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Viability Tests, Active Caspase-3 and -7, and Chromatin Structure in Ram Sperm Selected Using the Swim-Up Procedure

MARÍA E. AYALA-ESCOBAR

From the ^* Laboratorio de Biología de la Reproducción, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México, El Cerrillo, México; and the Unidad Multidisciplinaria de Investigación, Laboratorio de Pubertad, Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Distrito Federal, México.

Correspondence to: Dr Andrés Aragón-Martínez, Laboratorio de Biología de la Reproducción, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México, CP 50090, El Cerrillo, Piedras Blancas, Toluca, Estado de México (e-mail: armandres{at}gmail.com, andresammx{at}yahoo.com).

Received for publication October 24, 2008; accepted for publication May 18, 2009.

	Abstract

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Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 ± 4.6 vs 54.2 ± 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 ± 3.1 vs 297.2 ± 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicatethat the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.

     Key words: Semen analysis, apoptosis markers, membrane integrity, mitochondrial function, semen processing

Early and late markers of apoptosis, such as phosphatidylserine translocation (Paasch et al, 2003; Kotwicka et al, 2008; Martí et al, 2008) and DNA fragmentation (Younglai et al, 2001; Sakkas et al, 2002; Martí et al, 2008), have been used to evaluate sperm quality under different conditions (Sakkas et al, 2000; Paasch et al, 2003; Ricci et al, in press). Phosphatidylserine translocation has been associated with maturation (Barroso et al, 2000; de Vries et al, 2003) and capacitation of sperm (Kotwicka et al, 2002). However, DNA fragmentation may originate from processes other than apoptosis (Sakkas et al, 1999), and because intrinsic and extrinsic paths of apoptosis converge in caspase activation, it is a more reliable marker of cell death. Caspase activity has been detected in the sperm of humans (Weng et al, 2002; Kotwicka et al, 2008) and rams (Martí et al, 2006, 2008), and both procaspases and caspases have been immunolocated in the ram (Martí et al, 2008); however, the specific location of active caspases is unknown (Martí et al, 2006, 2008). Previous studies have reported that ejaculates with a high number of sperm positive for caspases are negatively correlated with fertilization rate after in vitro fertilization (Marchetti et al, 2004), but this relationship has not been described following intracytoplasmic sperm injection (Said et al, 2006).

The objective of this study was to analyze ram sperm before and after SU to 1) assess cell viability by detecting cytoplasmic esterase and mitochondrial dehydrogenase activity, 2) evaluate chromatin integrity, and 3) detect the presence of active caspases.

	Materials and Methods

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Sample Collection and Sperm Preparation

All experiments were performed using neat or SU-processed semen. Semen samples were collected from 4 mature Suffolk rams using an artificial vagina. The rams were 1.5 years of age, weighed 100–115 kg, and were kept at the Facultad de Medicina Veterinaria y Zootecnia under uniform nutritional conditions. Ejaculates were obtained from each ram every second day. An aliquot of each ejaculate was processed by SU as described below. The protocol of this work was approved by the Bioethics Committee of our faculty.

Sperm SU Processing

We designed a micro SU assay based on the SU procedure from García-López et al (1996). Aliquots (100 µL) of neat semen were carefully pipetted in the bottom of a conical tube of 1.5 mL containing 400 µL of dextran, 30 mg/mL human tubal fluid (HTF) medium, 200 mM sucrose, 50 mM NaCl, 18.6 mM sodium lactate, 21 mM HEPES, 10 mM KCl, 2.8 mM glucose, 0.4 mM MgSO₄, 0.3 mM sodium pyruvate, 0.3 mM K₂HPO₄, 1.5 UI/mL penicillin, and 1.5 mg/mL streptomycin, pH 6.4, then overlaid with 300 µL of bovine serum albumin (5 mg/mL in HTF medium). The tubes were kept at 37°C in a vertical position for 60 minutes. Aliquots of 100 µL each were carefully obtained from the top of the tube.

Sperm Motility

Fresh samples were diluted in HTF medium at 37°C before evaluation of sperm motility. Sperm motility was subjectively assessed with aid of a clear field microscope (ECLIPSE 90i; Nikon Instruments, Melville, New York) at x 100. Twenty-five microliters of fresh or SU sperm suspension was put in a clean slide prewarmed to 37°C and covered gently with a coverslip. Only sperm swimming in a progressive manner were considered as motile. Two hundred sperm cells were counted and the percentage of motile cells was obtained. Motility of fresh and SU samples was evaluated at almost the same time and the same person performed the evaluation throughout the study.

