1 Department of Genetics, Faculty of Medicine, University of Porto, 2 Department of Population Studies and 3 Lab Cell Biology, ICBAS, University of Porto and 4 Centre for Reproductive Genetics Alberto Barros, Porto, Portugal
5 To whom correspondence should be addressed at: Lab Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Lg Prof. Abel Salazar 2, 4099-003 Porto, Portugal. Email: msousa{at}icbas.up.pt
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Abstract |
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Key words: apoptosis/asthenoteratozoospermia/caspase-3 activity/male infertility/semen analysis parameters
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Introduction |
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Apoptosis regulates normal spermatogenesis. In the postnatal period, it is responsible for the death and phagocytosis of premeiotic germ cells in order to adjust their number to the number of Sertoli cells, and for the removal of damaged and abnormal germ cells during active spermatogenesis in the post-pubertal period (Print and Loveland, 2000; Sinha Hikim et al., 2003
). Germ cell apoptosis has been shown to increase after testicular injury, such as exposure to toxics, varicocele, testicular torsion, hormonal deprivation and genetic abnormalities (Francavilla et al., 2000
; Kim et al., 2001
; Said et al., 2004
), as well as a response to freezethawing (Glander and Schaller, 1999
; Schuffner et al., 2001
; Paasch et al., 2004
).
In the present investigation we quantitatively analysed caspase-3 activity in sperm retrieved from the semen and swim-up fractions of males with normal and abnormal semen parameters.
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Materials and methods |
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Caspase-3 activity detection
Fractions used for caspase-3 activity determination were centrifuged at 1300 g for 2 min at room temperature. The pellet was resuspended and incubated for 30 min (37°C, 5% CO2 in humidified air) in previously warmed 50 µl of medium containing (1:1) SPM and PhiPhiLux D2G2 (membrane permeant profluorogenic caspase-3 substrate; OncoImmunin Inc., USA; stock solution: 10 µmol/l in Roswell Park Memorial Institute 1640 medium with 25 mmol/l HEPES, kept in aliquots at 20°C). Semen and swim-up sperm fractions were then washed (1300 g for 2 x 1 min at room temperature) with 500 µl of phosphate-buffered saline (Sigma, Barcelone, Spain), and the pellet spread onto poly-L-lysine (Sigma)-coated glass slides. After air-drying, slides were mounted with 10 µl Vectashield antifade medium (Vector Laboratories, USA), containing 1.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) to stain the DNA (blue), and sperm counted in an epifluorescence microscope (Eclipse, E-400; Nikon, Japan) fitted with a CCD camera (Sony, Japan) and an automated karyotyper software (Cytovision Ultra; Applied Imaging International, UK). In the presence of activated caspase-3, the specific substrate PhiPhiLux G2D2 is cleaved and emits red light. In all cases, sperm positive for caspase-3 activity showed an intense fluorescent red colour in the mitochondrial midpiece (Figure 1).
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Statistical analysis
Descriptive statistics were used for the initial characterization of the population, considering each male as an individual and a two-sided significance level of 5%. In this approach, Wilcoxon and KruskalWallis tests were used to compare the percentages of caspase-3-positive sperm between semen and swim-up fractions, as well as between the defined levels of each parameter in each fraction. This approach is considered the traditional way for analysing this kind of outcome, where a number of cells are observed for each subject and the result is represented as a percentage of positive cells. A limitation of this traditional approach is that the total number of cells counted is not considered, and the characteristics observed for each cell is addressed only to the subject to which it belongs. Thus, data were then analysed at the cellular level, by which each spermatozoon scored was considered independently from other cells (Zhao et al., 2001). In this approach, the cells are considered as the individuals, and for each one the outcome, caspase-3-positive or caspase-3 negative sperm, corresponds to a binary variable. When data were analysed at the cellular level, the size of the sample studied and the power of the hypothesis tested were greatly increased. To control the possibility of committing a type I error, hypotheses were tested at a probability of 0.01 rather than 0.05, and thus a stringent significance level of 1% was chosen for statistical significance. The presence or absence of positive caspase-3 sperm was studied with logistic regression models and the odds ratio (OR) for risk of presence and their associated 99% confidence interval (CI) are presented. Firstly, a comparative analysis between semen and swim-up fractions was performed. To find any possible association between the presence of caspase-3 and the different spermiogram parameters, semen and swim-up fractions were then analysed separately. All analyses were performed with the SPSS statistical package (version 11.0) for Windows.
