Mammalian Sex-determining Gene Research Articles

Two rather than one

Sex Determination For several decades, it has been believed that the mammalian sex-determining gene Sry contains a single exon. Miyawaki et al. have now identified a cryptic second exon of mouse Sry . Loss- and gain-of-function analyses revealed that the two-exon SRY (SRY-T), not the canonical single exon–encoded SRY (SRY-S), is the bona fide testis-determining factor. Sry exon2 is composed of retrotransposon-derived sequences. The SRY-S carboxyl terminus contains a degradation sequence (degron), whereas the SRY-T carboxyl terminus encoded in the Sry exon2 is degron free, thereby conferring protein stability on SRY-T. Science , this issue p. [121][1] [1]: /lookup/doi/10.1126/science.abb6430

Role of epigenetic regulation in mammalian sex determination.

Mammalian sex determination is triggered by activation of the mammalian sex-determining gene, Sry, in a spatially and temporally controlled manner. Because reduced or delayed Sry expression results in male-to-female sex reversal, male development is highly dependent on the accurate transcription of Sry. SRY dysregulation is a potential cause of human disorders of sex development (DSD). In addition to changes in DNA sequences, gene expression is regulated by epigenetic mechanisms. Epigenetic regulation ensures spatial and temporal accuracy of the expression of developmentally regulated genes. Epigenetic regulation such as histone tail modification, DNA methylation, chromatin remodeling, and non-coding RNA regulation engages several biological processes in multicellular organisms. In recent years, it has been revealed that various types of epigenetic regulation are involved in accurate gonadal differentiation in mammals. In particular, histone modification plays an integral part in sex determination, which is the first step of gonadal differentiation. Here, we focus on the findings on the epigenetic modifications that regulate Sry expression. Finally, we discuss the role of metabolism that potentially alters the epigenetic state in response to environmental cues.

Of sex and determination: marking 25 years of Randy, the sex-reversed mouse.

On Thursday 9 May 1991, the world awoke to front-page news of a breakthrough in biological research. From Washington to Wollongong, newspapers, radio and TV were abuzz with the story of a transgenic mouse in London called Randy. Why was this mouse so special? The mouse in question was a chromosomal female (XX) made male by the presence of a transgene containing the Y chromosome gene Sry This sex-reversal provided clear experimental proof that Sry was the elusive mammalian sex-determining gene. Twenty-five years on, we reflect on what this discovery meant for our understanding of how males and females arise and what remains to be understood.

Evolution of the sex-determining gene in the teleostean genus Oryzias

In the genetic sex determination of vertebrates, the gonadal sex depends on the combination of sex chromosomes that a zygote possesses. Despite the discovery of the sex-determining gene (SRY/Sry) in mammals in 1990s, the sex-determining gene in non-mammalian vertebrates remained an enigma for over a decade. In most mammals, the male-inducing master sex-determining gene is located on the Y chromosome and is therefore absent from XX females. A second sex-determining gene, Dmy, was described in the Oryzias latipes in 2002 and has a DNA-binding motif that is different from the motif in the mammalian sex-determining gene SRY or Sry. Dmy is also located on the Y chromosome and is therefore absent in XX females. Seven other sex-determining genes, including candidate genes, are now known in birds, a frog species, and 5 fish species. These findings over the past twenty years have increased our knowledge of sex-determining genes and sex chromosomes among vertebrates. Here, we review recent advances in our understanding of sex-determining genes and genetic sex determination systems in fish, especially those of the Oryzias species, which are described in detail. The facts suggest some patterns of how new sex-determining genes emerged and evolved. We believe that these facts are common not only in Oryzias but also in other fish species. This knowledge will help to elucidate the conserved mechanisms from which various sex-determining mechanisms have evolved.

Pronuclear microinjection is not suitable for RNA polymerase III promoter driven constitutive RNAi transgenesis in mice for XY male-to-female sex reversal by <i>Sry</i> gene knockdown

Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.

SRY protein function in sex determination: thinking outside the box

Even though the mammalian sex-determining gene Sry has been intensively studied for the two decades since its discovery, the regions outside the conserved HMG box DNA-binding domain have received less attention due to a lack of sequence conservation and of obvious structural/functional motifs. Here, we summarize the available evidence for function beyond the HMG box, identify the known and postulated biochemical functions of the non-HMG-box domains in sex determination, and present possible explanations for the puzzling diversity of these non-HMG-box domains.

