Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n = 3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1 h (T1) and 24 h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r = 0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

Tissue-specific DNA methylation in Haemanthus katharinae Bak. (Amaryllidaceae)

The level of DNA methylation was compared in root meristem, adult leaf and endosperm of monocotyledonous species, <em>Haemanthus katharinae</em>, with the use of HPLC, restriction analysis, Southern blot hybridization and in situ nick-translation driven by restriction enzyme HhaI The highest level of 5-methylcytosine was observed in adult leaf whose nuclear chromatin is particularly condensed, and the lowest in endosperm. The level of DNA methylation of repetitive HaeIII 4006p sequence follows that of the total DNA.

Patterns of DNase I sensitivity in the holocentric chromosomes of the aphid Megoura viciae

Using the in situ nick translation technique, we looked for the presence of DNase I sensitive sites in Megoura viciae chromosomes, to study the distribution of active or potentially active genes in aphids, a group of insects possessing holocentric chromosomes. Cytological preparations obtained by the spreading of embryo cells were treated in situ with increasing concentrations (ranging from 5 to 200 ng/mL) of DNase I. At DNase I concentrations below 50 ng/mL, only one hypersensitive site was observed, and this was located on a telomeric region of the X chromosome that contains transcriptionally active nucleolar organizing regions, as assayed by silver staining. Interestingly, at intermediate concentrations of DNase, the incorporation of biotinylated nucleotide occurred uniformly throughout all chromosomes, whereas at concentrations above 100 ng/mL, a C-like banding pattern was produced. Our data differ from results obtained with mammalian, frog, and grasshopper chromosomes, where it was found that DNase I nicking is concentrated at the distal regions of all chromosomes.Key words: aphids, holocentric chromosomes, DNase I sensitivity, nick translation.

Major DNA Fragmentation Is a Late Event in Apoptosis

Apoptosis, the terminal morphological and biochemical events of programmed cell death, is characterized by specific changes in cell surface and nuclear morphology. In addition, DNA fragmentation in an internucleosomal pattern is detectable in mass cultures of apoptotic cells. However, DNA fragmentation and nuclear morphological changes may not necessarily be associated events. In this study, we examined OVCAR-3 and KB human carcinoma cells using time-lapse video phase-contrast microscopy to characterize the surface and nuclear morphological features of apoptosis in response to treatment with either taxol or ricin. The surface morphological features of apoptosis were the same in both cell types and with both drugs. Using an in situ nick-translation histochemical assay, these single cells were also examined for DNA strand breaks during apoptosis. Surface morphological changes demonstrated discrete stages of cell rounding, surface blebbing, followed by cessation of movement and the extension of thin surface microspikes, followed much later by surface blistering and cell lysis. Nuclear features examined by DAPI cytochemistry demonstrated apoptotic nuclear condensation very early in this sequence, usually at the time of initial surface blebbing. The nick-translation assay, however, demonstrated DNA strand breaks at a much later time, only after the formation of separated apoptotic bodies or after final cell lysis. This study points out the differences between surface and nuclear morphological changes in apoptosis, and the large temporal separation between nuclear morphological changes and major DNA fragmentation detectable by this in situ technique. This result suggests caution in using in situ nick-translation as a direct correlate of internucleosomal DNA fragmentation in apoptosis.