserves during larval development of turbot
(Scophthalmus maximus) and emphasized that the apoE
gene expression in the yolk cell is parallel to very lowdensity lipoprotein particles exocytosis into a perisyncytial space. However, a few investigators have addressed
the cytological basis of endogenous feeding, particularly
utilization of yolk reserves in teleost fish embryos (Shimizu and Yamada, 1980; Sire et al., 1994) and larvae
(Vernier and Sire, 1977a,b; Sire and Vernier, 1979;
Walzer and Schönenberger, 1979a,b; Kjørsvik and
Reiersen, 1992; Mani-Ponset et al., 1994). As much as
we know about the roles of the ysl in early development
and organogenesis, the structure that correlates with its
function in larval/yolksac juvenile of fish development
remains unexplored, especially in the most ancient teleost fish, Osteoglossomorpha (Assheton, 1907).
Among the representatives of family Osteoglossidae
just a few species exhibit parental care by mouthbrooding. It occurs in fish from genus Osteoglossum and
Scleropages (Nelson, 2006). Female lays approximately
200 large eggs (1–1.5 cm diameter), which male takes
into the mouth for completion of embryonic and yolksac
juvenile development. Egg size affects the pattern of
cleavage (Colazzo et al., 1994) and early development,
which is associated with differences in molecular organization of embryonic patterning (Buchholz et al., 2007).
Silver arowana (Osteoglossum bicirrhosum) has large
eggs and yolksac juvenile stage of development lasts
nearly 2 months when young are incubated in parent’s
buccal cavity (Aragão, 1984). There is no mixed feeding
phase during this time (our observation; Maupin, 1967).
It can be assumed that, following the discussion on the
early life history in cichlids (Balon, 1999), in silver arowana, an increased yolk density and volume, combined
with a prolonged incubation in parents mouth, rendered
juveniles to become larger and better developed, prepared for independent exogenous feeding. In comparison
with silver arowana, in all precocial mouthbrooding
cichlids, the endoexogenous feeding was described. For
example, in Labeotropheus trewavasae, with the largest
eggs (4.4 mm of diameter), the young ones are released
from the protection of the buccal cavity of parents and
start to feed exogenously at 15 mm of body length when
they are 24-day-old (Balon, 1999).
The aim of this study was to describe the yolk structure during later yolksac juvenile stages in O. bicirrhosum. By using the unique morphology of arowana
juveniles’ yolksac, we aimed to understand the ysl structure and cellular processes involved in endogenous
reserves utilization. We investigated highly structured
yolk cytoplasmic zone (ycz) of the ysl, populated, among
other organelles, by vacuoles filled with yolk material in
different stages of digestion. This is the tissue devoid of
nuclei and equipped with the exocytosis machinery at
the microvillar layer of the ycz. Based on those results,
it is postulated that the term yolk syncytial layer (ysl)
should be limited to an early development and organogenesis in fish and be renamed and called yolk trophoblastic layer (ytl) in the later development to manifest
the link between endotrophic nutrition and growth of
the metamorphosing, differentiating larval or yolksac
possessing juvenile fish.
This article presents new findings on the subcellular
level related to utilization of the yolk. We concentrated
on an interphase between the yolk and the delivery
system of exocytosed lipoproteins into the vesicular
vitelline system that are then distributed into target tissues. On the basis of the provided transmission electron
microscopy (TEM) data, we suggested that the ytl is the
functional equivalent of the secretory membrane that
can be used as a convenient model to study exosomes,
vesicles released in cell-to-cell communication, antigen
presentation, and many other functions in animal cells
(Marsh and van Meer, 2008).
MATERIAL AND METHODS
The healthy silver arowana’s, O. bicirrhosum, yolksac
juveniles used in this study were obtained in January
2007 and 2008 through ornamental trade outlets and,
according to the dealer, were originally from Colombia.
Based on the illustration in the literature (Aragão, 1984)
and total body length of yolksac juveniles that we
obtained (50–70 mm), we estimated that fishes were
aged approximately 1 month after hatching. Fishes were
kept in 40 L glass aquaria (12–15 fish/tank) supplied
with freshwater and maintained under constant flowthrough conditions. Water temperature was between
YOLK TROPHOBLASTIC LAYER OF SILVER AROWANA
Fig. 1. Silver arowana individual with an extended external part of
the yolksac. With yolksac juvenile of size 6–7 cm, these are some of the
largest larvae and yolksac juveniles among teleosts. Portions of the
yolksac examined under light and TEM are indicated by boxes with
numbers referring to corresponding histological pictures, Fig. 2a–c.
