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.     

Isolation and development of a molecular sex marker for <Emphasis Type="Italic">Bassiana duperreyi</Emphasis>, a lizard with XX/XY sex chromosomes and temperature-induced sex reversal

Sex determination in the endemic Australian lizard Bassiana duperreyi (Scincidae) is influenced by sex chromosomes and incubation temperature, challenging the traditional dichotomy in reptilian sex determination. Analysis of those interactions requires sex chromosome markers to identify temperature-induced sex reversal. Here, we report the isolation of Y chromosome DNA sequence from B. duperreyi using amplified fragment length polymorphism PCR, the conversion of that sequence to a single-locus assay, and its combination with a single-copy nuclear gene (C-mos) to form a duplex PCR test for chromosomal sex. The accuracy of the assay was tested on an independent panel of individuals with known phenotypic sex. When used on offspring from field nests, our test identified the likely occurrence of a low rate of natural sex reversal in this species. This work represents the first report of Y chromosome sequence from a reptile and one of the few reptile sex tests.     

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.     

Evolutionary transition to XY sex chromosomes associated with Y-linked duplication of a male hormone gene in a terrestrial isopod

Sex chromosomes are highly variable in some taxonomic groups, but the evolutionary mechanisms underlying this diversity are not well understood. In terrestrial isopod crustaceans, evolutionary turnovers in sex chromosomes are frequent, possibly caused by Wolbachia, a vertically-transmitted endosymbiont causing male-to-female sex reversal. Here, we use surgical manipulations and genetic crosses, plus genome sequencing, to examine sex chromosomes in the terrestrial isopod Trachelipus rathkei. Although an earlier cytogenetics study suggested a ZZ/ZW sex chromosome system in this species, we surprisingly find multiple lines of evidence that in our study population, sex is determined by an XX/XY system. Consistent with a recent evolutionary origin for this XX/XY system, the putative male-specific region of the genome is small. The genome shows evidence of Y-linked duplications of the gene encoding the androgenic gland hormone, a major component of male sexual differentiation in isopods. Our analyses also uncover sequences horizontally acquired from past Wolbachia infections, consistent with the hypothesis that Wolbachia may have interfered with the evolution of sex determination in T. rathkei. Overall, these results provide evidence for the co-occurrence of multiple sex chromosome systems within T. rathkei, further highlighting the relevance of terrestrial isopods as models for the study of sex chromosome evolution.Subject terms: Evolutionary genetics, Genome evolution     

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.     

XY FEMALES DO BETTER THAN THE XX IN THE AFRICAN PYGMY MOUSE,MUS MINUTOIDES

All therian mammals have a similar XY/XX sex‐determination system except for a dozen species. The African pygmy mouse, Mus minutoides, harbors an unconventional system in which all males are XY, and there are three types of females: the usual XX but also XX* and X*Y ones (the asterisk designates a sex‐reversal mutation on the X chromosome). The long‐term evolution of such a system is a paradox, because X*Y females are expected to face high reproductive costs (e.g., meiotic disruption and loss of unviable YY embryos), which should prevent invasion and maintenance of a sex‐reversal mutation. Hence, mechanisms for compensating for the costs could have evolved in M. minutoides. Data gathered from our laboratory colony revealed that X*Y females do compensate and even show enhanced reproductive performance in comparison to the XX and XX*; they produce significantly more offspring due to (i) a higher probability of breeding, (ii) an earlier first litter, and (iii) a larger litter size, linked to (iv) a greater ovulation rate. These findings confirm that rare conditions are needed for an atypical sex‐determination mechanism to evolve in mammals, and provide valuable insight into understanding modifications of systems with highly heteromorphic sex chromosomes.     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

Induction of female-to-male sex reversal by high temperature treatment in Medaka, Oryzias latipes

Medaka, Oryzias latipes, has a firm XX-XY sex-determining system with the sex-determining gene, DMY, on the Y chromosome. However, previous studies have suggested that high water temperature might affect sex determination in Medaka. In the present study, the influence of high water temperature on sex reversal was examined. Fertilized eggs of two inbred strains of Medaka were developed at high water temperature (32 degrees C) until hatching. The hatched fry were kept at normal water temperatures (27 degrees C) until adulthood, and the phenotypic and genotypic sex was examined. As a result, 24% (N=105) and 50% (N=36) of XX fish developed a male phenotype in the Hd-rR and HNI inbred strains, respectively. These XX sex-reversed males had a normal testis and were fully fertile. On the other hand, all XY fish were male in the both strains. These results demonstrate that high water temperatures can induce XX sex reversal and that elevated water temperatures during the embryonic stage is a simple and useful method for getting XX males in Medaka.     

