The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, USA
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Abstract |
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Key words: cryopreservation/human spermatozoa/lipid peroxidation/phosphatidylserine externalization
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Introduction |
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Human spermatozoa have a high concentration of polyunsaturated fatty acids in their plasma membrane (Jones et al., 1979) and lack adequate antioxidant protection (Alvarez et al., 1987
). Polyunsaturated fatty acids are required for membrane fusion events associated with fertilization (Storey, 1997
); however, their presence renders membranes vulnerable to peroxidative damage (Jones et al., 1979
). Under conditions of oxidative stress, initiation of a LPO cascade results in the loss of membrane fluidity and impairment of sperm function (Aitken et al., 1989
, Aitken, 1999
).
Lipid diffusion through the plasma membrane is significantly compromised in frozenthawed compared with fresh human spermatozoa (James et al., 1999). The membrane phospholipid content decreases after cryopreservation with loss of phosphatidylcholine and phosphatidylethanolamine being the more pronounced (Alvarez and Storey, 1992
). Cryopreservedthawed ram spermatozoa depicted translocation of diphosphatidylglycerol (Hinkovska-Galcheva et al., 1989
) and phosphatidylserine (Muller et al., 1999
) to the outer membrane and instability leading to transbilayer phospholipid asymmetry.
Under different biological scenarios, disturbance of membrane function is associated with translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane (Fadok et al., 1992), identified by annexin V binding (Koopman et al., 1994
; Martin et al., 1995
; Vermes et al., 1995
). It has been demonstrated that in bovine and human spermatozoa, cryopreservation is associated with phosphatidylserine translocation as measured by annexin V binding either by flow cytometry or fluorescent microscopy (Glander and Schaller, 1999
; Anzar et al., 2000
; Duru et al., 2001a
, b
).
Here, we aimed to assess further the sub-lethal effects of human sperm cryopreservationthawing on plasma membrane integrity. For this purpose we examined pre-freeze and post-thaw changes of LPO and phosphatidylserine externalization, as well as their relationships with motility impairment. Translocation of phosphatidylserine was detected using annexin V binding, LPO was measured spectrophotometrically, and motion parameters were monitored using a computer-assisted semen analyser (CASA).
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Materials and methods |
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After semen liquefaction for 30 min at 37°C, a basic semen analysis (concentration, motility, and morphology) was performed. This was immediately followed by discontinuous Percoll gradient centrifugation in order to isolate the purified population of highly motile spermatozoa. Pre-freeze evaluation of motion parameters, annexin V binding, and LPO were performed. The remainder of each separated fraction was cryopreserved for 4872 h. Post-thaw analyses of motion parameters, annexin V binding and LPO were repeated within 30 min.
Preparation of spermatozoa and basic semen analysis
For each sample, the fraction with high sperm motility was isolated using discontinuous Percoll (Sigma Chemical Co, St Louis, MO, USA) gradient separation (90 and 40% layers). Two ml of semen were carefully placed on a gradient top and centrifuged at 380 g for 20 min. The cell population pellet at the base of the 90% Percoll was collected and washed with human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) at 380 g for 10 min. The supernatant was discarded and the pellet was resuspended in HTF. No human serum albumin or other protein source was used in order to avoid confounding effects on LPO measurement as shown previously (Alvarez and Storey, 1983).
Sperm concentration and motion parameters were objectively evaluated with fixed parameter settings using the HTM-IVOS semen analyser (Hamilton Thorne Research, Beverly, MA, USA). Sperm concentration readings were manually monitored and corrections were made as appropriate. Sperm parameters were assessed according to the World Health Organization (World Health Organization, 1999) criteria, except for sperm morphology, which was examined according to strict criteria (Kruger et al., 1988
). The concentration of motile spermatozoa, percentage of progressive motility, average path velocity (VAP, µm/s) and linearity were determined as motion parameters in these experiments. The post-thaw motion parameters were assessed in the freezing medium described below.
Cryopreservationthawing technique
The technique for cryopreservationthawing has been previously described (Morshedi 1996; Srisombut et al., 1998
). Briefly, the motile sperm suspensions were mixed slowly with freezing medium containing TES-TRIS citrate and 20% egg yolk, with 12% glycerol as cryoprotectant (TYB-G) at a final concentration of 4.5% (Irvine Scientific) in a drop-wise fashion until a 1:1/2 volume ratio of sperm suspension to freezing medium was attained in 10 min. Aliquots were then placed in 2 ml cryo vials at a volume of 0.4ml/vial, and refrigerated at 4°C for 1 h. Subsequently, the vials were immersed in liquid nitrogen vapour for 30 min before finally being plunged in liquid nitrogen at 196°C for a storage period of 4872 h. The specimens were thawed in a water bath at 40°C for 3 min.
