Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

Estrogen rescues masculinization of genetically female medaka by exposure to cortisol or high temperature

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by exposure to high water temperature (HT) during gonadal sex differentiation, possibly by elevation of cortisol, the major glucocorticoid produced by the interrenal cells in teleosts. Yet, it remains unclear how the elevation of cortisol levels by HT causes female‐to‐male sex reversal. This paper reports that exposure to cortisol or HT after hatching inhibited both the proliferation of female‐type germ cells and the expression of ovarian‐type aromatase (cyp19a1), which encodes a steroidogenic enzyme responsible for the conversion of androgens to estrogens, and induced the expression of gonadal soma‐derived growth factor (gsdf) in XX gonads during gonadal sex differentiation. In contrast, exposure to either cortisol or HT in combination with 17β‐estradiol (E2) did not produce these effects. Moreover, E2 completely rescued cortisol‐ and HT‐induced masculinization of XX medaka. These results strongly suggest that cortisol and HT cause female‐to‐male sex reversal in medaka by suppression of cyp19a1 expression, with a resultant inhibition of estrogen biosynthesis. This mechanism may be common among animals with temperature‐dependent sex determination. Mol. Reprod. Dev. 79: 719–726, 2012. © 2012 Wiley Periodicals, Inc.     

Sex reversal of the newt Triturus cristatus reared at extreme temperatures

Crested newt larvae were reared at defined temperatures, either from uncleaved eggs or from early feeding larvae, until metamorphosis when sexual differentiation had occurred. Trials at 18-24 degrees C showed a 1:1 sex ratio. A higher temperature trial produced more males than females, including some XX neomales. Lower temperatures resulted in a significant excess of females, including XY neofemales. Sex reversal only occurred in about half the possible cases on average. Extreme temperatures must perturb the normal XX/XY system of sex determination, to reveal either an ancestral ZZ/ZW system or a still more primitive environmental control. It is suggested that neofemales (but not neomales) could occur in nature.     

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.     

Genetic risk involved in stock enhancement of fish having environmental sex determination

Environmental sex determination is reported in various fish species, including some fishes subject to stock enhancement. We studied the influence of stock enhancement on the sex-determining system of fish with both genotypic and environmental sex determination. We constructed and analyzed recurrence formula models for the dynamics of gene frequency in the male heterogametic (XX female and XY male) sex-determining system of the Japanese flounder (Paralichthys olivaceus). In this species, an XX individual can develop as a phenotypic male (sex-reversed male) depending on the conditions experienced by a juvenile. We show that the release of sex-reversed males may result in the extinction of the Y gene that determines sex. We also studied how the risk depends on the kind of hatchery broodstock used (e.g., fish collected from the wild or fish from a lineage established in a hatchery), sex-reversal rates in the hatchery and in the wild, and the relative contribution of released fish to reproduction in the wild. We propose some ways to reduce the risk. Received: October 10, 2001 / Accepted: December 17, 2001     

Mapping and Validation of the Major Sex-Determining Region in Nile Tilapia (Oreochromis niloticus L.) Using RAD Sequencing

Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the “female” genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the “female” genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture.     

Sex reversal of pejerrey (Odontesthes bonariensis), a species with temperature-dependent sex determination,in a seminatural environment

This study examined the changes in sex ratios and sex reversal rates in pejerrey Odontesthes bonariensis that occur with the progression of the spawning season in a seminatural setting. Four groups of hatchery-produced pejerrey larvae were stocked in floating cages in La Salada de Monasterio lake (Pampas region), a natural habitat of this species, and reared from hatching beyond gonadal sex determination with minimum human interference. Cage 1 was stocked at the beginning of the spring spawning season and the other cages were stocked with monthly delays until cage 4 in early summer. The genotypic (amhy+, XY/YY; amhy−, XX) and phenotypic (testis, male; ovary, female) sex ratios and proportions of genotype/phenotype mismatched individuals were estimated and their relation to water temperature and daylength during the experiment was analysed by generalized linear modelling. Water temperature varied between 11 and 30.5°C, and daylength duration between 11 h 22 min and 14 h 35 min. Sex genotyping revealed nearly balanced sex ratios of XY/YY (46%–49.1%) and XX (50.9%–54%) fish in cages 2–4 whereas the genotypic sex ratio in cage 1 was clearly biased towards XY/YY fish (60.6%). Phenotypic males ranged from 42% to 54.4% in cages 1–3. Cage 4, in turn, had significantly more phenotypic males (66%). The percentage of XX males (phenotypic male/genotypic female) was 23.1% in cage 1, decreased to a minimum of 5.4% in cage 2 and gradually increased in cages 3 and 4 to a maximum of 40.7% in the latter. The percentages of XY/YY females (phenotypic female/genotypic male) were highest in cage 1 (30%) and decreased progressively in the other cages to a significantly lower value (4.3%) in cage 4. These results generally support the findings of laboratory studies on the effect of temperature on the sex determination of this species and also provide novel evidence of a XX genotype-specific masculinizing effect of short daylength.     

