Hatching envelope formation in the egg of the black tiger shrimp

Transcription

Hatching envelope formation in the egg of the black tiger shrimp
Aquaculture Research, 2012, 1–12
doi:10.1111/j.1365-2109.2012.03141.x
Hatching envelope formation in the egg of the black
tiger shrimp, Penaeus monodon (Decapoda,
Penaeidae)
Pattira Pongtippatee1, Wanita Putthawat2, Pornsawan Dungsuwan2,
Wattana Weerachartyanukul3 & Boonsirm Withyachumnarnkul1,3,4,5
1
Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla
University, Surat Thani campus, Surat Thani campus, Surat Thani, Thailand
2
Department of Anatomy, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkla, Thailand
3
Department of Anatomy, Mahidol University, Bangkok, Thailand
4
Center of Excellence for Shrimp Biotechnology and Molecular Biology (Centex Shrimp), Faculty of Science, Mahidol
University, Bangkok, Thailand
5
Shrimp Genetic Improvement Center, The National Center for Genetic Engineering and Biotechnology (Biotec), Surat
Thani, Thailand
Correspondence: B Withyachumnarnkul, Centex Shrimp, Chalerm Prakiat Building, 4th Floor, Faculty of Science, Mahidol University, 272 Rama 6th Rd., Bangkok 10400, Thailand. E-mail: wboonsirm@yahoo.com; P Pongtippatee, Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani 84100, Thailand.
E-mail: pattirataweepreda@yahoo.com
Abstract
The aim of this study was to reveal the process of
hatching envelope (HE) formation in eggs of the
black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti-FITC-conjugated gold-lectin labelling. Following lectin
binding screening tests, Concanavalin A (Con A)
and wheat germ agglutinin (WGA) were chosen to
trace movements of specific sugar-associated components of the HE. This revealed that both Con A
and WGA-binding components migrated from the
ooplasm to the HE. Using TEM, it was revealed that
membranous materials in the ooplasm were
released at the time of spawning, that these became
associated with granular structures outside the
oocyte and that they together developed into an
outer layer of the HE. Contents of flocculent vesicles
and dense vesicles in the ooplasm were exocytosed
and formed the inner layer of the HE. The TEM
with gold-labelled Con A and WGA revealed that
the dense and flocculent vesicles and the inner
layer of the HE contained components associated
with mannose (sugar affinity to Con A) and N-acetyl-b-D-glucosamine (sugar affinity to WGA).
© 2012 Blackwell Publishing Ltd
Keywords: Penaeus monodon, hatching envelope,
egg activation, perivitelline space, lectin binding
Introduction
Eggs of several crustacean species are morphologically and biochemically modified immediately after
spawning. The process, termed egg activation,
includes extrusion of cortical rods to form a jelly
layer covering the oocyte, followed by formation of
the hatching envelope (HE) or egg shell. In the
process of HE formation, several types of vesicles,
initially evenly distributed in the ooplasm, move
towards the cortical area of the egg, and were
thus named cortical vesicles. These vesicles fuse
with the oolemma and release their contents to
bind with other components outside the oocyte
and together form the HE (Clark, Lynn & Persyo
1980; Clark, Yudin, Griffin & Shigekawa 1984;
Pillai & Clark 1988, 1990; Yano 1988; Clark,
Yudin, Lynn, Griffin & Pillai 1990; Blades-Eckelbarger
& Marcus 1992; Hirose, Toda, Saito & Watanabe
1992; Lynn, Glas & Green 1992).
