Cytological evidence that the Sxr fragment of XY,Sxr mice pairs homologously at meiotic prophase with the proximal testis-determining region

Self-pairing of the Y chromosome at prophase of meiosis in XY,Sxr male mice appears to take place in many cells to the exclusion of pairing between the Y and the X. This phenomenon offers an explanation for the high level of X-Y separation seen in these males at prophase of meiosis, additional separations being evident, however, when metaphase I (MI) cells are examined. A minority of prophase cells show the Y paired both autologously and with a sub-terminal region of the X which could be the normal pairing region. The balloon-like configurations observed when self-pairing occurs suggest that the distal Sxr fragment is inverted on the Y chromosome of Sxr carrier males in relation to the normal proximal testis-determining (Td)-containing region.     

Recombination between the X and Y chromosomes and the Sxr region of the mouse.

The Sxr (sex-reversed) region that carries a copy of the mouse Y chromosomal testis-determining gene can be attached to the distal end of either the Y or the X chromosome. During male meiosis, Sxr recombined freely between the X and Y chromosomes, with an estimated recombination frequency not significantly different from 50% in either direction. During female meiosis, Sxr recombined freely between the X chromosome to which it was attached and an X-autosome translocation. A male mouse carrying the original Sxra region on its Y chromosome, and the shorter Sxrb variant on the X, also showed 50% recombination between the sex chromosomes. Evidence of unequal crossing-over between the two Sxr regions was obtained: using five markers deleted from Sxrb, 3 variant Sxr regions were detected in 159 progeny (1.9%). Four other variants (one from the original cross and three from later generations) were presumed to have been derived from illegitimate pairing and crossing-over between Sxrb and the homologous region on the short arm of the Y chromosome. The generation of new variants throws light on the arrangement of gene loci and other markers within the short arm of the mouse Y chromosome.     

The tricky path to recombining X and Y chromosomes in meiosis

Sex chromosomes are the Achilles' heel of male meiosis in mammals. Mis-segregation of the X and Y chromosomes leads to sex chromosome aneuploidies, with clinical outcomes such as infertility and Klinefelter syndrome. Successful meiotic divisions require that all chromosomes find their homologous partner and achieve recombination and pairing. Sex chromosomes in males of many species have only a small region of homology (the pseudoautosomal region, PAR) that enables pairing. Until recently, little was known about the dynamics of recombination and pairing within mammalian X and Y PARs. Here, we review our recent findings on PAR behavior in mouse meiosis. We uncovered unexpected differences between autosomal chromosomes and the X-Y chromosome pair, namely that PAR recombination and pairing occurs later, and is under different genetic control. These findings imply that spermatocytes have evolved distinct strategies that ensure successful X-Y recombination and chromosome segregation.     

Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis

In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.     

Synapsis and obligate recombination between the sex chromosomes of male laboratory mice carrying the Y* rearrangement.

The synaptic and recombinational behavior of the sex chromosomes in male laboratory mice carrying the Y* rearrangement was analyzed by light and electron microscopy. Examination of zygotene and pachytene X-Y* configurations revealed a surprising paucity of the staggered pairing configuration predicted from the distal position of the X pseudoautosomal region and the subcentromeric position of the Y* pseudoautosomal region. When paired at pachynema, the X and Y* chromosomes usually assumed configurations similar to those of typical sex bivalents from normal male laboratory mice. The X and Y* chromosomes were present as univalents in more than half of the early- and mid-pachytene nuclei, presumably as a result of steric difficulties associated with homologous alignment of the pseudoautosomal regions. When paired at diakinesis and metaphase I, the X and Y* chromosomes exhibited an asymmetrical chiasmatic association indicative of recombination within the staggered synaptic configuration. Both pairing disruption and recombinational failure apparently contribute to diakinesis/metaphase I sex-chromosome univalency, as most cells at these stages possessed X and Y* univalents lacking evidence of prior recombination. Recombinant X or Y* chromosomes were detected in all metaphase II complements examined, thus substantiating the hypothesis that X-Y recombination is a prerequisite for the normal progression of male meiosis.     

Physical mapping of the human pseudo-autosomal region; comparison with genetic linkage map.