26 C and 30 C. A feeding was attempted several times
during observation of yolk absorption, but there was no
evidence of active feeding at this stage until the yolksac
remnants were visible protruding outside of the abdomen. Fishes were anesthetized in water with MS 222
(Tricaine methanesulfonate; Argent Chemical Laboratories, WA) at the concentration of 75 mg/L.
All procedures and handling of animals during the
experiments were conducted in compliance with the
guidelines of the Institutional Laboratory Animal Care
and Use Committee, The Ohio State University (Animal
Protocol 2007A0221).
Histological Studies (Light Microscopy)
Twelve individuals were fixed for histological analyses.
Fishes were ranging from 50 to 66 mm of total body
length, with yolksac extended up to 11.8 mm (Fig. 1) to
almost remnants, not visible outside. Some individuals
were observed at the stage of almost complete yolk
absorption. Immediately after anesthesia yolksac juvenile were fixed in 10% buffered formalin and kept for 72
hr. After fixation, specimens were rinsed in water and
transferred to 75% ethanol for 2 weeks. Body length and
yolk dimensions were measured before histological procedures and after fixation and storage in alcohol. All
yolksac juveniles were paraffin-embedded after dehydratation with a graded ethanol series and treated with xylene. Histological sections of the thorax with inclusion of
the yolksac were cut transversally (8 individuals) or longitudinally (4 individuals) into a complete series of serial
sections (5 lm). Sections were mounted on albumincoated glass slides, processed for staining with Meyer’s
hematoxilin and eosin. Alcian blue/periodic acid-Schiff
(AB/PAS, pH 2.5; counterstained with Gill’s hematoxylin) procedure was performed to identify glycogen
(Merck) on transverse section with the liver. All obtained
sections, on average 6 per 80–90 slides for one individual, were observed under the light microscopy. Micrographs were taken with a Zeiss Axioscope microscope
(Carl Zeiss) and the Olympus MagnaFire Digital camera
(Olympus).
Transmission Electron Microscopy Studies
Six arowana yolksac juveniles were used for TEM.
The total fish body length was from 60 to 70 mm and
was inversely proportional to the yolksac length (12.5–
1747
17.4 mm; external yolksac mass amounted to 50% of the
body weight).
Whole fish were fixed for 3 hr in room temperature in
(1) 0.15 M cacodylate-buffered 2% glutaraldehyde with
4% paraformaldehyde at pH 7.3 and (2) 0.15 M cacodylate-buffered 2.5% glutaraldehyde at pH 7.2 with 2.5%
sucrose to adjust osmolarity to 350 mOsm according to
Diaz et al. (2002). Then, the yolksac membrane was
removed, and the small pieces of yolk surface (1 mm in
depth) were taken and kept in adequate fixative overnight in 4 C. The samples were rinsed several times in
buffer, postfixed with 1% osmium tetroxide in 0.15 M
cacodylate buffer, dehydrated in a graded series of ethanol and propylene oxide, and embedded in Eponate 12
(Ted Pella). To recognize the required section, blocks
were cut (1 lm) transversally, placed on slides, and
stained with 1% methylene blue with 1% azure II in 1%
borax (sodium tetraborate ) or 2% toluidine blue with
2% borax and 1% pyronin B. Then, the section (0.5 lm),
obtained by a Leica EM UC6 ultramicrotome, were
stained with 2% uranyl acetate and Reynold’s lead citrate and examined with a FEI Tecnai G2 Spirit Biotwin operated at 80 KV transmission electron
microscope.