Increase of cortisol levels after temperature stress activates dmrt1a causing female‐to‐male sex reversal and reduced germ cell number in medaka

In vertebrates, there is accumulating evidence that environmental factors as triggers for sex determination and genetic sex determination are not two opposing alternatives but that a continuum of mechanisms bridge those extremes. One prominent example is the model fish species Oryzias latipes which has a stable XX/XY genetic sex determination system, but still responds to environmental cues, where high temperatures lead to female‐to‐male sex reversal. However, the mechanisms behind are still unknown. We show that high temperatures increase primordial germ cells (PGC) numbers before they reach the genital ridge, which, in turn, regulates the germ cell proliferation. Complete ablation of PGCs led to XX males with germ cell less testis, whereas experimentally increased PGC numbers did not reverse XY genotypes to female. For the underlying molecular mechanism, we provide support for the explanation that activation of the dmrt1a gene by cortisol during early development of XX embryos enables this autosomal gene to take over the role of the male determining Y‐chromosomal dmrt1bY.     

Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes

The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession number AY623815.     

高温和皮质醇对黄颡鱼性别分化的影响

研究以性染色体类型已确定且已有性别特异分子标记的黄颡鱼为研究对象, 开展高温与皮质醇诱导黄颡鱼(Tachysurus fulvidraco Richardson)XX个体雄性化组织学进程研究, 以期为环境应激诱导鱼类雄性化提供研究基础。通过对每尾鱼采用性别特异性标记鉴定遗传性别(XX或XY)及组织学鉴定生理型性别, 仅经过24d的处理(12—35日龄), 高温或皮质醇便能诱导XX遗传型个体雄性化。在此过程中, 部分XX遗传型个体卵母细胞受到抑制, 之后发育成带有卵巢腔的精巢结构。62日龄时, XX伪雄鱼性腺较正常XY雄鱼大, XX伪雄鱼体重与正常XY雄鱼相近, 而显著大于未发生性逆转的XX雌鱼。122日龄时, XX伪雄鱼从62日龄带有卵巢腔的精巢结构发育成具有典型的精小叶结构样精巢, 且都具有生理性雄鱼特有的生殖突, 推测这些雄鱼可能具有与正常雄鱼类似的生殖能力。部分XX个体对高温处理不敏感, 没有发生性逆转, 温度处理反而加快了卵巢发育的进程, 这些个体对高温的耐受性和另外一些发生性逆转的个体对温度的敏感性值得进一步研究。     

High temperature causes masculinization of genetically female medaka by elevation of cortisol

In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by high water temperature (HT; 32–34°C) treatment during the sex differentiation period. Here we report that cortisol caused female‐to‐male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT‐induced masculinization of XX medaka. HT treatment caused elevation of whole‐body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33°C treatments inhibited female‐type proliferation of germ cells as well as expression of follicle‐stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression. Mol. Reprod. Dev. 77: 679–686, 2010. © 2010 Wiley‐Liss, Inc.     

GENETIC AND ENVIRONMENTAL BASIS OF VARIABLE SEX RATIOS IN LABORATORY STRAINS OF POECILIOPSIS LUCIDA

Skewed sex ratios are common among several species of Poeciliopsis, a viviparous fish from northwestern Mexico. Since previous, unrelated studies from this laboratory (Angus and Schultz, 1983) suggested that deviation from a 1:1 sex ratio might be influenced by temperature, two inbred strains of P. lucida were tested for temperature-dependent sex determination by comparing sex ratios of offspring from pregnant females held at different water temperatures. Different sex ratios were produced by the two strains at the same temperature: one strain produced almost all-male offspring at 30°C and female-biased sex ratios at 24°C, while the other strain produced a 1:1 sex ratio at both temperatures. At intermediate temperatures, the labile strain produced sequentially fewer males with decreasing temperatures. The other strain produced a consistent sex ratio regardless of temperature. Poeciliopsis lucida apparently has a genetic polymorphism for temperature-influenced sex determination. An hypothesis is offered for the evolutionary origin of environmental sex determination.     

Hormonal induction and stability of monosex populations in the medaka (Oryzias latipes): expression of sex-specific marker genes