Almost all animal eggs undergo activation
following fertilization and morphological and biochemical events during egg activation have been
described in several species, and some basic
1
Hatching envelope of Penaeus monodon P Pongtippatee et al.
similarity are revealed. For example, in the sea
urchin Strongylocentrotus purpuratus, there have
been many publications on the types and contents
of its cortical granules, on mRNA encoding their
proteins and on the release of these proteins and
other substance from the ooplasm for incorporaton into the fertilization envelope (Anstrom,
Chin, Leaf, Parks & Raff 1988; Somers, Battaglia
& Shapiro 1989; Cheng, Glas & Green 1991; Laidlaw & Wessel 1994; Murray, Reed, Marsden, Rise,
Wang & Burke 2000). These details have yet to
be revealed in crustaceans, especially in penaeid
shrimp, some of which are of considerable
economical importance. In the marine shrimp
Sicyonia ingentis, the cortical vesicles are divided
into dense and ring types that release their contents at different times after spawning (Clark et al.
1980, 1984, 1990; Pillai & Clark 1988, 1990).
At the beginning, the dense vesicles release their
N-acetyl-b-D-glucosamine-containing contents that
become part of the outer layer of the HE. Subsequently, the ring vesicles released their mannosecontaining materials to form the inner layer of
the HE (Pillai & Clark 1990). In the black tiger
shrimp Penaeus monodon, the steps in egg activation have been timed and described at the
microscopic level from cortical rod extrusion to
the complete formation of the HE (PongtippateeTaweepreda, Chavadej, Plodpai, Pratoomchart,
Sobhon, Weerachatyanukul & Withyachumnarnkul
2004). However, details of the process in terms of
biochemical analysis and ultrastructural changes
are still lacking.
In this study, lectin-based assays were utilized to
study the distribution of sugar-rich materials in
specific types of cortical vesicles of spawned eggs
and to trace their movement from intracellular
to extracellular locations at the microscopic and
ultrastructural levels.
Materials and methods
Egg collection
At the Shrimp Genetics Improvement Center, Surat
Thani, Thailand, mated P. monodon female broodstock at stage IV of ovarian maturation were
allowed to spawn into a plastic tank containing
200 L of clean seawater (salinity 30 g L 1, alkalinity 150 mg L 1, pH 8.2, 28°C). Fertilized eggs
were collected at 15-s intervals during the first
15 min postspawning and at 15-min intervals
2
Aquaculture Research, 2012, 1–12
thereafter for 1 h. The collected eggs were used
(1) for lectin binding tests carried out using fluorescence microscopy and (2) for transmission electron microscopy (TEM) with and without goldlabelling of lectins. Initial egg collections at
45 min postspawning (the time at which HE formation in most eggs has been completed) (Pongtippatee-Taweepreda et al. 2004) were used for
preliminary screening tests to determine the types
of lectins that would be suitable for the study.
Initial lectin screening tests
Screening tests were carried out according to the
procedure described by Pillai and Clark (1990).
Briefly, egg suspensions were mixed with a lysis
buffer (10 mM Tris-HCl, 2 mM EDTA, 0.4 M NaCl
and 0.01% Nonidet P-40, pH 8, plus 1 mM PMSF)
and gently ground to release naked HE that were
removed and re-suspended in artificial seawater
(460 mM NaCl, 55 mM MgCl2, 10 mM KCl and
10 mM CaCl2) at 4°C. The HE were washed in
phosphate buffered saline (PBS) and incubated in
PBS containing 4% bovine serum albumin (BSA)
to block non-specific reactions. The suspension
was then centrifuged (200 9 g, 5 min) and the
naked HE, thus obtained were washed with PBS
before incubation (1 h at room temperature) with
fluorescein isothyocyanate (FITC)-labelled lectins
(Vector Laboratories, Burlingame, CA, USA),
referred to hereafter as F-lectins at the concentration of 5 lg mL 1. After several washes with PBS,
treated envelopes were observed under an Olympus (Shinjuku, Japan) BX50 microscope fitted with
an Olympus DP50 digital camera. Lectin binding
competitions were carried out by pre-incubation of
the F-lectins with appropriate, specific sugars at a
concentration of 1 mg mL 1 prior to incubation
with the HE samples.
