Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia

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1 Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations Gerardo Barroso, M.D., M.S., a Steve Taylor, Ph.D., b Mahmood Morshedi, Ph.D., b Félix Manzur, M.D., a Fernando Gaviño, M.D., a and Sergio Oehninger, M.D., Ph.D. b a Assisted Reproductive Division, Instituto Nacional de Perinatología, Mexico City, Mexico; and b The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Objective: To examine whether a relationship exists between loss of mitochondrial transmembrane potential and plasma membrane translocation of phosphatidylserine (PS) in subpopulations of human spermatozoa of men consulting for infertility. Setting: A tertiary institutional research center. Design: Prospective observational study. Patient(s): Twelve infertile men and five fertile controls were compared. Intervention(s): Sperm subpopulations were compared after density gradient separation. Main Outcome Measure(s): Mitochondrial membrane potential was measured with a cationic dye, translocation of PS was evaluated with Annexin-V binding, and motion parameters were assessed manually. Result(s): In both the study and control groups and compared with the high-motility fraction, the low-motility fraction had significantly lower sperm motility and normal morphology, and significantly higher percentage of cells with disrupted mitochondrial membrane potential and plasma membrane PS translocation. There was a positive and significant correlation in both subfertile and control groups between the percentages of Annexin-V live cells and cells with mitochondrial membrane potential disruption (r 0.82 and r 0.85, respectively). Conclusion(s): The correlation of plasma membrane PS translocation and loss of mitochondrial membrane potential is suggestive of an early apoptosis phenotype, as is typically observed in somatic cells identified in sperm subpopulations with percentage of low-motile cells. We speculate that such changes might be used as diagnostic markers of sperm dysfunction(s) and that increased levels found in subfertile men might be indicators of reduced fertility potential. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Apoptosis, mitochondrial membrane potential, male infertility, phosphatidylserine translocation, spermatozoa Several features that are characteristic of apoptosis in somatic cells have been described in human spermatozoa. These include, principally, plasma membrane externalization of phosphatidylserine (PS) and DNA fragmentation. Such markers are observed with higher frequency in ejaculates of infertile men compared with fertile controls (1 5). In addition, key components of the somatic cell apoptosis pathways, such as presence and activation of caspases, have recently been described in purified populations of ejaculated sperm from the high- and low-motility fractions (5 8). The identification of plasma membrane and mitochondrial dysfunctions, sensitive and early markers of programmed cell death in somatic cells (9 11), might allow us to gain further insight into this process and its clinical significance Received January 6, 2005; revised and accepted June 21, Reprint requests: Gerardo Barroso, M.D., Instituto Nacional de Perinatología, Assisted Reproductive Division, Montes Urales 800, Col. Lomas de Virreyes, Mexico City 11000, Mexico (FAX: ; asistida@servidor.inper.edu.mx). in human ejaculated spermatozoa. Here, we investigated the presence and relationship between PS externalization and disturbances of mitochondrial transmembrane potential in purified sperm populations obtained from ejaculates of men consulting for infertility. MATERIALS AND METHODS Semen Collection and Processing Semen samples of 12 infertile men (consecutive samples matching the set criteria for acceptance) undergoing infertility workup were analyzed. The ethics committee of the Instituto Nacional de Perinatolog&iacutea (Mexico City, Mexico) approved the study. Inclusion criteria were infertility 1 year duration, no history of smoking, and total motile sperm in the original (unprocessed) sample. We also studied five fertile donors with a history of fatherhood within a 1-year period. All patients had normal physical examination and endocrine profiles (serum FSH, LH, and T determinations). Patients collected semen samples in sterile containers after a 3 5-day period of sexual abstinence. Sem /06/$32.00 Fertility and Sterility Vol. 85, No. 1, January 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 149

