1 Dipartimento di Biologia Animale, Laboratorio di Biologia dello Sviluppo e Centro di Eccellenza in Biologia Applicata, Universita di Pavia, Piazza Botta, 9 27100 Pavia, Italy
2 Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Universita di Parma, Via Volturno, 39 43100 Parma, Italy
3 Department of Obstetrics and Gynaecology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA
4 Laboratorio de Cariobiología y Citogenética, Programa de Genética Humana, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70061, Santiago 7, Chile
5 Dipartimento di Biologia Animale e dellUomo, Universita "La Sapienza" Roma, Via Alfonso Borelli, 50 00161 Roma, Italy
Author for correspondence (e-mail: silvia.garagna{at}unipv.it)
Accepted September 7, 2001
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SUMMARY |
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Key words: Nuclear architecture, Germ cells, Chromosome translocation, Subfertile mice, Sertoli cell, X and Y chromosomes
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INTRODUCTION |
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Our knowledge, however, of large-scale chromosomal and nuclear architecture is limited and little is known about the influence of karyotype restructuring on nuclear spatial organization and on the regulation of gene activity. Making use of dual-colour FISH (fluorescence in situ hybridisation) for whole-chromosome painting, we have analysed the spatial arrangements of chromosomes 5, 11, 13, 15, 16, 17, X and Y in the nuclei of Sertoli, spermatogonia, pachytene spermatocytes and spermatid cells in mice exhibiting karyotype variation owing to the presence or absence of Robertsonian (Rb) fusion chromosomes. The analysis of male germ-cell differentiation throughout the cycle of the seminiferous epithelium made it possible: (1) to follow the changes in the nuclear territories occupied by each single telocentric chromosome when in an all-telocentric karyotype or when that same chromosome fuses with another to form an Rb metacentric in either a homozygous or a heterozygous state; and (2) to try and relate these changes of chromosome localisation (nuclear architecture) to the associated changes in spermatogenesis. We show that the presence of Rb heterozygosities in the nucleus has a marked impact on the spatial arrangements of chromosomes, producing large-scale changes in chromosome territories, which correlate with detrimental effects on male germ-cell differentiation. Interestingly, the Sertoli cells of subfertile mice show X and Y chromosome FISH-signals condensed as in human patients with the Sertoli-cell-only syndrome.
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MATERIALS AND METHODS |
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Testicular histology, isolation and countings of germ cells
One testis from each animal was used for histology; the other was minced to separate the germ cells. Isolated germ cells were released from the seminiferous epithelium in 2.2% sodium citrate, pelleted by centrifugation and resuspended in 0.56% KCl. After the hypotonic treatment, cells were fixed in 3:1 (v/v) methanol:acetic acid, dropped onto slides and air-dried. The criteria to assess the dividing line between diakinesis and MI-spermatocytes [degree of condensation and opening out of the bivalents and trivalents, as in (Odorisio et al., 1998)] were established before the blind scoring by two operators on two Giemsa-stained slides per animal. Using a low-power objective, the number of diakinesis, MI and MII-spermatocytes was assessed for 100 microscopic fields per slide by each operator. Analysis of variance was performed to determine the significance of the differences between the means.
Testes were fixed in Bouins fixative for 24 hours, embedded in paraffin wax (Paraplast) and sectioned (6 µm). Ten sections per slide were prepared, five sections being discarded of each of those collected. Four slides were prepared for each mouse. The twelve stages of the seminiferous epithelium cycle were scored after periodic acid-Schiff and haematoxylin staining according to (Oakberg, 1956). The number of primary spermatocytes and spermatids, evaluated by three operators, was corrected according to Abercrombie (Abercrombie, 1946); no significant intra- or inter-animal variation was recorded by the three operators.
