Developmental features of Japanese eels, Anguilla japonica, from the late leptocephalus to the yellow eel stages: an early metamorphosis to the eel-like form and a prolonged transition to the juvenile.

Organogenesis of Japanese eels (Anguilla japonica) was investigated histologically from the late leptocephalus to the yellow eel stages. Early organogenesis, such as the formation of inner ears and the appearance of round blood cells that might be larval erythrocytes, had already begun at the late leptocephalus stage. During the first developmental phase (M1-M3 stages) of metamorphosing into early glass eels (G1 stage), the formation of gills and lateral muscles progressed conspicuously with a drastic body shape change from leaf-like to eel-like. In contrast, obvious regression in oesophageal muscle and pancreas occurred during metamorphosis. Formation of lateral line canals advanced continuously until the yellow eel stage. When the second developmental phase was initiated at the G1 stage, cone photoreceptor cells appeared, and the formation of oesophageal, stomach and intestinal muscles was initiated. Differentiation of gastric glands began at 1 week after metamorphosis. Erythrocytes increased continuously in density in glass eels and elvers (G1-E2 stages), and the morphological features of cone cells and olfactory epidermal cells became clearer with stage progression. In early elvers (E1 stage), the swimbladder initiated inflation, the stomach fully expanded and the rectal longitudinal fold changed to a circle. Swimbladder gas glands appeared in late elvers (E2 stage). In the yellow eels (juvenile stage), almost all organ structures were formed. These observations indicate that the organogenesis of A. japonica is ongoing after metamorphosis into glass eels, and the M1-E2 stages are considered to be a homologous phase to first metamorphosis, which is a transformation from the larval to the juvenile stages in other teleosts. In comparison to conger eels, the completion of the body shape change to eel-like occurs at the G1 stage, when organogenesis is still in progress, being followed by a prolonged duration of the G1-E2 stages before reaching the yellow eel juvenile stage, which may be a unique characteristic that is related to the early migratory life history of A. japonica.

Coccidian Parasite in Sea Cucumber (Apostichopus japonicus) Ovaries.

We investigated an unknown ellipsoidal body that is sometimes found in the ovaries of the sea cucumber Apostichopus japonicus. Its external morphology, comprising an ellipsoidal dark central body (about 150 µm in length) and a surrounding transparent layer (about 50 µm in thickness), resembled that of a protozoan cyst, particularly an oocyst. Histological observations of the developing A. japonicus ovaries clarified that a small mass of organisms appeared in the cytoplasm of young oocytes, proliferated in these cells through budding, became rod shaped and arranged radially, and, finally, formed an outer layer. These processes were considered to be the formation of a cyst by a protozoan parasite. The small subunit ribosomal RNA (18S rRNA) gene was amplified from the DNA extracted from unknown ellipsoidal bodies by using polymerase chain reaction with universal primers for eukaryote 18S rRNA. The determined sequence was not identical to any of the known sequences in DNA databases, but it clustered in a clade of coccidian species belonging to Eucoccidiorida in phylogenetic analyses. From these results, we concluded that the unknown ellipsoidal body is a cyst (possibly an oocyst) of a coccidian parasite (order Eucoccidiorida) that is formed in the A. japonicus oocyte, though its lower taxonomic position is uncertain. In a survey of the gonads of wild A. japonicus at Esashi, Hokkaido, during the reproductive season, these cysts were detected in more than 50% of females but were never found in males. We consider that the cysts of this parasite can only be formed in A. japonicus ovaries.

Accumulation of the major yolk protein and zinc in the agametogenic sea urchin gonad.

Sea urchins of both sexes store the nutrients necessary for gametogenesis in nutritive phagocytes of the agametogenic gonad. A zinc-binding protein termed the major yolk protein (MYP) is stored here as two isoforms: the egg-type (predominant in egg yolk granules) and the coelomic fluid-type (a precursor with greater zinc-binding capacity). MYP is used during gametogenesis as material for synthesizing gametic proteins and other components. We investigated its accumulation and relationship to zinc contents in gonads during the non-reproductive season in Pseudocentrotus depressus. MYP constituted most of the protein in coelomic fluid and gonads. Both ovaries and testes grew gradually, accumulating MYP and zinc during the year. Total zinc contents and the ratio of coelomic fluid-type to egg-type protein were higher in ovaries than in testes as gametogenesis approached. Most of the zinc in the coelomic fluid was bound to MYP, and the concentrations of MYP and zinc were elevated toward the onset of oogenesis in the female coelomic fluid. Thus, MYP accumulates in the agametogenic ovaries and testes during the non-reproductive season, playing a role as a carrier to transport zinc to the gonad. Transportation of zinc by MYP is more active in females than in males.

