DNA alterations detected in the progeny of paternally irradiated Japanese medaka fish (Oryzias latipes).

A nonmammalian test system for germ-cell mutagenesis has been developed by using the Japanese medaka fish. We describe a system for detecting DNA alterations in F1 progeny descended from the gamma-irradiated male medaka that uses an arbitrarily primed polymerase chain reaction and fingerprinting. A combination of these two methods has some advantages for screening changes in genomic DNA of individual progeny because this detection system can (i) screen for mutational events before embryos with dominant lethal mutations are eliminated during development and (ii) detect DNA changes in the progeny of irradiated males without functional selection and bias, such as resistance to chemicals. DNA alterations are detected as changes in patterns (i.e., band loss and/or band gain) of DNA fingerprints of progeny descended from males whose spermatozoa or spermatids were gamma-irradiated (4.75 or 9.50 Gy). We determined the frequency of gamma-irradiation-induced band loss in arbitrarily primed polymerase chain reaction fingerprints of DNA from severely malformed embryos with dominant lethal mutations and hatched viable embryos. The frequency of band loss in both dominant lethal embryos and hatched viable embryos increased with increasing gamma-ray dose, although more so in the former. We detected a new band in the fingerprints as a heritable DNA alteration but not a viability- or phenotype-affecting DNA alteration in two viable mutants recovered after gamma-irradiation experiments. A cloned amplified fragment of the new band contained a repeated sequence of p(ATGT)n.     

Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes)

Fertile and diploid nuclear transplants were successfully generated by using embryonic cells as donors in a small laboratory fish, medaka (Oryzias latipes). Embryonic cell nuclei from transgenic fish carrying the green fluorescent protein (GFP) gene were transplanted into unfertilized eggs enucleated by x-ray irradiation. In this study, 1 out of 588 eggs transplanted in the first experiment and 5 out of 298 eggs transplanted in the second experiment reached the adult stage. All of these nuclear transplants were fertile and diploid, and the natural and GFP markers of the donor nuclei were transmitted to the F(1) and F(2) offspring in a Mendelian fashion. This systematic study proves the feasibility of generating nuclear transplants by using embryonic cells from fish as donors, and it is supported by convincing evidence.     

Development of the steroidogenic capacity of medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation

Developmental changes in the steroidogenic capacity of medaka, Oryzias latipes, ovarian follicles at 12 different stages during vitellogenesis and oocyte maturation were examined using 18-hr incubations. Medaka were acclimated to conditions of 26 degrees on a lighting regime of 14 hr light and 10 hr dark. Under these conditions, females usually spawn daily within 1 hr of the onset of light. The process of vitellogenesis and oocyte maturation occurs within 72 hr, the breakdown of the germinal vesicle (GVBD) and ovulation being completed at 6 and 1 hr, respectively, before the expected time of spawning. Vitellogenic follicles between 32 and 16 hr before spawning produced large amounts of estradiol-17 beta spontaneously and in response to partially purified chum salmon gonadotropin (SGA) or pregnant mare's serum gonadotropin (PMSG). However, postvitellogenic follicles between 12 and 4 hr before spawning showed very little evidence of estradiol-17 beta production. By contrast, basal concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) remained very low in follicles during vitellogenesis and were elevated in those collected during oocyte maturation; there was a close relationship between the medium concentration of 17 alpha,20 beta-diOH-prog and the percentage GVBD in the oocytes. 17 alpha,20 beta-DiOHprog production in response to PMSG was very low in follicles during early and mid-vitellogenesis and increased in those collected at 28 hr before spawning, a time which coincided with the first acquisition of the ability of the follicles to undergo maturation in response to gonadotropin. These results clearly demonstrate that a distinct shift from the secretion of predominantly estradiol-17 beta to the secretion of 17 alpha,20 beta-diOHprog occurs in the medaka ovarian follicle immediately prior to oocyte maturation. Considering the potency of 17 alpha,20 beta-diOHprog for the induction of oocyte maturation in vitro, these results further suggest that 17 alpha,20 beta-diOHprog is a naturally occurring steroidal mediator of oocyte maturation in the medaka.     

