X-linked gene expression and sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of X chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two X chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one X chromosome and diploid hermaphrodites have two X chromosomes. However, the difference in X-chromosome number between the sexes is not reflected in general levels of X-linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X-linked gene expression appears to be 'turned down' in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X-chromosome duplications that elevate X-linked gene expression also feminize triploid males. One way that this relationship between sex determination and X-linked gene expression may be operating is discussed.     

Autonomy and nonautonomy of sex determination in triploid intersex mosaics of C. elegans

The primary sex-determining signal in Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio), normally 1.0 in hermaphrodites (XX) and 0.5 in males (XO). XX triploids (X/A = 0.67) are males, but if these animals carry a partial duplication of the X chromosome such that X/A approximately equal to 0.7, they develop as intersexes that are sexually mosaic. We have analyzed these mosaics using Nomarski microscopy and in situ hybridization to obtain information on whether sex determination decisions can be made independently in different cells and tissues, and when these commitments are made. The observed patterns of male and female cells in individual animals indicate that sex determination decisions can be influenced by anterior-posterior position and that sex determination decisions can be made as late as the third larval stage of postembryonic development. Although these decisions clearly can be made independently in different lineages, they show substantial biases toward one sex or the other in individual animals. We interpret these results to suggest that sex determination in C. elegans is not entirely cell autonomous.     

Selection and maintenance of androdioecy in Caenorhabditis elegans

Caenorhabditis elegans is an androdioecious nematode composed of selfing hermaphrodites and rare males. A model of male maintenance demonstrates that selfing rates in hermaphrodites cannot be too high or else the frequency of males will be driven down to the rate of spontaneous nondisjunction of the X chromosome. After their outcrossing ability is assessed, males are found to skirt the frequency range in which they would be maintained. When male maintenance is directly assessed by elevating male frequency and observing the frequency change through time, males are gradually eliminated from the population. Males, therefore, appear to reproduce at a rate just below that necessary for them to be maintained. Populations polymorphic for a mutation (fog-2) that effectively changes hermaphrodites into females demonstrate that there is strong selection against dioecy. Factors such as variation in male mating ability and inbreeding depression could potentially lead to the long-term maintenance of males.     

Identification of X Chromosome Regions in Caenorhabditis Elegans That Contain Sex-Determination Signal Elements

The primary sex-determination signal of Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). This signal coordinately controls both sex determination and X chromosome dosage compensation. To delineate regions of X that contain counted signal elements, we examined the effect on the X/A ratio of changing the dose of specific regions of X, using duplications in XO animals and deficiencies in XX animals. Based on the mutant phenotypes of genes that are controlled by the signal, we expected that increases (in males) or decreases (in hermaphrodites) in the dose of X chromosome elements could cause sex-specific lethality. We isolated duplications and deficiencies of specific X chromosome regions, using strategies that would permit their recovery regardless of whether they affect the signal. We identified a dose-sensitive region at the left end of X that contains X chromosome signal elements. XX hermaphrodites with only one dose of this region have sex determination and dosage compensation defects, and XO males with two doses are more severely affected and die. The hermaphrodite defects are suppressed by a downstream mutation that forces all animals into the XX mode of sex determination and dosage compensation. The male lethality is suppressed by mutations that force all animals into the XO mode of both processes. We were able to subdivide this region into three smaller regions, each of which contains at least one signal element. We propose that the X chromosome component of the sex-determination signal is the dose of a relatively small number of genes.     

The proximate determinants of sex ratio in C. elegans populations

The soil nematode Caenorhabditis elegans is an example of a species in which self-fertilizing hermaphrodites predominate, but functional males continue to persist--allowing outcrossing to persevere at low levels. Hermaphrodites can produce male progeny as a consequence of sex chromosome non-disjunction or via outcrossing with males. Consequently, the genetics of sex determination coupled with the efficiency by which males find, inseminate and obtain fertilizations with hermaphrodites will influence the frequency at which males and outcrossing occurs in such populations. Behavioural and physiological traits with a heritable basis, as well as ecological characters, may influence male reproductive success and therefore sex ratio. Because sex ratio is tied to male reproductive success, sex ratio greatly affects outcrossing rates, patterns of genetic variation, and the ability of natural selection to act within populations. In this paper we explore the determinants of male frequency in C. elegans with a mathematical model and experimental data. We address the role of the genetic machinery of sex determination via sex chromosome non-disjunction on sex ratio and the influence of physiological components of C. elegans' life history that contribute to variation in sex ratio by way of male reproductive success. Finally, we discuss the short-term and long-term factors that are likely to affect sex ratio and breeding system evolution in species like C. elegans.     

