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Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous heterogeneous nuclear ribonucleoprotein (hnRNP) that is expressed abundantly in the testis. DAZAP1 deficiency in mice results in growth retardation and spermatogenic arrest. Previous reports on DAZAP1's binding to several naturally occurring splicing mutations support a role for DAZAP1 in RNA splicing. To elucidate the biological function(s) of DAZAP1 and to search for its natural RNA substrates, we used microarrays to compare the expression profiles and exon usages of wild-type and Dazap1 mutant testes and identified three genes (Crem, Crisp2 and Pot1a) with aberrant RNA splicing in the mutant testes. We further demonstrated that DAZAP1, but not DAZAP1 mutant proteins, promoted the inclusion of Crem exon 4, Crisp2 exon 9 and Pot1a exon 4 in splicing reporter transcripts in cultured cells. Additional studies on the binding of DAZAP1 to the exons and their flanking intronic sequences and the effects of minigene deletions on exon inclusion identified regulatory regions in Crem intron 3, Crisp2 intron 9 and Pot1a intron 4 where DAZAP1 bound and regulated splicing. Aberrant splicing of the Pot1a gene, which encodes an essential protein that protects telomere integrity, may partially account for the growth retardation phenotype of DAZAP1-deficient mice.

Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We examined mouse epididymal sperm with immunostaining and transmission electron microscopy (EM) and identified a previously unknown structure on the apical hook. The structure has a coiled configuration around 11 nm in thickness and is present at the tip of each corner of the triangular-rod shaped perforatorium. Surveying sperm isolated from various regions of the epididymis indicated that mouse sperm acquire the hook rim (HR) structure during its passage through the proximal two-thirds of the caput epididymidis. The structure withstands vigorous sonication and harsh chemical treatments and remains intact after the acrosome reaction. Its location and sturdiness suggest a function in protecting the apical hook from mechanical wear during fertilization. Our EM images of epididymal sperm also revealed additional novel structures as well as lateral asymmetry of the sperm head, indicating that mouse sperm head has a structure more complex than previously recognized.

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

Summary Ubiquitin Specific Protease 26 (USP26) is a little studied ubiquitin-specific protease that is expressed specifically in the testis. In humans, some USP26 polymorphisms have been reported to be associated with impaired male fertility. However, how USP26 affects male reproduction remains unclear. We generated an antibody that stained specifically cultured cells expressing an epitope-tagged USP26 and used it to elucidate the biological function of USP26. Immunostaining of mouse testis sections as well as dispersed germ cells showed the presence of USP26 at the blood–testis barrier, near the Sertoli cell–germ cell interface of post-step 7 spermatids, and coating the dorsal surface of sperm head. Further RT-PCR assays detected the expression of Usp26 in germ cells, but not in primary Sertoli cell lines. In addition, USP26 immunoprecipitated from testis lysates exhibited deubiquitinating activities. The localization of USP26 in the testis suggests a possible role in the movement of germ cells along the seminiferous epithelium.

DAZAP1 (Deleted in Azoospermia Associated Protein 1) is a ubiquitous hnRNP protein that is expressed most abundantly in the testis. Its ability to shuttle between the nucleus and the cytoplasm and its exclusion from the transcriptionally inactive XY body in pachytene spermatocytes implicate it in mRNA transcription and transport. We generated Dazap1 mutant alleles to study the role of DAZAP1 in mouse development. Most mice homozygous for the null allele as well as a hypomorphic Fn allele died soon after birth. The few Dazap1Fn/Fn mice that survived could nonetheless live for more than a year. They appeared and behaved normally but were much smaller in size compared to their wild-type and heterozygous littermates. Both male and female Dazap1Fn/Fn mice were sterile. Males had small testes, and the seminiferous tubules were atrophic with increased numbers of apoptotic cells. The tubules contained many germ cells, including pachytene spermatocytes with visible XY-bodies and diplotene spermatocytes, but no post-meiotic cells. FACS analyses confirmed the absence of haploid germ cells, indicating spermatogenesis arrested right before the meiotic division. Female Dazap1Fn/Fn mice had small ovaries that contained normal-appearing follicles, yet their pregnancy produced no progeny due to failure in embryonic development. The phenotypes of Dazap1 mutant mice indicate that DAZAP1 is not only essential for spermatogenesis, but also required for the normal growth and development of mice.

