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.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

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.     

Artificial fertilization by intracytoplasmic sperm injection in a teleost fish, the medaka (Oryzias latipes)

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.     

Construction of a BAC library for Haplochromis chilotes,a cichlid fish from Lake Victoria

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.     

Growth and maturation of melanosomes in the melanophores of a teleost,Oryzias latipes

Summary Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent hollows in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.     

Circus movement in dissociated embryonic cells of a teleost, Oryzias latipes.

The dissociated early embryonic cells of the fresh water fish, Oryzias latipes, protrude hyaline lobopodia, which tend to rotate around the cell circumference in a propagating wave. Cells from late blastula or gastrula continuously show this "circus movement", while most cells up to early blastula are rounded. The linear velocity of the lobopodium was estimated by means of time-lapse cinemicrography. The velocity increases slightly as cell diameter increases. The effects of pH, temperature and osmotic pressure of the immersion media on the movement were also quantitatively investigated. Cells become rounded and do not form lobopodial blebs when immersed in media below pH 5. The velocity is reduced by decreasing temperature, but the movement continues even at 5 degrees C. Cells placed in hypertonic salt solutions become crenated and do not continuously demonstrate the circus movement.     

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.     

Acetylcholine in embryos of Oryzias latipes,a teleost: Gas chromatographic-mass spectrometric assay

1. A gas chromatographic-mass spectrometric assay was used to acetycholine in extracts of embryos of the medaka Oryzias latipes, a teleost fish.2. Acetylcholine is present during cleavage and in blastulae and gastrulae, the mean amount per embryo ranging from 0.49 to 1.43 pmol.3. A substantial proportion of the total acetylcholine in the embryo and yolk is present in cells dissociated from the embryo.     

Allelic expression changes in Medaka (Oryzias latipes) hybrids between inbred strains derived from genetically distant populations

Variations in allele expressions between genetically distant populations are one of the most important factors which affects their morphological and physiological variations. These variations are caused by natural mutations accumulated in their habitats. It has been reported that allelic expression differences in the hybrids of genetically distant populations are different from parental strains. In that case, there is a possibility that allelic expression changes lead to novel phenotypes in hybrids. Based on genomic information of the genetically distant populations, quantification and comparison of allelic expression changes make importance of regulatory sequences (cis-acting factors) or upstream regulatory factors (trans-acting modulators) for these changes clearer. In this study, we focused on two Medaka inbred strains, Hd-rR and HNI, derived from genetically distant populations and their hybrids. They are highly polymorphic and we can utilize whole-genome information. To analyze allelic expression changes, we established a method to quantify and compare allele-specific expressions of 11 genes between the parental strains and their reciprocal hybrids. In intestines of reciprocal hybrids, allelic expression was either similar or different in comparison with the parental strains. Total expressions in Hd-rR and HNI were tissue-dependent in the case of HPRT1, with high up-regulation of Hd-rR allele expression in liver. The proportion of genes with differential allelic expression in Medaka hybrids seems to be the same as that in other animals, despite the high SNP rate in the genomes of the two inbred strains. It is suggested that each tissue of the strain difference in trans-acting modulators is more important than polymorphisms in cis-regulatory sequences in producing the allelic expression changes in reciprocal hybrids.     

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.     

Uptake and distribution of cadmium in the egg of the teleost, Oryzias latipes

Eggs of Oryzius latipes in the blastula stage were exposed to M/100 artificial sea water which contained cadmium at the concentrations of 0.1, 1.0, 10.0, 20.0 or 50.0 mg 1⁻¹. The 96 h TL₅₀, value for cadmium was estimated to be 20 5 mg 1⁻¹. When the eggs were incubated for 24 h in the M/100 sea water with 10.0 mg Cd 1⁻¹ and then rinsed in glycine buffer solution (pH; 2.0), the cadmium content of the egg decreased markedly. Cadmium levels were determined in parts of the embryonic body, the chorion and the yolk sac. The most cadmium was detected in the chorion (94.6%). Prolonged cadmium exposure revealed that most of the cadmium was absorbed by the chorion and little was detected in the embryonic body and the yolk sac.     

Pacemaker region in a rhythmically contracting embryonic epithelium, the enveloping layer of Oryzias latipes, a teleost

The primary objectives of this study were to determine the embryonic stage at which the Oryzias latipes enveloping layer (EVL) begins to contract rhythmically, and to determine where these contractions arise within the EVL. Using time-lapse video recording, we showed that the contractions begin at stage 14 (the stage of the embryonic shield) and arise in the ventral region of the EVL, which is centered at 180 degrees longitude from the embryonic shield. We have called this the pacemaker region for the contractions. Using fluorescein diacetate as a vital stain, we showed that the ventral region of the EVL continues to act as a pacemaker even after the EVL is detached from the rest of the egg. Rhythmic contractile activity ceased when we removed a group of about 130 cells--10% of the total EVL--from the pacemaker region; comparably large wounds elsewhere had no effect on the contractions. When we cut detached EVLs into ten pieces, only 2.4 +/- 1.8 (mean +/- SD, N = 11) of them contracted rhythmically, even though a considerably larger proportion of the EVL cells participate in the contractions in undisturbed blastoderms. We conclude that the pacemaker cells are necessary for rhythmic contractile activity and that cells outside this region do not contract spontaneously. The contractile waves are propagated at a velocity of 14-54 microns sec-1. This value, which is two to three orders of magnitude slower than the propagation of epithelial action potentials, is similar to the rate of propagation of waves of increased cytosolic Ca2+ in other systems. We propose that the medaka EVL is a good system in which to study certain aspects of epithelial morphogenesis.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.