Hum. Reprod. Advance Access originally published online on September 1, 2007
Human Reproduction 2007 22(11):2947-2955; doi:10.1093/humrep/dem273
Phagocytosis of human post-capacitated spermatozoa by macrophages
1 Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel 2 Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA
3 To whom correspondence should be addressed. Tel: +972-8-9343923; Fax: +972-8-9472722; E-mail: m.eisenbach{at}weizmann.ac.il
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Abstract |
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BACKGROUND: Earlier studies demonstrated that macrophages phagocytize spermatozoa in the female genital tract of mammals. In spite of this phagocytosis, fecundity is not affected, raising questions of how the resulting decrease in the number of spermatozoa does not reduce the fertilization rate and of the role of this phagocytosis. We hypothesized that its role is to rid the female genital tract of spermatozoa past their fertilizing stage (post-capacitated spermatozoa). Here we examined whether, indeed, phagocytosis is restricted to post-capacitated spermatozoa.
METHODS: Spermatozoa were incubated for 22 h either in a medium that allows them to become capacitated and then post-capacitated, or in a medium that prevents them from acquiring these states. These sperm populations were compared for their susceptibilities to macrophage phagocytosis.
RESULTS: Phagocytosis was significantly higher (P << 0.001) in the sperm population containing post-capacitated spermatozoa. Vitality, motility, the acrosomal status and the proportion of capacitated cells did not affect phagocytosis.
CONCLUSION: Post-capacitated spermatozoa are, probably, preferentially phagocytized by macrophages.
Key words: acrosome reaction/macrophages/post-capacitated sperm/sperm capacitation/sperm phagocytosis
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Introduction |
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A long-standing question in mammalian reproduction is why spermatozoa that enter the female genital tract are attacked and phagocytized by leukocytes and epithelial cells (Austin, 1957
; Bedford, 1965; Chakraborty and Nelson, 1975; Phillips and Mahler, 1977a,b; Tyler, 1977; Pandya and Cohen, 1985; Woelders and Matthijs, 2001; Suarez, 2002). This question is especially intriguing for two reasons. First, this phagocytosis apparently does not interfere with fecundity. No loss of fertilizing ability was observed when female rabbits, whose vaginas had been induced by an earlier mating to contain leukocytes, were mated with a second male (Taylor, 1982). Second, successful fertilization depends, among other factors, on the number of motile spermatozoa (Olds-Clarke, 1996); one could, therefore, expect that the female genital tract would protect and preserve the motile spermatozoa residing in it, rather than reduce their number by phagocytosis.
In an attempt to reconcile these apparently conflicting facts, a hypothesis was put forward, according to which the role of phagocytosis is to selectively remove, in a ‘silent and clean’ way, spermatozoa that have finished being functional, i.e. ended being capacitated (Eisenbach, 2003). Capacitated spermatozoa are cells that reached the maturation stage allowing them to bind to the oocyte, penetrate and fertilize it (Jaiswal and Eisenbach, 2002; De Jonge, 2005). The ‘problem’ is that the capacitated state is short [only 1–4 h in humans in vitro (Cohen-Dayag et al., 1995)] and, subsequent to being at this state, such spermatozoa have no role. Therefore, like any other superfluous cells, these post-capacitated spermatozoa should be removed. The hypothesis (Eisenbach, 2003) thus conferred an important role on sperm phagocytosis and, at the same time, explained how it does not interfere with fertilization. In spite of being an appealing hypothesis, there has been no evidence in the literature to support or negate it. The aim of the current study was to put this hypothesis to the test and examine its validity.
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Chemicals and media
The medium that allows capacitation, termed medium A, was essentially Biggers–Whitten–Whittingham medium—95 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 20 mM sodium lactate, 5 mM glucose and 0.25 mM sodium pyruvate, pH 7.4 (Biggers et al., 1971
), supplemented with HEPES (50 mM, pH 7.4) and 0.3% human serum albumin (Irvine Scientific, Santa Ana, CA, USA). The medium that does not allow capacitation and, therefore, prevents the formation of post-capacitated spermatozoa, termed medium B, was medium A from which components required for capacitation [CaCl2, NaHCO3 and human serum albumin (Yanagimachi, 1994)] were omitted. The medium used for macrophage growth was RPMI (Gibco; Invitrogen Corporation, Paisley, UK), supplemented with 1% serum from human male AB plasma (Sigma Medical Company, St. Louis, MO, USA), 2 mM pyruvate (Biological Industries, Beit Haemek, Israel), 2 mM glutamine (Biological Industries) and a mixture of 1% essential amino acids (Biological Industries). Sperm Freezing Medium was obtained from MediCult (Jylinge, Denmark), DIFF-QUIK staining kit from Dade Behring (Eschborn, Germany), Dulbecco's phosphate-buffered saline from Gibco, Ficoll from Amersham Biosciences (Uppsala, Sweden), boric acid from Merck (Darmstadt, Germany), Pisum sativum agglutinin labeled with fluorescein isothiocyanate (PSA-FITC), as well as Trypan Blue, A23187
[GenBank]
, eosin Y, nigrosin, poly-D-lysine (PDL), and all other chemicals, for which the source was not mentioned, were from Sigma.
