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.

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

“…Previous studies showed that cells of a same cellular lineage can undergo apoptosis without demonstrating the concomitant phosphatidylserine translocation [34,35]. It appears unlikely that phospholipids asymmetry, if lost at the very beginning of the process, could be restored later, because of the depletion of energetic resources occurring in the late stages of apoptosis.…”

“…A previous study in different adherent cells, as PC 12 and NIH3T3, demonstrated that acetoxymethyl ester of calcein (calcein-AM) and ethidium homodimer (EthD-1) were more sensitive in earlier detection of apoptosis when compared to annexin V-FITC/propidium iodide [35]. In fact, the present study demonstrated that calcein-AM/ethidium homodimer was equally useful and more sensitive in mononuclear cell apoptosis quantification, when compared to annexin V-FITC/propidium iodide; besides allowing clarity when defining the viable and apoptotic lymphocyte populations.…”

In Vitro Evaluation of Acrosomal Status and Motility in Rat Epididymal Spermatozoa Treated with .ALPHA.-Chlorohydrin for Predicting Their Fertilizing Capacity.

“…The FITC-ConA labeling method in o u r r e p o r t a l s o a s s e s s e d t h e v i a b i l i t y o f spermatozoa by means of supravital staining with CAM and EthD-1. CAM and EthD-1 fluorescent labeling clearly distinguishes viable from dead spermatozoa [20]. This FITC-ConA labeling procedure combined with CAM and EthD-1 is therefore a useful method for accurate evaluation of acrosome reaction in rat spermatozoa without degenerative acrosome loss of dead sperm.…”

“…Spermatozoa labeled with CAM and EthD-1 displaying green fluorescence on the midpiece w itho ut red fluor escence on the hea d w ere identified as live sperm, and those spermatozoa displaying red fluorescence on the head were identified as dead sperm. This procedure to discriminate the viable and dead spermatozoa in the rat was described by Kato et al [20].…”

Perfil oxidativo e avaliação funcional de sêmen criopreservado de touros (Bos taurus taurus e Bos taurus indicus) criados em clima tropical

“…Assim como a integridade estrutural das membranas, a motilidade da célula espermática também exerce um papel fundamental na fertilização (KATO et al, 2001).…”