title: Evaluation of mitochondrial function and membrane integrity by dual fluorescent staining for assessment of sperm status in rats.
abstract: Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.
authors.affiliation: Safety Study Department, Nihon Bioresearch Inc., 6-104 Majima, Fukuju-cho, Hashima, Gifu 501-6251, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Sachiko
authors.initials: S
authors.lastname: Makino
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kimura
authors.forename: Takao
authors.initials: T
authors.lastname: Ota
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Furuhashi
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nagamura
mesh_terms: D000284:Administration, Oral; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009578:Nitrobenzenes / Q000008:administration & dosage / Q000633:toxicity; D017830:Octoxynol / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013089:Spermatocidal Agents / Q000633:toxicity; D013094:Spermatozoa / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D013194:Staining and Labeling; D018675:Toxicity Tests
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; D013089:Spermatocidal Agents; C085925:calcein AM; C018533:ethidium homodimer; D017830:Octoxynol; C036077:nitrobenzene; D004996:Ethidium