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MicroRNA (miRNA) is a posttranscriptional downregulator that plays a vital role in a wide variety of biological processes. In this study, we constructed five ovarian and testicular small RNA libraries using two somatic libraries as reference controls for the identification of sex-biased miRNAs and gonadal differentially expressed miRNAs (DEMs) of the Chinese mitten crab, Eriocheir sinensis. A total of 535 known and 243 novel miRNAs were identified, including 312 sex-biased miRNAs and 402 gonadal DEMs. KEGG pathway analysis showed that DEM target genes were statistically enriched in MAPK, Wnt, and GnRH signaling pathway, and so on. A number of the sex-biased miRNAs target genes associated with sex determination/differentiation, such as IAG, Dsx, Dmrt1, and Fem1, while others target the genes related to gonadal development, such as P450s, Wnt, Ef1, and Tra-2c. Dual-luciferase reporter assay in vitro further confirmed that miR-34 and let-7b can downregulate IAG expression, miR-9-5p, let-7d, let-7b, and miR-8915 can downregulate Dsx. Taken together, these data strongly suggest a potential role for the sex-biased miRNAs in sex determination/differentiation and gonadal development in the crab.

Largemouth bass (Micropterus salmoides) is an economically important fish. It can spawn many times during a breeding season, and there are no obvious morphological characteristics to distinguish male and female juvenile fish. So far, little is known about the genes regulating their sexual development in this species. Here, we performed RNA sequencing (RNA-Seq) analysis of the testis, ovary, and somatic tissue to identify sex-related genes in the largemouth bass. A total of 51,672 unigenes were obtained via the transcriptome analysis, and 5900 differential expression genes (DEGs), including 3028 up-regulated and 2872 down-regulated DEGs, were obtained in the somatic tissue, testis, and ovary. DEGs were retrieved by making comparisons: somatic tissue vs testis (1733-up and 1382-down), testis vs ovary (841-up and 807-down), and ovary vs somatic tissue (454-up and 683-down). Finally, functional annotation identified 22 key sex-related DEGs, including 13 testis-biased DEGs (dmrt1, cyp11b1, sox9, spata4, spata22, spata17, fshr, fem-1a, wt1, daz1, amh, vasa, and piwi1) and 9 ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, bmp15, fem-1b, fig. la, and piwi2). This result was further confirmed by the tissue expression detection via RT-PCR and RT-qPCR. Protein-protein interacting (PPI) network analysis revealed that the testis-specific dmrt1 interacts directly with the testis-biased DEGs (cyp11b1 and spata4) and the ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, and bmp15), suggesting that the dmrt1 as a sex-determining gene can play a dual role through inducing the testis-biased DEGs and inhibiting the ovary-biased DEGs during the testicular development. Our present results provide useful molecular data for a better understanding of sexual development in the largemouth bass.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4 kb, corresponding to ∼ 4× coverage of the estimated 5.38 Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

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.

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 < 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 < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

As post-transcriptional regulators, microRNAs (miRNAs) play an important role in growth and reproductive processes. So far, there is limited information regarding crustacean miRNAs. To explore the potential role of miRNAs in the gonadal development of the prawn Macrobrachium rosenbergii, we constructed seven small RNA libraries from ovarian and testicular tissues at various stages using somatic tissue as the control. A total of 1,954 known and 129 novel miRNAs were retrieved. By comparing differentially expressed miRNAs (DEMs) between testes and ovaries, forty-one miRNAs were identified with sex-biased expression patterns, including 17 ovary-biased and 24 testis-biased patterns. Furthermore, the putative target genes of the sex-biased miRNAs, such as cyclin L1, mitogen-activated protein kinase 7 (MAPK 7), heat shock protein (HSP), and zinc finger protein, were significantly enriched in many reproduction-related pathways including the Gonadotropin-releasing hormone (GnRH) pathway, glycolysis, gluconeogenesis pathway, ovarian steroidogenesis, estrogen signaling pathway, MAPK pathway, Wnt pathway, and insulin signaling pathway, implicating potential regulatory roles of miRNAs in reproduction. These data aid in the further investigation of the mechanism of gonadal development and reproductive regulation mediated by miRNA in M. rosenbergii.

As an essential mediator in the Gonadotropin-releasing hormone (GnRH) signaling pathway, GnRH receptor (GnRHR) coupled to GnRH, plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction. To detect the existence of GnRHR and potential GnRH signaling pathway, we cloned and characterized GnRHR in the Chinese mitten crab, Eriocheir sinensis (named EsGnRHR). The full-length EsGnRHR cDNA is 2038 bp in length, including an open reading frame (ORF) of 1566 bp, a 57 bp 5′-untranslated region (5′-UTR) and a 415 bp 3′-UTR. Prediction of transmembrane domains in protein sequence revealed that the EsGnRHR protein contained seven hydrophobic transmembrane regions (TMs). Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary, and weakly distributed in the testis and brain. In situ hybridization further demonstrated that EsGnRHR mRNA was localized at the protocerebrum and deutocerebrum. In the ovary and testis, the hybridization signal was dominantly at the earlier developmental stages. The signal was mainly localized in the cytoplasm cell in the ovary, and in the epithelium cell in the testis. During the different stages of gonadal development, EsGnRHR displayed increasing trends in both female and male when analyzed by quantitative real-time PCR, suggesting that EsGnRHR was involved in controlling gonadal development. Our study provides important information for further research on the molecular mechanisms underlying crab development.

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.

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex-reversed females (neomales). To provide molecular evidence for the proposed system, novel sex-linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male-specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female-specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female-specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female-linked SCAR markers can be applied for rapid detection of prawn gender. These sex-specific SCAR markers and sex-associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).