Molecular cloning and expression analysis of the Mitogen-activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Molecular cloning and expression of progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp Penaeus monodon.

Knowledge on molecular mechanisms of steroid hormonal induction on oocyte development may lead to the possible ways to effectively induce ovarian maturation in shrimp. In this study, progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp (Penaeus monodon) initially identified by EST analysis was further characterized. The full-length cDNA of Pgmrc1 was 2015bp in length containing an ORF of 573bp corresponding to a polypeptide of 190 amino acids. Northern blot analysis revealed a single form of Pgmrc1 in ovaries of P. monodon. Quantitative real-time PCR indicated that the expression level of Pgmrc1 mRNA in ovaries of both intact and eyestalk-ablated broodstock was greater than that of juveniles (P<0.05). Pgmrc1 was up-regulated in mature (stage IV) ovaries of intact broodstock (P<0.05). Unilateral eyestalk ablation resulted in an earlier up-regulation of Pgmrc1 since the vitellogenic (II) ovarian stage. Moreover, the expression level of Pgmrc1 in vitellogenic, early cortical rod and mature (II-IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock (P<0.05). Pgmrc1 mRNA was clearly localized in the cytoplasm of follicular cells, previtellogenic and early vitellogenic oocytes. Immunohistochemistry revealed the positive signals of the Pgmrc1 protein in the follicular layers and cell membrane of follicular cells and various stages of oocytes. Taken the information together, Pgmrc1 gene products seem to play the important role on ovarian development and may be used as the bioindicator for monitoring progression of oocyte maturation of P. monodon.

Molecular characterization of the major yolk protein of the Japanese common sea cucumber (Apostichopus japonicus) and its expression profile during ovarian development.

The most abundant protein in the coelomic fluid of the Japanese common sea cucumber (Apostichopus japonicus) was purified through two steps of liquid chromatography. Subsequent peptide sequencing and cDNA cloning demonstrated that the purified fraction contained two similar but distinct proteins. Deduced amino acid sequences of these proteins revealed about 30% identity with those of sea urchin major yolk protein (MYP) and therefore they were designated AjMYP1 and AjMYP2. The full-length cDNAs for AjMYP1 and AjMYP2 consisted of 4600 and 4420bp, with predicted protein lengths of 1365 and 1345 amino acid residues, respectively. RT-PCR detected transcripts for both types of AjMYPs in all the tissues and organs examined. The transcript levels of both AjMYPs in the ovary were apparently elevated at late stages of ovarian development whereas the MYP content of the ovary examined by SDS-PAGE remained stable throughout ovarian development.

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis.

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real-time reverse-transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad.

Molecular cloning and expression analysis of ovary-specific transcript 1 (Pm-OSTI) of the giant tiger shrimp, Penaeus monodon.

Isolation and characterization of genes specifically expressed in ovaries are necessary for understanding sex differentiation and ovarian development processes in the giant tiger shrimp, Penaeus monodon. In this study, a transcript that significantly matched the polehole precursor was further characterized by RACE-PCR. The sequence obtained was 5151 bp in length and contained a coding region of 5031 bp corresponding to 1677 amino acids. This transcript was only expressed in ovaries but not in testes of Juveniles (N = 10) and broodstock (N = 22) of P. monodon. A tissue distribution analysis further confirmed ovary-specific expression of this transcript (called P. monodon ovary-specific transcript 1, Pm-OST1) in female broodstock. Expression levels of Pm-OST 1 in ovaries of juvenile P. monodon upon 5-HT Injection (33.9+/-6.40 g; 50 microg/g body weight) were significantly higher at 12-72 hours post Injection (P<0.05). Quantitative real-time PCR Indicated that Pm-OST1 was comparably expressed throughout ovarian development in normal P. monodon broodstock (P>0.05). However, the expression level of Pm-OST1 was significantly higher in stage-III ovaries in eyestalk-ablated broodstock (P<0.05). Pm-OST1 was clearly localized in the ooplasm of previtellogenic and vitellogenic oocytes. Our results suggest that Pm-OST1 plays a functionally Important role in promoting the development of female germ cells and oocytes in P. monodon.

Expression and purification of active recombinant cathepsin C (dipeptidyl aminopeptidase I) of kuruma prawn Marsupenaeus japonicus in insect cells.

Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37 degrees C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-beta-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae.

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus.

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 muM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.

Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai.

Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai.

Zinc-binding property of the major yolk protein in the sea urchin - implications of its role as a zinc transporter for gametogenesis.

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus.

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.

Expressed sequence tags from eyestalk of kuruma prawn, Marsupenaeus japonicus.

