Ewa Wiland

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).

Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22) with high rates of unbalanced sperm and embryos: a case report.

Regarding the literature on the results of preimplantation genetic diagnosis (PGD) in reciprocal chromosomal translocation carriers seems to prevail a view that this method reduces the frequency of miscarriages, and the pregnancy rate is directly proportional to the number of normal spermatozoa. Therefore, we compared the results of sperm karyotype analysis of a carrier of familial t(2;7)(p11.2;q22) with PGD results. The carrier was ascertained as his wife had had two miscarriages.

Risk evaluation of carriers with chromosome reciprocal translocation t(7;13)(q34;q13) and concomitant meiotic segregation analyzed by FISH on ejaculated spermatozoa

We performed the segregation analysis of a relatively large pedigree of t(7;13)(q34;q13) carriers together with the sperm karyotype analysis of the one carrier using a tri‐color fluorescence in situ hybridization (FISH) method. The risk assessments for unfavorable pregnancy outcomes in a series of 36 pregnancies in eight reciprocal chromosome translocation (RCT) couples of carriers were estimated directly from a pedigree after ascertainment correction. The individual probability rate for unbalanced child was predicted according to Stengel‐Rutkowski and co‐workers. The unbalanced karyotypes in the form of monosomy 7q34 → qter and trisomy 13q13 → qter were detected among stillborn/early death newborns with holoprosencephaly (HPE), cyclopia and other malformations. Based on clinical description of unkaryotyped stillbirth progeny, it can be assumed that the phenotype distinctions were connected with the unbalanced karyotype from 2:2 segregation (monosomy 7q with trisomy 13q) and 3:1 segregation as interchange trisomy 13 (Patau syndrome). Probability rates for miscarriages, stillbirth/early death were 12.9 ± 6% (4/31) and 29 ± 8.2% (9/31), respectively. The results of the meiotic segregation pattern indicated the rate of unbalanced spermatozoa for about 60%, with the unusual high rate (29.4%) of 3:1 segregant (i.e., 13.4% of the tertiary segregation and 16% of the interchange segregation). Adjacent‐1 segregation followed with 23.5% and adjacent‐2 followed with 7.2% of analyzed spermatozoa. The high rate of unbalanced gametes in comparison to the number of stillborn/early death and miscarriages detected in pedigree suggests a strong selection against unbalanced chromosomal constitutions during fetal development. It corresponds to a very small probability rate (about 0.3%) of viable unbalanced progeny from 3:1 meiotic segregation predicted for maternal carriers. This knowledge can be used in genetic counseling of families with similar RCT ascertained in a different way.

Interindividual differences and alterations in the topology of chromosomes in human sperm nuclei of fertile donors and carriers of reciprocal translocations

Recently it has been shown that the nucleus of the human spermatozoon appears to possess a specific architecture. The current prevailing view is that spatial organization of the male genome contains information critical for the spermatozoon’s function as well as for early embryonic development. The purpose of this study was to determine whether there are alterations in intranuclear localization of centromeres in spermatozoa of chromosomes associated with particular reciprocal chromosome translocations (RCT). We analyzed the longitudinal and spatial localization of centromeres of selected chromosomes in sperm nuclei of four control males with normal karyotypes as well as in six carriers of reciprocal chromosome translocations: t(1;7), t(7;2), t(7;13), t(7;9), t(9;14), and t(4;13). Our study revealed that chromosomes with translocations may have shifted their intranuclear localization and that these translocations may influence the localization of other chromosomes in sperm nuclei. The chromocenter in sperm nuclei of translocation carriers was widened toward the apical side in comparison with chromocenter sites visible in control males. Our study also revealed interindividual differences in the localization of the Y chromosome centromere in the chromocenter area of sperm from fertile individuals.

