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Ovary |
Division of Farming Biology,3 National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan
Fisheries College,4 Shanghai Fisheries University, Shanghai, 200090, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CR is also remarkable from the viewpoint of practical applications in the artificial production of prawn juveniles for farming. Large numbers of juveniles of various penaeid shrimps are artificially produced around the world. The kuruma prawn (Marsupenaeus japonicus) is a particularly important species for shrimp culture in Japan because of its high commercial value. In captivity, however, the kuruma prawn is mostly incapable of forming CRs and therefore has a poor reproductive potential. Most female prawn used for artificial production of juveniles are collected from the wild. Therefore, the juvenile production is possible only during the natural reproductive season; in this species, moreover, strains with some desirable potentials for cultivation have not been developed so far. Consequently, an effective way of inducing maturation in the kuruma prawn has long been sought. This study was thus conducted to elucidate the mechanism of CR formation, with the aim of applying that knowledge to the development of the artificial production of juvenile kuruma prawn.
Biochemical analyses have shown that CR are composed 25–30% of carbohydrate and 70–75% protein on dry matter basis [2]. Two related cDNA sequences encode CR proteins with a molecular mass of about 30 kDa in Penaeus semisulcatus [3]. Given a sequence homology to insect intestinal peritrophin, the CR proteins were designated shrimp ovarian peritrophin (SOP)1 and SOP2. The expression of SOP proteins was controlled at the translational level [4]. A CR protein with a larger molecular mass has been described [5]; however, its identity is obscure because the sequences of the amino acid and the nucleotide have neither been described nor deposited in the data bank, although its cDNA and a recombinant protein have been produced. In our previous study [6], SDS-PAGE analysis of CR isolates from the kuruma prawn has demonstrated five major components, of which 130-, 140-, and 150-kDa proteins displayed quite similar immunological characteristics. The CR proteins were immunohistochemically detectable even in early vitellogenic oocytes, although CRs appear only after the completion of vitellogenesis, suggesting that synthesis of the CR proteins and formation of the CR structure occur at different phases.
In this study, to further understand the nature of CR proteins and their expression profiles during ovarian development, cDNAs encoding the 150-, 140-, and 130-kDa CR proteins were isolated from the ovary of the kuruma prawn. Those proteins were estimated to be isoforms generated from a single gene. Deduced amino acid sequences of the cDNAs shared characteristic features with those of a thrombospondin (TSP) 3,4/cartilage oligomeric matrix protein (COMP) family. Moreover, transcripts of the gene, intensively identified in the early stages of previtellogenic oocytes disappeared from vitellogenic oocytes, whereas the proteins were prominent in oocytes after vitellogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The kuruma prawn (M. japonicus) was purchased from a commercial source in Mie, Japan, or provided from the Momoshima Branch of the National Farming Center, Hiroshima, Japan. Ovarian stages were determined from histological observation of paraffin sections as follows: immature stage—yolk accumulation has not yet occurred, and therefore the ovary contains only oogonia and basophilic previtellogenic oocytes (gonad somatic index [GSI] 1.1–1.8); oil globule stage—the ovary contains yolk- accumulating oocytes, the ooplasm of which is stained by the Shiffs reagent but weakly or not stained by eosin, and small vacuoles in the ooplasm are notable (GSI 2.0–4.0); yolk granule stage—the ovary contains oocytes, the ooplasm of which is full of eosinophilic yolk granules (GSI 3.2–11.8); and CR stage—the ovary contains oocytes that develop CRs peripherally (GSI 8.6–14.1).
Peptide Sequencing
CRs were isolated from the mature ovary and subjected to SDS-PAGE as described [6]. The proteins were transferred onto a polyvinyl difluoride (PVDF) membrane, and then bands of 150- and 130-kDa proteins were subjected to N-terminal sequencing by Edman degradation on an HP G1005A sequencer (Hewlett Packard, Palo Alto, CA) in the pulse-liquid mode. To determine the internal sequences, the 150-kDa protein was fragmented with lysyl endopeptidase, and then peptide fractions selected by reverse-phase HPLC were analyzed on a Procise 494 HT protein sequencing system (PE Applied Biosystems, Foster City, CA).
cDNA Sequencing
Total RNA was extracted from the ovary at the CR stage, and then mRNA was purified on an oligo(dT) cellulose column. Full-length cDNA was constructed with a Marathon cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. Primers for polymerase chain reaction (PCR) were designed from the amino acid sequences of the 150-kDa protein and our unpublished expressed sequence tag (EST) sequences encoding the amino acid sequences determined by peptide sequencing. Both 5'- and 3'-end sequences were determined by the rapid amplification of cDNA ends (RACE) method. The cloning strategy is shown in Figure 1, and the primers used are listed in Table 1.
