Phenotypic Sex Reversal Research Articles

DAX1 and its network partners: exploring complexity in development

DAX1 encoded by NR0B1, when mutated, is responsible for X-linked adrenal hypoplasia congenita (AHC). AHC is due to failure of the adrenal cortex to develop normally and is fatal if untreated. When duplicated, this gene is associated with an XY sex-reversed phenotype. DAX1 expression is present during development of the steroidogenic hypothalamic–pituitary–adrenal–gonadal (HPAG) axis and persists into adult life. Despite recognition of the crucial role for DAX1, its function remains largely undefined. The phenotypes of patients and animal models are complex and not always in agreement. Investigations using cell lines have proved difficult to interpret, possibly reflecting cell line choices and their limited characterization. We will review the efforts of our group and others to identify appropriate cell lines for optimizing ex vivo analysis of NR0B1 function throughout development. We will examine the role of DAX1 and its network partners in development of the hypothalamic–pituitary–adrenal/gonadal axis (HPAG) using a variety of different types of investigations, including those in model organisms. This network analysis will help us to understand normal and abnormal development of the HPAG. In addition, these studies permit identification of candidate genes for human inborn errors of HPAG development.

Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad.

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.

Successful Sex Reversal of Brook Trout with 17α‐Methyldihydrotestosterone Treatments

AbstractMonosex female stocks are widely used in the commercial production of rainbow trout Oncorhynchus mykiss. The potential for commercial production of brook trout Salvelinus fontinalis, however, is constrained by the lack of published protocols for producing the sex‐reversed males required to create monosex female stocks. Immersion and immersion plus feeding treatments with 17α‐methyltestosterone (MT) and 17α‐methyldihydrotestosterone (MDHT) were applied to genotypically female gynogenetic brook trout to induce phenotypic sex reversal. The fry were exposed to a 6‐h immersion in a solution of MT or MDHT on day 10 following completion of hatch and/or to a steroid‐treated diet for 60 d beginning at first feeding. Immersion dosages were 0.5 or 1.0 mg/L, and feeding dosages were 1.0 or 2.0 mg/kg of feed for MT and 0.5 or 1.0 mg/kg for MDHT. Phenotypic sex of the fish was determined 19 or 22 months after first feeding. Control gynogenetic fish were 100% phenotypic females. Treatments with MT had minimal effect: most fish remained female, with only a low incidence of phenotypic males (1–3% in four of the treatments), intersex fish, or sterile fish. In contrast, a substantial number of phenotypic males were observed in several of the MDHT treatments, with the highest proportion (45%) occurring in the 0.5 mg/L immersion plus 0.5 mg/kg feeding treatment. Sperm was obtained from 29 males from five MDHT treatment groups and one MT treatment group examined at maturity (22 months) and was used in progeny tests of these males. The progeny were 100% female, confirming the male parents to be genotypically female. These protocols may be used to create sex‐reversed brook trout males for the production of monosex female progeny, although additional trials are ongoing to test similar MDHT immersion dosages applied once or multiple times, with or without feeding treatments, to identify protocols with increased efficacy.

DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation.

The unusual orphan member of the nuclear hormone receptor superfamily DAX-1 (NR0B1) owes its name to its double role in human pathology. On one side, duplications in Xp21, containing the DAX-1 gene, cause phenotypic sex reversal in XY individuals. On the other side, DAX-1 gene mutations are responsible for adrenal hypoplasia congenita, invariably associated with hypogonadotropic hypogonadism. DAX-1 functions as a global negative regulator of steroid hormone production by repressing the expression of multiple genes involved in the steroidogenic pathway. Here we review the mechanism of DAX-1 function in adrenal and gonadal differentiation, with special emphasis on recent results showing the critical role of DAX-1 protein misfolding in the pathogenesis of adrenal hypoplasia congenita.