Detection of membrane phosphatidylserine translocation
During early stages of membrane disturbance phosphatidylserine is translocated to the outer portion of plasma membrane, eventually becoming available for binding to annexin V. In the present experiments we used Annexin V Cy3.18 (Apoptosis Detection Kit; Annexin V-Cy3; Sigma, USA). In order to differentiate between live cells with and without phosphatidylserine translocation and necrotic cells, we used 6-carboxyfluorescein diacetate (6-CFDA) in combination with Annexin V-Cy3. The non-fluorescent 6-CFDA enters the cell and it is converted to the fluorescent compound 6-carboxyfluorescein (6-CF). This conversion is a function of the esterases present only in living cells. Thus, no fluorescence can be observed in the necrotic (dead) cells.
By fluorescence microscopy, 6-CF is observed as green fluorescence and Annexin V-Cy3 as red. Three patterns of fluorescence are observed: (i) normal cells (annexin V, live) that stain only with 6-CF (green); (ii) cells with translocation of membrane phosphatidylserine (annexin V+, live) that stain with both 6-CF (green) and Annexin V-Cy3 (red); and (iii) necrotic or dead cells (annexin V+, dead) that stain only with Annexin V-Cy3 (red).
A total of 25 µl of each sperm suspension of highly motile spermatozoa at concentration of 10x106 cells/ml were placed on a poly-L-lysine-coated slide and stained with 6-CFDA/Annexin V-Cy3 solution in the presence of calcium. After 10 min incubation in the dark an anti-quench solution of immersion oil was used to minimize the loss of fluorescence. The slide was then covered with a 24x50 mm cover slip and immediately read blindly by two investigators at a magnification of x600 by epifluorescence microscopy with an ultraviolet filter. Our laboratory has reported an intra-observer variability of <6% and inter-observer variability of <3% for the technique (Barroso et al., 2000; Duru et al., 2000
; 2001a
).
Lipid peroxidation
The purified sperm fractions with high motility at a concentration of 20x106spermatozoa/ml were assessed for LPO pre-freezing and post-thaw using the LPO-586 assay Kit (LPO-586; Byotech SA, Portland, OR, USA) according to the manufacturer's instructions. The assay involves a spectrophotometric endpoint and is capable of detecting both malondialdehyde (MA) and 4-hydroxyalkenals (4HA). The amount of LPO in the pre-freezing samples was evaluated in parallel aliquots with and without TYB-G. This was performed due to the fact that in preliminary experiments we observed LPO upon incubation of TYB-G for 30 min at room temperature (data not shown). The amount of MA and 4HA in pre-freeze and post-thaw samples was also performed following incubation of the purified populations of motile sperm with and without a ferrous ion promoter (0.64 mmol ferrous sulphate plus 20 mmol ascorbate) used to stimulate the LPO cascade (Gomez et al., 1998) in Ca2+- and Mg2+-free HBSS (Hank's balanced salt solution). In the post-thaw samples, sensitization with the ferrous ion promoter was performed following a double step washing at 380 g for 10 min (Alvarez and Storey, 1992
) in order to remove the TYB-G.
Statistical analysis
Non-parametric paired (Wilcoxon signed-rank) and non-paired (MannWhitney) tests, and Spearman's rank correlation analyses were used as appropriate. P values 0.05 were considered significant. Statistical computations were calculated using SPSS 10 for Windows software (SPSS Inc, Chicago, IL, USA).
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Results |
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Results of pre-freeze and post-thaw motion parameters as well as cryosurvival rates are given in Table I. In both groups (patients and controls), and after separation of the fractions with high sperm motility (90% Percoll layers), all post-thaw motion parameters (except linearity) were significantly lower than the corresponding pre-freezing values. The cryosurvival rate (expressed as post-thaw/pre-freeze ratio of percentage of motility) did not differ between patients and donors.
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Discussion |
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Recently, the clinical use of intrauterine insemination-ready processed samples (i.e. following wash or gradient separation of the motile sperm fraction) has been advocated (Sharma and Agarwal, 1996; Larson et al., 1997
; Morshedi et al., 2001
). Consequently, we elected to test the effect of freezing on LPO and membrane integrity in purified populations of highly motile spermatozoa of patients and donors. According to Gomez et al. (1998) the use of the commercial LPO-586 kit in motile sperm fractions offers the most sensitive and reliable method to assess LPO, including the lack of dependency of removal of contaminating leukocytes (Gomez et al., 1998
).
Our results showed that pre-freeze LPO was significantly lower in donors than in patients, which might indicate better sample quality in this group. Cryopreservationthawing appeared to increase LPO but the differences were not significant (in patients and donors) when corrected for the effect of TYB-G in the spontaneous and iron-catalyzed measurements.
