Reconstructing the evolution of vertebrate sex chromosomes.

Sex chromosomes and their evolution have captivated researchers since their discovery. For more than 100 years, the dominant model of sex chromosome evolution has held that differentiated sex chromosomes, such as the X and Y chromosomes of mammals or the Z and W chromosomes of birds, evolved from ordinary autosomes, primarily through the degeneration of the sex-specific Y or W chromosome. At the same time, the sex chromosomes shared between sexes, the X and Z chromosomes, are expected to remain essentially untouched. This model was based on limited cytogenetic and genetic data. Only in the last decade, with the advent of genomics, has the complete sequence of any sex chromosome pair become available. High-quality finished sequences of the human and chimpanzee Y chromosomes, as well as the human X chromosome, have revealed sequence features unanticipated by the traditional model of sex chromosome evolution. Large, highly identical, tandem and inverted arrays of testis-expressed genes are major sources of innovation in gene content on sex-specific chromosomes as well as sex-shared chromosomes. Accounting for the emergence of these ampliconic structures presents a challenge for future studies of sex chromosome evolution.

The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.

Two closely related human nuclear export factors utilize entirely distinct export pathways.

Nuclear mRNA export mediated by the human protein TAP requires a carboxy-terminal domain that directly interacts with components of the nuclear pore complex. Here we demonstrate that NXF3, a human RNA binding protein related to TAP, lacks this domain yet retains the ability to export tethered RNA transcripts and to shuttle between the nucleus and the cytoplasm. NXF3 contains a novel Crm1-dependent nuclear export signal that compensates in cis for the loss of the nuclear pore targeting domain. NXF3-dependent RNA export is therefore blocked by Crm1-specific inhibitors that do not affect TAP function. Thus, while the related TAP and NXF3 proteins are both capable of mediating nuclear RNA export, they do so via unrelated export pathways.

A physical map of the human Y chromosome.

The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.

An abundance of X-linked genes expressed in spermatogonia.

Spermatogonia are the self-renewing, mitotic germ cells of the testis from which sperm arise by means of the differentiation pathway known as spermatogenesis. By contrast with hematopoietic and other mammalian stem-cell populations, which have been subjects of intense molecular genetic investigation, spermatogonia have remained largely unexplored at the molecular level. Here we describe a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. We identified 25 genes (19 of which are novel) that are expressed in only male germ cells. Of the 25 genes, 3 are Y-linked and 10 are X-linked. If these genes had been distributed randomly in the genome, one would have expected zero to two of the genes to be X-linked. Our findings indicate that the X chromosome has a predominant role in pre-meiotic stages of mammalian spermatogenesis. We hypothesize that the X chromosome acquired this prominent role in male germ-cell development as it evolved from an ordinary, unspecialized autosome.

Breastfeeding is early functional jaw orthopedics (an introduction).

Breastfeeding places beneficial orthopedic forces on the jaws, similar to the forces of Functional Jaw Orthopedics--the newest form of orthodontics. To date most breastfeeding benefits have been attributed to the content of mother's milk. The true orthopedic benefits of breastfeeding, suckling, deserve more definitive attention and research. Breastfeeding is early preventive Functional Jaw Orthopedics because breastfeeding forces impact the jaws during a very rapid period of infant jaw growth. Breast suckling aids proper development of the jaws which form the gateway to the human airway. Bottle, pacifier and digit sucking deform jaws and airways. Forward forces of suckling clearly oppose the backward forces of sucking. Dentists who understand the positive impact of forward orthopedic forces on the jaws should support and advocate exclusive breastfeeding for about 6 months.

How to promote & provide functional jaw orthopedics.

This article outlines how to promote and provide Functional Jaw Orthopedic (FJO) treatment, which is unique medical dentistry. FJO treatment is valuable preventive and therapeutic medical dentistry because jaw treatment impacts the extensive structures that surround the human airway and adjoin the skull. To best provide the broad range of unique FJO treatments, dentists need extensive knowledge, skills and experience. In order to promote FJO treatments, dentists and other interested health care providers need a wide assortment of educational materials that clearly show FJO treatment options and benefits.

DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice.

The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and Drosophila. Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.

Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses.

