Effect of paternal age on the frequency of cytogenetic abnormalities in human spermatozoa.

Many surveys have been performed to find etiological relationships between pregnancy outcome and specific risk factors, such as exposure to chemicals and radiation or parental age. Advanced maternal age is a strong risk factor for trisomic pregnancies, albeit there are considerable variations among the different chromosomes. The definite incidence of the various structural and numerical chromosome aberrations in spontaneous abortions and liveborns is well known, as well as the rate of maternally and paternally derived rearrangements. Nevertheless studies have failed to assert an age-dependent risk for men fathering chromosomally abnormal children. New techniques using fluorescence in situ hybridization render it possible to analyze spermatozoa directly for numerical and, to some extent, for structural aberrations. This article compiles the findings of studies on human spermatozoa over the last few years.

Comparative chromosome painting of chicken autosomal paints 1-9 in nine different bird species.

In a Zoo-FISH study chicken autosomal chromosome paints 1 to 9 (GGA1-GGA9) were hybridized to metaphase spreads of nine diverse birds belonging to primitive and modern orders. This comparative approach allows tracing of chromosomal rearrangements that occurred during bird evolution. Striking homologies in the chromosomes of the different species were noted, indicating a high degree of evolutionary conservation in avian karyotypes. In two species, the quail and the goose, all chicken paints specifically labeled their corresponding chromosomes. In three pheasant species as well as in the American rhea and blackbird, GGA4 hybridized to chromosome 4 and additionally to a single pair of microchromosomes. Furthermore, in the pheasants fission of the ancestral galliform chromosome 2 could be documented. Hybridization of various chicken probes to two different chromosomes or to only the short or long chromosome arm of one chromosome pair in the species representing the orders Passeriformes, Strigiformes, and Columbiformes revealed translocations and chromosome fissions during species radiation. Thus comparative analysis with chicken chromosome-specific painting probes proves to be a rapid and comprehensive approach to elucidate the chromosomal relationships of the extant birds.

Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. Conventional banding techniques, FISH and SKY.

Permanent Sertoli cell lines provide an ideal system for the in vitro analysis of function and responsiveness to biochemical/hormonal factors of this particular cell type. In general, cytogenetic analyses of cell lines often reveal remarkable chromosomal changes that may be associated with functional characteristics. In the present study we investigated the mouse Sertoli cell line TM4 by C-banding, silver staining, FISH and spectral karyotyping (SKY). A highly increased chromosome number (average 85-95) as well as five stable marker chromosomes were detected by the conventional staining techniques. SKY identified the markers as a translocation chromosome T(1;3), isochromosomes 11 and 18 and two different-sized microchromosomes. The results show the usefulness of combining SKY and conventional banding methods for the evaluation of chromosome alterations in widely used cell lines.

Heterochromatin is not an adequate explanation for close proximity of interphase chromosomes 1--Y, 9--Y, and 16--Y in human spermatozoa.

Analysis of human spermatozoa and lymphocytes using C-banding techniques and in situ hybridization has shown a higher order packaging of the human genome. Chromosomes are not distributed entirely at random within the nucleus. In particular, chromosomes 1, 9, and 16, carrying large blocks of pericentromeric heterochromatin, and the Y chromosome, carrying heterochromatin in Yq12, are in close proximity to each other within the nucleus and are involved in somatic pairing with nonhomologous chromosomes. In order to determine whether the close proximity of these chromosomes in any way is attributable to the distribution of heterochromatin, double in situ hybridization was performed on chromosomes 1--Y, 9--Y, and 16--Y as well as on 1--X, 9--X, and 16--X-with chromosome X as the other gonosome carrying less heterochromatin-in human spermatozoa. Each pair was found to have a nonrandom spatial distribution. However, comparison of the arrangement of chromosomes 1--Y versus 1--X and 9--Y versus 9--X revealed that heterochromatin cannot be the only cause for the tendency of chromosome fusion, because only the results of the chromosome pair 1--Y/1--X could support this proposition. In conclusion, the heterochromatin effect cannot be, in itself, an adequate explanation for chromosome association, implicating as well other mechanisms.

Meiotic nondisjunction of chromosomes 1, 17, 18, X, and Y in men more than 80 years of Age.

In order to evaluate a possible paternal age effect, testicular sperm cells from three men aged 81, 82, and 83 yr were analyzed by two-color- and three-color-fluorescence in situ hybridization for disomy rates of chromosomes 1, 17, 18, X, and Y as well as for diploidy frequencies. A minimum of 1500 sperm cells per donor and probe was evaluated due to the low number of spermatozoa in the preparations. Diploidy and disomy frequencies were in the same range as found in men aged <30 yr, a slight increase only being noticed for XY nuclei.