An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae

The free-living nematode worm Caenorhabditis elegans reproduces primarily as a self-fertilizing hermaphrodite, yet males are maintained in wild-type populations at low frequency. To determine the role of males in C. elegans, we develop a mathematical model for the genetic system of hermaphrodites that can either self-fertilize or be fertilized by males and we perform laboratory observations and experiments on both C. elegans and a related dioecious species C. remanei. We show that the mating efficiency of C. elegans is poor compared to a dioecious species and that C. elegans males are more attracted to C. remanei females than they are to their conspecific hermaphrodites. We postulate that a genetic mutation occurred during the evolution of C. elegans hermaphrodites, resulting in the loss of an attracting sex pheromone present in the ancestor of both C. elegans and C. remanei. Our findings suggest that males are maintained in C. elegans because of the particular genetic system inherited from its dioecious ancestor and because of nonadaptive spontaneous nondisjunction of sex chromosomes, which occurs during meiosis in the hermaphrodite. A theoretical argument shows that the low frequency of male mating observed in C. elegans can support male-specific genes against mutational degeneration. This results in the continuing presence of functional males in a 99.9% hermaphroditic species in which outcrossing is disadvantageous to hermaphrodites.     

Somatic damage to the X chromosome of the nematode Caenorhabditis elegans induced by gamma radiation

Summary Wild-type male embryos and young larvae of the nematode Caenorhabditis elegans were more sensitive than wild-type hermaphrodites to inactivation by gamma rays; wild-type males have one X chromosome per cell (XO), whereas wild-type hermaphrodites have two (XX). Furthermore, after transformation into fertile hermaphrodites by a her-1 mutation, XO animals were more radiosensitive than XX her-1 animals; and XX animals transformed into fertile males by a tra-1 mutation did not show increased radiosensitivity. It is concluded that wild-type males are more radiosensitive than wild-type hermaphrodites because they have one X chromosome rather than two, and the predominant mode of inactivation of XO animals involves damage to the single X chromosome. No sex-specific differences in survival were observed after UV irradiation.     

Evolution of male longevity bias in nematodes

Many animal species exhibit sex differences in aging. In the nematode Caenorhabditis elegans, under conditions that minimize mortality, males are the longer-lived sex. In a survey of 12 independent C. elegans isolates, we find that this is a species-typical character. To test the hypothesis that the C. elegans male longevity bias evolved as a consequence of androdioecy (having males and hermaphrodites), we compared sex-specific survival in four androdioecious and four dioecious (males and females) nematode species. Contrary to expectation, in all but C. briggsae (androdioecious), males were the longer-lived sex, and this difference was greatest among dioecious species. Moreover, male lifespan was reduced in androdioecious species relative to dioecious species. The evolutionary theory of aging predicts the evolution of a shorter lifespan in the sex with the greater rate of extrinsic mortality. We demonstrate that in each of eight species early adult mortality is elevated in females/hermaphrodites in the absence of food as the consequence of internal hatching of larvae (matricide). This age-independent mortality risk can favour the evolution of rapid aging in females and hermaphrodites relative to males.     

Dosage compensation: making 1X equal 2X

Animals that have XX females and XY or XO males have differing doses of X-linked genes in each sex. Overcoming this is the most immediate and vital aspect of sexual differentiation. A number of systems that accurately compensate for sex-chromosome dosage have evolved independently: silencing a single X chromosome in female mammals, downregulating both X chromosomes in hermaphrodite Caenorhabditis elegans and upregulating the X chromosome in male Drosophila all equalize X-linked gene expression. Each organism uses a largely non-overlapping set of molecules to achieve the same outcome: 1X = 2X.     

Sperm status regulates sexual attraction in Caenorhabditis elegans

Mating behavior of animals is regulated by the sensory stimuli provided by the other sex. Sexually receptive females emit mating signals that can be inhibited by male ejaculate. The genetic mechanisms controlling the release of mating signals and encoding behavioral responses remain enigmatic. Here we present evidence of a Caenorhabditis elegans hermaphrodite-derived cue that stimulates male mating-response behavior and is dynamically regulated by her reproductive status. Wild-type males preferentially mated with older hermaphrodites. Increased sex appeal of older hermaphrodites was potent enough to stimulate robust response from mating-deficient pkd-2 and lov-1 polycystin mutant males. This enhanced response of pkd-2 males toward older hermaphrodites was independent of short-chain ascaroside pheromones, but was contingent on the absence of active sperm in the hermaphrodites. The improved pkd-2 male response toward spermless hermaphrodites was blocked by prior insemination or by genetic ablation of the ceh-18-dependent sperm-sensing pathway of the hermaphrodite somatic gonad. Our work suggests an interaction between sperm and the soma that has a negative but reversible effect on a hermaphrodite-derived mating cue that regulates male mating response, a phenomenon to date attributed to gonochoristic species only.     

