Sex Chromosome Divergence Research Articles

How to make a sex chromosome.

Sex chromosomes can evolve once recombination is halted between a homologous pair of chromosomes. Owing to detailed studies using key model systems, we have a nuanced understanding and a rich review literature of what happens to sex chromosomes once recombination is arrested. However, three broad questions remain unanswered. First, why do sex chromosomes stop recombining in the first place? Second, how is recombination halted? Finally, why does the spread of recombination suppression, and therefore the rate of sex chromosome divergence, vary so substantially across clades? In this review, we consider each of these three questions in turn to address fundamental questions in the field, summarize our current understanding, and highlight important areas for future work.

Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida.

The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.

Sex chromosome synapsis and recombination in male guppies.

Guppy X and Y chromosomes represent an early stage in sex chromosome divergence. Synapsis and recombination between X and Y chromosomes attract special attention because recombination suppression promotes their differentiation, but previous studies have given contradictory results. Linkage analysis indicated that recombination between X and Y was extremely rare (<10%) and occurred in the medial part of the Y chromosome, while cytological analysis demonstrated regular association between the distal ends of the X and Y at diakinesis. In this study, we examine pairing and recombination between X and Y chromosomes using immunolocalization of MLH1 to mark recombination nodules, and genomic in situ hybridization with a male DNA probe to identify the Y-specific heterochromatic region. Pairing between X and Y is initiated distally. Single crossovers were detected in 87% of XY synaptonemal complexes, most often in the distal region and less frequently in a median position indicating that end-to-end associations between X and Y are chiasmatic. Thus, we suggest that the very low frequency of recombination detected by linkage analysis in a previous study resulted from a lack of informative markers in distal regions.

Independent stratum formation on the avian sex chromosomes reveals inter-chromosomal gene conversion and predominance of purifying selection on the W chromosome.

We used a comparative approach spanning three species and 90 million years to study the evolutionary history of the avian sex chromosomes. Using whole transcriptomes, we assembled the largest cross-species dataset of W-linked coding content to date. Our results show that recombination suppression in large portions of the avian sex chromosomes has evolved independently, and that long-term sex chromosome divergence is consistent with repeated and independent inversions spreading progressively to restrict recombination. In contrast, over short-term periods we observe heterogeneous and locus-specific divergence. We also uncover four instances of gene conversion between both highly diverged and recently evolved gametologs, suggesting a complex mosaic of recombination suppression across the sex chromosomes. Lastly, evidence from 16 gametologs reveal that the W chromosome is evolving with a significant contribution of purifying selection, consistent with previous findings that W-linked genes play an important role in encoding sex-specific fitness.

Globally Divergent but Locally Convergent X- and Y-Chromosome Influences on Cortical Development.

Owing to their unique evolutionary history, modern mammalian X- and Y-chromosomes have highly divergent gene contents counterbalanced by regulatory features, which preferentially restrict expression of X- and Y-specific genes. These 2 characteristics make opposing predictions regarding the expected dissimilarity of X- vs. Y-chromosome influences on biological structure and function. Here, we quantify this dissimilarity using in vivo neuroimaging within a rare cohort of humans with diverse sex chromosome aneuploidies (SCAs). We show that X- and Y-chromosomes have opposing effects on overall brain size but exert highly convergent influences on local brain anatomy, which manifest across biologically distinct dimensions of the cerebral cortex. Large-scale online meta-analysis of functional neuroimaging data indicates that convergent sex chromosome dosage effects preferentially impact centers for social perception, communication, and decision-making. Thus, despite an almost complete lack of sequence homology, and opposing effects on overall brain size, X- and Y-chromosomes exert congruent effects on the proportional size of cortical systems involved in adaptive social functioning. These convergent X-Y effects (i) track the dosage of those few genes that are still shared by X- and Y-chromosomes, and (ii) may provide a biological substrate for the link between SCA and increased rates of psychopathology.

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

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.

Molecular cytogenetic analysis for TFE3 rearrangement in Xp11.2 renal cell carcinoma and alveolar soft part sarcoma: validation and clinical experience with 75 cases

Renal cell carcinoma with TFE3 rearrangement at Xp11.2 is a distinct subtype manifesting an indolent clinical course in children, with recent reports suggesting a more aggressive entity in adults. This subtype is morphologically heterogeneous and can be misclassified as clear cell or papillary renal cell carcinoma. TFE3 is also rearranged in alveolar soft part sarcoma. To aid in diagnosis, a break-apart strategy fluorescence in situ hybridization (FISH) probe set specific for TFE3 rearrangement and a reflex dual-color, single-fusion strategy probe set involving the most common TFE3 partner gene, ASPSCR1, were validated on formalin-fixed, paraffin-embedded tissues from nine alveolar soft part sarcoma, two suspected Xp11.2 renal cell carcinoma, and nine tumors in the differential diagnosis. The impact of tissue cut artifact was reduced through inclusion of a chromosome X centromere control probe. Analysis of the UOK-109 renal carcinoma cell line confirmed the break-apart TFE3 probe set can distinguish the subtle TFE3/NONO fusion-associated inversion of chromosome X. Subsequent extensive clinical experience was gained through analysis of 75 cases with an indication of Xp11.2 renal cell carcinoma (n=54), alveolar soft part sarcoma (n=13), perivascular epithelioid cell neoplasms (n=2), chordoma (n=1), or unspecified (n=5). We observed balanced and unbalanced chromosome X;17 translocations in both Xp11.2 renal cell carcinoma and alveolar soft part sarcoma, supporting a preference but not a necessity for the translocation to be balanced in the carcinoma and unbalanced in the sarcoma. We further demonstrate the unbalanced separation is atypical, with TFE3/ASPSCR1 fusion and loss of the derivative X chromosome but also an unanticipated normal X chromosome gain in both males and females. Other diverse sex chromosome copy number combinations were observed. Our TFE3 FISH assay is a useful adjunct to morphologic analysis of such challenging cases and will be applicable to assess the growing spectrum of TFE3-rearranged tumors.

Transcriptome Sequencing Reveals the Character of Incomplete Dosage Compensation across Multiple Tissues in Flycatchers

Sex chromosome divergence, which follows the cessation of recombination and degeneration of the sex-limited chromosome, can cause a reduction in expression level for sex-linked genes in the heterozygous sex, unless some mechanisms of dosage compensation develops to counter the reduction in gene dose. Because large-scale perturbations in expression levels arising from changes in gene dose might have strong deleterious effects, the evolutionary response should be strong. However, in birds and in at least some other female heterogametic organisms, wholesale sex chromosome dosage compensation does not seem to occur. Using RNA-seq of multiple tissues and individuals, we investigated male and female expression levels of Z-linked and autosomal genes in the collared flycatcher, a bird for which a draft genome sequence recently has been reported. We found that male expression of Z-linked genes was on average 50% higher than female expression, although there was considerable variation in the male-to-female ratio among genes. The ratio for individual genes was well correlated among tissues and there was also a correlation in the extent of compensation between flycatcher and chicken orthologs. The relative excess of male expression was positively correlated with expression breadth, expression level, and number of interacting proteins (protein connectivity), and negatively correlated with variance in expression. These observations lead to a model of compensation occurring on a gene-by-gene basis, supported by an absence of clustering of genes on the Z chromosome with respect to the extent of compensation. Equal mean expression level of autosomal and Z-linked genes in males, and 50% higher expression of autosomal than Z-linked genes in females, is compatible with that partial compensation is achieved by hypertranscription from females’ single Z chromosome. A comparison with male-to-female expression ratios in orthologous Z-linked genes of ostriches, where Z–W recombination still occurs, suggests that male-biased expression of Z-linked genes is a derived trait after avian sex chromosome divergence.

A Large Pseudoautosomal Region on the Sex Chromosomes of the Frog Silurana tropicalis

Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged (“homomorphic”) sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used “RAD-tags” and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.

Diversity of Mating-Type Chromosome Structures in the Yeast Zygosaccharomyces rouxii Caused by Ectopic Exchanges between MAT-Like Loci

We investigated sex chromosome diversity in Zygosaccharomyces rouxii (Z. rouxii). In the current study, we show that the organization of the mating-type (MAT) locus is highly variable in the Z. rouxii population, indicating the MAT, HML, and HMR loci are translocation hotspots. Although NBRC1130 and CBS732 were originally two stocks of the type strain of the species, only NBRC1130 retains the original karyotype. A reciprocal translocation between the MAT and HMR loci appears to have occurred during the early passage culture of CBS732, which was used for genome sequencing. In NBRC1733, NBRC0686, NBRC0740 and NBRC1053, the terminal region of the chromosome containing the HMR locus was replaced with the chromosomal region to the left of the MAT or HML loci. The translocation events found in NBRC1733, NBRC0686, NBRC0740, and NBRC1053 were reconstructed under our experimental conditions using the DA2 background, and the reconstruction suggests that the frequency of this type of translocation is approximately 10−7. These results suggest that the MAT and MAT-like loci were the susceptible regions in the genome, and the diversity of mating-type chromosome structures in Z. rouxii was caused by ectopic exchanges between MAT-like loci.

