Human Reproduction vol.6 no.3 pp.401-404, 1991
Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction
Patricio Morales1, David Vantman2, Claudio Barros and Pilar Vigil Unit of Reproduction and Development, Faculty of Biological Sciences, P. Catholic University of Chile, P.O. Box 114-D, Santiago, Chile and 2Mother and Child Institute, University of Chile, Paula Jaraquemada Hospital, Santiago, Chile 'To whom correspondence should be addressed
Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swimup, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swimup and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 107 spermatozoa/ml, the suspensions were incubated at 37°C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P < 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method. Key words: Percoll/swim-up/human reaction/human zona pellucida
spermatozoa/acrosome
Introduction Several techniques have been suggested as procedures for selecting motile spermatozoa, including albumin gradients (Ericsson, 1977; Broer and Dauber, 1978; Koper et al., 1979), © Oxford University Press
Percoll gradients (Gorus and Pipeleers, 1981; Forster etal., 1983; Lessley and Gamer, 1983; Bolton and Braude, 1984; Kaneko et al., 1986; Suarez et al., 1986; Siiteri et al., 1988), swim-up procedures (S-U) (Drevius, 1972; Lopata et al., 1976; Overstreet et al., 1980; Aitken, 1988), and glass wool filtration (Paulson and Polakoski, 1977; Paulson et al., 1979; Jeyendran et al., 1986). A practical method is designed to give a high yield of motile spermatozoa as well as enhanced motility. Another important feature in the assessment of these techniques is whether or not they tend to select the functionally normal and most competent spermatozoa. Several reports have indicated that the Percoll method permits the recovery of a high number of motile spermatozoa (Berger etal., 1985; McClure etal., 1989). In addition, it has been reported that the cells selected by this method were more fertile than swim-up selected spermatozoa (Bongso et al., 1989). However, Lannou and Blanchard (1988) did not find differences in the nuclear maturity or in the morphology of the spermatozoa selected by either method. In the present report we compared the fertilizing ability of the spermatozoa selected by swim-up, (S-U) or Percoll gradient (P) methods. The parameters compared were: (a) sperm number and motility; (b) percentage of sperm recovery; (c) percentage of acrosome reactions (either spontaneous or induced by human follicular fluid, HFF); (d) number of spermatozoa able to bind to the human zona pellucida (HZP); and (e) number of spermatozoa able to fuse with the zona-free hamster oocytes (ZFHO). It was concluded that although the percentage of sperm recovery was higher with Percoll gradient than with the swimup method, both procedures produced functionally normal and equivalent cells.
Materials and methods Semen Normal semen samples were obtained from fertile men attending the Centre for the Study of Reproductive Biology (CEBRE), of the P.Catholic University of Chile. The samples were used within 1 h of collection. Each semen sample was divided into two aliquots of equal volume. One aliquot was processed by the S - U method and other aliquot was processed by the P method. Swim-up The semen sample was washed with 4 ml of modified Tyrode's medium (Suarez et al., 1986) supplemented with 3 mg/ml of bovine serum albumin (mTyr-3B, Sigma) by centrifugation for 10 min at 600 g. After discarding the supernatant, the pellet was resuspended in 1.5 ml of mTyr-3B and centrifuged for 5 min 401
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at 300 g. The supernatant was carefully removed and the pellet was covered with medium supplemented with 26 mg/ml bovine serum albumin (mTyr-26B). At the end of 1 h the upper fraction, containing the motile cells, was collected in 0.5 ml of mTyr-26B and the concentration of spermatozoa was determined and adjusted to 1 x 107 spermatozoa/ml. The tubes were incubated at 37°C in an atmosphere of 5% CO2 in air. All the media were filtered through a filter of 0.22 /tm pore size before use. The percentage of motile spermatozoa was determined at the beginning of the incubation (TO), after 5 h of incubation (T5), and after 24 h of incubation (T24).
on its effectiveness in inducing acrosome reactions. The spermatozoa were incubated with the supravital dye H258 (Hoechst 33258, Sigma) to label dead cells, and with the FITCconjugated lectin, Pisum sativum agglutinin (PSA) (Vector Laboratories, Inc) to label the acrosome. H258 and FITC-PSA fluorescences were inspected using an Olympus BH2 microscope. For each spermatozoa, the presence or absence of acrosomal fluorescence and the presence or absence of H258 supravital staining was noted (Cross et al., 1986). In each experiment the effect of HFF was tested in duplicate.