RESULTS
Histological Studies (Light Microscopy)
The yolk of silver arowana is divided into two parts:
an external part present in the yolksac, and an internal
part distributed among organs in the abdominal cavity
(Figs. 1, 2a–c). The yolksac is composed of external part
of the yolk, which protrudes from the body cavity, and
the covering integument (Fig. 2c). The internal compartment of the yolk is a directly connected part of the external yolk, and its inner extension can be found around
the liver, between the liver and the stomach, and the
gas bladder (Figs. 2a,b, 3a–e). It was evident in transverse sections of the liver tissue that both the internal
and external yolk are provided by the liver with blood
vessels, especially associated with a yolk trophoblastic
layer (ytl) in the external yolk (Fig. 2b,c). In all examined individual fish, the hepatocytes were filled with
yolk droplets. It was revealed by the light microscopy
techniques, when staining with hematoxylin and eosin,
as well as with AB/PAS, that the particles in the internal yolk, blood, and the content of hepatocytes had the
same color. This result suggests presence of the same
substances in these tissues (Fig. 3d–e). No glycogen was
found in hepatocytes. TEM analysis of hepatocytes confirmed the presence of yolk platelets in the cytoplasm of
cells lying close to the blood vessels (Fig. 3f) and the internal yolk (Fig. 3d inset, 3g).
There are differences in the structure of the external
and internal part of the yolk, including the appearance
of blood vessels on their surface. The internal part of the
yolk is vascularized mainly by capillary vessels, whereas
the external part of the yolk is supplied by an extensive
vitelline circulation of large blood vessels (Fig. 4a,b).
There was no direct evidence of the different rate of yolk
utilization in those two different compartments. Based
on the results of the latest yolksac-juvenile stages, and
appearance of remnants of the external and internal
part of the yolk (Figs. 2a–c, 5a–d), the similar rate of
the nutrient utilization for both parts of the yolk is
1748
JAROSZEWSKA AND DABROWSKI
suggested. When yolk reserves are exhausted, the ytl is
resorbed (Fig. 5a–d) and does not take a part in the
formation of the fish body. In all individual fish
examined, the alimentary tract was found highly
differentiated into esophagus, stomach, and intestine
(Figs. 3a–3c, 5b, and 6a–b).
Transmission Electron Microscopy Studies
of the Yolk
Fig. 2. Overview (transverse sections) of the body cavity and yolksac
(ys) of silver arowana juvenile; (a) inner part of the yolk (iy) above the liver
(liv), surrounding gas bladder (gb) and oesophagus (oe); there are fluid
filled spaces (arrows) might have been formed as the results of yolk digestion, where the yolk-blood capillaries are close to liver; (b) note the close
apposition of the liver (liv) and internal part of the yolk (iy). Liver spreads
into the external part (ey) of the yolk (arrowheads on the right) and provides blood vessels (asterisk) neighboring with the ytl (arrowheads on the
left); (c) the inner compartment of the yolk is an extension of the external
part. Fig. 2a and 2b are the discontinuous presentation of the yolk. The
connecting fragment, the liver, and the yolk, is shown at higher magnification in Fig. 3a–c. Rectangles in (a) illustrate the region when micrograph
for Fig.8a [\\\] and Fig.8c [///] were taken; rectangle in (b) refers to Fig. 3f–
g, where the hepatocytes located between internal yolk and blood vessels
are shown; rectangle in (c) refers to Fig.4b and Fig.7f–i where the ytl is presented. Other abbreviations: (#) shows the artifact, present in all examined
individuals, as the result of different shrinking of soft tissue and massive
yolk fluid in the fixatives. Stained with hematoxylin and eosin.
Microscopic studies of the surface of the external yolksac in silver arowana showed that the yolk cytoplasmic
zone (ycz; nomenclature used according to Walzer and
Schönenberger, 1979a,b) of the ytl is the highly organized structure that, as a thin layer, encompasses the
acellular yolk mass (Fig. 7a–f). The ycz adjoins ventrally
to the vitellolysis zone (nomenclature according to
Walzer and Schönenberger, 1979a,b), containing yolk
platelets. The ycz is sequentially composed of three
domains (zones): (1) an inner domain, localized basally
in the ycz (Fig. 7a), consists mainly the rough endoplasmic reticulum (RER) and some mitochondria and Golgi
complexes that appear locally close to the RER system
and mitochondria apparatus (Fig. 7b–d); (2) the middle
domain is composed of an abundance of mitochondria
between numerous lipoprotein-filled secretory vesicles
(Fig. 7e); and (3) the most external microvillar layer
(Fig. 7f–i ). No nuclei were found in the ycz surrounding
the visible outside part of the external yolk. Throughout
all domains of the ycz, there are vacuoles filled with yolk
material in different stages of digestion (Fig. 7b,f–i). The
RER system is composed of long and flat cysternae
(trabeculae), bordered by osmiophilic granules, and is
orientated in a parallel direction to the yolk sphere
(Fig. 7b). The microvillar layer constitutes a zone of
intense exocytosis of yolk material. The secretory
vesicles are transported toward the yolk trophoblastic
layer membrane, fuse with the membrane, and their
content is exocytosed in the vicinity of blood capillaries
of a vitelline circulation. The endothelium of capillaries
is involved in the transport of lipoprotein particles
through the microvillar interspace into the lumen of
blood vessels (Fig. 7f,g).
