You are on page 1of 6

ZOOLOGICAL SCIENCE 22: 675680 (2005) 2005 Zoological Society of Japan

Localization and Developmental Expression of mRNA for Cortical


Rod Protein in Kuruma Prawn Marsupenaeus japonicus
Yi Kyung Kim1*, Naoaki Tsutsui2, Ichiro Kawazoe1, Takuji Okumura3,
Toyoji Kaneko1 and Katsumi Aida1
1
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
2
Japan International Research Center for Agricultural Sciences, Tsukuba,
Ibaraki 305-8686, Japan
3
National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Mie 516-0193, Japan

ABSTRACTThe mature penaeid oocytes possess cortical rods that contain two related cortical rod pro-
teins (CRP, 28.6 kDa and 30.5 kDa). In the present study, localization of CRP mRNA and gene expression
profiles of CRP and vitellogenin (Vg) during ovarian development were examined in kuruma prawn, Mar-
supenaeus japonicus, an economically important species for shrimp and prawn farming. Northern blot
analysis revealed that CRP mRNA was expressed in the ovary. In situ hybridization showed strong signals
for CRP transcripts in the oocytes at early developmental stages in both immature and mature ovaries.
Quantitative analysis by real-time polymerase chain reaction revealed that CRP mRNA levels were higher
in the previtellogenic and endogenous (primary) vitellogenic stages than in more advanced stages. Unlike
CRP mRNA, Vg mRNA levels were low in the ovary and hepatopancreas in previtellogenic females. When
the ovary developed into the endogenous vitellogenic stage, ovarian Vg mRNA levels increased signifi-
cantly, followed by rapid decrease in more advanced stages. The Vg mRNA levels in the hepatopancreas,
on the other hand, tended to be high in the exogenous (secondary) vitellogenic and maturation stages, in
which ovarian Vg mRNA levels were decreased. Our findings indicate that CRP mRNA is highly expressed
before the onset of vitellogenesis, suggesting that the transcription, translation, and cortical-rod formation
of CRP occur at different phases of oocyte development. The endogenous vitellogenic stage is a crucial
stage for the initiation of CRP and Vg syntheses. The coincidence of these protein syntheses suggests
that CRP and Vg syntheses are regulated by closely-related mechanisms.

Key words: Marsupenaeus japonicus, oogenesis, cortical rod protein, vitellogenin

and then transported to maturing oocytes via hemolymph.


INTRODUCTION
Eventually, it is internalized into the developing oocytes, and
In the female penaeid prawn, gonadal maturation subsequently forms vitellin (Vt), which serves as a principal
involves the two crucial phases, vitellogenesis and cortical nutrient reserve for embryonic growth and development
rod formation. In the developing ovary, primary oocytes (Tsukimura, 2001).
released from the germinative zone stay in their primary By the end of vitellogenesis, cortical rods first appear as
vitellogenic stage until the onset of secondary vitellogenesis, spherical bodies in the periphery of the oocyte 12 days
which is marked by oocyte growth and rapid accumulation before spawning, and then elongate toward the nucleus as
of yolk proteins (Meusy and Payen, 1988; Avarre et al., maturation proceeds, forming club-shaped structures. The
2001). Vitellogenin (Vg), the major egg yolk protein precur- cortical rods are overlain by the vitellin envelope, the out-
sor, is a high-density lipoglycoprotein associated with caro- most oocyte extracellular investment coat. When the oocyte
tenoid pigments. As in other oviparous animals, the crusta- is spawned into seawater, cortical rods are rapidly released
cean Vg is generally synthesized by extraovarian tissues into the surrounding medium through disaggregation of the
* Corresponding author. Phone: +81-3-5841-5289;
vitellin envelope, then forming a jelly layer around the oocyte
Fax : +81-3-5841-5289; (Clark et al., 1990). The jelly layer may act to block
E-mail: kimyk@marine.fs.a.u-tokyo.ac.jp polyspermy or to provide a microenvironment for developing
676 Y. K. Kim et al.