Sperm preparation
Human semen samples were obtained from healthy donors after three days of sexual abstinence. Semen samples with normal sperm density, motility and morphology [according to WHO guidelines (World Health Organization, 1993)] were allowed to liquefy for 30–60 min at room temperature. Semen samples were divided into two portions; one portion was washed twice (120 x g, 20 min, room temperature) with medium A, and the other was similarly washed with the medium B. The sperm concentration was adjusted to 5 x 106 cells/ml (for capacitation assays), 107 cells/ml (for motility assays) and 3 x 107 cells/ml (for phagocytosis and vitality assays), followed by incubation at 37°C under an atmosphere of 5% CO2 for the indicated time periods.
In the experiments with seminal fluid, described in the Appendix, the semen samples were first centrifuged (without addition of any medium) at 120 x g for 10 min; under these conditions only a fraction of the sperm population are precipitated. The supernatant, containing seminal fluid and spermatozoa, was adjusted to a sperm concentration of 5 x 106 cells/ml (for capacitation assays) and 3 x 107 cells/ml (for phagocytosis and vitality assays) in medium A. The pellet of spermatozoa was washed twice in medium A as described above. Both sperm samples, the one obtained from the supernatant (i.e. spermatozoa suspended in a seminal fluid-containing medium) and the one obtained from the pellet (i.e. spermatozoa suspended in a seminal fluid-free medium), were incubated at 37°C under an atmosphere of 5% CO2 for the indicated time periods.
Determination of sperm motility
The sperm samples were analysed for the percentage of motile cells (from a population of 250 counted cells in each determination) using a Makler counting chamber (Sefi Medical Instruments, Israel) and a computerized-sperm analysis software program (Hobson Tracking System, UK). The measured kinetic parameters (Davis and Siemers, 1995; Mortimer et al., 1997) were: VCL, curvilinear velocity (the time-average velocity of sperm head along its actual trajectory); VSL, straight line velocity (also termed progressive velocity; the time-average velocity of sperm head along a straight line from its first position to its last position); LIN, percentage linearity (the ratio VSL/VCL x 100); STR, percentage straightness (the ratio between the straight line from the first point on the smoothed path to the last point on this path and the total distance along the smoothed path, multiplied by 100); MOT, the percentage of motile cells (calculated as the number of motile cells within the analysis field divided by the sum of the motile and immotile cells within this field, multiplied by 100) and HYP, the percentage of hyperactive spermatozoa [spermatozoa with a motility pattern characterized by increased velocity, decreased linearity, increased amplitude of lateral head displacement and flagellar whiplash movement (Yanagimachi, 1970; Burkman, 1990)].
Determination of the level of capacitated spermatozoa
The percentage of capacitated spermatozoa was determined from the difference between the levels of acrosome-reacted spermatozoa before and after an acrosome-reaction induction with the calcium ionophore A23187
[GenBank]
(Mortimer and Fraser, 1996; Jaiswal and Eisenbach, 2002). Acrosome-reacted spermatozoa were identified by the acrosomal marker PSA-FITC, essentially as described by Jaiswal et al. (1998, 1999) with the following few modifications. The sperm dry smears were washed with cold methanol and then incubated at room temperature with 50 µg/ml PSA-FITC for 30 min in a humid chamber. The washes of the slides with distilled water after this incubation and after incubation with 2% formaldehyde lasted much longer (15 min instead of a few seconds). Two hundred spermatozoa on each slide were examined for two patterns of fluorescence; a pattern with the acrosome completely fluorescent (acrosome intact) and a pattern showing only an equatorial fluorescent band (acrosome reacted). Rarely observed spermatozoa that were completely fluorescent or non-stained (dead cells) were not included in the counting.
Determination of sperm vitality
Sperm vitality was evaluated by the one-step eosin–nigrosin staining technique (Mortimer et al., 1990; Bjorndahl et al., 2003). The evaluation was carried out as described in the Manual on Basic Semen Analysis (NAFA and ESHRE-SIGA, 2002).
