Comprehensive Transcriptome Analysis of Gonadal and Somatic Tissues for Identification of Sex-Related Genes in the Largemouth Bass Micropterus salmoides.

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.

Identification of a novel germ cell marker MnTdrd from the oriental river prawn Macrobrachium nipponense.

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.

Cyclin B protein undergoes increased expression and nuclear relocation during oocyte meiotic maturation of the freshwater prawn Macrobrachium rosenbergii and the Chinese mitten crab Eriocheir sinensis.

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.