Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

Kaneko, Satoru

doi:10.4172/2155-9929.s5-005

Cited by 6 publications

(13 citation statements)

References 62 publications

(99 reference statements)

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“…The repair of DSB is intrinsically difficult compared with that of other DNA lesions, and the critical threshold of DNA fragmentation may be extremely low [30,31]. We developed the single-cell pulsed-field gel electrophoresis to visualize the early stage of DNA fragmentation [32,33]. We observed that even after separating motile sperm through the same procedures in the present study, some of motile sperm have undergone an early stage of DNA fragmentation, the frequency of which varied among ejaculates.…”

Section: Discussionmentioning

confidence: 58%

Low Density Regions of DNA in Human Sperm Appear as Vacuoles after Translucent Staining With Reactive Blue 2

Kaneko¹,

Yoshida²,

Takamatsu³

2013

J Med Diagn Meth

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Human sperm often contain vacuoles, and it is therefore necessary to determine whether vacuoles in the chromosomal region are responsible for DNA deterioration. To that end, the present study developed a novel clinical examination technique, translucent staining, for the visualization of vacuoles. Highly diluted Reactive Blue 2 (RB2, 100 pmol/L) faintly stained the sperm head to reveal a translucent bluish body with toneless spots. Differential interference contrast showed the vacuoles as shaded-relief images. In the same, field of view the localization and the shape of the toneless spots were coincided with the shaded-relief images. Dual staining with RB2 and DNA fluorescent dye suggested that the vacuoles might include vacant or lowdensity DNA. The present results revealed important issues in clinical Assisted Reproductive Technology (ART), that is, a variety of vacuoles were involved regardless of the normalcy of outline or the motility. Features were classified into three categories: zero or one small vacuole; multiple sporadic vacuoles of various sizes; and one large vacuole. Moreover, in vitro processing steps such as density gradient centrifugation and subsequent swim up could not isolate sperm without vacuoles. Since it is still unknown whether the vacuoles merely push out DNA to the surroundings without damaging it, or if the empty region is a consequence of DNA degradation, the translucent staining for simultaneous visualization of the outline and vacuoles constitutes a major step for pre-ART cyto-diagnosis to determine whether the sperm population is competent for use in clinical ICSI.

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Section: Discussionmentioning

confidence: 58%

Low Density Regions of DNA in Human Sperm Appear as Vacuoles after Translucent Staining With Reactive Blue 2

Kaneko¹,

Yoshida²,

Takamatsu³

2013

J Med Diagn Meth

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“…Bovine pancreatic trypsin (EC.3.4.21.4) 22 , which is used for in-gel digestion of nucleoproteins 14 , 15 , specifically cleaves lysine and arginine residues. It is inactive at pH 4.7 during gelation and activated at pH 8.2 in cell-lytic medium.…”

Section: Discussionmentioning

confidence: 99%

“…The present study developed a novel angle modulated two-dimensional single cell pulsed-field gel electrophoresis (2D-SCPFGE), it revealed unexpected results that 1D-SCPFGE 14 , 15 also produced false negative for analysis of the early stage of fragmentation. After 1D-SCPFGE, a fraction of long chain fibers remained at the origin and long segments were tangled in the bundle of elongated fibers.…”

Section: Introductionmentioning

confidence: 89%

“…Mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFG) with in-gel tryptic digestion has revealed that human semen contains a heterogeneous population of sperm with various stages of DNA fragmentation. Initially, a few large fibrous segments appear beyond a bundle of elongated long chain fibers, and cleavage proceeds until all DNA fibers are degraded to granular segments 14 , 15 . This observation demonstrated that the comet tail might be derived from the sperm at the end stage of fragmentation.…”

Section: Introductionmentioning

confidence: 99%

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Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei

Kaneko,

Takamatsu

2024

Sci Rep

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We here developed a novel angle-modulated two-dimensional single cell pulsed-field gel electrophoresis (2D-SCPFGE). Variations in current-application-time and rotation angle generated different alignments of DNA fibers and segments. After the first run, the specimen was turned by 150° (2D-SCPFGE-0–150) to detect naturally occurring the earliest stage of DNA fragmentation or 75° (2D-SCPFGE-0–75) to analyze artificially induced cleavage. The former revealed that a part of long chain fibers remained at the origin and long segments were still tangled in the bundle of elongated fibers after the first run. The latter visualized the dose-dependent cleavage of DNA by EcoR1. Multicycle 2D-SCPFGE was useful for generating 2D-alignments of single nuclear DNA fibers, which is the first step for visualization of single-strand breaks on stretched fibers. To date, many articles have accepted the pathogenetic significances of DNA fragmentation in human sperm for male infertility and congenital anomaly. It is necessary to perform multivariate analyses of not only earliest-stage DNA fragmentation but also other types of damage, including single-strand breaks, in sequential DNA fibers. 2D-SCPFGE is the fundamental tool for understanding single nuclear DNA damages.

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“…Previously, we reported three technical problems of SCSA, SCD, and CA in the analysis of DNA fragmentation in human sperm nuclei [14][15][16]. First, there is a lack of validation using the comparative standards, leading to inconsistencies regarding the underlying principles.…”

Section: Discussionmentioning

confidence: 99%

Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei

Kaneko,

Takamatsu

2023

Preprint

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We here developed a novel angle-modulated two-dimensional single cell pulsed-field gel electrophoresis (2D-SCPFGE). Variations in current-application-time and rotation angle generated different alignments of DNA fibers and segments. After the first run, the specimen was turned by 150 degrees (2D-SCPFGE-0-150) to detect naturally occurring the earliest stage of DNA fragmentation or 75 degrees (2D-SCPFGE-0-75) to analyze artificially induced cleavage. The former revealed that a part of long chain fibers remained at the origin and long segments were still tangled in the bundle of elongated fibers after the first run. The latter visualized the dose-dependent cleavage of DNA by EcoR1. Multicycle 2D-SCPFGE was useful for generating 2D-alignments of single nuclear DNA fibers, which is the first step for visualization of single-strand breaks on stretched fibers. To date, many articles have accepted the pathogenetic significances of DNA fragmentation in human sperm for male infertility and congenital anomaly. It is necessary to perform multivariate analyses of not only earliest-stage DNA fragmentation but also other types of damage, including single-strand breaks, in sequential DNA fibers. 2D-SCPFGE is the fundamental tool for understanding single nuclear DNA damages.

show abstract

Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

Cited by 6 publications

References 62 publications

Low Density Regions of DNA in Human Sperm Appear as Vacuoles after Translucent Staining With Reactive Blue 2

Low Density Regions of DNA in Human Sperm Appear as Vacuoles after Translucent Staining With Reactive Blue 2

Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei

Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei

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