A long-range restriction map of the pseudo-autosomal or exchange pairing region (corresponding to the terminal parts of the short arms of the human sex chromosomes) has been established using pulsed field gel electrophoresis. A total of seven loci have been located on this physical map based essentially on the analysis of 45,X Turner genomes. The region spans a total of 2600 kb. The 5' end of the MIC2 gene maps at less than 80 kb from the proximal pseudo-autosomal boundary. Since the total pseudo-autosomal linkage interval represents approximately 50% of recombination at male meiosis, 1 cM corresponds to 50-60 kb. This is consistent with the almost 20-fold increase in recombination frequency observed in male versus female meiosis in this region. The present data show no distortion between both physical and linkage maps. The distribution of the CpG-rich restriction sites is notably disequilibrated. A large subset of these sites is concentrated within the 500 kb closest to the telomere whereas others appear in clusters (probably HTF islands) scattered in the rest of the pseudo-autosomal region.     

Meiotic studies in mice carrying the sex reversal (Sxr) factor

A sex reversal factor (Sxr) that causes mice having apparently normal X chromosomes to become phenotypically male is transmitted in an autosomal pattern. The origin of the Sxr factor is still unknown. It seems most likely that it has originated from an autosomal gene mutation or is the result of a translocation of part of the Y chromosome to one of the autosomes. Chromosomes from four XY and six XO mice carrying this sex reversal factor were examined in the diakinesis stage of meiosis. The following unusual observations were noted: (1) in XY males carrying the Sxr factor, the X and Y chromosomes were separated more often than in controls. (2) The Y chromosome tends to be closer to an autosome when the X and Y are separate than when the X and Y are attached. (3) A chromosome fragment was present in 4/226 cells from two XO males and a single cell from an XY, Sxr carrier. Although there is no direct evidence, these observations seem to favor the possibility that the Sxr factor involves a chromosomal rearrangement rather than a single gene mutation.     

Spermatogenesis in XO,Sxr mice: role of the Y chromosome

The goal of this investigation was to evaluate the role of the Y chromosome in spermatogenesis by a quantitative and qualitative analysis of spermatogenesis as it occurs in the absence of a significant portion of the Y chromosome, i.e., in XO,Sxr male mice. Although these mice have the testis-determining portion of the Y chromosome on their single X chromosome, they lack most of the Y chromosome. Since it was found that all sperm-specific structures were assembled in a normal spatial and temporal pattern in spermatids of XO,Sxr mice, the genes controlling these structures cannot be located on the Y chromosome outside of the Sxr region, and are more likely to be on autosomes or on the X chromosome. In spite of the assembly of the correct sperm-specific structures, spermatogenesis was not quantitatively normal in XO,Sxr mice and significantly reduced numbers of spermatids were found in the seminiferous tubules of these mice. Furthermore, two size classes of spermatids were found in the testes of XO,Sxr mice, normal and twice-normal size. These findings are suggestive of abnormalities of meiosis in XO,Sxr spermatocytes, which lack one of the two sex chromosomes, and may not implicate function of specific genes on the Y chromosome. Morphological abnormalities of spermatids, which were not unique to XO,Sxr mice, were observed and these may be due to either a defective testicular environment because of reduced numbers of germ cells or to the lack of critical Y chromosome-encoded products. Since pachytene spermatocytes of XO,Sxr mice exhibited a sex vesicle, it can be concluded that the assembly of this structure does not depend on the presence of either a complete Y chromosome or the pairing partner for the X chromosome.     

Sex-chromosome pairing: evidence that the behavior of the pseudoautosomal region differs during male and female meiosis.

In humans, recombination in the pseudoautosomal region is approximately 10-fold higher in males than in females. This difference is thought to reflect the fact that, in females, there is opportunity for genetic exchange along the entire length of the X chromosome, resulting in a relative reduction in the likelihood of exchange in the pseudoautosomal region. In two instances in the laboratory mouse where X-chromosome pairing and exchange in females are limited to the pseudoautosomal region, a significant level of X-chromosome pairing failure was observed at diakinesis/metaphase I. Further analysis indicated that, in female meiosis, the inability of the X chromosome to consistently form a pairing configuration via the pseudoautosomal region alone is not a property of the pseudoautosomal region per se but is due to the fact that it resides on an X chromosome. Thus previously reported sex-linked differences in recombination rate in the pseudoautosomal region may actually reflect differences in pairing and/or recombination of the pseudoautosomal region on an X chromosome undergoing male versus female meiosis.     

