Egg activation in the black tiger shrimp Penaeus...
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Aquaculture 234 (2004) 183–198
Egg activation in the black tiger shrimp
Penaeus monodon
Pattira Pongtippatee-Taweepredaa,b, Jittipan Chavadeja,Pornthep Plodpaic, Boonyarath Pratoomchartd, Prasert Sobhona,Wattana Weerachatyanukula, Boonsirm Withyachumnarnkula,b,*
aDepartment of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, ThailandbCentex Shrimp, Chalerm Prakiat Building, 4th Floor, Faculty of Science, Mahidol University,
Rama 6th Road, Bangkok 10400, ThailandcShrimp Culture Research Center, Charoen Pokphand Foods Company (Public),
Nakorn Srithamaraj 80170, ThailanddDepartment of Marine Biology, Faculty of Science, Burapha University, Cholburi 20131, Thailand
Received 24 July 2003; received in revised form 3 October 2003; accepted 7 October 2003
Abstract
This report describes morphological changes in the eggs in the black tiger shrimp Penaeus
monodon upon contact with seawater, the process known as egg activation. Eggs from wild P.
monodon broodstock were collected at 15-s intervals post-spawning during the first 15 min, and
at 15-min intervals thereafter for 2 h. The samples were fixed and processed for light, scanning
and transmission electron microscopy. As soon as the egg was released into seawater, the
cortical rods began to emerge from the crypts on the periphery of the egg, and elevated the thin
investment coat that covered the surface of the egg. Sperm in the first phase of the acrosome
reaction were observed on both the egg and the surface of the investment coat. The rods
protruded from the surface and were completely expelled out within 45 s. I0mmediately after
complete extrusion, the cortical rods began to break up and formed the jelly layer around the
egg. By this time, the interaction between the sperm at the second phase of the acrosome
reaction and egg began. The hatching envelope had started formation at 1-min post-spawning,
and was completed within 13–15-min post-spawning. The first and second polar bodies
extruded from the egg at 3–5- and 10–15-min post-spawning, respectively. It was apparent that
after the hatching envelop had formed, additional sperm could not enter the egg. This study
0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2003.10.036
* Corresponding author. Centex Shrimp, Chalerm Prakiat Building, 4th Floor, Faculty of Science, Mahidol
University, Rama 6th Road, Bangkok 10400, Thailand. Tel.: +66-2-201-5866; fax: +66-2-247-7051.
E-mail address: [email protected] (B. Withyachumnarnkul).
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198184
suggests that the critical period for the egg–sperm interaction in P. monodon is within 45-s
post-spawning.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Egg activation; Penaeus monodon; Broodstock; Cortical rod; Polar body; Acrosome reaction;
Fertilization
1. Introduction
Morphological changes of eggs of penaeid shrimp and several marine species occur as
soon as the eggs are released from the gonopore and come into contact with seawater. This
event, termed egg activation, comprises a release of the jelly precursor from the cortical
crypts, transformation of the precursor material into a layer of jelly, exocytosis of cortical
vesicles, and formation of the hatching envelope (Lynn et al., 1992). Egg activation is
believed to be responsible for the prevention of polyspermy, and may also establish a
microenvironment inside the egg suitable for embryo development (Clark et al., 1980).
Morphological sequences of egg activation have been described in lobsters (Homarus
americanus and H. gammarus), penaeoidean shrimp (Trachypenaeus similes and Sicyonia
ingentis), and penaeid shrimp (Penaeus japonicus, P. monoceros, P. setiferus and P.
aztecus) (Hundinaga, 1942; Duronslet et al., 1975; Clark and Lynn, 1977; Clark et al.,
1980, 1990; Pillai and Clark, 1987, 1990; Tabot and Goudeau, 1988; Yano, 1988; Lynn et
al., 1992), but not in P. monodon, which is the most important economic species among
cultured shrimp. One problem in P. monodon breeding programs is that fertilization and
hatching rates are unpredictable, especially for domesticated brooders (Withyachumnarnkul
et al., 2002). Knowledge of egg activation could lead to improved fertilization rates, and
therefore, the morphological sequences of egg activation in this species have been studied
and reported.
2. Materials and methods
Ten mated wild P. monodon brooders caught from the Andaman Sea (6jN, 99jE) wereallowed to spawn and eggs were collected at 15-s intervals post-spawning during the first
15 min, and at 15-min intervals thereafter for two more hours. The samples were fixed in
2% glutaraldehyde, followed by two rinses in 0.2 M cacodylate, pH 7.8, and were
examined as whole-mount preparations under light microscopy (LM).