Testosterone content of developing eggs and sex reversal in the medaka (Oryzias latipes)

To understand the effect of testosterone on sex differentiation, the quantities of testosterone (T) and estradiol-17beta (E2) in developing eggs of medaka (Oryzias latipes) were measured by radioimmunoassay, and the influence on sex differentiation of treating embryos with exogenous androgens was also examined. Endogenous T of eggs dispersed into the environmental water at spawning, and precipitously declined to a minimum level during incubation for 2 days post-fertilization (dpf). It did not significantly increase during development. The E2 content of fertilized eggs increased when eggs were incubated in medium containing exogenous T at the concentrations of 100 and 500 ng/ml, but not in low concentrations of 10 ng/ml or less. The presence of 500 ng/ml 17alpha-methyltestosterone (MT) in the incubation medium also induced an increase in the E2 content of embryos. Exposure of embryos to exogenous 1 ng/ml T that corresponded with the level of T in eggs shortly after fertilization was enough to induce sex reversal of genotypic females to functional males. The co-existence of T and aromatase inhibitor in incubation medium inhibited not only the T-induced increase in the embryonic E2 content, but also the estrogenic effect of T in causing the paradoxical sex reversal from genotypic males to phenotypic females. However, treatment of embryos with the non-aromatizable androgen, 17alpha-methyldihydrotestosterone, induced no detectable increase in the E2 content of embryos, but still brought about sex reversal of genotypic males into females. This contradictory result suggests that the conversion of androgens to E2 may not always be the cause for induction of paradoxical sex reversal by T treatment. Consequently, these results on sex reversal induced by treatment of embryos with exogenous androgens suggest that endogenous T of developing medaka embryos may not act as the natural andro-inducer, and that genotypic sex can be modified by exogenous sex steroids at early developmental stages long before gonadal differentiation in the medaka.     

Identification and characterization of two distinct GnRH receptor subtypes in a teleost, the medaka Oryzias latipes

We report the identification and characterization of two distinct GnRH receptor (GnRH-R) subtypes, designated GnRH-R1 and GnRH-R2, in a model teleost, the medaka Oryzias latipes. These seven-transmembrane receptors of the medaka contain a cytoplasmic C-terminal tail, which has been found in all other nonmammalian GnRH-Rs cloned to date. The GnRH-R1 gene is composed of three exons separated by two introns, whereas the GnRH-R2 gene has an additional intron and therefore consists of four exons and three introns. The GnRH-R1 and GnRH-R2 genes, both of which exist as single-copy genes in the medaka genome, were mapped to linkage groups 3 and 16, respectively. Inositol phosphate assays using COS-7 cells transfected with GnRH-R1 and GnRH-R2 demonstrated that they had remarkably different ligand sensitivities, although both receptors showed highest preference for chicken-II-type GnRH. Phylogenetic analysis showed the presence of three paralogous lineages for vertebrate GnRH-Rs and indicated that neither GnRH-R1 nor GnRH-R2 is the medaka ortholog to mammalian GnRH-Rs that lack a cytoplasmic tail. This, together with an observation that medaka-type GnRH had low affinity for GnRH-R1 and GnRH-R2, suggests that a third GnRH-R may exist in the medaka.     

Growth, energetics and the cortisol-hepatic glucocorticoid receptor axis of medaka (Oryzias latipes) in various salinities

We examined growth of euryhaline Japanese medaka (Oryzias latipes) after transfer to freshwater or seawater from isotonic saline. Growth was unaffected by the different salinities for 1week, but the body weight increase and BMI of fish kept in freshwater for 2-3weeks were significantly higher than those in the isotonic controls. These results may reflect the usual habitat of this species. To assess the basis for the difference in growth, energetics and the hepatic stress axis were evaluated 1week after the transfer. Unexpectedly, despite the higher growth rate, the rate of routine oxygen consumption was significantly higher in freshwater. Plasma cortisol levels in freshwater were significantly higher than those in seawater, and the mRNA levels of the glucocorticoid receptor (GR1) in the liver were significantly lower in freshwater and seawater, compared to that in isotonic saline. Branchial Na(+)/K(+)-ATPase activities were also reduced significantly in freshwater and seawater, compared to that in isotonic saline. The higher levels of hepatic GR1 expression and branchial Na(+)/K(+)-ATPase activity in isotonic salinity than those in freshwater and seawater for 1week may account for the lower growth rate under the isotonic condition. After 3weeks, however, the Na(+)/K(+)-ATPase activity in seawater was significantly higher than that in freshwater. No significant difference in growth rate between freshwater and seawater groups indicates that medaka is a good model for studies of hypo- and hyperosmotic adaptations, since osmoregulation is not strongly associated with size and growth.     