Sequence comparison of avian interferon regulatory factors and identification of the avian CEC-32 cell as a quail cell line.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.

Chromosomal localization of the genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma, and VIM in chicken by fluorescence in situ hybridization.

Six structural genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma and VIM were mapped in the chicken by fluorescence in situ hybridization (FISH) using genomic and cDNA clones as probes. The genes were found to be located on four different macrochromosomes: chromosome 1 (IFNG and FABP), chromosome 2 (VIM and ALDH), chromosome 3 (BMP2) and a smaller macrochromosome, most probably chromosome 7 (RXRG). With the exception of IFNG none of the newly mapped sites corresponds to known orthologous regions between chicken and human chromosomes.

Ectopic NORs on human chromosomes 4qter and 8q11: rare chromosomal variants detected in two families.

Two different NOR bearing non-acrocentric chromosomes were detected during prenatal diagnosis performed on two probands because of advanced maternal age. In the first case, a chromosome 4 carried a NOR in the telomeric region of the long arm (4qs), while in the second case a NOR was inserted into chromosome 8q11. Family analysis showed the variant chromosomes to be transmitted through at least three generations in each family. There were no reports of reproductive problems or phenotypic effects in the carriers of these chromosomes, indicating the benign character of the aberrant chromosomes. In order to characterise the chromosomal variants more precisely, various differential banding techniques were applied.

An interstitial nucleolus organizer region in the long arm of human chromosome 7: cytogenetic characterization and familial segregation.

An unusual NOR-bearing chromosome 7 was detected in a phenotypically normal, healthy 29-year-old male proband. Differential banding techniques as well as in situ hybridization employing various DNA-probes were performed in order to characterize the chromosome in detail. The nucleolus organizer region was found to be located between bands 7q21.3 and 7q22.1. No further rearrangements were detected in this chromosome. Analysis of spontaneously occurring micronuclei revealed 9% of them to contain a 7q fragment distal to (or including) the inserted NOR, suggesting that the inserted secondary constriction represents a potential hot spot for chromosomal breakage and rearrangement. Segregation analysis of the variant chromosome 7 in 51 members of the probands' family showed transmission in a Mendelian fashion. 27 individuals were found to be heterozygous for the inserted chromosome. A three-year-old child in the consanguineous marriage of two heterozygous carriers exhibited the NOR-insertion in both of his chromosome 7 homologues. To our knowledge, this is the first report on a homozygous carrier of a non-acrocentric NOR-bearing chromosome.

Genetic counseling in a patient with XXY/XXXY/XY mosaic Klinefelter's syndrome: estimate of sex chromosome aberrations in sperm before intracytoplasmic sperm injection.

OBJECTIVE: To report the sex chromosome aberrations in the sperm of a patient with mosaic Klinefelter's syndrome before ICSI. DESIGN: Case report. SETTING: Institute of Human Genetics, University Hospital PATIENT(S): A patient with an XXY/XXXY/XY mosaic Klinefelter's syndrome and extreme oligozoospermia. INTERVENTION(S): Skin biopsy, buccal smear, hair root sampling, and semen sampling. MAIN OUTCOME MEASURE(S): The karyotypes of three additional somatic cell systems and the ratio of sex chromosome aberrations in sperm. RESULT(S): After two-color fluorescence in situ hybridization of 202 interphase sperm nuclei, both the proportion of hyperhaploid 24, XY and 25, XXY sperm (5.0% and 0.5%, respectively) and of hyperhaploid 24, XX sperm (2.0%) were elevated. In contrast with peripheral lymphocytes, 93.9% of which showed sex chromosome aberrations, in the present patient only 7.5% of sperm proved to be hyperhaploid with an extra sex chromosome. CONCLUSION(S): The determination of sex chromosome aberrations in the sperm of a patient with mosaic Klinefelter's syndrome may provide additional information to estimate the transmission risk to his offspring.

Pregnancy after intracytoplasmic sperm injection with sperm from a man with a 47,XXY Klinefelter's karyotype.