Ultramicroscopic studies have revealed that the internal part of the yolk is devoid of the ytl and its structure.
The internal yolk is not organized into zones when its
surface is in close proximity to alimentary tract (not
shown), blood capillaries, and liver (Fig. 8a–c). Besides
yolk platelets and very rare mitochondria, no other cell
organelles were found in this yolk compartment.
DISCUSSION
The morphological analysis of the yolksac structure in
the juveniles of silver arowana revealed the presence of
external and internal parts of the yolk. These results
confirmed morphological observations of ‘‘migrating
yolk’’ in the body cavity of the juveniles of this species
(Trilleras, 2005). It is hypothesized that the division of
the yolk into two parts is an adaptation during arowana
development that highlights both the ytl and liver as the
distinguished sites of mobilization and release of
nutrients in a quantitatively significant manner. It can
be summarized that, in silver arowana, its large yolk is
used efficiently as the result of combined effect of
YOLK TROPHOBLASTIC LAYER OF SILVER AROWANA
1749
Fig. 3. Transverse sections of the body cavity show the internal
yolk (iy) to be in close proximity to the liver (liv), when sieve-like places
are representative of the yolk utilization; (a,b) the inner yolk compartment is very often found between ‘‘liver lobes;’’ (c) the liver tissue (liv)
provides a surrounding to establish direct contact of blood vessels
(bv) or d–e) is surrounded by the internal part of the yolksac; arrows in
(d) and inset (9,300) indicate yolk droplets visible inside hepatocytes
and in (e) the blood in color of yolk substance; In (f) the hepatocytes
protruding into the internal yolk are seen. (g; 6,800) is TEM image
showing yolk material (arrowheads) inside the hepatocytes, like in Fig.
3d inset and 3f. Other abbreviations: asterisks show lipid vacuoles
emptied following alcohol treatment during tissue processing, nucleus
(n), oesophagus (oe), pyloric part of the stomach (pc), (#) shows the
artifact, present in samples, as the result of different shrinking of soft
tissue and massive yolk fluid in the fixatives; Rectangle in 3c refers to
3f–g. Stained with hematoxylin and eosin (3a–d) and (3e) with AB/PAS.
prolonged parental care, yolk division into two compartments, which are mobilized simultaneously by the ytl
and the liver during the juvenile stage.
The examination of the external yolk in silver arowana juveniles revealed the cytoplasmic zone of the ytl,
above the vitellolysis zone, similar to that reported by
Walzer and Schönenberger (1979a,b) in brown trout
(Salmo trutta morpha fario) immediately after hatching.
The presence of cellular organelles in the cytoplasmic
region in the silver arowana corresponds to those in the
larvae of trout, Atlantic halibut (Hippoglossus hippoglossus), sea bass (Dicentrarchus labrax), and pike-perch
(Sander lucioperca; Walzer and Schönenberger, 1979a,b;
Kjørsvik and Reiersen, 1992; Sire et al., 1994; Mani-
1750
JAROSZEWSKA AND DABROWSKI
Fig. 4. (a) The internal yolk is surrounded mainly by capillary vessels (arrow) and rare collecting blood vessels (asterisk); (b) the external part of the yolk is supplied with amount of larger blood vessels
(black arrows), laying under the very thin dermis covered by multilayered, stratified epidermis (ep and white arrows). mfys ¼ massive fluid
yolk sphere; ytl ¼ yolk trophoblastic layer. Stained with methylene
blue with azure II in borax (a) and hematoxylin and eosin (b).