New evidence for the involvement of prostaglandin receptor EP4b in ovulation of the medaka, Oryzias latipes

A cDNA for a prostaglandin E(2) (PGE(2)) receptor subtype 4, EP4b (Ptger4b), was cloned from the medaka ovary. The effect of PGE(2) was examined using COS-7 cells expressing the recombinant Ptger4b protein. An increase in intracellular cAMP levels was observed when the cells were incubated with PGE(2), but the increase in cAMP levels was nullified by the addition of the EP4 antagonist GW627368X. The expression of ptger4b mRNA was drastically induced by the addition of pregnant mare serum gonadotropin to the in vitro culture of large preovulatory follicles. In in vitro ovulation studies of the effect of GW627368X addition on follicle ovulation, the critical timing of the PGE(2)/Ptger4b interaction was suggested to be between -1 and 0h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary.     

Age related changes in morphology of the thymus of the fish,Oryzias latipes

The thymus of the teleost fish Oryzias latipes is a paired structure found at the dorsoposterior part of the gill chamber. In 3-month-old fish, the thymus shows a great development. The thymus displays atrophy during aging, and the thymus involution continues until 5 years of age. Male thymus shows heavier involution than female thymus of the same age. Emigration of thymus cells takes place at all ages but increases with age.     

Effect of x-irradiation during embryonic stage on life span in the fish, Oryzias latipes

Embryos of Oryzias latipes were irradiated with 0, 10, 25, 50, 100, 250, 500 and 1000 R of X-rays, respectively. If 100 R or more were given, mortality rate increased even 450 days or later and mean life span of the irradiated fish became short.     

A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka,Oryzias latipes

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene.     

Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes)

Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380 kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17beta (E2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (相似文献   

Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes)

Endogenous opioid peptides (EOPs) are an important class of modulators of the hypothalamo-pituitary axis; treatment with opiates leads to inhibition of GnRH and LH secretion and suppression of reproductive functions. However, little work has been done to investigate the effect of opiates on the electrical activity of GnRH neurons, which ultimately controls GnRH secretion. The purpose of the present study was to investigate the effects of the EOP beta-endorphin on electrical activity of GnRH neurons located in the terminal nerve (TN) associated with the olfactory bulb. We used an excised intact brain preparation from transgenic medaka in which green fluorescent protein (GFP) is genetically expressed in TN-GnRH neurons. These GFP-expressing neurons were then targeted for whole-cell current clamp recordings. Treatment with beta-endorphin led to changes in several characteristics of electrical activity, including depolarization of membrane potential and a decrease in spike amplitude--similar to that observed in response to depolarizing high K(+) treatment. This finding suggests a model in which beta-endorphin depolarizes membrane potential leading to Na(+)-channel inactivation, and subsequent suppression of action-potential amplitude. On the other hand, beta-endorphin had no effect on membrane potential in synaptically isolated GnRH neurons. These results suggest that beta-endorphin is acting indirectly on TN-GnRH neurons to inhibit action potential firing.     

Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicle in response to exogenous substrates

Changes in the capacity of medaka, Oryzias latipes, ovarian follicles to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) or testosterone to testosterone, estradiol-17 beta, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were examined using 18-hr incubations. Under a constant long photoperiod (14 hr light-10 hr dark) at 26 degrees medaka spawn daily within 1 hr of the onset of light. Under these conditions, vitellogenesis and oocyte maturation of individual follicles occur within 72 hr, allowing accurate determination of the time of oocyte maturation and ovulation. In the absence of substrates, vitellogenic follicles isolated between 28 and 16 hr before spawning produced increased amounts of estradiol-17 beta, while postvitellogenic follicles between 14 and 8 hr produced a large amount of 17 alpha,20 beta-diOHprog. However, under the same conditions, testosterone levels were very low in follicles from all stages of development. The capacity of follicles to produce estradiol-17 beta in response to 17 alpha-OHprog or testosterone increased as follicles developed from the early to late vitellogenic stage, but declined during oocyte maturation. Maximum estradiol-17 beta production was observed in follicles at 20 hr before spawning. In contrast, the conversion of 17 alpha-OHprog to 17 alpha,20 beta-diOHprog was low in vitellogenic follicles and increased in follicles isolated immediately prior to or during oocyte maturation, with maximum 17 alpha,20 beta-diOHprog production occurring in follicles isolated at 14 hr before spawning. These results demonstrate a distinct shift in the activities of steroidogenic enzymes from C17-20 lyase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and aromatase to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) occurring in the medaka ovarian follicles immediately prior to oocyte maturation.