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 Spo11ß 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 C57Spo11ßki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11ßki/- 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 SPO11ß 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.

Assembly of 43 human Y chromosomes reveals extensive complexity and variation.

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.

Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes.

BACKGROUND: The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and mutation processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes, and two archaic human genome sequences. RESULTS: We find that PAR1 is fast evolving and closer to evolutionary nucleotide equilibrium than autosomal telomeres. We detect a difference between substitution patterns and extant diversity in PAR1, mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. We detect excess C-to-G mutations in PAR1 of all great apes, specific to the mutagenic effect of male recombination. Despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence. We find differences between substitution spectra of these archaics suggesting rapid evolution of PAR1 in recent hominin history. Frequency analysis of alleles segregating in females and males provided no evidence for recent sexual antagonism in this region. We study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. CONCLUSIONS: Our study provides an unprecedented quantification of population genetic forces governing PAR1 biology across extant and extinct hominids. PAR1 evolutionary dynamics are predominantly governed by recombination processes with a strong impact on mutation patterns across all species.

The genes from the pseudoautosomal region 1 (PAR1) of the mammalian sex chromosomes: Synteny, phylogeny and selection.

Sex chromosomes recombine restrictly in their homologous area, the pseudoautosomal region (PAR), represented by PAR1 and PAR2, which behave like an autosome in both pairing and recombination. The PAR1, common to most of the eutherian mammals, is located at the terminus of the sex chromosomes short arm and exhibit recombination rates ~20 times higher than the autosomes. Here, we assessed the interspecific evolutionary genomic dynamics of 15 genes of the PAR1 across 41 mammalian genera (representing six orders). The strong negative selection detected in most of the assessed groups reinforces the presence of evolutionary constraints, imposed by the important function of the PAR1 genes. Indeed, mutations in these genes are associated with various diseases in humans, including stature problems (Klinefelter Syndrome), leukemia and mental diseases. Yet, a few genes exhibiting positive selection (ω-value >1) were depicted in Rodentia (ASMT and ZBED1) and Primates (CRLF2 and CSF2RA). Rodents have the smallest described PAR1, while that of simian primates/humans underwent a 3 to 5 fold size reduction. The assessment of the PAR1 genes synteny revealed differences among the mammalian species, especially in the Rodentia order where chromosomic translocations from the sex chromosomes to the autosomes were observed. Such syntenic changes may be an evidence of the rapid evolution in rodents, as previous referred in other papers, also depicted by their increased branch lengths in the phylogenetic analyses. Concluding, we suggest that genome migration is an important factor influencing the evolution of mammals and may result in changes of the selective pressures operating on the genome.

Evolutionary dynamics of the human pseudoautosomal regions.

Recombination between the X and Y human sex chromosomes is limited to the two pseudoautosomal regions (PARs) that present quite distinct evolutionary origins. Despite the crucial importance for male meiosis, genetic diversity patterns and evolutionary dynamics of these regions are poorly understood. In the present study, we analyzed and compared the genetic diversity of the PAR regions using publicly available genomic sequences encompassing both PAR1 and PAR2. Comparisons were performed through allele diversities, linkage disequilibrium status and recombination frequencies within and between X and Y chromosomes. In agreement with previous studies, we confirmed the role of PAR1 as a male-specific recombination hotspot, but also observed similar characteristic patterns of diversity in both regions although male recombination occurs at PAR2 to a much lower extent (at least one recombination event at PAR1 and in ≈1% in normal male meioses at PAR2). Furthermore, we demonstrate that both PARs harbor significantly different allele frequencies between X and Y chromosomes, which could support that recombination is not sufficient to homogenize the pseudoautosomal gene pool or is counterbalanced by other evolutionary forces. Nevertheless, the observed patterns of diversity are not entirely explainable by sexually antagonistic selection. A better understanding of such processes requires new data from intergenerational transmission studies of PARs, which would be decisive on the elucidation of PARs evolution and their role in male-driven heterosomal aneuploidies.

