Smcy transgene does not rescue spermatogenesis in sex-reversed mice.

In mouse, the Sxr(b) deletion interval (delta Sxr(b)) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1-2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr(b) interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr(b)/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.

Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, or a limited, role in spermatogenesis but underlines a recent African origin for human populations.

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provided a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences were observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gave a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. We therefore hypothesize that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55 000-200 000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations showed that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race.

Transposition of RhoA to the murine Y chromosome.

In an effort to produce a more complete transcription map of the short (approximately 5 Mb) arm of the mouse Y chromosome, we have initiated exon trapping from Yp-derived YACs. Sequence analysis of the trapped products has identified exons of previously cloned mouse Y-located genes Zfy and SSty and potential exons homologous to the human Y-located Tspy gene family. In addition, a family of three Yp-located transcripts that show close homology to human RHOA (locus designation ARHA), a member of the Ras family of small GTPases, has been identified. To determine whether these Yp sequences had been transposed from an autosomal ancestor, we used this trapped product to isolate a full-length autosomal mouse RhoA cDNA that is 80% identical at the nucleotide level and 98% identical at the amino acid level to human RHOA and maps to mouse Chromosome 2 (locus designation ArhA). Sequence analysis indicates that the Y-linked copies have diverged from the autosomal form, with small deletions precluding maintenance of a significant open reading frame in all Yp copies. Yet RT-PCR analysis indicates that two of these pseudogenes, RhoAy1 and 3, are expressed in a testis-specific manner, in sharp contrast to the nearly ubiquitous expression pattern of the autosomal ancestor. The data indicate that the Y copies of RhoA have been transposed from an autosome, followed by subsequent duplication, sequence divergence, and acquisition of a testis-specific promoter/enhancer.

A murine TSPY.

Sequences homologous to human and bovine TSPY were isolated from M. musculus testicular cDNA, and a nearly full-length gene was polymerase chain reaction (PCR) amplified from mouse genomic DNA. This gene is apparently non-functional. Contrary to the situation encountered in species along the primate and artiodactyl lineages, in which TSPY is moderately repetitive, murine Tspy appears to be single copy. Murine Tspy is located on Yp, i.e. in the same syntenic group as in man. Sequence comparisons of murine, human and bovine TSPY exons suggest that TSPY became non-functional during rodent evolution.

The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition.

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven.

Mammalian evolution is believed to be male driven because the greater number of germ cell divisions per generation in males increases the opportunity for errors in DNA replication. Since the Y Chromosome (Chr) replicates exclusively in males, its genes should also evolve faster than X or autosomal genes. In addition, estimating the overall male-to-female mutation ratio (alpha m) is of great importance as a large alpha m implies that replication-independent mutagenic events play a relatively small role in evolution. A small alpha m suggests that the impact of these factors may, in fact, be significant. In order to address this problem, we have analyzed the rates of evolution in the homologous X-Y common SMCX/SMCY genes from three different species--mouse, human, and horse. The SMC genes were chosen because the X and Y copies are highly homologous, well conserved in evolution, and in all probability functionally interchangeable. Sequence comparisons and analysis of synonymous substitutions in approximately 1kb of the 5' coding region of the SMC genes reveal that the Y-linked copies are evolving approximately 1.8 times faster than their X homologs. The male-to-female mutation ratio alpha m was estimated to be 3. These data support the hypothesis that mammalian evolution is male driven. However, the ratio value is far smaller than suggested in earlier works, implying significance of replication-independent mutagenic events in evolution.

Absence of DAZ gene mutations in cases of non-obstructed azoospermia.

Sequenced-tagged site (STS) analysis of the Y chromosome long arm (Yq) of azoospermic males has identified a minimum common deleted region of several hundred kilobases in approximately 13% of cases. A candidate azoospermia gene, DAZ (deleted in azoospermia), has been isolated from this region. DAZ has also been shown to be absent in severely oligozoospermic males albeit at a much lower frequency. These data, although highly suggestive, do not constitute formal proof that DAZ actually plays a role in azoospermia, as no small intragenic deletions, rearrangements or point mutations in the gene have been found. In this study we report the screening of DNA from 168 azoospermic/oligospermic males for the presence of the DAZ gene. Deletions involving DAZ were detected in five out of 43 (11.6%) azoospermic males whereas none were found in the remaining 125 oligospermic patients. We present the genomic structure of the 5' end of the DAZ gene together with its sequence analysis in 30 non-obstructed azoospermic males. No mutations in DAZ were found in any of the patients sequenced. These data provide no formal proof that DAZ is AZF. Thus the possibility is still valid that another gene(s) mapping to the deletion interval may be responsible for, or contribute to, the observed phenotypes. Alternatively, if DAZ is AZF, they suggest that the most frequent cause of gene inactivation is via large deletions possibly mobilized by Y chromosome repetitive sequences.

Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein.

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by HLA-B7 was identified as an 11-residue peptide derived from SMCY, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, SMCX, differs by two amino acid residues in the same region. The identification of H-Y may aid in transplantation prognosis, prenatal diagnosis, and fertilization strategies.

Identification of a mouse male-specific transplantation antigen, H-Y.

The male-specific transplantation antigen, H-Y, causes rejection of male tissue grafts by genotypically identical female mice and contributes to the rejection of human leukocyte antigen-matched male organ grafts by human females. Although first recognized 40 years ago, the identity of H-Y has remained elusive. T cells detect several distinct H-Y epitopes, and these are probably peptides, derived from intracellular proteins, that are presented at the cell surface with major histocompatibility complex (MHC) molecules. In the mouse, the gene(s) controlling H-Y expression (Hya) are located on the short arm of the Y chromosome between the zinc-finger genes Zfy-1 and Zfy-2. We have recently identified Smcy, a ubiquitously expressed gene, in this region and its X-chromosome homologue, Smcx. Here we report that Smcy encodes an H-YKk epitope that is defined by the octamer peptide TENSGKDI: no similar peptide is found in Smcx. These findings provide a genetic basis for the antigenic difference between males and females that contributes towards a tissue transplant rejection response.

Phenotypic expression of the fused (Fu) gene in chimaeric mice.

The dominant gene Fused (Fu) produces skeletal abnormalities during embryonic development. It was previously shown that C57BL/6 mice contain a suppressor of Fu, which acts after fertilization. Chimaeras were used to study whether this gene would suppress the Fu phenotype after the 8-cell stage of embryo development. We found no effect of the suppressor gene on Fu phenotype (its degree and frequency of expression) in chimaeric mice. We conclude that either the suppressor gene from C57BL/6 mice can only influence Fu expression at the intracellular level or Fu expression is determined before the 8-cell embryonic stage.

A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific minor histocompatibility antigens.

A new mouse Y chromosome gene, Smcy, has been isolated from the region encoding Spy, a spermatogenesis gene and Hya and Sdma, the genes that, respectively, control the expression of the male specific minor histocompatibility antigen H-Y, as measured by specific T-cell assays and the serologically detected male antigen SDMA. Smcy is well conserved on the Y in mouse, man and even marsupials. It is expressed in all adult male tissues tested and can also be detected during mouse development from as early as two cells. In addition, its human Y homologue, SMCY, is expressed in multiple tissues and maps to the same Yq deletion interval as the human H-Y antigen controlling locus, HY.

A novel X gene with a widely transcribed Y-linked homologue escapes X-inactivation in mouse and human.

A new gene, designated Smcx, was cloned from the mouse X chromosome by its homology to the Y located gene Smcy. Using direct in situ hybridisation Smcx was mapped to the distal end of the mouse X chromosome (XF2-XF4) and its human homologue, SMCX, was mapped to proximal Xp (Xp11.1-Xp11.2). Further meiotic mapping in the mouse placed Smcx in the Plp-Pdha1 interval. As Smcx/SMCX have widely expressed homologues on the Y chromosome, they appeared good candidates for genes that escape X-inactivation. In the human we show this to be the case as SMCX is expressed in hamster-human hybrids containing either an active or inactive human X chromosome. Two alleles of Smcx were found to be expressed in T(16;X)16H female mice despite the intact X chromosome being inactive in all cells. This indicates that Smcx is also not subject to X-inactivation and provides the first example of a gene that is expressed from inactive and active X chromosomes in the mouse.

Chromosome pairing and recombination in mice heterozygous for different translocations in chromosomes 16 and 17.