In situ hybridization and hybridization probes
Mouse whole-chromosome specific probes (CAMBIO, UK) for chromosomes 5, 11, 13, 15, 16, 17, X and Y were employed for FISH on fixed isolated germ cells. Probes for chromosomes 5, 11, 16 and X were fluorescein-isothiocyanate-(FITC)-labelled; those for chromosomes 13 and 17 were biotin-labelled, and those for chromosomes 15 and Y were Cy3 labelled. For each of the two animals employed, three slides for each karyotype (CR, F1 and C3H) were subjected to chromosome painting (for chromosome pairs 5/15, 11/13, 16/17 and X/Y) according to the manufacturers instructions. Stringency washes were in 50% formamide in 0.5xSSC at 42°C. FITC signals were amplified by the use of the FITC Amplification kit (CAMBIO catalogue number 1084-KF); biotin-labelled probes with the Biotin (Texas Red) painting kit (CAMBIO, catalogue number 1082-KT). Slides were counterstained with 4'-6-diamino-2-phenylindole (DAPI, 0.05 µg/ml) and mounted with an antifade (DABCO, 2%).
Scoring of Sertoli and germ cells hybridization patterns
Thirty spermatogonia, spermatocytes, spermatids and Sertoli cells from each of the three DAPI-stained slides for each of the two animals (to give a total number of 540 scorings per germ-cell type) were independently analysed by the three observers. Before the blind scoring, common criteria were adopted to identify and stage the cells on the basis of the nuclear and the cellular characteristics of the mouse testis cells as described (Meistrich et al., 1973). The hybridization patterns were evaluated after focusing on the brightest area of the fluorescent signal. A signal was regarded as decondensed, as opposed to condensed, if expanded over a relatively large area. The nuclear position occupied by a probe signal was seen to vary from being close to the nuclear membrane (peripheral) or spread across the nucleus (central). The proximity between chromosomes, overlapping versus opposite ends, was recorded as well. During pilot runs, we performed image analysis of the signals on selected cells both by printing photos and converting to µm the area and linear distances of the signals and by using the NIH Image program (http://rsb.info.nih.gov/nih-image/about.html). It was obvious that for our purposes the signals can be classified equally well by the subjective evaluation of the observer. It must, however, be recalled that caution must be exercised when drawing conclusions from 2D FISH analysis because, when air-dried onto a slide, a nucleus with a peripheral signal might become one with a central signal and vice versa. Regarding the reliability of the conclusions we draw, it should be considered that several systematic comparisons of 2D versus 3D FISH analysis support the validity of the assumption that the relative organization of the nucleus is not significantly altered by the hypotonic treatment of the cells and its subsequent squashing [(Csink and Henikoff, 1998) in Drosophila; (Croft et al., 1999) in humans; (Mayer et al., 2000), in mouse; (Metzler-Guillemain et al., 2000), in man, chimpanzee and mouse; (Nikiforova et al., 2000) in man; (Cremer and Cremer, 2001) in mouse]. The analysis of the frequency of the detectable hybridization patterns theoretically expected (on the basis of the combined possibilities of the three categories we employed to classify the signals: position, condensation and proximity) showed one pattern remarkably being 15-20 times commoner than any other (Fig. 2). This most frequent pattern showed negligible variations among observers (the greatest variation ranged from 76-82% for observers 2 and 3 in the pachytene cells of testes from Rb(16.17)/+ mice to 81-88% for observers 3 and 1 in the spermatids of C3H mice with chromosome pairs 11 and 13 in the homozygous state), and therefore we called it typical. Images were captured using an Olympus (Provis) epifluorescence microscope equipped with a thermoelectronically cooled charge-coupled device camera (Photometrics CH350). Grey-scale images were recorded separately for each fluorescent dye (viewed with the appropriate filters) using the IPLab Spectrum Imaging software (Signal Analytics Corporation) and then pseudocoloured (red for Cy3 and Texas Red, yellow-green for FITC and blue for DAPI) and superimposed.