Xp;Yq Unbalanced Translocation with Pseudoautosomal Region Aberrations in a Natural Two-Generation Transmission.

Translocations involving X and Y chromosomes rarely occur in humans and may affect reproductive function. We investigated an Xp:Yq unbalanced translocation with pseudoautosomal region (PAR) aberrations in a natural two-generation transmission. We report the case of an azoospermic male and his fertile mother without any other abnormal clinical phenotypes, except for short stature. Cytogenetic methods, including karyotyping and fluorescence in situ hybridization (FISH), revealed the translocation. Chromosomal microarray comparative genomic hybridization (array-CGH) was used to investigate the regions of Xp partial deletion and Yq partial duplication. Final chromosome karyotypes in the peripheral blood of the infertile male and his mother were 46,Y,der(X)t(X;Y)(p22.33;q11.22) and 46,X,der(X)t(X;Y)(p22.33;q11.22), respectively. Short-stature-homeobox gene deletion was responsible for the short stature in both subjects. PAR aberrations and AZFc duplication may be a direct genetic risk factor for spermatogenesis. This report further supports the use of routine karyotype analysis, FISH-based technology, and array-CGH analysis to identify derivative chromosomes in a complex rearrangement.

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

Human Spermatogenesis Tolerates Massive Size Reduction of the Pseudoautosomal Region.

Mammalian male meiosis requires homologous recombination between the X and Y chromosomes. In humans, such recombination occurs exclusively in the short arm pseudoautosomal region (PAR1) of 2.699 Mb in size. Although it is known that complete deletion of PAR1 causes spermatogenic arrest, no studies have addressed to what extent male meiosis tolerates PAR1 size reduction. Here, we report two families in which PAR1 partial deletions were transmitted from fathers to their offspring. Cytogenetic analyses revealed that a ∼400-kb segment at the centromeric end of PAR1, which accounts for only 14.8% of normal PAR1 and 0.26% and 0.68% of the X and Y chromosomes, respectively, is sufficient to mediate sex chromosomal recombination during spermatogenesis. These results highlight the extreme recombinogenic activity of human PAR1. Our data, in conjunction with previous findings from animal studies, indicate that the minimal size requirement of mammalian PARs to maintain male fertility is fairly small.

Rare and de novo duplications containing SHOX in clubfoot.

INTRODUCTION: Congenital clubfoot is a common birth defect that affects at least 0.1% of all births. Nearly 25% cases are familial and the remaining are sporadic in inheritance. Copy number variants (CNVs) involving transcriptional regulators of limb development, including PITX1 and TBX4, have previously been shown to cause familial clubfoot, but much of the heritability remains unexplained. METHODS: Exome sequence data from 816 unrelated clubfoot cases and 2645 in-house controls were analysed using coverage data to identify rare CNVs. The precise size and location of duplications were then determined using high-density Affymetrix Cytoscan chromosomal microarray (CMA). Segregation in families and de novo status were determined using qantitative PCR. RESULTS: Chromosome Xp22.33 duplications involving SHOX were identified in 1.1% of cases (9/816) compared with 0.07% of in-house controls (2/2645) (p=7.98×10-5, OR=14.57) and 0.27% (38/13592) of Atherosclerosis Risk in Communities/the Wellcome Trust Case Control Consortium 2 controls (p=0.001, OR=3.97). CMA validation confirmed an overlapping 180.28 kb duplicated region that included SHOX exons as well as downstream non-coding regions. In four of six sporadic cases where DNA was available for unaffected parents, the duplication was de novo. The probability of four de novo mutations in SHOX by chance in a cohort of 450 sporadic clubfoot cases is 5.4×10-10. CONCLUSIONS: Microduplications of the pseudoautosomal chromosome Xp22.33 region (PAR1) containing SHOX and downstream enhancer elements occur in ~1% of patients with clubfoot. SHOX and regulatory regions have previously been implicated in skeletal dysplasia as well as idiopathic short stature, but have not yet been reported in clubfoot. SHOX duplications likely contribute to clubfoot pathogenesis by altering early limb development.

