XY Recombination Research Articles

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11β splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11βki/− mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11βki/− males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11β and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

Genome Complexity Reduction High-Throughput Genome Sequencing of Green Iguana (Iguana iguana) Reveal a Paradigm Shift in Understanding Sex-Chromosomal Linkages on Homomorphic X and Y Sex Chromosomes

The majority of lizards classified in the superfamily Iguanoidea have an XX/XY sex-determination system in which sex-chromosomal linkage shows homology with chicken (Gallus gallus) chromosome 15 (GGA15). However, the genomics of sex chromosomes remain largely unexplored owing to the presence of homomorphic sex chromosomes in majority of the species. Recent advances in high-throughput genome complexity reduction sequencing provide an effective approach to the identification of sex-specific loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA), and a better understanding of sex chromosome dynamics in Iguanoidea. In this study, we applied Diversity Arrays Technology (DArTseqTM) in 29 phenotypic sex assignments (14 males and 15 females) of green iguana (Iguana iguana). We confirmed a male heterogametic (XX/XY) sex determination mode in this species, identifying 29 perfectly sex-linked SNP/PA loci and 164 moderately sex-linked SNP/PA loci, providing evidence probably indicative of XY recombination. Three loci from among the perfectly sex-linked SNP/PA loci showed partial homology with several amniote sex chromosomal linkages. The results support the hypothesis of an ancestral super-sex chromosome with overlaps of partial sex-chromosomal linkages. However, only one locus among the moderately sex-linked loci showed homology with GGA15, which suggests that the specific region homologous to GGA15 was located outside the non-recombination region but in close proximity to this region of the sex chromosome in green iguana. Therefore, the location of GGA15 might be further from the putative sex-determination locus in green iguana. This is a paradigm shift in understanding linkages on homomorphic X and Y sex chromosomes. The DArTseq platform provides an easy-to-use strategy for future research on the evolution of sex chromosomes in Iguanoidea, particularly for non-model species with homomorphic or highly cryptic sex chromosomes.

Identifying sex-linked markers in Litoria aurea: a novel approach to understanding sex chromosome evolution in an amphibian

Few taxa exhibit the variability of sex-determining modes as amphibians. However, due to the presence of homomorphic sex chromosomes in many species, this phenomenon has been difficult to study. The Australian frog, Litoria aurea, has been relatively well studied over the past 20 years due to widespread declines largely attributable to chytrid fungus. However, it has been subject to few molecular studies and its mode of sex determination remained unknown. We applied DArTseq™ to develop sex-linked single nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 44 phenotypically sexed L. aurea individuals from the Molonglo River in NSW, Australia. We conclusively identified a male heterogametic (XX-XY) sex determination mode in this species, identifying 11 perfectly sex-linked SNP and six strongly sex-linked PA markers. We identified a further 47 moderately sex-linked SNP loci, likely serving as evidence indicative of XY recombination. Furthermore, within these 47 loci, a group of nine males were found to have a feminised Y chromosome that significantly differed to all other males. We postulate ancestral sex-reversal as a means for the evolution of this now pseudoautosomal region on the Y chromosome. Our findings present new evidence for the ‘fountain of youth’ hypothesis for the retention of homomorphic sex chromosomes in amphibians and describe a novel approach for the study of sex chromosome evolution in amphibia.

Introgression across Hybrid Zones Is Not Mediated by Large X-Effects in Green Toads with Undifferentiated Sex Chromosomes.

Divergence between incipient species remains an incompletely understood process. Hybrid zones provide great research potential, reflecting natural organismal genomic interactions and gene evolution in a variety of recombinants over generations. While sex chromosomes are known evolutionary drivers of reproductive isolation, empirical population genetics has mostly examined species with heteromorphic sex chromosomes. We recently reported restricted introgression at sex-linked markers in an amphibian system with homomorphic sex chromosomes (Hyla), consistent with a large X-effect, designating a greater role of sex chromosomes in driving hybrid incompatibilities. Here, using a similar approach, we examined two hybrid zones of Palearctic green toads (Bufo viridis subgroup), involving several lineages that arose at different times and form secondary contacts. We find no evidence for differential introgression of sex-linked versus autosomal markers across both zones. This absence of large X-effects in Bufo indicates that, unlike in Hyla, hybrid incompatibilities may not result from the faster-heterogametic sex and faster-male aspects of Haldane's rule. The recent suppression of XY recombination in Hyla but not in Bufo may have driven greater divergence between Hyla sex chromosomes, causing stronger reproductive isolation. Alternatively, stronger linkage among Hyla's sex-linked markers could restrict introgression. We hypothesize that the degree of sex-specific recombination may condition the importance of homomorphic sex chromosomes in speciation.

Tissue Specificity and Dynamics of Sex-Biased Gene Expression in a Common Frog Population with Differentiated, Yet Homomorphic, Sex Chromosomes.

