The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability  Frédérique Gallon, M.D., Carole Marchetti, M.D., Nathalie Jouy, Ph.D., Philippe Marchetti, M.D., Ph.D.  Fertility and Sterility  Volume 86, Issue 5, Pages 1526-1530 (November 2006) DOI: 10.1016/j.fertnstert.2006.03.055 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 (A) Flow cytometric measurement of ΔΨm and viability. Spermatozoa were stained with 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) and propidium iodide (left panel) or with CMX-ros and YOPRO-1 (right panel) to have compatible emission wavelengths. Abscissa indicates fluorescence intensity of spermatozoa stained with ΔΨm-sensitive dye (dihexyloxacarbocyanine iodide or CMX-ros), and ordinate indicates fluorescence intensity of spermatozoa stained with impermeant vital dye (propidium iodide or YOPRO-1). Note that all nonviable spermatozoa (propidium iodide– or YOPRO-1–positive cells) had a low ΔΨm (dihexyloxacarbocyanine iodide or CMX-roslow). These flow-cytometric parameters were used for sorting. Red squares depict the viable ΔΨmlow subpopulation, sorted flow cytometrically, and blue squares represent the ΔΨmhigh counterparts. Numbers indicate percentages of spermatozoa in each quadrant. (B) Comparison of motility values (left histogram) and morphological evaluation (right histogram) in subpopulations of viable spermatozoa with high and low ΔΨm. Samples were collected immediately after ejaculation, stained, and sorted. Percentage of progressive motile sperm (a+b-type motility according to 1999 World Health Organization criteria) and forward motile sperm (a-type motility according to 1999 World Health Organization criteria) were estimated immediately after sorting (n = 15), and progressive motility also was estimated 24 hours after sorting (n = 8). Assessment of morphology was conducted on each population (n = 8). Data are mean ± SEM of the percentage of spermatozoa. *Statistically significant. NS = not significant. (C) Spontaneous acrosome reaction in spermatozoa with high and low ΔΨm (left panel). Typical distribution of the percentage of CD46+ spermatozoa in relation to ΔΨm detected by flow cytometry. Spermatozoa were double-stained with fluorescein isothiocyanate–conjugated antibodies against CD46 and CMX-ros. Spontaneous acrosome reaction was defined by percentage of CD46+ spermatozoa under classical in vitro conditions. Right histogram, percentage of CD46+ spermatozoa in viable purified ΔΨmlow and ΔΨmhigh subpopulations from five different samples. Values are means ± SEM. *Statistically significant between purified ΔΨmlow and ΔΨmhigh subpopulations. (D) Ionophore-induced acrosome reaction in subpopulations of viable spermatozoa with high and low ΔΨm. Typical distribution of percentage of CD46+ spermatozoa detected by flow cytometry in viable purified ΔΨmlow subpopulation or ΔΨmhigh subpopulation (left graphs). Spermatozoa from each subpopulation were kept either untreated (spontaneous acrosome reaction; white outlines) or treated with the calcium ionophore A 23187 (induced acrosome reaction; gray outlines). Acrosome-response-to-ionophore-challenge score in viable purified ΔΨmlow and ΔΨmhigh subpopulations from five different samples (right histogram). Values are means ± SEM. *Statistically significant between purified ΔΨmlow and ΔΨmhigh subpopulations. Gallon. Mitochondrial functionality and sperm quality. Fertil Steril 2006. Fertility and Sterility 2006 86, 1526-1530DOI: (10.1016/j.fertnstert.2006.03.055) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions