Apoptosis-related phenotype of ejaculated spermatozoa in patients with varicocele

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1 Apoptosis-related phenotype of ejaculated spermatozoa in patients with varicocele Gwo-Jang Wu, M.D., Ph.D., a,c Fung-Wei Chang, M.D., a Shang-Sen Lee, M.D., b Ya-Yuan Cheng, M.Sc., a Chi-Huang Chen, M.Sc., a and I-Ching Chen, M.D. a a Department of Obstetrics and Gynecology, b Department of Urology, and c Department of Biomedical Engineering, Reproductive Medicine Center, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Objective: To determine the integrity of the sperm plasma membrane, mitochondria, and DNA, which are essential for accurate transmission of genetic material to offspring, and to quantify possible apoptosis and investigate any relationship between these parameters in ejaculated sperm from men with or without varicoceles. Design: Retrospective study. Setting: University teaching hospital. Patient(s): Twenty-five patients with varicocele and 10 normal, fertile controls. Intervention(s): Apoptosis-related phenotype activations including the plasma membrane translocation of phosphatidylserine, mitochondrial dysfunction, and nuclear DNA damage, were assessed by using the annexin- V/propidium iodide double staining assay, 3,3 -dihexloxacarbocyanine iodide staining assay, and single-cell gel electrophoresis assay (comet assay). Main Outcome Measure(s): Apoptosis-related phenotype. Result(s): Patients with varicocele had statistically significantly more annexin V live sperm cells and nuclear DNA fragmentation than did the control men. In contrast, their numbers of 3,3 0 -dihexloxacarbocyanine iodide live cells were statistically significantly less than those in control men. Conclusion(s): The increased externalization of phosphatidylserine, mitochondrial dysfunction, and nuclear DNA damage occurred in the sperm of men with varicoceles, suggesting that certain apoptotic mechanisms may relate to the condition of varicocele, originating in the mitochondria of spermatocytes and then functioning within the nucleus of the cell. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Apoptosis, DNA fragmentation, phosphatidylserine, sperm, varicocele. Varicocele compromises the countercurrent system of scrotal thermal regulation that allows for heat exchange between arterial and venous blood (1). It is characterized by venous stasis, heat stress, hypoxia, and accumulation of toxic metabolites in the testes (2), leading to increased reactive oxygen production and apoptosis during specific stages of spermatogenesis (3 5). Not all men with varicoceles are infertile. Interestingly, the varicocele incidence in that subset of men with secondary infertility (those who were previously fertile but now are infertile) is about 70% 80% (6 8). Gorelick et al.(7) postulated that men with prior fertility may have varicocele-mediated secondary infertility and that the presence of a varicocele may cause a progressive decline in fertility. Nonetheless, it remains unclear why some men with varicoceles can father children and produce relatively normal semen, whereas other similarly affected men fail to improve despite corrective surgery (9). Received September 25, 2007; revised and accepted December 18, G.-J.W. has nothing to disclose. F.-W.C. has nothing to disclose. S.-S.L. has nothing to disclose. C.-H.C. has nothing to disclose. Supported by grant TSGH-C93-03-S05 from Tri-Service General Hospital and grant NSC B from the National Science Council (both, Taipei, Taiwan). Reprint requests: Gwo-Jang Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, Reproductive Medicine Center, Tri-Service General Hospital, National Defense Medical Center, 5F, No. 325, Sec. 2, Cheng-Kung Rd, Niheu, Taipei 114, Taiwan (FAX: ; gwojang@yahoo.com). Successful fertilization requires a sperm plasma membrane with normal integrity and function (10). The numerous functions of the membrane are related to cell metabolism, for maintaining sperm motility, capacitation, acrosome reaction, and sperm oocyte interaction (11). In vital sperm with intact plasma membranes, the negatively charged membrane phosphatidylserine (PS) is located on the inner leaflet of the plasma membrane. The disturbance of membrane function starts with translocation of PS from the inner to the outer leaflet of the plasma membrane and results in exposure of PS on the external cell surface (12). Thus, the basal apoptotic cell population in the ejaculated sperm may result from abortive apoptosis of sperm that escaped the elimination mechanism that operates during spermatogenesis (13, 14). Because externalized PS is involved in the recognition of apoptotic cells by phagocytes, ejaculated sperm exhibiting PS externalization may represent apoptotic sperm cells that escaped phagocytosis during spermatogenesis (15). Sperm showing evidence of PS exposure are found at higher levels in semen samples from infertile men than in those from normal sperm donors (16 19). The early phase of disruption of plasma membrane integrity is characterized by the loss of phospholipid asymmetry, caused by the translocation of PS from the inner to the outer leaflet of the plasma membrane (12). This translocation of PS is one of the earliest features of cells undergoing apoptosis (20) /09/$36.00 Fertility and Sterility â Vol. 91, No. 3, March doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 Mitochondria ensheath the midpiece of spermatozoa and are required for efficient energy metabolism, production of membrane lipids, and cell growth but are also the primary determinants of cellular life or death. They deliver adenosine triphosphate to the axoneme, where it is utilized for flagellar propulsion (21). Oxidative phosphorylation is the process whereby protons are pumped from inside the mitochondrial membrane to outside it, creating an electrochemical gradient. This process is termed the mitochondrial membrane potential and is used to estimate cell metabolic function through measurement of differences in potential from the inner to the outer mitochondrial membrane. The changes in mitochondrial transmembrane potential reflect the initial apoptotic phenomena. Dysfunctional mitochondria may result in reduction of sperm movement (21). Integrity of DNA in sperm is essential for the accurate transmission of genetic information to offspring. In human studies, recent reports demonstrated that varicoceles are associated with high levels of DNA-damaged spermatozoa, even in the presence of a normal semen profile (22 24). The presence of a varicocele is thought to lead to an increase in reactive oxygen species (ROS) production and to apoptosis in the testes and semen (4, 5). Seminal oxidative stress and testicular apoptosis both are well-documented causes of increased sperm nuclear DNA fragmentation (22, 25, 26). Higher rates of sperm nuclear DNA fragmentation lead to lower fertility rates, both in natural and assisted reproduction (25, 27, 28). Varicocele-associated apoptosis is recognized as a cause of male infertility (9). A number of previous studies determined that the incidence of apoptosis appeared to be remarkably more frequent within the testicular tissue of patients who have varicoceles than in control individuals and that the concentration of sperm in semen increased subsequent to varicocelectomy (5, 29, 30). However, we reported elsewhere that we were unable to determine any relationship between semen quality and apoptosis (9). Semen quality and sperm motion characteristics did not appear to differ significantly, although the varicocele group had a significantly greater apoptotic index than was the case for the fertile control group. The possible detrimental effects of varicoceles on sperm cells are of great interest, but the data available to support this relationship are somewhat elusive. To approach these problems, we assessed sperm motility and morphology by using conventional semen parameters and membrane translocation of PS by annexin V propidium iodide (PI) double staining of the plasma membrane, diminution of mitochondrial membrane potential by using the 3,3 0 -dihexloxacarbocyanine iodide (DiOC 6 )assay, and sperm DNA fragmentation by using the comet assay. These are among the main signs of apoptosis in a group of men with varicoceles and among normally fertile men. MATERIALS AND METHODS Subjects and Semen Preparation A total of 40 men were recruited for this study, including 30 patients (20 35 y of age) with varicoceles and 10 volunteers who participated as normal control (semen) donors (30 37 y of age). The study protocol was reviewed and approved by the Institutional Review Board of Tri-Service General Hospital, National Defense Medical Center in Taipei, Taiwan. The diagnosis of varicoceles (grade II III) was assessed clinically in these patients by genital examination, which was performed by a urologist. Sperm donor selection required healthy fertile individuals with no serious chronic or contagious diseases and with normal genital examinations. Donors were required as well not to work with toxic substances. Participating subjects were required to collect semen by masturbation into a sterile specimen container, subsequent to a 