Sperm Viability Assays

Esterase and mitochondrial dehydrogenase activity was evaluated by flow cytometry in aliquots of neat or SU-processed ejaculates. Semen samples were placed in TNE buffer (0.01 mol/L Tris-HCl, 0.15 mol/L NaCl, 1 mmol/L EDTA, pH 7.4) and the sperm number was adjusted to 1 x 10⁶ cells/mL.

Metabolic function in sperm was evaluated by mitochondrial dehydrogenase activity by using resazurin. The nonfluorescent resazurin is reduced by dehydrogenases in functional mitochondria to the fluorescent resofurin molecule (Zrimsek et al, 2006). The LIVE/DEAD cell viability assay kit (Molecular Probes Inc, Eugene, Oregon) was used as follows: 100 µL of sperm suspension was incubated with 1 µL of resazurin (50 µM) and 1 µL of cell permeable fluorochrome SYTOX green (1 µM) for 15 minutes at room temperature. The samples were adjusted to 500 µL and maintained in ice prior to flow cytometry.

Calcein acetomethylester (calcein AM) is a nonfluorescent membrane-permeable molecule that is reduced to fluorescent calcein by intracellular esterases (Uggeri et al, 2004). In this study, calcein AM and ethidium homodimer (EthD1) contained in the LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Molecular Probes) were used as follows: 100 µL of sperm suspension, 2 µL of calcein AM (50 µmol/L), and 4 µL of EthD1 (2 µmol/L) were mixed and incubated for 15 minutes in the dark at room temperature prior to flow cytometry.

Sperm Chromatin Structure Assay

Fresh or SU-processed ejaculates were placed in TNE buffer, frozen, and kept in liquid nitrogen until analysis. All of samples were thawed and processed for sperm chromatin structure assay (SCSA) as described by Evenson and Jost (2000). Briefly, the frozen sperm samples were thawed in a water bath at 37°C and aliquots of the sperm suspension (200 µL) were mixed with 400 µL of acid solution (Triton X-100 0.1%, NaCl 0.15 mol/L, HCl 0.08 N, pH 1.4). Thirty seconds later, 1.2 mL of a solution containing 6 µg/mL of acridine orange (Molecular Probes) in staining buffer (citric acid 0.1 mol/L, Na₂HPO₄ 0.2 mol/L, EDTA 1 mmol/L, NaCl 0.15 M, pH 6.0) was added, and flow cytometry began 3 minutes after adding the acridine orange. All samples were evaluated the same day and a recalibration with a standard sample was performed after 5 to 10 samples.

A FACScan flow cytometer (Becton Dickinson, Immunocytometry Systems, San Jose, California) equipped with an argon laser (488 nm) was used to evaluate the sperm parameters. A live gate was used in the forward light scatter and side light scatter parameters to exclude aggregates and debris, thereby restricting data acquisition to an almost pure population of sperm. List mode data of 10 000 events were collected for each sample using CellQuest software (Becton Dickinson, Immunocytometry Systems). The DNA fragmentation index (DFI) was obtained with WinList 3.0 (Verity Software House, Inc, Topsham, Maine) software in a PC with Windows XP.

View larger version (8K): [in this window] [in a new window]   Figure 1. Percentage of motility in neat semen samples among individual rams. Dotted line separates values above or below 25% motility.

To inhibit the caspases, semen samples were incubated for 20 minutes with Z-DEVD-FMK (Sigma), a cell-permeable inhibitor of caspase-3, -6, -7, -8, and -10, which competitively and irreversibly inhibits the caspases. The inhibitor was diluted in dimethyl sulfoxide and added to the samples to reach a final concentration of 40 µM and then processed as described above.

Statistics

Results are expressed as mean ± SEM. A 2-tailed Wilcoxon rank sum test was performed to compare the neat and SU-processed sperm groups. The relationships among the viability tests, caspase activity, and SCSA were analyzed by Pearson regression. All analyses were performed with R 1.16 software (The R Foundation for Statistical Computing, http://www.R-project.org). P < .05 was considered significant.

View larger version (16K): [in this window] [in a new window]   Figure 2. Distribution of the percentage of viable sperm as evaluated by the resazurin/SYTOX assay (A) and calcein/ethidium homodimer (B) of neat or swim-up–processed ejaculates. Values are medians (horizontal bar in boxes) with 25%–75% interquartile ranges (boxes); notch indicates confidence interval; dotted vertical lines indicate minimum and maximum values; circles indicate outliers.

