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Key Points Sex determination in vertebrates is a highly plastic process that centres on the decision within the gonad to develop as a testis or ovary. Regulation of this process has been co-opted by a diverse array of genetic and environmental upstream signals in mammals, birds, reptiles and fish. Although many of the same genes are involved, their order in the cascade is not conserved. Multiple upstream and downstream elements may feed into a threshold decision process driving a bistable outcome. A common theme is the existence of antagonistic signals that ensure canalization of one pathway or the other once a threshold bias for testis or ovary fate exists. The differentiation of an organism into a male or female phenotype is a critical developmental process, but the mechanisms that control this decision are remarkably evolutionarily labile. This Review discusses the wide diversity of vertebrate sex-determination mechanisms, their rapid evolution under different forms of genetic and environmental control and the over-arching principles that are shared despite this mechanistic diversity. The discovery of the Sry gene in 1990 triggered a revolution in our understanding of sex determination. More recently, advances in non-model organisms have been fuelled by the rapid evolution of affordable genome and transcriptome technologies. This Review considers the unusual plasticity in the bipotential system of sex determination and some of the diverse mechanisms that have evolved to control this critical developmental decision, including strong genetic pathways, environmental influences and epigenetic regulation. Ideas emerging from model and non-model organisms that suggest that sex determination operates as an antagonistic network with the emergent property of bistability are discussed.

According to the simple scenario, the presence or absence of a Y chromosome is what counts: with it, you are male, and without it, you are female. Gene mutations affecting gonad development can result in a person with XY chromosomes developing typically female characteristics, whereas alterations in hormone signalling can cause XX individuals to develop along male lines.

The first report of reptile sex reversal in the wild and rapid transition between genetic and environmental sex determination in the Australian bearded dragon ( Pogona vitticeps ) Climate-induced sex reversal There have been repeated evolutionary transitions in reptiles between genetic and temperature-dependent sex determination, the regulatory process that initiates differentiation of the gonads in the early embryo to form either testes or ovaries. Various mechanisms have been proposed to explain the transition, including a role for sex reversal. Clare Holleley et al . present the first report of reptile sex reversal in the wild, associated with rapid transition between genetic and environmental sex determination. In a study of the Australian bearded dragon ( Pogona vitticeps ), they observe sex reversal at the warmer end of the animals geographic range. When sex-reversed females mate with normal males, the chromosomal sex determination system is lost and temperature-dependent sex determination is established. It is not known whether climate-induced changes in sex determination are advantageous or detrimental to the process of evolutionary adaptation. Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination) 1 . Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles 2 . These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene–environment interactions 3 . Most mechanistic models of transitions invoke a role for sex reversal 3 , 4 , 5 . Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish 6 and rarely in amphibians 7 , 8 . Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon ( Pogona vitticeps ), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Zebrafish have emerged as a major model organism to study vertebrate reproduction due to their high fecundity and external development of eggs and embryos. The mechanisms through which zebrafish determine their sex have come under extensive investigation, as they lack a definite sex-determining chromosome and appear to have a highly complex method of sex determination. Single-gene mutagenesis has been employed to isolate the function of genes that determine zebrafish sex and regulate sex-specific differentiation, and to explore the interactions of genes that promote female or male sexual fate. In this review, we focus on recent advances in understanding of the mechanisms, including genetic and environmental factors, governing zebrafish sex development with comparisons to gene functions in other species to highlight conserved and potentially species-specific mechanisms for specifying and maintaining sexual fate.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena , and find that the locus on the Y chromosome contains a cis -regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3 Y transgenic fish, and Sox3 Y loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf , which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component. Sex chromosomes harbour specific sequences that determine the sexual development of the organism; yet these sequences remain unknown for many species. Here, Takehana et al. show that, similarly to mammals, Sox3 on the Y chromosome is the male-determining factor in the medaka-related fish Oryzias dancena .

Anti-Müllerian hormone (Amh, or Müllerian-inhibiting substance, Mis), a member of TGF-β superfamily, has been well documented in some vertebrates as initiator or key regulator in sexual development, and particularly in fish. However, its functional role has not yet been identified in reptiles. Here, we characterized the Amh gene in the Chinese soft-shelled turtle Pelodiscus sinensis, a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The messenger RNA of Amh was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited a male-specific expression pattern throughout embryogenesis. Moreover, Amh was rapidly upregulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Amh loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker Sox9, and upregulation of the ovarian regulator Cyp19a1. Conversely, overexpression of Amh in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of a testis structure, ectopic activation of Sox9, and a remarkable decline in Cyp19a1. Collectively, these findings provide the first solid evidence that Amh is both necessary and sufficient to drive testicular development in a reptilian species, P. sinensis, highlighting the significance of the TGF-β pathway in reptilian sex determination.

Background The American alligator ( Alligator mississippiensis ) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. Results Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B , and transcription factor C/EBPA . Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX . Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9 , and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. Conclusions Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

DMRT1 loss is cue for sex swap The presence or absence of the Y-chromosome gene Sry determines whether precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells in the mammalian fetus. Loss of the transcription factor FOXL2 in the adult ovary can lead to transdifferentiation of granulosa cells into Sertoli cells, but in males the sex-determining decision was thought to be stable. This study shows that this is not the case: adult mouse testicular cells become ovarian cells if the Dmrt1 gene is lost. In the absence of transcription factor DMRT1, FOXL2 is activated and Sertoli cells are reprogrammed as granulosa cells. Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry , which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells 1 . This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/β-catenin signalling 2 . In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells 2 . Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor 3 in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates 4 , 5 and Dmrt1 -related sexual regulators are conserved throughout metazoans 3 . Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1 , including disorders of sexual differentiation 6 and testicular cancer 7 .

CITATION: Valenzuela, N., et al. 2019. Extreme thermal fluctuations from climate change unexpectedly accelerate demographic collapse of vertebrates with temperature-dependent sex determination. Scientific Reports, 9:4254, doi:10.1038/s41598-019-40597-4.