Sex chromosome pairing patterns in male mice of novel Sxr genotypes

The influence of the sex-reversal factor (Sxr) on X and Y chromosome pairing was examined by comparing males with novel and standard Sxr genotypes. The novel Sxr males were exceptional in carrying Sxr on their X rather than their Y chromosome, or homozygously on both their X and Y chromosomes, or on a Y chromosome of different origin to that on which the factor arose. Regardless of its chromosomal location, Sxr was found to elevate the frequency of X-Y separation. Univalent X and Y chromosomes were observed to undergo self-association in a variable proportion of spermatocytes of all Sxr-carrying males. There was a suggestion that chromosomal location of the factor could influence the frequency of univalent self-association. Our observations do not support the published hypothesis of Y self-pairing as the cause of the elevated rate of X-Y separation at pachytene in Sxr-carrying males. Rather, they suggest that heterozygosity due to the presence of Sxr in the XY pairing region may be sufficient to disrupt pairing and cause univalence, or alternatively, that Sxr is an inefficient promoter of X-Y pairing initiation.     

Heterochromatin variation and spermatogenesis in Nesokia

A probable role of heterochromatin variation in male meiosis has been evaluated using fertile and infertile Indian mole rat males (Nesokia) with polymorphic X and/or Y chromosomes. A comprehensive study of tubular histology, meiotic progression, and X-Y chromosome pairing was undertaken. Despite heterochromatin variation, spermatogenesis was found to be complete in all individuals. Patterns of X-Y synaptonemal complex pairing varied considerably from extensive synapsis in individuals with a normal heterochromatin complement, through end-to-end synapsis, to X and Y univalents in those with different degrees of loss of heterochromatin. Changes in the gonadal histology corresponding to heterochromatin variation were also observed. Loss of some coding DNA sequences in polymorphic X-chromosomes otherwise located at specific sites in the X-chromosome heterochromatin have been linked directly to modifications of the reproductive process. This is thought to be mediated by an altered X-chromosome activity during spermatogenesis or regulation of other locus/loci involved in fertility or reproduction.     

Male sterility and meiotic drive associated with sex chromosome rearrangements in Drosophila. Role of X-Y pairing.

In Drosophila melanogaster, deletions of the pericentromeric X heterochromatin cause X-Y nondisjunction, reduced male fertility and distorted sperm recovery ratios (meiotic drive) in combination with a normal Y chromosome and interact with Y-autosome translocations (T(Y;A)) to cause complete male sterility. The pericentromeric heterochromatin has been shown to contain the male-specific X-Y meiotic pairing sites, which consist mostly of a 240-bp repeated sequence in the intergenic spacers (IGS) of the rDNA repeats. The experiments in this paper address the relationship between X-Y pairing failure and the meiotic drive and sterility effects of Xh deletions. X-linked insertions either of complete rDNA repeats or of rDNA fragments that contain the IGS were found to suppress X-Y nondisjunction and meiotic drive in Xh-/Y males, and to restore fertility to Xh-/T(Y;A) males for eight of nine tested Y-autosome translocations. rDNA fragments devoid of IGS repeats proved incapable of suppressing either meiotic drive or chromosomal sterility. These results indicate that the various spermatogenic disruptions associated with X heterochromatic deletions are all consequences of X-Y pairing failure. We interpret these findings in terms of a novel model in which misalignment of chromosomes triggers a checkpoint that acts by disabling the spermatids that derive from affected spermatocytes.     

Adding the heterochromatic YL arm to an X chromosome reduces reproductive fitnesses in Drosophila melanogaster: implications for the evolution of rDNA, heterochromatin, and reproductive isolation

For X-Y exchange to be of importance in the coevolution of X and Y rDNA, there must be a mechanism to maintain cytologically normal X chromosomes in the face of continual infusions of X.YL chromosomes produced by X-Y exchanges. Replicated populations were founded with different frequencies of isogenic X and X.YL chromosomes. The X.YL chromosome declined in frequency over time in all lines. Relative fitnesses, estimated from chromosome frequency trajectories, were 0.40, 1.01, and 1.0 for X.YL/X.YL, X.YL/X, and X/X females and 0.75 and 1.0 for X.YL/Y and X/Y males, respectively. The equilibrium frequency for the X.YL chromosome due to the balance between X-Y exchange and selection was predicted to be 4-16 x 10(-4). The results strengthen the evidence for the involvement of X-Y exchange in the coevolution of X and Y rDNA arrays. Conditions for the evolution of reproductive isolation by sex-chromosome translocation are much less probable than previously supposed since the X.YL translocation chromosome is at a selective disadvantage to cytologically normal X chromosomes. Additional heterochromatin was not neutral but was only deleterious beyond a threshold, as one dose of the heterochromatic XL arm did not reduce female reproductive fitness, but two doses did.     

