Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male

BACKGROUND: There is little information regarding the behaviour of the extra Y chromosome during meiosis I in men with 47,XYY karyotypes and the segregation of the sex chromosomes in sperm. We applied immunofluorescent and FISH techniques to study the relationship between the sex chromosome configuration in meiotic germ cells and the segregation pattern in sperm, both isolated from semen samples of a 47,XYY infertile man. METHODS: The sex chromosome configuration of pachytene germ cells was determined by immunostaining pachytene nuclei for synaptonemal complex protein 3 (SCP3) and SCP1. FISH was subsequently performed to identify the sex chromosomes and chromosome 18 in pachytene cells. Dual- and triple-color FISH was performed on sperm to analyse aneuploidy for chromosomes 13, 18, 21, X, and Y. RESULTS: 46,XY/47,XYY mosaic pachytene cells were observed (22.2% vs. 77.8%, respectively). The XYY trivalent, and X+YY configurations were most common. While the majority of sperm were of normal chromosomal constitution, an increase in sex and autosome disomy was observed. CONCLUSIONS: The level of germ cell moscaicism and their meiotic sex chromosome configurations may determine sperm aneuploidy rate and fertility status in 47,XYY men. Our approach of immunostaining meiotic cells in the ejaculate is a novel method for investigating spermatogenesis in infertile men.     

The prevalence of a YY synaptonemal complex over XY synapsis in an XYY man with exclusive XYY spermatocytes

An infertile XYY man was studied by synaptonemal complex analysis of microspread spermatocytes and by quantitation of germ cells in semithin sections. All the 74 spermatocytes micrographed have an XYY constitution, and the biopsy shows a homogeneous arrest of spermatogenesis at the spermatocyte/young spermatid stages. The overwhelming majority (86%) of spermatocytes showed a Y—Y bivalent plus a univalent X. The Y—Y bivalent is totally synapsed in 48% of the cells. In the remaining cells, the YY bivalent has an average synaptic segment covering 43% of its length that always includes Yp. Another 9% of the spermatocytes showed an XYY trivalent and 4% of the spermatocytes showed univalence of the three gonosomes. Progression through all the pachytene substages was observed in cells with the two main synaptic configurations, but a high level of germ cell death was observed at or immediately after the meiotic divisions. The prevalence of Y—Y synapsis arises from the longer homologous region and the higher speed of pairing between the two Y chromosomes. Germ cell death is probably related to the univalence of the X chromosome. Synaptic competition between three gonosomes seems to be similar to that found in triploid birds but is somewhat different from that of XYY mice.This revised version was published online in November 2005 with corrections to the Cover Date.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

Meiotic behaviour of the sex chromosomes in three patients with sex chromosome anomalies (47,XXY, mosaic 46,XY/47,XXY and 47,XYY) assessed by fluorescence in-situ hybridization

Meiotic studies using multicolour fluorescent in-situ hybridization (FISH) and chromosome painting were carried out in three patients with sex chromosome anomalies (47,XXY; 46,XY/47,XXY and 47,XYY). In the two patients with Klinefelter syndrome, although variable percentages of XXY cells (88.5 and 28.3%) could be found in the pre-meiotic stages, none of the abnormal cells entered meiosis, and all pachytenes were XY. However, the abnormal testicular environment of these patients probably resulted in meiotic I non-disjunction, and a certain proportion of post-reductional cells were XY (18.3 and 1.7%). The fact that none of the spermatozoa were XY also suggests the existence of an arrest at the secondary spermatocyte or the spermatid level. In the XYY patient, most (95.9%) premeiotic cells were XYY. The percentage of XYY pachytenes was 57.9%. The sex chromosomes were either in close proximity (XYY) or the X chromosome was separated from the two Ys (X + YY). A high proportion (42.1%) of post-reductional germ cells were XY. However, only 0.11% of spermatozoa were disomic for the sex chromosomes. In this case, the data suggest the existence of an arrest of the abnormal cells at the primary and the secondary spermatocyte or the spermatid level, giving rise to the continuous elimination of abnormal cells in the germ-cell line along spermatogenesis. The fact that the proportion of diploid spermatozoa was only increased in one of the three cases (XXY) is also suggestive of an arrest of the abnormal cell lines in these patients. The two apparently non-mosaic patients were, in fact, germ-cell mosaics. This suggests that the cytogenetic criteria used to define non-mosaic patients may be inadequate; thus, the risk of intracytoplasmic sperm injection in apparently non-mosaics may be lower than expected.     