BACKGROUND: The role of the Y chromosome-encoded Deleted in Azoospermia (DAZ) gene family in spermatogenesis remains unclear. The ability of men without the DAZ gene to produce sperm, as well as the lack of selective pressure on DAZ exon sequences during evolution, casts doubts on its functional significance. Most men have four DAZ genes encoding protein isoforms that differ significantly in size. However, published western blots showed only a single “DAZ” band, raising the possibility that not all four DAZ genes are expressed. METHODS: RT‐ PCR, western blotting and immunostaining were used to study the expression of the four DAZgenes and the autosomal DAZL gene in human testes and in tissue culture cells. RESULTS: RNA transcripts of all four DAZ genes were found in the testis, but at much lower levels than that of the DAZL transcripts. Expression in cultured somatic cells showed that DAZ transcripts encoding multiple DAZ repeats were translated inefficiently. No DAZ proteins could be unambiguously identified on western blots when the testicular samples from three patients without the DAZ genes were used as negative controls. Nonetheless, low levels of DAZ were detected in the cytoplasm of spermatogonia by immunostaining. CONCLUSIONS: The expression of DAZ proteins in adult human testes is restricted to the spermatogonia and suggests a premeiotic role.

The AZFc region on the human Y chromosome consists mainly of very long direct and inverted repeats and is prone to rearrangement. Although deletion of the entire AZFc is found only in subfertile men, duplications and deletions of portions of AZFc as well as inversions are quite common and represent major polymorphisms of the Y chromosome. Several methods are available to detect these rearrangements, and each has its own advantages and limits. We designed a two-step PCR protocol to study the polymorphic structure of AZFc. The first PCR determines the copy number of the Deleted in Azoospermia (DAZ) genes within AZFc using the autosomal DAZ-Like gene as a dosage control, and the results could be verified by dosage Southern blot analyses. The second PCR simultaneously detects five sequence tagged sites (STSs) that are either present or absent in the various AZFc partial deletions. One of the STSs, sY1291, was found to be polymorphic in size due to varying lengths of a poly-T stretch. A combination of the DAZ dosage PCR and the 5-STS multiplex PCR reaction detects most, if not all, deletions and duplications at AZFc. It offers a simple and reliable way to screen large populations for AZFc rearrangements and study their effects on male fertility.

Short-term exposure (43 C for 15 min) of the rat testis to mild heat results within 6 h in stage- and cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Here we show that the relocation of Bax is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7 and cleavage of poly(ADP) ribose polymerase. Furthermore, early in apoptosis, a significant amount of Bax also accumulates in endoplasmic reticulum, as assessed by Western blot analyses of fractionated testicular lysates. In additional studies using the FasL-defective gld mice, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also endoplasmic reticulum-dependent pathways are the key apoptotic pathways for heat-induced germ cell death in the testis.

Using fluorescence in-situ hybridization on interphase chromatin fibers (fiber-FISH), we have constructed an overlapping fiber-FISH contig spanning the non-recombining region of the human Y chromosome (NRY). We first established a standard FISH-signal pattern for a distinct panel of DNA clones on prometaphase Y chromosomes in six healthy fertile men. Clones in the panel were selected from all R-bands as well as deletion intervals 1 through 7 plus PAR1 and PAR2 of the human Y chromosome. We next used signals of these marker clones to build a fiber-FISH contig for the multicopy gene families, CDY, DAZ, RBMY, TSPY and XKRY, along the NRY. Our fiber-FISH contig of human NRY may help to close the four gaps that still exist in the current physical map of the human Y chromosome. Furthermore, it provides a more complete picture with respect to the positions and arrangements of the multicopy gene families along the human NRY.

Klinefelter syndrome (47,XXY) is the most common sex chromosome aneuploidy in men. Thus, it is important to establish an experimental animal model to explore its underlying molecular mechanisms. Mice with a 41,XXY karyotype were produced by mating wild-type male mice with chimeric female mice carrying male embryonic stem cells. The objectives of the present study were to characterize the testicular phenotype of adult XXY mice and to examine the ontogeny of loss of germ cells in juvenile XXY mice. In the first experiment the testicular phenotypes of four adult XXY mice and four littermate controls (40,XY) were studied. XXY mice were identified by either Southern hybridization or karyotyping and were further confirmed by fluorescence in situ hybridization. The results showed that the testis weights of adult XXY mice (0.02 +/- 0.01 g) were dramatically decreased compared with those of the controls (0.11 +/- 0.01 g). Although no significant differences were apparent in plasma testosterone levels, the mean plasma LH and FSH levels were elevated in adult XXY mice compared with controls. The testicular histology of adult XXY mice showed small seminiferous tubules with varying degrees of intraepithelial vacuolization and a complete absence of germ cells. Hypertrophy and hyperplasia of Leydig cells were observed in the interstitium. Electron microscopic examination showed Sertoli cells containing scanty amounts of cytoplasm and irregular nuclei with prominent nucleoli. The junctional region between Sertoli cells appeared normal. In some tubules, nests of apparently degenerating Sertoli cells were found. In the second experiment the ontogeny of germ cell loss in juvenile XXY mice and their littermate controls was studied. Spermatogonia were found and appeared to be morphologically normal in juvenile XXY mice. Progressive loss of germ cells occurred within 10 days after birth. This resulted in the absence of germ cells in the adult XXY mice. We conclude that a progressive loss of germ cells occurring in early postnatal life results in the complete absence of germ cells in adult XXY mice. The XXY mouse provides an experimental model for its human XXY counterpart, Klinefelter syndrome.

