University of Padova, Department of Medical and Surgical Sciences, Padova, Italy
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Abstract |
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Key words: calcium/cryopreservation/membrane potential/progesterone/spermatozoa
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Introduction |
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In this study we have evaluated the effects of freezing and thawing procedures on progesterone-induced [Ca2+]i, plasma membrane potential variations and acrosome reaction induction in human spermatozoa from fertile donors.
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Materials and methods |
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Sperm cryopreservation
Sperm aliquots to be frozen were diluted (1:1, v:v) with human sperm cryopreservation medium (Irvine Scientific, Santa Ana, CA, USA). The diluted aliquots were drawn into 0.25 ml sterile plastic straws (130x2 mm, CryoBiosystem, L'Aigle Cedex, France) and placed in the chamber of a programmable biological freezer (Biomed Kryo 10/1.7 III, Planer Bloxed, Sunbury, UK) and cooled with the freezing programme described previously (Serafini and Marrs, 1986). In brief: from room temperature to 5°C at a rate of 0.5°C/min; from 54°C at a rate of 1°C/min; from 43°C at a rate of 2°C/min; from 32°C at a rate of 4°C/min; from 21°C at a rate of 8°C/min; from 1°C to 80°C at a rate of 10°C/min. Sperm straws were then maintained at this temperature for 10 min and then plunged into liquid nitrogen (196°C) for 60 min.
Thawing procedure
Sperm samples were maintained in liquid nitrogen for 60 min and then thawed by placement of the straws upright on a rack at room temperature until all visible ice was gone.
Sperm preparation
The non-frozen sperm aliquot from each donor remained in seminal plasma at room temperature until the frozen aliquot was placed into liquid nitrogen. After this time and after thawing of frozen spermatozoa, motile cells from each aliquot were isolated by the swim-up technique as previously described (Foresta et al., 1992). Before and after isolation, motile spermatozoa were washed in BiggersWhittenWhittingham (BWW) medium containing (in mmol/l): 95 NaCl, 4.8 KCl, 1.7 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 20 HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), 5.6 fructose, 0.25 sodium pyruvate, 3.7 ml/l sodium lactate syrup (60%) to pH 7.4 and resuspended at a concentration of 10x106 spermatozoa/ml. Aliquots of fresh and thawed spermatozoa isolated as above were incubated in the absence and presence of progesterone (1.0 µg/ml) for 120 min at 37°C in a controlled atmosphere (5% CO2 and 95% O2). After incubation sperm samples were evaluated for motility, viability and acrosome reaction.
Acrosome reaction evaluation
Sperm aliquots from fresh and thawed sperm suspensions were retrieved before and after incubation with progesterone in the different experimental conditions as described above. After fixation with formaldehyde the percentage of acrosome reacted spermatozoa was assayed using an indirect fluorescence technique with FITCconjugated lectins from pisum sativum which selectively binds to intact acrosome (Morales and Cross, 1989). Then non-fluorescent spermatozoa were scored as acrosome reacted while fluorescent spermatozoa were scored as acrosome intact. Two hundred spermatozoa were scored in each sample to evaluate the percentage of acrosome reacted spermatozoa.
Measurement of spermatozoa [Ca2+]i
[Ca2+]i was measured utilizing the fluorescent probe fura-2/AM (Foresta et al., 1996): spermatozoa isolated as above were incubated for 30 min at 37°C in the presence of fura-2/AM (2 µmol/l). After loading, spermatozoa were washed to remove extracellular fura-2/AM by centrifugation at 800 g for 10 min, resuspended in BWW and maintained at room temperature until used. One ml sperm aliquots were used to measure [Ca2+] in a LS50B Perkin Elmer fluorometer equipped with a thermostat and magnetically-stirred cuvette holder (Perkin Elmer, Norwalk, CT, USA). The excitation wavelength was alternated between 350 and 380 nm and emission fluorescence was continuously monitored at 505 nm.
Evaluation of sperm plasma membrane potential changes
Sperm plasma membrane changes were monitored utilizing the potential sensitive fluorescent dye bis-oxonol as previously described (Foresta et al., 1996). Briefly, 1.5x106 spermatozoa were placed in a cuvette containing the bis-oxonol solution (200 nmol/l) in BWW at 37°C. After stabilization of the fluorescent signal, progesterone was added to sperm suspension at the final concentration of 1.0 µg/ml. Excitation and emission wavelengths were 540 and 580 nm respectively.
