Transmission of three radiation-induced translations in the Syrian hamster. I. Chromosome studies of male meiosis: pachytene to metaphase II.

Chromosome behaviour during meiosis in male Syrian hamsters heterozygous for one of three translocations was analysed as part of a study of the transmission of these structural changes. Synapsis was studied using preparations of synaptonemal complexes, and chiasmate associations and the results of anaphase I segregation were studied in air-dried preparations of metaphases I and II respectively. The main findings were: (i) that, at least in the two trivalent-forming translocations, there is no simple relationship between either the frequency or the extent of synapsis and chiasma formation between the chromosomes involved in the translocation; (ii) that the presence of a univalent in a substantial proportion of metaphase I cells does not necessarily lead to irregular segregation as judged by analysis of metaphase IIs; and (iii) conversely, that in translocation heterozygotes in which metaphase I contains the chromosomes involved in the translocation as a quadrivalent or as two bivalents, with no univalents or trivalents, unexpected numerical segregation can be found. The observations of meiotic chromosomes behaviour reported here show that it is not always possible to predict the effects of structural change, or to determine the basis of these effects, from an analysis of any stage of meiosis taken in isolation, or from an analysis of an apparently similar change.

The cytological and histological effects of photoperiodism and X-irradiation on the testes of the Syrian hamster.

The radiosensitivity of meiotic stem cells from photoregressed testes of the Syrian hamster was studied after in vivo irradiation with 2.67 Gy (1.77 Gy/min). The testes of Expt I animals were irradiated at the time of transfer from short days (11 weeks at 6L:18D (6 hours light: 18 hours) darkness) to long days (14L:10D) and sampled at weekly intervals between 5 and 10 weeks and at 15 weeks postirradiation. The Ext II animals received the same X-ray dose, 2, 4, 6, 8 weeks after the transfer to long days and were sampled 10 weeks postirradiation. In Expt I X-irradiation delayed the recovery of testis structure and function by 4 weeks. Animals in Expt II were homogeneous with regard to testicular weight, sperm count, tubule diameter and the level of pachytene, MI and MII damage. The incidence of structural chromosomal changes in primary spermatocytes at pachytene (synaptonemal complexes) and MI and MII was determined. In Exp I and II and data from X-irradiated testes (2.6 Gy) not exposed to short days (Control B, see Cawood and Breckon 1983), these cell types show a difference in sensitivity to the induction of structural damage. The lowest level of pachytene damage was in Expt II, and the ratio of the number of pachytene to MI cells showing structural change was 1.23:1 (P = 0.41), not significantly different from 1:1. In Expt I the pachytene, MI structural change ratio was 1.60:1 (P = 0.010), significantly different from 1:1. The control B ratio was 1.85:1 (P = 0.0037) which was significantly different from 1:1. The preliminary results indicate that testes fully regressed to the 'stem' cell level by exposure to short day length have a similar radiosensitivity to normal (non-regressed) testes. However, testes irradiated during the recovery phase from photoperiod-induced regression have increased sensitivity to radiation damage. The relative testis weight was reduced to a mean of 1.25 g by the sequential X-ray treatment--this may indicate that the effects of the X-rays (3 Gy) on a recovering photoregressed testis are independent of the time of the X-ray treatment during the recovery period. Recovery of tubule diameter is not perturbed and may be slightly increased, with an associated elevation of MI-MII activity--but a low spermatogonia count.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA fingerprinting.

Hypervariable tandem-repetitive minisatellite regions of human DNA can be used to generate individual-specific DNA fingerprints. Validation studies have demonstrated the reliability of the analysis, the mode of inheritance of the minisatellites, and the unparalleled degree of individual specificity. The uses of hypervariable probes in forensic biology, paternity testing, and the resolution of a wide range of problems in genetics, molecular biology, population biology, and medicine are illustrated.

Induced structural changes in chromosomes of the Syrian hamster after X-irradiation of spermatogonia: comparison of dose-response curves derived from synaptonemal complexes and from air-dried preparations of metaphase I.

The dose-response relationships of the induction of structural change in chromosomes after X-irradiation of spermatogonia have been determined from analyses of synaptonemal complexes in pachytene spermatocytes and from air-dried preparations of metaphase I. The dose-response curves were superficially the same shape, with a peak yield of cells containing a multivalent at 4 Gy, although only in the pachytene data was there any statistically significant hump. The pachytene preparations were much more sensitive, revealing nearly twice the proportion of cells containing multivalents than found at metaphase.