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.

The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.

Using the technique of ‘fibre-FISH’ (fluorescence in situ hybridization), we describe the direct visualization of seven longer DAZ signal stretches and in addition a maximum of four isolated single DAZ signals on Y-chromatin fibres of four different individuals. These seven longer DAZ signal stretches may represent seven DAZ genes or pseudogenes, whereas the single DAZ signals may represent truncated DAZ genes.

Several genes located within or proximal to the human PAR in Xp22 have homologues on the Y chromosome and escape, or partly escape, inactivation. To study the evolution of Xp22 genes and their Y homologues, we applied multicolour fluorescence in situ hybridization (FISH) to comparatively map DNA probes for the genes ANT3, XG, ARSD, ARSE (CDPX), PRK, STS, KAL and AMEL to prometaphase chromosomes of the human species and hominoid apes. We demonstrate that the genes residing proximal to the PAR have a highly conserved order on the higher primate X chromosomes but show considerable rearrangements on the Y chromosomes of hominoids. These rearrangements cannot be traced back to a simple model involving only a single or a few evolutionary events. The linear instability of the Y chromosomes gives some insight into the evolutionary isolation of large parts of the Y chromosomes and thus might reflect the isolated evolutionary history of the primate species over millions of years.

The DAZ (Deleted in AZoospermia) and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. We report the isolation of the human DAZLA gene by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologues. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility.

Although the human steroid sulfatase (STS) gene has been cloned and characterized in detail, several attempts to clone its mouse homologue, with either anti-human STS antibodies or human STS cDNA probes, have failed, suggesting a substantial divergence between these genes. However, partial amino-terminal sequence from purified rat liver STS is very similar to its human counterpart, and sequence comparisons have revealed several domains that are conserved among all the sulfatases characterized to date. Thus, we used a degenerate-primer RT-PCR approach to amplify a 321-bp fragment from rat liver cDNA, which was used as a probe to clone and characterize the complete cDNA. Comparison of the protein coding region between the rat and human genes showed 66% homology both at the DNA and the protein levels. STS activity was conferred to STS(-) A9 cells upon transfection with a rat Sts expression construct, indicating the authenticity of the cloned cDNA. While Sts has been shown to be located in the mouse pseudoautosomal region, both physical and genetic mapping demonstrate that Sts is not pseudoautosomal in the rat. The overall genomic organization of rat Sts and human STS is very similar, except that the insertion site for intron 1 in the rat is 26 bp upstream from that in the human. Rat Sts is only 8.2 kb long, while the human STS spans over 146 kb.

Steroid sulphatase (STS) is an important enzyme in steroid metabolism. The human STS gene has been cloned and mapped to Xp22.3, proximal to the pseudoautosomal region (PAR). Using quantitative differences in STS activity among various mouse strains, a segregation pattern consistent with autosomal linkage was first reported, but more recent studies have linked Sts to the mouse PAR. Failed attempts to clone the mouse Sts gene using human reagants (STS cDNA and anti-STS antibodies) suggest a substantial divergence between these genes. However, partial amino-terminal sequence from purified rat liver Sts is very similar to its human counterpart, and several domains are conserved among all the sulphatases. We followed a degenerate-primer reverse transcriptase-PCR (RT-PCR) approach to amplify a conserved fragment of the rat Sts cDNA that was then used to clone the mouse Sts cDNA. This 2.3-kb cDNA revealed 75% similarity with rat Sts cDNA, while it was only 63% similar to human STS cDNA. Transfection of STS(-) A9 cells with the mouse Sts cDNA restored STS enzymatic activity. Sts was also mapped physically to the distal end of the mouse sex chromosomes, and our backcross studies placed Sts distal to the 'obligatory' cross-over in male meiosis.