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Homology search and sequence analysis were carried out on a FASTA program [7] provided by the Web site of the DNA data bank of Japan (http://www.ddbj.nig.ac.jp/Welcome.html) and on a Genetyx-Mac, version 9 (Genetyx Co. Ltd., Tokyo, Japan).
Real-Time PCR
RNA was extracted from the ovaries at various maturational stages with the TRIzol reagent (Invitrogen, La Jolla, CA), followed by treatment with DNaseI (Promega, Madison, WI) to eliminate possible contamination of genomic DNA. First-strand cDNA was synthesized from total RNA with Powerscript reverse transcriptase (Invitrogen) and an oligo(dT) primer. Aliquots of the first-strand cDNA solution, as well as dilution series of known amounts of control template, were subjected to polymerse chain reaction (PCR) with a QuantiTect Probe PCR kit (Qiagen, Alameda, CA). The PCR reactions were initiated from an enzyme activation step at 95°C for 15 min, followed by 30 or 40 cycles of amplification comprising denaturing at 94°C for 15 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec. Primers TSPa1f and TSPa2r were used for the amplification of common sequences for three forms of mjTSPs; primers GAPDHf and GAPDHr were used for the amplificaion of kuruma prawn glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table 1). In the preliminary experiments under these conditions, a single-band product was confirmed by gel electrophoresis. Each control template for TSP and GAPDH was prepared by the PCRs with the above primer set, and the dilution of the templates were 1/10 at each step. Quantities of PCR products were monitored on an iCycler iQ by detecting the intercalating dye that binds to double-stranded DNA (Bio-Rad Laboratories, Richmond, CA). All measurements were made in duplicate. The data obtained by the iCycler iQ were analyzed on an iCycler program (Bio-Rad). Briefly, the threshold cycles, at which the fluorescence rises appreciably above background, were determined for both cDNA samples and known amounts of controls (Fig. 2). A standard curve was then constructed by plotting the log of starting amount of control versus the corresponding threshold cycle (inset in Fig. 2). The standard curves were drawn at every run, and correlation coefficients of the standard curves ranged from 1.000 to 0.997. The standard curves were then used to determine the starting amount of the target in each sample. The amounts of TSP and GAPDH in the same cDNA sample were measured separately. Results are indicated as the molar ratio of TSP to GAPDH.
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In Situ Hybridization
Small pieces of specimens were cut from the ovaries, fixed in Davidsons fixative (30% ethanol, 22% formalin, and 11.5% acetic acid), embedded in paraffin, and sectioned onto silane-coated slides. After removal of the paraffin, the sections were incubated at 50°C overnight with either digoxigenin (DIG)-labeled sense or antisense cRNA probe. The sections were rinsed in 4x SSC (67 mM NaCl; 67 mM sodium citrate, pH 7.0) at 50°C for 5 min twice, and in 2x SSC containing 50% formamide at 50°C for 20 min. After equilibration in a RNase buffer (0.5M NaCl; 10 mM Tris-HCl, pH 8.0; 1mM EDTA) at 37°C for 30 min, single-strand RNA was digested with 10 µg/ml of RNase A at 37°C for 10 min. The sections were again rinsed in 2x SSC at 50°C for 15 min four times and in 0.2x SSC at 50°C for 20 min twice. DIG was immunologically detected with an alkali phosphatase-labeled antibody to DIG and BM purple, a substrate for alkali phosphatase, according to the manufacturer's instructions (Roche, Mannheim, Germany).
For the preparation of cRNA probes, PCR II in Figure 1 were subcloned into pCRscript II (Stratagene, La Jolla, CA). After linearizing the plasmid with BamHI or NotI (Takara, Shiga, Japan), cRNA was synthesized by either T3 or T7 RNA polymerase using a DIG cRNA synthesis kit (Roche).