OBJECTIVE: To report the initiation of a pregnancy that was achieved by intracytoplasmic sperm injection (ICSI) with sperm from a patient with Klinefelter's syndrome. DESIGN: Case report. SETTING: University women's hospital IVF center. PATIENT(S): A couple with primary infertility and nonmosaic 47,XXY karyotype of the male partner. INTERVENTION(S): Intracytoplasmic sperm injection after ovarian stimulation and transvaginal ultrasound-guided oocyte pick-up with sperm from a hypergonadotropic man with a nonmosaic 47,XXY karyotype. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Despite a 47,XXY karyotype in all 50 analyzed lymphocyte metaphases, the sperm of the patient led to a clinical pregnancy with the first attempt of ICSI and intrauterine transfer of three embryos. The pregnancy stopped developing in the ninth week. Cytogenetic investigation of the abortion material revealed a numerical normal 46,XXY karyotype. CONCLUSION(S): Sperm from a patient with hypergonadotropic nonmosaic Klinefelter's syndrome, when used for ICSI, can lead to a pregnancy.

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). II. Molecular cytogenetics, organization and evolutionary conservation of a chromosome-specific satellite DNA from Leporinus obtusidens.

A chromosome-specific satellite DNA from the South American fish species Leporinus obtusidens has been isolated and characterized. Sequence analysis and Southern hybridization studies indicate that the cloned 483-bp fragment is 60% AT rich and appears to comprise two diverged monomers. A highly variable low-copy number polymorphism was detected and, thus, this satellite DNA may serve as a valuable genetic marker. Using a Southern blot approach, the cloned satellite DNA cross-hybridized strongly to the DNA of Leporinus elongatus but failed to detect homologous sequences in the genomes of other closely related Leporinus species and higher vertebrates. Using fluorescence in situ hybridization to mitotic metaphase spreads of L. obtusidens and L. elongatus, this satellite DNA was located to the (peri)centromeric region of one single chromosome pair in both species. As the cloned satellite DNA sequence clearly evolved along a chromosomal lineage and is highly variable, it may serve as a very useful marker in further genetic, molecular and cytogenetic studies of the genus Leporinus.

Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review.

Sperm chromosome analysis offers the opportunity to gather information about the origin of chromosome aberrations in human germ cells. Over the last 20 years more than 20,000 sperm chromosome complements from normal donors and almost 6000 spermatozoa from men with constitutional chromosome aberrations (inversions, translocations) have been analyzed for structural and numerical chromosome abnormalities, as well as for segregation of the constitutional chromosome aberrations after the sperm had penetrated hamster oocytes. On the other hand, it took only 6 years to screen more than 3 million mature spermatozoa from healthy probands for disomy rates of 20 autosomes (chromosomes 19 and 22 not evaluated) and the sex chromosomes, and for diploidy rates by in situ hybridization techniques. In the present paper the results arising from both methods are compiled and compared.

Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter's karyotype: a FISH analysis.

Meiotic segregation of the sex chromosomes was analysed in sperm nuclei from a man with Klinefelter's karyotype by three-colour FISH. The X- and Y-specific DNA probes were co-hybridized with a probe specific for chromosome 1, thus allowing diploid and hyperhaploid spermatozoa to be distinguished. A total of 2206 sperm nuclei was examined; 958 cells contained an X chromosome, 1077 a Y chromosome. The ratio of X:Y bearing sperm differed significantly from the expected 1:1 ratio (chi2 = 6.96; 0.001 < P < 0.01). Sex-chromosomal hyperhaploidy was detected in 2.67% of the cells (1.22% XX, 1.36% XY, 0.09% YY) and a diploid constitution in 0.23%. Although the frequency of 24,YY sperm was similar to that detected in fertile males, the frequencies of 24,XX, 24,XY and diploid cells were significantly increased. A sex-chromosomal signal was missing in 4.26% of the spermatozoa. This percentage appeared to be too high to be attributed merely to nullisomy for the sex chromosomes and was considered, at least partially, to be the result of superposition of sex-chromosomal hybridization signals by autosomal signals in a number of sperm nuclei. The results contribute additional evidence that 47,XXY cells are able to complete meiosis and produce mature sperm nuclei.

Incidence of diploid and disomic sperm nuclei in 45 infertile men.

Sperm cells from 45 infertile patients were investigated for disomy rates of chromosomes 1, 7, 10, 17, X and Y as well as for diploidy by single- and double-target in-situ hybridization. The patients who attended the infertility clinic were aged 23-46 years. Semenograms showed that the patients had oligo-, astheno-, oligoastheno-, oligoterato-, oligoasthenoterato-, or asthenoteratozoospermia. The average disomy rates determined in the patients were similar for all chromosomes, ranging from 0.10% (chromosome Y) to 0.14% (chromosomes 10 and X). Diploidy was detected with a mean incidence of 0.1%. With the exception of two patients who exhibited significantly increased diploidy rates of 0.35 and 1.6%, neither disomy nor diploidy was increased in the group of infertile patients as compared to healthy, fertile males.