The human X-linked steroid sulfatase gene (STS) was among the first genes shown to escape X inactivation. At least fourteen genes regulated in this fashion have now been recognized. They are dispersed into several regions of the X chromosome and may be controlled in a locus specific manner. Studies of the promoters of these genes could provide insights into the mechanism of X inactivation, however little information of this nature is currently available. For this reason we examined 5' flanking sequences of the human STS gene for promoter function. Four transcription start sites scattered over a 50bp region were identified. Functional domains of this TATA-less and GC poor promoter were identified by study of a series of terminal and internal deletions. A putative promoter sequence was identified which by itself exhibits little or no basal activity. However when combined with upstream regulatory elements, this segment showed weak but reproducible activity in a CAT (chloramphenicol acetyltransferase) reporter assay. Several regulatory domains acting as enhancers and repressors were subsequently identified. The relationship of this 5' sequence to the ability of the STS gene to escape X-inactivation is discussed.

Klinefelter syndrome (KS), characterized by the presence of more than one X-chromosome in men, is a major genetic cause of male infertility. Germ cell degeneration in KS patients is thought to be the consequences of overexpression of some genes on the X-chromosome. However, the identity of these genes and the underlying mechanisms remain unclear. Testis-expressed 11 (TEX11) is an X-chromosome-encoded germ-cell-specific protein that is expressed most abundantly in spermatogonia and early spermatocytes in the testes. In our search for TEX11-interacting partners using the yeast two-hybrid system, we identified hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP), which anchors estrogen receptors (ER) to the cytoskeleton and modulates their functions. We found that mouse spermatogonial stem cells expressed Tex11, Hpip, and Esr2 but not Esr1. In cultured cells, TEX11 competed with ERβ for binding to HPIP. Upon treatment with 17β-estradiol or an ERβ agonist diarylpropionitrile, TEX11 promoted the nuclear translocation of ERβ and enhanced its transcriptional activities. On the other hand, TEX11 suppressed the nongenomic activities of ERβ in the cytoplasm, as indicated by reduced phosphorylation of AKT and ERK signaling molecules. Overexpression of TEX11 in mouse germ-cell-derived GC-1 and GC-2 cells suppressed the cell proliferation and the expression of cFos, Ccnd1, and Ccnb1 that were stimulated by 17β-estradiol or diarylpropionitrile and elevated the expression level of the proapoptotic Bax gene. The negative effect of TEX11 on cell proliferation suggests that increased expression of TEX11 in the germ cells may partially contribute to the spermatogenic defect observed in KS patients.

Among a number of genes that escape X-chromosome inactivation in humans, three have been evaluated in mice and unexpectedly all three are subject to X-inactivation. We report here the cloning and expression studies of a novel mouse gene, Xe169, and show that it escapes X-inactivation like its human homologue. Xe169 was assigned to band F2/F3 on the mouse X chromosome by fluorescent in situ hybridization and Southern analysis indicates that the gene is located outside the pseudoautosomal region. Homologous, but divergent, sequences exist on the Y chromosome. In vitro and in vivo studies show that Xe169 is expressed from both the active and the inactive X chromosomes. Xe169 is the first cloned non-pseudoautosomal gene that escapes X-inactivation in mice.

A gene, GS2 (DXS1283E), was isolated from a CpG island located approximately midway between the steroid sulfatase (STS) and the Kallmann syndrome (KAL1) loci on the distal short arm of the human X chromosome. DNA sequencing of a GS2 cDNA clone revealed an open reading frame for a basic protein of 253 amino acid residues. A polymorphic CT dinucleotide repeat was found in the 3' untranslated region. The GS2 gene is expressed in all human tissues examined, including heart, brain, placenta, lung, liver, muscle, kidney, pancreas, and spleen. Several GS2 transcripts, ranging in size from 1.1 to 5.8 kb, were found among different tissues, suggesting tissue-specific processing of the GS2 transcript. Characterization of GS2 genomic clones revealed that the gene consists of 7 exons spanning over 26 kb, with a CpG island located in the first intron. The GS2 gene is transcribed toward Xpter, in the same direction as KAL1 but opposite to that of STS.

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specially to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders. © 1993 Wiley-Liss, Inc.

The expression of mouse Zfx, Rps4, Ube1x, and Xist was evaluated in hamster-mouse somatic cell hybrids containing either an active or an inactive mouse X chromosome using polymerase chain reaction of reverse transcribed RNA (RT-PCR). The results showed that Zfx, Rps4, and Ube1x are expressed exclusively from the active mouse X, while Xist is expressed exclusively from the inactive X. These findings confirm the pattern of X inactivation for these mouse genes reported previously based on expression in somatic tissues of F1 females from interspecific crosses. These results demonstrate the existence of differences between human and mouse X inactivation, as the corresponding human genes, ZFX, RPS4X, and UBE1 escape X inactivation.