Lack of Dosage Compensation Accompanies the Arrested Stage of Sex Chromosome Evolution in Ostriches

Sex chromosome evolution is usually seen as a process that, once initiated, will inevitably progress toward an advanced stage of degeneration of the nonrecombining chromosome. However, despite evidence that avian sex chromosome evolution was initiated >100 Ma, ratite birds have been trapped in an arrested stage of sex chromosome divergence. We performed RNA sequencing of several tissues from male and female ostriches and assembled the transcriptome de novo. A total of 315 Z-linked genes fell into two categories: those that have equal expression level in the two sexes (for which Z–W recombination still occurs) and those that have a 2-fold excess of male expression (for which Z–W recombination has ceased). We suggest that failure to evolve dosage compensation has constrained sex chromosome divergence in this basal avian lineage. Our results indicate that dosage compensation is a prerequisite for, not only a consequence of, sex chromosome evolution.

Contrasting Patterns of X/Y Polymorphism Distinguish Carica papaya from Other Sex Chromosome Systems

The sex chromosomes of the tropical crop papaya (Carica papaya) are evolutionarily young and consequently allow for the examination of evolutionary mechanisms that drive early sex chromosome divergence. We conducted a molecular population genetic analysis of four X/Y gene pairs from a collection of 45 wild papaya accessions. These population genetic analyses reveal striking differences in the patterns of polymorphism between the X and Y chromosomes that distinguish them from other sex chromosome systems. In most sex chromosome systems, the Y chromosome displays significantly reduced polymorphism levels, whereas the X chromosome maintains a level of polymorphism that is comparable to autosomal loci. However, the four papaya sex-linked loci that we examined display diversity patterns that are opposite this trend: the papaya X alleles exhibit significantly reduced polymorphism levels, whereas the papaya Y alleles maintain greater than expected levels of diversity. Our analyses suggest that selective sweeps in the regions of the X have contributed to this pattern while also revealing geographically restricted haplogroups on the Y. We discuss the possible role sexual selection and/or genomic conflict have played in shaping the contrasting patterns of polymorphism found for the papaya X and Y chromosomes.

THE GENOMIC SIGNATURE OF SEXUAL SELECTION IN THE GENETIC DIVERSITY OF THE SEX CHROMOSOMES AND AUTOSOMES

Genomic levels of variation can help reveal the selective and demographic forces that have affected a species during its history. The relative amount of genetic diversity observed on the sex chromosomes as compared to the autosomes is predicted to differ among monogamous and polygynous species. Many species show departures from the expectation for monogamy, but it can be difficult to conclude that this pattern results from variation in mating system because forces other than sexual selection can act upon sex chromosome genetic diversity. As a critical test of the role of mating system, we compared levels of genetic diversity on the Z chromosome and autosomes of phylogenetically independent pairs of shorebirds that differed in their mating systems. We found general support for sexual selection shaping sex chromosome diversity because most polygynous species showed relatively reduced genetic variation on their Z chromosomes as compared to monogamous species. Differences in levels of genetic diversity between the sex chromosomes and autosomes may therefore contribute to understanding the long-term history of sexual selection experienced by a species.

Karyotype Differentiation between Two Stickleback Species (Gasterosteidae)

The stickleback family (Gasterosteidae) of fish is less than 40 million years old, yet stickleback species have diverged in both diploid chromosome number (2n) and morphology. We used comparative fluorescence in situ hybridization (FISH) on 2 stickleback species, Gasterosteus aculeatus (2n = 42) and Apeltes quadracus (2n = 46), to ascertain the types of chromosome rearrangements that differentiate these species. The A. quadracus karyotype contains more acrocentric and telocentric chromosomes than the G. aculeatus karyotype. By using bacterial artificial chromosome probes from G. aculeatus in our FISH screen, we found that 6 pericentric inversions and 2 chromosome fusions/fissions are responsible for the greater number of acrocentric and telocentric chromosomes in A. quadracus. While most populations of G. aculeatus have an XX/XY sex chromosome system, A. quadracus has a ZZ/ZW sex chromosome system, as previously reported. However, we discovered that a population of A. quadracus from Connecticut lacks heteromorphic sex chromosomes, providing evidence for unexpected sex chromosome diversity in this species.