	Results

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Sperm Viability

There was no significant difference in esterase activity, that is, membrane integrity, after SU processing between the <25% group and the >25% group, although a nonsignificant reduction was observed in the >25% group (58.4 ± 4.7 vs 43.0 ± 5.4; Figure 2A). Similarly, there was no difference in mitochondrial dehydrogenase activity between the groups after the SU procedure (Figure 2B).

The dehydrogenase and esterase activity were highly correlated in the <25% group of the neat and SU-processed sperm samples (Table). The parameters of the viability tests were not significantly correlated with caspase activity or chromatin structure.

Caspase Activity

Caspase-3 and -7 activity were evaluated in aliquots of neat and SU-processed semen samples with the SR-DEVD-FMK FLICA reagent. In each group the percentage of sperm with active caspases was lower in SU samples, but the reduction was greater in the <25% group than in the >25% group (12.1% and 6.4%, respectively; Figure 3A). The number of sperm with active caspases was significantly reduced in the <25% group. For graphical purposes a flow cytometry experiment of caspase inhibition was conducted (Figure 4). Caspase-3 and -7 activity was located in the equatorial, middle, and principal sections, but not in the apical region of the sperm. There was a very limited amount of activity in the postacrosomal region (Figure 5). In all cases staining was observed in the implantation fossa region (Figure 5, inserts). In most of the sperm cells the active caspases were located in the middle piece or in the middle piece plus the principal piece.

View larger version (17K): [in this window] [in a new window]   Figure 3. Distribution of the percentage of sperm expressing active caspase-3 and -7 (A) and DNA fragmentation index values (B) detected in sperm of neat or swim-up–processed ejaculates. Values are medians (horizontal bar in boxes) with 25%–75% interquartile ranges (boxes); notch indicates confidence interval; dotted vertical lines indicate minimum and maximum values; circles indicate outliers. *, P < .05, Wilcoxon rank sum test.

View larger version (18K): [in this window] [in a new window]   Figure 4. Representative histograms of the active caspases in sperm of neat or swim-up (SU)–processed semen. (A) Control (without FLICA), neat, and SU semen (one ejaculate). (B) Controls for the FLICA experiment. Control (without FLICA), neat semen plus FLICA, neat semen plus H2O2 plus FLICA, neat plus z-DEVD-FMK (20 minutes before adding H2O2) plus FLICA.

View larger version (59K): [in this window] [in a new window]   Figure 5. Location of active caspase-3 and -7 in ram sperm. The montage illustrates the fluorescence view (SR-DEVD-FMK FLICA), the clearfield view, and an overlay of both. Head arrows indicate the sperm segments with active caspases. Note in overlay that staining is always present in the implantation fossa region (arrows, inserts in overlay column). Row B is an example of the quantity of sperm with active caspases in a whole sample. mp+, middle piece staining positive; pp+, principal piece staining positive; pp–, principal piece staining negative; pa+, postacrosomal region staining positive; pa–, postacrosomal region staining positive; asterisks indicate the sperm head augmented in the inserts. Color figure available online at www.andrologyjournal.org.

Sperm Chromatin Structure Assay

After the SU procedure a small but significant increase of chromatin structural abnormalities in the <25% group was observed; DFI values were significantly higher than in the neat samples (287.3 ± 3.1 vs 297.2 ± 2.4; Figure 3B). There were no changes in the >25% motility group after the SU procedure.

	Discussion

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The SU technique is used to prepare motile sperm for assisted reproductive techniques (Inaudi et al, 2002; Said et al, 2006). Because sperm motility is an indicator of cell viability, it is assumed that SU-processed sperm are of good quality. Some studies have demonstrated that membrane integrity and mitochondrial function are high, whereas DNA fragmentation and caspase expression decrease after SU processing (Sakkas et al, 2000; Martí et al, 2006, 2008; Kotwicka et al, 2008). We evaluated in neat and SU samples cytoplasmic esterases and mitochondrial dehydrogenases as indicators of cytoplasmic membrane integrity and mitochondrial function by using calcein AM and resazurin, respectively. Moreover, chromatin integrity and active caspases were assessed by SCSA assay and caspase inhibitors.