Gonadal differentiation and effects of temperature on sex determination in southern flounder ( Paralichthys lethostigma)

Southern flounder ( Paralichthys lethostigma) support valuable North American fisheries and show great promise for aquaculture. Because females grow faster and reach larger adult sizes than males, monosex culture of females is desirable for commercial operations. A detailed understanding of sexual development and its timing is critical to control sex and optimize culture. Structural and cellular sex-distinguishing markers were identified histologically, and then used to describe ovarian development in female and testicular development in male flounder. In presumptive ovaries of southern flounder, development of an ovarian cavity first occurs in fish ranging from 75 to 100 mm total length (TL). This is considerably delayed relative to that observed in the Japanese congener, Paralichthys olivaceus, where an ovarian cavity is seen in fish as small as 40 mm TL. The smallest southern flounder that possessed primary oocytes in the early perinucleolus stage was 115 mm TL. In presumptive testes, the formation of seminiferous tubules first occurs in fish of approximately 100 mm TL. Spermatogonia remained quiescent until most fish were over 100 mm TL. Overall, gonads from southern flounder greater than 120 mm TL commonly possess gonial cells undergoing meiosis, clearly differentiating sex. The effect of temperature on sex determination in southern flounder was addressed in a separate experiment. Juvenile southern flounder were grown at 18, 23, or 28°C for 245 days. High and low temperatures induced phenotypic sex reversal in juvenile southern flounder, producing a higher proportion of males (96% males at high temperature, P<0.001, 78% males at low temperature, P<0.01). Raising southern flounder at the midrange temperature held sex ratios close to 1:1. Sex ratios from these trials suggest that southern flounder possess a temperature-sensitive mechanism of sex determination similar to that shown for P. olivaceus, but possibly shifted towards warmer temperatures. These findings indicate that sex differentiation in southern flounder is distinguishable in most fish by 100–120 mm TL and that sex determination is sensitive to temperature. This information is critical to the development of strategies to maximize the number of faster-growing females for commercial flounder culture.

A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination.

In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.

Up-Regulation of WNT-4 Signaling and Dosage-Sensitive Sex Reversal in Humans

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

Two unbalanced translocations involving a common 6p25 region in two XY female patients

We report two 46,XY female patients with two different de novo unbalanced translocations, each involving the chromosomal region 6p25. The patient with a 46,XY,der(6)t(X;6)(p21.2;p25) karyotype had a sex reversal phenotype. The patient with a 46,XY,der(13)t(6;13)(p25;q33) karyotype had a male pseudohermaphrodite phenotype. Multi-paint fluorescent in situ hybridization was performed to determine the origin of the derivative material on 6p and 13q. The association of abnormalities of the 6p25 region with either an Xp duplication or a 13q deletion is reported here for the first time.

High Incidence of a Male-Specific Genetic Marker in Phenotypic Female Chinook Salmon from the Columbia River

Numerous populations of anadromous salmonids in the northwestern United States have been declining for many years, resulting in Endangered Species Act listings and in some cases extinction. The degradation of river ecosystems has been proposed as one of the major reasons for the inability of salmon to maintain their populations. However, the specific factors interfering with the reproduction and survival of salmon during the freshwater phase of their life cycle have not been fully described. This study was initiated to determine the incidence of phenotypic sex reversal in wild, fall chinook salmon (Oncorhynchus tshawytcha) that returned to spawn in the Columbia River. Fish were sampled at different locations within this watershed to determine whether they were faithfully expressing their genotype. We report a high incidence (84%) of a genetic marker for the Y chromosome in phenotypic females sampled from the wild, which was not observed in female fish raised in hatcheries. It appears likely that female salmon with a male genotype have been sex reversed, creating the potential for an abnormal YY genotype in the wild that would produce all-male offspring and alter sex ratios significantly.

High incidence of a male-specific genetic marker in phenotypic female chinook salmon from the Columbia River.

Numerous populations of anadromous salmonids in the northwestern United States have been declining for many years, resulting in Endangered Species Act listings and in some cases extinction. The degradation of river ecosystems has been proposed as one of the major reasons for the inability of salmon to maintain their populations. However, the specific factors interfering with the reproduction and survival of salmon during the freshwater phase of their life cycle have not been fully described. This study was initiated to determine the incidence of phenotypic sex reversal in wild, fall chinook salmon (Oncorhynchus tshawytcha) that returned to spawn in the Columbia River. Fish were sampled at different locations within this watershed to determine whether they were faithfully expressing their genotype. We report a high incidence (84%) of a genetic marker for the Y chromosome in phenotypic females sampled from the wild, which was not observed in female fish raised in hatcheries. It appears likely that female salmon with a male genotype have been sex reversed, creating the potential for an abnormal YY genotype in the wild that would produce all-male offspring and alter sex ratios significantly.

Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus).

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex-reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.

SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated ( SRY, SOX9) or over-expressed ( DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.

An in vivo model fish system to test chemical effects on sexual differentiation and development: exposure to ethinyl estradiol

A model system was characterized which may be used as an in vivo screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE 2), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5–2.5 ng EE 2/egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as 7 days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE 2-exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No instances of gonadal intersexes were observed. Ethinyl estradiol also appeared to reduce growth but not condition (weight-at-length) and exposed XX females appeared to have a higher incidence of atretic follicles relative to controls. Our results suggest that estrogenic chemicals may influence sexual differentiation and development and that the medaka model is well suited to assessing these effects.

Expression of steroidogenic factor 1 and Wilms' tumour 1 during early human gonadal development and sex determination

The transcription factors SF-1 and WT1 play pivotal roles in mammalian gonadal development and sexual differentiation. In human embryos, both SF-1 and WT1 are expressed when the indifferent gonadal ridge first forms at 32 days post-ovulation. As the sex cords develop – providing morphological evidence of testis differentiation – SF-1 localises predominantly to developing Sertoli cells in the sex cords, whereas WT1 retains a broader pattern of expression. Later, SF-1 localises predominantly to steroidogenic Leydig cells, and WT1 localises to the sex cords. In the ovary, SF-1 and WT1 transcripts persist in the gonadal ridge from the earliest developmental stages throughout the critical period of sex determination. These studies, which delineate for the first time the sequential expression profiles of SF-1 and WT1 during human gonadal development, provide a framework for understanding human sex reversal phenotypes associated with their mutations.

Ineffectiveness of Steroid Immersion Treatments for Sex Reversal of Brook Trout

Abstract Immersion applications of androgenic steroids to sac fry of brook trout Salvelinus fontinalis were ineffective in inducing phenotypic sex reversal. Four experiments were conducted to test the effects of paired 2-h immersions in solutions of 17α-methyldihydrotestosterone (MDHT) at 1 or 3 mg/L and applied at times ranging from 1 to 25 d after completion of hatch. In a fifth experiment, similar tests were conducted with 0.4 mg/L solutions of MDHT, 17α-methyltestosterone, and trenbolone acetate. The experimental fish were of two types: normal mixed-sex fish and all-female gynogenetic fish. Phenotypic sex was determined by visual examination of the gonads when fish were sacrificed 6–8 months after initiation of feeding. In no experiment was there a significant increase in the percentage of males or a significant incidence of intersex gonads among treated fish. A generally low level of sterile and partially sterile fish was observed among control fish of both types, and these percentages tended to incr...

DAX-1, an 'antitestis' gene.

The DAX-1 gene has been involved in the dosage sensitive sex reversal (DSS) phenotype, a male-to-female sex-reversal syndrome due to the duplication of a small region of human chromosome Xp21. Dax-1 and Sry have been shown to act antagonistically in the mouse system, where increasing expression of the former leads to female development and increasing activity of the latter to male development. Although these data strongly implicate DAX-1 in sex determination, the mouse and human proteins appear to behave differently. Absence of DAX-1 is responsible for adrenal hypoplasia congenita, a human inherited disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Unlike human patients, Dax-1-deficient XY mice have normal levels of corticotropins and adrenal hormones but are sterile. Dax-1-deficient females are fertile. The DAX-1 protein, an unusual member of the nuclear hormone receptor, may act as a transcriptional repressor. It has been shown to both repress transcriptional activators by direct protein-protein interactions and to bind DNA hairpin structures and repress target genes.

Mammalian sex determination: a molecular drama.

Our knowledge of mammalian sex determination is based on two main areas of study. First, the characterization of the biological events that determine the sexual development of the individual, including patterns of gene expression, and second, the study of genetic mutations in humans and mice that lead to abnormal sexual phenotypes. In the search for molecular components of this process, the identification of genes in loci involved in human disease has been especially fruitful. Serendipity has also played a hand, where in several cases targeted mutations in mouse genes, being studied for other reasons, have led to unexpected sex reversal phenotypes. The collection of molecular candidates implicated in sex determination is now quite extensive. We are not able to fit all of these into simple pathways, where one gene acts on the next and so on in a linear fashion, as seems possible in the invertebrate model organisms, Caenorhabditis elegans and Drosophila. In part this is due to gaps in our knowledge, as we are clearly missing several key components, but it is looking increasingly likely that the system is much better described as a network of factors. In fact, the story so far is like some partly recovered script for a play. Thus some gene products are main characters with roles at several different stages, some act as a chorus, in a combinatorial fashion with others, whereas a few play a critical role in one scene and then disappear. The mechanism presumably evolved to be delicately poised to respond to the initial trigger to be male or female and then to amplify this decision while avoiding development of intersex phenotypes. It is therefore likely to be a system full of back-ups and functional redundancy. The complexity may also follow from the relatively late embryonic stage at which the decision is reached. This means that events occurring in one cell lineage have to be coordinated with others in the context of a developing organ and eventually the whole organism. We review here the story that is beginning to emerge, focusing mostly on the set of transcription factors that appear to play important leading and supporting roles. In mammals, the genetic sex of the embryo is established at fertilization with the inheritance of an X or Y chromosome from the father. However, the sex-determining process is set in motion only during the period of organogenesis when the gonads develop. The Y chromosome, through the testis-determining gene Sry, acts dominantly to trigger differentiation of testes from the indifferent gonads (or genital ridges) that would otherwise develop as ovaries (Gubbay et al. 1990; Sinclair et al. 1990). Sry is the only gene needed from the Y chromosome to establish male development, as shown by transgenic experiments where XX mice carrying the Sry gene develop as males (Koopman et al. 1991). Conversely, mutations in Sry can lead to relatively normal female development of XY mice and humans (Gubbay et al. 1992; Hawkins et al. 1992). Once the gonads begin to differentiate as testes or ovaries, they secrete factors, notably anti-Mullerian hormone (AMH, otherwise known as Mullerian-inhibiting substance, or MIS) and testosterone from the testes, which determine the sexual development of the rest of the embryo. There are many steps in this process that, when affected, will give rise to different degrees of sex reversal. The challenge has been to correlate the phenotype of affected individuals with the appropriate step and translate this into a molecular mechanism.

A Gene Involved in XY Sex Reversal Is Located on Chromosome 9, Distal to Marker D9S1779

The genetic mechanisms involved in sex differentiation are poorly understood, and progress in identification of the genes involved has been slow. The fortuitous finding of chromosomal rearrangements in association with a sex-reversed phenotype has led to the isolation of SRY and SOX9, both shown to be involved in the sex-determining pathway. In addition, duplications of the X chromosome, deletions of chromosomes 9 and 10, and translocations involving chromosome 17 have been reported to be associated with abnormal testicular differentiation, leading to male-to-female sex reversal in 46,XY individuals. We present the cytogenetic and molecular analyses of four sex-reversed XY females, each with gonadal dysgenesis and other variable malformations, and with terminal deletions of distal chromosome 9p, resulting from unbalanced autosomal translocations. PCR amplification and DNA sequence analysis of SRY revealed no mutations in the high-mobility-group domain (i.e., HMG box) in any of the four patients. Conventional and molecular cytogenetic analyses of metaphase chromosomes from each patient suggest that the smallest region of overlap (SRO) of deletions involves a very small region of distal band 9p24. Loss-of-heterozygosity studies using 17 highly polymorphic microsatellite markers, as well as FISH using YAC clones corresponding to the most distal markers on 9p, showed that the SRO lies distal to marker D9S1779. These results significantly narrow the putative sex-determining gene to the very terminal region of the short arm of chromosome 9.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Evidence to exclude SOX9 as a candidate gene for XY sex reversal without skeletal malformation.

The skeletal malformation syndrome campomelic dysplasia (CMD1) is caused by mutations within the SOX9 gene or chromosomal rearrangement breakpoints outside SOX9. Approximately three quarters of cases of CMD1 in XY...