Ovarian germline stem cells in the teleost fish, medaka (Oryzias latipes)

In the mammalian testis germline stem cells keep producing many sperms, while there is no direct evidence for the presence of germline stem cells in the ovary. It is widely accepted in mammals that the mature oocytes are supplied from a pool of primordial follicles in the adult ovary. In other vertebrates, such as fish, however, there has been no investigation on the mechanism underlying the high egg-producing ability. In this review, we introduce the recently identified ovarian germline stem cells and the surrounding unique structure in teleost fish, medaka (Oryzias latipes) [Nakamura S et al. Science. 2010; 328: 1561-1563]. We also discuss about the expression and function of sox9 that characterizes this unique structure.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.     

Development of the endoderm and gut in medaka, Oryzias latipes

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.     

Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes)

This study was designed to determine the estrogenic effect of the phytoestrogen genistein on several measures of endocrine function in adult Japanese medaka (Oryzias latipes) relative to 17-beta-estradiol. Adult animals of both sexes were exposed to 75, 750 and 30,000 ng/fish (average fish weight equals 0.26 g) of genistein by i.p. injection, with a positive control group treated with 300 ng/fish of 17-beta-estradiol, while a negative control group received a vehicle-only (corn oil) injection. Content of vitellogenin, the yolk glycoprotein made in the liver in response to estradiol stimulation, was measured using Western blots. Circulating estradiol and testosterone levels were measured using a steroid-enzyme immunosorbant assay. The ability of ovaries and testes to synthesize and release estradiol and testosterone was determined by ex vivo incubation of gonads with 25-hydroxycholesterol. Vitellogenin, while induced by 17-beta-estradiol, was not increased in the liver of individuals treated with genistein. In females, genistein treatment at 750 and 30,000 ng increased the estradiol production of ovaries more than the 17-beta-estradiol treatment. In males, genistein treatment resulted in decreased testosterone production from ex vivo testis and a comparable reduction in circulating testosterone level. The changes in vitellogenin, circulating steroids and ex vivo steroidogenesis in medaka in response to genistein are similar to that of 17-beta-estradiol. However, some endpoints are more sensitive to estradiol treatment (vitellogenin), while others are more sensitive to genistein (male testosterone and ovarian estrogenesis).     

Gills of the medaka (Oryzias latipes): A scanning electron microscopy study

The general morphology and surface ultrastructure of the gills of adult and larvae medaka (Oryzias latipes) were studied in freshwater and seawater using scanning electron microscopy. The gills of all examined fish were structurally similar to those of other teleosts and consisted of four pairs of arches supporting (i) filaments bearing lamellae and (ii) rakers containing taste buds. Three cell types, specifically pavement cells, mitochondria‐rich cells (MRCs), and mucous cells, constituted the surface layer of the gill epithelium. Several distinctive characteristics of medaka gills were noted, including the presence of regularly distributed outgrowth on the lamellae, enlarged filament tips, the absence of microridges in most pavement cells in the filament and lamellae and the presence of MRCs in the arch at the filament base. A rapid mode of development was recorded in the gills of larval fish. At hatching, the larvae already had four arches with rudimentary filaments, rakers, and taste buds. The rudimentary lamellae appeared within 2 days after hatching. These results suggest the early involvement of larval gills in respiratory and osmoregulation activities. The responses of the macrostructures and microstructures of gills to seawater acclimation were similar in larvae and adult fish and included modification of the apical surface of MRCs, confirming the importance of these cells in osmoregulation. The potential roles of these peculiarities of the macrostructures and microstructures of medaka gills in the major functions of this organ, such as respiration and osmoregulation, are discussed.     

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.     

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.     

Sex determination in fish: Lessons from the sex-determining gene of the teleost medaka, Oryzias latipes

Although sex determination systems in animals are diverse, sex-determining genes have been identified only in mammals and some invertebrates. Recently, DMY (DM domain gene on the Y chromosome) has been found in the sex-determining region on the Y chromosome of the teleost medaka fish, Oryzias latipes. Functional and expression analyses of DMY show it to be the leading candidate for the male-determining master gene of the medaka. Although some work is required to define DMY as the master sex-determining gene, medaka is expected to be a good experimental animal for investigating the precise mechanisms involved in primary sex determination in non-mammalian vertebrates. In this article, the process of identification of DMY and is summarized and the origins of DMY and sexual development of the medaka's gonads are reviewed. In addition, putative functions of DMY are discussed.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.     