For transmission electron microscopy (TEM), the eggs were fixed for 3 h in 2%
glutaraldehyde in artificial seawater and rinsed twice in artificial seawater (Collins and
Epel, 1977), followed by two rinses in 0.2 M cacodylate, pH 7.8 for 30 min. Fixed samples
were dehydrated in a series of graded ethanol, embedded in a low-viscosity epoxy resin, and
sectioned with diamond knives in an ultramicrotome (MT-XL RMC). Semi-thin plastic
sections (0.5–1.0 Am) for LM were stained with 1% toluidine blue. Thin sections (60–90
nm) were stained with saturated methanolic uranyl acetate, counter-stained with lead citrate,
and examined under an electron microscope (Jeol, JEM-100 CXII).
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For scanning electron microscopy (SEM), eggs were fixed as in the TEM process,
dehydrated in a series of graded ethanol, and critical-point dried. Samples were coated
with gold and examined in a scanning electron microscope (Hitachi, S-2500).
3. Results
The sequence of changes in eggs, from the time of spawning to the first mitotic
division, can be rapidly visualized by observing the whole-mount preparations of the egg
using LM (Fig. 1). Mature eggs at the time of spawning (0 s) are not absolutely round
and are approximately 270–280 Am in diameter (Fig. 1a). Light striations of cortical rods
are apparent in the peripheral cytoplasm. Within 15 s, the egg becomes rounded and the
cortical rods begin to emerge from the egg (Fig. 1b). Once the cortical rods are expelled,
they form a corona around the egg (Fig. 1c,d) which becomes dissipated within 45–60 s
(Fig. 1d,e). After the disappearance of the cortical rods, a small spherical first polar body
was observed and extruded from the oolemma at 2 min, but it was observed more clearly
at 3–5 min (Fig. 1f). At 5 min, a transparent hatching envelope began to form around the
egg surface (Fig. 1f), and was completed at 13–15 min (Fig. 1h). At 10–15 min, the
second polar body was extruded closed to the first one (Fig. 1h). The two polar bodies
remained located between the hatching envelope and ooplasm, and appeared to move in
and out of the hatching envelope for at least 2-h post-spawning, which was at eight-cell
stage. The observation was stopped at 2 h and the fate of the polar bodies had not been
followed afterward. After the completion of egg activation (15 min), the eggs became
smaller with diameters of 200 Am. The first mitotic stage toke place at 30–60-min post-
spawning (Fig. 1i).
Details of ultrastructural changes during egg activation are depicted in Figs. 2–8. At 0
s, when the egg was spawned directly into the fixative, many round pits or cortical crypts
(10–15 Am in diameter) appeared on the egg surface (Fig. 2a). The most superficial layer
of the egg was covered by membranous investment, which was torn and stretched between
the apical surfaces of the cortical rods (Fig. 2b). The cortical rods, located in the crypts,
were clearly separated from the oolemma. Yolk granules, mitochondria and different kinds
of vesicles were dispersed throughout the ooplasm (Fig. 2c,d). The investment coat was
comprised of three layers: the outer (200 nm), middle (30 nm) and inner (300 nm) layers
(Fig. 2d). The middle layer was a row of discontinuous electron-dense granules. In certain
areas, the inner and the middle layers were separated, and clusters of electron-dense
granules (about 100 nm) were observed between the two layers (Fig. 2e,f). In other areas,
either the outer layer (Fig. 2h), or both the outer and middle layers were lost (Fig. 2c,g). In
the latter case, several electron-dense granules were observed above the inner layer. Parts
of the investment layer were torn from the egg surface as soon as the egg came from the
female gonopore.
The cortical rod was composed of numerous, tightly packed, bottle-brush like elements
embedded within the electron-dense matrix. Each element had a width of about 150 nm
and a variable length of 500 nm or longer (Fig. 3a,b,c). The surface of the cortical rod was
partially covered by patches of electron dense granular material (Fig. 3b), which was also
found in the investment layer covering the oolemma and the apical surface of the cortical
Fig. 1. Light micrographs showing the cortical reaction of P. monodon eggs from the unreacted stage (a), through
the release of polar body and formation of a hatching envelope (f, g and h), to the first mitotic stage (i). PB, polar
body; HE, hatching envelope, Bar = 100 Am.
