Journal des méthodes de diagnostic médical
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Reactive Blue Dye: Highlights of Vacuoles in Human Sperm

Satoru Kaneko*, Yuki Okada, Satoshi Yokota, Kiyoshi Takamatsu

Objectives: The WHO reference manual for andrology considers the outline of sperm head as a key parameter for assessing male infertility and classifies internal vacuoles as head defects. The features of vacuoles were heterogeneous among interindividual as well as inter-sperm. The present study reported some unique properties of Reactive Blue 2 (RB2). The dye exhibited pH-dependent cellular specificity for human sperm and spermatid.

Materials and Methods: Human sperm and spermatid were stained with RB2 at pH 10.0.

Results: RB2 stained human sperm and spermatids, which appeared as translucent bluish bodies, but did not stain lymphocytes, while inner vacuoles appeared as toneless spots. RB2 staining also revealed degraded spermatids that had undergone protamination but were arrested prior to tail elongation. The majority of azoospermic semen specimens (16/23 cases) included sperm or degraded spermatids. Although the features of the head outline and vacuoles were heterogenous among interindividual, RB2 staining in the presence of 2.0 mmol/L Dithiothreitol universally re-formed the outline into an oval shape and led to the disappearance of the vacuoles, regardless of their original features.

Conclusion: Three sulfate residues in RB2 interacted selectively with guanidyl residues in Arg of protamines at pH 10. The cellular specificity was due to the Arg content of the nucleoproteins. RB2 revealed the head outline and internal vacuoles of the sperm and arrested spermatids those had undergone protamination. Local failure of disulfide cross-linkage might play a critical role in vacuole formation. RB2 staining opened new methods for exploring spermiogenesis.