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Germ cell-specific genes play an important role in establishing the reproductive system in sexual organisms and have been used as valuable markers for studying gametogenesis and sex differentiation. Previously, we isolated a vasa transcript as a germ cell marker to trace the origin and migration of germ cells in the oriental river prawn Macrobrachium nipponense. Here, we identified a new germ cell-specific marker MnTdrd RNA and assessed its temporal and spatial expression during oogenesis and embryogenesis. MnTdrd transcripts were expressed in high abundance in unfertilized eggs and embryos at cleavage stage and then dropped significantly during late embryogenesis, suggesting that MnTdrd mRNA is maternally inherited. In situ hybridization of ovarian tissue showed that MnTdrd mRNA was initially present in the cytoplasm of previtellogenic oocyte and localized to the perinuclear region as the accumulation of yolk in vitellogenic oocyte. Whole-mount in situ hybridization of embryos showed that MnTdrd-positive signals were only localized in one blastomere until 16-cell stage. In the blastula, there were approximately 16 MnTdrd-positive blastomeres. During embryonized-zoea stage, the MnTdrd-positive cells aggregated as a cluster and migrated to the genital rudiment which would develop into primordial germ cells (PGCs). The localized expression pattern of MnTdrd transcripts resembled that of the previously identified germ cell marker vasa, supporting the preformation mode of germ cell specification. Therefore, we concluded that MnTdrd, together with vasa, is a component of the germ plasm and might have critical roles in germ cell formation and differentiation in the prawn. Thus, MnTdrd can be used as a novel germ cell marker to trace the origin and migration of germ cells.

Germ plasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germ plasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germ plasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germ plasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germ plasm that functions in early germ cell specification.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M−phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial–temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Characterized by ankyrin repeat motifs, the feminization-1 (fem-1) gene plays an essential role in sex determination/differentiation in Caenorhabditis elegans. However, there are only a few reports on fem-1 in crustaceans. In this study, a fem-1 gene (Mrfem-1) was first isolated from the giant freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of Mrfem-1 was 2607 bp long, containing an open reading frame encoding 615 amino acids, and presenting eight ankyrin repeats. The full-length cDNA has been submitted to GenBank with the accession no. MT160093. According to the RT-PCR results, Mrfem-1 was exclusively expressed in the ovary. The expression level of Mrfem-1 had increased with ovarian maturation and reached the highest peak at vitellogenic stage. In situ hybridization results showed that positive signals were concentrated in the cytoplasm of previtellogenic stage, and scattered in the cytoplasm and follicular cells at vitellogenic stage, suggesting that Mrfem-1 might be associated with ovarian maturation. Moreover, two effective siRNAs targeting Mrfem-1 were found and their effectiveness verified in vitro. These results on Mrfem-1 will help us to better understand the fem family and provide a new resource for subsequent investigations of siRNA-mediated regulation on ovarian development in M. rosenbergii.

During recent years, China has become a hotspot for the domestication of mandarin fish, and this is of great commercial value. Although the food preference of domesticated mandarin fish has been studied, little is known about genes regulating their growth. We raised hybrid mandarin fish on artificial feed for 3 months, the results showed that the survival rate of hybrid mandarin fish was 60.00%. Their total length and body weight were 18.34 ±0.43 cm and 100.44 ±4.87 g. The absolute length and weight gain rates were 0.14 cm/d and 1.08 g/d, respectively. Finally, RNA sequencing (RNA-Seq) was performed to identify potential genes and pathways activated in response to growth performance. The transcriptome analysis generated 68, 197 transcripts and 45,871 unigenes. Among them, 1025 genes were up-regulated and 593 genes were down-regulated between the fast- and slow-growth fish. Finally, we obtained 32 differentially expressed genes, which were mainly related to fatty acid biosynthesis (e.g. FASN and ACACB), collecting duct acid secretion (e.g. ATP6E and KCC4), cell cycle (e.g. CDC20 and CCNB), and the insulin-like growth factor (IGF) system (IGFBP1). These pathways might be related to the growth of hybrid mandarin fish. In addition, more potential single nucleotide polymorphisms (SNPs) were detected in the fast-growth fish than in the slow-growth fish. The results suggest that the interaction of metabolism and abundant alleles might determine the growth of hybrid mandarin fish after food conversion.

Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.