embryos (see Guraya, 1982, for review). site of CRP has not yet been determined.
In penaeid prawns, ovarian Vt was purified and the The kuruma prawn is an economically important spe-
complete sequence of Vg cDNA was determined (Kawazoe cies for shrimp and prawn farming. In captivity, kuruma
et al., 2000; Tsutsui et al., 2000). In kuruma prawn, Marsu- prawn is mostly incapable of forming cortical rods, and thus
penaeus japonicus, ovarian Vt was composed of three their reproductive potential decreases under culture condi-
polypeptide subunits of 91 kDa, 128 kDa and 186 kDa, the tions (Medina et al., 1996). The understanding of CRP-gene
precursors of which are produced in the ovarian follicles and expression patterns would give some insights into the mech-
hepatopancreas. In the giant freshwater prawn, Macro- anism of cortical rod formation and successful artificial pro-
brachium rosenbergii, Vg mRNA is expressed only in the duction of prawn juveniles for culture. In the present study,
hepatopancreas (Yang et al., 2000; Okuno et al., 2002; we first examined tissue and cellular distribution of CRP
Wilder et al., 2002), while the ovarian follicle cells are not transcripts in kuruma prawn, and then clarified the gene
the sites of Vg synthesis. expression profile of CRP during ovarian development, com-
Despite the prominence of these structures, information paring with those of Vg in the ovary and hepatopancreas.
regarding the nature of cortical rods is largely unknown.
Recently, there are several reports on cDNA cloning of cor-
MATERIALS AND METHODS
tical rod proteins in penaeid prawns. In Penaeus semisulca-
tus, two related cDNAs encoding cortical rod proteins have Animals
been cloned. These proteins were named shrimp ovarian Immature and mature female kuruma prawns were obtained
from the Momoshima National Center for Stock Enhancement,
peritrophin (SOP) because of its structural homology with
Fisheries Research Agency in Hiroshima Prefecture, Japan. At the
insect intestinal peritrophin (Khayat et al., 2001). We have time of sampling, a part of the ovary was fixed in Bouins solution
previously reported that two related proteins (28.6 kDa and for histological observation in order to determine its developmental
30.5 kDa) are localized in cortical rods in the mature ovary stage.
of kuruma prawn, and subsequently corresponding cDNAs In the present study, ovarian development was divided into five
stages according to Tsutsui et al. (2000). Since the kuruma prawn
were cloned based on their N-terminal amino acid
is a multiple spawner in a single breeding season, the mature ovary
sequences (Kim et al., 2004). These proteins were termed contains various stages of oocytes including previtellogenic ones.
cortical rod proteins (CRP), of which deduced amino acid Therefore, the ovarian developmental stages were defined accord-
sequences showed high similarities to those of SOP. The ing to the most advanced oocytes in the ovary. The immature ovary
western blot analysis has revealed that these proteins are including chromatin nucleolus and perinucleolus stage oocytes is
classified into the previtellogenic ovary; the ovary where PAS-pos-
present in the endogenous (primary) vitellogenic ovary,
itive granule stage oocytes are dominant is grouped as the endog-
when cortical rod structures are not yet formed. Yamano et enous (primary) vitellogenic stage; the ovary characterized by early
al. (2003, 2004) have reported the presence of other com- yolk globule stage oocytes is grouped as the early exogenous (sec-
ponents with molecular mass of 130 kDa, 140 kDa and 150 ondary) vitellogenic stage; the ovary including dominant late yolk
kDa in cortical rods of kuruma prawn. Based on a sequence globule stage oocytes is grouped as the late exogenous (second-
ary) vitellogenic stage; and the ovary including early and late corti-
homology to extracellular matrix proteins in a thrombospodin
cal alveoli stage oocytes, characterized by appearance of cortical
(TSP), the cDNAs encoding those three proteins were des- rods, is classified into the maturation stage.
ignated as mjTSP; i.e., marsupenaeus japonicus TSP.
Moreover, a 200 kDa protein has been found in cortical rods Northern blot analysis
of Litopenaeus vannamei, although its cDNA and amino acid For Northern blot analysis, we used immature (body weight,
20.1 g) and mature (87.3 g) kuruma prawns, the gonadosomatic
sequences have not been published nor deposited in data-
index (GSI) being 0.3 and 3.7%, respectively. The ovaries from both
bases (Bradfield et al., 1989). immature and mature prawns, and hepatopancreas, muscle, intes-
Little is known about regulatory mechanisms of CRP tine and fan blade from the mature kuruma prawn were dissected
and Vg syntheses during ovarian development. Our previ- out, frozen in liquid nitrogen, and stored at 80C until total RNA
ous study (Kim et al., 2004) has shown that accumulation of extraction. Total RNA was isolated from these tissues using RNA
extraction solution (ISOGEN, Nippongene, Toyama, Japan). Ten
CRP and Vt in the oocytes starts at the endogenous (pri-
micrograms of total RNA was subjected to electrophoresis on a
mary) vitellogenic stage in kuruma prawn. This suggests 1.2% formaldehyde-agarose gel and transferred onto a BIODYNE
that the onset of CRP and Vt accumulation is linked to each B membrane (Pall, East Hills, NY, USA). Because of the high iden-
other and that those biological events are regulated by tity between two CRPs (Kim et al., 2004), Northern blot and the fol-
closely-related mechanisms. On the other hand, crustacean lowing analyses were performed without distinguishing the two pro-
teins. A cDNA fragment, corresponding to nucleotides 461813 in
hyperglycemic hormone (CHH) family peptides produced in
both 28.6 kDa and 30.5 kDa CRPs, was labeled with [-32P] dCTP
the X-organ have been implicated in inhibiting vitellogenesis (Amersham Biosciences, Upaasala, Sweden) using the
and cortical rod protein synthesis (De Kleijin and Van Herp, Megaprime DNA labeling system (Amersham Biosciences). The
1998; Khayat et al., 1998; Avarre et al., 2001). Thus, a membrane was prehybridized in 6 SSC containing 50% forma-
decline in secretion of CHH peptide is likely to stimulate mide, 2 Denhardts solution, 0.4% SDS and calf thymus DNA (40
g/ ml) at 42C for 1 hr. The membrane was then labeled with the
CRP and Vg syntheses. However, relationship has not yet
radiolabeled probe (9.1105 cpm/ml) in prehybridization buffer at
been examined between CRP and Vg gene expressions 42C at 3 hr. After washing in 2 SSC containing 0.1% SDS at 60C
during ovarian development. Furthermore, the production
Cortical Rod Protein in Kuruma Prawn 677

