Reactive blue 2 labels protamine in late-haploid spermatids and spermatozoa and can be used for toxicity evaluation

Background Reactive blue 2 (RB2) dye specifically binds to the nuclei of human spermatozoa under weakly alkaline conditions, thus providing a new method to assess sperm quality. However, this technique has not yet been applied to other mammalian species, such as well-established rodent models, which could enable evaluation of the male reproductive toxicity of drug candidates in non-clinical studies. Objectives We aimed to evaluate the usefulness of RB2 staining in assessing testicular and epididymal sperm toxicity in mice using a busulfan-induced infertility model. Methods Male C57BL/6J mice were intraperitoneally administered 40 mg/kg of busulfan. After 28 days, the testes and epididymis were collected and stained with RB2 at pH 10. In vitro evaluations were conducted on uncoated glass slides with RB2 mixed with either protamines extracted from the spermatozoa or intracellular protein components from somatic cells without protamines. Results Following peanut agglutinin (PNA) lectin histochemistry, RB2-positive cells were observed in elongating and elongated spermatids at all stages except for stages IX–XI of the seminiferous epithelium. After busulfan administration, the proportion of RB2-positive germ cells in the seminiferous tubules decreased significantly, and no RB2-positive spermatozoa were found in the caput epididymis of treated mice. Aggregates were observed in the mixture of RB2 dye (pH 10) with protamines but not in the mixture of intracellular protein components without protamines, and this specificity was lost at neutral pH. Discussion and Conclusion Our study demonstrates that RB2 specifically stains steps 12–16 spermatids, indicating specific binding to protamine expressed in these spermatids. The RB2 staining technique has potential as a biomarker for male reproductive toxicity, allowing for the rapid visualization of protamination. ### Competing Interest Statement The authors have declared no competing interest.