Diversity in mating behavior of hermaphroditic and male-female Caenorhabditis nematodes

In this study, we addressed why Caenorhabditis elegans males are inefficient at fertilizing their hermaphrodites. During copulation, hermaphrodites generally move away from males before they become impregnated. C. elegans hermaphrodites reproduce by internal self-fertilization, so that copulation with males is not required for species propagation. The hermaphroditic mode of reproduction could potentially relax selection for genes that optimize male mating behavior. We examined males from hermaphroditic and gonochoristic (male-female copulation) Caenorhabditis species to determine if they use different sensory and motor mechanisms to control their mating behavior. Instead, we found through laser ablation analysis and behavioral observations that hermaphroditic C. briggsae and gonochoristic C. remanei and Caenorhabditis species 4, PB2801 males produce a factor that immobilizes females during copulation. This factor also stimulates the vulval slit to widen, so that the male copulatory spicules can easily insert. C. elegans and C. briggsae hermaphrodites are not affected by this factor. We suggest that sensory and motor execution of mating behavior have not significantly changed among males of different Caenorhabditis species; however, during the evolution of internal self-fertilization, hermaphrodites have lost the ability to respond to the male soporific-inducing factor.     

Sex Determination in Polyploids of Caenorhabditis Elegans

In Caenorhabditis elegans triploid animals with two X chromosomes (symbolized 3A;2X) are males. However, these triploid males can be feminized by making them mutant for recessive dosage compensation mutations, by adding X chromosome duplications or by microinjecting particular DNA sequences termed feminizing elements. None of these treatments affects diploid males. This study explores several aspects of these treatments in polyploids. The dosage compensation mutants exhibit a strong maternal effect, such that reduction of any of the dosage compensation gene functions in the mother leads to sex reversal of 3A;2X animals. Likewise, all X chromosome duplications tested cause both sex reversal and intersexual development of many 3A;2X animals. Microinjected feminizing element DNA does not cause extensive sex reversal, but does result in intersexual development in 3A;2X animals. Neither X chromosome duplications nor microinjected feminizing elements show the extreme maternal effect of the dosage compensation mutants, although there is indirect evidence for a maternal effect of the feminizing elements. In particular, very little feminizing element DNA needs to be microinjected in order to feminize triploid males, far less than what is needed for stable inheritance, implying that feminizing elements can work within the mother's gonad. However, even very high concentrations of microinjected feminizing elements do not affect sex determination in diploid males, suggesting that they are not part of the numerator of the X/A ratio. In addition, no pair of X chromosome duplications feminizes diploid males, suggesting that none of these duplications contains a numerator of the X/A ratio. Instead, I infer that an X-linked locus, as yet undefined, must be present in two copies for hermaphrodite development to ensue or that the two X chromosomes might interact.     

Complex mosaicism in sex reversed SRY+ male twins

Sex reversal is characterized by discordance between genetic and phenotypic sex. Most XX males result from an unequal interchange between X and Y chromosomes during paternal meiosis, therefore transferring SRY to the X chromosome, which explains the male development in the presence of an otherwise normal female karyotype. We present here the case of sex reversed SRY+ male twins with several cell lines. They consulted for infertility. The presence of SRY on an X chromosome was demonstrated by FISH. Their respective karyotypes were: 46,X,der(X)t(X;Y)(p22.3;p11.2)[249]/45,X [12]/45,der(X)t(X;Y)(p22.3;p11.2)[11]/47,XX,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1]/50, XX,der(X)t(X;Y)(p22.3;p11.2)x4[1]/46,XX[1] for the first twin (SH-1) and 46,X,der(X)t(X;Y)(p22.3;p11.2)[108]/45,X [3]/47,XX,der(X)t(X;Y)(p22.3;p11.2)[2]/45,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1] for the second twin (SH-2). There are three different types of XX males: 1) with normal genitalia, 2) with genital ambiguity, and 3) XX true hermaphrodites. The phenotype of the twins presented in this report is consistent with what is generally seen in XX SRY+ males: they have normal genitalia.     