for 10 min, the membrane was exposed to a X-ray film (Fuji Film, Statistics
Tokyo, Japan). RNA ladder (GIBCOBRL, Gaithersburg, MD, USA) The results are expressed as meansstandard errors of the
was used as molecular mass markers. means. CRP mRNA levels in the ovary and Vg mRNA levels in the
ovary and hepatopancreas were tested for significance (P<0.05)
In situ hybridization using one-way ANOVA, followed by Newman-Keuls test.
For in situ hybridization, an immature ovary (body weight, 35.4
g; GSI, 0.6%) and a mature ovary (body weight, 62.5 g; GSI, 11.9
%) were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate RESULTS
buffer (PB, pH 7.4). The ovaries were dehydrated through an etha-
nol series, and embedded in Paraplast. Sections (6 m) were cut Tissue distribution of CRP mRNA
and mounted on MAS-coated glass slides (Matsunami, Osaka, Tissue-specific expression of CRP mRNA was exam-
Japan). The localization of CRP mRNA was examined using a ined by Northern blot analysis (Fig. 1). Hybridization signals
digoxigenin (DIG)-labeled oligonucleotide probe. The oligonucle- were observed in the ovaries from immature and mature
otides for antisense and sense probes corresponding to nucleotides
kuruma prawns, but no signal was detected in the other tis-
401-444 in CRP (Kim et al., 2004) were synthesized by Hokkaido
System Science (Sapporo, Japan). sues examined. The size of the mRNA (approximately 1 kb)
Hybridization was carried out according to the method reported corresponded to that of cDNA for CRP obtained previously
previously (Uchida et al., 1998) with some modifications. After (Kim et al., 2004).
deparaffinization and hydration, sections were processed as fol-
lows: (1) 0.2 M HCl for 15 min, (2) 5 g/ml proteinase K (Wako,
Osaka, Japan) in phosphate-buffered saline (PBS, pH 7.4) for 10
min, (3) 4% PFA in 0.1 M PB (pH 7.4) for 10 min, and (4) 0.2% gly-
cine in PBS for 15 min. Following preincubation with a solution con-
sisting of 40% formamide and 2 SSC for 1 hr at room temperature,
sections were hybridized with either the antisense or sense probe
(1 g/ml) at 37C for 20 hr in hybridization buffer consisting 40%
formamide, 2 SSC, 20 mM Tris-HCl (pH 7.6), 1 Denhardts, 0.1%
Tween-20, 10% dextran sulfate, and 25 g/ml calf thymus DNA.
Sections were washed sequentially with: (1) 2 SSC for 30 min at
room temperature, (2) 2 times in 1 SSC (15 min each) at 40C,
and (3) 2 SSC for 30 min. Sections were then incubated with an
anti-digoxigenin-gold (Roche Diagnostics GmbH, Mannheim, Ger-
many) and visualized using a Silver Enhancing kit (British BioCell
International).