SDS-PAGE and Western Blotting
Tissues were homogenized in eight volumes (v/w) of an extraction solution (9 M urea, 2% Triton-X, 1% dithiothreitol, 1 mM PMSF). Two volumes of 10% SDS were added to the homogenate, and the mixture was sonicated for several seconds and centrifuged; the supernatant was subjected to SDS-PAGE. Monoclonal antibodies (P3B10) raised to CR proteins were used for immunodetection. The procedure of Western blotting and characterization of monoclonal antibodies were described previously [6].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine a sequence similarity of CR proteins that displayed immunological similarity in our previous study [6], N-terminal sequences of the 150- and 130-kDa proteins were analyzed by microsequencing. The sequences were identical in the two proteins: SVTTAYHLYSYLCEGQ. The 150-kDa protein was treated with lysyl endopeptidase, and then N-terminal sequences of selected fragments were also examined. Seven to 14 residues were determined on five fragments: fragment 1, ADVFVRDMYDPQ; fragment 2, TDFTNLQMVN; fragment 3, GVQLRLVNSATG; fragment 4, IIWSNMK; fragment 5, YSCNRDIPQAIFDD.
cDNA Cloning of CR Proteins
A schematic approach for determining a whole cDNA sequence is shown in Figure 1, and primer sequences used for PCR amplification are listed in Table 1. First, we searched through our EST data that encode amino acid sequences determined by microsequencing of the 150-kDa protein (ESTs have not been published). EST I encoded the N-terminal sequence of fragment 2, whereas EST II encoded those of fragments 3, 4, and 5 (Fig. 1). These two ESTs overlapped each other. To confirm these EST sequences and to obtain undetermined downstream sequences, PCR products II and III were amplified using primers based on EST and Marathon adaptor sequences. On the other hand, PCR I obtained by PCR using an adaptor primer and a degenerate primer based on fragment 1 included a 5'-untraslated sequence, an initiation codon, and the N-terminus of the150-kDa protein (Fig. 1). Based on these sequences, the region bridging the gap between PCR products I and II was amplified by PCR. Interestingly, PCR simultaneously amplified three products of different sizes, PCR IV-VI (inset in Fig. 1). These products contained the same sequences except for an internal region in which sequences of the medium and short products lacked 246- and 369-bp fragments, respectively, when compared with the longest sequence. As shown in Figure 3, a merged sequence of the long form was composed of 3545 bp, which contained 3342 bp of an open reading frame (ORF), whereas the medium and short forms consisted of 3299 and 3176 bp with 3096 and 2973 bp of ORFs, respectively. A polyadenylation signal, AATAAA, was located 15 bp upstream of the poly(A) sequence (Fig. 3). The cDNA sequences are deposited in the DNA data bank of Japan under accession numbers AB121209, AB121210, and AB121211.
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Characterization of Deduced Amino Acid Sequence
The deduced amino acid sequence included all the sequences obtained by peptide microsequencing of the 150- kDa protein (Fig. 3). The N-terminal sequence of the 150- kDa protein was found at 65 residues downstream from the first methionine in the deduced amino acid sequence (Fig. 3). Molecular masses of the long, medium, and short forms of the polypeptides (from Ser66 to the end) were calculated at 110 945, 102 697, and 98 630 daltons, respectively. Two potential N-glycosylation sites were located at positions 220 and 545.
