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The human Y chromosome. Right: schematic representation of FISH mapping of selected DNA clones on an RBG-banded human prometaphase Y. Left: Y chromosome showing the basic deletion intervals 1À7 and positions of the two pseudoautosomal regions, PAR1 and PAR2. The azoospermia factor (AZF) regions aÀc are indicated by square brackets. Correspondence between deletion intervals and R-bands on the ideogram is only schematic.
Source publication
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 me...
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Context 1
... should be mentioned that no microscopically visi- ble structural alterations, especially para-or peri- centric inversions, could be detected for the Y chromosomes of our probands investigated. Figure 2 depicts a synopsis of our mapping results on an RBG-banded prometaphase human Y chromosome. In addition to our panel of DNA clones used from NRY, the locations for SHOX, SYBL1 and SRY as markers for PAR1, PAR2, and the sex-determining region are also indicated. ...
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Prometaphase preparations have been used to construct a ‘standard’ RBA-banded karyotype of the swamp buffalo (Bubalus bubalis L., 2n = 48) and to compare the banding homologies between buffalo and cattle (Bos taurus L.). A double numbering system, one progressive and one corresponding to the standardized nomenclature of cattle chromosomes, has been...
A comparison of high resolution G- and R-banding in cattle and river buffalo chromosomes was performed at the 500 band level. Close banding homologies between the two species were apparent. However a loss of pericentromeric G-positive band in chromosome arms lp, 2q, 4p and 5q and a retention of pericentromeric G-positive bands in chromosome 3 and R...
Citations
... FISH followed essentially the methods described in Schempp et al. [1995]. To characterize both Y markers in detail, we FISH-mapped a panel of Y chromosomal probes originally developed to present a standard FISH-signal pattern for the human prometaphase Y chromosome [Röttger et al., 2002]. Chromosome in situ suppression (CISS) was applied to the following DNA clones: SHOX cosmid 34F05 [Rao et al., 1997], TSPY cosmid 2.2133 [Taylor et al., 1996], UTY PAC RP5-1035N09 (Human Male Genome PAC library [RPCI5] of P.J. de Jong and P.A. Iaonnou), RBMY cosmid A5F [Taylor et al., 1996], XKRY BAC 357E16 (AC007742), DAZ cosmid 6B7 [Taylor et al., 1996], CDY cosmid CDY-2A49 [Kühl et al., 2001], and SYBL1 cosmid LLycos130G4 [G. ...
... In order to confirm the presence of 2 isoforms of the Y chromosome and for their detailed characterization, we used the panel of Y chromosomal DNA probes proposed by Röttger et al. [2002]. As shown in figure 1 a after DAPI staining, the bigger marker (arrowhead) presents 2 blocks of chromatin showing bright DAPI fluorescence. ...
A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.
... Adaptations of the approach might also be used for the determination of the order of individual sequences (e.g. BACs in interphase nuclei [7,44] or for fibre FISH [10,32,48,55,56]). ...
From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos.
... TSPY is evolutionarily conserved as moderately repetitive sequences on the Y chromosome and is preferentially expressed in the testis of many mammals, including bovine and primates [Zhang et al. 1992;Vogel et al. 1997;Rottger et al. 2002], suggesting that it might serve a conserved function(s), as hypothesized above, in spermatogenesis. Significantly, Tspy genes in the laboratory mouse and Mongolian gerbil are incapable of encoding any Tspy proteins [Mazeyrat and Mitchell 1998;Schubert et al. 2000a;2000b;Karwacki et al. 2006]. ...
The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G(2)/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.
... In a former study, we established a standard FISH signal pattern for Y chromosomal DNA probes on prometaphase Y chromosomes of healthy, fertile human males [Röttger et al., 2002]. With respect to our re-investigation of 9 cases of inv(Y) with seemingly unvarying inversion breakpoints, the male-specific ampliconic fertility genes DAZ and CDY turned out to be most informative for a closer definition by FISH of the inversion breakpoints in the Y chromosome long arm. ...
