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Sperm Specific Two-Step Dye Exclusion Assays to Evaluate Integrity of Plasma and Organelle Membranes-New Approach for Quality Assurance of the Sperm for Intra-Cytoplasmic Sperm Injection

Satoru Kaneko, Yuki Okada, Kiyoshi Takamatsu

Objective: The comprehensive assessments of the plasma and organelle membranes are new approach to understand human sperm fertility. The present study developed 2 types of the sperm specific 2-step dye exclusion assays to evaluate the plasma and organelle membranes in human sperm.

Methods: Sperm specific 2-step dye exclusion assay: Sperm was incubated with reactive red 195 (RR195) or Cy3- conjugated concanavalin A (Cy3-con A) in isotonic culture medium, they were counterstained with reactive blue 222 or Alexa 488 conjugated concanavalin A after exclusion of the plasma membrane with methanol, respectively. Integrity of the organelle membrane in the mitochondria was observed by retention of Mito Tracker FM. The cellROX Orange produced orange fluorescence upon oxidation by reacted reactive oxygen species.

Results: The sperm specific 2-step dye exclusion assay is composed of permeation and bind of the first dye to the nuclei through damaged plasma and orgnelle membranes, after membrane exclusion, newly exposed nuclei or the inner acrosomal membrane were counterstained with the second dye. The intact in plasma and acrosomal membranes in human motile sperm (live sperm; LS) excluded RR195 and Cy3-con A, but those denatured allowed permeations. LS colored bule or produced green fluorescence, on the one hand, those denatured did red or red fluorescence, respectively. The fluorescence of Mito Tracker FM and cellROX Orange in the mitochondria certified the normality of oxidative phosphorylation in the mitochondria with intact organelle membrane. The derivative single-step RR195 exclusion assay is a viable candidate for intra-operative non-destructive assessment of the motile sperm in clinical ICSI.

Conclusion: The dye exclusion and retention assays contribute as the pre-operative assessments of sperm qualities and the modified single-step dye exclusion assay may be useful for intra-operative sperm selection in clinical intracytoplasmic sperm injection, respectively.