Statistical analysis
Experimental data were analysed using the Stat View II (Abacus Concepts, Berkeley, CA, USA) statistical package. Data are reported as mean ± SD of 10 separate experiments performed in triplicate. Statistical analysis was carried out using analysis of variance (ANOVA) and Student's t-test. A P value of < 0.05 was chosen as the limit for statistical significance.
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Results |
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Discussion |
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In this study we have evaluated the effects of freezingthawing procedures on the progesterone-induced effects in human spermatozoa. The results of this study demonstrated that human sperm cryopreservation modified the ability of spermatozoa to respond to progesterone in terms of [Ca2+]i increase, plasma membrane depolarization and acrosome reaction induction. In fact in frozenthawed spermatozoa, the [Ca2+]i rise induced by this steroid was significantly lower than that observed in fresh spermatozoa. Furthermore freezingthawing procedures deeply modified the action of progesterone on plasma membrane potential variations blunting the depolarizing effects of this steroid in all thawed sperm samples. The lack of any depolarizing effect of progesterone in human spermatozoa after freezing and thawing procedures was not due to sperm plasma membrane collapse since gramicidin D was promptly able to depolarize it. Cryopreservation induced also a reduction of the percentages of sperm acrosome reaction induced by progesterone, probably due to a reduction of the effects on [Ca2+]i rise and plasma membrane depolarization observed in frozenthawed spermatozoa after progesterone addition. These results are in agreement with that previously demonstrated, i.e. that both [Ca2+]i rise and Na+-dependent plasma membrane depolarization are important events in the biological pathway leading to acrosome reaction induced by progesterone in human spermatozoa (Foresta et al., 1993; Garcia and Meizel, 1996
). The reasons for these effects are not known but it is possible that the freezingthawing procedures may have induced damage to the sperm plasma membrane leading to a reduction of Ca2+ influx and the inhibition of Na+ influx followed by plasma membrane depolarization. Thus, in agreement with the existence of at least two progesterone receptors on human sperm plasma membrane, one coupled to Ca2+ influx and [Ca2+]i rise and the other coupled to Na+ influx and plasma membrane depolarization (Foresta et al., 1993
, 1995
; Sabeur et al., 1996
), it could be postulated that the Na+ channel or the progesterone-receptor coupled to its activation is more sensitive to freezingthawing procedures than that coupled to Ca2+ influx activation. The small increase in acrosome reaction percentages induced by progesterone in frozenthawed spermatozoa (although not significantly different from controls) may be due to the small rise of [Ca2+]i induced by the steroid, and that might have been sufficient (Foresta et al., 1993
) for acrosome reaction induction in a small number of spermatozoa. Another possibility is that, in a small number of spermatozoa, progesterone may also have induced a plasma membrane depolarization that could not be detected by our system.
The reduction of progesterone induced [Ca2+]i rise observed in spermatozoa after freezingthawing may be due (i) to the lack of voltage-operated calcium channels (VOCC) activation induced by progesterone-dependent plasma membrane depolarization that can be observed only in fresh spermatozoa and is completely absent in thawed spermatozoa or (ii) to the lack of Ca2+ influx through a Na+-permeable channel that is not functional in thawed spermatozoa and that is permeable also to Ca2+ (Foresta et al., 1993). Further studies are needed to confirm these hypotheses.
All these data put together demonstrate that cryopreservation modifies the responsiveness of human spermatozoa to putative physiological sperm activators such as progesterone. These results could explain the reduced fertilizing potential of frozenthawed spermatozoa. At present it is difficult to understand what the precise effects of freezingthawing procedures on human spermatozoa are but it is possible to postulate that cryopreservation alters plasma membrane receptors for progesterone thus modifying sperm responsiveness to this steroid. Further studies will be necessary to evaluate the effects of cryopreservation on human sperm functions and to develop new strategies for freezing and thawing that preserve the functional integrity of spermatozoa.
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Notes |
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References |
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Submitted on March 13, 2000; accepted on May 19, 2000.