The model teleost medaka (Oryzias latipes, d-rR.YHNI strain) was used to produce offspring of a defined sex (monosex populations) by crossing experimentally produced YY and XX males to normal females. These monosex populations had the predicted chromosomal constitution as shown by a sex chromosome-specific DNA sequence. However, in XX populations the spontaneous development of males without previous exposure to androgens was observed. Differences in the percentage of male offspring from individual XX breeding pairs indicate a possible variation of unknown genetic factors to be responsible for the development of XX males. The expression of two gonadal genes that are involved in sex differentiation, Dmrt1b(Y) and Fig1a (factor in the germ line alpha), was analyzed in monosex populations. Dmrt1b(Y) expression correlated strictly with the genotype but not the sexual phenotype. When XY juvenile fish were exposed to 17 alpha-ethynylestradiol at concentrations that induce sex reversal, Dmrt1b(Y) expression was not repressed. However, Dmrt1b(Y) was expressed in XY or YY gonads regardless of the sex and could not be detected in XX individuals. In contrast, the expression of Fig1a correlated with the phenotypic sex: Fig1a was expressed in male juvenile fish exposed to 17 alpha-ethynylestradiol and repressed in fish exposed to 17 alpha-methyltestosterone. The Dmrt1b(Y) expression appears to reflect an early and important event in sex determination and lends support to the suggested key regulatory role of the Dmrt1b(Y) gene in sex determination. This process is apparently hormone insensitive, and the expression of further downstream acting genes can be regulated (directly or indirectly) by sex steroids.     

Common spontaneous sex-reversed XX males of the medaka Oryzias latipes

In the medaka, a duplicated version of the dmrt1 gene, dmrt1bY, has been identified as a candidate for the master male sex-determining gene on the Y chromosome. By screening several strains of Northern and Southern medaka we identified a considerable number of males with normal phenotype and uncompromised fertility, but lacking dmrt1bY. The frequency of such males was >10% in some strains and zero in others. Analysis for the presence of other Y-linked markers by FISH analysis, PCR, and phenotype indicated that their genotype is XX. Crossing such males with XX females led to a strong female bias in the offspring and also to a reappearance of XX males in the following generations. This indicated that the candidate male sex-determining gene dmrt1bY may not be necessary for male development in every case, but that its function can be taken over by so far unidentified autosomal modifiers.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Sex determination in hydra: control by a subpopulation of interstitial cells in Hydra oligactis males

The stability of sexual phenotype was examined in a single clone of Hydra oligactis males maintained at two culture temperatures, 18 and 22 degrees C. At these temperatures animals of this species do not reproduce sexually, but reproduce asexually by budding, and males and females are morphologically indistinguishable. When the temperature is lowered to 10 degrees C gametogenesis is induced and sexual phenotype can be assayed. Males cultured for several years at 18 degrees C expressed a stable sexual phenotype when induced to undergo gametogenesis; males remained male. Those cultured at 22 degrees C for 1 year, however, expressed a low frequency of sex reversal from male to female; males ceased sperm differentiation and began producing eggs. Male sex reversal in cultures maintained at the higher temperature was correlated with the loss of a specific subpopulation of interstitial cells, those that bind the monoclonal antibody, AC2, which labels cells specific to the spermatogenic pathway in H. oligactis males. When interstitial cells restricted to this pathway were reintroduced into sex-reversed males (phenotypic females), the male phenotype was reestablished and animals reverted to sperm production. To further investigate the role of AC2+ cells in the masculinization of females, normal males (containing AC2+ cells) and sex-reversed males (lacking AC2+ cells) were grafted to females. In grafts between normal males and females, egg production ceased and sperm differentiation ensued, whereas those between sex-reversed males and females continued to produce eggs. Thus, the presence of AC2+ interstitial cells is strictly correlated with male sexual phenotypes and it is only in their absence that the female phenotype is expressed.     

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.     

Treatment with an aromatase inhibitor suppresses high-temperature feminization of genetic male (YY) Nile tilapia

High temperature (36° C) treatment during sexual differentiation caused significant changes in sex ratio in YY male Nile tilapia Oreochromis niloticus fry (64.5% males compared to 100.0% males at 28° C), while dietary treatment with a chemical aromatase inhibitor (AI: Fadrozole™ CGS16949A) during this period suppressed the high temperature feminization (98.9% males). This implies that cytochrome P450 aromatase is mechanistically associated with temperature-dependent sex determination (TSD) in this species. XY male fry did not show significant sex reversal at 36° C. In XX female fry, high temperature treatment resulted in significant masculinization (62.5% males compared with 21.9% males at 28° C), while treatment with AI at either temperature resulted in very high proportions of males (100.0% males at 36° C; 99.0% males at 28° C). These results confirm the importance of aromatase in sexual differentiation in the Nile tilapia below the TSD threshold and suggest that it also plays a role in TSD, at least in the YY genotype.     

Sex-determining mechanisms in animals

Biological mechanisms leading to the development of males and females are extremely varied. In the XX/XY system, the male has an unequal pair of chromosomes, while in the ZZ/ZW system, the unequal pair is in the female. Sex can also be determined by the temperature of incubation. Recent research has focused on the identification of sex-determining genes, culminating in the demonstration that the Sry gene on the Y chromosome of mice can induce male development in genetically female XX mouse embryos. Nevertheless, the occurrence of phenotypes in apparent contrast to the genotype suggests that the genetic male/female switch is not simple, and there may be common features linking different sex-determining mechanisms. There is increasing evidence that such a link may be provided by the induction of growth differences, and that the primary sex difference may result from the distinction between fast versus slow growth.