Offspring from inseminations with mammalian sperm stained with Hoechst 33342, either with or without flow cytometry

The technique of vital staining with a fluorochrome and subsequent flow cytometry has been employed to investigate the DNA content of living sperm. There have been several reports of chromosomal damage caused by staining cells with a bis-benzimidazole dye. Hoechst 33342, either with or without subsequent flow cytometry. Therefore the use of this technique might be expected to affect adversely the fertilising capacity of inseminated sperm, or to result in the production of congenital deformities in the young. Insemination experiments to date have resulted in the production of more than 400 offspring in four species, including 5 successive generations of rabbits, where stained or sorted sperm were used. All progeny were observed to be normal by anatomical criteria. There was no evidence to suggest that sperm treated in this manner were unable to fertilise oocytes in vivo or that embryonic development was affected.     

Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342

Bovine spermatozoa were stained with Hoechst 33342. The fluorescence distribution of stained spermatozoa was complex. Non-motile spermatozoa displayed a higher fluorescence than did motile spermatozoa. The fluorescence profile of the motile spermatozoa was bimodal. Sort and reanalysis, and orientation experiments suggested that there are two distinct populations of motile spermatozoa.     

Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm

Hoechst 33342 is the fluorophore used routinely to measure DNA in X- and Y-chromosome-bearing mammalian sperm so they can be separated by flow sorting. A difference of <3% in DNA mass can be detected. This synthetic dye consists of two adjacent benzimidazole rings with N-methyl-piperazine and phenolic groups at the ends. The molecule permeates the cell membrane of living cells and binds selectively to A-T base pairs exposed in the minor-groove of double stranded DNA. Capability to distinguish and separate X- and Y-chromosome-bearing sperm has led to artificial insemination of somewhere around a million female mammals. Offspring with obvious abnormalities are no more frequent than after insemination of unsorted sperm into cows, horses, humans, pigs, sheep, rabbits, dolphins and other mammals. There is no apparent genotoxic effect from exposure of sperm to Hoechst 33342, although information on cellular toxicity or development of embryos resulting from Hoechst 33342-stained sperm is less reassuring. Little is known about the fate of sperm-delivered Hoechst dye in the female reproductive tract or on progeny of resultant offspring.     

Different Hoechst 33342 and DAPI fluorescence of the human Y chromosome in bivariate flow karyotypes

Summary The staining properties of AT-specific dyes Hoechst 33342 and DAPI as revealed by Hoechst 33342/mithramycin and mithramycin/DAPI bivariate human flow karyotype patterns are different for chromosomes rich in heterochromatin. The peak corresponding to chromosome Y of a given cell line is higher on the A/T axis with mithramycin/DAPI staining than with mithramycin/Hoechst. The chromosome 1 was found slightly more fluorescent in mithramycin/DAPI than in mithramycin/Hoechst as judged by the slight displacement of its area on the Hoechst-DAPI axis. The other peaks did not show major differences. On the same flow karyotypes, chromosomal rearrangements were detected.     

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Microscopic and flow cytophotometric analysis of parasitemia in cultures of Plasmodium falciparum vitally stained with Hoechst 33342--application to studies of antimalarial agents

Conditions for rapid vital staining of Plasmodium falciparum infected human erythrocytes were 1 microgram/ml of the dye Hoechst 33342 for 15 min in the standard culture medium at 37 degrees C. Fixed and stained cultures were analyzed by fluorescence microscopy and flow cytophotometry. The usefulness of this type of analysis for in vitro studies of antimalarial agents was demonstrated using three such agents--cyclosporin A, chloroquine, and pyrimethamine.     

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.     

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.     

Comparison of Hoechst 33342 and propidium iodide as fluorescent markers for sperm fusion with hamster oocytes.

Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 mumol Hoechst 333421(-1) showed intracellular fluorescence within 10-20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultra-structural examination, no bound, acrosome-intact hamster spermatozoa (n = 311) were found to be fused. By contrast, oocytes incubated with 10 mumol propidium iodide l-1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5-45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm-oocyte fusion following adhesion. This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm-oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.     

Significance of structural chromosome aberrations in human sperm: analysis of induced aberrations

Summary A significant increase in the incidence of structural chromosome anomalies has been observed in the sperm of patients treated with radio and/or chemotherapy for different types of cancer when analyzed by the interspecific fertilization of hamster eggs. The analysis of these aberrations shows that while in controls only 9.4% of structural abnormalities are of the stable type, in treated patients this figure increases to 39.3%, thus indicating that the anomalies have not been produced during the fertilization of the hamster egg. However, it is possible that part, or even most, of the breaks appear as a result of a reduced repair capacity of sperm chromosomes in the cytoplasm of the hamster egg.     

Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342

A new and improved method of preparing mammalian spermatozoa for high resolution flow cytometric DNA analysis and flow sorting is described. Ejaculated or cryopreserved sperm were briefly sonicated to remove tails and then stained with Hoechst 33342. This simple procedure was found superior to more severe treatments of dimethylsulfoxide washes, fixation in 80% ethanol, and protease digestion of the sperm membranes and tails by papain. Flow cytometric DNA analyses of sperm samples subjected to varying sonication times indicated that X and Y chromosome-bearing sperm populations could be well resolved with as little as 15-sec sonication. In addition, a comparison of sonicated samples stained with four concentrations of bisbenzimide (Hoechst 33342) or 4′,6-diamidino-2-phenylindole (DAPI) indicated that 2.5 or 5.0 μg/ml of Hoechst was sufficient to resolve the X and Y sperm populations. In order to quantitatively describe the flow cytometric data, several indices (sample quality, orientation and splitting) were developed.     

Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC₆ staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC₆ (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC₆-positive organelles. Sea urchin and surf clam sperm stained with DIOC₆ fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC₆ were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.     

Repair of human sperm chromosome aberrations in the hamster egg

Summary In order to study the repair capacity of fertilized hamster eggs for the lesions present or induced in human sperm, we have examined the potentiating effect of caffeine, a DNA repair inhibitor, on the frequency and types of sperm chromosome aberrations. Sperm samples were donated by an individual treated with chemotherapy for a testicular cancer 3 years previously. Exposure of spermatozoa and inseminated oocytes to caffeine led to an increase of sperm chromosome aberrations, indicating that the damage to human sperm can be repaired in untreated hamster egg cytoplasm. The potentiating effect of caffeine was mainly reflected in an increase of unrejoined aberrations, indicating that the formation of chromosomal rearrangements is also inhibited. Since both chromatid-type and chromosome-type aberrations increase after treatment with caffeine, damage to human sperm can probably be repaired inside the hamster egg cytoplasm by pre and post-replication repair mechanisms.     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

The formation of chromosome aberrations from single-strand damage in irradiated DNA

Consideration is given to molecular structures by which chromosome aberrations can arise from single-strand damage in irradiated DNA.