Defective fin regeneration in medaka fish (Oryzias latipes) with hypothyroidism

Wild-type medaka are known to have remarkable capabilities of fin, or epimorphic, regeneration. However, a hypothyroid mutant, kamaitachi (kmi), frequently suffers from injury in fins, suggesting an important role of thyroid hormone in fin regeneration. This led us to examine the relationship between thyroid hormone and fin regeneration using medaka as a model. For this, we first set up a medaka experimental system in which the rate of regeneration was statistically analyzed after caudal fin amputation under normal and hypothyroid conditions. As expected, the regeneration of amputated caudal fins was delayed in hypothyroid kmi -/- mutants. We then examined wild-type medaka with thiourea-induced hypothyroidism to evaluate the requirement of thyroid hormone during epimorphic fin regeneration. The results demonstrate that the growth rate of regenerates was much reduced in severely hypothyroid medaka throughout the regeneration period. This reduction in regenerative rate was recovered by exogenous administration of L-thyroxine. The present study is thus the first to report the direct involvement of thyroid hormone in teleost fin regeneration, and provides a basic framework for future molecular and genetic analyses.     

Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes

Spermatocytes of the teleost, Oryzias latipes , at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n = 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro . The size and shape of the flagellum were the same as those seen in vivo . The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.     

Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): use of diploidized eggs as recipients

Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish ( Oryzias latipes ). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene ( GFP )-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.     

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.     

Icariin reduces bone loss in a Rankl-induced transgenic medaka (Oryzias latipes) model for osteoporosis

Given the limitations and side effects of many synthetic drugs, natural products are an important alternative source for drugs and medications for many diseases. Icariin (ICA), one of the main flavonoids from plants of the Epimedium genus, has been shown to ameliorate osteoporosis and improve bone health in preclinical studies. Those studies have used different in vivo models, mostly rodents, but the underlying mechanisms remain unclear. The present study shows, for the first time, that ICA reduces bone damage in a Rankl-induced medaka fish (Oryzias latipes), a non-rodent osteoporosis model. Live imaging was previously performed in this model to characterize antiresorptive and bone-anabolic properties of drugs. Here, a new quantification method (I_M) was established based on the length of mineralized neural arches to quantify levels of bone mineralization damage and protection in early post-embryonic fish. This method was validated by quantification of three levels of bone damage in three independent Rankl fish lines, and by the determination of different degrees of severity of osteoporosis-like phenotypes in one Rankl line exposed to variable Rankl induction schemes. I_M was also used to quantify the efficacy of alendronate and etidronate, two common anti-osteoporotic bisphosphonates, and revealed comparable bone protective effects for ICA and alendronate in this fish osteoporosis model. This study's data support the value of the medaka fish model for bone research and establish a method to screen for novel osteoprotective compounds.     

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta‐actin promoter. The aNLS‐EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time‐lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.     

Molecular phylogeny and geography of Korean medaka fish (Oryzias latipes)

The phylogeny and geography of the medaka (Oryzias latipes) populations of Korea were investigated by analyzing sequence data for the mitochondrial control region. From the 41 haplotypes including 25 Korean haplotypes detected in 64 Korean specimens and data for the Japanese and Chinese populations, phylogenetic and nested clade analyses were executed to examine the phylogeny of haplogroups and the relation of the genetic architecture of the haplotypes to the historical geography of the Korean medaka fish. The analyses suggest that there are two very distinct lineages of Korean medaka, and that these result from reproductive isolation mechanisms due to geographic barriers. The southeastern lineage has experienced recent range expansion to the western region. The northwestern lineage, sister to Chinese populations, showed evidence of internal range expansion with shared haplotypes.     

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes)

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.