Steroid sulfatase gene in XX males.

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage of the Steroid Sulfatase Gene to the Sex-Reversed Mutation in the Mouse

Dosage studies and the inheritance pattern of the gene for steroid sulfatase (Sts) in the mouse have previously provided indirect evidence for a functional Y-linked allele which recombines obligatorily with its X-linked allele in male meiosis. In this study, we have investigated the linkage relationship of Sts and the sex-reversed mutation (Sxr), a gene which is known to reside in the pairing region of the Y chromosome. The results clearly demonstrate that Sxr and Sts are linked in a region of obligatory recombination and Sts maps proximal to Sxr with most recombinants occurring proximal to the two genes.     

Genetic Analysis of Sex Chromosomal Meiotic Mutants in DROSOPHILA MELANOGASTER

A total of 209 ethyl methanesulfonate-treated X chromosomes were screened for meiotic mutants that either (1) increased sex or fourth chromosome nondisjunction at either meiotic division in males; (2) allowed recombination in such males; (3) increased nondisjunction of the X chromosome at either meiotic division in females; or (4) caused such females, when mated to males heterozygous for Segregation-Distorter (SD) and a sensitive homolog to alter the strength of meiotic drive in males.-Twenty male-specific meiotic mutants were found. Though the rates of nondisjunction differed, all twenty mutants were qualitatively similar in that (1) they alter the disjunction of the X chromosome from the Y chromosome; (2) among the recovered sex-chromosome exceptional progeny, there is a large excess of those derived from nullo-XY as compared to XY gametes; (3) there is a negative correlation between the frequency of sex-chromosome exceptional progeny and the frequency of males among the regular progeny. In their effects on meiosis these mutants are similar to In(1)sc(4L)sc(8R), which is deleted for the basal heterochromatin. These mutants, however, have normal phenotypes and viabilities when examined as X/0 males, and furthermore, a mapping of two of the mutants places them in the euchromatin of the X chromosome. It is suggested that these mutants are in genes whose products are involved in insuring the proper functioning of the basal pairing sites which are deleted in In(1)sc(4L)sc(8R), and in addition that there is a close connection, perhaps causal, between the disruption of normal X-Y pairing (and, therefore, disjunction) and the occurrence of meiotic drive in the male.-Eleven mutants were found which increased nondisjunction in females. These mutants were characterized as to (1) the division at which they acted; (2) their effect on recombination; (3) their dominance; (4) their effects on disjunction of all four chromosome pairs. Five female mutants caused a nonuniform decrease in recombination, being most pronounced in distal regions, and an increase in first division nondisjunction of all chromosome pairs. Their behavior is consistent with the hypothesis that these mutants are defective in a process which is a precondition for exchange. Two female mutants were allelic and caused a uniform reduction in recombination for all intervals (though to different extents for the two alleles) and an increase in first-division nondisjunction of all chromosomes. Limited recombination data suggest that these mutants do not alter coincidence, and thus, following the arguments of Sandler et al. (1968), are defective in exchange rather than a precondiiton for exchange. A single female mutant behaves in a manner that is consistent with it being a defect in a gene whose functioning is essential for distributive pairing. Three of the female meiotic mutants cause abnormal chromosome behavior at a number of times in meiosis. Thus, nondisjunction at both meiotic divisions is increased, recombinant chromosomes nondisjoin, and there is a polarized alteration in recombination.-The striking differences between the types of control of meiosis in the two sexes is discussed and attention is drawn to the possible similarities between (1) the disjunction functions of exchange and the process specified by the chromosome-specific male mutants; and (2) the prevention of functional aneuploid gamete formation by distributive disjunction and meiotic drive.     