Mammalian X–chromosome inactivation is thought to be controlled by the X inactivation centre (XIC, X–controlling element –Xce– in mice). A human gene, XIST and its mouse counterpart, Xist, which map to the XIC/Xce, are expressed exclusively from inactive X chromosomes, suggesting their involvement in the process of X–inactivation. We now report the presence of Xist/XIST transcripts in newborn and adult mouse testes, and in human testicular tissue with normal spermatogenesis, but not in the testes of patients who lack germ cells. Our results indicate that while the X chromosome in males is active in somatic cells, it undergoes inactivation during spermatogenesis.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Background The Azoospermia Factor c (AZFc) region of the human Y chromosome is a unique product of segmental duplication. It consists almost entirely of very long amplicons, represented by different colors, and is frequently deleted in subfertile men. Most of the AZFc amplicons have high sequence similarity with autosomal segments, indicating recent duplication and transposition to the Y chromosome. The Deleted in Azoospermia (DAZ) gene within the red-amplicon arose from an ancestral autosomal DAZ-like (DAZL) gene. It varies significantly between different men regarding to its copy number and the numbers of RNA recognition motif and DAZ repeat it encodes. We used Southern analyses to study the evolution of DAZ and AZFc amplicons on the Y chromosomes of primates. Results The Old World monkey rhesus macaque has only one DAZ gene. In contrast, the great apes have multiple copies of DAZ, ranging from 2 copies in bonobos and gorillas to at least 6 copies in orangutans, and these DAZ genes have polymorphic structures similar to those of their human counterparts. Sequences homologous to the various AZFc amplicons are present on the Y chromosomes of some but not all primates, indicating that they arrived on the Y chromosome at different times during primate evolution. Conclusion The duplication and transposition of AZFc amplicons to the human Y chromosome occurred in three waves, i.e., after the branching of the New World monkey, the gorilla, and the chimpanzee/bonobo lineages, respectively. The red-amplicon, one of the first to arrive on the Y chromosome, amplified by inverted duplication followed by direct duplication after the separation of the Old World monkey and the great ape lineages. Subsequent duplication/deletion in the various lineages gave rise to a spectrum of DAZ gene structure and copy number found in today's great apes.

Substantial DNA deletions appear to be the molecular basis of several human genetic disorders but rarely account for the majority of observed mutations at any given locus. Exceptions in which deletions do account for the majority of observed abnormalities include the alpha-thalassemias, Duchenne muscular dystrophy, and steroid sulfatase deficiency. Variable deletion breakpoints have been recognized at the alpha-globin and dystrophin loci, but no information is available regarding STS deletions. We have found that these STS alterations usually involve breakpoints within highly similar sequence elements situated approximately 1.9 megabases apart on the X chromosome. It is surprising that these very large deletions produce such mild clinical abnormalities. These results may provide insight into the molecular mechanism of a number of human genetic defects.

X chromosome rearrangements usually convey clinical manifestations in the hemizygous males and are, thus, readily ascertained. They are found in all parts of the X chromosome and are associated with more than 20 disorders. Some of the rearrangements are the results of homologous recombination between low-copy repeats (LCRs) on the X chromosome or between large homologous regions on the X and Y chromosome, whereas others are caused by nonhomologous end-joining (NHEJ). For most large deletions associated with contiguous gene syndromes, the deletion breakpoints remain uncharacterized. The deletions, as well as inversions and duplications on the X chromosome, occur mainly in male germ cells, indicating intrachromatid or sister chromatid exchange as the underlying mechanism.

The Azoospermia Factor c (AZFc) region on the Y chromosome long arm is one of the least stable regions in the human genome. It consists almost entirely of very long repeats and is prone to rearrangement. Numerous structures at AZFc have been identified, and some of them have been reported to be associated with male infertility. We screened 580 Han Chinese in Taiwan for AZFc deletion and duplication using three PCR assays, and characterized the DAZ genes in selected subjects with additional Southern analyses. About 9.5% of our subjects have AZFc partial deletion, 2.8% have partial deletion followed by duplication, and 1.7% have partial duplication. The overall rearrangement frequencies vary significantly between different Y chromosome haplogroups (Yhgs), ranging from 2.9% in O3e to 100% in N and Q. All individuals in Yhg-N lack the sY1191 marker, but one out of three of them actually have four DAZ genes, indicating further duplication after the b2/b3 deletion. Our additional screening of 142 oligospermic men and 107 fertile controls found no significant difference in the frequencies of the gr/gr and the b2/b3 deletion. However, the frequency of AZFc partial duplication in the infertile group (7.0%) was significantly higher than that in the fertile control group (0.9%) and the general Taiwanese population (1.7%). Our results indicate that AZFc partial deletion and partial duplication are common polymorphisms in Han Chinese, and that the AZFc partial duplication, but not the AZFc partial deletion, is a risk factor for male infertility in the Taiwanese population. Hum Mutat 28(5), 486–494, 2007. © 2007 Wiley-Liss, Inc.