Comparison of the deduced amino acid sequence of the short form with the database on a FASTA program revealed high similarities to those of the proteins in the TSP 3, 4/ COMP family, with such scores as 1715 for mouse COMP, 1700 for equine COMP, 1694 for human TSP 4, 1689 for human COMP, and 1684 for mouse TSP 3. Quite similar results were obtained by comparing the sequences of the medium and long forms. As for crustacean TSP 3, 4/COMP, only ESTs [8] and a partial cDNA sequence were deposited in the data bank. TSP 3, 4/COMP is known as an extracellular matrix protein and shares a unique domain structure that holds an N-terminal domain, four type II (epidermal growth factor-like) repeats, seven type III (Ca2+-binding) repeats, and C-terminal domain [9–13]. These domains were compared separately (Table 2). The N-terminal domain was a hypervariable region showing no homologies, even among proteins in the same family. The second domain exhibited considerable homology to the known TSP 3, 4/COMP. A fitting comparison was not valid, however, because sequence alignment for comparison was accessible only in parts of the areas because of relatively low homologies (Table 2), despite which the prawn sequence conserved to a great extent the structural features of type II repeats (Fig. 4). The sequence was rich in cysteine residues that appeared at regular intervals. Sequence variation in the three forms resulted in differences in the repeat numbers of the type II sequence; each had 7, 5, or 4 type II repeats (Fig. 4). The type III region showed the strongest similarities among domains, sharing 54.9% to 68% identities with comparable sequences (Table 2). The type III sequence, as well as type II, contained abundant cysteine that was conserved even in the human and the prawn. Sequence similarities extended into the C-terminal domain in which 42.7– 66.5% identities were observed (Table 2). The TSP of the tiger prawn clearly showed higher similarities to the kuruma prawn sequence than to the other animals, although the tiger prawn sequence was only partly available. The TSP 3, 4/COMP of the other animals including the fruit fly exhibited equivalent percent identities to the kuruma prawn sequence. In considering that the deduced amino acid sequences displayed high identities and structural similarities to those of the TSP 3, 4/COMP family, but were not capable of being attributed to a specific type, these three forms were designated as mjTSPa, mjTSPb, and mjTSPc, respectively.
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The N-terminal domain that exhibited no significant homology to that of the TSP3, 4/COMP family showed a high homology to the SOP, another component of cortical rod proteins (Fig. 5). This domain was rich in cysteine, and the position was completely conserved in mjTSP and SOPs. The positions of N-terminus in mature proteins demonstrated by peptide sequencing were also conserved between mjTSP and SOP [3].
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Expression Profiles of mjTSP Gene During Gonadal Development
The expression levels of the three forms of mjTSP transcripts, as well as GAPDH, a housekeeping gene, were measured by the real-time PCR. The values in Figure 6 are indicated as relative molar ratio of mjTSPs to GAPDH. Transcripts of mjTSP were observed, even in the immature ovary. Although more than threefold variation in the level existed among individuals at the same stages, no notable changes were found in relation to ovarian maturational stages or GSI (Fig. 6).
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The localization of mjTSP transcripts in the ovaries at different stages was visualized by in situ hybridization (Fig. 7). In the germinative zone in which oogonia proliferate mitotically and meiotically (Fig. 7a), the oogonia did not display positive signals for mjTSP transcripts (Fig. 7b). Oocytes at quite young stages that localize close to the germinative zone showed the earliest expression of mjTSP transcripts (Fig. 7b). The absence of yolk substances in positive oocytes was confirmed by periodic acid-Schiff (PAS), which stains yolk and connective tissues in the ovary (Fig. 7c). In the immature ovary, strong signals were observed in oocytes at the early perinucleolus stage; they decreased slightly in perinucleolus oocytes (Fig. 7, d and e). In the vitellogenic ovaries, oocytes at the oil globule stage showed much weaker signals than previtellogenic oocytes (Fig. 7, e and f), and the signals disappeared completely from the oocytes at yolk granule (Fig. 7, g and h) and CR stages. In the vitellogenic ovaries, however, previtellogenic oocytes demonstrated the same abundant signals as observed in the immature ovary: because the kuruma prawn is a multispawner in a single breeding season, the mature ovary contains various stages of oocytes including previtellogenic ones. The expression of mjTSP transcripts was observed in oocytes, not in follicle cells, of ovaries at all stages. The sense probe used as a negative control demonstrated no staining at any stage of the ovaries.
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Expression Profiles of mjTSP Protein During Gonadal Development and in Various Organs
Amount of proteins extracted from the same weight of the ovary increased after the yolk granule stage (Fig. 8a, lanes 4–7), when GSI also increased greatly, indicating rapid accumulation of yolk substances in the ovary. The developmental transition of the expression of the mjTSP protein in the ovary was further demonstrated by Western blotting (Fig. 8b). In the immature ovary, no mjTSP proteins were detected (Fig. 8b, lane 2). Three immunoreactive bands became manifest at the oil globule stage (Fig. 8b, lane 3), when CR structure had not yet formed. The mjTSP- positive immunosignals increased at the yolk granule stage (Fig 8b, lanes 4 and 5) and maintained a high level at the CR stage (Fig. 8b, lanes 6 and 7). At spawning, CRs are released outward to encompass the eggs in a layer of jelly. The mjTSP proteins were detected in a remnant of jelly materials that were found on the walls of the aquarium after the disappearance of the jelly layer (Fig.8b, lane 8).