Sperm separation by chemotaxis
Sperm separation by chemotaxis for obtaining a sperm population enriched with capacitated cells was carried out as described by Cohen-Dayag et al. (1994, 1995), except that the chemoattractant used for the separation was progesterone (10–6 M in medium A) instead of follicular fluid [Note that we used a higher progesterone concentration than the one used by Teves et al. (2006) for chemotaxis assays because of the much higher distance between the wells in the separation chamber].
Sperm freezing and thawing
Semen samples were allowed to liquefy for 30–60 min at room temperature and washed twice with 10 ml medium A (120 x g for 20 min). The pellet was suspended in 1 ml medium A and divided into two portions. One portion was diluted with medium A to 30 x 106 cells/ml and incubated for 2 h under an atmosphere of 5% CO2 at 37°C. The other portion was frozen and thawed in a Sperm Freezing Medium according to the manufacture's instructions, as follows. For freezing, Sperm Freezing Medium was added dripwise to the spermatozoa (1:1 final v/v ratio) in a cryo-tube, and then sequentially incubated for 10 min at room temperature, for 30 min just above the surface of liquid nitrogen and for 30 min in liquid nitrogen. For thawing, the cryo-tube was kept under running water for 5 min and incubated for 10 min at room temperature. Non-motile spermatozoa were removed by a discontinuous Percoll gradient (95 and 47.5% Percoll), as described by Draveland and Mortimer (1985) and Jaiswal et al. (1998). The lower sperm layer, containing motile cells, was washed with (120 x g for 10 min) and resuspended in medium A.
Isolation and growth of macrophages
Macrophages were isolated and grown according to a protocol obtained from Proneuron Biotechnologies (Israel) (Knoller et al., 2005). Human peripheral blood was obtained from healthy donors. The blood was diluted 1:1 with PBS and was separated on Ficoll to obtain the mononuclear cells. The mononuclear cells were further separated on a Percoll gradient to obtain the monocyte-enriched fraction. Cell count and viability were evaluated using Trypan blue staining. Cells were transferred to 12-mm diameter coverslips in a 24-well culture dish (106 cells/ml). The coverslips were pretreated with 0.5% PDL (0.05 mM PDL in 0.1 M borate buffer, pH 8) for 24 h. The tissue culture dishes were kept in incubation under an atmosphere of 5% CO2 at 37°C.
Sperm phagocytosis assay
The growth medium in the macrophage-coated wells was discarded, and a sperm suspension in medium A or B (30 x 106 cells/ml) was added to each well. To improve sperm motility in the phagocytosis assay, the wells that contained spermatozoa in medium B were supplemented with human serum albumin (3 mg/ml, as is present in medium A). Following 30-min incubation under an atmosphere of 5% CO2 at 37°C (a time period insufficient for complete sperm phagocytosis, thus allowing the visualization of spermatozoa within the macrophages), the coverslip at the bottom of each well was removed, washed well with PBS to remove non-bound spermatozoa, and then treated, according to the manufacturer's instructions, as follows. The coverslip was sequentially washed in DIFF-QUIK fixative solution for 10 s, DIFF-QUIK stain solution I for 20 s, DIFF-QUIK solution II for 20 s and, finally, in distilled water for 10 s. The sample-containing coverslips were observed under a light microscope at x400 magnification, and the number of spermatozoa inside 200–700 macrophages in each treatment was counted.
Statistical analysis
All statistical analyses were carried out by employing InStat 3 and Prism 4 software packages (Graph Pad Software, USA). To test for the significance of the difference between phagocytosis in medium A and B, the Chi-square test was applied. To test the significance of the difference in phagocytosis of spermatozoa incubated in medium A for 0 and 22 h, the unpaired two-tailed t-test was applied. Because of the large variation between the responses of different sperm samples (even when obtained from the same sperm donor) and different macrophage samples, each test was first performed for each of the four experiments separately, followed by the Fisher's Combined Probability test (Fisher, 1932) for obtaining the combined P-value for all the experiments together. To test for the significance of the difference between the sperm vitality at the 4 and 22 h time points in medium B, the paired t-test was employed. In all other cases, the significance of the difference between medium A and B was tested by two-way ANOVA (factors: medium and time).
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Our working hypothesis, outlined above, predicts that a sperm population, enriched with post-capacitated cells, would be more susceptible to macrophage phagocytosis than would be a non-enriched sperm population. Our aim was, therefore, to obtain a sperm population enriched with post-capacitated cells, and to use it to test this prediction and, thereby, the validity of the hypothesis.