Photoperiodic control of meiosis in the male Syrian hamster (Mesocricetus auratus).

Male Syrian hamsters exposed to short photoperiods of 6 h light/day (6L:18D) show regression of the testes within 12 weeks. Chromosome preparations of the meiotic stages (pachytene, metaphase I (MI) and metaphase II (MII)), testicular weights relative to body weights, sperm counts, seminiferous tubule diameter and histological appearance were examined at intervals during regression and subsequent recovery in a long photoperiod (14L:10D). The fall of testicular weight was associated with the decrease in tubule diameter. Spermatogenesis and sperm count were reduced rapidly and finally ceased after 10 weeks in short days. The numbers of MI and MII cells relative to 100 pachytene cells progressively decreased during the short-day treatment, although the ratio of MI:MII stayed constant whenever there was meiotic activity (except in the first week of the recovery phase). This suggests that an increasing proportion of pachytene cells did not progress to MI with increased time in short days, but cells which did reach MI progressed to MII in the same proportions as in the control testes. Meiosis ceased after 10 weeks in short days. Recovery in the long days was marked by a peak in the number of MI and MII cells/100 pachytene cells soon after the return to long days. This preceded the return (to control values) of the sperm count by 10 weeks. Initial recovery in the first 3 weeks was very rapid in all the determined values.

Uninterrupted DNA synthesis during S-phase in untransformed diploid hamster fibroblasts.

Untransformed Syrian hamster fibroblasts in exponential growth were exposed to a pulse of [3H]-thymidine for 5 min, followed immediately by bromodeoxyuridine, and serial samples were taken up to 16 h. Preparations were autoradiographed and stained for replication banding. No cell with replication bands was found without significant [3H]-thymidine uptake, although the extent of uptake varied between sub-phases of S. Thus there is no indication of a total cessation of synthesis at any period during S-phase.

Synaptonemal complexes as indicators of induced structural change in chromosomes after irradiation of spermatogonia.

The incidence of multivalent formation has been compared in primary spermatocytes at pachytene, using synaptonemal complex preparations and at diakinesis/metaphase, using air-dried preparations, from testes of Syrian hamsters several weeks after exposure to an acute dose of X-rays. The pachytene preparations revealed nearly twice the number of multivalents found at diakinesis/metaphase I, and showed the ability to reveal types of structural alteration undetectable in conventional air-dried techniques.

The disparity between homologous chromosomes during DNA replication.

When the patterns of replicating chromosome bands of homologous chromosomes within a diploid cell during DNA synthesis phase are compared, the frequency of disparity (i.e. a band present on only one homologue) is less than expected on the basis of chance. This could be taken as implying some "link" between homologues which constrains their programmes of replication to keep in step. This paper develops a model showing that the observed disparities can be accommodated within a framework of homologue independence. Differences between cells in the time of appearance of bands lead in any sample to the summation of an infinitude of binomial distributions and hence to over-dispersion. The model fits observed data for bands replicating in euchromatic and heterochromatic chromosome regions obtained from Syrian hamster fibroblast cells growing in vitro.

A comparison of the use of bromodeoxyuridine and [3H]thymidine in studies of the cell cycle.

Untransformed Syrian hamster fibroblasts in short-term culture were studied by means of [3H]thymidine autoradiography combined with differential staining after continuous exposure to bromodeoxyuridine. Fractions of 3H-labelled metaphases (FLM) and of differentially stained metaphases (FDM) were determined in sequential samples. The resulting FLM and FDM curves are in close agreement, in sequential samples. The resulting FLM and FDM curves are in close agreement, except at later sample times (10 hr onwards), when the FLM is greater than the FDM. This inefficiency on the part of the bromodeoxyuridine technique in the detection of cells in early S-phase leads to an underestimate of the average duration of S compared with that derived from autoradiography. Grain counts suggest that only a small proportion of total DNA synthesis is missed. FLM and FDM curves from both diploid and tetraploid cells are very similar. Despite the inefficiency in the detection of cells in early S phase, the bromodeoxyuridine technique has a number of useful features in studies of the cell cycle. It is possible to distinguish pre-S cells from G2 cells unambiguously, and cells in their first cycle can be distinguished from those in their second with a much greater efficiency than by autoradiography. The technique is consistent and has no problems from background or the consequent need for thresholds in scoring. As is demonstrated here, the two methods can be combined, the differential staining being little affected by the autoradiographic procedures.