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation1,2. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.

Gene mapping and genomic prediction of bull fertility using sex chromosome markers.

Service sire has been recognized as an important factor affecting dairy herd fertility. Our group has reported promising results on gene mapping and genomic prediction of dairy bull fertility using autosomal SNP markers. Little is known, however, about the genetic contribution of sex chromosomes, which are enriched in genes related to sexual development and reproduction. As such, the main goal of this study was to investigate the effect of SNP markers on X and Y chromosomes (BTAX and BTAY, respectively) on sire conception rate (SCR) in US Holstein bulls. The analysis included a total of 5,014 bulls with SCR records and genotypes for roughly 291k SNP located on the autosomes, 1.5k SNP located on the pseudoautosomal region (PAR), 13.7k BTAX-specific SNP, and 24 BTAY-specific SNP. We first performed genomic scans of the sex chromosomes, and then we evaluated the genomic prediction of SCR including BTAX SNP markers in the predictive models. Two markers located on PAR and 3 markers located on the X-specific region showed significant associations with sire fertility. Interestingly, these regions harbor genes, such as FAM9B, TBL1X, and PIH1D3, that are directly implicated in testosterone concentration, spermatogenesis, and sperm motility. On the other hand, BTAY showed very low genetic variability, and none of the segregating markers were associated with SCR. Notably, model predictive ability was largely improved by including BTAX markers. Indeed, the combination of autosomal with BTAX SNP delivered predictive correlations around 0.343, representing an increase in accuracy of about 7.5% compared with the standard whole autosomal genome approach. Overall, this study provides evidence of the importance of both PAR and X-specific regions in male fertility in dairy cattle. These findings may help to improve conception rates in dairy herds through accurate genome-guided decisions on bull fertility.

Evolution of the Autism-Associated Neuroligin-4 Gene Reveals Broad Erosion of Pseudoautosomal Regions in Rodents.

Variants in genes encoding synaptic adhesion proteins of the neuroligin family, most notably neuroligin-4, are a significant cause of autism spectrum disorders in humans. Although human neuroligin-4 is encoded by two genes, NLGN4X and NLGN4Y, that are localized on the X-specific and male-specific regions of the two sex chromosomes, the chromosomal localization and full genomic sequence of the mouse Nlgn4 gene remain elusive. Here, we analyzed the neuroligin-4 genes of numerous rodent species by direct sequencing and bioinformatics, generated complete drafts of multiple rodent neuroligin-4 genes, and examined their evolution. Surprisingly, we find that the murine Nlgn4 gene is localized to the pseudoautosomal region (PAR) of the sex chromosomes, different from its human orthologs. We show that the sequence differences between various neuroligin-4 proteins are restricted to hotspots in which rodent neuroligin-4 proteins contain short repetitive sequence insertions compared with neuroligin-4 proteins from other species, whereas all other protein sequences are highly conserved. Evolutionarily, these sequence insertions initiate in the clade eumuroidea of the infraorder myomorpha and are additionally associated with dramatic changes in noncoding sequences and gene size. Importantly, these changes are not exclusively restricted to neuroligin-4 genes but reflect major evolutionary changes that substantially altered or even deleted genes from the PARs of both sex chromosomes. Our results show that despite the fact that the PAR in rodents and the neuroligin-4 genes within the rodent PAR underwent massive evolutionary changes, neuroligin-4 proteins maintained a highly conserved core structure, consistent with a substantial evolutionary pressure preserving its physiological function.

A 105 kb interstitial insertion in the Xq27.1 palindrome from pseudoautosomal region PAR1 causes a novel X-linked recessive compound phenotype.