Sex-biased genes are central to the study of sexual selection, sexual antagonism, and sex chromosome evolution. We describe a comprehensive de novo assembled transcriptome in the common frog Rana temporaria based on five developmental stages and three adult tissues from both sexes, obtained from a population with karyotypically homomorphic but genetically differentiated sex chromosomes. This allows the study of sex-biased gene expression throughout development, and its effect on the rate of gene evolution while accounting for pleiotropic expression, which is known to negatively correlate with the evolutionary rate. Overall, sex-biased genes had little overlap among developmental stages and adult tissues. Late developmental stages and gonad tissues had the highest numbers of stage- or tissue-specific genes. We find that pleiotropic gene expression is a better predictor than sex bias for the evolutionary rate of genes, though it often interacts with sex bias. Although genetically differentiated, the sex chromosomes were not enriched in sex-biased genes, possibly due to a very recent arrest of XY recombination. These results extend our understanding of the developmental dynamics, tissue specificity, and genomic localization of sex-biased genes.

Sex-antagonistic genes, XY recombination and feminized Y chromosomes.

The canonical model of sex-chromosome evolution predicts that sex-antagonistic (SA) genes play an instrumental role in the arrest of XY recombination and ensuing Y chromosome degeneration. Although this model might account for the highly differentiated sex chromosomes of birds and mammals, it does not fit the situation of many lineages of fish, amphibians or nonavian reptiles, where sex chromosomes are maintained homomorphic through occasional XY recombination and/or high turnover rates. Such situations call for alternative explanatory frameworks. A crucial issue at stake is the effect of XY recombination on the dynamics of SA genes and deleterious mutations. Using individual-based simulations, we show that a complete arrest of XY recombination actually benefits females, not males. Male fitness is maximized at different XY recombination rates depending on SA selection, but never at zero XY recombination. This should consistently favour some level of XY recombination, which in turn generates a recombination load at sex-linked SA genes. Hill-Robertson interferences with deleterious mutations also impede the differentiation of sex-linked SA genes, to the point that males may actually fix feminized phenotypes when SA selection and XY recombination are low. We argue that sex chromosomes might not be a good localization for SA genes, and sex conflicts seem better solved through the differential expression of autosomal genes.

Trans-species variation in Dmrt1 is associated with sex determination in four European tree-frog species.

Empirical studies on the relative roles of occasional XY recombination versus sex-chromosome turnover in preventing sex-chromosome differentiation may shed light on the evolutionary forces acting on sex-determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex-determination systems that keep sex-chromosome identity): both models predict X and Y alleles at sex-linked genes to cluster by species. However, the XY-recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex-determining region (SDR) with trans-species polymorphism associated to sex. Here we combine a candidate-gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N-terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex-chromosome homomorphy in this clade results only from XY recombination, and take an important step toward the identification of the sex-determining locus. Moreover, the sex-diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.

Sex Chromosome Conservation, DMRT1 Phylogeny and Gonad Morphology in Diploid Palearctic Green Toads (Bufo viridis Subgroup)

Due to the prevailing sex chromosome homomorphy and large genome size, the knowledge on sex determination systems, sex chromosomes and sex-determining genes in amphibians remains scarce. Using 3 cross-amplifying sex-linked microsatellite markers, we uncover sex determination systems and sex chromosomes in purebred, diploid Palearctic green toads (Bufo viridis subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in B. balearicus, B. viridis and B. variabilis. While females show recombination, it is strongly suppressed (or not detectable) in males. Markers corroborate the largest chromosome pair 1 (homologous to linkage group 1 of Xenopus tropicalis) to represent the sex chromosomes in diploid species of the B. viridis subgroup (B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis, and probably B. boulengeri). This chromosome harbors DMRT1, a key gene of the sexual pathway in deeply divergent animal taxa. However, our phylogenetic analysis of a 600-bp fragment of that gene in diploid green toad taxa reveals that X and Y alleles cluster by species and not by gametolog. This suggests that XY-sequence similarity stems from occasional XY recombination within DMRT1, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire DMRT1 gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of X. tropicalis appears to be maintained as the largest chromosome (1), and thus is homologous in anuran karyotype evolution from pipid to hylid, bufonid and ranid anurans.

Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man

Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P < 0.001) and decrease in the pachytene spermatocytes (P < 0.001) were observed in the t(5;7;9;13) carrier compared with controls. It was further observed that 93% of octavalents were found partially asynapsed between homologous chromosomes. A significant decrease in the recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

Sex-chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea).

Occasional XY recombination is a proposed explanation for the sex-chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW-European Hyla arborea populations identified male-specific alleles at sex-linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex-linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex-chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY-recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).

Range-wide sex-chromosome sequence similarity supports occasional XY recombination in European tree frogs (Hyla arborea).