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A high content of TSP was identified only in the ovary (Fig. 9). Faint immunoreactivity to mjTSP was observed in the heart, gut, muscle, ganglions, stomach, and hemolymph but not in the hepatopancreas or the eye (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous study has demonstrated the presence of five major components of CR proteins in the kuruma prawn [6]. In the present study, we cloned the cDNA of the 150-kDa protein, one of the CR proteins, on the basis of its partial amino acid sequences. The sequences contained a long ORF and 5'- and 3'-untranslated regions (UTRs). The deduced amino acid sequences included all the partial amino acid sequences determined by peptide sequencing, ensuring successful cDNA cloning of the 150-kDa CR protein. The deduced amino acid sequence as well as the structural features speculated from the conserved cysteine residues showed strong similarities to those of extracellular matrix proteins of the TSP 3, 4/COMP family, but the particular member of the family was difficult to define. The CR protein was therefore designated as mjTSP. This is the first report of overall cDNA sequence of crustacean TSP.
Extracellular matrix proteins are known to play a role in maintaining tissue and organ structures. Recent increasing knowledge of the extracellular matrix proteins has proved its function as a signal modulator involved in cell adhesion, migration, proliferation, differentiation, and so forth [14– 16]. Mammalian TSP was expressed during embryogenesis in a specific temporal and spatial manner, suggesting the role of a signal modulator [17–19]. Prawn TSP found in CRs probably functions mainly as a classical extracellular matrix protein because the contents of CRs are released outward to surround the eggs with a layer of jelly that is believed to function as a physical barrier against polyspermy and mechanical damage. Some specific molecular interactions between sperm and matrix proteins may also be possible. In the mouse and the sea urchin, proteins released from cortical granules block polyspermy by modulating the sperm receptor, the egg surface, or the fertilization membrane [20, 21], although the presence of TSP 3, 4/COMP in cortical granules has not been reported in these animals.
During the cloning procedure, the same primer set amplified three PCR products of different sizes. The medium and short nucleotide sequences lacked 246 and 369 bp, respectively, when compared with the longest one. The deletion resulted in a decrease in the repeat numbers of the type II sequence at the protein level. The three forms designated as mjTSPa, mjTSPb, and mjTSPc thus had seven, five, and four repeats, respectively. The variation in the number of repeats in the type II sequence in the kuruma prawn is unique because all TSP 3, 4/COMP reported so far have four repeats. Considered with previous immunological data, the three forms were estimated to correspond to 150-, 140-, and 130-kDa CR proteins. Thus, three forms of mjTSP transcripts might be generated from one gene by alternative splicing.
Molecular sizes estimated on an SDS-PAGE gel and calculated from the deduced amino acid sequences differed considerably, and potential glycosylation sites were found in the deduced amino acid sequences. CRs contained 25– 30% carbohydrate, demonstrating a PAS-positive stain [2]. Post-translational modification of mjTSP might account for the difference in the sizes estimated by SDS-PAGE and deduced amino acid sequences.
CR Proteins Are of Oocyte Origin
During ovarian development in penaeid shrimp, oocytes accumulate a large amount of yolk proteins in which vitellin and CR proteins are major constitutions. The production sites of such proteins are not identical, however. This study demonstrated that the mjTSP gene expressed intensively in oocytes. Although the mjTSP protein was detected faintly in various organs, it probably is not related to ovarian TSP because TSP is reported to be widely distributed in tissues and organs [17–19]. Based on its immunohistochemistry, SOP, another CR component in P. semisulcatus, is also thought to be produced in the ovary [3]. On the other hand, vitellogenin, a vitellin precursor, of penaeid shrimp has been demonstrated to be produced by follicle cells surrounding oocytes and by parenchymal cells in the hepatopancreas [22]. In fresh-water shrimp, Macrobranchium rosenbergii, a single source of vitellogenin from the hepatopancreas has been reported [23]. The allopatric origin of yolk proteins has been widely recognized in various groups of animals. Vitellogenin has a mostly an extraovarian origin: liver in fish [24], fat body in insects [25], intestine in nematodes [26], nutritive phagocyte in echinoids [27]. On the other hand, components of cortical granules are produced in oocytes [28–30].