Early-replication DNA patterns in a derivative chromosome in a Syrian hamster with only 42 chromosomes.

From crosses within a 2n=43 line of Syrian hamsters (Mesocricetus auratus) lacking one derivative (der 11) of an 11;20 reciprocal translocation we have obtained homozygotes with only 42 chromosomes. These animals are homozygous deficient (nullisomic) for the centromere and short arm of chromosome 11 and for the bulk of the long arm of chromosome 20. -During cytogenetic studies, we investigated the frequency patterns of early-replicating bands in the surviving derivative (der 20) at two cytologically defined sub-phases of S using short-term fibroblast cultures. These patterns were compared with those observed in the component, untranslocated arms in normal 2n=44 cells at the same two sub-phases. -Very close agreement was found, indicating that neither the nullisomy, nor the new arm combination has interfered detectably with the pattern or programme of early band replication.

Chromosome replication in fibroblasts of the Syrian hamster (Mesocricetus auratus).

The BrdU/Hoechst 33258/Giemsa method for sub-dividing S-phase in asynchronous cell populations has been re-evaluated and modified to give better definition and more even distribution of sub-phases. A reference pattern of early-replicating euchromatic bands is given for all chromosomes at Sk2 in primary cultures of skin fibroblasts. The overall band patterns at each sub-phase have allowed more objective definitions of "early" and "late" replication for these cells, and show that in both classes of chromatin light G-bands preceed dark G-bands. Asynchrony between homologous bands is observed at all stages of S, albeit with a variable frequency. The observed in vitro replication patterns and programme for the chromosomes of skin fibroblasts does not appear to be affected by the age or sex of the source.

Cell-specific variation of X chromosome replication in the Syrian hamster (Mesocricetus auratus).

The sub-division of S-phase in Syrian hamsters, on the basis of Brd/U/Hoechst 33258/Giemsa banding, has allowed a quantitative comparison of the replication of individual chromosome bands within defined subphases of S. This analysis has shown that in hamsters, as has been reported in humans, there are distinct patterns of early replication in vitro in the early X, the late X in fibroblasts, and the late X in lymphocytes. In addition, it has been possible to show that, although the pattern of replication of the late X in fibroblasts differs from that in lymphocytes, the time in S at which bands first appear on this chromosome is the same in the two cell types. No significant heterogeneity can be ascribed to differences between individuals, adult or embryonic sources, culture media, or time of exposure to BrdU. The absence of any detectable heterogeneity in the replication band frequencies in autosomal heterochromatic arms suggests that the cell-specific variability of the late-replicating X is a feature of facultative rather than constitutive heterochromatin.

The development of nuclear vacuoles during meiosis in plants.

Vacuoles formed by the invagination of the inner membrane of the nuclear envelope have been observed during meiotic prophase in a wide range of plants. In the angiosperm Lycopersicon their formation was found to coincide with the completion of synaptonemal complex formation, and this timing is analogous to that observed during this stage in the silkworm Bombyx. The implications of this activity in relation to the process of chromosome movement are discussed. In the gymnosperm Pinus, the heterosporous fern Marsilea and homosporous ferns Pteridium and Dryopteris the formation of nuclear vacuoles begins much earlier, coinciding with the condensation of chromatin during leptotene. They enlarge and become more elaborate as meiosis proceeds, and may eventually become detached from the nuclear envelope. It is therefore thought unlikely that theyfulfil functions connected with chromosome movement in the manner proposed for the silkworm and the tomato. During diplotene/diakinesis they contain electron-opaque granules and fibrils, and the possible origin and significance of this material is discussed.

An evaluation of Coomassie Brilliant Blue as a stain for quantitative microdensitometry of protein in section.

The specificity and stoichiometry of the binding of Coomassie Brilliant Blue (CBB) to protein in section has been examined using both frozen protein matrices and plant material. The maximum adsorbance of the stain, bound and in solution, was found to be 620 nm although variation in the results at this wavelength necessitated measurements to be made at 600 nm. After enzyme treatments of sectioned plant material embedded in resin, all CBB-binding biological material was shown to be sensitive to non-specific protease. The relationship between optical density at 600 nm and section thickness was tested statistically against the Lambert-Beer law, using microdensitometry of cryostat-sectioned, frozen genatine solution. The analyses showed conclusively that, under these conditions, CBB adheres strongly to the Lambert-Beer relationship. CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.