In contrast with mammals and birds, most poikilothermic vertebrates feature structurally undifferentiated sex chromosomes, which may result either from frequent turnovers, or from occasional events of XY recombination. The latter mechanism was recently suggested to be responsible for sex-chromosome homomorphy in European tree frogs (Hyla arborea). However, no single case of male recombination has been identified in large-scale laboratory crosses, and populations from NW Europe consistently display sex-specific allelic frequencies with male-diagnostic alleles, suggesting the absence of recombination in their recent history. To address this apparent paradox, we extended the phylogeographic scope of investigations, by analyzing the sequences of three sex-linked markers throughout the whole species distribution. Refugial populations (southern Balkans and Adriatic coast) show a mix of X and Y alleles in haplotypic networks, and no more within-individual pairwise nucleotide differences in males than in females, testifying to recurrent XY recombination. In contrast, populations of NW Europe, which originated from a recent postglacial expansion, show a clear pattern of XY differentiation; the X and Y gametologs of the sex-linked gene Med15 present different alleles, likely fixed by drift on the front wave of expansions, and kept differentiated since. Our results support the view that sex-chromosome homomorphy in H. arborea is maintained by occasional or historical events of recombination; whether the frequency of these events indeed differs between populations remains to be clarified.

THE EVOLUTION OF XY RECOMBINATION: SEXUALLY ANTAGONISTIC SELECTION VERSUS DELETERIOUS MUTATION LOAD

Recombination arrest between X and Y chromosomes, driven by sexually antagonistic genes, is expected to induce their progressive differentiation. However, in contrast to birds and mammals (which display the predicted pattern), most cold-blooded vertebrates have homomorphic sex chromosomes. Two main hypotheses have been proposed to account for this, namely high turnover rates of sex-determining systems and occasional XY recombination. Using individual-based simulations, we formalize the evolution of XY recombination (here mediated by sex reversal; the "fountain-of-youth" model) under the contrasting forces of sexually antagonistic selection and deleterious mutations. The shift between the domains of elimination and accumulation occurs at much lower selection coefficients for the Y than for the X. In the absence of dosage compensation, mildly deleterious mutations accumulating on the Y depress male fitness, thereby providing incentives for XY recombination. Under our settings, this occurs via "demasculinization" of the Y, allowing recombination in XY (sex-reversed) females. As we also show, this generates a conflict with the X, which coevolves to oppose sex reversal. The resulting rare events of XY sex reversal are enough to purge the Y from its load of deleterious mutations. Our results support the "fountain of youth" as a plausible mechanism to account for the maintenance of sex-chromosome homomorphy.

Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier

Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance. In infertile men who are carriers of chromosome abnormalities, adequate sperm production can be impaired. Meiosis, a cell division process in which spermatozoa are produced, can be impaired by the interaction of the abnormal chromosome with its homologous pair or with other chromosomes. In meiosis, a process called ‘recombination’ occurs, where homologous chromosomes pair up and exchange genetic material. Recombination is important in ensuring equal distribution of chromosomes during meiosis. When these processes are disrupted, sperm production can be impaired. In this study, we investigated meiosis in an infertile man carrying an inversion on chromosome 1, a particular type of chromosome abnormality. We used fluorescence microscopy techniques to examine recombination, the fidelity of chromosome pairing (synapsis) and chromosome distribution in the meiotic cells obtained from testicular tissue. Spermatozoa was also studied for proper chromosome distribution. Meiotic cells and spermatozoa from five fertile men that did not have any chromosome abnormalities were used as a reference group. Our results showed that recombination involving the affected chromosome, and between the sex-determining chromosomes were reduced compared with the reference. The abnormal chromosome associated with the sex chromosomes in 43.4% of the meiotic cells, in which recombination was noticeably absent between the sex chromosomes. Despite these findings, the frequency of chromosomally abnormal spermatozoa was not greater than the reference. Our findings suggest that certain chromosome inversions may have impaired recombination on the involved and the sex chromosomes. However, this impairment may be a protective factor against the production of chromosomally abnormal spermatozoa.

Preimplantation genetic diagnosis (PGD) for SHOX-related haploinsufficiency in conjunction with trisomy 21 detection by molecular analysis

Development of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring. We developed a 13 marker multiplex fluorescent PCR protocol: 3 markers within the deleted SHOX region, 5 flanking markers, 3 informative markers on chromosome 21 (advanced maternal age) and 2 markers for sex determination. Of four embryos, two wild type males, diploid for chromosome 21 were transferred resulting in twin boys. One embryo was an affected female and another embryo was Turner. Amniocentesis confirmed the implanted embryos were males (46XY), with no recombinations. While many X-linked disorders can be analyzed by sexing, genes located in the pseudoautosomal regions have high XY recombination rates, requiring multiple markers to enable an accurate diagnosis.

Mathematical glimpse on the Y chromosome degeneration

The Y chromosomes are genetically degenerate and do not recombine with their matching partners X. Non-recombination of XY pairs has been pointed out as the key factor for the degeneration of the Y chromosome. The aim here is to show that there is a mathematical asymmetry in sex chromosomes which leads to the degeneration of Y chromosomes even in the absence of XX and XY recombination. A model for sex-chromosome evolution in a stationary regime is proposed. The consequences of their asymmetry are analyzed and lead us to a couple of conclusions. First, Y chromosome degeneration shows up \(\sqrt{2}\) more often than X chromosome degeneration. Second, if nature prohibits female mortalities from beeing exactly \(50\%\), then Y chromosome degeneration is inevitable.