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Fig. 2. SEM and TEM micrographs of the egg spawned directly into the fixative, showing cortical crypts, cortical
rods and investment coat. CC, cortical crypt; CR, cortical rod; C, investment coat; G, granules; I, inner layer; M,
middle layer; Mi, mitochondria; O, outer layer; Y, yolk granule.
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Fig. 3. TEM micrographs showing, ‘‘bottle-brush’’ structures of the cortical rod at 0 s, upon contact with seawater.
Exocytosis of low electron density material was observed (a and d, arrows). BB, bottle-brush; C, investment coat;
CC, cortical crypt; CR, cortical rod; Gf, fine granules; Y, yolk granule.
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Fig. 4. SEM and TEM micrographs of the egg at 15 s, showing extension of the cortical rod. Beads of granules
with a size of 10–30 nm (d, arrow) were observed between the surface of the ooplasm and the cortical rods. BB,
bottle-brush; C, investment coat; CR, cortical rod.
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Fig. 5. SEM micrographs showing the extrusion of cortical rods at 30 s (a) and 45 s (b– f). The cortical crypts
became shallow as soon as the rods detached from the crypts (e, arrows). CR, cortical rod; S, sperm.
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198190
Fig. 6. TEMmicrographs showing membrane vesiculations at the base of the cortical crypts, following detachment
of cortical rods. Several vesicles (a, arrow) were observed between the cortical rod and oolemma. Some vesicles
contained electron-dense material (c, arrow), whereas most of them contain electron-lucent material. Membrane
vesiculation is clearly observed (d, arrow) before the oolemma is smoothed out (e). CR, cortical rod.
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198 191
Fig. 7. TEM micrographs showing formation of the hatching envelop, starting from oolemma thickening (a,
arrow). At 15 min, the hatching envelop was strengthened by electron-dense material in dense vesicles from the
ooplasm (d, arrow). CV, clear vesicle; DV, dense vesicle; HE, hatching envelop; PS, perivitelline space.
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198192
Fig. 8. SEM micrograph showing the hatching envelop surrounding one (a and b) or two (c, arrow) eggs, at 15-
min post-spawning. Wrinkles on the egg surface are fixation artifacts. S, sperm.
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P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198194
rod (Fig. 3a). Vesicles (1.0–1.5 Am in diameter) containing low electron-density material
were also observed in the ooplasm (Fig. 3a,d), and some of them were exocytosed from the
ooplasm into a space beneath the investment layer, as well as into the cortical crypt (Fig.
3d). This released material apparently became the inner layer of the investment coat.
Patches of electron dense granular material were frequently observed at the base of the
cortical crypts (Fig. 3e).
At 15 s, the cortical rods began to protrude from the cortical crypts, with no apparent
changes in the substructure of the rods (Fig. 4). This protrusion coincided with the lifting
and partial loss of the integrity of the investment coat from the egg surface (Fig. 4b).
Following protrusion, the club-shaped cortical rods slightly expanded at the top to about
10 Am wide and 35–40 Am long (Figs. 4b and 5b). Membranous vesicles (10–30 nm)
were observed between the surface of the ooplasm and the cortical rods (Fig. 4d). These
vesicles disappeared at later stages (45 s). Round-shaped structures (4–5 Am), possibly
sperm, were seen closely associated with the egg surface (Fig. 4b).
From 30 to 45 s, cortical rods were further extruded from the egg (Fig. 5). The cortical
crypts became shallow as soon as the rods detached from the crypts (Fig. 5e). Sperm (4–5
Am) were observed on the surface of the egg, recognizable by a slightly electron-lucent area
on their surface (Fig. 5f). These sperm showed the first phase of acrosome reaction, being
evidenced by disappearance of the spike, and more than one sperm may have penetrated the
eggs (Fig. 5f). As the cortical rods were extruded from the crypts, patches of electron dense
granules (Fig. 6a,b,c) were observed between the cortical rods and the oolemma. These
patches were a discontinuous array of vesicles of variable sizes and shapes, some were
tubular and some were oval. Most of them contained electron-lucent material but some also
carried electron-dense material. These vesicular elements (30–50 nm) disappeared after the
cortical rods detached from the crypts, which occurred within 1 min. These vesicular
elements were formed by membrane vesiculation (Fig. 6d), after which the surface became
smooth (Fig. 6e).