Pericentric inversions of the human Y chromosome (inv(Y)) are the result of breakpoints in Yp and Yq. Whether these breakpoints occur recurrently on specific hotspots or appear at different locations along the repeat structure of the human Y chromosome is an open question. Employing FISH for a better definition and refinement of the inversion breakpoints in 9 cases of inv(Y) chromosomes, with seemingly unvarying metacentric appearance after banding analysis, unequivocally resulted in heterogeneity of the pericentric inversions of the human Y chromosome. While in all 9 inv(Y) cases the inversion breakpoints in the short arm fall in a gene-poor region of X-transposed sequences proximal to PAR1 and SRY in Yp11.2, there are clearly 3 different inversion breakpoints in the long arm. Inv(Y)-types I and II are familial cases showing inversion breakpoints that map in Yq11.23 or in Yq11.223, outside the ampliconic fertility gene cluster of DAZ and CDY in AZFc. Inv(Y)-type III shows an inversion breakpoint in Yq11.223 that splits the DAZ and CDY fertility gene-cluster in AZFc. This inversion type is representative of both familial cases and cases with spermatogenetic impairment. In a further familial case of inv(Y), with almost acrocentric morphology, the breakpoints are within the TSPY and RBMY repeat in Yp and within the heterochromatin in Yq. Therefore, the presence of specific inversion breakpoints leading to impaired fertility in certain inv(Y) cases remains an open question.
... One member locates on the Y chromosome in the form of four highly related copies that are collectively known as chromodomain protein on the Y (CDY). To date, the CDY gene has been identified only in the primates, including chimpanzees, Old World Monkeys and New World Monkeys (Lahn & Page 1999;Kostova et al. 2002;Rottger et al. 2002;Wimmer et al. 2002;Dorus et al. 2003). Two members, the CDYL and CDYL2 genes, map to autosomes, and exist in most mammalian species (Lahn & Page 1999;Dorus et al. 2003). ...
... The CDY gene instead arose by retroposition and subsequent amplification of a processed messenger RNA derived from an autosomal CDYL gene (Lahn & Page 1999;Dorus et al. 2003;Bhowmick et al. 2006). In the simian lineage the CDY gene was retained and subsequently amplified into many copies (Lahn & Page 1999;Kostova et al. 2002;Rottger et al. 2002;Wimmer et al. 2002;Dorus et al. 2003). But in other mammals this gene has been lost (Lahn & Page 1999;Dorus et al. 2003). ...
The human chromodomain protein, Y-like (CDYL) gene family consists of three members, one on the Y chromosome (CDY) and two on autosomes (CDYL and CDYL2). Studies in the human and mouse showed that genes in the CDYL family are abundantly expressed in testis and play an important role in spermatogenesis. In this study, we have characterized the bovine CDYL (bCDYL) and CDYL2 (bCDYL2) genes. We found that bCDYL and bCDYL2 are very similar to the human orthologues at both mRNA (79% and 85%) and protein (89% and 93%) levels. However, the similarity between the bCDYL and bCDYL2 proteins is low (41%). The bCDYL gene is composed of nine exons, and the bCDYL2 has seven exons. The bCDYL and bCDYL2 genes were mapped by radiation hybrid mapping to bovine chromosomes (BTA) 24 and 18 respectively. The bCDYL gene has four transcript variants that produce four protein isoforms. RT-PCR expression analysis in 12 bovine tissues showed that bCDYL variant 2 was expressed in the testis only, bCDYL variants 1, 3 and 4 were expressed predominantly in the testis and at very low or undetectable levels in the remaining tissues and bCDYL2 was expressed ubiquitously. Examination of bovine testis with in situ hybridization revealed that the bCDYL and bCDYL2 transcripts were found mainly in spermatids, though the amounts of transcripts varied among genes/variants. In addition, antisense transcripts were detected in bCDYL variants 2/3 and 4, as well as in the bCDYL2 gene.
... It has also been observed that, even for the DNA fibers, the detection resolution is greatly influenced by the stage of the cell cycle. S-phase fibers provide much higher FISH resolution compared to G 1 or G 2 fibers ( Rottger et al. 2002). Because the existing protocols in plants use DNA fibers released from interphase nuclei isolated from mechanically crushed leaves ( Fransz et al. 1996), the efficiency of fiber FISH may be limited to some extent because the DNA fibers used are mainly at G 1. No success has since been reported in plants in obtaining fibers from active nuclei isolated from meristematic tissues. ...
... This could be best compared by performing FISH on DNA fibers released from the nuclei at different stages of the cell cycle ranging from G 1 to S to G 2 through the division cycle. It may be pertinent to note here that, using a nonrecombining region of the human Y chromosome, it has been unequivocally demonstrated that the resolution efficiency of fiber FISH is best on the DNA fibers obtained from the nuclei at S-phase compared to G 1 or G 2 , and is enhanced by over threefold compared to fibers from G 2 nuclei ( Rottger et al. 2002). ...
The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G(1) nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency.
... Extended Y chromatin structures in interphase nuclei were generated according to previously published protocols (Fidlerovà et al., 1994) with the following modification. After sodium hydroxide treatment we applied classical methanol:acetic acid (3:1) fixation to improve the reproducibility of the release technique (Röttger et al., 2002). ...
Applying fluorescence in situ hybridisation (FISH), six cosmid clones of rhesus macaque origin containing the genes SACM2L, RING1, BAT1 and MIC2, MIC3, MICD, and MOG of the major histocompatibility complex (MHC) were localised to the long arm of the rhesus macaque chromosome 6 in 6q24, the orthologous region to human 6p21.3. Furthermore, centromere to telomere orientation of the rhesus macaque MHC as well as the internal order of the MHC genes tested are the same as in human. Fiber-FISH allows a rough estimate of distances between these MHC genes in the rhesus macaque, and, as in the human, the rhesus macaque MHC comprises about 3 to 4 Mb.
... Such difficulties have also been reported during the mapping of Y chromosomes in other species especially humans (Tilford et al. 2001). In humans a combination of fingerprinting (Tilford et al. 2001), optical mapping (Giacalone et al. 2000), fiber-FISH mapping (Rottger et al. 2002) and STS content mapping (Foote et al. 1992; Ferlin et al. 2003) was used to overcome these problems. Similarly in analyzing the horse Y chromosome integrating RH map along with various FISH approaches allowed us to verify marker, order, orientation, number of copies and distribution. ...
Stallion fertility is of significant economic importance in the multibillion dollar equine industry. Presently, the underlying genetic causes of infertility in stallions are unknown. Analysis of the human genome has shown that in more than 25% of cases, male infertility is associated with deletions/rearrangements in the Y chromosome. Presently there is no gene map for the Y chromosome in the horse. Therefore, the primary aim of this study is to build a detailed physical map of the chromosome with a long-term aim to identify and analyze Y-specific factors affecting fertility in stallions. To materialize this, we constructed the first radiation hybrid and FISH map of the euchromatic region of the horse Y chromosome. This basic map was used to obtain Y-specific BAC clones that provided new STS markers from the end sequences. Chromosome walking provided 73 BACs comprising 7 contigs that were built across the euchromatic region using 124 markers for content mapping. The results were validated by restriction fingerprinting and Fiber FISH. The map is presently the most informative among the domestic species and second to only human and mouse Y chromosome maps. The construction of this map will pave the way for isolation and functional characterization of genes critical for normal male fertility and reproduction and will in the future lead to the development of a diagnostic test to facilitate early identification of deletions/rearrangements on the Y chromosome of potentially affected foals/stallions. The second part of the study comprised the first extended investigation to assess genetic variation in the horse Y chromosome. Approximately 4.5Mb of the euchromatic region was screened for polymorphic microsatellite markers. Of the 27 markers that were characterized and screened for polymorphism in 14 breeds of the domestic horse and eight extant equids, only one was polymorphic in the domestic horse, suggesting a low level of genetic variation on the chromosome. However, 21 of the markers showed noteworthy variation (on average four alleles/marker) among the eight equids. These markers will be vital in future studies aimed at elucidating the genetic relationships between the various equids through phylogenetic analysis.
According to the latest data, globally 15% of couples have infertility and male infertility contributes to 10% of all cases. Infertility can be caused by certain biological changes in the gonads and the reproductive system like azoospermia, oligospermia, asthenospermia, teratozoospermia and hypospermatogenesis. Genetic causes of azoospermia include chromosomal abnormalities, Y chromosome microdeletions and deletion or other mutations of Y-linked genes. The maximum number of the genes are located in the azoospermia factor region of the long arm (Yq) of the Y chromosome. Y chromosome microdeletion is known as the second major genetic cause of spermatogenetic failure. This article aims to review the latest updates on the involvement of Yq microdeletions in male infertility. The diagnostics, prevalence and phenotypic spectrum related to Yq gene microdeletions are discussed.