Real-time quantitative PCR analysis


Real-time quantitative PCR was performed using an ABI
PRISM 7700 Sequence Detector (Perkin-Elmer, Branchburg, NJ,
Fig. 1. Tissue specific expression of CRP mRNA examined by
USA) to examine changes in gene expressions of CRP and Vg dur-
Northern blot analysis. Total RNA (10 g) was electrophoresed,
ing oocyte maturation. From prawns weighing 59.587.7 g with GSI
transferred to a nylon membrane, and hybridized with 32P-labeled
values ranging 1.611.4%, total RNA was isolated from the ovary
CRP cDNA corresponding to nucleotides 461813. A single CRP
and hepatopancreas, and treated with DNase (Invitrogen, Carlsbad,
transcript is detected in immature (lane 1) and mature (lane 2) ova-
CA, USA). First strand cDNA was synthesized from 1 g total RNA
ries, but not detected in the hepatopancreas (lane 3), muscle (lane
using the Superscript first-strand synthesis system for RT-PCR
4), intestine (lane 5), and fan blade (lane 6). The molecular markers
with random hexamers according to manufacturers instructions
are presented on the left.
(Invitrogen). For CRP, a specific primer pair, F (5'-GAATGACTTTC-
GACCAAGGCA-3')/R (5'-AAGGTACCCGACATCGTGCA-3') and a
Taqman probe (5'-AAGGGTGGAAGTAGCGATGCGTCACA-3')
were designed (Kim et al., 2004). Primers for Vg mRNA, F (5'-
In situ hybridization
AACATCGCCCAAATCCTCTCT-3')/R (5'- GGCACTCAAAGCCCA-
CAAAA-3') and a Taqman probe (5'- CCTGGCAATTTGCAGACCG- The cellular expression of CRP mRNA was determined
GCA-3') were designed based on Vg cDNA (Tsutsui et al., 2000). in the ovaries of immature and mature kuruma prawns by in
The PCR products amplified with the primers were used for the situ hybridization (Fig. 2). The immature ovary consisted
quantification of CRP and Vg transcripts. Primers and probes were mostly of previtellogenic oocytes, whereas the mature ovary
designed using PRIMER EXPRESS software version 1.0 (Perkin-
contained oocytes at different developmental stages simul-
Elmer). The plasmid DNAs containing the amplified parts of target
mRNAs were prepared as standard samples, which were serially taneously. In the immature ovary, intense signals for CRP
diluted. The PCR mixture consisted of 0.2 M of each primer, 0.2 transcripts were observed in the oocytes at the previtello-
M Taqman probe and 12.5 l Platinum Quantitative PCR Super- genic stage (Fig. 2A). In the mature ovary, signals for CRP
mix-UDG (Invitrogen) in a final volume of 25 l. The cycling param- mRNA were evident in the previtellogenic oocytes, but not
eter was as follows: 2 min at 95C followed by 40 cycles at 95C
detected in more advanced oocytes (Fig. 2B). The CRP
for 15 s and 60C for 1 min. Results were analyzed using PRIZM
software (GraphPad Software, San Diego, CA, USA) and transcripts were found in the cytoplasm of the previtello-
expressed as the copy number of the target mRNA per 1 g total genic oocytes, but not in follicle cells surrounding the
RNA. oocytes. No signal was seen in the control sections incu-
bated with the sense probe (data not shown).
678 Y. K. Kim et al.