3 5-day period of sexual abstinence. Subsequent to ejaculation of semen, all semen samples were allowed to liquefy for a period of minutes at room temperature. After liquefaction, on the basis of the World Health Organization criteria, semen-sample analysis was performed by using computeraided sperm analysis to determine sperm concentration and motility and by using manual analysis to determine sperm morphology. Samples with detectable leukocytes in the semen were excluded from the study. Flow Cytometry Analysis For each experimental set of double staining (annexin V fluorescein isothiocyanate [FITC]/PI) and single staining (DiOC 6 FITC), different sperm suspensions were prepared for instrument settings and data analyses. Analyses were performed by using the CyFlow SL (Partec GmbH, M unster, Germany), a fully equipped desktop flow cytometer with five optical parameters plus time parameters that performs both fluorescence analyses and absolute cell counting. Fluorescence of samples was detected by using FL1 (525 nm) for the annexin V FITC and DiOC 6 assays and FL3 (630 nm) for the PI detectors; 10,000 events were analyzed for each sample. The FL1 and FL3 fluorescence signals were recorded after logarithmic amplification. Evaluation of Membrane PS Translocation and Sperm Viability Annexin V is a Ca 2þ -dependent, phospholipid-binding protein with high affinity for PS, which is expressed on the outer leaflet of sperm plasma membrane in a calcium-dependent manner. Annexin V staining was used to detect PS translocation from the inner to the outer leaflet of the plasma membrane of sperm cells. This test identifies cells with deteriorated membranes, which is one of the earliest features of cells undergoing apoptosis (12). Propidium iodide (red staining) is a vital dye that is used to measure viability and to distinguish apoptotic cells from necrotic cells. Staining with annexin V FITC PI was performed by using a commercial kit (Sigma-Aldrich, St. Louis, MO). As directed in the manufacturer s instructions, each sample was resuspended in 1 phosphate-buffered saline (PBS; sperm per ml of PBS). All samples were washed twice with cold 1 PBS by centrifugation (10 min, 300 g, 4 C), the supernatant was decanted, and the pellet was resuspended in 500 mlof1 binding buffer, labeled with 5 mlof 832 Wu et al. Sperm apoptosis in varicoceles Vol. 91, No. 3, March 2009

3 annexin V plus 10 ml of PI. The mixture was incubated at room temperature for exactly 10 minutes and protected from light. Cell fluorescence immediately was determined by using flow cytometry. The different labeling patterns used in the annexin V PI analyses identified the different cell populations. Annexin V-FITC and PI-negative sperm were designated as live cells, annexin V-FITC negative and PI-positive sperm were designated as necrotic cells, annexin V-FITC positive and PI-negative sperm were designated as apoptotic cells, and annexin V-FITC and PI-positive sperm were designated as late apoptotic cells or necrotic cells. Detection of Mitochondrial Membrane Potential The functional integrity of sperm mitochondria, in which poor mitochondrial integrity is indicative of apoptosis, was assessed by using a commercial DiOC 6 assay kit (Calbiochem, Darmstadt, Germany). 3,3 -Dihexloxacarbocyanine iodide is a cationic, cell-permeable, voltage-sensitive, lipophilic, and fluorescent carbocyanine dye that is used as a membrane potential probe that fluoresces differently in apoptotic cells than in nonapoptotic cells. In nonapoptotic cells, DiOC 6 is taken up in the mitochondria and exhibits green fluorescence. However, in apoptotic cells, DiOC 6 cannot enter the mitochondria because of the altered mitochondrial membrane potential, and the dye cannot be detected. This kit allows detection of changes in mitochondrial transmembrane potential that occur during the early stages of apoptosis. As directed in the instructions of the kit manufacturer, the sample was resuspended in 1 PBS (10 6 sperm per ml of PBS). All samples were washed twice with cold 1 PBS by centrifugation (10 min, 300 g, 4 C), then resuspended in 1 ml of 1 PBS, labeled with 1 ml of 40 mm DiOC 6, which was incubated at 37 C for 15 minutes while protected from light. Cell fluorescence immediately was determined by flow cytometry. Evaluation of Sperm Nuclear DNA Integrity We used the residual semen samples (15 patients and 5 controls) to complete the subsequent comet assay. The comet assay is a visual fluorescent technique for measurement of DNA strand breakage in individual cells. The assay is based on the ability of denatured, cleaved DNA