The lectins (and their affinities for oligosaccharides) used in this study were: (1) Concanavalin A
agglutinin (Con A, glucose/mannose), (2) Lens
culinaris agglutinin (LCA, mannose), (3) wheat
germ agglutinin (WGA, N-acetyl-b-D-glucosamine/
sialic acid), (4) Griffonia simplicifolia lectin II (GSL
II), Bauhinia purpurea lectin (BPL), Dolichos biflorus
agglutinin (DBA) and peanut agglutinin (PNA), all
with affinity for N-acetyl-b-D-glucosamine, (5)
Griffonia simplicifolia lectin I (GSL I), soybean agglutinin (SBA) and Maclura pomifera lectin (MPL), all
with affinity for D-galactose and (6) Ulex europaeus
agglutinin (UEA I, L-fucose).
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
Aquaculture Research, 2012, 1–12
Lectin binding observed using fluorescence
microscopy
Egg samples were fixed for 2 h in seawater-buffered 4% paraformaldehyde at 4°C, washed in
PBS, dehydrated in an ethanol series and embedded in LR-White (London Resin Company, England, UK). Thick sections (400 nm) on glass slides
were incubated in blocking solution consisting of
4% BSA for 20 min and treated for 1 h at room
temperature with 5 lg mL 1 F-lectins identified in
the screening binding test. Control sections were
Hatching envelope of Penaeus monodon P Pongtippatee et al.
those incubated with F-lectins that had been preincubated with appropriate sugars. The sections
were washed in PBS, air-dried, mounted in 75%
glycerol and observed with a fluorescence microscope (DP 50; Olympus, Tokyo, Japan).
Transmission electron microscopy
For conventional TEM, egg samples were fixed for
2 h in seawater-buffered 4% paraformaldehyde at
4°C, washed in PBS, postfixed with OsO4, dehydrated in an ethanol series, embedded in resin
(a)
(b)
(c)
(d)
(e)
(f)
Figure 1 Isolated hatching envelopes (HE) incubated with FITC-labelled Con A (a), LCA (c) and WGA (e), together
with their negative control counterparts [(b), (d) and (f)] respectively, pre-incubated with mannose (for Con A and
LCA) and N-acetyl-b-D-glucosamine (for WGA). The scale bar in (a) applies to all of the photomicrographs.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
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Hatching envelope of Penaeus monodon P Pongtippatee et al.
(London Resin Company, England, UK) and viewed
using TEM (JEM-100 CXII, JEOL, Tokyo, Japan).
For TEM with gold-labelling, thin sections (90 nm)
prepared from the LR-White blocks were incubated
in blocking solution for 20 min and treated with
5 lg mL 1 of either F-Con A or F-LCA or F-WGA,
for 1 h. The sections were then treated with
mouse monoclonal anti-FITC gold conjugate
(10 nm particles) (Electron Microscopy Science,
Hatfield, PA, USA) for 30 min at room temperature, before staining with uranyl acetate and lead
citrate, and viewed under TEM (100-CX II, JEOL).
Control sections were those incubated with F-lectins that were pre-incubated with appropriate sugars, as well as those incubated with gold-labelled
antibodies without prior lectin treatment.
Aquaculture Research, 2012, 1–12
Results
Lectin binding tests
The isolated HEs showed strong affinity for Con A
(Fig. 1a) and LCA (Fig. 1c), weak affinity with
WGA (Fig. 1e) and only a slight affinity with GSL
II (data not shown). Other lectins showed no
affinity. The affinity of Con A and LCA was inhibited by addition of mannose to the lectin solution,
before incubation with the isolated HE, whereas
WGA affinity was inhibited using similar treatment with N-acetyl-b-D-glucosamine (Fig. 1b, d
and f). Based on high levels of affinity, Con A
and WGA were chosen for tests with thick sections of the eggs sampled at various intervals
(a)
(b)
(c)
(d)
(e)
(f)
Figure 2 Penaeus monodon eggs at different times [(a)–(e)] after spawning and incubation with FITC-labelled Con A.
The control (f) was prepared by pre-incubation with mannose and photographed at 20 min postspawning. Insets
show magnifications of the fluorescent structures. The scale bar in (f) applies to all of the photomicrographs. PVS,
perivitelline space.
