Germ cell-specific expression of dead end (dnd) in rare minnow (Gobiocypris rarus).

Rare minnow (Gobiocypris rarus) is an emerging model fish in China, and the development of its gonads is still elusive. Germ cell-specific genes are conserved in animals. Dead end (Dnd) was first documented as a germ granule component in zebrafish. Here, we report the cloning and expression profile of dnd in rare minnow. RT-PCR results showed that dnd is expressed specifically in the gonads of both sexes, is maternal in origin and is expressed continuously during embryogenesis. Dnd mRNA could be detected exclusively in the germ cells of the testis and ovary. Temporal expression of dnd mRNA is similar to that of vasa and dnd in zebrafish during embryogenesis. Taken together, dnd mRNA is restricted to the germ cells of rare minnow.

Identification and expression profiles of prdm1 in medaka Oryzias latipes.

Mouse Prdm1, also known as Blimp1, plays important roles in maturation and survival of lymphoid cells, as well as in organogenesis of muscle, limb, sensor organs and primordial germ cells. The homologues of mouse prdm1 have been identified in a diverse of animals including zebrafish and fugu. Here, we report the identification and expression profiles of two homologues of prdm1, namely prdm1a and prdm1b in medaka, Oryzias latipes. The transcripts of prdm1a and prdm1b were detectable in all the tissues including immune organs such as gill, spleen, kidney, liver and intestine that we have checked on. The transcripts of prdm1a could be detected in the embryonic shield at mid-gastrula stage and later in the somite, eye, otic vesicle, branchial arches, fin, intestine and cloaca during embryogenesis using in situ hybridization. Moreover, the expression of prdm1a in the liver of both medaka and zebrafish could be up-regulated by the immune stimuli including lipopolysaccharide, polyI:C and the grass carp reovirus, similarly to the up-regulation of IL1B. These results indicate that Prdm1a may play important roles in embryogenesis and also in immune response in fish.

Ol4E-T, a eukaryotic translation initiation factor 4E-binding protein of medaka fish (Oryzias latipes), can interact with nanos3 and vasa in vitro.

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka.

Specific expression of Olpiwi1 and Olpiwi2 in medaka (Oryzias latipes) germ cells.

Piwi is necessary for germ stem cell survival in Drosophila and homologues have been identified in a diverse range of organisms. Here, we identify and characterize two homologous genes of piwi, Olpiwi1 and Olpiwi2, in the model fish medaka (Oryzias latipes). Olpiwi1 is similar to Ziwi in zebrafish or Miwi in the mouse, and Olpiwi2 is similar to Zili in zebrafish or Mili in the mouse. Moreover, Olpiwi2 mRNA is produced from two different chromosomes. RT-PCR showed expression of Olpiwi1 and Olpiwi2 predominantly in the gonads. In situ hybridization revealed germ cell-specific expression of Olpiwi1 and Olpiwi2 throughout the development of oocytes from oogonia to mature oocytes in the ovary, and from spermatogonia to spermatocytes in the testes of adults. RT-PCR and whole mount in situ hybridization showed that both Olpiwi1 and Olpiwi2 were maternally deposited in the embryo. Olpiwi1 and Olpiwi2 were detected in primordial germ cells during embryonic development. These results suggest that both Olpiwi1 and Olpiwi2 are germ cell specific, and may play important roles in germ cell development and gametogenesis in this model species.

Germ cell specific expression of Vasa in rare minnow, Gobiocypris rarus.

Germ cells are set aside early with somatic cells and take roles for reproduction of species from one generation to the next generation. Vasa, a member of DEAD family is well documented as germ cell marker in the animal kingdom. Rare minnow, Gobiocypris rarus, is an emerging model fish in China to study development and toxicology, etc. A suitable germ cell marker will benefit the studies of the factors that may influence germ cell development. Here, we report the cloning and characterization of G. rarus vasa named Grvas whose protein product has the typical characteristics of Vasa proteins. RT-PCR results showed that Grvas is expressed specifically in the gonads of male and female, it is maternally deposited into the eggs for embryos and is continuously expressed in the embryos from the zygote to larvae and adult. Grvas mRNA and/or protein is restricted to the germ cells of ovary and testis. Temporal expression of Grvas mRNA is similar to that of zebrafish vasa during embryogenesis. Grvas signals are coincident with primordial germ cells. These results mean that a germ cell marker, Grvas is isolated from rare minnow and its expression is exclusively in germ cells.

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus.

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.

Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus.

Gobiocypris rarus is an emerging fish model for aquatic toxicology in China as it is sensitive to environmental hormone disruptors. Exogenous sex steroids can affect sex differentiation and the expression of sex-related genes. Foxl2, a member of forkhead-box transcription factor family, is the key gene for ovary development and its mutation causes the blepharophimosis ptosis epicanthus inversus syndrome in human. We find that two foxl2 genes exist in fish genome, one is foxl2, and the other is foxl2b. Here, we reported the isolation and expression of foxl2 in G. rarus. G. rarus foxl2 cDNA is 1700bp in length with a 921bp of open reading frame encoding 306 amino acids containing the typical FH-domain. Semi-quantitative RT-PCR revealed its predominant expression in the eye, brain, gill and gonads. Moreover, the expression level in the ovary was significantly higher than that in the testis. Quantitative RT-PCR showed that foxl2 was up regulated after treatment with estradiol and was down regulated with 2-methyl-testosterone. These results suggested that Foxl2 plays an important role in female development of G. rarus, foxl2 mRNA expression is regulated by downstream sex hormones, and foxl2 can be used as a molecular indicator monitoring the environmental endocrine disruptors.