In vitro, in a medium that allows spontaneous capacitation, human spermatozoa undergo capacitation asynchronously, they remain capacitated for 1–4 h, and then they become post-capacitated—acrosome-intact but functionless (Cohen-Dayag et al., 1995; Eisenbach, 1999). The limited life span of the capacitated state coupled to the continuous replacement of spermatozoa at this stage results in a steady-state level of capacitated spermatozoa, maintained for many hours (Cohen-Dayag et al., 1995; Giojalas et al., 2004) as well as a continuously increasing level of post-capacitated spermatozoa (Fig. 1). We, therefore, incubated spermatozoa in a medium that allows capacitation (medium A) for relatively long time periods, thus producing a sperm population that, in all probability, is enriched with post-capacitated cells (Fig. 1). We compared its susceptibility to phagocytosis with that of another portion of the same sperm sample, incubated for the same time periods in a medium that does not allow capacitation (medium B) and, therefore, expected to be poor in post-capacitated spermatozoa (We say ‘in all probability’ and ‘expected’ because, currently, there is no way to visually identify post-capacitated spermatozoa. We also tried other approaches for obtaining sperm populations enriched with post-capacitated spermatozoa. However, we found each of these additional approaches unsatisfactory—see Appendix). As expected, at any time point, the fraction of capacitated spermatozoa in medium B was lower than that in medium A (Fig. 2).
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Sperm phagocytosis by macrophages was easily detectable, with single, or groups of, spermatozoa being seen within macrophages either through their heads or through their flagella (Fig. 3; the microscope slides were vigorously washed to remove spermatozoa that are not internalized by the macrophages or tightly bound to them). This is consistent with earlier studies, demonstrating that sperm phagocytosis starts either at the head or at the tail (Blanco et al., 1992
; Matthijs et al., 2000). We compared this susceptibility to phagocytosis at three time points: zero time (no incubation for capacitation), as a negative control; 4 h, a time point at which the steady-state level of capacitated spermatozoa is expected to be maximal and at which the level of post-capacitated spermatozoa is expected to be relatively low (Cohen-Dayag et al., 1995; Giojalas et al., 2004) and 22 h, a time point at which the level of post-capacitated spermatozoa is expected to be higher due to their gradual accumulation (Fig. 1). The level of capacitated cells at this point was not different from the level at 4 h (Fig. 2), in line with our earlier studies (Cohen-Dayag et al., 1995; Giojalas et al., 2004). We observed a large variability between experiments carried out with different macrophage batches and different sperm samples (Fig. 4). Nevertheless, in all the experiments, the highest extent of phagocytosis of spermatozoa incubated in medium A was at 22 h (gray columns in Fig. 4). In contrast, the extent of phagocytosis of spermatozoa that had been incubated for the same time periods in medium B decreased, non-significantly, with time. These results are consistent with the working hypothesis.
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Medium B was different from medium A in three components: albumin, Ca2+ and bicarbonate. However, in all phagocytosis assays, medium B was supplemented with albumin just before the assay. The time-dependendence of phagocytosis in medium A and the observation that, at time zero, the extent of phagocytosis was similar in both media (Fig. 4) argue that the absence of Ca2+ and bicarbonate was not the cause of the observed differences in phagocytosis. We substantiated this conclusion by repeating the experiment, this time with Ca2+ and bicarbonate (additional to albumin) added to medium B just before the assay. The results (not shown) were very similar to those shown in Fig. 4.
Following the above-mentioned 22 h incubation, we observed a drastic drop in sperm motility, in most cases being more prominent in medium B and expressed primarily in the percentage of motile spermatozoa (Table 1). Our efforts to prevent this motility loss by refreshing the medium were not successful. This drop in motility was not responsible for the changes in phagocytosis because we did not find any correlation between the two events (Fig. 5). The percentage of motile spermatozoa dropped with time in both media (Table 1), whereas the extent of phagocytosis increased with time in medium A and slightly decreased in medium B (Fig. 4). If only motile spermatozoa were phagocytized, one would expect to see time-dependent decreases in phagocytosis in both media. If, on the other hand, mainly non-motile spermatozoa were phagocytized, one would expect to see increased phagocytosis with time in both media, with the increase being steeper in medium B, where the motility loss was faster. None of these possibilities were observed (Fig. 4), suggesting that the changes in the extent of phagocytosis were not due to changes in the fraction of motile spermatozoa. Moreover, there were sperm samples that lost motility at 22 h in both media; the trend of phagocytosis in these samples (e.g. the lower left sperm sample in Fig. 4A) were consistent with the other samples.