4
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
Aquaculture Research, 2012, 1–12
after spawning and for gold-labelling studies
using TEM.
Lectin binding observed using fluorescence
microscopy
With F-Con A treatment, strong fluorescence was
observed in the cortical rods extruded from oocytes, and as several fluorescent dots distributed
evenly in the ooplasm immediately upon spawning
(Fig. 2a). As in previous study (Pongtippatee-Taweepreda et al. 2004), the cortical rods dispersed
out and became the jelly layer surrounding the
Hatching envelope of Penaeus monodon P Pongtippatee et al.
egg within 45 s of spawning. The fluorescent dots
moved from the entire area of the ooplasm
towards the cortical area within 2–5 min
(Fig. 2b). Within 8–15 min postspawning, the
accumulated fluorescent dots formed an intense
irregular line of fluorescence within the peripheral
cytoplasm, with several small projections and blebs
extending out from the oocytic surface (Fig. 2c).
During 20–30 min, a fluorescent line, presumably
a newly formed HE, separated from the oocyte,
resulting in a space (referred to as the perivitelline
space) between the oolemma and HE (Fig. 2d).
During this separation event, florescent dots were
(a)
(b)
(c)
(d)
(e)
Figure 3 Penaeus monodon eggs at different times [(a)-(d)] after spawning and incubation with FITC-labelled WGA.
The control (e) was prepared by pre-incubation with N-acetyl-b-D-glucosamine. The scale bar in (e) applies to all of
the photomicrographs.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
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Hatching envelope of Penaeus monodon P Pongtippatee et al.
observed scattered in the cortical area of the
ooplasm, in the perivitelline space and contacting
the newly formed HE. At 30–45 min, most of fluorescent dots had disappeared from the ooplasm
and the perivitelline space, and fluoresence of the
HE became very intense (Fig. 2e). In the control
preparation, using F-Con A pre-incubated with
mannose, no fluorescence was observed at 20 min
postspawning (Fig. 2f).
With F-WGA treatment, diffused fluorescent dots
were distributed throughout the ooplasm at the
time of spawning. Within 1–5 min, they had
migrated to the oolemma forming a continuous
fluorescent line (Fig. 3a). The line became intensely
Aquaculture Research, 2012, 1–12
fluorescent as time passed, indicating an accumulation of F-WGA in the cortical area (Fig. 3b). The
fluorescent dots migrated out of the ooplasm into
the perivitelline space during the 15–20 min interval postspawning (Fig. 3c). During this interval, the
fluorescent dots in the ooplasm and in the fluorescent line in the oolemma were visually reduced in
amount and intensity. At 30 min postspawning,
the fluorescent dots in the perivitelline space
migrated to the fully formed HE and those in the perivitelline space disappeared completely (Fig. 3d). In
the control preparation using F-WGA pre-incubated
with N-acetyl-b-D-glucosamine, no fluorescence was
observed at any time point after spawning (Fig. 3e).
(a)
(b)
Figure 4 Transmission electron micrograph (TEM) of an egg at the time of spawning. The inset shows a magnification of the flocculent vesicles. CR, cortical rod; DV, dense vesicle; GM, granular materials; Mb, membranous structures; Mi, mitochondria; FV, flocculent vesicles; Y, yolk granules.
6
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
Aquaculture Research, 2012, 1–12
Transmission electron microscopy
At the time of spawning, cortical rods containing
remarkable ‘bottle brush’ structures were observed
in the cortical crypts (Fig. 4a). In the ooplasm,
mitochondria, yolk granules (2 lm), dense vesicles
(500 nm), flocculent vesicles (200–1000 nm) and
membranous structures were observed (Fig. 4a–b).
Yolk granules were variable in size and were semielectron-dense. The flocculent vesicles contained
ring- or donut-shaped granules and some of them
(a)
(b)
Hatching envelope of Penaeus monodon P Pongtippatee et al.
coalesced to form irregular-shaped sacs. The membranous structures were like tangled membranes
or empty vesicles.