Ejaculates from the same animal often have distinct cellular features, and our results showed that grouping the motility of neat semen samples was an effective strategy to analyze other cell properties. In our study, esterase activity did not change after SU processing and was not related to sperm motility, which indicates that a semen sample with good membrane integrity will not necessarily have a high motility. Similarly to asthenozoospermic samples from humans (Piasecka et al, 2007), we have observed that membrane integrity of ram semen samples with low motility resembles that of ram semen samples with normal motility.

Different types of dehydrogenases as well as nicotinamide adenine dinucleotide and flavin adenine dinucleotide in the matrix and inner mitochondrial membranes are responsible for adenosine triphosphate production. When proteins lose their structure, they also lose their activity. In our case, a sperm negative for resazurin is a cell with no mitochondrial function because dehydrogenases have not converted the resazurin to the fluorescent form. The fact that SU processing does not increase the percentage of mitochondrially active sperm but does increase the percentage of motile cells indicates that energy sources other than from oxidative phosphorylation are being used for flagellar activity. That energy is generated by glycolysis in flagella, as demonstrated by Miki et al (2004).

Similar to a previous report (García-López et al, 1996), we observed that SU is more effective for neat samples with low motility than for samples with high motility.

Caspases comprise a family of highly specific proteases that contain the amino acid cysteine in their active sites. Some studies have reported the presence of active caspases in sperm of different species (Paasch et al, 2004; Martí et al, 2008), and distribution along the sperm appears to be specific in human (Paasch et al, 2004) and ram (Martí et al, 2008). We do not know whether the patterns of caspase activity observed in this study correspond to caspase-3 or -7 because the FLICA reagent interacts with the enzymatic reactive center of an active caspase through a specific recognition sequence (aspartic acid–glutamic acid–valine–aspartic acid, for caspase-3 and -7) and subsequently attaches covalently to a cysteine through the fluoromethyl ketone moiety (Ekert et al, 1999). Nevertheless, the presence of active caspases in neat ejaculates and the diminished presence of active caspases in ejaculates after SU processing suggest a role for caspases in motility and possibly in fertility. Martí et al (2006, 2008) reported reduced caspase activity in SU-processed ram sperm, but there were no statistical differences. These findings agree with our results that there is a reduction in the percentage of sperm with active caspases after SU processing in samples with low or high motility.

In a previous study, immunofluorescent labeling of caspase-3 and -7 in ram sperm showed positive staining of caspase-3 in the apical region, the tail, the equatorial region, and the postacrosomal region, and positive staining of caspase-7 in the neck, the apical region, the tail, and the postacrosomal region (Martí et al, 2008). However, that does not identify caspases and procaspases. In our work, active caspases were located principally in the middle and principal pieces, and this is the first report that describes active caspases in the implantation fossa region. There are 2 possible causes for the discrepancy in the location of caspases between the work of Martí et al (2008) and ours: 1) that, once activated, the caspases translocate to the cytoplasm, as observed for germ cells in the testis (Lysiak et al, 2007); and 2) that the FLICA reagent associates with other components in the scarce cytoplasm of the sperm cell, making molecules other than active caspases fluoresce. Recently, 2 studies (Pozarowski et al, 2003; Kuzelová et al, 2007) indicated that FLICA inhibitors could associate with no identified target(s) other than cysteine of active caspases, although the expression follows the pattern of apoptosis. In this sense, we have observed intense FLICA signals in sperm with a cytoplasmic drop (ie, atypical components of sperm cytoplasm).

We observed that SU processing reduced the number of sperm expressing active caspases. Other authors have obtained similar results using variants of the SU technique (Martí et al, 2006, 2008; Kotwicka et al, 2008) or other sperm separation techniques (Martí et al, 2006). However, we found that the neat and SU ejaculates had a high percentage of sperm expressing caspases, which did not correlate with motility. Recently, Kotwicka et al (2008) identified motile sperm expressing active caspase-3 and Perticarari et al (2007) did not find a correlation between sperm apoptosis and motility. These results may explain the lack of an association between motility and active caspases in spermatozoa. Cebrian's group (Martí et al, 2008) observed an even lower percentage of sperm expressing caspases-3 and -7 than observed by us in this study. We do not know why the percentage of sperm expressing active caspases is high in our study, but there are 2 possibilities: 1) we obtained false positives from a nonspecific chemical used to identify the active caspases, and 2) caspase expression is related to strain. We discard the specificity of FLICA reagent as specified above, because the percentage of sperm with active caspases in assays using caspase inhibitor (z-DEVD-FMK) and H₂O₂ as activator of caspases is significantly lower than that found in neat or SU samples.

