Project description:Gonadal soma-derived factor (gsdf) has been demonstrated as essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag “pull-down” assay coupled with shotgun LC-MS/MS to identify a group of potential interaction partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1), and actin filaments in mature medaka testis. Alike the interaction with TGF-dynein critical for spermatogonial division in Drosophila melanogaster, the physical interaction of Gsdf & dynein and Gsdf & eEF1 were identified through a yeast 2-hybrid (Y2H) screening of an adult testis cDNA library using Gsdf as bait, and were verified by a paired Y2H assay. Co-immunoprecipitation of Gsdf and eEF1was defined in adult testes, as supporting the requirement of Gsdf and eEF1 interaction in testis development. Proteomics analysis and ultrastrutural observation showed that Gsdf deficiency activated eEF1-mediated protein synthesis and ribosome biogenesis, which in turn led to the differentiation of undifferentiated germ cell. Thus, our results provide a framework and new insight into the coordination of Gsdf (TGF and eEF1complex in the basic processes of germ cell proliferation, and transcriptional and translational control of sexual RNA, which may be fundamentally conserved across phyla during sex differentiation.
Project description:We assess gene expression patterns upon 17beta-estradiol (E2) exposure to Japanese medaka (Oryzias latipes) in order to appere the E2 effects using DNA microarray analysis. Larval medaka were exposed to 0, 3, 30 and 100 ng/L of E2 and concentration-dependent changes in gene expressions were examined using Agilent medaka DNA microarrays. At 7 day, fish were sacrificed and mRNA was extracted for gene expression analysis. In an effort to link gene expression changes to effects on higher levels of biological organization, sex characteristics, gonadal histology, GSI, and egg production and fertility were examined. In microarray experiments, the correlation factors between the controls were from 0.91 to 1.00 among control samples. We observed highly induced O. latipes Gene Indices (OLGI) related to egg-yolk protein such as vitellogenin and L-SF precursor etc., which were significantly affected in a concentration-dependent manner by E2 exposure. To clarify the function of expressed genes by E2 treatments, we selected statistically expressed genes from the microarray experiments. We found 190 genes and 72 genes which were statistically expressed in E2 treatment as induced and repressed genes, respectively. In the induced gene list, there were characteristic induced-genes in the categories of lipid metabolism, stress (oxidative stress, DNA and protein damage), and apoptosis with MAPK pathway. On the other hands, there were characteristic repressed genes in the categories of heat shock protein. Our result may suggest that gene expressions in yolk medaka is able to be used for detection of E2 effect by DNA microarray analysis. Larval medaka were exposed to 0, 3, 30 and 100 ng/L of E2 and concentration and time-dependent changes in gene expressions were examined using Agilent medaka DNA microarrays. At 7 day, three independent samples (one sample contained thirty whole medakas) were sacrificed and mRNA was extracted for gene expression analysis.
Project description:We assembled transcription activator-like effector nucleases (TALENs) targeting the DMY gene and generated XYdmy- mutants to investigate gonadal dysgenesis in medaka. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XYdmy- female medaka (TA_F_OV) gonad libraries. Differential expression analysis identified 144 up- and 293 downregulated genes in TA_F_OV compared to WT_F_OV, 387 up- and 338 downregulated genes in TA_F_OV compared to WT_M_TE.
Project description:Here we analyze key epigenetic marks from medaka (O. latipes) during the vertebrate phylotypic period (stage 24). Chromatin immuno-precipitation with histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation antibodies.
Project description:Microarray analysis was performed using germ cells and gonadal somatic cells isolated by Fluorescence Activated Cell Sorter (FACS) from XX and XY embryos at st.33 and st.35. Medaka germ cells (olvas-EGFP) and somatic cells (sox9b-DsRed) were isolated by FACS. The microarray experiments were done using three independent biological replicates.
Project description:We assess gene expression patterns upon 17beta-estradiol (E2) exposure to Japanese medaka (Oryzias latipes) in order to appere the E2 effects using DNA microarray analysis. Larval medaka were exposed to 0, 3, 30 and 100 ng/L of E2 and concentration-dependent changes in gene expressions were examined using Agilent medaka DNA microarrays. At 7 day, fish were sacrificed and mRNA was extracted for gene expression analysis. In an effort to link gene expression changes to effects on higher levels of biological organization, sex characteristics, gonadal histology, GSI, and egg production and fertility were examined. In microarray experiments, the correlation factors between the controls were from 0.91 to 1.00 among control samples. We observed highly induced O. latipes Gene Indices (OLGI) related to egg-yolk protein such as vitellogenin and L-SF precursor etc., which were significantly affected in a concentration-dependent manner by E2 exposure. To clarify the function of expressed genes by E2 treatments, we selected statistically expressed genes from the microarray experiments. We found 190 genes and 72 genes which were statistically expressed in E2 treatment as induced and repressed genes, respectively. In the induced gene list, there were characteristic induced-genes in the categories of lipid metabolism, stress (oxidative stress, DNA and protein damage), and apoptosis with MAPK pathway. On the other hands, there were characteristic repressed genes in the categories of heat shock protein. Our result may suggest that gene expressions in yolk medaka is able to be used for detection of E2 effect by DNA microarray analysis.