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous RNA-binding protein highly expressed in the human and the mouse testes. It shows a dynamic subcellular localization during spermatogenesis, present predominantly in the nuclei of late-stage spermatocytes and round spermatids and translocated to the cytoplasm during spermatid elongation. To test the hypothesis that DAZAP1 shuttles between the nucleus and the cytoplasm, we studied the nuclear transport of DAZAP1 in somatic cells using immunostaining, heterokaryon formation, and mutagenesis. DAZAP1 is detected exclusively in the nucleus and has the ability to shuttle between the nucleus and the cytoplasm using a highly conserved 25 amino acid segment, designated ZNS, at its C terminus. ZNS shares no sequence homology with other known nuclear localization or export signals. Attachment of ZNS to a red fluorescent protein DsRed2 confers the nucleocytoplasmic shuttling ability to that protein. The nuclear localization of DAZAP1 depends on active transcription. In the presence of an RNA polymerase II inhibitor, DAZAP1 is retained in the cytoplasm. DAZAP1 colocalizes with hnRNP A1 and hnRNP C1 in the nucleus and is a component of the heterogeneous nuclear ribonucleoprotein particles. Our results suggest that DAZAP1 plays a key role in mRNA transport during spermatogenesis.

The human Y chromosome is unique in that it does not engage in pairing and crossing over during meiosis for most of its length. Y chromosome microdeletions, a frequent finding in infertile men, thus occur through intrachromosomal recombination, either within a single chromatid or between sister chromatids. A recently identified polymorphism associated with increased risk for spermatogenic failure, the gr/gr deletion, removes two of the four Deleted in Azoospermia (DAZ) genes in the AZFc region on the Y-chromosome long arm. We found the likely reciprocal duplication product of gr/gr deletion in 5 (6%) of 82 males using a novel DNA-blot hybridization strategy and confirmed the presence of six DAZ genes in three cases by FISH analysis. Additional polymorphisms identified within the DAZ repeat regions of the DAZ genes indicate that sister chromatid exchange plays a significant role in the genesis of deletions, duplications, and polymorphisms of the Y chromosome.

Summary The Deleted in Azoospermia (DAZ) gene belongs to a gene family that consists of three members: BOULE, DAZ-Like (DAZL) and DAZ. Members of the DAZ family are expressed exclusively in the germ cells, and their protein products contain a highly conserved RNA-binding motif and a unique DAZ repeat. Null mutations of the DAZ family members affect the fertility of either male or female, or both sexes. DAZ and DAZL are expressed throughout most of the life of germ cells and are required for the development of primordial germ cells and the differentiation and maturation of the germ cells. BOULE is expressed around the time of meiosis and has a more limited function. The DAZ proteins bind to RNAs in vitro and in vivo and are probably involved in the post-transcriptional regulation of mRNA expression. Their downstream RNA targets remain largely unknown, although members of the CDC25 gene family are potential candidates.

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 degrees C for 15 min) of the testis to mild heat results, within 6h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr(cg) mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.

Using fluorescence in-situ hybridization on interphase chromatin fibers (fiber-FISH), we have constructed an overlapping fiber-FISH contig spanning the non-recombining region of the human Y chromosome (NRY). We first established a standard FISH-signal pattern for a distinct panel of DNA clones on prometaphase Y chromosomes in six healthy fertile men. Clones in the panel were selected from all R-bands as well as deletion intervals 1 through 7 plus PAR1 and PAR2 of the human Y chromosome. We next used signals of these marker clones to build a fiber-FISH contig for the multicopy gene families, CDY, DAZ, RBMY, TSPY and XKRY, along the NRY. Our fiber-FISH contig of human NRY may help to close the four gaps that still exist in the current physical map of the human Y chromosome. Furthermore, it provides a more complete picture with respect to the positions and arrangements of the multicopy gene families along the human NRY.

DAZAP1 (Deleted in Azoospermia Associated Protein 1) was originally identified through its interaction with a putative male azoospermia factor, DAZ (Deleted in Azoospermia). It contains 2 RNA-binding domains (RBDs) and a proline-rich C-terminal portion and is expressed most abundantly in testes. We used RNA in situ hybridization and immunocytochemistry to study the expression of Dazap1 in mouse testes. Dazap1 messenger RNA (mRNA) was present predominantly in immature germ cells, between the intermediate spermatogonia and preleptotene spermatocyte stages. The DAZAP1 protein was more abundant in germ cells of later stages of development and showed a dynamic subcellular distribution. High expression of DAZAP1 was first detected in midpachytene spermatocytes in stage VII tubules. In these cells, DAZAP1 was present in both the cytoplasm and the nuclei and was clearly excluded from the sex vesicles. In round spermatids, DAZAP1 was localized mainly in the nuclei, whereas in elongated spermatids, it redistributed to the cytoplasm. The subcellular distribution of DAZAP1 suggests that it shuttles between the nucleus and the cytoplasm and may play a role in mRNA transport and/or localization.