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The observed motility loss was not due to vitality loss. We stained the spermatozoa with eosin–nigrosin, known to stain dead cells only (Bjorndahl et al., 2003
). The percentage of vital spermatozoa was essentially constant (Fig. 6). It, thus, appears that the changes in phagocytosis were neither due to loss of motility nor due to cell death.
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The term ‘post-capacitated’ has been used here to indicate spermatozoa that did not undergo the acrosome reaction but ended being capacitated (Jaiswal and Eisenbach, 2002
). To validate that the observed changes in phagocytosis were due to changes in the levels of post-capacitated spermatozoa rather than due to changes in acrosome-reacted spermatozoa, we studied the effect of the acrosome reaction on sperm phagocytosis. We incubated spermatozoa in medium A and added, to a portion of them, the Ca2+-ionophore A23187
[GenBank]
, known to induce the acrosome reaction (Aitken et al., 1984; Cummins et al., 1991; Jaiswal et al., 1999). There was no significant difference between the susceptibilities of the acrosome-reacted and acrosome-intact spermatozoa to phagocytosis (Fig. 7), suggesting that the level of acrosome-reacted spermatozoa in the sperm populations did not contribute to the changes in phagocytosis observed in Fig. 4.
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Discussion |
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The working hypothesis underlying this study is that the role of sperm phagocytosis in the female genital tract is to remove spermatozoa that have lost their fertilizing capacity, i.e. to eradicate post-capacitated spermatozoa (Eisenbach, 2003
). Putting this working hypothesis to the test is not at all trivial, because there is no way to visually identify the physiological stage of the phagocytized cells. There is also no way to obtain a sperm population consisting exclusively of post-capacitated spermatozoa. Therefore, the best that we could attempt was to obtain a sperm population enriched with post-capacitated cells and to compare its susceptibility to phagocytosis with that of a sperm population that was poor in these cells. The results clearly demonstrated that a sperm population, incubated long enough to contain a substantial number of post-capacitated spermatozoa, were significantly more susceptible to phagocytosis than a parallel population, incubated for the same time length but under conditions that repress the formation of post-capacitated spermatozoa. Proper controls indicated that the higher degree of phagocytosis in the former population was not related to changes in motility, vitality or acrosomal status.
Although we could not demonstrate, due to the unavailability of proper markers, that the long-incubated sperm population indeed contained higher levels of post-capacitated spermatozoa, this seems to be an inevitable outcome of the long incubation in medium A. Thus, as soon as the process of capacitation is initiated by seminal fluid removal (time zero in Fig. 1), the level of human capacitated spermatozoa rapidly rises (Cohen-Dayag et al., 1995; Giojalas et al., 2004). Because the life span of the capacitated state in humans is limited, and different spermatozoa acquire this state at different time points (Cohen-Dayag et al., 1995; Jaiswal and Eisenbach, 2002), a steady-state level of capacitated spermatozoa is reached soon after (Cohen-Dayag et al., 1995; Giojalas et al., 2004), when the rate of cells becoming capacitated equals the rate of cells ending being capacitated and entering into their post-capacitated stage. Consequently, human capacitated spermatozoa do not accumulate (Cohen-Dayag et al., 1995; Giojalas et al., 2004). The only spermatozoa that appear to accumulate under these conditions are those at the post-capacitated state (Fig. 1). Thus, the results of this study, taken together with these considerations, strongly suggest that post-capacitated spermatozoa are preferentially phagocytized by macrophages, as predicted by the hypothesis. This conclusion is consistent with the observation that phagocytosis in the uterus occurs several hours after insemination (Austin, 1957). This delay may reflect the time elapsed from insemination to the initial acquirement of the post-capacitated state.
These results and conclusions are in harmony with studies of boar spermatozoa, demonstrating that intact cells are better phagocytized than damaged or dead ones [e.g. Matthijs et al. (2000); for a review, see Woelders and Matthijs (2001)], and that capacitated spermatozoa are not phagocytized (Matthijs et al., 2000) [This latter observation of Matthijs et al. (2000) involved induction of capacitation in vitro for up to 1.5 h. If in boars, as in humans, the life span of the capacitated state of individual spermatozoa is longer than 1.5 h, post-capacitated spermatozoa cannot be formed and accumulate within 1.5 h incubation].
The mechanism by which post-capacitated spermatozoa recruit macrophages waits to be revealed. It is likely that they secrete macrophage-activating substances or chemoattractants recognized by macrophages, and that macrophage-specific markers are exposed on the sperm surface during the process of terminating the capacitated stage.