Regulation of DCC localization by HTZ-1/H2A.Z and DPY-30 does not correlate with H3K4 methylation levels

Dosage compensation is a specialized form of gene regulation that balances sex-chromosome linked gene expression between the sexes. In C. elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC binds along both X chromosomes in hermaphrodites to down-regulate gene expression by half, limiting X-linked gene products to levels produced in XO males. Sequence motifs enriched on the X chromosome play an important role in targeting the DCC to the X. However, these motifs are not strictly X-specific and therefore other factors, such as the chromatin environment of the X chromosome, are likely to aid in DCC targeting. Previously, we found that loss of HTZ-1 results in partial disruption of dosage compensation localization to the X chromosomes. We wanted to know whether other chromatin components coordinated with HTZ-1 to regulate DCC localization. One candidate is DPY-30, a protein known to play a role in DCC localization. DPY-30 homologs in yeast, flies, and mammals are highly conserved members of histone H3 lysine 4 (H3K4) methyltransferase Set1/MLL complexes. Therefore, we investigated the hypothesis that the dosage compensation function of DPY-30 involves H3K4 methylation. We found that in dpy-30 animals the DCC fails to stably bind chromatin. Interestingly, of all the C. elegans homologs of Set1/MLL complex subunits, only DPY-30 is required for stable DCC binding to chromatin. Additionally, loss of H3K4 methylation does not enhance DCC mislocalization in htz-1 animals. We conclude that DPY-30 and HTZ-1 have unique functions in DCC localization, both of which are largely independent of H3K4 methylation.     

Sex determination and sex reversal: genotype,phenotype, dogma and semantics

Summary The genetic terminology of sex determination and sex differentiation is examined in relation to its underlying biological basis. On the assumption that the function of the testis is to produce hormones and spermatozoa, the hypothesis of a single Y-chromosomal testis-determining gene with a dominant effect is shown to run counter to the following observed facts: a lowering in testosterone levels and an increase in the incidence of undescended testes, in addition to sterility, in males with multiple X chromosomes; abnormalities of the testes in autosomal trisomies; phenotypic abnormalities of XX males apparently increasing with decreasing amounts of Y-chromosomal material; the occurrence of patients with gonadal dysgenesis and XY males with ambiguous genitalia in the same sibship; the occurrence of identical SRY mutations in patients with gonadal dysgenesis and fertile males in the same pedigree; and the development of XY female and hermaphrodite mice having the same genetic constitution. The role of X inactivation in the production of males, females and hermaphrodites in T(X;16)16H mice has previously been suggested but not unequivocally demonstrated; moreover, X inactivation cannot account for the observed bilateral asymmetry of gonadal differentiation in XY hermaphrodites in humans and mice. There is evidence for a delay in development of the supporting cells in XY mice with ovarian formation. Once testicular differentiation and male hormone secretion have begun, other Y-chromosomal genes are required to maintain spermatogenesis and to complete spermiogenesis, but these genes do not function effectively in the presence of more than one X chromosome. The impairment of spermatogenesis by many other chromosome abnormalities seems to be more severe than that of oogenesis. It is concluded that the notion of a single testis-determining gene being responsible for male sex differentiation lacks biological validity, and that the genotype of a functional, i.e. fertile, male differs from that of a functional female by the presence of multiple Y-chromosomal genes in association with but a single X chromosome. Male sex differentiation in XY individuals can be further impaired by a euploid, but inappropriate, genetic background. The genes involved in testis development may function as growth regulators in the tissues in which they are active.     

Genetic flexibility in the convergent evolution of hermaphroditism in Caenorhabditis nematodes

The self-fertile hermaphrodites of C. elegans and C. briggsae evolved from female ancestors by acquiring limited spermatogenesis. Initiation of C. elegans hermaphrodite spermatogenesis requires germline translational repression of the female-promoting gene tra-2, which allows derepression of the three male-promoting fem genes. Cessation of hermaphrodite spermatogenesis requires fem-3 translational repression. We show that C. briggsae requires neither fem-2 nor fem-3 for hermaphrodite development, and that XO Cb-fem-2/3 animals are transformed into hermaphrodites, not females as in C. elegans. Exhaustive screens for Cb-tra-2 suppressors identified another 75 fem-like mutants, but all are self-fertile hermaphrodites rather than females. Control of hermaphrodite spermatogenesis therefore acts downstream of the fem genes in C. briggsae. The outwardly similar hermaphrodites of C. elegans and C. briggsae thus achieve self-fertility via intervention at different points in the core sex determination pathway. These findings are consistent with convergent evolution of hermaphroditism, which is marked by considerable developmental genetic flexibility.