Somatic Sex Research Articles

Molecular evolution of a sex determination protein. FEM-2 (pp2c) in Caenorhabditis.

Somatic sex determination in Caenorhabditis elegans involves a signal transduction pathway linking a membrane receptor to a transcription factor. The fem-2 gene is central to this pathway, producing a protein phosphatase (FEM-2) of the type 2C (PP2C). FEM-2 contains a long amino terminus that is absent in canonical PP2C enzymes. The function of this domain is difficult to predict, since it shows no sequence similarity to any other known proteins or motifs. Here we report the cloning of the fem-2 homologue from Caenorhabditis briggsae (Cb-fem-2). The sequence identity is much higher than that observed for other C. briggsae homologues of C. elegans sex determination proteins. However, this level is not uniform across the entire lengths of the proteins; it is much lower in the amino termini. Thus, the two domains of the same protein are evolving at different rates, suggesting that they have different functional constraints. Consistent with this, Cb-FEM-2 is able to replace some, but not all, of the Ce-FEM-2 in vivo function. We show that removal of the amino terminus from Ce-FEM-2 has no effect on its in vitro phosphatase activity, or its ability to replace the in vivo function of a yeast PP2C enzyme, but that it is necessary for proper FEM-2 function in worms. This demonstrates that the amino terminus is not an extended catalytic domain or a direct negative regulator of phosphatase activity.

Sex-lethal, the master sex-determining gene in Drosophila, is not sex-specifically regulated in Musca domestica.

Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila melanogaster. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, we isolated the homologous Sxl gene in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. Similar results were obtained with the Sxl homologue of Ceratitis capitata (Saccone, G., Peluso, I., Artiaco, D. , Giodano, E., Bopp, D. and Polito, L. C. (1998) Development 125, 1495-1500) suggesting that, in these non-drosophilid species, Sxl performs a function different from that in sex determination.

Regulation of sexual dimorphism in mammals.

Sexual dimorphism in humans has been the subject of wonder for centuries. In 355 BC, Aristotle postulated that sexual dimorphism arose from differences in the heat of semen at the time of copulation. In his scheme, hot semen generated males, whereas cold semen made females (Jacquart, D., and C. Thomasset. Sexuality and Medicine in the Middle Ages, 1988). In medieval times, there was great controversy about the existence of a female pope, who may have in fact had an intersex phenotype (New, M. I., and E. S. Kitzinger. J. Clin. Endocrinol. Metab. 76: 3-13, 1993.). Recent years have seen a resurgence of interest in mechanisms controlling sexual differentiation in mammals. Sex differentiation relies on establishment of chromosomal sex at fertilization, followed by the differentiation of gonads, and ultimately the establishment of phenotypic sex in its final form at puberty. Each event in sex determination depends on the preceding event, and normally, chromosomal, gonadal, and somatic sex all agree. There are, however, instances where chromosomal, gonadal, or somatic sex do not agree, and sexual differentiation is ambiguous, with male and female characteristics combined in a single individual. In humans, well-characterized patients are 46, XY women who have the syndrome of pure gonadal dysgenesis, and a subset of true hermaphrodites are phenotypic men with a 46, XX karyotype. Analysis of such individuals has permitted identification of some of the molecules involved in sex determination, including SRY (sex-determining region Y gene), which is a Y chromosomal gene fulfilling the genetic and conceptual requirements of a testis-determining factor. The purpose of this review is to summarize the molecular basis for syndromes of sexual ambiguity seen in human patients and to identify areas where further research is needed. Understanding how sex-specific gene activity is orchestrated may provide insight into the molecular basis of other cell fate decisions during development which, in turn, may lead to an understanding of aberrant cell fate decisions made in patients with birth defects and during neoplastic change.

Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination.

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL is maintained by a positive autoregulatory feedback loop in which Sxl protein insures its continued synthesis by binding to Sxl pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. We also show that female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu which control female-specific processes in the ovary.