A reasonable, though simplified, sequence of events in vivo is that human spermatozoa become capacitated asynchronously, each cell for a limited period of time, resulting in a steady-state level of cells at this state (Cohen-Dayag et al., 1995; Giojalas et al., 2004). Since a human spermatozoon can be capacitated only once, it becomes irreversibly non-functional as soon as its capacitated state is terminated (Cohen-Dayag et al., 1995; Eisenbach, 1999; Jaiswal and Eisenbach, 2002). To prevent accumulation of these functionless, post-capacitated spermatozoa, which may interfere with the approach and binding of freshly capacitated spermatozoa to the oocyte, macrophages are recruited to phagocytize them. This suggests that sperm phagocytosis not only does not interfere with fecundity (Taylor, 1982), but may be a process essential for normal fertilization.
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Appendix |
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Other attempts to form a sperm population enriched with post-capacitated cells
The notion behind all our other attempts to form a sperm population enriched with post-capacitated cells was, first, to generate a sperm population enriched with capacitated cells and, then, by
4 h incubation, to turn it into a population enriched with post-capacitated cells. We attempted to generate two sperm populations that would significantly differ from each other in their levels of capacitated cells (and then can be converted into sperm populations that differ from each other in their levels of post-capacitated cells) by the following means:
Enrichment by separation
This approach was based on the fact that only capacitated spermatozoa are chemotactically and thermotactically responsive (Cohen-Dayag et al., 1995; Bahat et al., 2003). We tried, as described in Materials and Methods, to preferentially accumulate capacitated spermatozoa in a chemoattractant-containing well and, thereby, to obtain a sperm population enriched with capacitated spermatozoa, as was successfully done by Cohen-Dayag et al. (1995). However, this approach turned out to be impractical because the total number of spermatozoa, with which we ended up after this treatment, was 1–2 orders of magnitude lower than the number required for a phagocytosis assay. Enrichment by thermotaxis [i.e. by accumulating capacitated spermatozoa in a warmer well (Bahat et al., 2003)] suffers from the same drawback (A. Bahat, unpublished observations).
Freezing and thawing
In this approach, we employed the finding that human spermatozoa, which undergo freezing and thawing, have elevated levels of capacitated cells (Esteves et al., 2000) [Similar results were reported for equine (Neild et al., 2003), dog (Rota et al., 1999), ram (Maxwell and Watson, 1996), boar (Maxwell and Johnson, 1997), bovine (Cormier et al., 1997) and monkey (Okada et al., 2001) spermatozoa]. Because the freezing and thawing procedure rendered a large fraction of the cells non-motile, we removed them by a Percoll gradient. The percentage of capacitated cells in this sperm population was significantly higher than that in the original population (24 ± versus 10 ± 2%, respectively; mean ± SEM of three experiments). However, again, the number of spermatozoa obtained by this procedure was too low for the phagocytosis assay.
Inhibition of capacitation by seminal fluid
In this approach we tried comparing a normal sperm population, incubated long enough to contain post-capacitated spermatozoa, with a sperm population treated with seminal fluid to inhibit capacitation. Decapacitating factors in the seminal fluid are well known to inhibit the ability of human spermatozoa to undergo capacitation (Cross, 1996; Jaiswal and Eisenbach, 2002; Chiu et al., 2005). Following verification that the fraction of capacitated cells was significantly higher in the seminal fluid-free sperm population than in the seminal fluid-containing population (15 ± 3 versus 5 ± 2% after 5 h incubation), we compared both sperm populations for their susceptibility to phagocytosis by macrophages. The extent of sperm phagocytosis was higher in the seminal fluid-free medium than in the seminal fluid-containing medium at any measured time point (Fig. 8), consistent with the study of Troedsson et al. (2005), published during the present study, demonstrating reduced phagocytosis (measured after 30 min) in the presence of seminal fluid. However, when we added seminal fluid to the sperm population incubated for 5 h in the seminal fluid-free medium, the extent of phagocytosis dropped to the low level of phagocytosis observed when seminal fluid was present throughout the incubation (data not shown). In line with earlier publications (Saxena et al., 1985; Quayle et al., 1989; Nocera and Chu, 1993), these results suggest that human seminal fluid per se suppresses phagocytosis and that, consequently, this approach was inadequate for our purpose.
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Acknowledgements |
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We thank Dr E. Yoles and her team in Proneuron Biotechnologies for the initial supply of macrophages and the protocol of macrophage isolation, Dr A. Gakamsky for the statistical analysis, L. Armon for her assistance and Dr R.M. Johnstone for reading the manuscript prior to submission. M.E. is an incumbent of Jack and Simon Djanogly Professorial Chair in Biochemistry.