fragments to migrate out of the cell under the influence of an electric field, whereas undamaged DNA migrates more slowly and remains within the confines of the nucleoid when a current is applied. Sperm nuclear DNA integrity can be quantified by using image analysis software. The comet assay was performed by using a commercial kit (Trevigen, Gaithersburg, MD). As directed in the manufacturer s instructions, the sperm sample was suspended in 1 PBS ( sperm/1 ml of PBS). All samples were washed twice with cold 1 PBS by centrifugation (10 min, 300 g, 4 C), then resuspended in 1 ml of 1 PBS. A 100- ml aliquot of cells was combined with 5 ml of molten LMAgarose (Trevigen), and 75 ml immediately was pipetted onto the Comet Slide (Trevigen); the slides were placed flat at 4 C in the dark for 30 minutes. After gel solidification, the slides were immersed in cold lysis solution (Trevigen) at 4 C in the dark for 60 minutes. Excess lysis solution was tapped from the slide, and the slide was immersed in fresh alkaline electrophoresis solution, containing 300 mm NaOH and 1 mm ethylenediaminetetraacetic acid, ph >13, at room temperature in the dark for 60 minutes, to allow the DNA to unwind. The slides then were immersed in 1 TBE buffer (0.089 M Tris base, M boric acid, and M EDTA-2 Na) for 5 minutes, twice. Then, the slides were placed on a horizontal electrophoresis apparatus and were covered with 1 TBE buffer until the level just covered the slides. Electrophoresis then was performed for 10 minutes at 13 V (1 V/cm). After electrophoresis, the slides were immersed in 70% ethanol for 5 minutes and then air dried. Next, 50 ml of SYBR Green I (Trevigen) was placed onto each dried sample and evaluated by using epifluorescence microscopy, with an excitation filter of nm under 200 magnification. A total of 100 sperm cells was randomly measured by using Komet imaging software (Kinetic Imaging Ltd., Liverpool, UK). The percentage of DNA in the sperm tail is a measurement of the degree of DNA damage in proportion to the total DNA present in the tail. The Olive tail moment is an integrated value that accounts for both the distance and intensity of the comet fragments. Statistical Analyses The experimental data were analyzed by using statistical procedures available in the Statistical Package for the Social Sciences for Windows (version 13.0; SPSS, Chicago, IL). All differences in apoptosis-related protein values were determined by using unpaired Student s t-tests, and statistical significance was set at P<.05. Data are expressed as mean SD. RESULTS The demographic characteristics and semen parameters in both groups did not differ significantly (Tables 1 and 2, P>.05). Phosphatidylserine externalization of sperm cell surface often is associated with the early stages of apoptosis. We studied PS externalization by using the annexin V probe. Typical cytograms of cells labeled with annexin V FITC PI are shown in Figure 1. Patients with varicoceles and normal sperm donors are compared. Figure 1A and B compares the different percentages for normal donors and patients with varicoceles by quadrant. The percentage of spermatozoa with PS translocation is located in the lower right quadrant and was significantly higher in patients with varicoceles than in normal sperm donors (P<.02; Fig. 1C). The accumulation of the cationic lipophilic fluorochrome DiOC 6 in the inner membrane of mitochondria enables detection of membrane potential variations (31). Among men with varicoceles, the accumulation of DiOC 6 in living sperm mitochondria was less than that in the controls. This characteristic Fertility and Sterility â 833

4 TABLE 1 Comparison of parameters, flow cytometry, and mitochondrial transmembrane potential between patients with varicocele and normal controls. Parameters Control group (n [ 10) Varicocele group (n [ 30) P value Age (y) Sperm concentration (10 6 /ml) Motile sperm (grade 3þ4, 10 6 /ml) Sperm morphology (%) Flow cytometry (%) Necrosis AVII Live AV a Membrane potential (%) a Note: Data are mean SD. AV ¼ early apoptosis; AVII ¼ late apoptosis. a P<.05. suggests a trend toward apoptosis and mitochondrial dysfunction (Fig. 2A). The lower mitochondrial membrane potential in the spermatozoa populations of patients with varicoceles differed significantly from that of controls (P<.02; Fig. 2B). There were no statistically significant differences between the varicocele and control groups regarding participant age, sperm concentration, and sperm motility. However, there were significant differences in percentages of tail DNA and Olive tail moment between the groups (Table 