Chromosome constitution and apoptosis of immature germ cells present in sperm of two 47,XYY infertile males

BACKGROUND: In order to assess sperm alterations observed in some XYY males, we analysed the chromosome constitution as well as apoptosis expression in germ cells from two oligozoospermic males with high count of immature germ cells in their semen. METHODS: Sex chromosome number and distribution were assessed at pachytene stage by fluorescence in situ hybridization (FISH). Immature germ cells and spermatozoa were examined by FISH and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) assay, combined with immunocytochemistry using the proacrosin-specific monoclonal antibody (mAb 4D4). RESULTS: For patients 1 and 2, two Y chromosomes were present in respectively 60.0 and 39.6% of pachytenes. The three sex chromosomes were always in close proximity and partially or totally condensed in a sex body. XYY spermatocytes I escape the pachytene checkpoint and achieve meiosis. Nevertheless, nuclear division and/or cytokinesis were often impaired during meiosis leading to diploid (mainly 47,XYY cells) and tetraploid (94,XXYYYY) meiocytes. The presence of binucleated (23,Y)(24,XY) immature germ cells resulting from cytokinesis failure agree with a preferential segregation of the two Y chromosomes during meiosis I. In addition, 69.6% (patient 1) and 53.12% (patient 2) of post-reductional round germ cells were XY. However, high level of apoptotic round germ cells (94.9% for patient 1 and 93.3% for patient 2) was detected and may explain the moderate increase of hyperhaploid XY spermatozoa. Segregation errors also occurred in the XY cell line responsible for disomic 18 and X, as well as 46,XY diploid spermatozoa. CONCLUSIONS: Our data are in agreement with the persistence of the extra Y chromosome during meiosis in XYY oligozoospermic males responsible for spermatogenesis impairment and a probable elimination via apoptosis of most XYY germ cells not solely during but also after meiosis.     

Three non-mongoloid patients of similar phenotype with an extra G-like chromosome

Three mentally retarded non-mongolid children with very similar clinical signs and an extra G-like chromosome are presented. In one of these patients, Case I, an attempt to trace the origin of the extra chromosome has been made by QM fluorescence analysis and autoradiography. Visually the QM pattern of the extra chromosome differs from that of chromosomes 21 and 22 by showing a diffuse fluorescence of intermediate intensity in the long arm, not unlike that found in the proximal part of the long arm of chromosome no. 13. However, whether these are sufficiently similar to indicate homologous regions cannot be decided. The autoradiographic pattern is not decisive as to the origin of the extra chromosome; it shows early labelling, as do no. 22 and the short arm and proximal part of the long arm of no. 13.
Case II, a sibling of Case I, died in 1962 at the age of 6 years (Gustavson et al. 1962). In Case III the autoradiographic pattern of the five G-group chromosomes is similar to that found in Case I.
The clinical and cytogenetic features of these 3 patients are compared with those of 9 other previously reported patients with a similar phenotype and an extra small acrocentric chromosome. These 12 patients seem to constitute a specific clinical syndrome.     

Molecular cytogenetic evaluation of the aneugenic effects of teniposide in somatic and germinal cells of male mice

The ability of topoisomerase II inhibitor, teniposide, to induce aneuploidy and meiotic delay in somatic and germinal cells of male mice was investigated by fluorescence in situ hybridisation (FISH) assay using labelled DNA probes and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, respectively. Colchicine and mitomycin C were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. Using FISH assay with centromeric and telomeric DNA probes for erythrocyte, micronuclei (MN) showed that teniposide is not only clastogenic but also aneugenic in somatic cells in vivo. The assay also showed that chromosomes can be enclosed in the MN before and after centromere separation. By using the BrdU incorporation assay, it could be shown that the meiotic delay caused by teniposide in germ cells was ～48 h. Disomic and diploid sperms were shown in epididymal sperm hybridised with DNA probes specific for chromosomes 8, X and Y after teniposide treatment. The prevalence of autodiploid (XX88 and YY88) sperm and disomic XX8 or YY8 sperm indicates that the second meiotic division was more sensitive to teniposide than the first meiotic division. The results also suggest that earlier prophase stages contribute relatively less to teniposide-induced aneuploidy. Both the clastogenic and the aneugenic potential of teniposide can give rise to the development of secondary tumours and abnormal reproductive outcomes in cured cancer patients and medical personnel exposing to drug regimens that include teniposide. Thus, genetic counselling of these patients should take place before the start of chemotherapy and should take the present results into consideration.     