DNA damage does not block fertilization and early embryonic development but induces apoptosis after the first cleavages (Fatehi et al, 2006). There is a strong relationship between the results of SCSA and other tests designed to identify DNA damage (Sailer et al, 1995; Chohan et al, 2006). SU increased the percentage of motile sperm in neat semen samples with low motility and reduced the number of gametes with active caspases; however, we found an increase in the DFI values in the same samples. Some studies have indicated low DFI values after SU processing (Spano et al, 2000), whereas others have indicated no change (Sakkas et al, 2000). We do not know the cause of the high DFI values in the sperm samples with <25% motility after the SU procedure; however, the fact that DFI was high after SU processing and there was no change in DFI in the samples with >25% motility emphasizes the importance of initial motility as an indicator of semen quality. Although SU processing can improve motility, the chromatin is damaged, which can result in a risk for early development (Fatehi et al, 2006).

In summary, individual neat ram semen samples had different degrees of motility, and viability did not change after the SU procedure. We have demonstrated the location of active caspase-3 and -7 in ram sperm and that SU significantly reduces the percentage of sperm expressing caspases, which is accompanied by an increase in chromatin abnormalities of samples with low motility.

	Acknowledgments

 
The authors are grateful to Leticia Morales, José Vázquez, and Luis Becerril for their technical assistance.

	Footnotes

 
Supported by UAEMex Grant 2332/2006U.

	References

Top Abstract Materials and Methods Results Discussion References

 
Almeida C, Cardoso MF, Sousa M, Viana P, Gonçalves A, Silva J, Barros A. Quantitative study of caspase-3 activity in semen and after swim-up preparation in relation to sperm quality. Hum Reprod. 2005;20: 1307 –1313.[Abstract/Free Full Text]

Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod. 2000; 15: 1338 –1344.[Abstract/Free Full Text]

Bejarano I, Lozano GM, Ortiz A, García JF, Paredes SD, Rodríguez AB, Pariente JA. Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril. 2007;90: 1340 –1347.[Medline]

Chang HY, Yang X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev. 2000; 64: 821 –846.[Abstract/Free Full Text]

Chohan KR, Griffin JT, Lafromboise M, Jonge CJD, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl. 2006; 27: 53 –59.[Abstract/Free Full Text]

Chohan KR, Hunter AG. In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes. Theriogenology. 2004; 61: 373 –380.[CrossRef][Medline]

de Vries KJ, Wiedmer T, Sims PJ, Gadella BM. Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells. Biol Reprod. 2003; 68: 2122 –2134.[Abstract/Free Full Text]

Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell Death Differ. 1999;6: 1081 –1086.[CrossRef][Medline]

Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci. 2000; 22: 169 –189.[CrossRef][Medline]

Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science. 1980; 210: 1131 –1133.[Abstract/Free Full Text]

Fatehi AN, Bevers MM, Schoevers E, Roelen BAJ, Colenbrander B, Gadella BM. DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages. J Androl. 2006;27: 176 –188.[Abstract/Free Full Text]

García-López NM, Ollero T, Muiño-Blanco T, Cebrián-Pérez JA. A Dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma. Theriogenology. 1996; 46: 141 –151.[CrossRef]

Grasa P, Pérez-Pé R, Báguena O, Forcada F, Abecia A, Cebrián-Pérez JA, Muiño-Blanco T. Ram sperm selection by a dextran/swim-up procedure increases fertilization rates following intrauterine insemination in superovulated ewes. J Androl. 2004;25: 982 –990.[Abstract/Free Full Text]

Inaudi P, Petrilli S, Joghtapour A, Trusso P, Petraglia F. Reduction of steps in the preparation of motile sperm for intrauterine insemination does not reduce efficacy of the procedure: simplified one-step swim-up method versus classic swim-up. Hum Reprod. 2002; 17: 1288 –1291.[Abstract/Free Full Text]

Johnson VL, Ko SC, Holmstrom TH, Eriksson JE, Chow SC. Effector caspases are dispensable for the early nuclear morphological changes during chemical-induced apoptosis. J Cell Sci. 2000; 113: 2941 –2953.[Abstract/Free Full Text]

Kotwicka M, Filipiak A, Jedrzejczak P, Warchol J. Caspase-3 activation and phosphatidylserine membrane translocation in human spermatozoa: there is a relationship? Reprod Biomed Online. 2008; 16: 657 –663.[Medline]