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Results |
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Semen fractions
Considering each male as an individual, the percentage values of caspase-3-positive sperm appeared much dispersed in the different groups of spermiogram parameters, thus suggesting that no relationship exists (Figure 2); nor were there significant differences with respect to subgroups (P0.070) for sperm concentration, normal morphology or rapid progressive motility.
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When data were treated at the cellular level using comparisons to the relative controls, all main spermiogram parameters (concentration, normal morphology, rapid progressive motility) appeared significantly associated (P<0.001) with the presence of caspase-3-positive sperm (Table II). No significant differences from the relative controls were found in the 5% subgroups of normal morphology (P=0.062) and rapid progressive motility (P=0.404). The probability of the spermatozoon being caspase-3-positive was significantly lower than the reference category in all other subgroups, except for a significant increase in the proportion of caspase-3-positive sperm relative to controls in the subgroups >5<10%,
10<15% and <15% of normal morphology, and >5<10% of rapid progressive motility (OR
1, P<0.001). To further consolidate the present results, a similar analysis was performed using comparisons to the absolute control group (Table II). In this case, significant differences (P<0.001) were found for the presence of caspase-3-positive sperm regarding all main spermiogram parameters. No significant differences from the absolute control were found in the
1% subgroup of sperm concentration (P=0.033), in the
5% (P=0.038) and <15% (P=0.010) subgroups of normal morphology, and in the
5% (P=0.012),
10<25% (P=0.158) and <25% (P=0.364) subgroups of rapid progressive motility. With the exception of a significant increase in the proportion of caspase-3-positive sperm relative to the control in the subgroups>5<10% and
10<15; of normal morphology, and >5<10% of rapid progressive motility (OR
1, P<0.001), the probability of the spermatozoon being caspase-3 positive was significantly lower than the reference category in all other subgroups. In the analysis of dichotomous study groups (Table III), comparisons with the relative controls or the absolute control showed significant differences in all subgroups, with the exception of the astheno- and oligoasthenozoospermic subgroups (P>0.01). This also demonstrated a higher risk for the presence of caspase-3-positive sperm in the terato- and asthenoteratozoospermic subgroups (OR
1, P<0.001).
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Discussion |
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Although it remains to be established whether sperm defects are a cause or a consequence of increased rates of apoptosis, several studies have shown an increase of DNA fragmentation in human sperm with low motility (Barroso et al., 2000; Weng et al., 2002
), low sperm counts (Oosterhuis et al., 2000
) or decreased normal sperm concentration, morphology and motility (Gandini et al., 2000
; Shen et al., 2002
). This is of clinical relevance, as semen samples showing increased rates of sperm DNA fragmentation have been associated with decreased rates of fertilization (Marchetti et al., 2002
) and pregnancy (Tomlinson et al., 2001
). Other studies have also shown that mitochondrial integrity is decreased in samples with low sperm motility (Donnelly et al., 2000
; Weng et al., 2002
), and that phosphatidylserine exposure is increased in samples exhibiting low sperm motility (Weng et al., 2002
), decreased normal sperm motility and morphology (Shen et al., 2002
), or decreased normal sperm concentration and motility (Oosterhuis et al., 2000
).