Our finding of no difference in LPO levels between pre-freeze and post-thaw human spermatozoa is in agreement with the results of Wang et al. (Wang et al., 1997a, b
) but contrary to those of Bell et al. (Bell et al., 1993
). The former reported no increase in LPO when comparing pre-freeze samples without TYB-G and post-thaw levels following a washing step to remove TYB-G. It has also been shown (Huszar and Vigue, 1994
) that a washing procedure of up to 3 cycles decreased sperm motility but had no effect on LPO measurements.
In agreement with earlier reports (Wang et al., 1997a, b
), we also found no significant correlation between pre-freeze morphology or LPO and post-thaw motility loss. Engel et al. found a negative correlation between motility loss and LPO in fresh human spermatozoa, attributing the decreased maintenance of motility to an enhanced LPO (Engel et al., 1999
).
Alvarez and Storey (1992) hypothesized that sperm LPO is enhanced by cryopreservation and this enhancement was mediated at least in part through loss of peroxidation protective enzyme activity. Nevertheless, these authors concluded that peroxidative contribution is negligible and that the membrane stress contribution to the cryodamage is substantial (Alvarez and Storey, 1993). According to Giraud et al. (2000), cryopreservation was associated with a loss of plasma membrane fluidity in human spermatozoa, and a higher recovery rate of motile or viable sperm cells was associated with higher membrane fluidity in the fresh sample (Giraud et al., 2000
).
In other cell systems (neuronal and glial cells), changes in lipid composition do not prevent the low temperature-induced changes in membrane structure. On the contrary, they may permit reversibility of these membrane-structure changes and counteract their potentially deleterious effect (Azzam et al., 2000).
It has been reported that annexin V binding combined with propidium iodide staining is more sensitive in detecting a deterioration of membrane functions than the vital stain propidium iodide alone (Glander and Schaller, 1999). Propidium iodide is normally a membrane-impermeable dye. However, dead cells lose their resistance to the influx of propidium resulting in intracellular staining. In our study, we used annexin V in combination with 6-CFDA; this compound is converted to 6-CF by esterases in the living cells, which allows for a clear distinction under epifluorescent microscopy of live-normal cells, live cells with altered membranes (here depicting phosphatidylserine translocation) and necrotic cells. We observed that annexin V stained the entire spermatozoon (head, mid-piece and flagellum) in more than 90% of the annexin V+ live cells on a slide (data not shown). We postulate that phosphatidylserine is therefore translocated to the outer leaflet at different domains of the cell membrane. In other cell systems, such a phenomenon is considered an early sign of programmed cell death (Koopman et al., 1994
; Martin et al., 1995
; Vermes et al., 1995
).
Our results have confirmed and extended our previous observations relating to the increased percentage of annexin V+, live cells following freezingthawing (Duru et al., 2001a, b
). These cells are alive, but they demonstrate a membrane alteration with a perturbed transbilayer asymmetry evidenced by externalization of phosphatidylserine. Additionally, the cryopreservationthawing process also induced necrosis (cell death). Therefore, freezingthawing significantly reduced the percentage of normal cells. Whether this is true also for different cryopreservation techniques and for samples from infertile men with more severe degrees of oligoasthenoteratozoospermia needs to be evaluated.
It has been suggested that an aminophospholipid translocase mediates specifically a rapid ATP-dependent translocation of aminophospholipids from the exoplasmic to the cytoplasmic leaflet (Muller et al., 1999). In cryopreserved ram spermatozoa, the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. Furthermore, in this system, post-thaw activity of the putative aminophospholipid translocase was significantly reduced in intact cells (Muller et al., 1999
). If the activity of this putative enzyme is inhibited in cryopreserved human spermatozoa, an increased externalization of phosphatidylserine is to be expected.
The inner leaflet location would make phosphatidylethanolamine (PE) and phosphatidylserine vulnerable to initiation of peroxidative attack from active intracellularly-reactive oxygen species (Storey, 1997). Considering that cryopreservationthawing is associated with an increase of phosphatidylserine externalization, we might speculate that these phospholipids will not be susceptible to peroxidative damage.
In summary, freezingthawing of motile spermatozoa of patients and donors did not result in a significant increase of LPO but was associated with plasma membrane translocation of phosphatidylserine and severe impairment of motion parameters. Sperm membrane change induced during cryopreservationthawing may be therefore related to membrane stress induced by the freezethaw process rather than oxidative stress. The significant correlation between pre-freeze LPO and post-thaw induction of phosphatidylserine externalization found in the control group may reflect the presence of a sub-population of dysfunctional sperm among the separated fractions of highly motile spermatozoa.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 30, 2001; accepted on June 28, 2001.