Ponset et al., 1994, 1996). To the contrary, some other
fish species do not seem to have stratified ‘‘periblast,’’
such as Atlantic cod (Gadus morhua), Atlantic halibut,
gilthead sea bream (Sparus aurata), sea bass, and pikeperch (reviewed and summarized by Hoehne-Reitan and
Kjørsvik, 2004). We showed for the first time in a teleost’s yolksac juvenile, silver arowana, that the ycz is a
very complex structure involved in well-organized processes including yolk hydrolysis, synthesis of lipoprotein
particles and their transport, and, in particular, exocytosis. Vacuoles filled with yolk granules in different stages
of digestion move from the vitellolysis zone through the
ycz to be emptied into the microvillar interspace. The
area of the ycz with the abundance of the mitochondria
must play an important role in providing energy for the
transport of vacuoles and for an intense exocytosis. Despite the fact that the Walzer and Schönenberger
(1979b) have postulated the polarization and secretory
function of the cytoplasmic zone, no exocytosis was
described in any previous work on the development and
ultrastructural organization of the ysl/ytl before or after
hatching in teleost larvae (Vernier and Sire, 1977a,b;
Walzer and Schönenberger, 1979a,b; Long, 1980; Shimizu and Yamada, 1980; Sire et al., 1994). Krieger and
Fleig (1999) have postulated that, in European perch
Perca fluviatilis embryos, the exocytosis, endocytosis,
and intarcellular digestion of yolk proteins and lipid in
the ysl and inner cell mass is the pathway of yolk utilization. However, they did not provide proof for this
statement and suggested that the transportation mechanism of yolk and lipid from the yolk cell to the embryo
in European perch is different in comparison to embryos
with other teleosts, such as salmonids (Walzer and Schönenberger, 1979a,b). Mani-Ponset et al. (1996) suggested
even presence of endocytotic vesicles involved in
exchanges with the perisyncytial space. No pinocytotic
or phagocytic vacuoles were observed close to the ysl microvilli in Prochilodus lineatus (Ninhaus-Silveira et al.,
2007). Our findings documented exocytotic vesicles and
active secretion process in the ytl.
Fishelson (1995) was the first author who hypothesized that microvilli described in the ysl of the three species of cichlid (tilapias) larvae are involved in transport
of nutrients across the monolayered capillary vessel endothelium into the embryonic blood, calling it a microvillar food transporting yolksac syncytial surface. The
report on the process of the exocytosis of very low-density lipoprotein particles into the perisyncytial space in
turbot (Scophthalamus maximus) does not provide evidence of microvillar structure of the plasma membrane
of the yolk cell (Poupard et al., 2000). In fish embryos
microvilli were observed in Fundulus (Trinkaus, 1984b)
and Prochilodus lineatus (Ninhaus-Silveira et al., 2007).
It was postulated by Mani-Ponset et al. (1994, 1996)
that the microvilli protruded into the perisyncytial
(‘‘perivitelline space" according to Mani-Ponset et al.,
1994, 1996) circulatory space in gilthead sea bream, sea
bass, and pike-perch larvae. However, in these cases, a
more folding-like structure was observed, considerably
different from the regular, elaborate, high-density microvillar structure described here in arowana. Diversity of
the microvilli appearance in teleost embryo was reported
by Trinkaus (1984b) who described that, during epiboly,
the long microvilli on the ysl surface are gradually
replaced by much shorter ones, but he gave no explanation regarding the purpose of this change.
The literature on the preimplantation of the yolksacpossesing shark (i.e., Rhizoprionodon terraenovae) provided evidence of highly organized yolk syncytium, populated by cellular organelles, and involved in systems of
yolk nutrient mobilization (Hamlett et al., 1987). The
authors described yolk droplets in the shark being
released from the membrane-limited vesicles and then
fusing with the membrane enveloping the yolk syncytium. The yolk droplets are released after the fusion
with syncytium membrane into the yolk syncytial-endoderm interspace (Hamlett, 1987; Hamlett et al., 1987).
Subsequently, the endoderm endocytoses the yolk to
digest platelets. However, there is no evidence of exocytosis at the vascular surface of the endodermal cells,
which mediate the transfer of metabolites from the yolk
mass to the extraembryonic circulation (Hamlett and
Wourms, 1984). This study in silver arowana revealed a
microvillar structure of the ycz with a massive secretory
YOLK TROPHOBLASTIC LAYER OF SILVER AROWANA
1751
Fig. 5. Longitudinal (a) and transverse (b) sections through body of arowana with yolk digested in 70%
and with yolk remnants, respectively, illustrating the similar proportion of external part of the yolk (ey) to
the internal compartment (iy), when used. alt ¼ alimentary tract; liv ¼ liver, musc ¼ muscles, pan ¼
pancreas. Stained with hematoxylin and eosin.
