Kay Taylor

A Y chromosome gene family with RNA-binding protein homology : Candidates for the azoospermia factor AZF controlling human spermatogenesis

We have previously mapped the human azoospermia factor to a deletion in Y chromosome interval 6 (subinterval XII-XIV). We now report the isolation and characterization of a gene family located within this deletion. Analysis of the predicted protein products suggests a possible role in RNA processing or translational control during early spermatogenesis. The Y chromosome RNA recognition motif (YRRM) family includes a minimum of three members expressed specifically in the testis. Interphase in situ results and Southern blot analysis indicate that several further YRRM sequences map within interval 6. Several mammalian species show Y chromosome conservation of YRRM sequences. We have detected deletions of YRRM sequences in two oligospermic patients with no previously detectable mutation.

Comparative mapping ofYRRM- andTSPY-related cosmids in man and hominoid apes

Using chromosomalin situ hybridization it has been demonstrated that specific members of theYRRM and theTSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with theYRRM-related cosmid A5F and theTSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and seconary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution ofYRRM andTSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.

Degeneracy in human multicopy RBM (YRRM), a candidate spermatogenesis gene.

In order to search for mutations in the multicopy RBM genes that might be associated with male infertility, we have used sequence data from the reported cDNA clone to determine the intron exon boundaries of the YRRM 1 gene. This gene has 12 exons, three of which encode the putative RNA binding domain of the protein. Different copies of the gene contain sequence variations and, additionally, give rise to transcripts with different numbers of copies of the repeated SRGY motif. Since mutations in the RNA binding domain would seem likely to have an effect on the activity of the protein, we have scanned these exons for mutations by SSCP on DNA from normal and infertile men. Sequence differences in the exon encoding the N-terminal part of the RNA binding domain account for at least four different classes of the gene and give rise to different SSCP conformers. Sequence analysis shows that one of these classes is a pseudogene and that the members of another class are nonfunctional. RT-PCR shows that all classes are transcribed and that the A class is most abundant. We have found a point mutation that alters the highly conserved RNP2 motif in one infertile patient. This mutation is also found in his father. We have used PCR followed by SSCP analysis to map RBM on a Y Chromosome (Chr) YAC contig and have demonstrated a distribution that spans a major part of this chromosome’s euchromatin.

Simian Y Chromosomes: species-specific rearrangements of DAZ, RBM, and TSPY versus contiguity of PAR and SRY

The three human male specific expressed gene families DAZ, RBM, and TSPY are known to be repetitively clustered in the Y-specific region of the human Y Chromosome (Chr). RBM and TSPY are Y-specifically conserved in simians, whereas DAZ cannot be detected on the Y chromosomes of New World monkeys. The proximity of SRY to the pseudoautosomal region (PAR) is highly conserved and thus most effectively stabilizes the pseudoautosomal boundary on the Y (PABY) in simians. In contrast, the non-recombining part of the Y Chrs, including DAZ, RBM, and TSPY, was exposed to species-specific amplifications, diversifications, and rearrangements. Evolutionary fast fixation of any of these variations was possible as long as they did not interfere with male fertility.

Tsga10 Encodes a 65-Kilodalton Protein That Is Processed to the 27-Kilodalton Fibrous Sheath Protein

Abstract We had previously reported the isolation of the testis-specific human gene Tsga10, which is not expressed in testes from two infertile patients. To study its role and function, we cloned the mouse homologue Mtsga10. Mtsga10 localizes to mouse chromosome 1, band B. This region is syntenic with human chromosome 2q11.2, where Tsga10 is located. We demonstrate that Mtsga10 mRNA is expressed in testis, but not in other adult tissues, and in several human fetal tissues and primary tumors. We uncovered that different species use different first exons and, consequently, different promoters. Using several antibodies, we discovered that, in mouse testis, Mtsga10 encodes a 65-kDa spermatid protein that appears to be processed to a 27-kDa protein of the fibrous sheath, a major sperm tail structure, in mature spermatozoa. Mtsga10 protein contains a putative myosin/Ezrin/radixin/moesin (ERM) domain. Transfection of fibroblasts with GFP-Mtsga10 fusion protein results in formation of short, thick filaments and deletion of the myosin/ERM domain abolished filament formation. Our results suggest the possibility that Tsga10 plays a role in the sperm tail fibrous sheath.

Identification and characterisation of a novel gene, TSGA10, expressed in testis

We describe the isolation of a novel gene, TSGA10, by differential mRNA display which is expressed solely in adult human testis. It seems likely that the gene is expressed during spermatogenesis possibly in spermatocytes. The gene is composed of 19 exons extending over more than 80 kb. The complete cDNA contains an open reading frame of 2094 nucleotides, which appears to encode a novel protein. It has been mapped by polymerase chain reaction on a panel of somatic cell hybrids and by fluorescence in situ hybridization to chromosome 2q11.2.