Fig. 2. Localization of CRP mRNA in the ovary examined by in situ


hybridization. Hybridization signals for CRP mRNA are detected
only in previtellogenic oocytes in both immature (A) and mature (B)
ovaries, but not in more advanced oocytes. An arrow indicates an
oocyte at the late exogenous stage. Bar, 50 m.

Changes in CRP and Vg gene expressions during ova-


rian maturation
The expression levels of CRP and Vg mRNAs at vari-
ous stages of ovarian development were determined by
real-time quantitative RT-PCR (Fig. 3). The copy number of
CRP mRNA per 1 g total RNA in the ovary was higher in
previtellogenic and endogenous vitellogenic stages than in
the later developmental stages (Fig. 3A). The levels of CRP Fig. 3. Changes in gene expressions of CRP in the ovary (A) and
mRNA in previtellogenic and endogenous vitellogenic Vg in the ovary (B) and hepatopancreas (C) during ovarian develop-
females were approximately 3 times higher than those in the ment. Ovaries were classified into five developmental stages: previ-
tellogenic stage (1), endogenous vitellogenic stage (2), early
maturation stage. However, no significant changes were
exogenous vitellogenic stage (3), late exogenous vitellogenic stage
seen when re-calculated on the basis of the copy number (4), and maturation stage (5). Values are meansSEM (n=35). Sig-
per ovary (data not shown). The amount of Vg transcripts in nificant dfference (P<0.05) is indicated by different letters.
the ovary was significantly (P<0.05) greater in the endoge-
nous vitellogenic stage than in the other developmental DISCUSSION
stages (Fig. 3B). In contrast, Vg gene expression in the
hepatopancreas showed a tendency to increase as the The oocyte synthesizes and stores a variety of proteins
oocyte maturation proceeded (Fig. 3C). during its development. While some proteins found in devel-
oping oocytes are produced by themselves, other molecules
Cortical Rod Protein in Kuruma Prawn 679

