Mass transport kinetics of the DNA-binding dye Hoechst-33342 into bovine spermatozoa

Weisenfeld, Robert B.

doi:10.1016/j.bmc.2007.06.061

Cited by 7 publications

(8 citation statements)

References 15 publications

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“…However, since H342-induced apoptosis is partially blocked by H258, this implies that Hoechst dyes at least partially share the same transport system. Previous literature has suggested that H342 enters the cell by an unmediated diffusion transport mechanism through the cell membrane prior to DNA binding [48], [49]. In addition, our initial data shows that the components of the cell culture media affect the potency of H342-induced apoptosis [41].…”

Section: Discussionmentioning

confidence: 59%

“…However, the mechanism by which a cell readily takes up Hoechst dyes remains unclear. The present results show that pretreatment of H258 for one hour fails to protect H2373 cells from H342-induced apoptosis, and H342 approaches its maximum peak emission spectra for the formation of H342-DNA complexes in spermatozoa after a 30 minute incubation period [48], thus indicating the antagonistic effect of H258 on H342-induced apoptosis is not associated with pre-occupied AT-rich DNA sequences by H258 (Figure 3A). However, since H342-induced apoptosis is partially blocked by H258, this implies that Hoechst dyes at least partially share the same transport system.…”

Section: Discussionmentioning

confidence: 67%

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New Insight into the Molecular Mechanisms of the Biological Effects of DNA Minor Groove Binders

Zhang

Sun³

et al. 2011

PLoS ONE

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BackgroundBisbenzimides, or Hoechst 33258 (H258), and its derivative Hoechst 33342 (H342) are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear.Methodology/Principal FindingsTo determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects.Conclusions/SignificanceA unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug development process. Further analysis of H258-upregulated genes of transcription regulation may identify the genes that enhance transgene overexpression in gene therapy and promote recombinant protein products in biopharmaceutical companies.Data DepositionThe microarray data reported in this article have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE28616).

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Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 67%

New Insight into the Molecular Mechanisms of the Biological Effects of DNA Minor Groove Binders

Zhang

Sun³

et al. 2011

PLoS ONE

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“…A pH of 7.45 optimizes H33342 membrane permeation (Weisenfeld, 2007), but as seen in Figure 2.3, the highest medium pH was 7.35, and pH decreased during incubation. Therefore, it is possible that increasing the pH of the staining medium may improve sort efficiency.…”

Section: Discussionmentioning

confidence: 97%

“…The use of the lipophilic cell membrane permeable dye Hoechst 33342 allowed sexing of sperm by flow cytometry to become a viable technology, because sperm were able to survive the staining process, plus this dye had no mutagenic properties (Garner, 2009 (Weisenfeld, 2007).…”

Section: Sex Sorting Sperm -Stainingmentioning

confidence: 99%

Removing Seminal Plasma Improves Sex-Sorting of Bovine Sperm.

Burroughs

Graham

Lenz

et al. 2011

Biology of Reproduction

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REMOVING SEMINAL PLASMA IMPROVES SEX-SORTING OF BOVINE SPERMExperiments for this thesis evaluated how characteristics of bovine ejaculates affect efficiency of sorting X-from Y-chromosome bearing sperm by flow cytometry/cell sorting, and further, document that removal of seminal plasma improves efficacy of sexsorting bovine sperm. In Experiment I, ejaculates were collected by artificial vagina, two each from 10 bulls with an average of one hour between collections. Semen was centrifuged to separate sperm from seminal plasma, and then sperm were re-diluted to 160x10 6 sperm per ml with staining TALP (Tyrode's albumin, lactate, pyruvate) and 0, 5, 10 or 20% seminal plasma from the same ejaculate or reciprocally from first/second ejaculates. Following incubation with Hoechst 33342, sperm were sorted and analyzed with a flow cytometer/cell sorter. The % live-oriented sperm was higher for treatments with 0% seminal plasma (64.4%) than 5 (59.6%), 10 (59.0%) and 20% (57.8%) seminal plasma (p<0.01). The % live-oriented sperm was higher for second (63.0%) than first ejaculates (56.2%). Sort rate was higher for samples with 0% seminal plasma (p<0.05).The percentages of membrane-impaired sperm were lower for 0% (16.5%) than 5 (21.9%), 10 (23.6%) or 20% (23.4%) seminal plasma (p<0.003), and for second iii ejaculates (18.2%) compared to first ejaculates (25.9%). Whether seminal plasma was from first versus second ejaculates had no effect (p>0.1). Effects of seminal plasma originating from bulls different from those whose sperm were sorted was evaluated in Experiment II. Semen collection and initial analysis were performed as in Experiment I and for all successive experiments. Seminal plasma from ejaculates of 6 bulls (3 that sorted well; 3 that sorted poorly) used in Experiment I was used with sperm collected for Experiment II as well as seminal plasma from sperm donors. Sperm were stained with Hoechst 33342 as in Experiment I, but with 10% seminal plasma in staining TALP.Only seminal plasma from one bull resulted in differences for any sorting parameters; 31% of sperm were membrane-impaired with seminal plasma from this bull compared to 16-19% for seminal plasma from other bulls (p<0.05). Sort rate was decreased (3.02x10 3 sperm per sec) compared to a range of 3.64-3.97x10 3 sperm per sec with the other seminal plasmas (p<0.05). This may have been due to contamination of the original semen sample, since this effect was only seen with one of six bulls.Experiment III investigated effects of adding bovine serum albumin (BSA) during staining with Hoechst 33342 using semen from 10 bulls. BSA was added at 0, 0.3 or 0.9% to staining TALP with either 0 or 10% seminal plasma. Although BSA has been shown to be beneficial to sperm, there was a possibility that BSA would bind Hoechst 33342, thereby reducing sorting efficiency. There was no evidence of either effect; however, sperm with 0% seminal plasma had higher % live oriented cells (65%) than sperm incubated with 10% seminal plasma (61%; p<0.05). In addition, samples conta...

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“…The measurement was repeated three times for each condition and the room temperature was controlled at 22 ± 2°C during the experiment to minimize the influence of temperature fluctuations (Guilbault ; Liu et al ; Weisenfeld ). For each measurement, the spectrofluorometer was zero‐cleared to avoid the effects of unwanted background signals.…”

Section: Methodsmentioning

confidence: 99%

Optimal fluorescent‐dye staining time for the real‐time detection of microbes: a study of Saccharomyces cerevisiae

et al. 2020

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Aims To provide information on the time‐dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. Methods and Results For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4′,6‐diamidino‐2‐phenylindole) and Auramine‐O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2–3 s with increasing Auramine‐O concentration, for example at a yeast concentration of 100 µg ml−1. Conclusions For the conditions of yeast >10 µg ml−1, DAPI >1 µg ml−1 and Auramine‐O >0·1 µg ml−1, τ could be adjusted to below 5 s to achieve a rapid and stable staining. Significance and Impact of the Study Design and operating conditions for rapid and online measurements of microbes can be optimized.

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Mass transport kinetics of the DNA-binding dye Hoechst-33342 into bovine spermatozoa

Cited by 7 publications

References 15 publications

New Insight into the Molecular Mechanisms of the Biological Effects of DNA Minor Groove Binders

New Insight into the Molecular Mechanisms of the Biological Effects of DNA Minor Groove Binders

Removing Seminal Plasma Improves Sex-Sorting of Bovine Sperm.

Optimal fluorescent‐dye staining time for the real‐time detection of microbes: a study of Saccharomyces cerevisiae

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