activity. Therefore, we may reasonably assume a similarity between preimplantation ultrastructure of the yolk
and utilization in the sharks and in most teleosts (with
the exception of Poecillidae; Hamlett and Wourms 1984;
Hamlett et al., 1987). However, the exocytosis in arowana occurs on the vascularized surface of the ytl. The
secretory vesicles appear identical in arowana and in
turbot (Poupard et al., 2000). However, in early stages of
turbot, like in gilthead sea bream, sea bass, or pikeperch, the vesicles are released into the perisyncytial
space, that is devoid of vascular network within the
walls of the yolksac (Mani-Ponset et al., 1996; Poupard
et al., 2000). In fact, there is no mediating endodermal
layer in arowana, which is a difference between Osteoglossidae, and most likely, the majority of teleosts, in
comparison with viviparous Elasmobranches. In addi-
tion, the process of exocytosis in silver arowana larvae
contradicts the theory that the acellular structure of the
ysl in the trout proves a more ancient evolutionary stage
than that attained by endodermal cells in birds (Walzer
and Schönenberger, 1979a). It was also previously
postulated that the ysl or ytl, as we suggested based on
our results, microvillar structure in tilapia larvae is
analogous to the folding of the yolksac of avian eggs
(Fishelson, 1995).
The fish liver arises and remains in the close vicinity
to the yolk. Because liver is well vascularized, this organ
plays an important role in circulation of yolk nutrients
to the gut and other organs (Morrison et al. 2001; Kunz
2004). The liver also regulates lipid mobilization and
synthesis of lipoproteins in connection with the use of
yolk reserves (Hoehne-Reitan and Kjørsvik, 2004).
1752
JAROSZEWSKA AND DABROWSKI
Fig. 6. Cross-section of stomach fundus (a) and longitudinal section through middle intestine mucosal
folds (b) of silver arowana yolksac juvenile at 55 mm TL and large yolk at length of 8.12 mm; (a) arrow ¼
gastric epithelium; asterisks ¼ gastric glands; lm ¼ lamina priopria; mus ¼ muscles; arrowheads ¼
mucosal cells. AB/PAS stain.
Moreover, it is assumed that the developing liver is
involved in the internal yolk mobilization, as we often
observed and documented in this work, yolk platelets
are present in large number in hepatocytes. This function of the liver is particularly puzzling in respect to the
finding that in silver arowana, internal part of the yolk
does not appear to posses the ytl. Fishelson (1995)
argued, although without direct evidence, that the presence of yolk granules in the primordial liver cells of
cichlid fish larvae can be interpreted as being a result of
the similar absorption process as in avian embryos. In
other species, like gilthead sea bream larvae (Guyot
et al., 1995) and pike-perch larvae (Diaz et al. 2002),
during the endogenous phase of feeding, the liver accumulates mainly glycogen, until the endoexogenous
(mixed) feeding period starts. Results presented here
show no glycogen content in hepatocytes of silver arowana yolksac juveniles, and they are in agreement with
studies on the early yolksac stage in Atlantic cod and Atlantic halibut (Hoehne-Reitan and Kjørsvik, 2004). The
later authors emphasized that hepatic glycogen can only
be observed in these two species after endotrophic phase
is completed. Hoehne-Reitan and Kjørsvik (2004) concluded that the observed differences between fish species
in hepatic glycogen content during the endogenous
phase of feeding could be related to the initial (maternal)
origin of yolk lipid content and the preferred substrate
for larval energy metabolism at yolksac stage.