At 1 min, the oolemma became thickened and a layer lifted away becoming the hatching
envelop (Fig. 7). This envelope was completed within 15 min, covering the perivitelline
space (about 0.5–3.0 Am in width). The envelope was a layer of vesicular elements
strengthened by electron-dense material released from dense vesicles of the ooplasm (Fig.
7c). The perivitelline space was increasingly widened by the exocytosis of contents from
the clear vesicles of the ooplasm (Fig. 7e). The hatching envelope usually covered an
individual egg; however, some adjacent eggs shared one hatching envelope (Fig. 8).
During the study, it was found that unfertilized eggs (i.e. having no contact with sperm)
also underwent a similar sequence of changes to those in the fertilized eggs. They also
underwent mitotic division to the four-cell stage and became arrested after that.
4. Discussion
The egg activation process of P. monodon comprised the following sequence of events:
(1) unreacted stage; (2) cortical rod extrusion; and (3) formation of the hatching envelope.
The time from spawning to the beginning of hatching envelope formation was approxi-
mately 1 min. Sperm attachment and penetration into the egg occurred up to 1-min post-
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spawning, after which the thick hatching envelope prevented sperm penetration. The
fertilization process of P. monodon is therefore very fast, compared to that of P. aztecus
which occurs until 20–40-min post-spawning (Clark et al., 1980). This explains why
artificial fertilization in P. monodon occurs only if sperm are added as the eggs are spawned
(Lin and Ting, 1986).
4.1. The unreacted stage
An unreacted egg is approximately 275 Am in diameter, with a surface area of about
2.4� 105 Am2. Direct counts suggest an egg probably has 400 cortical crypts (Fig. 2a).
Assuming that each is a cylindrically shaped structure, 10 Am in diameter and 35 Am long,
each crypt’s surface area is therefore about 569 Am2, and the total crypt surface area is
2.3� 105 Am2. This is equal to the egg surface area. If all the crypt surface area were added
up with the egg surface area, the egg would have a surface of two times of the unreacted
egg. Therefore, a rapid loss of oolemma, by membrane vesiculation (Fig. 6d), is necessary
to prevent the increase in the egg size. In fact, the egg becomes smaller after the complete
cortical reaction (Fig. 1), suggesting that the amount of membrane depleted by vesiculation
was more than the total crypt surface area.
The eggs of many species (e.g., the brook lamprey, echinoderms, the anthozoan, cited
by Clark et al., 1980) including the penaeid shrimp P. aztecus (Clark et al., 1980) also
decrease in size after completion of the cortical reaction. Extrusion of the cortical rods,
however, can account for only 50% of the decrease (Clark et al., 1980). It is possible that
the release of the contents of cortical vesicles (Fig. 3e) to form part of the investment coat
may further contribute to the loss of volume.
The investment coat in this study is equivalent to the vitelline envelope of the egg of S.
ingentis (Clark et al., 1990; Wikramanayake and Clark, 1994). Since the vitelline envelop
of S. ingentis is a specific site for primary sperm binding (Wikramanayake and Clark,
1994), the investment coat of P. monodonmay also serve the same function. The investment
layer of P. monodon is composed of three layers, the outer amorphous, middle granular and
inner amorphous layers. The difference in morphology suggests differences in chemical
compositions. Proteins and glycoproteins have been identified in these layers in S. ingentis
(Wikramanayake and Clark, 1994). However, each layer may serve a different function. In
S. ingentis, the vitelline envelope (investment coat in this case) serves as the primary site for
sperm attachment, and the cortical surface of the egg further stimulates the sperm to
undergo the second phase of the acrosome reaction (Clark et al., 1990). By analogy, it is
possible that in P. monodon, the outer layer may be the site for primary sperm binding
(before the acrosome reaction) and the middle or inner layers may be for secondary sperm
binding (after the acrosome reaction). While this hypothesis awaits further proof, it can be
stated from the observation of sperm attachment to the egg at 15 s (Fig. 4b) that fertilization
begins very soon after the egg is released from the gonopore.
4.2. Cortical rod extrusion
The bottle-brush structure in the cortical rods of P. monodon has also been observed in
other crustacean species (Duronslet et al., 1975; Tabot and Goudeau, 1988; Clark et al.,
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198196
1990). After complete extrusion, the rods become a jelly coat, which is maintained for a
short period before dissipation at about 1-min after spawning. This 1-min dissipation
period is similar to that of P. japonicus (Hundinaga, 1942). In P. aztecus, however, the jelly
coat remains around the egg until the hatching envelope is formed (Clark et al., 1980).