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RESULTS |
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In the spermatogonia of heterozygous mice, the Rb metacentric FISH signals are clearly well apart from the homologous telocentrics, the latter being localized in separate nuclear domains. The telocentrics showed a location similar to that described for the all-telocentric homozygous condition; on the contrary, the signals pertaining to the Rb metacentrics show that one chromosomal arm is positioned near the spatial domain of the other, being sequestered there (when compared to the all-telocentric pattern). The patterns of chromosome spatial location described are similar for all the chromosome pairs analysed and in each of the cell types studied, and are clearly visible in gonia and pachytene nuclei. In the Rb heterozygotes, the spatial relationship of both chromosome arms of the Rb metacentric and that of its telocentric homologues differ from that of the homozygotes. This suggests that the Rb fusion leaves the position of one homologue of each chromosome pair involved in the translocation unchanged, inducing a change in the position of the other homologue. The typical pachytene spermatocyte FISHed for chromosomes 16/17 (second column, Fig. 1) shows one signal well separated from the other three, suggesting complete asynapsis of chromosome 16 in over 75% of pachytene cells. This is contrary to synaptonemal complex analyses of other heterozygotes of this genotype, which failed to detect such asynapsis, although the Rb-telocentric-telocentric trivalent associations did have limited, localised asynapsis (Redi and Capanna, 1988). Further work is needed to resolve this discrepancy. The typical (76%) hybridization pattern of the diploid interphase nuclei of the Sertoli cells is characterized by condensed and overlapping X and Y chromosomes located near one of the two heterochromatic blocks at the sides of the prominent nucleolus. In total, the hybridization patterns showing condensed signals for X and Y amount to 84% of the total number of cells.
As there are possible links between the changed chromatin domains in the Rb heterozygotes and their known spermatogenic impairment (Redi and Capanna, 1988), we have characterised the spermatogenic impairment in detail. A major feature of the impairment is that at least 80% of tubule cross-sections show such severe damage to the spermatogenic epithelium (Fig. 3, inserts on the right: top, fertile homozygotes; bottom, subfertile heterozygotes) that the tubules can not be staged using the Oakberg criteria. The quantitative assessment of the the remaining 20% of tubules shows that even in these tubules the spermatogenetic process is impaired, with the production of fewer spermatids at stage I (Fig. 3). The pachytene spermatocytes/spermatids ratio is 1:2.2 in C3H x CR males, which is far from the theoretical 1:4 ratio expected for fertile animals or the observed ratios of 1:3.7 and 1:3.8 for the all-telocentrics and Rb homozygotes, respectively. In order to pinpoint the stage of loss, we have scored the relative numbers of diakinesis, MI- and MII-spermatocytes for the fertile homozygotes and the Rb heterozygote (Table 1). The mean values, and the ratio of MI to diakinesis spermatocytes, do not differ significantly among the three karyotypes, whereas MII-spermatocytes are significantly fewer (P<0.05) in the Rb heterozygote compared to the other two karyotypes. This suggests that the germ cell losses in the 20% of relatively normal tubules of C3H x CR Rb heterozygous mice occur during the transition from MI to round spermatids, probably at the spermatocyte II stage (as the reduced numbers of MII-spermatocytes corroborate, Table 1). Those round spermatids that reach stage I are able to differentiate until stage VIIVIII (Fig. 3).
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DISCUSSION |
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The Sertoli cells of Rb homozygous fertile animals always show diffuse X and Y FISH signals, suggesting a decondensed chromatin organisation correlated with an active gene expression. It is well known that the condensation of the X and Y chromosomes in Sertoli cells of man and mouse is dynamic. For example, the X and Y chromosomes are condensed in pre-pubertal fertile individuals and infertile men with the Sertoli-cell-only syndrome, although they are expanded in adult fertile individuals [(Guttenbach et al., 1996; Chandley and Speed, 1995) in the house mouse; (Speed et al., 1993; Kofman-Alfaro et al., 1994) in man]. Our finding that the X and Y signals are condensed in the majority (84%) of the Sertoli nuclei of Rb subfertile mice, parallels similar findings in humans with the Sertoli-cell-only syndrome (Kofman-Alfaro et al., 1994). On the one hand this supports the view that at least some facets of the spermatogenesis process may not be autonomous to the germ cells, instead it is controlled by the Sertoli cells (Speed et al., 1993). On the other hand, it suggests that the subfertile Robertsonian mouse might be a useful model of human impaired spermatogenesis.
We suggest that large-scale changes in chromosome territories by changing the physical interactions of specific genome portions act as an epigenetic factor in controlling gene expression in somatic and germ cells. Future studies of gene expression in Sertoli cells, as well as in diploid and haploid germ cells of Rb heterozygotes, could help to highlight the cell-specific orchestration of gene activity that governs cell differentiation one of the crucial themes of present-day biomedical researches.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Abercrombie, M. (1946). Estimation of nuclear population microtome sections. Anat. Rec. 94, 238-248.