Deletion of any of three regions of the human Y chromosome results in spermatogenic failure and infertility. We previously sequenced one of these regions, azoospermia factor a (AZFa) and found that it spanned approximately 800 kb. By sequence-tagged site (STS) content mapping, we roughly defined deletion breakpoints in two unrelated, azoospermic men with AZFa deletions. The positions of proximal and distal breakpoints were similar in the two men. Hypothesizing that the deletions might have been generated by homologous recombination, we searched electronically for DNA sequence similarities between the proximal and distal breakpoint regions. These comparisons revealed the most striking sequence similarities anywhere along or near the AZFa region. In the proximal breakpoint region, we identified a 10 kb provirus of the recently defined HERV15 class of endogenous retroviruses. In the distal breakpoint region, we found a second HERV15 provirus, 94% identical in DNA sequence to the first and in the same orientation. (A partial LINE insertion in this distal provirus proved to be the basis of the previously described DYS11/p12f polymorphism.) The AZFa deletions in the two men differed slightly, but all breakpoints fell within the HERV15 proviruses. Indeed, sequencing of deletion junctions from the two men revealed that homologous recombination had occurred within large domains of absolute sequence identity between the proximal and distal proviruses. When combined with published STS mapping data for other AZFa-deleted men, our findings suggest that recombination between these two HERV15 proviruses could account for most AZFa deletions.

Optical mapping of BAC clones from the human Y chromosome DAZ locus.

The accurate mapping of clones derived from genomic regions containing complex arrangements of repeated elements presents special problems for DNA sequencers. Recent advances in the automation of optical mapping have enabled us to map a set of 16 BAC clones derived from the DAZ locus of the human Y chromosome long arm, a locus in which the entire DAZ gene as well as subsections within the gene copies have been duplicated. High-resolution optical mapping employing seven enzymes places these clones into two contigs representing four distinct copies of the DAZ gene and highlights a number of differences between individual copies of DAZ.

Unexpectedly similar rates of nucleotide substitution found in male and female hominids.

In 1947, it was suggested that, in humans, the mutation rate is dramatically higher in the male germ line than in the female germ line. This hypothesis has been supported by the observation that, among primates, Y-linked genes evolved more rapidly than homologous X-linked genes. Based on these evolutionary studies, the ratio (alpha(m)) of male to female mutation rates in primates was estimated to be about 5. However, selection could have skewed sequence evolution in introns and exons. In addition, some of the X-Y gene pairs studied lie within chromosomal regions with substantially divergent nucleotide sequences. Here we directly compare human X and Y sequences within a large region with no known genes. Here the two chromosomes are 99% identical, and X-Y divergence began only three or four million years ago, during hominid evolution. In apes, homologous sequences exist only on the X chromosome. We sequenced and compared 38.6 kb of this region from human X, human Y, chimpanzee X and gorilla X chromosomes. We calculated alpha(m) to be 1.7 (95% confidence interval 1.15-2.87), significantly lower than previous estimates in primates. We infer that, in humans and their immediate ancestors, male and female mutation rates were far more similar than previously supposed.

Four DAZ genes in two clusters found in the AZFc region of the human Y chromosome.

The DAZ genes are candidate fertility factors that lie within the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. The number of DAZ genes has been difficult to determine, in part because the nucleotide sequences of the DAZ genes are nearly identical. Here, fluorescence in situ hybridization and characterization of BAC clones revealed four full-length DAZ genes on the human Y chromosome. They exist in two clusters, each comprising an inverted pair of DAZ genes (3' <-- 5'::5' --> 3'). Analysis of genomic sequences and testicular transcripts suggested that three or four DAZ genes are translated. Each gene contains at least seven tandem copies of a previously described, 2.4-kb repeat unit that encodes 24 amino acids. In addition, two DAZ genes contain tandem copies of a 10.8-kb repeat unit that encodes the RNA-binding domain, which appears to be multimerized in some DAZ proteins. Combining our present results with previous studies, we can reconstruct several steps in the evolution of the DAZ genes on the Y chromosome. In the ancestral Y-chromosomal DAZ gene, amplification of both intragenic repeats began before the human and cynomolgus (Old World) monkey lineages diverged. During subsequent evolution, an inverted duplication of this modified gene occurred. Finally, the resulting two-gene cluster was duplicated, generating the two-cluster/four-gene arrangement found on modern human Y chromosomes.