Additional copies of CBX2 in the genomes of males of mammals lacking SRY, the Amami spiny rat (Tokudaia osimensis) and the Tokunoshima spiny rat (Tokudaia tokunoshimensis)

Tokudaia osimensis (the Amami spiny rat) and Tokudaia tokunoshimensis (the Tokunoshima spiny rat) have a sex chromosome composition of XO/XO, no Y chromosome. The mammalian sex-determining gene, SRY, is also absent in these species, which indicates that these spiny rats exhibit a novel sex-determining mechanism that is independent of SRY. To identify a candidate gene that controls this mechanism, the copy numbers and chromosomal locations of 10 genes with important functions in gonadal differentiation were determined: ATRX, CBX2 (M33), DMRT1, FGF9, NR0B1 (DAX1), NR5A1 (Ad4BP/SF1), RSPO1, SOX9, WNT4, and WT1. Multiple bands were detected for NR0B1 in Southern blot analysis, which suggested the presence of multiple copies of the gene in the genomes of these two species. CBX2 was localized to two loci in both sexes of the two species by fluorescence in situ hybridization mapping: 3q24 and 6p11.2 in T. osimensis and 10q25-q26 and 14q12-q13.1 in T. tokunoshimensis. Quantification of copy numbers in the two species by quantitative real-time PCR indicated that there were two or three more copies of CBX2 per haploid genome in males (T. osimensis, n = 3; T. tokunoshimensis, n = 2) than in females (T. osimensis, n = 4; T. tokunoshimensis, n = 2), whereas NR0B1 was present as a single copy in both. The results suggest that additional copies of CBX2 in males might be involved in a novel sex-determining mechanism in species that lack SRY.

Multiple copies of SRY on the large Y chromosome of the Okinawa spiny rat, Tokudaia muenninki

The Okinawa spiny rat, Tokudaia muenninki, is the only species with a Y chromosome in the genus Tokudaia. Its phylogenic relationship with two XO/XO species, Tokudaia osimensis and Tokudaia tokunoshimensis, lacking a Y chromosome and the mammalian sex-determining gene SRY, is unknown. Furthermore, there has been little cytogenetic analysis of the sex chromosomes in T. muenninki. Therefore, we constructed molecular phylogenetic trees with nucleotide sequences of cyt b, RAG1, and IRBP. All trees strongly supported that T. muenninki was the first to diverge from the Tokudaia ancestor, indicating that loss of the Y chromosome and SRY occurred in the common ancestor of the two XO/XO species after T. muenninki diverged. We found that the X and Y chromosomes of T. muenninki consisted of large euchromatic and heterochromatic regions by conducting G- and C-banding analyses. PCR, Southern blotting, and FISH revealed that T. muenninki males had multiple SRY copies on the long arm of the Y chromosome. At least three of 24 SRY sequences contained a complete open reading frame (ORF). A species-specific substitution from alanine to serine was found in all copies at the DNA-binding surface within the HMG-box, suggesting that it occurred in an original SRY.

The process of a Y-loss event in an XO/XO mammal, the Ryukyu spiny rat

The Ryukyu spiny rat, Tokudaia osimensis, has an XO/XO sex chromosome constitution, lacking a Y chromosome and the mammalian sex-determining gene SRY. To investigate the Y-loss event, we traced three proto-Y-linked genes, RBMY1A1, EIF2S3Y, and KDM5D, in the genome. The original Y-linked RBMY1A1 was lost as well as SRY, and the remaining RBMY1A1 was a processed pseudogene on autosome. In contrast, EIF2S3Y and KDM5D were conserved in genomes of both sexes as a result of their translocation from the Y chromosome to the X chromosome and/or autosomes. Furthermore, these genes were expressed in gonads and brains of both sexes. Our study indicated a loss of Y-linked genes with important male functions to be necessary for the Y chromosome to disappear. These functions might have been retained through the acquisition of new genes, and therefore, the Y-loss has had no harmful effect on the maintenance of this species.

Ubiquitous expression of Sry induces embryonic lethality related to suppression of Tie2/Tek expression

Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. The importance of the strict regulation of Sry expression remains unknown. Thus, we attempted to produce a Sry ubiquitous-expressing transgenic (Tg) mouse in which foreign Sry is driven by the CAG (cytomegalovirus immediate-early enhancer, chicken beta-actin promoter, and the fusion intron of chicken beta-actin and rabbit beta-globin)-Sry gene for ubiquitous expressing Sry. A low rate (2/127) of Tg pups was observed, whereas the rate of early-stage transgenic embryos before birth was 19.2% (5/26). The Sry ubiquitous-expressing embryos showed abnormal development. The results suggest that ubiquitous expression of Sry exerts a negative effect on embryonic development. One of the two adult Tg mice showed low levels of Sry expression. The other Tg mouse showed high Sry transgene expression, but was mosaic for the transgene. Developmental analysis of transgenic F1 embryos produced from the mosaic Tg mouse revealed that ubiquitous expression of Sry had a lethal effect on embryonic development around 12.5 dpc. The histological data indicated that ubiquitous expression of Sry induced abnormal cardiovascular development, resulting in embryonic death. Enhanced expression of Sry suppressed endogenous Tie2/Tek (tyrosine kinase with Ig and EGF homology domains 2/tunica interna endothelial cell kinase) expression in Sry-transfected primary cultured cells from wild type embryonic hearts. The results indicate that the tissue-specific and stage-specific expression of Sry is essential for normal embryogenesis.

SRY and the Standoff in Sex Determination

SRY was identified as the mammalian sex-determining gene more than 15 yr ago and has been extensively studied since. Although many of the pathways regulating sexual differentiation have been elucidated, direct downstream targets of SRY are still unclear, making a top down approach difficult. However, recent work has demonstrated that the fate of the gonad is actively contested by both male-promoting and female-promoting signals. Sox9 and Fgf9 push gonads towards testis differentiation. These two genes are opposed by Wnt4, and possibly RSPO1, which push gonads toward ovary differentiation. In this review, we will discuss the history of the field, current findings, and exciting new directions in vertebrate sex determination.

Sugar and spice or slugs and snails?

Over the past 500 million years or so, metazoans have evolved a bewildering diversity of genetic mechanisms for generating that most interesting of phenotypes — the difference between girls and boys. Despite intense effort, a conserved sex-determination effector gene has proven elusive. Until recently, the evidence indicated that completely different molecular mechanisms and proteins regulate sexual dimorphism in different phyla; sex-specific RNA splicing in flies, a signal transduction cascade in worms and a male-determining DNA-binding protein in mammals. Could it be that the quest is now over and a single unifying gene is at the bottom of it all?Recent evidence from studies on a class of transcription factors known as DM-domain proteins has implicated a common effector in the regulation of male-specific characteristics in flies, nematodes, reptiles, birds and mammals. Mutations in the Drosophila doublesex (dsx) and Caenorhabditis elegans mab-3 genes lead to defects in a variety of male-specific processes. As well as being functionally interchangeable, the genes regulate similar male-specific biological processes, such as repressing yolk-protein synthesis and eliciting the development of a sex-specific nervous system. Furthermore, a vertebrate DM-domain gene, DMRT1, which is associated with a small interval of chromosome 9 required for human testis development, exhibits male-specific expression in the gonads of chickens and alligators. This indicates that diverged sex-determining mechanisms might converge at a common effector gene that functions to promote male development.Raymond et al. 1xDmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Raymond, C.S. et al. Genes Dev. 2000; 14: 2587–2595Crossref | PubMed | Scopus (374)See all References1 have examined this possibility by generating mice with mutations in Dmrt1. They report that, whereas female development is normal in mouse Dmrt1 knockouts, the differentiation of the testis is obviously not. Testis differentiation is the key event in mammalian sex determination, in particular the formation of a somatic support-cell lineage known as Sertoli cells. It is thought that the mammalian sex-determining gene Sry acts to promote the differentiation of Sertoli cells, and that these influence the subsequent events in testis differentiation. In Dmrt1-mutant males, the testes appear normal at birth, but by the onset of germ-cell meiosis a few days later, abnormalities are apparent. After two weeks, the mutant testes are filled with immature Sertoli cells, a phenotype resembling that observed in human chromosome 9-deficient males.Thus, it appears that a DM-domain gene functions as a downstream effector in vertebrate sexual differentiation in a way analogous to that observed in invertebrates. Interestingly, the chromosome 9 interval associated with DMRT1 in humans contains at least two other DM-domain genes. The situation in the mouse is not clear, but it could be that multiple DM-domain genes have evolved in mammals to ensure correct male differentiation. Clearly, a functional analysis of DMRT1 in other organisms will go a long way to confirm whether the evolution of sexual dimorphism has, after all, been conserved.

From brain determination to testis determination: evolution of the mammalian sex-determining gene.