Trans‐species variation in Dmrt1 is associated with sex determination in four European tree‐frog species

Empirical studies on the relative roles of occasional XY recombination versus sex‐chromosome turnover in preventing sex‐chromosome differentiation may shed light on the evolutionary forces acting on sex‐determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex‐determination systems that keep sex‐chromosome identity): both models predict X and Y alleles at sex‐linked genes to cluster by species. However, the XY‐recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex‐determining region (SDR) with trans‐species polymorphism associated to sex. Here we combine a candidate‐gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N‐terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex‐chromosome homomorphy in this clade results only from XY recombination, and take an important step toward the identification of the sex‐determining locus. Moreover, the sex‐diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.     

Novel evolutionary pathways of sex‐determining mechanisms

Evolutionary transitions between sex‐determining mechanisms (SDMs) are an enigma. Among vertebrates, individual sex (male or female) is primarily determined by either genes (genotypic sex determination, GSD) or embryonic incubation temperature (temperature‐dependent sex determination, TSD), and these mechanisms have undergone repeated evolutionary transitions. Despite this evolutionary lability, transitions from GSD (i.e. from male heterogamety, XX/XY, or female heterogamety, ZZ/ZW) to TSD are an evolutionary conundrum, as they appear to require crossing a fitness valley arising from the production of genotypes with reduced viability owing to being homogametic for degenerated sex chromosomes (YY or WW individuals). Moreover, it is unclear whether alternative (e.g. mixed) forms of sex determination can persist across evolutionary time. It has previously been suggested that transitions would be easy if temperature‐dependent sex reversal (e.g. XX male or XY female) was asymmetrical, occurring only in the homogametic sex. However, only recently has a mechanistic model of sex determination emerged that may allow such asymmetrical sex reversal. We demonstrate that selection for TSD in a realistic sex‐determining system can readily drive evolutionary transitions from GSD to TSD that do not require the production of YY or WW individuals. In XX/XY systems, sex reversal (female to male) occurs in a portion of the XX individuals only, leading to the loss of the Y allele (or chromosome) from the population as XX individuals mate with each other. The outcome is a population of XX individuals whose sex is determined by incubation temperature (TSD). Moreover, our model reveals a novel evolutionarily stable state representing a mixed‐mechanism system that has not been revealed by previous approaches. This study solves two long‐standing puzzles of the evolution of sex‐determining mechanisms by illuminating the evolutionary pathways and endpoints.     

Phenotypic/genotypic sex mismatches and temperature‐dependent sex determination in a wild population of an Old World atherinid,the cobaltcap silverside Hypoatherina tsurugae

Several New World atheriniforms have been recognized as temperature‐dependent sex determined (TSD) and yet possess a genotypic sex determinant (amhy) which is primarily functional at mid‐range temperatures. In contrast, little is known about the sex determination in Old World atheriniforms, even though such knowledge is crucial to understand the evolution of sex determination mechanisms in fishes and to model the effects of global warming and climate change on their populations. This study examined the effects of water temperature on sex determination of an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae, in which we recently described an amhy homologue. We first assessed the occurrence of phenotypic/genotypic sex mismatches in wild specimens from Tokyo Bay for three years (2014–2016) and used otolith analysis to estimate their birth dates and approximate thermal history during the presumptive period of sex determination. Phenotypic sex ratios became progressively biased towards males (47.3%–78.2%) during the period and were associated with year‐to‐year increases in the frequency of XX‐males (7.3%–52.0%) and decreases in XY/YY‐females (14.5%–0%). The breeding season had similar length but was delayed by about 1 month per year between 2014 and 2016, causing larvae to experience higher temperatures during the period of sex determination from year to year. Larval rearing experiments confirmed increased likelihood of feminization and masculinization at low and high temperatures, respectively. The results suggest that cobaltcap silverside has TSD, or more specifically the coexistence of genotypic and environmental sex determinants, and that it affects sex ratios in wild populations.     

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.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

Development of an X-specific marker and identification of YY individuals in spinach

Spinach is a popular vegetable native to central and western Asia. It is dioecious with a pair of nascent sex chromosomes. The difficulties of working with the non-recombining sex determination region of XY individuals have hindered the progress toward sequencing sex chromosomes of most dioecious species. Here we present important advances toward characterizing the non-recombining sex chromosomes in spinach. Of nearly 400 spinach accessions screened, we identified a single accession of spinach in which androdioecious XY individuals segregate YY spinach. The male and female genomes of the spinach cultivar Shami and USDA accession PI 664497 were sequenced at 12–17?× coverage. X-specific sequences were identified by comparing the depth of coverage differences between male and female alignments to a female draft genome. YY individuals were used as a negative control to validate X-specific markers found by depth of coverage analysis. Of 19 possible X chromosome sequences found by depth of coverage analysis, one was verified to be X-specific by a PCR-based marker, SpoX, which amplified genomic DNA from XX and XY, but not YY templates. Androdioecious XY individuals of accession PI 217425 (Cornell #9) were used to develop inbred lines, and at S7 generation, all XY individuals were androdioecious and all YY individuals were pure male. The sex reversal of the XY mutant to hermaphrodite is strong evidence that the sex chromosomes in spinach have a two-gene sex determination system. These results are crucial towards sequencing the X and Y chromosomes to advance sex chromosome research in spinach.     