At 15–45 s postspawning, by which time the
cortical rods had completely extruded and formed
a jelly layer earlier described (Pongtippatee-Taweepreda et al. 2004), the membranous structures
observed in the ooplasm evaginated from the
oocyte (Fig. 5a). They formed membranous structures located just beneath the jelly layer that had
been formed by the extruded cortical rods (Fig. 5b)
and became associated with empty vesicles or
granular materials beneath the jelly layer
(Fig. 5c). At 1 min, the granular materials connected with one another in rows (Fig. 6a) and
coalesced to form tubular structures. The outer
and inner lines of the tubules thickened and gradually formed two dense lines separated by an electron-lucent space. This structure formed the outer
layer of the HE (Fig. 6b and c). Patches of electron-dense material were observed in the perivitelline space (Fig. 6b) and attached to the inner side
of the HE (Fig. 6c). These structures were the
contents released by exocytosis from the dense vesicles starting from 1 min postspawning and
(a)
(b)
(c)
Figure 5 TEM of an egg at 15–45 s postspawning
showing evagination of the membranous structure (a),
underneath the jelly layer (b) and associated with
granular materials (c). GM, granular materials; Mb,
membranous structure; CR, cortical rod; JL, jelly layer.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
(c)
Figure 6 TEM of an egg at 1 min postspawning showing formation of the outer layer of the hatching envelope. The granular materials have coalesced to form a
tubular structure (a) with its outer and inner walls
thickening (b). Dense materials from a dense vesicle in
the perivitelline space is attached to the inner side of
the newly formed hatching envelope (c). DM, dense
material; GM, granular material.
7
Hatching envelope of Penaeus monodon P Pongtippatee et al.
continuing for 45 min or more until the HE was
completely formed (Fig. 7a). The materials gradually accumulated on the inner side of the existing
HE (Fig. 6c) and formed its inner layer (Fig. 7b).
At 3 min postspawning, the newly formed HE
could be clearly seen to consist of two dense lines
separated by a space (Fig. 8a). During that period,
the flocculent vesicles moved towards the
oolemma and evaginated into the perivitelline
space (Fig. 8b). As the contents of the flocculent
vesicles disappeared as they bulged into the perivitelline space, their contents were presumably
released into the perivitelline space and became
homogenous material.
At 15 min postspawning, the HE was clearly
composed of an outer and inner layer (Fig. 9a).
The outer layer comprised two parallel dense lines
separated by an electron-lucent layer, altogether
about 25–30 nm thick. The inner layer was com-
Aquaculture Research, 2012, 1–12
posed of slightly electron-dense materials that
accumulated as time passed (Fig. 9b).
Gold-labelling of the Con A revealed that it
was bound with the dense vesicles (Fig. 10a).
Their contents were continuously exocytosed into
the perivitelline space from 15 min postspawning
(Fig. 10b). Gold-Con A was observed in the fully
formed HE, located mostly in the inner layer
of the HE. The whole HE measured approximately 200 nm in width, most of which consisted of the inner layer (Fig. 10c). In the
control section, Con A binding was inhibited by
pre-incubation with mannose, preventing goldlabelling (Fig. 10d).
Gold-labelling of WGA revealed that it bound
with flocculent vesicles (Fig. 11a). The vesicle contents were exocytosed into the perivitelline space
(Fig. 11b) and subsequently became located in the
inner layer of the HE (Fig. 11c). Control sections
using WGA pre-incubated with N-acetyl-b-D-glucosamine gave no gold-labelling (Fig. 11d), as did
sections incubated with gold-labelled antibodies
without prior lectin treatment (not shown).
Discussion
(a)
(b)
Figure 7 TEM of an egg at 1–2 min postspawning
showing exocytosis of dense vesicles (a) and attachment of their dense contents to the inner surface of the
hatching envelope [(b), arrows]. DV, dense vesicle.