Percoll gradient
Results
The P gradient was prepared as described previously (Siiteri et al., 1988). Briefly, this consisted of a lower layer of 80% Percoll (Sigma) and an upper layer of 40% Percoll. The semen was deposited on top of the upper layer and centrifuged for 20 min at 300 g. The pellet was washed twice in mTyr-3B by centrifugation for 10 min at 300 g. The final pellet was diluted with 0.5 ml of mTyr-26B and the sperm concentration was determined and adjusted to 1 x 107 spermatozoa/ml, as above. The tubes were incubated at 37 °C in an atmosphere of 5% CO2 in air. The percentage of motile spermatozoa was determined as above.
The concentration of motile spermatozoa and the percentage of spermatozoa recovered were higher when the P method was used than when the S - U method was used for selection (Table I). On the other hand, the percentage of motile spermatozoa at TO, T5, and T24 was not different between these two sperm suspensions (Figure 1). Both sperm populations, selected by the P or the S - U method, were equally able to undergo the AR. The percentage of AR spermatozoa, either spontaneous or induced by HFF, was not different between these two groups at any of the time intervals studied (Figure 2). There was a consistent increase in the percentage of spontaneous AR throughout the in-vitro incubation period, the percentage of AR spermatozoa at T24 being significantly higher than at TO (P < 0.05). At T24, HFF was able to increase the percentage of AR spermatozoa from 15 ± 1 (mean ± SEM) to 34 ± 3 in the P selected spermatozoa, and from 13 ± 1 to 33 ± 3 in the S - U selected spermatozoa (P < 0.001, Figure 2).
Oocyte recovery Hamster oocytes were recovered from superovulated female golden hamsters (Yanagimachi et al., 1976). Cumuli and zonae were removed with 0.1% hyaluronidase (Sigma) and 0.1% trypsin (Sigma), respectively, in mTyr-3B, followed by rinsing in mTyr-26B. A 100 yl sample of 4 h capacitated spermatozoa Was placed under mineral oil in a plastic Petri dish and 20 or more ZFHO were introduced into the sperm suspension. The concentration of the sperm suspension had been adjusted to 1 X 107 spermatozoa/ml. The preparations were incubated at 37°C with 5% CO2 for 4 h. The oocytes were then removed and rinsed in mTyr-3B and evaluated for the presence of swollen sperm heads using phase-contrast microscopy. Human oocytes were dissected from ovarian tissue from cadavers and stored at — 80°C as previously described (Cross et al., 1986). After thawing, the oocytes were freed of remaining cumulus cells by passing them through a narrow bore pipette. As a result of freezing and thawing these oocytes were not viable. A 100 /tl sample of 4 h capacitated spermatozoa was placed under mineral oil in a plastic Petri dish and 3 - 4 human oocytes were introduced into the sperm suspension. The sperm suspension had been adjusted to 1 X 107 spermatozoa/ml. The preparations were incubated at 37CC with 5% CO2 for 4 h. The oocytes were then removed and rinsed in medium mTyr-3B and the number of spermatozoa bound to the HZP was evaluated using phasecontrast microscopy. Induction and detection of acrosome reactions The procedure to induce and detect the acrosome reaction (AR) has been published in detail elsewhere (Cross et al., 1986, 1988; Fukuda et al., 1989). Briefly, to induce acrosome reactions the sperm suspensions were diluted with HFF (20% v/v) and incubated for 15 min. HFF was selected prior to the study based 402
Table I. Sperm characteristics before and after selection using Percoll gradient or swim-up procedure Sperm characteristics
Semen
Swim-up
Percoll
Number (x 10°/ml) % Motile Number of motile (x 10*/ml) % Recovery/total cells % Recovery/motile cells
173 ± 30 54 ± 4 102 ± 2
19 89 17 9 21
52 84 40 29 44
± ± ± ± ±
5 2 5 2 5
12* ± 3 ± 10* ± 5* ± 7
The data are the mean ± SEM of 12 experiments. 'Significantly higher than the corresponding value for the swim-up (P < 0.001).
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«
H
tu ^ tU
M
o TO
TS
T24
TIME (Hr) Fig. 1. Percentage of motile spermatozoa during in-vitro incubation of human spermatozoa selected by Percoll gradient or swim-up procedures. Results are the mean ± SEM of 12 experiments.
