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.     

Specificity of carotenoid transfer in the larval medaka, Oryzias latipes

Carotenoids were introduced into the egg yolk of the medaka by either injection or feeding. Carotenoids for injection were dissolved in either olive oil, a mixture of olive oil and castor oil or Tween 80. Capsanthin, lutein and canthaxanthin were readily transferred from the yolk to the larval xanthophores, but no β-apo-8′-carotenal and little β-carotene were transferred.     

Effects of kisspeptin1 on electrical activity of an extrahypothalamic population of gonadotropin-releasing hormone neurons in medaka (Oryzias latipes)

Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca(2+)/high Mg(2+) solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions.     

Developmental process of genital ducts in the medaka, Oryzias latipes

The morphological development of genital ducts both intra-gonadal (ovarian cavity and efferent duct) and extra-gonadal (oviduct and sperm duct) was investigated in a model teleost, medaka Oryzias latipes. The results showed that the extra-gonadal genital ducts contained two structural units, the anterior and posterior parts, in both sexes. Of special interest is a newly discovered process for the development of a posterior part of the oviduct. The anterior part of oviduct extended continuously from the ovarian cavity at the posterior end of the ovary. Then the posterior part of oviduct, which termed genital pore lip (GPL) in this study, was formed. This part results from invagination and cavitation of the cortex of urinogenital papillae (UGP) and forms the wall of the oviduct opening. We also suggest that the ventral region of urethra mesenchyme has an important role in extra-genital ducts formation.     

Stages of normal development in the medaka Oryzias latipes

Unfertilized eggs of Oryzias latipes were artificially inseminated and incubated at 26+/-1 degrees C. Careful observation of the process of embryonic development by light microscopy allowed division of the process into 39 stages based on diagnostic features of the developing embryos. The principal diagnostic features are the number and size of blastomeres, form of the blastoderm, extent of epiboly, development of the central nervous system, number and form of somites, optic and otic development, development of the notochord, heart development, blood circulation, the size and movement of the body, development of the tail, membranous fin (fin fold) development, and development of such viscera as the liver, gallbladder, gut tube, spleen and swim (air) bladder. After hatching, development of the larvae (fry) and young can be divided into six stages based on such diagnostic features as the fins, scales and secondary sexual characteristics.     

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes)

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.     

The ultrastructure of the ovarian follicle of medaka,Oryzias latipes

Summary The follicular cells in the oocytes of Oryzias latipes were studied by electron microscopy in order to clarify the fine structure, and the role of the cells during yolk formation and ovulation. The smallest follicles were observed during the early phase of peri-nucleolus stage of the oocyte. The cells have flattened nuclei, and perikarya with undeveloped organelles. But when the oocytes attain diameter of about 250 (yolk vesicle stage), both types of endoplasmic reticula are present. Moreover, the microvilli of the plasma membrane of oocyte as well as the follicles protrude into the pore canals of the zona radiata. In the oocytes of yolk stage the rough-surfaced endoplasmic-reticulum is typically developed and observed around the nuclei. Other organelles (lysosomes, mitochondria and Golgi) increase in number. The relation between the changes of cytoarchitecture in the follicles and yolk formation is discussed.At 17.00 p.m. on the day preceding ovulation the microvilli withdraw somewhat. Ribosomes are attached to the vesicular and cisternal endoplasmic reticula. When the oocytes attain complete maturation (24.00 p.m. at near ovulation), striking changes of the follicles are observed. The microvilli are almost withdrawn. In the degenerating follicles the lamellar structure is formed, and lipids are deposited at the center. At this time the contents of lysosomes have mostly disappeared.     

Classification of late embryonic stages of medaka,Oryzias latipes

To indicate more clearly the stages of development in late embryo of the medaka,Oryzias latipes, the quotients obtained from the length of tail (measured from the vent to distal end of caudal fin) in microns divided by 100 μm were adopted to nominate stage numbers. The 25 stages thus defined were checked against the calcification of 8 elements in the chondrocranium, and also 4 other structures which develop numerically. By the observations of development (cartilages and calcification indicated by alizarin red S staining) of the 8 elements—parasphenoid, clavicle, operculum, occipital arch, hyomandibular, ceratohyal, anterior otic process and mandibular, the stages defined here were found to classify the order and degree of development of bony elements more precisely than other stage classification in the past. The numerical structures—branchiostegal rays, pharyngeal teeth, vertebrae and mandibular teeth also demonstrated clearly their development corresponding to tail length. The “critical” stages in embryonic development of the medaka as shown by the length of notochord with vacuolized cells associated with development of bony elements were also noted.     

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.     

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.     

Characterization of mutations affecting embryonic hematopoiesis in the medaka, Oryzias latipes

In a genetic screen for mutations affecting organogenesis in the medaka, Oryzias latipes, we identified eight mutants with defects in embryonic hematopoiesis. These mutations were classified into seven complementation groups. In this paper, we characterize the five mutants that were confirmed in the next generation. The beni fuji mutant was defective in the generation of blood cells, exhibiting reduced blood cells at the initiation of circulation. Mutations in two genes, lady finger and ryogyoku, caused abnormal morphology of blood cells, i.e., deformation, along with a progressive decrease in the number of blood cells. The sekirei mutant exhibited photosensitivity with autofluorescent blood cells. Mutations in kyoho resulted in huge blood cells that were approximately three times longer than the wild-type blood cells. The spectrum of phenotypes identified in this study is similar to that of the zebrafish hematopoietic mutants except for the huge blood cells in kyoho. Our results demonstrate that medaka, as well as zebrafish, is a useful model to study hematopoiesis.     

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.     

Mesodermal metamerism in the teleost, Oryzias latipes (the medaka)

Previous studies of the metameric pattern in mesodermal tissues of chick, mouse, turtle, and amphibian embryos have indicated that segmental characteristics exist along the entire length of the embryo. This paper describes this phenomenon in a fish embryo, for some differences in the cranial segmental plan exist between the anamniote and the amniote embryos hitherto studied. Embryos of the cyprinodont, Oryzias latipes, were fixed at various times, the examined by means of stereo scanning electron microscopy. As in other vertebrate embryos, the first indication of mesodermal metamerism in this fish embryo is the occurrence of somitomeres, which are orderly, tandemly arranged units of uncondensed mesenchymal cells in the paraxial mesoderm. As many as ten somitomeres can be observed caudal to the last formed somite to the elongating tail region. In addition, 7 somitomeres are present rostral to the first definitive somite, which is segment number eight. As in other vertebrate embryos examined, somitomeres in Oryzias embryos are circular, bilaminar arrays of paraxial mesoderm that form before any indications of segmentation can be seen with the light microscope. In the trunk region these mesodermal units condense to give rise to definitive somites, but in the head they eventually disperse. Despite a fundamentally different mode of gastrulation and a relatively small number of cells in the newly formed somitomeres, cranial segmentation in Oryzias embryos was found to be more similar in number to the metameric pattern of the embryos of the bird, reptile, and mammal than to the situation found in the two amphibians studied thus far.     

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.