BACKGROUND: Genomic disorders present a wide spectrum of unrelated clinical entities that result from genomic rearrangements. Interstitial insertions requiring three points of breakage are rare genomic rearrangement events. The pseudoautosomal region PAR1, homologous between the Xp22 and Yp11 loci, has a high crossover and recombination rate. A 180 bp human-specific palindrome at Xq27.1 appears to be a hotspot for genomic rearrangement, and several genetic diseases/phenotypes associated with Xq27.1 palindrome-driven genomic rearrangement have been reported. Here we investigate a Chinese family with an extremely rare X-linked compound phenotype that remains undiagnosed. We attempt to identify underlying genetic causes by an integrated genome analysis. METHODS: A five-generation Chinese family with a distinct X-linked compound phenotype was recruited. Peripheral blood samples were collected and genomic DNA was extracted. Systemic physical and lab examinations were performed to evaluate the phenotype. An integrated genomic analysis was performed. Genotyping and linkage analysis were conducted to map the disease locus. Whole exome sequencing was performed to detect mutations in coding region. Whole genome sequencing was used to detect single nucleotide variations, small insertions, small deletions, or large structural variations. Copy number variation scanning was also performed on the genome scale. Interstitial insertion was confirmed by gap-PCR and quantitative-PCR, and breakpoint junctions were identified by genome walking and direct sequencing. Expression of products of genes nearby to the Xq27.1 palindrome was measured in peripheral blood from patients and unrelated controls via quantitative-PCR. RESULTS: The identified compound phenotype of genu varum, cubitus valgus, and everted lipsdoes not match any reported clinical entities. Fine mapping and linkage analysis identified a candidate interval of 4 Mb on the X chromosome. No potential coding region mutations were detected. A 105 kb genomic fragment of PAR1 containing no coding genes was duplicated and inserted into the center of a human-specific palindrome at Xq27.1. The interstitial insertion fully cosegregated with the family phenotype. No expression of FGF13 or SOX3 was detected in peripheral blood from the proband or unrelated controls. CONCLUSION: We report an extremely rare phenotype associated with an infrequently-seen genomic rearrangement. The novel compound phenotype is X-linked and characterized by genu varum, cubitus valgus, and everted lips. A 105 kb interstitial insertion of a PAR1 fragment into the Xq27.1 palindrome is associated with the phenotype in the family. The present study identified the underlying genetic cause of the phenotype, expanding the spectrum of known human-specific Xq27.1 palindrome insertion events and associated phenotypes.

Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes.

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.

Instability of the Pseudoautosomal Boundary in House Mice.

Faithful segregation of homologous chromosomes at meiosis requires pairing and recombination. In taxa with dimorphic sex chromosomes, pairing between them in the heterogametic sex is limited to a narrow interval of residual sequence homology known as the pseudoautosomal region (PAR). Failure to form the obligate crossover in the PAR is associated with male infertility in house mice (Mus musculus) and humans. Yet despite this apparent functional constraint, the boundary and organization of the PAR is highly variable in mammals, and even between subspecies of mice. Here, we estimate the genetic map in a previously documented expansion of the PAR in the M. musculus castaneus subspecies and show that the local recombination rate is 100-fold higher than the autosomal background. We identify an independent shift in the PAR boundary in the M. musculus musculus subspecies and show that it involves a complex rearrangement, but still recombines in heterozygous males. Finally, we demonstrate pervasive copy-number variation at the PAR boundary in wild populations of M. m. domesticus, M. m. musculus, and M. m. castaneus Our results suggest that the intensity of recombination activity in the PAR, coupled with relatively weak constraints on its sequence, permit the generation and maintenance of unusual levels of polymorphism in the population of unknown functional significance.

Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

Short communication: Identification of the pseudoautosomal region in the Hereford bovine reference genome assembly ARS-UCD1.2.