This study described the ultrastructure of the ytl–vascular complex in the yolksac juvenile of silver arowana
and provided new information that could be used in
studies on the mechanism by which nutrients from the
yolk are transformed into body tissues. The highly
organized yolk cytoplasmic zone (ycz) of the ytl, populated by cellular organelles and devoid of nuclei, as well
as equipped with the exocytosis machinery at the microvillar layer of the ycz was revealed for the first time in
Osteoglossomorpha. The silver arowana is the most ancient extant lineage of Teleostei, and for this reason, it
was assumed that results obtained in this species could
provide some arguments for comparative evolutionary
theories in a phylogenetic framework. From the phylogenetic point of view, the yolk trophoblastic layer of arowana marks the continuity of this structure in the
actinopterygian fish. This opinion is supported by studies on zebrafish (Danio rerio) that presented evidence of
the ysl structure is functioning in a very similar manner
to the higher vertebrates (mouse) yolk endoderm (Ho
et al., 1999). In birds, the hypoblast of the embryo that
becomes the origin of gut endoderm lies over the yolk
and serves the major role in yolk absorption (Yoshizaki
et al., 2004). Ho et al. (1999) alluded to the fact that the
Fig. 7. TEM micrographs of the yolk trophoblastic layer (ytl): (a;
30,000) shows the formation of small degradation yolk vesicles cut
out of the yolk platelets; in this process, which occurs within the vitellolysis zone, large yolk droplets are divided into smaller yolk vesicles
(arrows); (b; 49,000) in the inner domain RER cisterns are located
between yolk platelets (P); (c; 30,000) the middle domain composed
of mitochondria (m) with some RER elements and Golgi apparatus (d;
68,000, arrowheads); (e; 49,000) the ycz of silver arowana is characterized by a heterogenous population of lipoprotein-filled secreting
vesicles (sv) with yolk nutrients in different stages of digestion; (f;
11,000) the figure shows the microvillar layer (mv), attached to the
blood capillaries (bc); note the numerous mitochondria (m) located
close to the base of the microvillar layer below secreting vesicles; (g;
11,000) the microvilli adjoin the vitelline vessels endothelium (arrow);
(h; 30,000) the content of the yolk vesicle is released by exocytosis
(circles) into the microvillar interspace; (i; 49,000) micrograph in higer
magnification illustrates moment of exocytosis (circles) in the microvillar layer of the ycz.
1754
JAROSZEWSKA AND DABROWSKI
Fig. 8. (a; 9,300) and (b; 6,800) present TEM micrographs of the
internal yolk (IY) and blood vessels in close apposition; no ytl is distinguishable in internal yolk structure in its contact zone with endothelium (arrows); the samples are taken for different individuals in the
region, as it was showed in Fig. 2a; (c; 4,800) presentation of the
contact between hepatocytes (hep) and yolk platelets of the internal
yolk, where the ytl is also not present. Lipid vacuoles (asterisk) were
emptied after alcohol treatment during tissue processing.
ysl in teleosts is the ‘‘functional equivalent’’ of mammalian visceral endoderm. It could be assumed that the
nonplacental and placental vertebrate connection can be
portrayed at the level of the ysl and then the ytl. Our
results confirmed a postulate of Cooper and Virta (2007)
that studies on the presence and function of the ysl and
ytl in Osteoglossidae might provide the missing link
between different lower vertebrates. We suggest that the
cytoplasmic zone of the ytl in an ancient teleost appears
to be a unique model system for studies of membrane
transport and secretory activity that is still requiring
many explanations (Marsh and van Meer, 2008).
Based on the work by Finn et al. (2007a,b) who examined phylogenetic relationships in vitellogenin structures
among fish, it is postulated that further studies are
needed to characterize the vitellogenin (vtg) genes in silver arowana. The functional studies of lipoprotein substances in the context of their hydrolysis, their transport
through the yolk, and especially the ytl, as well as the
exocytosis into the vitelline circulation, are currently
under way in our laboratory.
Poland and were conducted in the Laboratory of Electron and Confocal Microscopy at NCU Torun, Poland by
the supervisor Janusz Niedojadlo with assistance and
technical support of Maria Kowalczyk.
ACKNOWLEDGMENTS
Authors of this work share Balon’s (1986, 1999) opinions in regard to the nomenclature of teleost ontogenic
stages, in particular fish species ‘life history’. However,
they decided that this is not the aim of this work to alter
the nomenclature used in the original form of cited
articles. Therefore, they encourage readers to critically
review of the existing evidence in favor of terminology
coined by E. Balon. The Aquaculture CRSP accession
number is 1350. They are grateful to Kathy Wolken and
Brian Kemmenoe from Imaging Facilities of The Ohio
State University, Columbus, USA for assistance in sampling and TEM observations, and Dr. Paul Baumann,
USGS, Columbus, for critical reading of the manuscript.
Some parts of this work were support by a grant 325-B
from Nicolaus Copernicus University (NCU) in Torun,
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exocytosis, silver, trophoblast, arowana, fisher, teleost, representation, ancient, yolk, nature, bicirrhosum, osteoglossum, layer, juvenile
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