Gene Expression and Protein Synthesis of mjTSP Occur Much Earlier than the Appearance of CR Structures
In this study, the gene expression of mjTSP in the ovary was examined semiquantitatively by real-time PCR and qualitatively by in situ hybridization. The results of the two different methods clearly exhibited mjTSP expression in the early stages of oocytes. Surprisingly, transcription of the mjTSP gene occurred in the oocytes soon after initiation of meiosis, and the most concentrated presence of mjTSP transcripts was observed in previtellogenic oocytes at the early perinucleolus stage. Gene expression of SOP has also been found in the immature ovary by Western blotting, although localization of SOP transcripts in the ovary has not been examined [3]. Protein synthesis of mjTSP started in the early vitellogenic ovary despite the development of CR structures in fully grown oocytes. Simultaneously with the start of the translation, mjTSP transcripts rapidly disappeared from the vitellogenic oocytes, suggesting that mjTSP protein synthesis is controlled mainly at the translational level not at the transcriptional one. Translational regulation has also been demonstrated in SOP [4]. Although quantitative analysis by real-time PCR demonstrated that the ovaries at the yolk granule and CR stages contained mjTSP transcripts to a similar extent as at the earlier stages of the ovaries, positive signals by in situ hybridization localized mostly in coexisting previtellogenic oocytes in the maturing and mature ovaries.
Considered together with data obtained so far, the process of CR formation can be separated into three phases. First, the transcription of genes for CR proteins in the previtellogenic oocytes and accumulation of the transcripts in oocytes occur in the quite early stage of oocytes, just after the initiation of meiosis. The transcription occurs only or mostly in previtellogenic oocytes. Second, the translation into proteins starts in oocytes at the oil globule stage, indicating that CR proteins are synthesized simultaneously with initiation of ovarian development. Third, in fully grown oocytes, CR proteins are rapidly assembled to form the CR structures. Electron microscopic observations have described this process in detail [31]. In the perinuclear cytoplasm of fully grown oocytes, small dense granules decondense into the fine fibrillar matrix that is contained in small vesicles that translocate peripherally, fuse with each other during the transition, and finally make a large CR. Thus, transcription, translation, and formation of CR structures take place at apparently distinct phases in the development of oocytes. Different regulatory mechanisms are probably involved in each step.
Two CR Proteins, mjTSP and SOP, Have Common Characteristics
In many aspects mjTSP exhibited characteristics similar to those of SOP: 1) a molecular similarity found in cDNA and deduced amino acid sequences, 2) a proteolytic site for post-translational modulation, 3) gene expression in oocytes in the immature ovary, 4) protein production in the early vitellogenic ovary, and 5) post-translational modification such as glycosylation. These findings suggest that TSP and SOP might be produced from a single gene through alternative splicing or post-translational modification or that they are encoded by distinct genes that originate in the same ancestral gene. Nevertheless, TSP and SOP appear to be governed by the same regulatory mechanisms from gene expression to post-translational modification. Electron microscopic observations of CR have disclosed that CRs contain numerous tightly packed feathery elements [1, 31], suggesting crystallization or polymerization of CR substances. Both mjTSP and SOP, which were quite rich in cysteine that can be utilized for an S-S bond, might cooperatively create the feathery structure.
Possible Cause of a Reduced Potential in Maturation in Captive Animal
The kuruma prawn in captivity has a reduced spawning potential as described in the introduction, and its degree of maturation varies among individuals and depends on maintaining conditions. In most cases, the ovaries start to develop in the reproductive season but fail to reach full-grown requisite to the formation of CRs; the ovaries then regress without spawning. As shown in the present study, hypoplasia of the CR structure does not always signify failure in the synthesis of CR proteins. The prawn can initiate production of CR proteins but cannot carry the progression through to the accumulation of yolk substances or cannot proceed to the next phase even when yolk accumulation is achieved. Further elucidation of mechanisms controlling each step of CR formation is required to control maturation of the kuruma prawn in captivity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Keisuke Yamano, Division of Farming Biology, National Research Institute of Aquaculture, Fisheries Research Agency, 422-1, Nakatsuhamaura, Nansei, Mie 516-0193, Japan. FAX: 81 599 66 1962;yamano{at}fra.affrc.go.jp
Received: 11 November 2003.
First decision: 11 December 2003.
Accepted: 30 January 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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