Genome Evolution in Reptilia, the Sister Group of Mammals

The genomes of birds and nonavian reptiles (Reptilia) are critical for understanding genome evolution in mammals and amniotes generally. Despite decades of study at the chromosomal and single-gene levels, and the evidence for great diversity in genome size, karyotype, and sex chromosome diversity, reptile genomes are virtually unknown in the comparative genomics era. The recent sequencing of the chicken and zebra finch genomes, in conjunction with genome scans and the online publication of the Anolis lizard genome, has begun to clarify the events leading from an ancestral amniote genome--predicted to be large and to possess a diverse repeat landscape on par with mammals and a birdlike sex chromosome system--to the small and highly streamlined genomes of birds. Reptilia exhibit a wide range of evolutionary rates of different subgenomes and, from isochores to mitochondrial DNA, provide a critical contrast to the genomic paradigms established in mammals.

Cytogenetic studies in Eigenmannia virescens (Sternopygidae, Gymnotiformes) and new inferences on the origin of sex chromosomes in the Eigenmannia genus

BackgroundCytogenetic studies were carried out on samples of Eigenmannia virescens (Sternopygidae, Gymnotiformes) obtained from four river systems of the Eastern Amazon region (Para, Brazil).ResultsAll four populations had 2n = 38, with ZZ/ZW sex chromosomes (Z, acrocentric; W, submetacentric). Constitutive heterochromatin (CH) was found at the centromeric regions of all chromosomes. The W chromosome had a heterochromatic block in the proximal region of the short arm; this CH was positive for DAPI staining, indicating that it is rich in A-T base pairs. The nucleolar organizer region (NOR) was localized to the short arm of chromosome pair 15; this result was confirmed by fluorescent in situ hybridization (FISH) with human 45S rDNA, and CMA3 staining indicated that the region is G-C rich. FISH with telomeric probes did not show any evidence of interstitial telomeric sequences (ITS).ConclusionPrevious studies have shown that the species Eigenmannia sp. 2 and E. virescens have differentiated sex chromosomes, and diverse sex chromosome systems have been described for E. virescens specimens obtained from different Brazilian rivers. A comparative analysis of the present data and prior reports suggests that the sex chromosomes of Eigenmannia may have arisen independently in the different populations.

Repetitive DNA probe linked to sex chromosomes in hybrids between Neotropical fish Leporinus macrocephalus and Leporinus elongatus (Characiformes, Anostomidae)

Interspecific hybrids of fish can offer important advances in the zootechnical exploration of this animal group. However, hybrids can pose serious biological risks to the environment and natural populations, making genetic characterization and monitoring of hybrids produced in fish culture important concerns. In the present study, individuals of the hybrid ‘Piaupara’ were characterized by fluorescence in situ hybridization (FISH) using sex-specific probes to differentiate the respective parental contributions from female Leporinus macrocephalus (Piauçu) and male Leporinus elongatus (Piapara). The application of this technique demonstrated a difference in hybridization patterns between males and females of L. elongatus and L. macrocephalus, mainly due to the fact that the NOR-bearing chromosomes of L. elongatus contain some homology with the probe. This allowed us to detect a clear sex-specific distinction of the hybrid ‘Piaupara’, demonstrating that this technique is well-suited for the characterization of these hybrids. Moreover, this technique offers an important methodological advance for the study of diverse sex chromosome systems of Leporinus, supplying valuable information about the origin and establishment of a recently described sexual system in this genus.

Turnover of Sex Chromosomes in the Stickleback Fishes (Gasterosteidae)