We analyzed the expressed sequence tags (ESTs) obtained from a cDNA library of the eyestalk of the kuruma prawn, Marsupenaeus japonicus, to examine gene expression profile with special focus on female reproduction. The assembly of 1988 ESTs created 136 contigs from 738 ESTs; however 1250 ESTs remained singletons. Significant similarities (blast score > or = 50 bits) to the DNA sequences in the databank were found for only 16.7% of the 1386 sequences (136 contigs plus 1250 singletons), suggesting that the eyestalk library contains many unknown genes. Ribosomal RNA and mitochondrial respiration enzymes with significant similarities were found abundantly in the ESTs, whereas genes related to maturation or endocrine systems were scarce. Three ESTs were assumed to encode novel eyestalk hormones with marked similarities to pigment-dispersing hormone, molt-inhibiting hormone and crustacean hyperglycemic hormone. Sequences encoding a product highly homologous to farnesoic acid O-methyltransferase, an enzyme that produces methyl farnesoate, were also found.

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus.

In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).

Three forms of cyclin B transcripts in the ovary of the kuruma prawn Marsupenaeus japonicus: their molecular characterizations and expression profiles during oogenesis.

Cyclin B is a well known regulatory factor that plays a crucial role in mitosis and meiosis. Although the existence of cyclin B has been reported to be universal in a wide variety of eukaryotic organisms, no molecular data are available on crustacean species. In this study, three forms of cyclin B transcripts were first identified and characterized in the ovary of the commercially important kuruma prawn Marsupenaeus japonicus. The three transcripts (2.4, 1.9 and 1.7 kb) shared the identical sequence, with variations only in the length of 3' untranslated regions (UTRs), and coexisted in the ovary as demonstrated by Northern blot analysis. The sequences of 3' UTRs indicated that the distinct length UTRs of the transcripts is attributed to an alternative usage of various polyadenylation signals in the 3' UTR. The open reading frame of 1203 bp encoded a putative 401 amino acid peptide. The deduced amino acid sequence shared 45-50% identities with the known B-type cyclin in other animals. Quantitative real-time RT-PCR revealed that the short transcript (1.7 kb) was the most abundant among the three transcripts, followed by the long (2.4 kb) and medium (1.9 kb), and the three forms of the transcripts displayed various expression profiles during oogenesis. In situ hybridization showed that the short transcript commenced expressing in the ova as early as the oogonia stage and accumulated largely at the perinucleolus (PN) stage, whereas almost no expression was found for the medium and long transcripts at the oogonia stage and moderate signals were detected at the PN stage. The differential expression of the three forms of transcripts suggested that various transcripts might perform different roles during oogenesis of the kuruma prawn.

Cathepsin C transcripts are differentially expressed in the final stages of oocyte maturation in kuruma prawn Marsupenaeus japonicus.

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn (Marsupenaeus japonicus), subtractive suppression hybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed by evaluation of the differential expression profile by macroarray and quantitative real-time RT-PCR analyses. The cathepsin C (dipeptidyl peptidase I) gene was thus found to exhibit a significantly higher expression around the onset of cortical rod (CR) formation (early CR stage, appearance of round CRs), progress to a higher mRNA level until the middle CR stage (elongation of CRs), then rapidly revert to a low expression level at the late CR stage (occurrence of germinal vesicle breakdown, GVBD), as also observed at the non-CR stage (previtellogenesis and vitellogenesis). In situ hybridization analyses revealed that the sites of the expression of cathepsin C transcripts in the ovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specific gene was subsequently determined by rapid amplification of the cDNA 3' and 5' ends (3' and 5' RACE). The deduced amino acid sequence of the 230-residue mature peptide shared 67-70% identity to the known cathepsin C in mammals. Western blot analysis showed that expression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs as revealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration of its involvement in the final stages of oocyte maturation in crustacean species.

Detection of white spot syndrome virus from stomach tissue homogenate of the kuruma shrimp (Penaeus japonicus) by reverse passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay was developed for detecting the white spot syndrome virus (WSSV), which was formally named as penaeid rod-shaped DNA virus (PRDV) in Japan, from stomach tissue homogenate of the kuruma shrimp (Penaeus japonicus). Using high-density latex particles and specific polyclonal antibody, WSSV was detectable after 4h incubation. The hemolymph, the stomach, and the gills were extracted from a shrimp that had been infected experimentally with WSSV, the virus contained in each sample was tested by the PRLA and PCR assay. It was possible to detect the WSSV only from stomach tissue homogenates by the RPLA assay. And there was an agreement between RPLA and PCR assays for WSSV detection. Considering that the RPLA assay does not require biochemical expertise and latex reagents and all apparatus can be provided as a kit, this assay can be used for virus detection in the culture pond of shrimps or in the field as a convenient method.

Molecular cloning and ovarian expression profiles of thrombospondin, a major component of cortical rods in mature oocytes of penaeid shrimp, Marsupenaeus japonicus.

In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.