Vertebrate gonadotropin-releasing hormone (GnRH) plays a central role in the regulation of gonadial development by binding with its specific membrane receptor, GnRH receptor (GnRHR), which is an incipient key node in the GnRH signaling pathway. To explore the potential GnRH pathway in invertebrates, in this study, we reanalyze the transcriptomic library data of the oriental prawn Macrobrachium nipponense achieved previously in our laboratory. A total of 26 transcripts representing the genes assigned to the vertebrate GnRH signaling cascade were identified, including a transcript encoding a GnRH-like receptor ( Mn-GnRHR ). The Mn-GnRHR transcript contains an open reading frame (ORF) of 1083 bp, encoding a protein of 360 amino acid residues. The putative protein is characterized by seven typical transmembrane (TM) domains of vertebrates GnRHR and is most closely related to the invertebrate Octopus vulgaris GnRHR as demonstrated by phylogenetic analysis. Reverse-transcription PCR revealed that the Mn-GnRHR mRNA was distributed predominantly in the brain, gonad, eyestalk, muscle and weakly or not in other tissues examined. In situ hybridization further demonstrated that the Mn-GnRHR mRNA was mainly distributed in the small- and medium-sized neurons located in the protocerebrum and deutocerebrum. In the ovary,the Mn-GnRHR mRNA was localized in the ooplasm of previtellogenic and vitellogenic oocytes and meiotically-mature oocytes as well as the follicular cells around oocytes. Quantitative real time PCR showed that the Mn-GnRHR transcript was expressed stably in ovaries at various stages. The above data provided the first evidence of the presence of GnRH-like receptors in the crustacean, which may function through GnRH signaling pathway to regulate ovarian development.

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P &lt, 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P &lt, 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH2-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.

Germ cells are specified by the inheritance of maternal germline determinants (preformation mode) or inductive signals from somatic cells (epigenesis mode) during embryogenesis. However, the germline specification in decapod crustaceans is unclear so far. Using vasa homolog (MnVasa) as a germ cell marker, here we probed the early events of germline specification in the oriental river prawn Macrobrachium nipponense. Quantitative RT-PCR analysis of unfertilized eggs and embryos demonstrated that the prawn MnVasa mRNA is a maternal factor. Whole-mount in situ hybridization further indicated that MnVasa transcripts are maternally supplied to only one blastomere at the very early cleavage stages. As cleavage proceeds, the MnVasa-positive blastomere undergoes proliferation and increases in number. During gastrulation, the MnVasa-positive cells are found to be around a blastopore and could migrate into an embryo through the blastopore. At the zoea stage, clusters of the MnVasa-positive cells distribute not only in the gonad rudiment in the cephalothorax but also at an extragonadic site, dorsal to the posterior hindgut in the abdomen, suggesting that MnVasa-positive cells could migrate anteriorly to the genital rudiment through the hindgut. Based on the dynamic localization and number of MnVasa-positive cells during embryogenesis, we concluded that the MnVasa-positive cells are primordial germ cells (PGC) or founder cells of PGC that are separated from soma at the early cleavage stage. MnVasa mRNA might have a key function in the specification of the prawn germline cells as a maternal determinant. These results provide the first evidence that the germline specification in decapod crustaceans follows a preformation mode.

SRY-related HMG box (Sox) genes are characterized by the presence of a DNA-binding HMG domain and involved in a diverse range of developmental processes. In this study, we identified a novel Sox gene, designated as EsSoxB2-1, from the Chinese mitten crab Eriocheir sinensis. The EsSoxB2-1 encodes a protein of 259 amino acids, sharing the highest identity with the beetle Tribolium castaneum SOX21b. Unlike insect Sox21b, however, EsSoxB2-1 is intronless and exhibits a gonad-specific expression pattern at both mRNA and protein level. Two core promoters in 5′ flanking region were demonstrated to be essential for inducing transcriptional regulatory activity. The transcription of EsSoxB2-1 mRNA begins in spermatogonia stage, while the translation of EsSOXB2-1 protein initiates at spermiogenesis stage. Interestingly, EsSOXB2-1 protein was exclusively localized in the nucleus of spermatid and spermatozoa even at the end of acrosome reaction, and was bound to the uncondensed chromatin in nucleoplasm of mature spermatozoa. Knockdown of EsSoxB2-1 by RNAi leads to abnormal transformation of the nucleus during spermiogenesis. Together, these findings demonstrated the requirement of EsSoxB2-1 for the spermatozoa nucleus maturation and also suggested that EsSoxB2-1 would be delivered into fertilized eggs along with chromatins as a paternal transcription factor for regulating early embryonic development.

Cdc2 kinase is a catalytic subunit of maturation-promoting factor (MPF), a central factor for inducing the meiotic 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 its spatial-temporal expression profiles were analyzed during oogenesis at RNA and protein levels. The crab Cdc2 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 protein level was found at previtellogenesis, late vitellogenesis and germinal vesicle breakdown (GVBD) stages. Two forms (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 previtellogenic oocyte, and then relocate into germinal vesicle at vitellogenesis stage and accumulate on meiotic spindle at oocyte maturation. These findings suggest that Cdc2 kinase has essential roles in inducing GVBD and generating meiotic apparatus during the crab oocyte maturation.