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). 1. Karyotype analysis, heterochromatin distribution and sex chromosomes.

Cytogenetic analyses (Giemsa staining, C-banding, AgNO3 labelling of nucleolus organizer regions (NORs) and staining with base-specific fluorochromes) were performed on the South American fish species Leporinus friderici, L. obtusidens and L. elongatus. The overall karyotypic structure, position of NORs, as well as the amount, distribution and composition of constitutive heterochromatin were determined. Particular attention was given to the highly differentiated ZZ/ZW sex chromosome system of L. obtusidens and L. elongatus. Sharing the apparently ancient macroscopic karyotype of Anostomidae, all three species have 2n = 54 meta- or submetacentric chromosomes. NORs were found exclusively on chromosome pair 2, which may represent the ancestral NOR-bearing chromosome of the anostomid karyotype. Observed differences in the relative position of NORs along chromosome 2 and variations in the amount and distribution of constitutive heterochromatin throughout the karyotype were most probably caused by heterochromatin-mediated chromosome rearrangements. Detailed analysis of the morphologically similar heteromorphic ZZ/ZW sex chromosomes of L. obtusidens and L. elongatus allowed detection of differences in the DNA composition of the largely heterochromatic W chromosomes. However, since these and the W chromosomes of three other Leporinus species exhibit homologies with respect to their relative size, centromere position and amount and distribution of heterochromatin, it is concluded that they evolved from the same ancestral W chromosome.

Interphase chromosome arrangement in Sertoli cells of adult mice.

Sertoli cells of adult male laboratory mice were examined with a number of banding techniques and by nonradioactive in situ hybridization applying different repetitive DNA probes. All banding methods revealed the typical features of mouse Sertoli cells, i.e., a central nucleolus, usually with two chromocenters associated at diametrically opposed sides in which the centromeric regions of the chromosomes are clustered. Silver staining as well as in situ hydridization with rDNA labeled part of the chromocenters and the nucleolus, indicating transcriptional activity of at least some of the nucleolus organizer regions. In situ hybridization with X- and Y-specific DNA probes showed both sex chromosomes to be undercondensed in Sertoli cells This decondensation suggests expression of sex chromosomal genes in Sertoli cells. While the X chromosome appeared to occupy a central position near one of the chromocenters, the Y chromosome was found at the periphery of the nucleus in the majority of cells. Hybridization with telomeric sequences resulted in strong labeling of the chromocenters and dispersed signals at the nuclear periphery.

Sex chromosome loss and aging: in situ hybridization studies on human interphase nuclei.

A total of 1,000 lymphocyte interphase nuclei per proband from 90 females and 138 males age 1 wk to 93 years were analyzed by in situ hybridization for loss of the X and Y chromosomes, respectively. Both sex chromosomes showed an age-dependent loss. In males, Y hypoploidy was very low up to age 15 years (0.05%) but continuously increased to a frequency of 1.34% in men age 76-80 years. In females, the baseline level for X chromosome loss is much higher than that seen for the Y chromosome in males. Even prepubertal females show a rate of X chromosome loss, on the order of 1.5%-2.5%, rising to approximately 4.5%-5% in women older than 75 years. Dividing the female probands into three biological age groups on the basis of sex hormone function (< 13 years, 13-51 years, and > 51 years), a significant correlation of X chromosome loss versus age could clearly be demonstrated in women beyond age 51 years. Females age 51-91 years showed monosomy X at a rate from 3.2% to 5.1%. In contrast to sex chromosomal loss, the frequency of autosomal monosomies does not change during the course of aging: Chromosome 1 and chromosome 17 monosomic cells were found with a constant incidence of 1.2% and 1%, respectively. These data also indicate that autosome loss in interphase nuclei is not a function of chromosome size.

Immunostaining and interphase arrangement of field bean kinetochores.

More than 100 sera from patients with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were tested in order to detect antigenic nuclear components of the field bean Vicia faba (2n = 12). Kinetochores of mitotic chromosomes and prekinetochores of interphase cells from root-tip meristems were specifically labelled via an indirect immunofluorescence procedure by antibodies of one of these sera. In 44% of interphase nuclei in which centromeres could be identified, only half (6) of the number of expected prekinetochores (12) was detected, circumstantially indicating at least transient association of homologous centromeres. Some nuclei showed clustering of centromeres at one pole (Rabl configuration). In metaphase chromosomes, each sister kinetochore contained a fluorescent spot. Western blotting of field bean nuclear proteins revealed four antigenic proteins of 28, 30, 64 and 68 kDa.