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References |
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Aitken RJ, Ross A, Hargreave T, Richardson D, Best F. Analysis of human sperm function following exposure to ionophore A23187. J Androl (1984) 5:321–329.
Austin CR. Fate of spermatozoa in the uterus of the mouse and rat. J Endocrinol (1957) 14:335–342.[Web of Science][Medline]
Bahat A, Tur-Kaspa I, Gakamsky A, Giojalas LC, Breitbart H, Eisenbach M. Thermotaxis of mammalian sperm cells: A potential navigation mechanism in the female genital tract. Nature Med (2003) 9:149–150.[CrossRef][Web of Science][Medline]
Bedford JM. Effect of environment on phagocytosis of rabbit spermatozoa. J Reprod Fertil (1965) 9:249–256.[Medline]
Biggers JD, Whitten WK, Whittingham DG. The culture of mouse embryos in vitro. In: Methods in Mammalian Embryology—Daniel JD, ed. (1971) San Francisco: Freeman. 86–116.
Bjorndahl L, Soderlund I, Kvist U. Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Hum Reprod (2003) 18:81381–81386.
Blanco AM, Palaoro L, Ahedo MI, Palamas M, Zanchetti F. Phagocytosis of ejaculated spermatozoa. Acta Cytol (1992) 36:251–258.[Web of Science][Medline]
Burkman LJ. Hyperactivated motility of human spermatozoa during in vitro capacitation and implications for fertility. In: Controls of Sperm Motility: Biological and Clinical Aspects—Gagnon C, ed. (1990) Boca Raton: CRC Press. 303–329.
Chakraborty J, Nelson L. Fate of surplus sperm in the fallopian tube of the white mouse. Biol Reprod (1975) 12:455–463.[Abstract]
Chiu PCN, Chung M-K, Tsang H-Y, Koistinen R, Koistinen H, Seppala M, Lee K-F, Yeung WS. Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa. J Biol Chem (2005) 280:25580–25589.
Cohen-Dayag A, Ralt D, Tur-Kaspa I, Manor M, Makler A, Dor J, Mashiach S, Eisenbach M. Sequential acquisition of chemotactic responsiveness by human spermatozoa. Biol Reprod (1994) 50:786–790.[Abstract]
Cohen-Dayag A, Tur-Kaspa I, Dor J, Mashiach S, Eisenbach M. Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors. Proc Natl Acad Sci USA (1995) 92:11039–11043.
Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl (1997) 18:461–468.
Cross NL. Human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist, progesterone: cholesterol is the major inhibitor. Biol Reprod (1996) 54:138–145.[Abstract]
Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome reaction following ionophore challenge - relationship to fertility and other seminal parameters. J Androl (1991) 12:98–103.
Davis RO, Siemers RJ. Derivation and reliability of kinematic measures of sperm motion. Reprod Fertil Dev (1995) 7:857–869.[CrossRef][Medline]
Jonge De C. Biological basis for human capacitation. Hum Reprod Update (2005) 11:205–214.
Draveland JE, Mortimer D. A simple discontinuous Percoll gradient procedure for washing human spermatozoa. IRCS Med Sci (1985) 13:16–17.
Eisenbach M. Mammalian sperm chemotaxis and its association with capacitation. Dev Genet (1999) 25:87–94.[CrossRef][Web of Science][Medline]
Eisenbach M. Why are sperm cells phagocytosed by leukocytes in the female genital tract? Med Hypoth (2003) 60:590–592.[CrossRef][Web of Science][Medline]
Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing. Hum Reprod (2000) 15:2173–2179.
Fisher RA. Statistical Methods for Research Workers (1932) Edinburgh: Oliver and Boyd.
Giojalas LC, Rovasio RA, Fabro G, Gakamsky A, Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract. Fertil Steril (2004) 82:247–249.[CrossRef][Web of Science][Medline]
Jaiswal BS, Cohen-Dayag A, Tur-Kaspa I, Eisenbach M. Sperm capacitation is, after all, a prerequisite for both partial and complete acrosome reaction. FEBS Lett (1998) 427:309–313.[CrossRef][Web of Science][Medline]
Jaiswal BS, Eisenbach M. Capacitation. In: Fertilization—Hardy DM, ed. (2002) San Diego: Academic Press. 57–117.
Jaiswal BS, Eisenbach M, Tur-Kaspa I. Detection of partial and complete acrosome reaction in human spermatozoa: which inducers and probes to use? Mol Hum Reprod (1999) 5:214–219.
