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 (DIC) microscopy. To explore the optimal dye for the visualization of 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 provided higher image resolution than DIC optics and visualized small sporadic vacuoles. Dissociation of the DNA–protamine complex with sodium dodecyl sulfate and reduction of disulfide bonds with dithiothreitol enlarged and transformed the head outlines into an oval shape and disappeared the vacuoles. RB 5 and 2221 are useful to analyze the local failure of protamine crosslinking for the deformation of the outlines and formation of the vacuoles.