DNA and chromatin structure: Analysis of the sex chromosomal equipment in spermatozoa of a 47, XYY male using two-colour fluorescence in-situ hybridization

The sex chromosomes in spermatozoa of a 47, XYY fertile malewere analysed simultaneously by dual fluorescence in-situ hybridization(FISH), with two probes (pHY2.1 and pXBR). Of the 100 000 cellsanalysed, 95 179 spermatozoa (95.18%) exhibited one or morehybridization signals. Of the hybridized nuclei, 85.37% showeda normal sex chromosome constitution (37.37% X-bearing cellsand 48.00% Y-bearing cells), with an X:Y ratio of 0.78:1. Atotal of 14.63% of the hybridized nuclei exhibited sex chromosomeaneuploidy with a majority of XY-and YY-bearing spermatozoa(9.37 and 4.65% respectively). Even if the majority of spermatozoahave chromosomal haploidy, a large proportion of them exhibitsnumerical errors for the sex chromosomes. These observationsraise questions about the commonly-admitted notions concerningthe absence of chromosomal risk for XYY male offspring. fluorescence interphase in-situ hybridization/sex chromosome/spermatozoa/XYY male     

The penta-X syndrome.

A child is presented with a 49,XXXXX chromosomal constitution bringing to 12 the total number of children described with this karyotype. Comparison of this child's features with previously reported cases indicates a clinically recognisable specific pattern of malformations referred to as the penta-X syndrome. X chromosome replication studies using BrdU labelling in the patient's cells clearly showed that the four presumably inactive X chromosomes were late replicating but not in a strictly synchronous fashion.     

Chromosomal aberrations in 130 patients with multiple myeloma studied by interphase FISH: diagnostic and prognostic relevance

The study described the molecular cytogenetic characterization of myeloma cells in 130 patients via interphase fluorescence in situ hybridization. Nine repetitive DNA probes (for chromosomes 3, 7, 9, 11, 15, 17, 18, X, and Y) as well as seven single-copy DNA probes (for chromosomes 13, 17, 21, and two each for chromosomes 5 and 22) were used for the hybridizations. Using this panel of probes, we were able to show aberrations in 86% of patients. Most of them had one to three aberrations. There was a distinct correlation between the number of aberrations per patient and the tumor stage. Thus, the proportion of patients with 8-12 aberrations increased from 16% in stage II to 26% in stage III. There were marked differences among the chromosomes with respect to the prevalence of genomic losses and gains and deletions of gene loci. Chromosomes 3, 5, 7, 9, 11, 15, and 21 showed a preference for genomic gains. Losses were most often found for chromosomes 13 and 17 (locus specific) as well as for the X and Y chromosomes. The frequency of monosomies and trisomies were approximately the same for chromosomes 15 and 18, which indicates a skewed pattern of distribution. We found two specific aberrations that caused distinct changes in the survival rates of the patients: deletion 13q14 (28% of patients) and translocation of the IGH locus 14q32 (79% of 39 patients who were analyzed separately). The results obtained in this study yielded data of extremely relevant prognostic value.     

The origin of the extra Y chromosome in males with a 47,XYY karyotype.

The presence of an extra Y chromosome in males is a relatively common occurrence, the 47,XYY karyotype being found in approximately 1 in 1000 male births. The error of disjunction must occur either during paternal meiosis II or as a post-zygotic mitotic error, both of which are rare events for other chromosomes. It is therefore of interest to determine when errors of Y chromosome disjunction occur. It is possible to distinguish between the different mechanisms of non-disjunction by analysing DNA polymorphisms at the distal tip of the Xp/Yp pseudoautosomal region in 47,XYY males, their parents and in some cases paternal grandparents. A cohort of 28 non-mosaic 47,XYY males was analysed. The results show that there are at least two mechanisms causing non-disjunction of the Y chromosome. In 16 of the 19 cases from which parents were available, the extra Y was generated by non-disjunction at meiosis II after a normal chiasmate meiosis I. Three cases were due to either a post-zygotic mitotic error or non-disjunction at meiosis II after a nullichiasmate meiosis I. Of the nine cases with no parental DNA available, at least four were due to meiosis II non-disjunction following a normal chiasmate meiosis I.     

Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation

A novel technique using the incorporation of 5-ethynyl-2''-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.     

Longer Y chromosome in criminals

In 84 male criminals without obvious psychiatric problems, the Y chromosomes were measured and compared with Y chromosomes of 38 staff men of a psychiatric hospital. There were significantly ( P < 0.025) more long Y chromosomes in prisoners than in controls. Both the fluorescent and non-fluorescent segment of Y were involved in the increase.
No case of XYY or Y chromosome with atypical structure was found.     

