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).

P. Pongtippatee-Taweepreda et al. / Aquaculture 234 (2004) 183–198 185

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.


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.


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.


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.


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.


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.


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.


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.



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-


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.,


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).


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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