Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Germ cell-less expression in medaka (Oryzias latipes)

The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH revealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15-18, 2004.     

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX‐XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species. genesis 47:289–299, 2009. © 2009 Wiley‐Liss, Inc.     

Spatial-temporal expression of pum1 and pum2 in medaka Oryzias latipes

Two pumilios, pum1 and pum2, were identified in medaka Oryzias latipes. Oryzias latipes pum1 and pum2 are ubiquitous in the adult tissues but with specific expression in the germ cells of gonads, ovary and testis. Pum1 is expressed in the spermatogonia to spermatocytes whilst pum2 presents in spermatocytes of testis only. Oryzias latipes pum1 and pum2 are maternally supplied RNA with ubiquitous expression in the early stages, and embryonic expression of pum1 and pum2 may begin from early gastrula. Both pum1 and pum2 are expressed in the tissues including brain, eye and trunk, and both are expressed in the gonads after hatching. Taken together, Pum1 and Pum2 may play important roles in embryonic and germ cell development of O. latipes.     

Influence of parental and developmental cadmium exposure on endocrine and reproductive function in Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubiquitous element and an important anthropogenic metal contaminant. A series of assays were modified or developed for Japanese medaka (Oryzias latipes), and used to compare the effects of Cd exposure on indicators of endocrine function in adult animals previously exposed in ovo or as hatchlings. Adults were raised either from eggs produced during a 2 week exposure to 0-10 microg/l Cd or from fry exposed for 2 weeks beginning 2 days after hatching. The reproductive capacity of the resulting adults was determined during a 2 week period during which half of the animals were re-exposed to Cd. Two week Cd exposure did not result in reproductive impairment despite producing some changes in circulating steroid concentration. In addition, 1 microg/l cadmium exposure in ovo elevated male hepatic vitellogenin (VTG) relative to controls. Hence, steroid parameters were a better biomarker of cadmium exposure than changes in VTG. However, reproductive impairment was not correlated to change in VTG or plasma steroids.     

Seasonal change in the locomotor activity rhythm of the medaka,Oryzias latipes

A group of the medaka,Oryzias latipes (Cyprinodontidae, orange-red variety, 25 males and 25 females), was kept in an aquarium, which was placed outdoors under natural conditions from December 1984 to January 1986. Locomotor activity at three layers (upper, middle, and lower layers) was recorded with a phototransistor system in each season. In summer, the fish showed typical diurnal activity at all three layers and the activity was greater than in other seasons. However, in autumn and winter, the fish became less active and showed relatively high activity at night at the upper or middle layer and diurnal activity at the lower layer. Nocturnal activity seemed to appear when the water temperature was decreased and the photoperiod was shortened. A free-running activity rhythm was also recorded under continuous darkness (DD) in each season; however, the fish showed clear free-running activity rhythms under DD only in summer.     

Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus

A Foxl2 cDNA was cloned from the Nile tilapia ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame and protein sequences, especially the forkhead domain and C-terminal region, while some homopolymeric runs of amino acids are found only in mammals but not in non-mammalian vertebrates. RT-PCR revealed that Foxl2 is expressed in the tilapia brain (B), pituitary (P), gill, and gonads (G), with the highest level of expression in the ovary, reflecting the involvement of Foxl2 in B-P-G axis. Northern blotting and in situ hybridization also revealed an evident sexual dimorphic expression pattern in the gonads. Foxl2 mRNA was mainly detected in the granulosa cells surrounding the oocytes. The ovarian expression of Foxl2 in tilapia begins early during the differentiation of the gonads and persists until adulthood, implying the involvement of Foxl2 in fish gonad differentiation and the maintenance of ovarian function.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

Water- and cryoprotectant-permeability of mature and immature oocytes in the medaka (Oryzias latipes)

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes/embryos. To identify a stage feasible for the cryopreservation of teleost oocytes, we investigated the permeability to water and various cryoprotectants of medaka (Oryzias latipes) oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. In sucrose solutions, the volume changes were greater in GV oocytes than MII oocytes. Estimated values for osmotically inactive volume were 0.41 for GV oocytes and 0.74 for MII oocytes. Water-permeability (microm/min/atm) at 25 degrees C was higher in GV oocytes (0.13+/-0.01) than MII oocytes (0.06+/-0.01). The permeability of MII oocytes to various cryoprotectants (glycerol, propylene glycol, ethylene glycol, and DMSO) was quite low because the oocytes remained shrunken during 2 h of exposure in the cryoprotectant solutions at 25 degrees C. When the chorion of MII oocytes was removed, the volume change was not affected, except in DMSO solution, where dechorionated oocytes shrunk and then regained their volume slowly; the P(DMSO) value was estimated to be 0.14+/-0.01x10(-3) cm/min. On the other hand, the permeability of GV oocytes to cryoprotectants were markedly high, the P(s) values (x10(-3) cm/min) for propylene glycol, ethylene glycol, and DMSO being 2.21+/-0.29, 1.36+/-0.18, and 1.19+/-0.01, respectively. However, the permeability to glycerol was too low to be estimated, because GV oocytes remained shrunken after 2 h of exposure in glycerol solution. These results suggest that, during maturation, medaka oocytes become less permeable to water and to small neutral solutes, probably by acquiring resistance to hypotonic conditions before being spawned in fresh water. Since such changes would make it difficult to cryopreserve mature oocytes, immature oocytes would be more suitable for the cryopreservation of teleosts.     