including yolk materials are synthesized by extraovarian tis- expressed in the immature oocytes of previtellogenic and
sues and incorporated into oocytes. During oogenesis in endogenous vitellogenic stages. This is also supported by
penaeid prawns, the oocytes accumulate vitellin and cortical our observation that in situ hybridization signals were
rod proteins as major proteins. Cortical rods contain several detected only in immature oocytes regardless of ovarian
proteins, including SOP, mjTSP and CRP. These proteins maturity.
exhibited similar characteristics in the following aspects: The Vg mRNA levels changed significantly during the
similarities in cDNA and deduced amino acid sequences, ovarian development. Vg mRNA levels were low in the ovary
gene expression in the young oocytes, and post-transla- and hepatopancreas in previtellogenic females. When the
tional modification such as glycosylation. In the present ovary developed into the endogenous vitellogenic stage,
study, a single transcript of CRP was detected in the ovary, ovarian Vg mRNA levels increased significantly, followed by
but not in the other tissues examined (Fig. 1). In our previ- a rapid decrease in more advanced stages. The Vg mRNA
ous study (Kim et al., 2004), CRP-immunopositive signals levels in the hepatopancreas, on the other hand, tended to
were detected in kuruma prawn oocytes. Taken together, it be high in exogenous vitellogenic and maturation stages,
is likely that the CRP mRNA originates from the oocytes, as when ovarian Vg mRNA levels were decreased. These
suggested for SOP in P. semisulcatus and for mjTSP in results indicate that the expression site of the Vg gene may
kuruma prawn (Khayat et al., 2001; Yamano et al., 2004). shift from the ovary to hepatopancreas, as the oocytes
Rankin and Davis (1990) have demonstrated that the develop toward the maturation stage. This is in accordance
oocytes of L. vannamei are responsible for synthesis of cor- with the Vg gene expression pattern of kuruma prawn
tical rod proteins, which are subsequently accumulated reported by Tsutsui et al. (2000, 2005).
within the developing extracellular cortical rods. In the present study, gene expressions of CRP in the
The preferential localization of CRP mRNA in the ovary and Vg in the ovary and hepatopancreas showed dif-
oocytes and change in CRP mRNA levels during the ovarian ferent patterns during ovarian development. The CRP
development suggest that CRP synthesis in the oocytes is mRNA levels in the ovary were higher in the previtellogenic
under translational control, as shown for SOP and mjTSP and endogenous (primary) vitellogenic stages than in the
(Avarre et al., 2001; Yamano et al., 2004). The previtello- advaneced stages; in contrast, Vg mRNA levels in the ovary
genic oocytes of kuruma prawn transcribed and accumu- increased at the endogenous (primary) vitellogenic stage
lated CRP mRNA in the cytoplasms, but protein synthesis of and levels in the hepatopancreas increased in the exoge-
CRP did not start until the endogenous vitellogenic stage nous (secondary) vitellogenesis and maturation stages. In
(Kim et al., 2004). In situ hybridization in the ovary of our previous study, western blot analysis revealed the pres-
kuruma prawn revealed intense signals of CRP transcripts ence of CRP in the endogenous vitellogenic stage (Kim et
in the oocytes at early developmental stages in both imma- al., 2004). Taken together, it is most probable that CRP
ture and mature ovaries (Fig. 2A and B); the CRP transcripts accumulation starts in the endogenous vitellogenic stage
were detectable in the previtellogenic oocytes, but not and CRP mRNA decreases between the endogenous and
detected in maturing and fully matured oocytes. As in mjTSP exogenous vitellogenic stages. On the other hand, Vg
and SOP, CRP was present in the endogenous vitellogenic mRNA begins to increase in the endogenous vitellogenic
ovary as revealed by the western blot analysis, although stage concomitant with Vt accumulation in the oocytes.
cortical rods were not yet formed in this early developmental From these results, the endogenous vitellogenic stage is
stage of the oocyte (Kim et al., 2004). With the onset of considered to be a crucial stage for CRP and Vg syntheses.
translation, CRP transcripts disappeared from vitellogenic Such profiles of CRP and Vg/Vt suggest closely-related reg-
oocytes, indicating that CRP synthesis is regulated at the ulatory mechanisms of their syntheses.
translational level. In general, protein synthesis is required for oocyte mat-
To further examine the expression patterns of CRP and uration and the following early embryonic development, but
Vg genes during ovarian development, real-time PCR was in many organisms protein synthesis utilizes transcripts that
performed (Fig. 3). In the ovary, there were significant had been synthesized during oogenesis and stored for later
changes in CRP and Vg mRNA levels during ovarian devel- utilization (Seydoux, 1996; Picton et al., 1998). Furthermore,
opment. As shown in Fig. 3A, CRP mRNA levels, calculated the utilization of transcripts during oocyte maturation and
on the basis of the copy number per 1 g total RNA, were development appears to be highly selective; activation of
higher in the previtellogenic and endogenous vitellogenic stored mRNA can occur at specific stages of oocyte matu-
stages than in more advanced stages. However, there was ration and development. This may also be the case with
no significant change in the levels throughout the five ova- CRP, SOP and mjTSP mRNAs. In view of data obtained so
rian stages, when re-calculated on the basis of the copy far, the transcription of CRP is most likely to take place only
number per ovary. These findings indicate that there was no in the previtellogenic and endogenous vitellogenic oocytes.
difference in the total CRP transcript content between small The protein synthesis of CRP may start in the endogenous
immature and enlarged mature ovaries. Considering that a vitellogenic stage, and the cortical rod structures appear
fully mature ovary contains immature oocytes in addition to only in fully mature oocytes. Based on these results, we
mature ones, it is most likely that CRP mRNA is intensively concluded that the transcription, translation and cortical-rod
680 Y. K. Kim et al.