In order to clarify the relationship between meiotic pairing and recombination, an electron microscopic (EM) study of synaptonemal complexes (SC) and an analysis of chiasma frequency and distribution were made in male mice singly and doubly heterozygous for Robertsonian [Rb(16.17)7Bnr] and reciprocal [T(16:17)43H] translocations and also in tertiary trisomics for the proximal region of chromosome 17. In all these genotypes an extensive zone of asynapsis/desynapsis around the breakpoints was revealed. At the same time a high frequency of non-homologous pairing was observed in precentromeric regions of acrocentric chromosomes. The presence in the proximal region of chromosome 17 of the t haplotype did not affect the synaptic behaviour of this region. Chiasma frequency in the proximal region of chromosome 17 in the T(16:17)43H heterozygotes and trisomics was increased when compared with that in Robertsonian heterozygotes.

Two doses of the paternal Tme gene do not compensate the lethality of the Thp deletion.

The hairpin-tail (Thp) deletion in chromosome 17 is lethal when it is inherited from the mother, whereas heterozygotes with Thp deletion that is paternal in origin are viable. The lethal effect of maternal Thp is due to a deficiency of the Tme gene that is located in the Thp-deleted region. In this article we describe analysis of the viability of mice with tertiary trisomy of chromosome 17, Ts(17(16]43H, with different doses of the paternal and maternal Tme alleles. We demonstrate that the presence of an additional copy of the region with the Tme gene in the female gamete entirely compensates maternal Thp lethality. We failed to compensate the absence of the Tme gene from the chromosome of maternal derivation by two doses of Tme derived from the father. Thus evidence was obtained indicating that there are significant differences between the activities of the paternal and maternal alleles of the Tme gene due to chromosome imprinting.

Meiotic drive of t haplotypes: chromosome segregation in mice with tertiary trisomy.

The properties of the t haplotypes, specific mutant states of the proximal region of chromosomes 17 in the house mouse, are of continuing interest. One such property is increased transmission of the t haplotype by heterozygous t/+ males to offspring. Using the reciprocal translocation T(16;17)43H we have constructed males with tertiary trisomy of chromosome 17 (+T43/+ +/Rb7+) carrying the Robertsonian translocation Rb(16.17)7Bnr. Only the progeny of these males which had inherited either T43/+ or Rb7 from their male parent were viable. The segregation patterns in the offspring of t-bearing trisomics were analysed on days 16-18 of embryonic development. It was found that, when the t12 haplotype is in the normal acrocentric (males+ +T43/+ t12 + /Rb7+ +), its presence in the gamete +t12+/+ + T43 does not produce meiotic drive. However, when t6 is in Rb7, meiotic drive was observed: 80% of offspring carried the t haplotype. It is concluded that the meiotic drive is probably inhibited by the presence of a normal homologue of chromosome 17 in the same sperm. Possible mechanisms for the t haplotype effect are discussed.

Meiotic drive in female mice heterozygous for the HSR inserts on chromosome 1.

Chromosome 1 with one or two long insertions has been previously found in natural mouse populations. The inheritance of chromosome 1 with two insertions from the Yakutsk population is analysed in this paper. It was demonstrated that heterozygous females transmit this chromosome to 80-85% of offspring. The observations made at M II, in conjunction with the recombination data, allowed us to conclude that preferential passage of the chromosome 1 with insertions to the oocyte and egg, rather than to the first and second polar bodies at meiosis, is the causative factor of the distorted segregation. A meiotic drive of such potency has not been previously reported for female mammals. The possible mechanism of the drive is discussed.

Gametic imprinting and the manifestation of the fused gene in the house mouse.

The phenomenon of gametic imprinting in mammals has raised developmentally relevant questions concerning the manifestation and inheritance of genes with variable penetrance. The dominant fused (Fu) gene located on chromosome 17 is one of the few good cases demonstrating the phenomenon in mice. The Fu mutation has a maternal effect. We have previously shown that the t12 haplotype significantly lowers the penetrance of Fu in female Fu/t12 offspring. Results of reciprocal matings of the heterozygotes for Fu indicated that the Fu of maternal origin has a lowered level of penetrance. The dominant suppressors located outside chromosome 17, in contrast to t12 residing in it, had stronger effects on the manifestation of Fu, decreasing its penetrance to 8-17%. Experimental evidence is presented that the pathway via which Fu passes to the zygote nucleus during gametogenesis through successive generations has a marked effect on its penetrance. Based on this evidence, patterns of genetic imprinting are described. A survey of genetic imprinting allowed us to distinguish two developmental phases, gametic and zygotic. The hypothesis for the gametic phase of the development of multicellular organisms suggests that it proceeds from initialization, a process thought to ensure the freeing of chromosomes from redundant epigenetic information and their preparation for the consecutive developmental cycle.