8
From previous work (Pongtippatee-Taweepreda
et al. 2004), it has been shown that spawned eggs
from the black tiger shrimp P. monodon must be fertilized within 30 s of release into seawater because
after that the cortical rods are exuded from the egg
and surround it as a jelly layer. Thereafter, the HE
is formed to protect the developing embryo. This
study has revealed that the HE of the black tiger
shrimp P. monodon consists of outer and inner layers. The outer layer was formed by the granular
materials originating from an unknown source
outside the oocyte. The inner layer was formed by
substances secreted from dense and flocculent vesicles in the ooplasm. Based on competitive binding
tests, the dense vesicles contained mannose-rich
materials, whereas the flocculent vesicles contained
N-acetyl-b-D-glucosamine-rich materials. By fluorescence studies, the release of these materials
occurred mainly within 30 min postspawning,
whereas complete formation of the HE required up
to 1 h postspawning. Regarding the release of these
materials from ooplasmic vesicles, the findings
herein are similar to those previously reported for
S. ingentis (Pillai & Clark 1990). However, some
differences were also detected for P. monodon. These
were as follows: (1) All types of cortical vesicles in
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
Aquaculture Research, 2012, 1–12
Hatching envelope of Penaeus monodon P Pongtippatee et al.
(a)
(b)
Figure 8 TEM of an egg at 3 min postspawning showing flocculent vesicles close to the oolemma (a) and evaginating into the perivitelline space (b). DV, dense vesicles; FV, flocculent vesicles; HE, hatching envelope; PVS, perivitelline space.
P. monodon were present within the oocyte at the
time of spawning, but in S. ingentis were not
observed until a time (approx. 30 min) postspawning. (2) In P. monodon, mannose was associated
with dense vesicles, but in S. ingentis with ring vesicles. (3) In P. monodon, N-acetyl-b-D-glucosamine
was associated with flocculent vesicles, whereas in
S. ingentis with dense vesicles. (4) In P. monodon,
both sugar-substances were associated mainly with
the inner layer of the HE, whereas in S. ingentis,
N-acetyl-b-D-glucosamine was associated with the
outer layer and mannose with the inner layer. (5)
In P. monodon, F-WGA binding to the HE gave only
weak fluorescence, whereas in S. ingentis, it gave
strong fluorescence. It should be noted that Pillai
and Clark (1990) reported association of N-acetylb-D-glucosamine with the HE outer layer in S.
ingentis based on their F-lectin fluorescence study,
but could not confirm it with gold-labelling using
TEM. Therefore, association of N-acetyl-b-D-glucosamine with the outer HE layer in S. ingentis may
be considered questionable. In any case, all the
differences enumerated could simply be species-spe© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
cific differences. On the other hand, Glas, Green
and Lynn (1996) reported weak fluorescence from
F-WGA binding in S. ingentis, similar to our study
in P. monodon. At the same time, they suggested
that different timing of samples between their study
that of Pillai and Clark (1990) might have
accounted for the discrepancy. We must also consider the possibility differences arising from variations in abundance or accessibility to label of
N-acetyl-b-D-glucosamine in the HE structure.
The finding that cortical rods stained intensely
with F-Con A suggests that they contain glucose
or mannose-containing materials. It has been
reported that the cortical rods of eggs of the marine shrimp Penaeus semisulcatus and Fenneropenaeus
merguiensis contains glycosylated protein, namely
shrimp ovarian peritrophin (SOP), that may protect the eggs against pathogens (Khayat, Babin,
Funkenstein, Sammar, Nagasawa, Tietz & Lubzens
2001; Loongyai, Phongdara & Chotigeat 2007). It
is likely that cortical rods of P. monodon also contain SOP, thus its glycosylated component should
be composed of glucose or mannose.
9
Hatching envelope of Penaeus monodon P Pongtippatee et al.
Aquaculture Research, 2012, 1–12
(a)
(b)
Figure 9 TEM of an egg at 30 min postspawning showing the structure of the hatching envelope (a), composed of
outer (O) and inner (I) layers (b). HE, hatching envelope; PVS, perivitelline space.
