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

Satrou Kaneko, Joji Yoshida and Kiyoshi Takamatsu

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.