1). Nuclear DNA damage is often associated with the late stages of apoptosis. The nuclear DNA integrity of normal donor sperm and that of patients with varicoceles is shown in Figure 3. The percentages of tail DNA and Olive tail moment were higher in patients with varicoceles than in normal donors (P<.02 and P<.05, respectively; Fig. 3C). The computer-aided sperm analysis assay and the DNA integrity are shown in Table 2. DISCUSSION Normal spermatogenesis depends on the efficiency of apoptosis, which is thought to ensure cellular homeostasis. Apoptosis is observed clearly in rat testes and affects spermatogonia, spermatocytes, and spermatids (32). Apoptosis is referred to as programmed cell death and is characterized microscopically by cell shrinkage, membrane blebbing, and nuclear fragmentation (33). Many pathways result in apoptosis, and these processes appear to be regulated by the plasma membrane externalization of PS, mitochondrial dysfunction, chromatin condensation, and DNA fragmentation (6 9). Externalized PS is involved in the recognition of apoptotic cells by phagocytes; ejaculated sperm exhibiting PS TABLE 2 Comparison of parameters and sperm nuclear DNA integrity (comet assay) between patients with varicocele and control group. Parameters Control group (n [ 5) Varicocele group (n [ 15) P value Age (y) Sperm concentration (10 6 /ml) Motile sperm (grade 3þ4, 10 6 /ml) Sperm morphology (%) Comet assay Tail DNA (%) a Olive tail moment (%) a Note: Data are mean SD. a P< Wu et al. Sperm apoptosis in varicoceles Vol. 91, No. 3, March 2009

5 FIGURE 1 Expression of annexin V PI staining sperm assay by flow cytometry between (A) normal controls and (B) patients with varicocele. Q1 ¼ percentage of necrotic cells, Q2 ¼ percentage of late apoptotic cells, Q3 ¼ percentage of live cells, and Q4 ¼ percentage of apoptotic cells. (C) Bar chart showing percentage of apoptosis. AV ¼ early apoptosis; AV II ¼ late apoptosis. *P<.05. externalization could represent apoptotic sperm cells that escaped phagocytosis during spermatogenesis (15). Thus, the apoptotic sperm population in the ejaculate could result from aborted apoptosis of sperm that escaped the elimination mechanism operating during spermatogenesis (13, 14). The ejaculated sperm in men with varicoceles undergo injury-mediated apoptosis and may reflect the apoptotic phenomenon on spermatogenesis of testes. In our experience, the increased apoptosis-related phenotype of sperm may associate with the testes afflicted with varicocele. Vitality and integrity of the plasma membrane are requirements for spermatozoa to fertilize oocytes. These functions are highly associated with the capacity to undergo the acrosome reaction. However, the significance of PS externalization is not fully understood. In our study, we demonstrated that varicocele injury mediated sperm in apoptosis induced high PS externalization in the living cell population. Increased incidence of apoptosis could have been remarkably more frequent within the testicular tissue of patients with varicocele and could have resulted in the high PS externalization in ejaculated sperm. Changes in mitochondrial transmembrane potential are a function of the functional integrity of sperm mitochondria. These changes can occur during the early stages of apoptosis FIGURE 2 Expression of mitochondrial membrane potential, using the DiOC 6 staining assay by flow cytometry. (A)The numbers of DiOC 6 -stained sperm cells were lower among patients with varicoceles than among normal controls, with a shift to the left. (B) Furthermore, the mitochondrial transmembrane potential was lower among patients with varicocele than among controls. *P<.05. Fertility and Sterility â 835

6 FIGURE 3 Sperm nuclear DNA integrity, assessed by single-cell gel electrophoresis (comet assay). (A) Cells with intact, undamaged DNA from normal controls and (B) cells with a high degree of DNA damage in patients with varicoceles. (C) The percentages of tail DNA and Olive tail moment (OTM) were higher in patients with varicoceles than in normal donor controls (P<.02 and P<.05, respectively). in the organelles that are believed to be the primary determinants of cellular life or death. Our results showed that normal sperm cell counts with DiOC6-stained mitochondrial membranes were significantly higher in the control group than in the varicocele group. In contrast, the sperm cells with dysfunctional mitochondria, a possible indicator of apoptosis, from the varicocele group were shifted to the left of the control group. This may be an indication that these spermatozoa are going through