无精和严重少精症患者的遗传缺陷分析——-附2例世界首报染色体异常核型

对217例无精和严重少精症患者外周血淋巴细胞染色体核型进行分析,并采用聚合酶链反应对7例Y染色体结构异常患者的AZFc区进行检测。发现187例无精症患者中检出异常核型77例（41.18%）（其中46,XY,t(6;14)(p21;p13),46,XY,t(8;12)(p21;q24)为世界首报核型）,主要涉及染色体异常（数目异常和结构异常）;染色体异态（Y染色体异态和9号染色体臂间倒位）及46,XX性反转;30例严重少精症患者中检出异常核型4例（13.33%）（结构异常和46,XX性反转）。由此可见,性染色体数目和结构异常是精子发生障碍的主要原因,其次常染色体的某些断裂点也可能影响精子发生。AZFc区的缺失与否与精子发生也有直接关系。     

High Temperature Increases the Masculinization Rate of the All-Female (XX) Rainbow Trout “Mal” Population

Salmonids are generally considered to have a robust genetic sex determination system with a simple male heterogamety (XX/XY). However, spontaneous masculinization of XX females has been found in a rainbow trout population of gynogenetic doubled haploid individuals. The analysis of this masculinization phenotype transmission supported the hypothesis of the involvement of a recessive mutation (termed mal). As temperature effect on sex differentiation has been reported in some salmonid species, in this study we investigated in detail the potential implication of temperature on masculinization in this XX mal-carrying population. Seven families issued from XX mal-carrying parents were exposed from the time of hatching to different rearing water temperatures ((8, 12 and 18°C), and the resulting sex-ratios were confirmed by histological analysis of both gonads. Our results demonstrate that masculinization rates are strongly increased (up to nearly two fold) at the highest temperature treatment (18°C). Interestingly, we also found clear differences between temperatures on the masculinization of the left versus the right gonads with the right gonad consistently more often masculinized than the left one at lower temperatures (8 and 12°C). However, the masculinization rate is also strongly dependent on the genetic background of the XX mal-carrying families. Thus, masculinization in XX mal-carrying rainbow trout is potentially triggered by an interaction between the temperature treatment and a complex genetic background potentially involving some part of the genetic sex differentiation regulatory cascade along with some minor sex-influencing loci. These results indicate that despite its rather strict genetic sex determinism system, rainbow trout sex differentiation can be modulated by temperature, as described in many other fish species.     

Genetics of dioecy and causal sex chromosomes in plants

Dioecy (separate male and female individuals) ensures outcrossing and is more prevalent in animals than in plants. Although it is common in bryophytes and gymnosperms, only 5% of angiosperms are dioecious. In dioecious higher plants, flowers borne on male and female individuals are, respectively deficient in functional gynoecium and roecium. Dioecy is inherited via three sex chromosome systems: XX/XY, XX/X0 and WZ/ZZ, such that XX or WZ is female and XY, X0 or ZZ are males. The XX/XY system generates the rarer XX/X0 and WZ/ZZ systems. An autosome pair begets XY chromosomes. A recessive loss-of-androecium mutation (ana) creates X chromosome and a dominant gynoecium-suppressing (GYS) mutation creates Y chromosome. The ana/ANA and gys/GYS loci are in the sex-determining region (SDR) of the XY pair. Accumulation of inversions, deleterious mutations and repeat elements, especially transposons, in the SDR of Y suppresses recombination between X and Y in SDR, making Y labile and increasingly degenerate and heteromorphic from X. Continued recombination between X and Y in their pseudoautosomal region located at the ends of chromosomal arms allows survival of the degenerated Y and of the species. Dioecy is presumably a component of the evolutionary cycle for the origin of new species. Inbred hermaphrodite species assume dioecy. Later they suffer degenerate-Y-led population regression. Cross-hybridization between such extinguishing species and heterologous species, followed by genome duplication of segregants from hybrids, give rise to new species.     

The Staurotypus Turtles and Aves Share the Same Origin of Sex Chromosomes but Evolved Different Types of Heterogametic Sex Determination

Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.