Enhancement by fibronectin of spreading of isolated melanophores of the medaka,Oryzias latipes

Summary Cell spreading of isolated melanophores in medium containing fibronectin was observed in the wild type and two mutants of the medaka, Oryzias latipes. Isolated and cultured melanophores of the wild type and the mm mutant were different in appearance from those within scales but dendritic in shape and with fully dispersed pigment granules. Isolated melanophores of the cm mutant were stellate with dispersed pigment granules, whereas in scales the pigment granules are condensed. In the presence of fibronectin, spreading of cultured melanophores of wild type and cm mutant was observed. Spreading of melanophores from the mm mutant was observed only among dendritic melanophores, but not among condensed melanophores. The increase of spreading was inhibited by antibody against fibronectin. To test the involvement of cytoskeletal elements, colchicine, vinblastine or cytochalasin B were added to the culture medium; spreading did not increase, even in the presence of fibronectin. These results suggest that fibronectin-induced melanophore spreading is correlated with the state of pigment granule dispersal and that microtubules and microfilaments may play a role in the mechanism of spreading.Department of Zoology NJ-15, University of Washington, Seattle, Wa 98195, USA.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Summary Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.This work contains part of the PhD thesis of C. Winkler     

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka,Oryzias latipes

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.     

Structural components of the synaptonemal complex, SYCP1 and SYCP3, in the medaka fish Oryzias latipes

The synaptonemal complex (SC) is a meiosis-specific structure essential for synapsis of homologous chromosomes. For the first time in any non-mammalian vertebrates, we have isolated cDNA clones encoding two structural components of the SC, SYCP1 and SYCP3, in the medaka, and investigated their protein expression during gametogenesis. As in the case of mammals, medaka SYCP1 and SYCP3 are expressed solely in meiotically dividing cells. In the diplotene stage, SYCP1 is diminished at desynaptic regions of chromosomes and completely lost on the chromosomes at later stages. SYCP3 is localized along the arm and centromeric regions of chromosomes at metaphase I, and its existence on the whole chromosomes persists up to anaphase I, a situation different from that reported in the mouse, in which SYCP3 is confined to the centromeric regions but lost on the arm regions at metaphase I. Thus, the expression patterns of SC components are different in mammals and fish despite the resemblance in morphological structure of the SC, suggesting divergence in the function of the SC in regulation of meiosis-specific chromosomal behavior. Since the antibody against medaka SYCP3 is cross-reactive to other fishes, it should be generally useful for a meiosis-specific marker in fish germ cells.     

Polyploidization of nuclei in the yolk syncytial layer of the embryo of the medaka, Oryzias latipes, after the halt of mitosis

It has been reported that nuclei repeat parasynchronous mitosis four or five times in the yolk syncytial layer (YSL) of the embryo of the medaka, Oryzias latipes , during the blastula stage and that no mitosis occurs in the YSL after the gastrula stage. The present investigation demonstrated the size of nuclei and the number of nucleoli and their staining properties with DNA binding dye. The results indicate that the YSL nuclei actively transcribe RNA and that their DNA content is greater than that of somatic nuclei. The onset and subsequent time course of polyploidization were examined in embryos stained with 4',6-diamidino-2-phenylindole (DAPI) by epifluorescence microspectrophotometry from the cessation of mitosis through hatching. Embryos included YSL nuclei whose DNA content spanned from diploid (2C), tetraploid (4C) to octaploid (8C) at the end of the late blastula stage. The last two populations are produced probably by their early cessation of mitosis and the subsequent duplication of DNA without mitosis or by endoreduplication. The frequency distribution of the DNA content examined during epiboly of the blastoderm suggests that each population is duplicated again until the beginning of the gastrula stage and then once more until the end of epiboly. Eventually these nuclei include polyploid DNA between 8C and 64C or more during later embryonic development.