a transition stage or may be in the very early stages of apoptosis. Sperm motility possibly relies on healthy mitochondria (21). Although there was no significant relationship finding between the percentage of spermatozoa with dysfunctional mitochondria and sperm motility in both varicocele and control groups, this indicates that varicocele-induced mitochondrial dysfunction is associated with early apoptosis and may be not reducing sperm movement. Subclinically, mitochondria are related to cell metabolism and contribute to maintaining sperm motility, which is important for successful fertilization. In our study published elsewhere, varicocele was associated with high levels of sperm nuclear DNA fragmentation according to TUNEL assay results, even when there were no changes in sperm quality as assessed by classic seminal analysis (9). This time, we detected DNA integrity by using the comet assay, which is more objective and accurate in describing the degree of DNA damage. Similarly, our data showed that the percentages of sperm tail DNA and Olive tail moment were higher in patients with varicocele than in normal donors. Sperm nuclear DNA fragmentation may be present in the socalled normal semen that is detected by conventional semen analysis. The determination of DNA integrity may be important as an adjunct to standard semen analysis. In conclusion, our observations present evidence for the presence of an apoptosis-associated phenotype, which is recognizable at an earlier level of PS externalization and mitochondrial dysfunction but is not being detected until a later stage, characterized by nuclear DNA fragmentation in sperm cells from both the varicocele and control groups. Varicoceleassociated injury increased the incidence of externalization of PS, increased mitochondrial dysfunction, and increased DNA fragmentation, the possible consequences of an apoptotic mechanism. These injuries can be detected by studying PS externalization and mitochondrial dysfunction as possible early apoptosis indicators, by using the annexin V PI and DiOC 6 staining assays. Sperm plasma membrane and mitochondria undergo apoptosis, and the membrane and mitochondrial markers appears to be a more sensitive and immediate indicator of this early process. Nuclear DNA fragmentation of ejaculated spermatozoa appears in late apoptosis, as seen when using the comet assay. The exact mechanism of programmed cell death of spermatozoa remains to be elucidated. Apoptosis may be a means of eliminating potentially harmful mutations to prevent the transmission of these abnormalities to the next generation. We revealed that sperm from the varicocele group exhibited a more substantial level of constitutive apoptosis than was the case for the control group. The sperm samples from participating patients afflicted with varicocele may have been considered normal when such samples were investigated by using routine semen analysis procedures. Furthermore, these sperm may undergo injury-mediated apoptosis. Plasma membrane PS externalization is associated with decreasing sperm oocyte interaction. Mitochondrial dysfunction may result in failure to maintain sperm motility. Furthermore, under certain conditions (in vivo, or after in vitro treatment, particularly with intracytoplasmic sperm injection treatment for male infertility), it may be that these sperm carry a damaged genome into the oocyte, resulting in serious consequences (34, 35). Our findings provide more complete information about apoptosis in 836 Wu et al. Sperm apoptosis in varicoceles Vol. 91, No. 3, March 2009

7 patients with varicocele and could be used as diagnostic tools to predict other diseases of sperm, including male infertility, representing functional changes that sperm undergo both in vivo and in vitro. The roles of apoptosis and its mechanisms in postejaculated human sperm require further analysis, especially in ICSI programs, which may increase some genetic or epigenetic effect from the patients afflicted with varicocele, even when those men can succeed in producing offspring through assisted reproductive technology (36). REFERENCES 1. Dahl EV, Herrick JF. A vascular mechanism for maintaining testicular temperature by counter-current exchange. Surg Gynecol Obstet 1959;108: Brown JS, Dubin L, Hotchkiss RS. The varicocele as related to fertility. Fertil Steril 1967;18: Goldstein M, Eid JF. Elevation of intratesticular and scrotal skin surface temperature in men with varicocele. J Urol 1989;142: Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr, Agarwal A. 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