In mammals, sex is determined by an XY male:XX female sex chromosome system in which a male-dominant gene on the Y chromosome (SRY) determines testis formation. Sex chromosomes evolved from an ordinary autosome pair as the Y chromosome was progressively degraded. The Y chromosome has lost nearly all of its 1500 original genes, and those that survived did so because they evolved a critical role in male determination or differentiation. SRY is typical of Y-borne genes. Comparative gene mapping and sequencing shows that SRY arose quite recently as a degraded version of the SOX3 gene on the X chromosome. SOX3 is expressed predominantly in brain, and so is more likely to be a brain-determining than a testis-determining gene. The male-dominant action of SRYmay be an illusion, as its structure suggests that it works by interfering with the action of a related gene, which in turn inhibits testis development. This hypothesis can give a good account of how a brain-determining gene acquired a role in testis determination via differential dosage of SOX3. SRYhas no central role in sex determination and it can be replaced as a trigger and loft, as have many other Y-borne genes in recent evolutionary history. The absence of SRY in two species of the mole vole (Ellobius) suggests that its useful life is already running out.

Whatever happened to SRY?

The mammalian sex-determining gene, SRY, was identified by positional cloning approximately 10 years ago. Since its discovery, intense research into this gene has been directed on two main fronts: elucidation of its function in development of the testis and examination of its singular evolutionary history. The role or SRY as the testis-determining factor (TDF) places it at a crucial point in the highly conserved morphogenetic process of vertebrate gonadogenesis. None of the genes that directly activate SRY nor any of its immediate downstream targets have yet been positively identified. Several genes, however, such as SF1, DAX1, and SOX9, whose spatial and temporal expression profiles overlap with that of SRY, are strongly implicated as co-regulators of gonadogenesis. Molecular genetic manipulation of these genes in mice has shown that they are indispensable to sexual development. Remarkably, its key position in this cascade of gene action has not protected SRY from strong yet poorly understood selective forces that have caused it to evolve rapidly in mammals. The evolution of SRY has been characterized not only by rapid sequence divergence within mammals, but also by structural changes such as intron insertion, gene amplification, and deletion.

HMG box containing transcription factors in lymphocyte differentiation

The identification of the mammalian sex-determining gene Sry has led to the discovery of a large family of related (`HMG box') transcription factors that control developmental events in yeast, C. elegans, Drosophila and vertebrates. In lymphocyte differentiation, several HMG box proteins play a decisive role. Sox-4 is important for very early B-cell differentiation, while TCF-1/LEF-1 play a crucial role in early thymocyte development. TCF/LEF proteins have recently been found to constitute a downstream component of the Wingless/Wnt signal transduction pathway. In flies, this pathway controls segment polarity; in Xenopus it controls the definition of the body axis. Deregulation of the pathway occurs in several human tumors. These insights in the molecular events that are involved in TCF/LEF function in these organisms may eventually lead to the understanding of the function of these HMG box proteins in lymphoid development.

The Schizosaccharomyces pombe mating-type gene mat-Mc encodes a sequence-specific DNA-binding high mobility group box protein.

The Schizosaccharomyces pombe gene mat-Mc plays a determinative role in the sexual differentiation of the fission yeast. The mat-Mc protein has been suggested to belong to a novel family of so-called high mobility group (HMG) box proteins, characterized by homology to high mobility group-1 and -2 proteins. Several HMG box proteins, including the mammalian sex-determining gene product SRY and the lymphoid transcription factors TCF-1 and LEF-1, have been shown to bind to DNA in a sequence-specific fashion. To analyze possible DNA-binding properties of mat-Mc, we have cloned and expressed its putative HMG box in Escherichia coli. Gel retardation analysis revealed that the mat-Mc HMG box recognizes the AACAAAG heptamer in a sequence-specific fashion. Combined T-->C and A-->I substitutions on both strands of the AACAAAG heptamer, which change the surface of the major groove while leaving the minor groove intact, did not interfere with sequence-specific binding of mat-Mc. Methylation interference analysis confirmed that the mat-Mc HMG box contacts adenine residues in the minor groove. By using a circular permutation assay, the mat-Mc HMG box was observed to bend DNA. These results indicate that mat-Mc is indeed a member of the HMG box family with DNA-binding characteristics assigned earlier to other members of this novel transcription factor family.

Rapid sequence evolution of the mammalian sex-determining gene SRY: Nature 364, 713–715

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.

Rapid sequence evolution of the mammalian sex-determining gene SRY.

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.

Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.

The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box.

CD3-epsilon expression is controlled by a downstream T lymphocyte-specific enhancer element. We report the identification of a T cell-specific transcription factor, TCF-1, binding to this element. The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer. Subsequent cloning of TCF-1 identified three splice alternatives. TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF-1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non-T cells, TCF-1 could transactivate through its cognate motif. These results identify TCF-1 as a T cell-specific transcription factor, which might play a role in the establishment of the mature T cell phenotype.