Here we present a strict statistical analysis of 67 semen samples, in which 119 850 sperm were quantified for the presence of caspase-3 activity, 67 488 from semen fractions and 52 362 from swim-up fractions. The large number of cases studied enabled the analysis of different subgroups of severity of sperm defects within major groups of spermiogram abnormalities. In the first approach, cases were divided according to decreased sperm normal concentration, morphology and rapid progressive motility, and each group was then subdivided into different levels of severity of each sperm defect. In a second approach, and to uncover the possible contribution of simultaneous sperm defects, cases were analysed for single abnormalities (oligo-, astheno- and teratozoospermia). In the semen fractions, no relationship was found between the presence of caspase-3-positive sperm and any of the abnormal spermiogram values. In accordance with previous results (Ricci et al., 2002), the proportion of caspase-3-positive sperm was shown to be unrelated to sperm quality. However, we first show that this is due to the presence of simultaneous sperm defects, as the analysis of single sperm abnormalities revealed a strong relationship to asthenozoospermia. On the contrary, results obtained from the swim-up fractions showed a significant increase of caspase-3-positive sperm with decreased normal morphology and rapid progressive motility values, which after analysis of single sperm abnormalities was only confirmed for teratozoospermia. In accordance with previous reports that have used several other markers for detecting sperm apoptosis (Donnelly et al., 2000
; Gandini et al., 2000
; Marchetti et al., 2002
), our current results confirm that gradient centrifugation and swim-up significantly deplete caspase-3-positive sperm. Taking into account that the presence of caspase-3-positive sperm was 4.2-fold higher in semen than in the swim-up fractions (OR=4.227, 99% CI=3.9754.495), that the swim-up fractions were associated only with teratozoospermia (OR
1 and P<0.001), and that samples with decreased normal sperm morphology (<15%) have 4.5±3.5% (mean±SD) of caspase-3-positive sperm in the swim-up fractions, the data suggest a low risk of selecting apoptotic sperm during clinical treatments.
Caspase activation is a well-defined point of no return for apoptotic progression in somatic cells (Wolf et al., 1999). Although procaspases are present in human mature sperm and might be activated by different stimuli such as cryopreservation (Paasch et al., 2004
), the role of caspases and apoptosis in ejaculated sperm is still an open question. Caspase activity in mature sperm may correspond to the activation of the apoptotic machinery leading to cell death as induced by different factors during spermiogenesis. In this mechanism, spermatids marked for elimination via apoptosis could somehow escape the removal mechanism and thus contribute to the poor sperm quality found in the ejaculate (Sakkas et al., 2002
; Said et al., 2004
). However, as active caspase-1 was demonstrated to be involved in the degradation of surplus spermatid cytoplasmic components (residual bodies) during spermiogenesis, caspase activity in ejaculated sperm may, alternatively, correspond to enzyme diffusion from residual bodies (Blanco-Rodriguez and Martinez-Garcia, 1999
). Caspase-3 is the major effector enzyme causing cell disruption during apoptosis. Caspase-3 activity has been previously detected in the midpiece of ejaculated human sperm (Weng et al., 2002
) and shown to be significantly associated with low sperm motility (Weng et al., 2002
) or with decreased normal sperm concentration, motility and morphology (Wang et al., 2003
). Our present results confirm that sperm caspase-3 activity is restricted to the mitochondrial sheath, and show no statistical association between the presence of caspase-3-positive sperm and decreased normal sperm concentration, motility or morphology in the neat semen. On the contrary, a significant increase of caspase-3-positive sperm is shown in the swim-up fractions from samples with decreased normal sperm morphology. These data also show that when the analysis is corrected for single sperm defects, caspase-3-positive sperm appear significantly associated with asthenozoospermia in the semen fractions and with teratozoospermia in the swim-up fractions. These data thus strongly favour the hypothesis that caspase activity is not the result of active enzyme diffusion from residual bodies and may correspond to the activation of the cell apoptotic machinery for discarding abnormal spermatids/sperm due to nuclear, mitochondrial and cytoskeleton structural defects.
In conclusion, no relationship was found between caspase-3-positive sperm and abnormal spermiogram subgroups in the semen fractions, whereas a significant relationship was found between caspase-3-positive sperm and decreased normal sperm morphology and rapid progressive motility in the swim-up fractions. When the analysis was restricted to single spermiogram defects, a significant increase in caspase-3-positive sperm was found in relation to asthenozoospermia in the semen and to teratozoospermia in the swim-up fraction.
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Acknowledgements |
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Submitted on April 5, 2004; resubmitted on August 5, 2004; accepted on December 10, 2004.
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