formation of CRP occur at different phases of oocyte devel- Kim YK, Kawazoe I, Tsutsui N, Jasmani S, Wilder MN, Aida K
opment, which could be regulated by different mechanisms. (2004) Isolation and cDNA cloning of ovarian cortical rod pro-
tein in kuruma prawn Marsupenaeus japonicus (Crustacea:
In contrast with CRP, gene transcription and translation of
Deapoda: Penaeida). Zool Sci 21: 11091119
Vg seem to occur consecutively in the ovary and hepatopan- Khayat M, Yang WJ, Aida K, Nagasawa H, Tietz A, Funkenstein B,
creas. The gene expression patterns, however, are appar- Lubzens E (1998) Hyperglycaemic hormones inhibit protein
ently different between the two organs, presumably being and mRNA synthesis in in vitro-incubated ovarian fragments of
under different control. the marine shrimp Peanaeus semisulcatus. Gen Comp Endo-
crinol 110: 307318
CHH peptides are considered to play important roles in
Khayat M, Babin PJ, Funkenstein B, Sammar M, Nagasawa H, Tietz
the ovarian development. CHH peptides inhibit ovarian pro- A, Lubzens E (2001) Molecular characterization and high
tein synthesis in P. semisulcatus (Khayat et al., 1998), and expression during oocyte development of a shrimp ovarian cor-
also decreased SOP synthesis at the translational level in tical rod protein homologous to insect intestinal peritrophins.
the same species (Avarre et al., 2001). CHH peptide levels Biol Reprod 64: 10901099
Medina A, Vila Y, Mourente G, Rodriguez A (1996) A comparative
in hemolymph and CHH peptide mRNA levels in the X-
study of the ovarian development in wild and pond-reared
organs decreased during yolk accumulation (De Kleijn et al., shrimp, Penaeus kerathurus (Forskal, 1775). Aquaculture 148:
1998). These results suggest that the reduction of CHH lev- 6375
els triggers the onset of the ovarian protein synthesis (De Meusy JJ, Payen GG (1988) Female reproduction in malacostracan
Kleijn and Van Herp, 1998). Considering the similarity Crustacea. Zool Sci 5: 217265
Okuno A, Yang WJ, Jayasankar V, Saido-Sakanaka H, Huong DTT,
between CRP and SOP, it is expected that CRP synthesis
Jasmani S, Atmomarsono M, Subramoniam T, Tsutsui N, Ohira
is also under the inhibitory control of CHH peptides. It would T, Kawazoe I, Aida K, Wilder MN (2002) Deduced primary
be of great interest to examine effects of CHH peptides on structure of vitellogenin in the giant freshwater prawn Macro-
CRP synthesis in kuruma prawn at both transcriptional and brachium rosenbergii, and yolk processing during ovarian mat-
translational levels. uration. J Exp Zool 292: 417429
Picton H, Briggs D, Gosden R (1998) The molecular basis of oocyte
growth and development. Mol Cell Endocrinol 145: 2737
ACKNOWLEDGMENTS Rankin SM, Davis RW (1990) Ultrastructure of oocytes of the
shrimp, Penaeus vannamei: Cortical specialization formation.
We are grateful to the Momoshima National Center for Stock Tissue Cell 22: 879893
Enhancement, Fisheries Research Agency for providing the exper- Seydoux G (1996) Mechanisms of translational control in early
imental animals. The present study was supported in part by a Fish- development. Curr Opin Genet Dev 6: 555561