2 inal volume and ph were normal; all samples had round cells per milliliter and negative cultures for microorganisms. Samples were prepared after liquefaction at 37 C for minutes, with a discontinuous Percoll gradient (90% and 40% layers) (Biotech AB Pharmacy, Uppsala, Sweden). The semen was placed in the upper part of the concentration gradient and centrifuged at 400 g for 15 minutes. After centrifugation, the two purified subpopulations of sperm with high (90%) and low (40%) motility were recovered. Human tubal fluid (Irvine Scientific, Santa Ana, CA) supplemented with 7.5% serum substitute supplement (Irvine Scientific) was used to resuspend each pellet at sperm per milliliter and kept in an incubator at 37 C in 5% CO 2 in humidified air. The separated samples were independently analyzed for sperm motility and morphology, mitochondrial transmembrane potential, and Annexin-V binding within the next 30 minutes. Evaluation of Basic Sperm Parameters: Concentration, Motility, and Morphology Sperm concentration, motility, and morphology were evaluated in the original raw sample and in both fractions with sperm of high and low motility. After liquefaction, 5 L of each sample were placed into a Makler Chamber (Sefi Medical Instruments, Haifa, Israel) and read under light microscopy. Sperm morphology was assessed according to strict criteria at 1,000 magnification (12). Each sample (10 L) was spread along the slide and allowed to dry for 20 minutes before staining with Diff-Quick staining (Baxter Dade Diagnostics, Dubingen, Switzerland). An average of 100 spermatozoa per slide was read under microscopy with a 100 oil immersion objective. Coefficients of interobserver and intraobserver variability were 5% (13). Determination of the Integrity of Mitochondrial Membrane Potential Oxidative phosphorylation is the process whereby protons are pumped from inside the mitochondrial membrane to outside, creating an electrochemical gradient. This process is termed mitochondrial membrane potential and helps to estimate the metabolic function through measure of differences in potential from the inner to the outer mitochondrial membrane. The functional integrity of the sperm mitochondria was analyzed with a commercial kit (Apoalert Mitochondrial Membrane Sensor Kit [MitoSensor]; Clontec Laboratories, Palo Alto, CA) (14). This allows the detection of changes in mitochondrial transmembrane potential reflective of initial apoptotic phenomena. MitoSensor is a cationic dye, which fluoresces differently in apoptotic and nonapoptotic cells. In healthy cells, MitoSensor is taken up in the mitochondria, where it forms aggregates. These aggregates exhibit intense red fluorescence. However, in apoptotic cells, MitoSensor cannot aggregate in the mitochondria because of the altered mitochondrial membrane potential. As a result, the dye remains in monomeric form in the cytoplasm, where it fluoresces green. Each sample (0.5 ml) was placed in an Eppendorf tube (Sigma-Aldrich, St. Louis, MO), centrifuged at 350 g for 5 minutes, and resuspended with 1 ml of diluted MitoSensor reagent before incubation at 37 C and 5% CO 2 in the dark for 30 minutes. MitoSensor (1 L) was added 10 minutes after incubation. The percentage of cells with mitochondrial dysfunction (disruption of transmembrane potential) was determined under fluorescence microscopy; an average of 200 cells was evaluated for each sample. Duplicate readings were performed and averaged. Samples were read under epifluorescence microscopy with a nm excitation filter. Evaluation of Externalization of PS with Annexin-V Annexin-V is a calcium-dependent phospholipidic union protein with a high affinity for PS, also reflective of early apoptotic events. Annexin-V binds to PS translocated from the internal membrane to the external membrane. Binding of Annexin-V to apoptotic cells typically shows a fluorescent green stain. Propidium iodide (PI) (red staining) was added to differentiate viable from necrotic cells escaping the apoptotic analysis; this allows the exclusion of those cells showing PS translocation that are dead and therefore the identification of live cells without (normal sperm) and with (live but apoptotic) PS translocation. After separation, samples were resuspended in phosphatebuffered saline (Sigma-Aldrich) and incubated with Annexin- V (Boehringer Mannheim, Indianapolis, IN) in N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid buffer solution (Sigma-Aldrich) containing PI. Samples were then read under epifluorescent microscopy. At least 200 cells per field were analyzed in a randomized way and identified as normal (negative for Annexin-V and PI), apoptotic (positive for Annexin-V and negative for PI) or necrotic (positive for PI). Previously established intraobserver and interobserver coefficients of variation for this technique were 7% (2, 15). Statistical Analysis Nonparametric analysis (Wilcoxon s signed ranks) was used to compare mitochondrial membrane potential changes (expressed as percentage of cells with disrupted potential) and PS externalization (expressed as percentage of normal, live apoptotic, and dead cells) in the purified fractions of sperm with high motility (90% layers) and low motility (40% layers). Correlations between translocation of PS and mitochondrial membrane potential in the different fractions were assessed with a linear correlation test. All values were expressed as mean SD. P levels.05 were considered significant. RESULTS The basal semen analysis in the raw samples for both groups showed no differences in sperm concentration ( Barroso et al. Early apoptotic markers in human spermatozoa Vol. 85, No. 1, January 2006

3 TABLE 1 Overall basal semen analysis characteristics in study and control groups. Group Sperm concentration (10 6 /ml) Progressive motility Normal morphology Study group (n 12) Control group (n 5) Note: Values are mean SD /ml vs /ml) and percent progressive motility (79% 3% vs. 81% 7%) for study and control groups, respectively. However, there was a significantly difference in percent normal morphology (5% 2% vs. 10% 1%) for subfertile and control groups, respectively (Table 1). In the study group, the percentages of progressively motile sperm in the high-motility fractions (Percoll gradient 90%) and low-motility fractions (Percoll gradient 40%) were 89% 3% vs. 7% 2%, respectively (P.005) and the percentages of sperm with normal morphology were 10% 5% and 2% 3%, respectively (P.01) (Table 2). Results of Annexin-V binding showed a significantly higher percentage of live cells with PS translocation in the low-motility fractions compared with the high-motility fractions (14% 5% vs. 6% 3%; P.005). Furthermore, the percentage of cells with disturbed mitochondrial membrane potential was also significantly higher in the low-motility (27% 3%) than in the high-motility (11% 2%) fractions (P.0005). The proportion of necrotic sperm (PI positive) was also significantly different between sperm fractions (Table 2). In the control group, a significant difference was observed in the percentages of progressively motile sperm (92% 4% vs. 8% 1%; P.0001) and normal-morphology sperm (11% 1% vs. 4% 2%) for high- and low-motility fractions, respectively (Table 3). Phosphatidylserine translocation in live cells showed a significantly difference (5% 1% vs. 12% 2%; P.0001) for the high- and lowmotility fractions, respectively. In addition, the impairment of mitochondrial membrane potential was also higher in the low- compared with the high-motility fraction (25% 4% vs. 10% 2%; P.0001) (Table 3). There was a highly significant and positive correlation between PS externalization (Annexin V live cells) and the degree of mitochondrial membrane dysfunction in the subfertile and control groups (r 0.82, P.0001 and r 0.85, P.0001, respectively) (Fig. 1). DISCUSSION These results confirmed previous studies demonstrating that a variable proportion of live, ejaculated spermatozoa under in vitro incubation conditions depict PS externalization (2, 15 17). The experiments also corroborated the presence of mitochondrial membrane potential anomalies (18 20). Furthermore, we demonstrated that in ejaculates of men consulting for infertility, such dysfunctions are less frequently observed in the purified populations of sperm from the 90% Percoll gradients, typically depicting higher progressive motility, best morphology, and an optimized fertilizing capacity. TABLE 2 Results from the separated high- and low-motility fractions in the subfertile population (study group): percentages of sperm motility, morphology, PS translocation in live ells, necrotic cells, and mitochondrial transmembrane potential disruption. Fraction Motility Morphology Annexin-V live Necrosis Mitochondrial potential disruption High-motility fraction (90%) Low-motility fraction (40%) P Note: Values are mean SD. Fertility and Sterility 151