Background DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast two-hybrid system through its interaction with a putative male infertility factor, DAZ (Deleted in Azoospermia). In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL. DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis. To understand the biological function of DAZAP1 and the significance of its interaction with DAZ and DAZL, we isolated and characterized the mouse Dazap1 gene, and studied its expression and the subcellular localization of its protein product. Results The human and mouse genes have similar genomic structures and map to syntenic chromosomal regions. The mouse and human DAZAP1 proteins share 98% identity and their sequences are highly similar to the Xenopus orthologue Prrp, especially in the RBDs. Dazap1 is expressed throughout testis development. Western blot detects a single 45 kD DAZAP1 protein that is most abundant in the testis. Although a majority of DAZAP1 is present in the cytoplasmic fraction, they are not associated with polyribosomes. Conclusions DAZAP1 is evolutionarily highly conserved. Its predominant expression in testes suggests a role in spermatogenesis. Its subcellular localization indicates that it is not directly involved in mRNA translation.

The human Y chromosome has long been recognized as being responsible for sex determination. In fact, it also encodes more than 30 genes and gene families that participate in a variety of cellular functions, including bone development, tooth growth, and spermatogenesis. De-novo deletion of Y chromosome segments that contain spermatogenesis genes occurs frequently, resulting in low sperm production and male infertility. This article reviews our current knowledge of the structure and function of the Y chromosome is reviewed.

Deletion interval 6 (DI6) of the human Y chromosome, located at the distal end of the long arm euchromatic region, is required for normal spermatogenesis. About 10% of males with idiopathic azoospermia or oligospermia have microdeletions in this region. Six gene families, including RBMY (RNA binding motif, Y chromosome), DAZ (deleted in azoospermia), and four recently isolated genes, have been mapped to this interval. Genes from all of these families show testis-specific expression and are thus candidates for azoospermic factor (AZF). DI6 is also rich in Y-specific repetitive sequences, which may be responsible for its frequent deletion. To understand the sequence organization of this region, a 5-Mb restriction map was constructed based on YAC clones and was partially verified on genomic DNA. The locations of five gene family members, as well as numerous STSs, were determined. The map shows several inverted and direct repeats several hundred kilobases in size. The restriction map of DI6 will facilitate future mapping of deletion breakpoints in infertile males and elucidation of mechanisms behind frequent deletions.

The RBMY (RNA-binding motif, Y chromosome) gene family encodes a germ-cell-specific nuclear protein implicated in spermatogenesis. It consists of approximately 30 genes and pseudogenes, found on both arms of the Y chromosome. RBMY shares high homology with an autosomal hnRNPG gene that contains an RNA-binding motif and one of the four SRGY repeats found in RBMY. One proposal is that RBMY represents an ancestral hnRNPG gene, transposed to the Y chromosome and then amplified. We characterized seven RBMY genes in interval 6 of the Y chromosome long arm. Four have the normal structure with 12 exons spanning 15 kb, whereas one lacks the first 3 exons, therefore representing a pseudogene. The remaining two genes belong to a different subfamily, resembling the autosomal hnRNPG gene with only one SRGY repeat. We also found that most RBMY genes in interval 6 are arranged in tandem. The structure and organization of the Y-linked RBMY genes support the transposition-amplification hypothesis.

The RBM (RNA-binding motif) gene family on the human Y chromosome encodes proteins with an RNA-binding domain. Its exclusive expression in germ cells and its partial deletion in some azoospermic or severely oligospermic males provide evidence of a role for RBM genes in spermatogenesis. There are approximately 30 RBM genes, found on both arms of the Y chromosome. Two RBM cDNA clones with slightly different sequences have been reported. To investigate the number of functional genes, we studied RBM expression by use of RT-PCR of RBM transcripts and by characterizing numerous RBM cDNA clones. A total of 27 RT-PCR and 19 cDNA clones were sequenced. Whereas the RT-PCR clones pointed to the existence of at least six RBM subfamilies (RBMI to RBMVI), the cDNA clones indicated that only RBMI is actively transcribed and encodes functional proteins. A total of six RBMI genes were identified, which produce four polypeptides due to some silent base substitutions. The transcripts of each gene are alternatively spliced to generate protein isoforms with three or four SRGY boxes, thus greatly increasing the complexity of the products of the RBM gene family. We also provide evidence suggesting that a 5-bp deletion in a previously reported RBM cDNA clone represents a processing irregularity.

The DAZ genes on the human Y Chromosome (Chr) are strong candidates for the azoospermia factor AZF. They are frequently deleted in azoospermic or severely oligospermic males and are expressed exclusively in germ cells. In addition, the DAZ genes share a high degree of similarity with a Drosophila male infertility gene, boule. The predicted DAZ proteins contain an RNA recognition motif (RRM), and multiple copies of a repeat (the DAZ repeat) in tandem array. To understand the DAZ gene family and its expression, the DAZ genomic structure and RNA transcripts in numerous males, as well as several DAZ cDNA clones were analyzed. The results of genomic Southern blot showed that each male contains multiple DAZ genes with varying numbers of DAZ repeats, and that the copy number of the DAZ repeats are polymorphic in the population. The presence of multiple species of DAZ transcripts with different copy number and arrangement of the DAZ repeats in an individual suggests that more than one DAZ gene are transcribed. The existence of multiple functional DAZ genes complicates the analysis of genotype/phenotype correlations among males with varying sperm counts.

Several human diseases have been mapped to Xp22.3 on the distal short arm of the human X chromosome, and many genes in this area have been found to be expressed from the inactive X chromosome. To facilitate physical mapping and characterization of this interesting region, we have constructed a battery of radiation hybrids containing human X chromosomal fragments, and isolated two hybrid clones A with overlapping fragments of Xp22.3. Alu-PCR on these hybrids and identification of sequences common to both hybrids allowed the isolation of six sequences-tagged sites (STSs) from Xp22.3. Five of the STSs were mapped+ to individual YACs comprising a recently constructed contig of this region. These novel STSs are useful markers for further physical characterization of this part of the genome.

The relative order of 11 loci in the distal half of the short arm of the human X chromosome was examined using a panel of somatic cell hybrids containing structurally rearranged X chromosomes. The results show that the gene for phosphoribosylpyrophosphate synthetase 2 (PRPS2) is located between ZFX (zinc finger protein, X-linked) and STS (steroid sulfatase). The results also confirm the localization of ZFX distal to POLA (alpha-DNA polymerase). Previous studies have shown that STS and ZFX escape X-inactivation whereas POLA undergoes inactivation. Evaluation of PRPS2 expression in somatic cell hybrids containing inactive human X chromosomes showed that PRPS2 undergoes X-inactivation. These results provide further evidence for interspersion of loci that do and do not undergo X-inactivation on the human X chromosome.

Several regions of sequence homology between the human X and Y chromosomes have been identified. These segments are thought to represent areas of these chromosomes that have engaged in meiotic recombination in relatively recent evolutionary times. Normally, the X and Y chromosomes pair during meiosis and exchange DNA only within the pseudoautosomal region at the distal short arms of both chromosomes. However, it has been suggested that aberrant recombination involving other segments of high homology could be responsible for the production of X/Y translocations. We have studied four X/Y translocation patients using molecular probes detecting homologous sequences on X and Y chromosomes. In one translocation the breakpoints have been isolated and sequenced. The mapping data are consistent with the hypothesis that X/Y translocations arise by homologous recombination. The sequencing data from one translocation demonstrate this directly.

Steroid sulfatase (STS), an important enzyme in the pathway of estrogen synthesis from sulfated steroid precursors, was localized to the syncytial trophoblast of human placentas during different periods of pregnancy by using a mouse monoclonal antibody and immunocytochemical techniques. Preembedding immunoelectron microscopy revealed STS immunoreactivity associated with the rough endoplasmic reticulum of the syncytial trophoblast. STS mRNA was also localized to this outermost layer of the human trophoblast. At a cellular level, both STS message and immunoreactivity seemed to be more abundant during the late first and early second trimesters than in term placentas. Although the syncytial trophoblast is known to originate from the cytotrophoblast, neither STS immunoreactivity nor STS mRNA was detected in the cytotrophoblast at any stage of placental development studied.

The majority of patients with steroid sulfatase deficiency have a deletion of the entire STS gene located on the distal short arm of the human X chromosome; however, two patients with partial gene deletions have been identified. We now report the sequences at the breakpoints of a deletion of the 3′ end of the STS gene. The deletion starts within intron 7 of the gene and extends over 150 kb downstream toward the centromere. Analysis of sequences flanking the deletion breakpoints revealed 3 bp of homology. The 3′ flanking sequence provides a new probe for isolation of YAC clones and for studying patients with deletions in this region of the X chromosome.