Cdc2 kinase is a catalytic subunit of maturation-promoting factor (MPF), a central factor for inducing themeiotic maturation of oocyte. To understand the role of Cdc2 kinase on the oocyte maturation in crustacean,a complete cDNA sequence of Cdc2 kinase was cloned from Chinese mitten crab Eriocheir sinensis and itsspatial-temporal expression profiles were analyzed during oogenesis at RNA and protein levels. The crabCdc2 cDNA (1364 bp) encodes for a 299 amino acids protein with calculated molecular weight of 34.7 kDa.The Cdc2 mRNAs level showed no significant change in the ovary during oogenesis, whereas higher proteinlevel was found at previtellogenesis, late vitellogenesis and germinal vesicle breakdown (GVBD) stages. Twoforms (35 kDa and 34 kDa) of Cdc2 proteins were simultaneously identified in ovary at all stages.Immunocytochemistry analysis revealed that Cdc2 proteins locate exclusively in ooplasm of previtellogenicoocyte, and then relocate into germinal vesicle at vitellogenesis stage and accumulate on meiotic spindle atoocyte maturation. These findings suggest that Cdc2 kinase has essential roles in inducing GVBD andgenerating meiotic apparatus during the crab oocyte maturation.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotionfactor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism ofoocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinaseduring oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and proteinlevels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greaterat previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during earlyembryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and earlyembryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1,CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogeniccells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein wasabsent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins werepresent throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes,but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expressionpatterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes andspermotocytes.

Ferritin, a major iron storage protein of most living organisms plays a crucial role in iron metabolism. A cDNAencoding a heavy-chain homologue of ferritin was isolated and sequenced in the freshwater giant prawn,Macrobrachium rosenbergii. The cloned cDNA was 1012 bp long containing an iron-responsive element(IRE) sequence in the 5′-untranslated region and a complete open-reading frame encoding for a 172 aminoacid residues protein with a conserved domain for the ferroxidase center characteristic for heavy chains ofvertebrate ferritin. Prawn ferritin transcripts are expressed in muscle, heart, hepatopancreas, stomach,hemocytes, ovary and testis. Quantitative real-time PCR revealed that the abundance of ferritin transcriptwas highest in the hepatopancreas, followed by the testis. The expression of the ferritin transcript in themuscle significantly increased 6-fold at 3 h after injection of iron. In the ovary, a high expression of ferritintranscript was first detected with nearly a 4-fold increase after 3 h post-injection that remained elevated for48 h. Heart ferritin mRNA expression increased up to 8-fold at 24 h. No significant difference was found in thehepatopancreas, hemocytes and testis. These results strongly suggested that the expression of prawn ferritinis regulated by iron at the transcriptional level as found in insects, and iron increased prawn ferritintranscripts in a tissue-specific manner.

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn (Marsupenaeus japonicus), subtractive suppressionhybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed byevaluation of the differential expression profile by macroarray and quantitative real-time RT–PCR analyses. The cathepsin C (dipeptidylpeptidase 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 lowexpression 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 theovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specificgene was subsequently determined by rapid amplification of the cDNA 3V and 5V ends (3V and 5V RACE). The deduced amino acid sequenceof the 230-residue mature peptide shared 67–70% identity to the known cathepsin C in mammals. Western blot analysis showed thatexpression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs asrevealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration ofits involvement in the final stages of oocyte maturation in crustacean species.

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

In penaeid shrimp, cortical rods (CRs) are formed in peripheralcrypts of the oocyte after completion of yolk accumulation;subsequently the CRs are utilized as a source of jelly materialsthat surround fertilized eggs. In our previous study, of five majorcomponents, three CR proteins displayed quite similar immunologicalcharacteristics. In this study, cDNA sequences and developmentalexpression profiles at both transcriptional and proteinlevels were examined to elucidate the molecular characteristicsof CR proteins and the process of CR formation. SequencingcDNAs exhibited the presence of three related forms thathave identical sequences except for the loss of 246 and 369 bpin medium and short forms, respectively, suggesting that a singlegene generates three transcriptional variants corresponding tothe three CR proteins. Their deduced amino acid sequences revealedsimilarities to those of extracellular matrix proteins in athrombospondin (TSP) 3,4/cartilage oligomeric protein family,and thereby the CR proteins were designated mjTSP. Semiquantitativeanalysis by real-time polymerase chain reaction revealedthe presence of mjTSP transcripts, at similar levels, in immature,vitellogenic, and mature ovaries. Furthermore, in situ hybridizationlocalized the majority of transcripts in previtellogenic oocytesin ovaries at all developmental stages. By theWestern blot,on the other hand, mjTSP proteins were undetectable in immatureovaries but became obvious at the early vitellogenicstage. The immunosignals were enhanced during vitellogenicstages and maintained a high intensity in mature ovaries. Thus,transcription, translation of mjTSP, and formation of the CRstructure occurred at different stages of ovarian development.

The penaeid shrimp develops cortical rods (CRs) in the oocyte after completing yolk accumulation during ovarianmaturation. In this study, CRs from the mature ovary of the kuruma prawn (Marsupenaeus japonicus) were isolated andtheir proteins were characterized. Under reducing conditions of SDS-PAGE, the CR demonstrated five major bands ofapproximately 210, 150, 140, 130 and 30 kDa. Of 32 clones of monoclonal antibodies raised against the CR proteins,21 were found to be immunoreactive by Western blotting, demonstrating 150, 140 and 130 kDa proteins simultaneously.This result suggested that the proteins were post-translational or post-transcriptional products from the same gene, orthat they originated from the same gene family. CRs were identified by immunohistochemical analysis by 31 clones ofmonoclonal antibodies in paraffin sections and by 26 clones in cryostatic ones. Most of the antibodies also stained theyolk in both developing and mature oocytes, suggesting that CR proteins accumulated in developing oocytes, thenassembled to form the CR structure.

根据人的睾丸决定因子（SRY）的高速泳动组蛋白区设计简并引物，以中华绒螯蟹雌雄个体的基因组DNA为模板进行PCR扩增，从雌雄体均扩出220bp左右的条带，说明中华绒螯蟹中存在SRY基因的同源体，且无性别差异。经克隆、测序后得到两条SRY盒区相关基因（Sox基因）片段，ES1和ES2，其序列与人相应Sox基因保守区的最高同源性分别为66%和93%。ESl和ES2在成体8种组织中的检测结果表明，ES1在精巢、肌肉、心脏、肝脏、鳃、胸神经团、不同时期的卵巢及排出的成熟卵等组织中均表达，而ES2只在精巢、成熟的卵巢以及排出的成熟卵中有表达，说明ES2基因可能参与性腺的发育和早期胚胎发育过程。