The influence of sex chromosomes on finger dermatoglyphic patterns

Finger pattern frequencies for patients exhibiting various sex chromosome aneuploidies were obtained from literature sources. The sample consisted of 141 XO, 500 XX, 68 XXX, 9 XXXX, 500 XY, 93 XYY, 30 XXYY and 6 XXXXY. Pattern frequencies were converted to radial and ulnar loop frequencies, and these in turn were used to construct four variables; pattern intensity; radial-ulnar difference; radial loop asymmetry; and ulnar loop asymmetry. The relationship between the dermatoglyphic variables on to the sex chromosomes was examined by regressing the dermatoglyphic variables on to the number of X and Y chromosomes. Radial-ulnar difference and radial loop asymmetry showed the strongest relationship with the number of X and Y chromosomes. The X and Y chromosomes had about equal influence on radial-ulnar difference, but the Y had a stronger effect on radial loop asymmetry. It is postulated that sex chromosomes influence dermatoglyphic development by controlling tissue sensitivity to fetal sex steroids.     

Cytogenetics in Brachidontes rodriguezi d'Orb (Bivalvia, Mytilidae)

The chromosomes of Brachidontes rodriguezi were analysed by means of direct Giemsa staining, silver staining, fluorescent in-situ hybridization (FISH) with 18S + 28S rDNA probes, replication banding and chromomycin A3 (CMA) and DAPI fluorescence banding techniques. The diploid chromosome number in this species is 32 and the karyotype is composed of two pairs of metacentric chromosomes, 2 pairs of telo/subtelocentric chromosomes and 12 pairs of subtelocentric chromosomes. 18S + 28S rDNA clusters were located on the short arms of the two pairs of telo/subtelocentric chromosomes. The replication band pattern induced in this species facilitates chromosome pairing and differentiation. The nucleolar organizing regions (NORs) replicate late in the S phase and were associated with bright CMA fluorescence and dull DAPI fluorescence, but not all the four NORs showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found for this character. This revised version was published online in August 2006 with corrections to the Cover Date.     

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.     

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.     

Chromosomal replication asynchrony of a human breast carcinoma cell line. I. Studied by continuous BrdU incorporation and G-banding analysis

Continuous BrdU incorporation and the Giemsa staining technique were used to study the cell cycle kinetics of a human breast tumor cell line. It was found that the interchromosomal replication pattern of the neoplastic cell was significantly different from that of normal cells in two respects. First, the pattern is highly asynchronous; within a single cell there are chromosomes at different replication cycles; that is, some chromosomes complete their DNA replication before others begin. Second, the replication schedule for the chromosomes, as identified by superimposing the BrdU-Giemsa technique on the trypsin G-banding technique, is relatively consistent within the cell line but differs from that of normal cells. Some chromosomes that replicate late in normal human lymphocytes and fibroblasts replicate early in this cell line. In contrast to the unusual interchromosomal replication pattern, gross analysis of the intrachromosomal replication schedule shows no apparent difference from that reported for normal cells. The asynchrony phenomenon reported here may be associated with the etiology of aneuploidy in neoplasia.     

Positioning of chromosome 15, 18, X and Y centromeres in sperm cells of fertile individuals and infertile patients with increased level of aneuploidy

Evidence has been accumulating that individual chromosomes in human sperm cells occupy defined, non-random positions. Our earlier study suggested that abnormal spermatogenesis in carriers of reciprocal translocations was reflected in the changes in the intranuclear topology of sperm chromosomes. The purpose of this study was to determine whether the increased level of disomy of sperm chromosomes may be the factor that can disturb topology within the sperm nuclei. The results obtained indicated that within the sperm nuclei of fertile individuals the centromeres of chromosomes 15, 18, X and Y were localized in a small area that may be a fragment of the chromocentre. When compared with the intranuclear positions of the same chromosomes in sperm nuclei of infertile patients with an increased level of aneuploidy, some disturbances in the centromere area were found. In disomic sperm cells (n + 1) centromeres 15,15 or 18,18 or YY (but not X,X) had a shifted average longitudinal position in comparison with normal sperm cells (n = 23).     

An XYY boy with short stature and a case of Klinefelter's syndrome (XXY) in a family with inversion 9

A patient with Klinefelter's syndrome and a boy with XYY sex chromosomes were both found to have a pericentric inversion of chromosome 9. An unusual feature of the XYY patient was that he presented because of short stature and disturbed behaviour. A family study showed that the patients were related and that there was an excess of males in the pedigree. Another member of the family was found to have some XYY cells in the blood.