Kotwicka M, Jendraszak M, Warchol JB. Plasma membrane translocation of phosphatidylserine in human spermatozoa. Folia Histochem Cytobiol. 2002;40: 111 –112.[Medline]

Kuzelová K, Grebenová D, Hrkal Z. Labeling of apoptotic JURL-MK1 cells by fluorescent caspase-3 inhibitor FAM-DEVD-fmk occurs mainly at site(s) different from caspase-3 active site. Cytometry A. 2007; 71: 605 –611.[Medline]

Lysiak JJ, Zheng S, Woodson R, Turner TT. Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2. Cell Tissue Res. 2007; 328: 411 –419.[CrossRef][Medline]

Marchetti C, Gallego MA, Defossez A, Formstecher P, Marchetti P. Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters. Hum Reprod. 2004; 19: 1127 –1134.[Abstract/Free Full Text]

Martí E, Pérez-Pé R, Colás C, Muiño-Blanco T, Cebrián-Pérez JA. Study of apoptosis-related markers in ram spermatozoa. Anim Reprod Sci. 2008;106: 113 –132.[CrossRef][Medline]

Martí E, Pérez-Pé R, Muiño-Blanco T, Cebrián-Pérez JA. Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa. J Androl. 2006;27: 746 –753.[Abstract/Free Full Text]

Miki K, Qu W, Goulding EH, Willis WD, Bunch DO, Strader LF, Perreault SD, Eddy EM, O'Brien DA. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proc Natl Acad Sci U S A. 2004; 101: 16501 –16506.[Abstract/Free Full Text]

Paasch U, Grunewald S, Agarwal A, Glandera HJ. Activation pattern of caspases in human spermatozoa. Fertil Steril. 2004; 81(suppl 1): 802 –809.[CrossRef][Medline]

Paasch U, Grunewald S, Fitzl G, Glander HJ. Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. J Androl. 2003;24: 246 –252.[Abstract/Free Full Text]

Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S, Sartore A, Presani G. A new multiparameter flow cytometric method for human semen analysis. Hum Reprod. 2007; 22: 485 –494.[Abstract/Free Full Text]

Piasecka M, Gaczarzewicz D, Laszczynska M, Starczewski A, Brodowska A. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia. Folia Histochem Cytobiol. 2007; 45(suppl 1): S127 –S136.

Pozarowski P, Huang X, Halicka DH, Lee B, Johnson G, Darzynkiewicz Z. Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation. Cytometry A. 2003; 55: 50 –60.[Medline]

Rasband WS. ImageJ (computer program). Bethesda, MD: US National Institutes of Health; 2005.

Ricci G, Perticarari S, Boscolo R, Montico M, Guaschino S, Presani G. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil Steril. 2009; 91(2): 632 –638.[CrossRef][Medline]

Said T, Agarwal A, Grunewald S, Rasch M, Baumann T, Kriegel C, Li L, Glander HJ, Thomas AJ, Paasch U. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biol Reprod. 2006; 74: 530 –537.[Abstract/Free Full Text]

Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl. 1995;16: 80 –87.[Abstract/Free Full Text]

Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, Bianchi U. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod. 2000; 15: 1112 –1116.[Abstract/Free Full Text]

Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999;4: 31 –37.[Abstract]

Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod. 2002; 66: 1061 –1067.[Abstract/Free Full Text]

Spano M, Bode J, Hjøllund H, Kolstad H, Cordelli E, Leter E. Sperm chromatin damage impairs human fertility. Fertil Steril. 2000;73: 43 –50.[CrossRef][Medline]

Uggeri J, Gatti R, Belletti S, Scandroglio R, Corradini R, Rotoli BM, Orlandini G. Calcein-AM is a detector of intracellular oxidative activity. Histochem Cell Biol. 2004; 122: 499 –505.[CrossRef][Medline]

Weng SL, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S. Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod. 2002; 8: 984 –991.[Abstract/Free Full Text]

Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up techniques and DNA fragmentation. Hum Reprod. 2001; 16: 1950 –1953.[Abstract/Free Full Text]

Zrimsek P, Kosec M, Kunc J, Mrkun J. Determination of the diagnostic value of the resazurin reduction assay for evaluating boar semen by receiver operating characteristic analysis. Asian J Androl. 2006;8: 343 –348.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 31/2/169    most recent Author Manuscript (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pichardo, A. I. Articles by Domínguez-Vara, I. A. Search for Related Content PubMed PubMed Citation Articles by Pichardo, A. I. Articles by Domínguez-Vara, I. A.