Knoller N, Auerbach G, Fulga V, Zelig G, Attias J, Bakimer R, Marder JB, Yoles E, Belkin M, Schwartz M, et al. Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: phase I study results. J Neurosurg Spine (2005) 3:173–181.[Web of Science][Medline]
Makler A. The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril (1980) 33:337–338.[Web of Science][Medline]
Matthijs A, Harkema W, Engel B, Woelders H. In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows. J Reprod Fertil (2000) 120:265–273.[Abstract]
Maxwell WMC, Johnson LA. Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation. Mol Reprod Dev (1997) 46:408–418.[CrossRef][Web of Science][Medline]
Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci (1996) 42:55–65.[CrossRef][Web of Science]
Mortimer D, Curtis EF, Camenzind AR. Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Hum Reprod (1990) 5:99–103.
Mortimer D, Fraser L. Consensus workshop on advanced diagnostic andrology techniques. Hum Reprod (1996) 11:1463–1479.
Mortimer ST, Schoevaert D, Swan MA, Mortimer D. Quantitative observations of flagellar motility of capacitating human spermatozoa. Hum Reprod (1997) 12:1006–1012.
NAFA and ESHRE-SIGA. Manual on Basic Semen Analysis (2002) wwww.andrology.dk/manual2002.pdf.
Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Aguero A. Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation. Theriogenology (2003) 59:1693–705.[CrossRef][Web of Science][Medline]
Nocera M, Chu TM. Transforming growth factor beta as an immunosuppressive protein in human seminal plasma. Am J Reprod Immunol (1993) 30:1–8.[Medline]
Okada A, Igarashi H, Kuroda M, Terao K, Yoshikawa Y, Sankai T. Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis). Hum Reprod (2001) 16:2139–2147.
Olds-Clarke P. How does poor motility alter sperm fertilizing ability? J Androl (1996) 17:183–186.
Pandya IJ, Cohen J. The leukocytic reaction of the human uterine cervix to spermatozoa. Fertil Steril (1985) 41:417–421.
Phillips DM, Mahler S. Leukocyte emigration and migration in the vagina following mating in the rabbit. Anat Rec (1977) a189:45–59.[CrossRef][Medline]
Phillips DM, Mahler S. Phagocytosis of spermatozoa by the rabbit vagina. Anat Rec (1977) b189:61–71.[CrossRef][Medline]
Quayle AJ, Kelly RW, Hargreave TB, James K. Immunosuppression by seminal prostaglandins. Clin Exp Immunol (1989) 75:387–391.[Web of Science][Medline]
Rota A, Pena AI, Linde-Forsberg C, Rodriguez-Martinez H. In vitro capacitation of fresh, chilled and frozen-thawed dog spermatozoa assessed by the chloretetracycline assay and changes in motility patterns. Anim Reprod Sci (1999) 57:199–215.[CrossRef][Web of Science][Medline]
Saxena S, Jha P, Farooq A. Immunosuppression by human seminal plasma. Immunol Invest (1985) 14:255–269.[Web of Science][Medline]
Suarez SS. Gamete transport. In: Fertilization—Hardy DM, ed. (2002) San Diego: Academic Press. 3–28.
Taylor NJ. Investigation of sperm-induced cervical leucocytosis by a double mating study in rabbits. J Reprod Fertil (1982) 66:157–160.
Teves ME, Barbano F, Guidobaldi HA, Sanchez R, Miska W, Giojalas LC. Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa. Fertil Steril (2006) 86:745–749.[CrossRef][Web of Science][Medline]
Troedsson MH, Desvousges A, Alghamdi AS, Dahms B, Dow CA, Hayna J, Valesco R, Collahan PT, Macpherson ML, Pozor M, et al. Components in seminal plasma regulating sperm transport and elimination. Anim Reprod Sci (2005) 89:171–186.[CrossRef][Web of Science][Medline]
Tyler KR. Histological changes in the cervix of the rabbit after coitus. J Reprod Fertil (1977) 49:341–345.
Woelders H, Matthijs A. Phagocytosis of boar spermatozoa in vitro and in vivo. Reprod Suppl (2001) 58:113–127.[Medline]
World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen-cervical Mucus Interaction (1993) New York: Cambridge University Press.
Yanagimachi R. The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil (1970) 23:193–196.
Yanagimachi R. Mammalian fertilization. In: The Physiology of Reproduction—Knobil E, Neill J, eds. (1994) Vol. 1. New York: Raven Press. 189–317.
Submitted on February 20, 2007; resubmitted on June 4, 2007; accepted on July 11, 2007.
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