Abney, J. R., Cutler, B., Fillbach, M. L., Axelrod, D. and Scalettar, B. A. (1997). Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J. Cell Biol. 137, 1459-1468.
Berezney, R. and Wei, X. (1998). The new paradigm: integrating genomic function and nuclear architecture. J. Cell Biochem. 31, 238-242.
Boyle, S., Gilchrist, S., Bridger, J. M., Mahy, N. L., Ellis, J. A. and Bickmore, W. A. (2001). The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells. Human Mol. Genetics 10, 211-219.
Cerda, M. C., Berrios, S., Fernandez-Donoso, R., Garagna, S. and Redi, C. A. (1999). Organisation of complex nuclear domains in somatic mouse cells. Biol. Cell 91, 55-65.[Medline]
Chandley, A. C. and Speed, R. M. (1995). A reassessment of Y chromosomal behaviour in germ cells and Sertoli cells of the mouse as revealed by in situ hybridisation. Chromosoma 104, 282-286.[Medline]
Cremer, T. and Cremer, C. (2001). Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2, 292-301.[Medline]
Croft, J. A., Bridger, J. M., Boyle, S., Perry, P., Teague, P. and Bickmore, W. A. (1999). Differences in the localization and morphology of chromosomes in the human nucleus. J. Cell Biol. 145, 1119-1131.
Csink, A. K. and Henikoff, S. (1998). Large-scale chromosomal movements during interphase progression in Drosophila. J. Cell Biol. 143, 13-22.
van Driel, R. and Otte, A. (1997). Nuclear organization, chromatin structure and gene expression. Oxford: Oxford University Press.
De Boni, U. (1994). The interphase nucleus as a dynamic structure. Int. Rev. Cytol. 150, 149-171.[Medline]
Ferguson, M. and Ward, D. C. (1992). Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei. Chromosoma 101, 557-565.[Medline]
Forejt, J. (1982). X-Y involvement in male sterility caused by autosome translocations A hypothesis. In Genetic control of gamete production and function (ed. P. G. Crosignani and B. L. Rubin), pp. 135-151. New York: Academic Press.
Gropp, A., Winking, H., Redi, C. A., Capanna, E., Britton Davidian, J. and Noack, G. (1982). Robertsonian karyotype variation in wild house mice from Rhaeto-Lombardia. Cytogenet. Cell Genet. 34, 67-77.[Medline]
Guttenbach, M., Martinez Esposito, M. J., Engel, W. and Schmid, M. (1996). Interphase chromosome arrangement in Sertoli cells of adult mice. Biol. Reprod. 54, 980-986.[Abstract]
Hamkalo, B. A. and Elgin S. C. R. (1991). Functional organization of the nucleus. A laboratory guide. Methods in Cell Biology. 35. New York: Academic Press.
Handel, M. A., Cobb, J. and Eaker, S. (1999). What are the spermatocytes requirements for successful meiotic division? J. Exp. Zool. 285, 243-250.[Medline]
Hotta, Y. and Chandley, A. C. (1982). Activities of X-linked enzymes in spermatocytes of mice rendered sterile by chromosomal alterations. Gamete Res. 6, 65-72.
Katsumata, M. and Lo, C. W. (1988). Organization of chromosomes in the mouse nucleus: analysis by in situ hybridization. J. Cell Sci. 90, 193-199.[Abstract]
Kofman-Alfaro, S., Speed, R. M., Boyle, S. and Chandley, A. C. (1994). Condensation behaviour of the human X chromosome in male germ cells and Sertoli cells examined by fluorescence in situ hybridization. Chrom. Res. 2, 439-444.[Medline]
Kozubek, S., Lukasova, E., Mareckova, A., Skalnikova, M., Kozubek, M., Bartova, E., Kroha, V., Krahulcova, E. and Slotova, J. (1999). The topological organization of chromosomes 9 and 22 in cell nuclei has a determinative role in the induction of t(9,22) translocations and in the pathogenesis of t(9,22) leukemias. Chromosoma 108, 426-435.[Medline]
Lamond, A. I. and Earnshaw, W. C. (1998). Structure and function in the nucleus. Science 280, 547-553.