Diverse sex-chromosome systems are found in vertebrates, particularly in teleost fishes, where different systems can be found in closely related species. Several mechanisms have been proposed for the rapid turnover of sex chromosomes, including the transposition of an existing sex-determination gene, the appearance of a new sex-determination gene on an autosome, and fusions between sex chromosomes and autosomes. To better understand these evolutionary transitions, a detailed comparison of sex chromosomes between closely related species is essential. Here, we used genetic mapping and molecular cytogenetics to characterize the sex-chromosome systems of multiple stickleback species (Gasterosteidae). Previously, we demonstrated that male threespine stickleback fish (Gasterosteus aculeatus) have a heteromorphic XY pair corresponding to linkage group (LG) 19. In this study, we found that the ninespine stickleback (Pungitius pungitius) has a heteromorphic XY pair corresponding to LG12. In black-spotted stickleback (G. wheatlandi) males, one copy of LG12 has fused to the LG19-derived Y chromosome, giving rise to an X1X2Y sex-determination system. In contrast, neither LG12 nor LG19 is linked to sex in two other species: the brook stickleback (Culaea inconstans) and the fourspine stickleback (Apeltes quadracus). However, we confirmed the existence of a previously reported heteromorphic ZW sex-chromosome pair in the fourspine stickleback. The sex-chromosome diversity that we have uncovered in sticklebacks provides a rich comparative resource for understanding the mechanisms that underlie the rapid turnover of sex-chromosome systems.

A highly polymorphic insertion in the Y-chromosome amelogenin gene can be used for evolutionary biology, population genetics and sexing in Cetacea and Artiodactyla

BackgroundThe early radiation of the Cetartiodactyla is complex, and unambiguous molecular characters are needed to clarify the positions of hippotamuses, camels and pigs relative to the remaining taxa (Cetacea and Ruminantia). There is also a need for informative genealogic markers for Y-chromosome population genetics as well as a sexing method applicable to all species from this group. We therefore studied the sequence variation of a partial sequence of the evolutionary conserved amelogenin gene to assess its potential use in each of these fields.Results and discussionWe report a large interstitial insertion in the Y amelogenin locus in most of the Cetartiodactyla lineages (cetaceans and ruminants). This sex-linked size polymorphism is the result of a 460–465 bp inserted element in intron 4 of the amelogenin gene of Ruminants and Cetaceans. Therefore, this polymorphism can easily be used in a sexing assay for these species.When taking into account this shared character in addition to nucleotide sequence, gene genealogy follows sex-chromosome divergence in Cetartiodactyla whereas it is more congruent with zoological history when ignoring these characters. This could be related to a loss of homology between chromosomal copies given the old age of the insertion.The 1 kbp Amel-Y amplified fragment is also characterized by high nucleotide diversity (64 polymorphic sites spanning over 1 kbp in seven haplotypes) which is greater than for other Y-chromosome sequence markers studied so far but less than the mitochondrial control region.ConclusionThe gender-dependent polymorphism we have identified is relevant not only for phylogenic inference within the Cetartiodactyla but also for Y-chromosome based population genetics and gender determination in cetaceans and ruminants. One single protocol can therefore be used for studies in population and evolutionary genetics, reproductive biotechnologies, and forensic science.

Characteristics, causes and evolutionary consequences of male-biased mutation

Mutation has traditionally been considered a random process, but this paradigm is challenged by recent evidence of divergence rate heterogeneity in different genomic regions. One facet of mutation rate variation is the propensity for genetic change to correlate with the number of germ cell divisions, reflecting the replication-dependent origin of many mutations. Haldane was the first to connect this association of replication and mutation to the difference in the number of cell divisions in oogenesis (low) and spermatogenesis (usually high), and the resulting sex difference in the rate of mutation. The concept of male-biased mutation has been thoroughly analysed in recent years using an evolutionary approach, in which sequence divergence of autosomes and/or sex chromosomes are compared to allow inference about the relative contribution of mothers and fathers in the accumulation of mutations. For instance, assuming that a neutral sequence is analysed, that rate heterogeneity owing to other factors is cancelled out by the investigation of many loci and that the effect of ancestral polymorphism is properly taken into account, the male-to-female mutation rate ratio, alpham, can be solved from the observed difference in rate of X and Y chromosome divergence. The male mutation bias is positively correlated with the relative excess of cell divisions in the male compared to the female germ line, as evidenced by a generation time effect: in mammals, alpham is estimated at approximately 4-6 in primates, approximately 3 in carnivores and approximately 2 in small rodents. Another life-history correlate is sexual selection: when there is intense sperm competition among males, increased sperm production will be associated with a larger number of mitotic cell divisions in spermatogenesis and hence an increase in alpham. Male-biased mutation has implications for important aspects of evolutionary biology such as mate choice in relation to mutation load, sexual selection and the maintenance of genetic diversity despite strong directional selection, the tendency for a disproportionate large role of the X (Z) chromosome in post-zygotic isolation, and the evolution of sex.