Protamine labeling with Reactive Black-5 and its molecularly designed derivatives visualize vacuoles in human sperm head

To date, numerous studies have examined the etiological relationship between vacuoles and DNA fragmentation in human sperm, with vacuoles typically assessed using differential interference contrast microscopy. To explore the optimal dye for visualizing vacuoles, we collected 12 commercially available reactive dyes and compared their staining performance. Labeling protamines with 9.0 μmol/L Reactive Black-5 (RB5) stained the nuclear body bluish and visualized vacuoles as unstained spots. Next, we synthesized 30 molecularly designed RB5 derivatives to identify an optimal molecular structure for vacuole observation, guided by structure-activity relationship analyses. Introducing 2-methylbenzenesulfonate into 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid (compound 2221) provided the minimal π-conjugation needed to produce a blue color. At 1.0 μmol/L, 2221 showed staining performance comparable to that of 9.0 μmol/L RB5. Both dyes visualized not only large vacuoles but also small sporadic vacuoles. Dissociation of the DNA-protamine complex with sodium dodecyl sulfate, reduction of disulfide bonds with dithiothreitol, and re-crosslinking of protamines with RB 5 transformed the outlines into an oval shape. RB5 staining gave enough contrast between the nuclear body and the vacuoles for quantitative morphometric analyses.