Reactive blue 2 labels protamine in late-haploid spermatids and spermatozoa and can be used for toxicity evaluation
Reactive blue 2 labels protamine in late-haploid spermatids and spermatozoa and can be used for toxicity evaluation
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DOI：
10.1101/2023.03.06.531276
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
Satoshi Yokota;T. Wakayama;H. Miyaso;Kousuke Suga;M. Fujinoki;Satoru Kaneko;S. Kitajima
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文献类型：
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作者：
Satoshi Yokota;T. Wakayama;H. Miyaso;Kousuke Suga;M. Fujinoki;Satoru Kaneko;S. Kitajima
背景活性蓝2（RB 2）染料在弱碱性条件下特异性结合人精子核，从而提供了一种新的评价精子质量的方法。然而，该技术尚未应用于其他哺乳动物种属，例如成熟的啮齿动物模型，这可以在非临床研究中评价候选药物的雄性生殖毒性。目的利用白消安诱导的小鼠不育模型，评价RB 2染色在评估睾丸和附睾精子毒性中的作用。方法雄性C57 BL/6 J小鼠腹腔注射白消安40 mg/kg。28天后，收集睾丸和附睾，并在pH 10下用RB 2染色。在未涂覆的载玻片上进行体外评价，其中RB 2与从精子中提取的鱼精蛋白或来自不含鱼精蛋白的体细胞的细胞内蛋白组分混合。结果花生凝集素（PNA）凝集素组织化学染色显示，除生精上皮的IX-XI期外，在所有期次的伸长和拉长精子细胞中均观察到RB 2阳性细胞。白消安给药后，睾丸曲细精管中RB 2阳性生殖细胞的比例显著降低，给药小鼠附睾头中未发现RB 2阳性精子。聚集体中观察到的混合物中的RB 2染料（pH 10）与鱼精蛋白，但不是在细胞内的蛋白质成分的混合物中没有鱼精蛋白，这种特异性在中性pH值丢失。讨论和结论我们的研究表明，RB 2特异性染色步骤12-16精子细胞，表明特异性结合鱼精蛋白在这些精子细胞中表达。RB 2染色技术具有作为雄性生殖毒性生物标志物的潜力，可以快速观察污染。
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.