In several species of sea urchin, the jelly coat comprises 80% sulfated fucosinic
polysaccharide and 20% sialoprotein. In P. aztecus, the egg jelly comprises 25–30%
carbohydrate and 70–75% protein, and contains a trypsin-like protease enzyme that is
involved in the release and dispersion of the jelly precursor; the enzyme may also act as an
antibacterial agent (Lynn and Clark, 1987). The jelly layer may also be involved in the
acrosome reaction of the sperm, as it is in sea urchin and S. ingentis (SeGall and Lennarz,
1979; Clark et al., 1984).
Extrusion of cortical rods from the cortical crypts in P. monodon was completed within
45 s, in contrast to 5–7 min in P. aztecus (Clark et al., 1980). In P. aztecus, the process is
Mg2 +-dependent (Clark and Lynn, 1977) while in P. monodon this dependency has not
been proven. One possible function of the extrusion of the cortical rods is to block
polyspermy. However, these cortical reactions occur upon contact with seawater and not
necessarily following contact with sperm. Additionally, polyspermy is thought to be a
physiological adaptation for many crustaceans (Clark et al., 1980). This suggests that the
cortical reaction does not require the presence of sperm and the two events are unrelated.
Even if polyspermy occurs, it does not appear to cause problems in fertilization. In P.
monodon, the extrusion of the cortical rods is less likely to prevent polyspermy since more
than one sperm have been observed to penetrate the egg surface at 45 s (Fig. 5f). It is
possible that contents of the cortical rod act, rather, as the stimulation of the sperm
acrosome reaction, and may provide a protective layer against environments unsuitable for
fertilization and developing embryos.
4.3. Formation of the hatching envelop
The hatching envelop is apparently composed of two layers, the outer vesicular and the
inner amorphous layers. In S. ingentis, the two layers are formed by two something less
anthropomorphic of cortical vesicles, i.e., the dense vesicles and the ring vesicles (Pillai
and Clark, 1987). Vesicles in the ooplasm that contain substances of various densities are
also observed in lobsters (Tabot and Goudeau, 1988). In this study, only the dense vesicles
are clearly observed. In all of these crustaceans, high-density vesicles are the first cortical
vesicles to be released after fertilization, and they may be involved in hatching envelope
formation.
The presence of vesicles in the ooplasm containing substances of different densities is
also reported in P. aztecus (Clark et al., 1980). In the sea urchin, these vesicles contain
protease that cleave proteins linking the surface coat to the oolemma (Chandler and
Heuser, 1979); and in S. ingentis, they play a role in the formation of the inner layer of the
hatching envelope (Pillai and Clark, 1990). Both S. ingentis and P. monodon have a
hatching envelope that is similarly strengthened by cortical vesicular contents and is
elevated from the egg surface. The only difference from S. ingentis is that the hatching
envelop of P. monodon was completed within 15 min, whereas that of S. ingentis is
completed within 45 min (Clark et al., 1990).
P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198 197
The first polar body was formed at 2-min post-spawning, after the formation of the
hatching envelope; and the second polar body was formed 3–5 min later. One of the
polar bodies was extruded out from the egg while the other was retained inside the
hatching envelope, but outside the dividing cell. This feature was also reported in S.
ingentis (Clark et al., 1990). It is likely that both polar bodies disintegrate at later stages
of embryonic development. Knowing the timing of polar body expulsion is important in
the manipulation of chromosome numbers in developing embryos, like creating poly-
ploided shrimp. For instance, to retain the first polar body, any interventions (e.g.,
temperature shock, chemical shock, pressure shock, etc.) have to be applied before 2-min
post-spawning.
5. Conclusions
1. The cortical reaction in P. monodon eggs is similar to other crustacean species, but the
process is one of the fastest, compared to other species.
2. Fertilization occurs within 1 min, beginning from the time of egg release into the water.
3. Cortical rod extrusion probably acts as an environment setting for proper fertilization
and embryo development.
4. Knowledge of the chemical nature and mechanisms of actions of the investment coat,
cortical rods, and other substances released during the cortical reaction will be very
important in understanding fertilization processes in P. monodon. Further studies may
lead to methods for improved and specialized fertilization practices (e.g., gynogenesis)
that improve the production of this economically important species.
Acknowledgements
This study was supported by the Center for Molecular Biology and Biotechnology of
the National Science and Technology Development Agency (NSTDA), Thailand, Grant
No. BT-B-06-SG-14-4505.
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