In cattle, the X chromosome accounts for approximately 3 and 6% of the genome in bulls and cows, respectively. In spite of the large size of this chromosome, very few studies report analysis of the X chromosome in genome-wide association studies and genomic selection. This lack of genetic interrogation is likely due to the complexities of undertaking these studies given the hemizygous state of some, but not all, of the X chromosome in males. The first step in facilitating analysis of this gene-rich chromosome is to accurately identify coordinates for the pseudoautosomal boundary (PAB) to split the chromosome into a region that may be treated as autosomal sequence (pseudoautosomal region) and a region that requires more complex statistical models. With the recent release of ARS-UCD1.2, a more complete and accurate assembly of the cattle genome than was previously available, it is timely to fine map the PAB for the first time. Here we report the use of SNP chip genotypes, short-read sequences, and long-read sequences to fine map the PAB (X chromosome:133,300,518) and simultaneously determine the neighboring regions of reduced homology and true pseudoautosomal region. These results greatly facilitate the inclusion of the X chromosome in genome-wide association studies, genomic selection, and other genetic analysis undertaken on this reference genome.

Dosage Compensation of the X Chromosomes in Bovine Germline, Early Embryos, and Somatic Tissues.

Dosage compensation of the mammalian X chromosome (X) was proposed by Susumu Ohno as a mechanism wherein the inactivation of one X in females would lead to doubling the expression of the other. This would resolve the dosage imbalance between eutherian females (XX) versus male (XY) and between a single active X versus autosome pairs (A). Expression ratio of X- and A-linked genes has been relatively well studied in humans and mice, despite controversial results over the existence of upregulation of X-linked genes. Here we report the first comprehensive test of Ohno's hypothesis in bovine preattachment embryos, germline, and somatic tissues. Overall an incomplete dosage compensation (0.5 < X:A < 1) of expressed genes and an excess X dosage compensation (X:A > 1) of ubiquitously expressed "dosage-sensitive" genes were seen. No significant differences in X:A ratios were observed between bovine female and male somatic tissues, further supporting Ohno's hypothesis. Interestingly, preimplantation embryos manifested a unique pattern of X dosage compensation dynamics. Specifically, X dosage decreased after fertilization, indicating that the sperm brings in an inactive X to the matured oocyte. Subsequently, the activation of the bovine embryonic genome enhanced expression of X-linked genes and increased the X dosage. As a result, an excess compensation was exhibited from the 8-cell stage to the compact morula stage. The X dosage peaked at the 16-cell stage and stabilized after the blastocyst stage. Together, our findings confirm Ohno's hypothesis of X dosage compensation in the bovine and extend it by showing incomplete and over-compensation for expressed and "dosage-sensitive" genes, respectively.

Recombination hotspots in an extended human pseudoautosomal domain predicted from double-strand break maps and characterized by sperm-based crossover analysis.

The human X and Y chromosomes are heteromorphic but share a region of homology at the tips of their short arms, pseudoautosomal region 1 (PAR1), that supports obligate crossover in male meiosis. Although the boundary between pseudoautosomal and sex-specific DNA has traditionally been regarded as conserved among primates, it was recently discovered that the boundary position varies among human males, due to a translocation of ~110 kb from the X to the Y chromosome that creates an extended PAR1 (ePAR). This event has occurred at least twice in human evolution. So far, only limited evidence has been presented to suggest this extension is recombinationally active. Here, we sought direct proof by examining thousands of gametes from each of two ePAR-carrying men, for two subregions chosen on the basis of previously published male X-chromosomal meiotic double-strand break (DSB) maps. Crossover activity comparable to that seen at autosomal hotspots was observed between the X and the ePAR borne on the Y chromosome both at a distal and a proximal site within the 110-kb extension. Other hallmarks of classic recombination hotspots included evidence of transmission distortion and GC-biased gene conversion. We observed good correspondence between the male DSB clusters and historical recombination activity of this region in the X chromosomes of females, as ascertained from linkage disequilibrium analysis; this suggests that this region is similarly primed for crossover in both male and female germlines, although sex-specific differences may also exist. Extensive resequencing and inference of ePAR haplotypes, placed in the framework of the Y phylogeny as ascertained by both Y microsatellites and single nucleotide polymorphisms, allowed us to estimate a minimum rate of crossover over the entire ePAR region of 6-fold greater than genome average, comparable with pedigree estimates of PAR1 activity generally. We conclude ePAR very likely contributes to the critical crossover function of PAR1.