X-Y chromosome synapsis and recombination in 3 vole species of Asian lineage of the genus Microtus (Rodentia: Arvicolinae)

The pattern of X-Y chromosome pairing in male meiosis is an important taxonomic feature of grey voles of the genus Microtus. Asynaptic sex chromosomes have been found in the majority of species of the Palearctic phylogenetic lineage of this genus, while normal X-Y synapsis has been observed in the species of subgenus Pallasiinus belonging to the Asian phylogenetic lineage. We analyzed sex chromosome pairing and recombination in M. maximowiczii, M. mujanensis and M. fortis which also belong to the Asian phylogenetic lineage (subgenus Alexandromys). Using immunostaining for the proteins of the synaptonemal complex (SCP3) and recombination nodules (MLH1) we demonstrated that X and Y chromosomes of these species paired and recombined in a short subtelomeric region. This indicates that the sex chromosomes of these species retain an ancestral fully functional pseudoautosomal region, which has been lost or rearranged in the asynaptic species of the genus Microtus.     

Mammalian sex chromosomes

The X and Y chromosomes of the musk shrew are the two largest in the complement and they regularly form a single chiasma during meiosis. This chiasma is located in the short arms of the X and Y, both of which show partial C-banding at meiosis. The in vitro incorporation of 5-bromodeoxyuridine/tritiated thymidine during late S reveals that the non-C-band region of the Y finishes replication later than the C-band positive heterochromatin. During meiosis, the sex bivalent opens out early in pachytene to reveal a single chiasma which persists until late metaphase-I. In surface-spread, silver-stained meiocytes, the sex bivalent morphology changes from a phase of extensive pairing to one which includes a visible chiasma through a brief diffuse stage. Observations on C-banded meiocytes show a shift in the sex pair from a C-band positive to a negative state as compared to their corresponding somatic pattern. Comparable changes are also observed in the sex bivalents of other mammals which undergo a chiasmatic exchange. This suggests that in addition to pairing homology, an alteration in the chromatin configuration may be necessary for crossing over to occur between the sex chromosomes.     

The Y-encoded gene zfy2 acts to remove cells with unpaired chromosomes at the first meiotic metaphase in male mice

During male but not female mammalian meiosis, there is efficient apoptotic elimination of cells with unpaired (univalent) chromosomes at the first meiotic metaphase (MI) [1]. Apoptotic elimination of MI spermatocytes is seen in response to the univalent X chromosome of XSxr(a)O male mice [2], in which the X chromosome carries Sxr(a) [3, 4], the Y-chromosome-derived sex-reversal factor that includes the testis determinant Sry. Sxr(b) is an Sxr(a)-derived variant in which a deletion has removed six Y short-arm genes and created a Zfy2/Zfy1 fusion gene spanning the deletion breakpoint [4, 5]. XSxr(b)O males have spermatogonial arrest that can be overcome by the re-addition of Eif2s3y from the deletion as a transgene; however, XSxr(b)OEif2s3y transgenic males do not show the expected elimination of MI spermatocytes in response to the univalent [6]. Here we show that these XSxr(b)OEif2s3y males have an impaired apoptotic response with completion of the first meiotic division, but there is no second meiotic division. We then show that Zfy2 (but not the closely related Zfy1) is sufficient to reinstate the apoptotic response to the X univalent. These findings provide further insight into the basis for the much lower transmission of chromosomal errors originating at the first meiotic division in men than in women [7].     

Sex Chromosome Meiotic Drive in DROSOPHILA MELANOGASTER Males

In Drosophila melanogaster males, deficiency for X heterochromatin causes high X-Y nondisjunction and skewed sex chromosome segregation ratios (meiotic drive). Y and XY classes are recovered poorly because of sperm dysfunction. In this study it was found that X heterochromatic deficiencies disrupt recovery not only of the Y chromosome but also of the X and autosomes, that both heterochromatic and euchromatic regions of chromosomes are affected and that the "sensitivity" of a chromosome to meiotic drive is a function of its length. Two models to explain these results are considered. One is a competitive model that proposes that all chromosomes must compete for a scarce chromosome-binding material in Xh(-) males. The failure to observe competitive interactions among chromosome recovery probabilities rules out this model. The second is a pairing model which holds that normal spermiogenesis requires X-Y pairing at special heterochromatic pairing sites. Unsaturated pairing sites become gametic lethals. This model fails to account for autosomal sensitivity to meiotic drive. It is also contradicted by evidence that saturation of Y-pairing sites fails to suppress meiotic drive in Xh(- ) males and that extra X-pairing sites in an otherwise normal male do not induce drive. It is argued that meiotic drive results from separation of X euchromatin from X heterochromatin.