The androgenic gland (AG), a male-specific endocrine organ in crustacean, is responsible for the maintenance of male characteristics and gender differentiation. In this study, an AG-specific gene, the Macrobrachium nipponesne insulin-like androgenic gland factor (MnIAG) was isolated from a transcriptome library of M. nipponesne and its full-length cDNA sequences were obtained by RACE method. The cDNA was 1,547 bp in length and encoded a precursor protein of 175 amino acids. The deduced precursor protein consisted of a signal peptide, B chain, C peptide and an A chain, which exhibited the same structural organization as that of previously identified insulin-like androgenic gland in crustacean. The mature peptide of the MnIAG owned two additional conserved cysteine residues, which were also found in the Palaemonidae species reported. Results of the tissue distribution and in situ hybridization showed the MnIAG expressed exclusively in androgenic gland. The quantitative RT-PCR results demonstrated that the MnIAG transcript was present at blastula stage and later developmental stages with low levels, which suggested that the primordial cells of the AG might form at these stages.

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.

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.

The feminization-1 (fem-1) gene is characterized by one of the most common protein-protein interaction motifs, ankyrin repeat motifs, displays many expression patterns in vertebrates and invertebrates, and plays an essential role in the sex-determination/differentiation pathway in Caenorhabditis elegans. In this study, a fem-1 homolog, designated as Mnfem-1, was first cloned from the oriental river prawn Macrobrachium nipponense. The prawn Mnfem-1 gene consists of six exons and five introns. The full-length cDNA (2603bp) of Mnfem-1 contains an open reading frame (ORF) encoding a protein of 622 amino acids. The Mnfem-1 RNA and protein are exclusively expressed in the ovary in adult prawns as revealed by RT-PCR and immunofluorescence analysis, respectively. In situ hybridization results showed that strong positive signals were concentrated at the edge of the previtellogenic and vitellogenic oocyte. During embryogenesis, Mnfem-1 is highly expressed in both unfertilized eggs and embryos at cleavage stage and thereafter dropped to a low level from blastula to zoea, indicating that the Mnfem-1 in early embryos is maternal. After hatching, the Mnfem-1 expression significantly increased in the larvae at length of 2cm, an important stage of sex differentiation. Yeast two hybridization results showed that the Mnfem-1 protein can be potentially interactive with cathepsin L and proteins containing the domains of insulinase, ankyrin or ubiquitin. Our results suggested that Mnfem-1 could have roles in prawn ovarian development and sex determination/differentiation.

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.

Background The oriental river prawn, Macrobrachium nipponense, is an economically and nutritionally important species of the Palaemonidae family of decapod crustaceans. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for M. nipponense using high-throughput sequencing technologies. Methodology and Principal Findings Total RNA was isolated from eyestalk, gill, heart, ovary, testis, hepatopancreas, muscle, and embryos at the cleavage, gastrula, nauplius and zoea stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, we generated a total of 984,204 high quality reads (338.59Mb) with an average length of 344 bp. Clustering and assembly of these reads produced a non-redundant set of 81,411 unique sequences, comprising 42,551 contigs and 38,860 singletons. All of the unique sequences were involved in the molecular function (30,425), cellular component (44,112) and biological process (67,679) categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literature, many putative genes involved in sex determination, including DMRT1, FTZ-F1, FOXL2, FEM1 and other potentially important candidate genes, were identified for the first time in this prawn. Furthermore, 6,689 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset. Conclusions The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in M. nipponense. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will be essential for accelerating aquaculture breeding programs with this species.

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 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.

Background MicroRNAs (miRNAs) are small non-coding RNA molecules that downregulate gene expression by base pairing to the 3′-untranslated region (UTR) of target messenger RNAs (mRNAs). Up to now, rare information for the miRNAs is available in decapod crustaceans. Our previous studies showed that many miRNA-binding sites are present in the 3′-UTR of the cyclin B in the Chinese mitten crab Eriocheir sinensis, suggesting that the translation or post-transcription of the crab cyclin B might be regulated by miRNAs during meiosis of oocyte. Results To identify ovarian miRNAs in the mitten crab, ovarian small RNAs were subjected to high-throughput sequencing using an Illumina Genome Analyzer. Of 14,631,328 reads, 55 known miRNAs representing 44 miRNA families were identified and 136 novel miRNA candidates were predicted. The 5′ seed sequences of four miRNAs, miR-2, miR-7, miR-79 and miR-133, were revealed to complementary to miRNA binding sites in 3′-UTR of the cyclin B. Quantitative real time PCR analysis showed that miR-2 and miR-133 are much more abundant in the first metaphase (MI) of meiosis than in germinal vesicle (GV) stage. But their increasing expressions are independent of induction of gonadotropin-releasing hormone (GnRH). Further expression analysis using double-luciferase reporter genes assay showed that miR-2 and miR-133 can downregulate the 3′-UTRs of the crab cyclin B gene, indicating that they could inhibit the translation of the cyclin B. Western blot analysis confirmed that cyclin B protein is completely disappeared in fertilized egg at the metaphase-anaphase transition of meiosis I, suggesting that miR-2 and miR-133 could function in destruction of cyclin B near the end of MI. Conclusions A high number of miRNAs have been identified from the crab ovarian small RNA transcriptom for the first time. miR-2 and miR-133 exhibit differential expression during the meiotic maturation of the oocytes and have activity in regulating the 3′-UTR of the crab cyclin B gene. This result is inconsistent with recent finding that miRNA activity is globally suppressed in mouse oocytes. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-547) contains supplementary material, which is available to authorized users.