The early appearance of cortical vesicles in
P. monodon as reported herein corresponded to the
observation by Kruevaisayawan, Vanichviriyakit,
Weerachatyanukul, Withyachumnarnkul, Chavadej and Sobhon (2010) that several types of vesicles exist in the mature oocytes in the ovary in this
species. Morphologically, the flocculent and dense
vesicles in this study were similar to the large
lightly electron-dense and small highly electrondense vesicles respectively, previously described in
the mature oocytes (Kruevaisayawan et al. 2010).
Therefore, unlike in S. ingentis, these two types of
cortical vesicles appear to be present in oocytes of
P. monodon prior to spawning. Pre-existence of cortical vesicles prior to spawning has also been found
in the lobster (Talbot & Goudeau 1988). This difference between P. monodon and S. ingentis may be
related to the fact that egg activation in P. monodon
takes place immediately after spawning and HE formation begins as early as 1 min postspawning
(Pongtippatee-Taweepreda et al. 2004), whereas
these processes in S. ingentis begin at 30–45 min
postspawning (Pillai & Clark 1988, 1990). Therefore, pre-existence of cortical vesicles in the mature
oocytes and newly spawned eggs in P. monodon
may be necessitated by the rapidity of HE formation
upon spawning.
10
The most detailed information on HE formation
is from the sea urchin S. purpuratus. However, the
ultrastructure of cortical granules of the sea
urchin are quite different from those of crustaceans. For example, a distinctive spiral lamellar
body is clearly associated with cortical granules in
S. purpuratus (Laidlaw & Wessel 1994; Murray
et al. 2000), but no such association has ever been
described in any crustacean. The closest structure
to the spiral lamellar body of S. purpuratus is the
membranous structures described in this study. In
S. purpuratus, enzymes, such as ovoperoxidase,
b-1,3-glucanase and proteoliaisin (Somers et al.
1989; Cheng et al. 1991) and integrins (Murray
et al. 2000) are released from the cortical granules, are incorporated into the HE and catalyse its
formation and hardening. There is less information
regarding the biochemical content and function of
cortical granules for crustaceans. In the shrimp S.
ingentis, chitin probably plays an important role in
HE assembly as chitinase and N-acetylglucosaminidase applied to the developing HE caused failure of
its formation (Glas et al. 1996). More studies on
HE formation in P. monodon and other economically important shrimp species are needed, especially for the purpose of finding genetic markers
for high fertilization and hatching rates that would
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
Hatching envelope of Penaeus monodon P Pongtippatee et al.
Aquaculture Research, 2012, 1–12
(a)
(b)
(c)
(d)
Figure 10 TEM of Con A gold-labelling of an egg at 30 min postspawning showing association of gold particles
with dense vesicles (a) being released into the perivitelline space and associated with the inner layer of the hatching
envelope (b). At 1 h postspawning, gold-Con A was associated with the inner layer of the fully formed hatching
envelope (c). The control section pre-incubated with mannose shows no gold-labelling (d). DM, dense material; DV,
dense vesicle; HE, hatching envelope; PSV, perivitelline space.
(a)
(b)
(c)
(d)
Figure 11 TEM of WGA gold-labelling of an egg at 30 min postspawning showing association of gold particles
with flocculent vesicles (a) with contents released into the perivitelline space (b). At 1 h postspawning, the gold particles were observed mostly in the inner layer of the hatching envelope (c). A control section pre-incubated with
N-acetyl-b-D-glucosamine shows no gold-labelling (d). FV, flocculent vesicle; HE, hatching envelope; PVS, perivitelline space.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–12
11
Hatching envelope of Penaeus monodon P Pongtippatee et al.
be beneficial in fry production and in breeding programmes.
Acknowledgments
This study was supported by Thailand Research
Fund, Thailand, Grant No. MRG-WI515S127. We
would like to thank Professor Tim Flegel for his
review and suggestions in writing the manuscript.
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