Swim-up vs. Percoll selected spermatozoa
The P and S - U selected sperm suspensions were further examined to evaluate their ability to bind to the HZP and to fuse with the ZFHO membranes. After 5 h of in-vitro capacitation, the number of spermatozoa bound to the HZP was 150 ± 27 and 163 ± 31 (mean ± SEM) with spermatozoa selected by S - U and P methods, respectively (Figure 3A). The percentage of zona-free hamster oocytes penetrated was 29 ± 4 and 28 ± 3 with spermatozoa selected by S - U and P, respectively (Figure 3B). In addition, the number of spermatozoa penetrated per oocyte was not significantly different between the two samples (1.3 ± 0.2 for the P selected spermatozoa versus 1.5 ± 0.3 for the S - U selected spermatozoa).
I I rntcon E 2 swiM-ur
T24 Control
T24 hFF
TIME (Hr)
Fig. 2. Percentage of spontaneous or induced acrosome reactions during in-vitro incubation of human spermatozoa selected by Percoll gradient or swim-up procedures. At T24 the acrosome reaction was stimulated by incubation with human follicular fluid (HFF). Results are the mean ± SEM of 12 experiments.
S-U
I" £
10
S-U
Fig. 3. A Number of sperm bound to the human zona pellucida of sperm suspensions selected using Percoll (P) gradient or the swimup (S—U) method. Results are the mean ± SEM of six experiments. B Percentage of zona-free hamster eggs penetrated by human sperm selected by P gradient or S - U method. Results are the mean ± SEM of 12 experiments.
Discussion The present results show that the P method produced the recovery of a higher number of both total and motile spermatozoa than the S - U method. The reason for this difference is still unclear but may be related to the fact that during P selection, centrifugal forces are applied to the sample. This is in contrast to the S —U technique where the motile cells have to 'rise' for themselves to the upper medium. Our results are in agreement with those published previously (Berger et al., 1985; McClure et al., 1989) and together suggest the use of the P method in those cases of oligozoospermic semen samples. In our study, however, the percentage of motile cells recovered by both techniques was not significantly different. In this study we have also compared the fertilizing ability of the sperm samples selected with the P and with the S - U methods. The functional competence of the sperm cells was evaluated by a series of biological assays that comprise the whole sequence of events involved in mammalian fertilization. These events, in chronological order, may be: sperm—zona pellucida binding, sperm acrosome reaction on the zona surface, and sperm fusion with the oocyte plasma membrane (Yanagimachi, 1988). These events were evaluated by analysing, respectively, the number of spermatozoa bound to the HZP, the percentage of AR spermatozoa, and the number of spermatozoa present in the vitellus of the ZFHO. It must be pointed out that in these experiments we determined not only the AR that took place spontaneously, but also the AR induced by a physiological inducer of the reaction such as HFF (Suarez et al., 1986; Fukuda et al., 1989). The earliest work demonstrating the ability of HFF to induce human sperm AR was carried out using Percoll selected spermatozoa (Suarez et al., 1986; Yudin et al., 1988). Washed, unselected sperm suspensions (Fukuda et al., 1989) or spermatozoa selected by the S—U procedure (Stock et al., 1989) were unable to respond to the HFF stimulus with an AR. Here, we present direct evidence suggesting that P and S —U both produce functionally normal and equivalent spermatozoa. This suggestion is based on the observation that both sperm populations were equally capable of binding to the HZP, undergoing the AR, and fusing with the ZFHO membranes. These results argue against a functional modification of the sperm cells due to their passage through the Percoll gradient (Fukuda et al., 1989) and suggest that Percoll may not interact with the sperm plasma membrane. In these experiments we evaluated spermatozoa from semen that was normal by clinical criteria. Different results have been reported for spermatoza from abnormal semen. It has been suggested that Percoll-separated spermatozoa from subfertile men have enhanced capacity to penetrate ZFHO (Serafini et al., 1990) and to fertilize in vivo (Pardo et al, 1989) and in vitro (Hyne etal., 1986). At the present time we do not have sufficient information to explain these discrepancies, although they may be related to the recovery of a higher total number of functional cells with P than with S-U. In summary, we have demonstrated a similar response of the P and S - U selected cells to the tests designed to measure the functions of human spermatozoa required for fertilization. These observations may not hold true in cases of abnormal semen samples. 403
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Acknowledgements We wish to thank Professor Dr Horatio B.Croxatto for his help with the preparation of the manuscript. This study was supported by research grants RF 85049/34 and FONDECYT 0823/90 to PM.
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