The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen.

The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) Ags that are recognized by female T cells in an MHC-restricted manner. Two human H-Y epitopes presented by HLA-A2 and HLA-B7, respectively, have been identified previously and both are derived from the SMCY gene. We previously isolated CD8+ CTL clones that recognized a male-specific minor histocompatibility Ag presented by HLA-B8. In contrast to the SMCY-encoded H-Y epitopes, the B8/H-Y Ag was not presented by fibroblasts from male donors, suggesting that it was encoded by a novel gene. We now report that the HLA-B8-restricted H-Y epitope is defined by the octameric peptide LPHNHTDL corresponding to aa residues 566-573 of the human UTY protein. Transcription of the UTY gene is detected in a wide range of human tissues, but presentation of the UTY-derived H-Y epitope to CTL by cultured human cells shows significant cell-type specificity. Identification of this CTL-defined H-Y epitope should facilitate analysis of its contribution to graft/host interactions following sex-mismatched organ and bone marrow transplantation.

Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated.

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.

The orthodontic shift towards functional jaw orthopedics.

FJO treatment is exceptionally unique and differs dramatically from traditional orthodontics. Parents are demanding earlier treatment, less bicuspid extractions, and broader smiles. A national trend towards FJO treatment methods will continue because FJO treatment has been shown to be medically effective for a number of common human disorders. Progressive dentists who provide cutting edge FJO therapy are uniquely positioned to impact human development and general health starting at birth, in ways yet to be charted. Early FJO treatment fulfills the ADA Code of Ethics which "calls upon dentists to follow high ethical standards which have the benefit of the patient as their primary goal: Do No Harm and Do Good."

A human sex-chromosomal gene family expressed in male germ cells and encoding variably charged proteins.

Approximately 12 X-Y homologous gene pairs have been identified in the non-recombining portions of human sex chromosomes. These X-Y gene pairs fall into two categories. In the first category, both X and Y homologs are ubiquitously expressed. In the second category, the X homolog is ubiquitously expressed, whereas the Y homolog is expressed exclusively in the testis. Here we describe a family of human X-Y genes that cannot be assigned to either category. Designated VCX / Y ( Variable Charge X / Y; VCY previously known as BPY1 ), this gene family has multiple members on both X and Y, and all appear to be expressed exclusively in male germ cells. Members of the VCX / Y family share a high degree of sequence identity, with the exception that a 30 nucleotide unit is tandemly repeated in X-linked members but is present only once in Y-linked members. These atypical features suggest that the VCX / Y family has evolved in a manner previously unrecognized for mammalian X-Y genes. We also found that a copy of VCX is present in CRI-S232, a previously described genomic fragment derived from the X chromosome. Studies have shown that aberrant recombination between arrays of CRI-S232-homologous repeats flanking the steroid sulfatase ( STS ) gene results in STS deletion, which is manifested clinically as X-linked ichthyosis. The revelation that CRI-S232 contains VCX offers a more precise description of the genetic etiology of X-linked ichthyosis: it results from aberrant recombination between VCX gene arrays that flank the STS locus.

The new dental-medical renaissance. Medically efficacious functional jaw orthopedics.

Functional Jaw Orthopedics (FJO) is medically efficacious, exceptionally unique and unequaled in speed of a cure for a number of very common medical conditions currently only "managed" by modern medicine. This publication combines scientific citations and case presentations to show how and why dentists who advocate and provide early FJO treatment will lead a New Dental-Medical Renaissance into some of the most profound areas of medicine. Nocturnal enuresis (chronic bed-wetting) otitis media (ear infections), cor pulmonare, cognitive thought, vision, hearing, cardio-pulmonary dysfunction, and myocardial infarction have FJO dental-medical relationships. Early FJO treatment fulfills the ADA Code of Ethics which "calls upon dentists to follow high ethical standards which have the benefit of the patient as their primary goal: Do No Harm and Do Good."

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y.

In humans, deletion of any one of three Y-chromosomal regions- AZFa, AZFb or AZFc-disrupts spermatogenesis, causing infertility in otherwise healthy men. Although candidate genes have been identified in all three regions, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.