Studies on frequency of Y chromatin in human sperm

Summary The preparation and evaluation of human ejaculate smears on cover glasses enables double-sided examination of the individual sperm heads in the fluorescence microscope. By this method of observation the frequency of Y chromatin in the ejaculates of 19 probands increased significantly to between 1.7 and 6.3%. However, the frequency of 50% Y chromatin-positive sperms theoretically to be expected is not found by applying this technique.Furthermore, sexual abstention of at least 14 days leads to a statistically significant decrease of the frequency of Y chromatin. These observations could explain why the data on Y chromatin frequency reported in the literature appear so inconsistent.Supported by the Deutsche Forschungsgemeinschaft (AZ Schw. 154/4).     

Differential cell division in human X chromosome mosaics

Summary Fibroblasts were grown from skin explants of 3 human females who are sex chromosome mosaics. The 3 cultures had the following chromosome complements: 45,X/46,XX, 45,X/46,XXqi and 45,X/47,XXX. Using thymidine labelling and Colcemid accumulation of metaphases it was found that in all 3 mosaic cultures the 45,X cells had a faster cell cycle than the second cell population. The difference in cell cycle duration was attributed to the longer G₁ phase in the cells with 2 or 3 X chromosomes. The 2 populations of cells in the mosaic only differ in the number of heterochromatic X chromosomes and it is argued that heterochromatin has a retardative effect on cell division.

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Zusammenfassung Kulturen von Fibroblasten der Haut dreier weiblicher Individuen mit Sexchromatin-Mosaiks zeigten folgende chromosomale Zusammensetzung: 45,X/46,XX; 45,X/46XXqi und 45,X/47,XXX. Mit Hilfe von Thymidinmarkierung und Akkumulation von Metaphasen durch Colcemid konnte gezeigt werden, daß in allen 3 Mosaikkulturen die 45,X-Zellen einen schnelleren Zellcyclus haben als die zweite Zellpopulation. Der Unterschied in der Dauer des Zellcyclus beruht auf der längeren G₁-Phase der Zellen mit 2 oder 3 X-Chromosomen. Die 2 Zellpopulationen im Mosaik differieren nur in der Zahl der heterochromatischen X-Chromosomen, so daß angenommen werden könnte, daß Heterochromatin einen retardierenden Effekt ausübt.

     

Differential structuring of human populations for homologous X and Y microsatellite loci.

The global pattern of variation at the homologous microsatellite loci DYS413 (Yq11) and DXS8174 and DXS8175 (Xp22) was analyzed by examination of 30 world populations from four continents, accounting for more than 1,100 chromosomes per locus. The data showed discordant patterns of among- and within-population gene diversity for the Y-linked and the X-linked microsatellites. For the Y-linked polymorphism, all groups of populations displayed high FST values (the correlation between random haplotypes within subpopulations, relative to haplotypes of the total population) and showed a general trend for the haplotypes to cluster in a population-specific way. This was especially true for sub-Saharan African populations. The data also indicated that a large fraction of the variation among populations was due to the accumulation of new variants associated with the radiation process. Europeans exhibited the highest level of within-population haplotype diversity, whereas sub-Saharan Africans showed the lowest. In contrast, data for the two X-linked polymorphisms were concordant in showing lower FST values, as compared with those for DYS413, but higher within-population variances, for African versus non-African populations. Whereas the results for the X-linked loci agreed with a model of greater antiquity for the African populations, those for DYS413 showed a confounding pattern that is apparently at odds with such a model. Possible factors involved in this differential structuring for homologous X and Y microsatellite polymorphisms are discussed.     

Sex chromatin and X chromosome replication patterns and incidence of sex chromatin in canine cell cultures

In order to provide evidence as to whether sex chromatin (SC) of interphase cells is equivalent to the late replicating X chromosome in female mammalian cells, time-lapse cinephotometric and autoradiographic methods were used to give precise data for comparison of the DNA replication patterns of SC with that of each of the X chromosomes throughout the S period. Canine kidney epithelial cells were selected because they have distinct large metacentric X chromosomes and typical SC. Time-lapse cinephotometry was used to avoid possible alteration of DNA synthesis by chemical cell synchronization agents. Determination of the incidence of SC during the stages of the cell life cycle of proliferating cells of the same origin was performed in order hopefully to clarify conflicting reports on the subject. Our results clearly show that time and intensity of the SC replication throughout S period is like that of the late replicating X chromosome and unlike that of the early replicating X chromosome. The incidence of SC in proliferating cells in culture was found to vary with the stage of the cell life cycle, increasing with increasing postmitotic interval — least in G1, greater in S, and greatest in G2. The SC incidence increased strikingly from G1 to S and a less marked increase was observed between S and G2.     