eries Research Agency project (Development of seed production Tsukimura B (2001) Crustacean vitellogenesis: Its role in oocyte
and releasing techniques for stock enhancement of marine development. Am Zool 41: 465476
resources considering the conservation of ecosystem). Tsutsui N, Kawazoe I, Ohira T, Jasmani S, Yang WJ, Wilder MN,
Aida K (2000) Molecular characterization of a cDNA encoding
vitellogenin and its expression in hepatopancreas and ovary
REFERENCES during vitellogenesis in the kuruma prawn, Penaeus japonicus.
Zool Sci 17: 651660
Avarre JC, Khayat M, Michelis R, Nagasawa H, Tietz A, Lubzens E Tsutsui N, Kim YK, Jasmani S, Ohira T, Wilder MN, Aida K (2005)
(2001) Inhibition of de novo synthesis of a jelly layer precursor The dynamics of vitellogenin gene expression differs between
protein by crustacean hyperglycemic hormone family peptides intact and eyestalk ablated kuruma prawn, Penaeus (Marsupe-
and posttranscriptional regulation by sinus gland extracts in naeus) japonicus. Fish Sci (in press)
Penaeus semisulcatus ovaries. Gen Comp Endocrinol 124: Uchida K, Kaneko T, Tagawa M, Hirano T (1998) Localization of
257268 cortisol receptor in branchial chloride cells in chum salmon fry.
Bradfield JY, Berlin RL, Rankin SM, Keeley LL (1989) Cloned cDNA Gen Comp Endocrinol 109: 175185
and antibody for an ovarian cortical granule polypeptide of the Wilder MN, Subramoniam T, Aida K (2002) Yolk protein of crusta-
shrimp Penaeus vannamei. Biol Bull 177: 344349 cea. In Reprod Biol of Invertebr Vol. XII Part A, Progress in
Clark WH Jr, Yudin Al, Lynn JW, Griffin FJ, Pillai MC (1990) Jelly Vitellogenesis Ed by AS Raikhel and TW Sappington, Science
layer formation in Penaeoidean shrimp eggs. Biol Bull 178: Publisher, Enfield, pp 131174
295299 Yamano K, Seto H, Qiu G, Unuma T (2003) Immunological charac-
De Kleijn DPV, Van Herp F (1998) Involvement of the hyperglyce- terization of cortical rod proteins of kuruma prawn, Marsupe-
mic neurohormone family in the control of reproduction in deca- naeus japonicus. Comp Biochem Physiol A 136: 371377
pod crustaceans. Invertebr Reprod Dev 33: 263272 Yamano K, Qiu G, Unuma T (2004) Molecular cloning and ovarian
De Kleijn DPV, Janssen KPC, Waddy SL, Hegeman R, Lai WY, expression profiles of thrombospodin, a major component of
Martens GJM, Van Herp F (1998) Expression of the crustacean cortical rods in mature oocytes of penaeid shrimp, Masupe-
hyperglycaemic hormones and the gonad-inhibiting hormone naeus japonicus. Biol Reprod 70: 16701678
during the reproductive cycle of the female American lobster Yang W, Ohira T, Tsutsui N, Subramoniam T, Huong DTT, Aida K,
Homarus americanus. J Endocrinol 156: 291298 Wilder MN (2000) Determination of amino acid sequence and
Guraya SS (1982) Recent progress in the structure, origin, composi- site of mRNA expression of four vitellins in the giant freshwater
tion and function of the cortical granules in animal eggs. Int Rev prawn, Macrobrachium rosenbergii. J Exp Zool 287: 413422
Cytol 78: 257360
Kawazoe I, Jasmani S, Shih TW, Suzuki Y, Aida K (2000) Purifica-
(Received December 27, 2004 / Accepted March 28, 2005)
tion and characterization of vitellin from the ovary of kuruma
prawn, Penaeus japonicus. Fish Sci 66: 390396