Leger-Silvestre, I., Trumtel, S., Noaillac-Depeyre, J. and Gas, N. (1999). Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae. Chromosoma 108, 103-113.[Medline]
Manuelidis, L. (1990). A view of interphase chromosomes. Science 250, 1533-1540.[Medline]
Manuelidis, L. (1997). Interphase chromosome positions and structure during silencing, transcription and replication. In Nuclear organization, chromatin structure, and gene expression (ed. R. Van Driel and A. P. Otte), pp.145-168. Oxford: Oxford University Press.
Manuelidis, L. and Borden, J. (1988). Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction. Chromosoma 96, 397-410.[Medline]
Mayer, W., Smith, A., Fundele, R. and Haff, T. (2000). Spatial separation of parental genomes in preimplantation mouse embryos. J. Cell Biol. 148, 629-634.
Meistrich, M. L., Bruce, W. R. and Clermont, Y. (1973). Cellular composition of fractions of mouse testis cells following velocity sedimentation separation. Exp. Cell Res. 79, 213-227.[Medline]
Metzler-Guillemain, C., Usson, Y., Mignon, C., Depretis, D., Dubreuil, G., Guichaoua, M. R. and Mattei, M. G. (2000). Organization of the X and Y chromosomes in human, chimpanzee and mouse pachytene nuclei using molecular cytogenetics and three-dimensional confocal analysis. Chrom. Res. 8, 571-584.[Medline]
Nagele, R. G., Freeman, T., Fazekas, J., Lee, K. M., Thomson, Z. and Lee, H. Y. (1998). Chromosome spatial order in human cells: evidence for early origin and faithful propagation. Chromosoma 107, 330-338.[Medline]
Nikiforova, M. N., Stringer, J. R., Blough, R., Medvedovic, M., Fagin, J. A. and Nikiforov, Y. E. (2000). Proximity of chromosomal loci that partecipate in radiation-induced rearrangements in human cells. Science 290, 138-141.
Oakberg, E. F. (1956). A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ renewal. Am. J. Anat. 99, 391-414.
Odorisio, T., Rodriguez, T. A., Evans, E. P., Clarke, A. R. and Burgoyne, P. S. (1998). The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis. Nat. Genet. 18, 257-261.[Medline]
van der Ploeg, M. (2000). Cytochemical nucleic acid research during the twentieth century. Eur. J. Histochem. 44, 7-42.[Medline]
Redi, C. A. and Capanna, E. (1988). Robertsonian heterozygotes in the house mouse and the fate of their germ cells. In The cytogenetics of mammalian autosomal rearrangements (ed. A. Daniel), pp. 315-359. New York: Alan R. Liss.
Redi, C. A., Hilscher, B. and Winking, H. (1983). Stage-dependent enzymatic activities in spermatogenesis of mice with the standard karyotype and of chromosomal variants with impaired fertility. Andrologia 15, 322-330.[Medline]
Redi, C. A., Garagna, S., Hilscher, B. and Winking, H. (1985). The effects of some Robertsonian chromosome combinations on the seminiferous epithelium of the mouse. Development 85, 1-19.
Rose, D. and Holm, C. (1993). Meiosis-specific arrest revealed in DNA topoisomerase II mutants. Mol. Cell Biol. 13, 3445-3455.[Abstract]
Schultz, R. M., Davis, W., Stein, P. and Svoboda, P. (1999). Reprogramming of gene expression during preimplantation development. J. Exp. Zool. 285, 276-282.[Medline]
Speed, R. M., Vogt, P., Kohler, M. R., Hargreave, T. B. and Chandley, A. C. (1993). Chromatin condensation behaviour of the Y chromosome in the human testis. Chromosoma 102, 421-427.[Medline]
Zink, D., Cremer, T., Saffrich, R., Fischer, R., Trendelenburg, M. F., Ansorge, W. and Stelzer, E. H. (1998). Structure and dynamics of human interphase chromosome territories in vivo. Hum. Genet. 102, 241-251.[Medline]
Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R. and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369-374.[Medline]