The transition in frequency of Y chromatin in males during the neonatal period

Summary The frequency of Y chromatin, visualized as fluorescent bodies in cell nuclei from lymphocytes in blood smears, was signficantly less in newborn males than in three-month-old male infants and adults. The frequency of Y chromatin-positive cells on day 0 was 36.16±9.11% and then increased daily. At one month after birth the frequency was 55.07±9.29%, which was not significantly different from that in adult males (57.08±5.97%).     

Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts

ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist.     

Swimming behavior of X and Y human sperm

Abstract. A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.     

Swimming behavior of X and Y human sperm

A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.     

Capacity for tumor cell implantation as a function of in vitro cell density

Implantation properties of two melanoma cell lines, line 26 (low rate of implantation) and line 37 (high rate of implantation) were studied as a function of the cell density of the cells grown in monolayer in vitro. Sparse cultures (collected at a density of 0.8 × 10³ cells cm^?2) of line 37 produced 7.7 times as many lung tumor foci as those of line 26. Confluent cultures (collected at a density of 40 × 10³ cells cm^?2) resulted in greater numbers of tumor foci for both cell lines, but line 37 produced only 3.1 times as many tumor foci as did line 26 cells. Thus the high implantation line (37) has a much greater ability to implant when grown in the sparse state and injected than the low implantation line (26), but both lines have high implantation rates when injected as confluent cells.     

A novel protein related to cell cycle-dependent alterations of chromatin structure

We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure. Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase. Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible. Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei. The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix. The proteins also occur in the midbody during cytokinesis. The immunogenic epitope is conserved between man and fission yeast.     

Differential sensitivity of human T helper cell pathways by in vitro exposure to cyclosporin A

Cyclosporin A (CsA) is a widely used agent for the prevention of tissue allograft rejection in human transplantation. As a result of the recent demonstration that the allospecific Th cell response of human PBL can be generated by three distinct pathways of Th cell and APC interactions, we investigated the sensitivity of these three Th-APC pathways, as well as the Th response to recall Ag, to different concentrations of CsA. PBL from healthy volunteer donors were set up as primary in vitro cultures either without antigenic stimulation, or with influenza A virus, tetanus toxoid, or HLA alloantigenic (ALLO) stimulation. Ag-stimulated IL-2 production and proliferation were measured to assess Th cell function. To study the effect of CsA on Th function, different concentrations of CsA (0.001 to 0.1 micrograms/ml final) were added to the cultures at the time of stimulation. Th responses to influenza A virus and tetanus toxoid were more sensitive to CsA than the Th response to ALLO. By selective depletion of either responder or stimulator APC and/or of CD4+ or CD8+ cells, we 1) verified that the human ALLO Th response can be mediated by three distinct Th-APC pathways; 2) demonstrated that the ALLO response mediated by CD4+ Th and self-APC (the same helper pathway used by recall Ag) is as sensitive to CsA as the responses to recall Ag; and 3) showed that there is a hierarchy of sensitivity of these three allospecific pathways. The results are discussed with respect to the potential significance of the differential sensitivity of these allospecific Th-APC pathways to CsA for prevention of tissue allograft rejection.     

Differential condensation of human chromosomes 9 and Y

Some differences were observed in the mitotic condensation of regions composing human chromosomes 9 and Y: regions 9p, 9h and Y nf are characterized by an intense condensation by the end of the spiralization interval studied (the length of the repair chromosome 3 varying from 5.4 to 2.9 mkm). At the same time, the condensation of regions 9q-h (region 9q without heterochromatic block) is slowing in the initial spiralization interval (the length of chromosome 3 varying from 16.6 to 5.5 mkm). The Yf-block of Y-chromosome is condensing faster than nf-region. The condensation parameters of Q-heterochromatic blocks are most variable while the euchromatic regions are most stable. The dynamics of 9h and of f-block condensation are independent within one karyotype. Based on the data obtained we doubt the correctness of studies on linear dimensions of the constitutive heterochromatin blocks for the evaluation of its quantity in the karyotype. A possible association of differential mitotic condensation with the chromosome segregation disturbances is discussed.     

Y to X translocation in man

Summary Five new cases are added to the single published instance of Yq to Xp translocation (X_t) in man. It is shown that the anomaly can occur as a mutational event during meiosis, and can be inherited from a parent, but also that it can arise in a 47,XXY embryo. In individuals with 46,XX_t karyotype the gonadal development, sexual differentiation, gonadal function and fertility are within the range of normal females. They do not present overt or discrete sings of virilisation. However, somatic stigmata, and more specifically short stature, are present in all patients. There is no uniform pattern of X_t inactivation which varies from random to apparently preferential inactivation. This phenomenon may be important for the better understanding of X-inactivation which for the X_t the authors believe is random but followed by differential proliferation of the resulting two types of cells.Aided by grant 20 122 F.G.W.O. — Belgium.