Characterisation of Nuclear Architectural Alterations during In Vitro Differentiation of Human Stem Cells of Myogenic Origin

Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

Non-histone chromatin proteins during various stages of activity of immunocompetent cells

SummaryQualitative and quantitative changes of non-histone chromatin proteins of spleen cells during the primary immune response to sheep red blood cells and aggregated human gamma-globulin were described. Synthesis of non-histone chromatin proteins was measured by labelling with3H-tryptophan during culture of spleen cellsin vitro. Chromatin was isolated and labelled proteins were analysed by polyacrylamide gel electrophoresis. During the immune response to both antigens in chromatin of spleen cells three new fractions of non-histone chromatin proteins were synthesized: fractions F1−M=12 000 and H−M=3 000, specific for sheep red blood cells and fractions I1−M<3 000 and B−M=120 000, specific for human gamma-globulin. The third antigen-non-specific fraction was synthesized at the time when the primary immune reaction was finishing. These new fractions were synthesized only in one of the analysed subpopulations of spleen cells. In thymocytes (non-fully-differentiated lymphoid cells) all these fractions were absent. Changes of non-histone chromatin proteins in thymocytes during the immune response were similar to those found during stimulation to proliferation by phytohemagglutininin vitro.

Binding of low mobility group protein from rat liver chromatin with histones studied by chemical cross-linking

The protein of molecular weight about 160 kD (designated LMG160) was isolated from purified low mobility group chromatin proteins. Polyclonal antibody directed against the LMG160 protein in mouse was raised. The specificity of the antibody was determined with the use of ELISA. Using chemical cross-linking procedure followed by immunoprecipitation with the antiLMG160 antibody complex formation with chromatin proteins was demonstrated. Among the proteins that form complexes with LMG160, histones H3, H2A, and H4 were identified (Western blotting technique).

Genetic dosage and position effect of small supernumerary marker chromosome (sSMC) in human sperm nuclei in infertile male patient

Chromosomes occupy specific distinct areas in the nucleus of the sperm cell that may be altered in males with disrupted spermatogenesis. Here, we present alterations in the positioning of the human chromosomes 15, 18, X and Y between spermatozoa with the small supernumerary marker chromosome (sSMC; sSMC+) and spermatozoa with normal chromosome complement (sSMC−), for the first time described in the same ejaculate of an infertile, phenotypically normal male patient. Using classical and confocal fluorescent microscopy, the nuclear colocalization of chromosomes 15 and sSMC was analyzed. The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat. Analysis of meiotic segregation showed a 1:1 ratio of sSMC+ to sSMC− spermatozoa, while evaluation of sperm aneuploidy status indicated an increased level of chromosome 13, 18, 21 and 22 disomy, up to 7 × (2.7 − 15.1). Sperm chromatin integrity assessment did not reveal any increase in deprotamination in the patient’s sperm chromatin. Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC. This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

FISH and array CGH characterization of de novo derivative Y chromosome (Yq duplication and partial Yp deletion) in an azoospermic male

This study presents a 28-year-old infertile male who was referred to the cytogenetic laboratory for chromosomal analysis after 4 years of regular unprotected intercourse in whom non-obstructive azoospermia was revealed. Standard cytogenetic G-banding was performed on metaphase spreads and a de-novo karyotype 46,X,der(Y)(q11.22;p11.3) was identified. This analysis was followed by flourescence in-situ hybridization(FISH) and array comparative genomic hybridization (aCGH). Finally, the patients karyotype was identified as 46,X,der(Y)(qter→q11.221::p11.31→qter).ish der(Y) (qter+,pter-,SHOX+,SRY+,Ycen+,DYZ3+;DYZ1+,qter+).arrYq11.221q12(14,448,863-59,288,511) x2, Yp11.32p11.31(104,062-266,388) x0. It is proposed that de-novo derivative monocentric Y chromosome with duplicated region Y qter→q11.221::p11.31→qter with partial deletion of Yp PAR1 region most probably can perturb the conjugation of sex chromosomes during first meiotic division of spermatogenic arrested differentiation (development).

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p=0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6-38.0%) and the frequency of genetically normal/balanced gametes (34.3-62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R=0.4524, p=0.2604; AB: R=0.5238, p=0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.