The meiotic maturation of oocyte in animals is regulated by maturation promotion factor (MPF), a complex of Cdc2 and cyclin B. Although the role of MPF during oocyte maturation has been well studied in a wide variety of eukaryotic organisms, little is known for crustacean species. In this study, a full-length cDNA of cyclin B was cloned from the Chinese mitten crab using degenerate RT-PCR and RACE methods. The crab cyclin B cDNA was 3,794 bp containing an unusually long 3' untranslation region (UTR) of 2,403 bp and an open-reading frame encoding for a protein of 410 amino acids, with a calculated molecular mass of 45 kDa. The long 3'UTR harbors many cytoplasmic polyadenylation elements (CPE), and the GY-box, Brd-box, K-box that are perfectly complementary to the 5'-ends of various Drosophila microRNAs. The crab cyclin B transcript was predominantly expressed in ovary and testis. Semi-quantitative RT-PCR analysis revealed that the amount of cyclin B mRNA was high at previtellogenesis and late vitellogenesis stages, while low at early and middle vitellogenesis, suggesting that differential expression of cyclin B is closely related to oogonial proliferation (mitosis) and oocyte meiotic maturation.

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.

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.

The penaeid shrimp develops cortical rods (CRs) in the oocyte after completing yolk accumulation during ovarian maturation. In this study, CRs from the mature ovary of the kuruma prawn (Marsupenaeus japonicus) were isolated and their proteins were characterized. Under reducing conditions of SDS-PAGE, the CR demonstrated five major bands of approximately 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, or that they originated from the same gene family. CRs were identified by immunohistochemical analysis by 31 clones of monoclonal antibodies in paraffin sections and by 26 clones in cryostatic ones. Most of the antibodies also stained the yolk in both developing and mature oocytes, suggesting that CR proteins accumulated in developing oocytes, then assembled to form the CR structure.

Dmrt is a family of genes related to the sexual regulators Doublesex of Drosophila melanogaster and Mab-3 of Caenorhabditis elegans. Dmrt genes are widely conserved and known for their involvement in sex determination and differentiation across phyla. In this study, we report here the identification of a novel Dmrt gene, named EsDmrt-like, from Chinese mitten crab Eriocheir sinensis. EsDmrt-like encodes a protein of 236 amino acids without intron. The protein contains a conserved DNA-binding DM domain that is characteristic of Dmrt genes. The DM domain shares 98% identity with that of Drosophila Dmrt99B and vertebrate Dmrt5, but outside the DM domain, there is little homology in sequence and no other conserved domain such as DMA specific to the Dmrt99B and Dmrt3-5. Interestingly, the expression pattern of EsDmrt-like is quite similar with that of vertebrate Dmrt1. Reverse transcription-polymerase chain reaction showed that EsDmrt-like transcripts were detectable only in testis with much higher expression at immature stage. In situ hybridization to gonad sections indicated that the EsDmrt-like mRNA was exclusively localized in Sertoli cells around the periphery of seminiferous tubules and developing germ cells including spermatogonium, spermatocyte, and spermatid, but absent in spermatozoa. This finding strongly suggests an essential role for EsDmrt-like in the male testicular development/differentiation of the crab.

Noncoding mRNA-like transcripts play important roles in a wide range of biological events such as cell differentiation and organogenesis. We report here the identification of a novel germ cell-specific mRNA-like transcript (RtGST-1) from a rainbow trout oocyte cDNA library. The novel transcript of 1,165 bp was confirmed to be full-length based on Northern blot and 5' RACE analyses. The transcript is polyadenylated but does not contain a significant open reading frame (ORF). The presumable ORF (encodes a peptide of 71 amino acids) has poor codon usage for rainbow trout and the AUG codon is in a poor context for translation initiation. RT-PCR showed that the novel gene is specifically expressed in ovaries of various stages and immature testis, whereas no transcripts of this gene were detected in mature testis and somatic tissues. Quantitative real-time PCR analysis revealed that the mRNA level of this novel gene is extremely high in early previtellogenesis stage ovaries relative to vitellogenesis stage ovaries. In situ hybridization analysis further demonstrated that the novel transcript is localized exclusively in early previtellogenic oocytes and spermatocytes. To our knowledge, this study represents the first report of a germline-specific mRNA-like transcript in fish.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis 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 spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present 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 expression patterns 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 and spermotocytes.

In this study a gonadotropin-releasing hormone (GnRH)-like peptide was partially isolated by reverse phase high performance liquid chromatography (RP-HPLC) from the brain extract of Chinese mit-ten crab Eriocheir sinensis. Immunological dot blot analyses from RP-HPLC fractions using the an-ti-octGnRH revealed that one of fractions is immune reaction positive, suggesting that the fractions of brain extract contain GnRH-like substance. Eight kinds of small peptides, with molecular weight ranging from 800 to 1 400 u, were further identified in this fraction by using matrix-assisted laser desorp-tion/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). One of peptides in MS component shared similar molecular weight with the octGnRH.