Sex determination and sexual differentiation

In humans and other mammals, sex is determined by the presence or absence of Y chromosome. If Y chromosome is present, it will channel the genital ridge of the embryo into the pathway of testis development, while in the absence of Y chromosome ovaries develop. Once testes have formed, they secrete anti-MA¼llerian hormone and testosterone, which masculinize the reproductive tract. By contrast, the female reproductive tract develops in the absence of fetal gonadal hormones. Testis development is brought about through the action of the sex determining region located on the short arm of the Y chromosome (Sry gene), but correct doses of other genes on autosomes as well as the X chromosome, are also required. Sry appears to be widely expressed in human fetuses, suggesting the possibility that its influence on development is not confined to the testes. There is additional evidence of a difference in developmental rates between XY and XX cleaving embryos, in which Sry and another gene in the sex-determining region named Zfy, for the zinc finger protein it encodes, are already expressed. These findings are consistent with the possibility that Y-chromosomal genes affect somatic sex differences prior to the formation of steroid hormones. Biomedical Reviews 1997; 7: 75-83.

Evolution of sex determination in caenorhabditis: unusually high divergence of tra-1 and its functional consequences.

The tra-1 gene is a terminal regulator of somatic sex in Caenorhabditis elegans: high tra-1 activity elicits female development, low tra-1 activity elicits male development. To investigate the function and evolution of tra-1, we examined the tra-1 gene from the closely related nematode C. briggsae. Ce-tra-1 and Cb-tra-1 are unusually divergent. Each gene generates two transcripts, but only one of these is present in both species. This common transcript encodes TRA-1A, which shows only 44% amino acid identity between the species, a figure much lower than that for previously compared genes. A Cb-tra-1 transgene rescues many tissues of tra-1(null) mutants of C. elegans but not the somatic gonad or germ line. This transgene also causes nongonadal feminization of XO animals, indicating incorrect sexual regulation. Alignment of Ce-TRA-1A and Cb-TRA-1A defines several conserved regions likely to be important for tra-1 function. The phenotypic differences between Ce-tra-1(null) mutants rescued by Cb-tra-1 transgenes and wild-type C. elegans indicate significant divergence of regulatory regions. These molecular and functional studies suggest that evolution of sex determination in nematodes is rapid and genetically complex.

Genetic mechanisms of sex differentiation.

Cloning of the SRY sex-determining locus on the Y chromosome has stimulated research on the genetic mechanisms of sex determination. It now appears that SRY is not the only gene required for testicular differentiation; X-linked and autosomal genes are also important. In parallel, mutations of genes involved in somatic sex differentiation shed light on the mechanisms involved in testis-dependent events.

The Biological Aspects of Puberty

Normal Pubertal Development SOMATIC CHANGES Puberty is characterized by an increase in growth rate and the appearance of striking somatic sex differences. The onset of these changes actually antedates the appearance of secondary sex characteristics by a few years. Thus, sexual maturation is a considerably longer process than the period of visible changes induced by marked incremental increases in gonadal hormones. Unlike boys, girls augment their body fat noticeably at 7 years of age; by 16 years of age girls have twice as much fat as boys. Total body water reflects lean body mass, which is made up primarily of muscle and skeletal tissues. At 9 years of age, total body water increases significantly in boys and signals the onset of more rapid growth in lean body mass. Muscle mass in boys doubles between the ages of 10 and 17 years, and skeletal mass doubles between ages 12 and 16 years. During adolescence boys exceed girls in all body measurements except hip width and body fat. The changes in body constituents during puberty are reflected more impressively by increases in height and weight. The initiation of the adolescent growth spurt precedes the onset of secondary sex characteristics by approximately 1 year in boys and girls.

Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila

In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination -- when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of the length except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently incapable of altering the splicing pattern of these pre-mRNAs.

The biological aspects of puberty.

Normal Pubertal Development SOMATIC CHANGES Puberty is characterized by an increase in growth rate and the appearance of striking somatic sex differences. The onset of these changes actually antedates the appearance of secondary sex characteristics by a few years. Thus, sexual maturation is a considerably longer process than the period of visible changes induced by marked incremental increases in gonadal hormones. Unlike boys, girls augment their body fat noticeably at 7 years of age; by 16 years of age girls have twice as much fat as boys. Total body water reflects lean body mass, which is made up primarily of muscle and skeletal tissues. At 9 years of age, total body water increases significantly in boys and signals the onset of more rapid growth in lean body mass. Muscle mass in boys doubles between the ages of 10 and 17 years, and skeletal mass doubles between ages 12 and 16 years. During adolescence boys exceed girls in all body measurements except hip width and body fat. The changes in body constituents during puberty are reflected more impressively by increases in height and weight. The initiation of the adolescent growth spurt precedes the onset of secondary sex characteristics by approximately 1 year in boys and girls.

Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila.

In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination -- when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of the length except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently incapable of altering the splicing pattern of these pre-mRNAs.

Genetic characterization of small ovaries, a gene required in the soma for the development of the Drosophila ovary and the female germline.

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.

Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes.

Transcription of the Drosophila yolk protein (Yp) genes is regulated by the somatic sex determination pathway. A gene at the bottom of this pathway, doublesex, encodes the female-specific DSXF and male-specific DSXM proteins that bind to and regulate transcription from several sites in the Yp genes. We report site-directed mutagenesis, protein binding and germline transformation experiments that identify and characterize the activity of a single binding site (dsxA) for the doublesex proteins and two binding sites for other regulatory proteins. A single copy of the three sites is sufficient to direct the sex and fat body specificities of Yp transcription. The sites form an enhancer with two strongly synergistic enhancer elements. One element (22 bp) consists of dsxA and an overlapping site, bzip1, that binds the DmC/EBP (slbo) protein, a member of the bZIP family of transcriptional activators. The other element is an 11 bp binding site (ref1) for an unknown protein. Tissue-specific activation requires strong cooperation between the ref1 site and the bzip1 or dsxA sites. Sex specificity is regulated exclusively by the dsxA site which connects the sex determination pathway to the target gene through DSXM repression and DSXF activation.

The somatic sex determines the requirement for ovarian tumor gene activity in the proliferation of the Drosophila germline

Gametogenesis in Drosophila requires sex-specific interactions between the soma and germline to control germ cell viability, proliferation, and differentiation. To determine what genetic components are involved in this interaction, we examined whether changes in the sexual identity of the soma affected the function of the ovarian tumor (otu) and ovo genes. These genes are required cell autonomously in the female germline for germ cell proliferation and differentiation. Mutations in otu and ovo cause a range of ovarian defects, including agametic ovaries and tumorous egg cysts, but do not affect spermatogenesis. We demonstrate that XY germ cells do not require otu when developing in testes, but become dependent on otu function for proliferation when placed in an ovary. This soma-induced requirement can be satisfied by the induced expression of the 98 x 10(3) M(r) OTU product, one of two isoforms produced by differential RNA splicing. These results indicate that the female somatic gonad can induce XY germ cells to become 'female-like' because they require an oogenesis-specific gene. In contrast, the requirement for ovo is dependent on a cell autonomous signal derived from the X:A ratio. We propose that differential regulation of the otu and ovo genes provides a mechanism for the female germline to incorporate both somatic and cell autonomous inputs required for oogenesis.

Dominant feminizing mutations implicate protein-protein interactions as the main mode of regulation of the nematode sex-determining gene tra-1.

The tra-1 gene is the terminal global selector of somatic sex in Caenorhabditis elegans: High tra-1 activity elicits female somatic development while low tra-1 activity elicits male development. Previous genetic studies defined a cascade of negatively interacting genes that regulates tra-1 activity in response to the primary sex-determining signal. Here, we investigate the last step in this regulatory cascade, by studying rare gain-of-function (gf) mutations of tra-1 that direct female somatic development irrespective of the upstream sex-determining signal. These mutations appear to abolish negative regulation of tra-1 in male tissues. We identify the lesions associated with 29 of these mutations and find that all affect a short stretch of amino acid residues present in both protein products of the tra-1 gene. Twenty-six alleles are associated with single nonconservative amino acid substitutions. Two alleles affect tra-1 RNA splicing and generate messages that omit part or all of the exon encoding this short stretch. These results suggest that sexual regulation of tra-1 is achieved post-translationally, by an inhibitory protein-protein interaction. The amino acid stretch altered by the tra-1(gf) mutations may define a site of interaction for negative regulators of tra-1. The stretch includes a potential phosphorylation site for glycogen synthase kinase 3 and may be conserved in the human gene GLI3, a homolog of tra-1 identified previously.

Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

Sex Determination and Dosage Compensation: Lessons from Flies and Worms

In both Drosophila melanogaster and Caenorhabditis elegans somatic sex determination, germline sex determination, and dosage compensation are controlled by means of a chromosomal signal known as the X:A ratio. A variety of mechanisms are used for establishing and implementing the chromosomal signal, and these do not appear to be similar in the two species. Instead, the study of sex determination and dosage compensation is providing more general lessons about different types of signaling pathways used to control alternative developmental states of cells and organisms.

Differential effects of Sex-lethal mutations on dosage compensation early in Drosophila development.

In response to the primary sex determination signal, X chromosome dose, the Sex-lethal gene controls all aspects of somatic sex determination and differentiation, including X chromosome dosage compensation. Two complementary classes of mutations have been identified that differentially affect Sxl somatic functions: (1) those impairing the "early" function used to set developmental pathway choice in response to the sex determination signal and (2) those impairing "late" functions involved in maintaining the pathway choice independent of the initiating signal and/or in directing differentiation. This "early vs. late" distinction correlates with a switch in promoter utilization from SxlPe to SxlPm at the blastoderm stage and a corresponding switch from transcriptional to RNA splicing control. Here we characterize five partial-loss-of-function Sxl alleles to explore a distinction between "early vs. late" functioning of Sxl in dosage compensation. Assaying for dosage compensation during the blastoderm stage, we find that the earliest phase of the dosage compensation process is controlled by products of the early Sxl promoter, SxlPe. Hence, in addition to triggering the sexual pathway decision of cells, products derived from SxlPe also control early dosage compensation, the first manifestation of sexually dimorphic differentiation. The effects of mutant Sxl alleles on early dosage compensation are consistent with their previous categorization as early vs. late defective with respect to their effects on pathway initiation. Results reported here suggest that the dosage compensation regulatory genes currently known to function downstream of Sxl, genes known as the "male-specific lethals," do not control all aspects of dosage compensation either at the blastoderm stage or later in development. In the course of this study, we also discovered that the canonical early defective allele, Sxlf9, which is impaired in its ability to establish the female developmental pathway commitment, is likely to be defective in the stability and/or functioning of products derived from SxlPe, rather than in the ability of SxlPe to respond to the chromosomal sex determination signal.

Control of sex determination in animals.

CONTENTS –Sex determination in the fruit fly and nematode–Somatic sex determination – Dosage compensation – Germ‐line sex determination–Mammalian sex determination signifies testis determination – The mammalian sex‐determining master‐regulatory gene, SRY–XX and XY sex reversal in man – Sex‐determining region Y chromosome (SRY) –Is SRY the primary testis determinant?– Biochemical properties of SRY – Sequence versus structure recognition – SRY: positive or negative regulation?– Evidence for other mammalian sex‐determining genes–Murine autosomal sex‐determining genes – Wilms' tumour gene WT1 –Chromosomal abnormalities associated with sex reversal – Müllerian inhibiting substance: a target of SRY?–Perspectives

Sex-lethal, master and slave: a hierarchy of germ-line sex determination in Drosophila

Female sex determination in the germ line of Drosophila melanogaster is regulated by genes functioning in the soma as well as genes that function within the germ line. Genes known or suspected to be involved in germ-line sex determination in Drosophila melanogaster have been examined to determine if they are required upstream or downstream of Sex-lethal+, a known germ-line sex determination gene. Seven genes required for female-specific splicing of germ-line Sex-lethal+ pre-mRNA are identified. These results together with information about the tissues in which these genes function and whether they control sex determination and viability or just sex determination in the germ line have been used to deduce the genetic hierarchy regulating female germ-line sex determination. This hierarchy includes the somatic sex determination genes transformer+, transformer-2+ and doublesex+ (and by inference Sex-lethal+), which control a somatic signal required for female germ-line sex determination, and the germ-line ovarian tumor genes fused+, ovarian tumor+, ovo+, sans fille+, and Sex-lethal+, which are involved in either the reception or interpretation of this somatic sex determination signal. The fused+, ovarian tumor+, ovo+ and sans fille+ genes function upstream of Sex-lethal+ in the germ line.