4 TABLE 3 Results from the separated high- and low-motility fractions in controls: percentages of sperm motility, morphology, PS translocation in live cells, necrotic cells, and mitochondrial transmembrane potential disruption. Fraction Motility Morphology Annexin-V live Necrosis Mitochondrial potential disruption High-motility fraction (90%) Low-motility fraction (40%) P Note: Values are mean SD. The percentages of live spermatozoa with PS translocation found in the separated fractions of high and low motility were similar to the percentages reported in fertile donors as published in our earlier studies (15, 21). Here, we elected to test a group of men consulting for infertility without significant anomalies of sperm concentration and motility to be able to recover enough cells in the gradient fractions to examine all the established parameters. Although sperm concentration and motility were within normal ranges, most of the samples presented moderate to severe teratozoospermia. Undoubtedly, it will be of interest to examine PS translocation and mitochondrial membrane alterations in men with more severe forms of oligoasthenoteratozoospermia. Alterations of mitochondrial transmembrane membrane potential, as well as PS externalization, are characteristic of early stages of apoptosis in somatic cells; these alterations precede other manifestations of programmed cell death, such as DNA fragmentation (22). Our studies demonstrated a significant positive correlation between translocation of PS and loss of mitochondrial membrane potential. Although mitochondrial alterations have been associated with sperm disorders in infertile men irrespective of apoptosis (23), and PS externalization might be related to other sperm conditions, such as capacitation (24), the observed strong correlation provides further evidence to suggest that early stages of apoptosis phenomena might be indeed present in ejaculated spermatozoa. Further studies are needed to determine the mechanisms leading to plasma and mitochondria membrane alterations in human sperm. FIGURE 1 Relationship between the percentage of Annexin-V live sperm and the percentage of sperm with mitochondrial transmembrane potential disruption in the subfertile population. 152 Barroso et al. Early apoptotic markers in human spermatozoa Vol. 85, No. 1, January 2006

5 We and others (2, 25, 26) have demonstrated a positive correlation between generation of reactive oxygen species and DNA fragmentation in ejaculated sperm. Marchetti et al. (20) concluded that analysis of mitochondrial membrane potential is a sensitive test to determine sperm quality when compared with the analysis of the basic sperm parameters, generation of reactive oxygen species, and presence of DNA fragmentation. Moreover, Wang et al. (19, 26) reported that increased oxidative stress is associated with alterations in mitochondrial membrane potential and increased spermatozoa DNA damage. In recent communications, it has been unequivocally demonstrated that inactive and active caspases are present in ejaculated sperm (6 8, 17). Active caspases are more frequently found in sperm from infertile than fertile men, and in the purified fractions of sperm with low motility compared with the ones with high motility (6, 8, 27). Paasch et al. (17) showed that deterioration of sperm plasma membrane (characterized by PS externalization) is associated with activated caspases. In addition, Wang et al. (26) reported a positive relationship between increased sperm DNA damage, production of reactive oxygen species, and higher levels of cytochrome c and caspases 9 and 3. Taken together, these and previously published results strongly suggest that [1] an apoptosis phenotype, similar in some fashion to the one observed in somatic cells, is found in ejaculated spermatozoa, more frequently in subfertile men with abnormal sperm parameters and also in the purified fractions of sperm obtained from the low gradient centrifugation layers (27), [2] examples of this conserved apoptotic phenotype include PS externalization and loss of mitochondrial membrane potential (early markers), as well as DNA damage (late marker) and presence of active caspases (4, 6, 8), and [3] although more studies are needed to elucidate the mechanisms and site of origin responsible for the induction of such apoptosis changes, previous studies support the involvement of oxidative stress and activation of caspases (6, 8, 19, 20). 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