Cytogenetic and molecular investigations of Y chromosome sequences and their role in Turner syndrome

It has been proposed that all live born females with Turner syndrome carry a cell line containing two sex chromosomes, which may be present at a low level of mosaicism (Hook & Warburton, 1983; Hassold et al. 1985; 1988; Connor & Loughlin, 1989). If the second sex chromosome is a Y, these patients are at risk of developing gonadoblastoma. In this study, 50 patients found to have a 45,X karyotype by conventional cytogenetic analysis, were screened by the polymerase chain reaction (PCR), for the presence of Y chromosome sequences. Two patients were positive for six of the eight Y chromosome loci tested and additional cytogenetic analysis confirmed the presence of a marker chromosome, in 8% and 3% of cells respectively. Fluorescence in situ hybridization (FISH) was used to confirm that the markers were of Y chromosome origin and helped to elucidate their structure. In addition, four other patients were found to have a Y chromosome by initial routine cytogenetic analysis. FISH, in conjunction with PCR, elucidated the structure of the Y chromosomes. This study illustrates the value of using a combination of cytogenetic and molecular techniques, to identify Y chromosome sequences in Turner syndrome.

High-resolution fluorescence in situ hybridization of human Y-linked genes on released chromatin

Genes within the differential region of the human Y chromosome do not recombine, and therefore the determination of their location depends on physical mapping. Yeast artificial chromosome (YAC) contigs spanning the euchromatic region of the human Y have become a powerful tool for the generation of an overlapping clone map. With this approach,however, complete physical mapping is difficult in Y euchromatic regions that are rich in repetitive sequences. We have, therefore, made use of the fluorescence in situ hybridization technique as an alternative strategy for physically mapping the PRKY and AMELY genes as well as the TSPY, RBM and DAZ gene families to human Y chromosomes in prometaphase and to extended Y chromatin in interphase. From our results, the following order of gene sequences in interval 3 of the short arm of the human Y chromosome is suggested: TSPY major with few RBM sequences interspersed-PRKY-AMELY-TSPY minor with few RBM sequences interspersed-cen. On the long arm, RBM sequences appear to be distributed over wide regions of intervals 5 and 6 with few TSPY sequences interspersed. Distal to an RBM signal cluster, a large cluster of DAZ signals is located with only a few DAZ and RBM signals overlapping in between the two clusters.

A comparative study between infertile males and patients with Turner syndrome to determine the influence of sex chromosome mosaicism and the breakpoints of structurally abnormal Y chromosomes on phenotypic sex

The Y chromosome is important for male development as it contains the sex determining gene SRY 1 and many spermatogenesis genes.2 Structural abnormalities of the Y chromosome include rings, deletions, inversions, and dicentrics.3,4 These types of abnormalities are common in infertile males (1.5%), especially those with azoospermia.5,6 However, such rearrangements are unstable and an additional 45,X cell line is frequently present.3 The 45,X cell line has been shown to influence phenotypic sex so that these chromosome constitutions may also be found in patients with ambiguous genitalia and in female patients with gonadal dysgenesis and Turner syndrome.4,7 In fact, from cytogenetic studies about 4-6.2% of female Turner patients show Y chromosome mosaicism8–10 irrespective of the presence of SRY .4,11,12 Mosaicism varies widely between tissues and accurate interpretation depends on the number of cells examined and the number and types of tissues studied.13,14 It has been reported that phenotypic sex is strongly influenced by the percentage and distribution of Y chromosome containing cells in the gonads.15,16 However, studies on gonadal tissue are hindered by the fact that it is rarely available for analysis and alternative, more easily accessible tissue is usually studied. It has also been suggested that the structure of the Y chromosome may indirectly affect phenotypic sex. The repetitive sequences at the euchromatin/heterochromatin boundary of the Y chromosome long arm are thought to have an important stabilising role and loss of this region loses this effect, resulting in mosaicism with a 45,X cell line.17 In dicentrics, which are the most common abnormality of the Y chromosome,3 it has been suggested that the position of the q arm breakpoint in dicentric Yp chromosomes can influence Y chromosome stability. The more proximal the …

Cytogenetic and Y chromosome microdeletion screening of a random group of infertile males.

OBJECTIVE To assess whether to perform routine cytogenetic and Y chromosome microdeletion screening on all infertile male patients. DESIGN A cytogenetic and Y microdeletion study of a random group of infertile men. SETTING University department. PATIENT(S) In total, 40 patients had azoospermia (21 nonidiopathic), 27 had severe oligozoospermia/oligoasthenozoospermia (<or=5 x 10(6)/mL) (5 nonidiopathic), 20 had oligozoospermia/oligoasthenozoospermia (5-20 x 10(6)/mL) (6 nonidiopathic), and 16 had asthenozoospermia (5 nonidiopathic). Many were candidates for intracytoplasmic sperm injection (ICSI). INTERVENTION(S) Collection of blood samples from all patients and buccal cells from one patient. MAIN OUTCOME MEASURE(S) Karyotype analysis, polymerase chain reaction (PCR) screening for Y chromosome microdeletions, and fluorescence in situ hybridization of abnormal chromosomes. RESULT(S) Ten (9.7%) subjects, including one nonidiopathic patient, were found to have an abnormal karyotype. Two idiopathic azoospermic patients were missing large portions of Y chromosome euchromatin, confirmed by PCR analysis and an additional idiopathic azoospermic patient had a Y chromosome microdeletion. CONCLUSION(S) Routine cytogenetic analysis of all infertile male patients is required but it may be advisable to limit routine Y chromosome microdeletion screening to patients with severe male factor infertility (<or=5 x 10(6)/mL).