1 Institute of Reproductive Medicine of the University Clinic, Münster, Germany and 2 Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
4 To whom correspondence should be addressed: Institute of Reproductive Medicine, Domagkstrasse 11, D-48129 Münster, Germany. Email: yeung{at}uni-muenster.de
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Abstract |
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Key words: epididymis/regulatory volume decrease/sperm function/sperm ion channels/sperm maturation
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Introduction |
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The physiological significance of sperm volume regulation is shown in c-ros knockout mice, where males are infertile because their spermatozoa exhibit inadequate volume regulation; as a consequence, flagellar angulation occurs and they fail to negotiate the uterotubal junction (Yeung et al., 1999). In mice, spermatozoa acquire this ability in the epididymis (Yeung et al., 1999
, 2002a
). Ejaculated human spermatozoa are able to regulate their volume when subjected to media of female tract hypotonicity (Yeung and Cooper, 2001
; Yeung et al., 2003
) but whether this is an epididymal-dependent process is not known, since living human epididymal spermatozoa are difficult to obtain for experimentation. Nevertheless, it has been demonstrated that inhibition of such volume regulation by the wide spectrum channel blocker quinine leads to failure in the penetration of surrogate mucus with a decrease in the forward progressive velocity, without hampering the vigour of beating (Yeung and Cooper, 2001
).
The cynomolgus monkey epididymis is a good primate model for that of the human, since the maturation of spermatozoa within the duct resembles that of man, with respect to acquisition of motility and development of kinematics (Yeung et al., 1993, 1996
), acquisition of ability to undergo acrosome reaction (Yeung et al., 1996
, 1997
), changes in sperm surface charge (Fain-Maurel et al., 1984
), modification of sperm antigens (Mahony et al., 1994
; Yeung et al., 1997
, 2000
, 2001
), development of the acrosomal response to second messengers (Mahony et al., 1996
), condensation of nuclear chromatin (Golan et al., 1997
) and diminution of sperm head size (Yeung et al., 1997
; Gago et al., 2000
Soler et al., 2000
).
In this study, immature, maturing and matured spermatozoa from the cynomolgus monkey were obtained from the epididymis and ejaculate, and examined for volume-regulating properties in relatively hypotonic medium mimicking that of the female tract. Cell volume of viable sperm was assessed by a flow cytometric method which has been validated for both murine (Yeung et al., 2002b) and human spermatozoa (Yeung et al., 2003
). This method has advantages over the more conventional electronic sizing technique, despite the disadvantage of not providing absolute values of volume, and data obtained from both methods have been shown to be well correlated. The flow cytometric method is preferred for its sensitivity, stability, simultaneous analysis of cell viability and independence of the electrolyte conductivity of the various media used. In the present study, inhibitors of channels involved in volume regulation of somatic cells were investigated for their ability to block volume regulation in spermatozoa, in an attempt to define the channels involved.
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Materials and methods |
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Epididymal spermatozoa
Six healthy adult cynomolgus monkeys (Cynomolgus fascicularis) involved in previous studies (Kamischke et al., 2003) were available for the present study, of which two had received testicular irradiation and fully recovered spermatogenesis and four had been used as non-irradiated controls. They were anaesthetized with Ketamine HCl (12 mg/kg) and the scrotal contents removed. The epididymis was severed from the testis and brought to the laboratory within 5 min. Spermatozoa were obtained from the caput, corpus and cauda epididymidis (regions 2, 4 and 6 from Yeung et al., 1996
) by exposing and then cutting the tubule to release spermatozoa directly into the relevant medium (see below). Sperm concentration was adjusted to
20 x 106/ml with relevant media (see below). The order in which spermatozoa from different regions were incubated in different media was alternated between experiments, to take into account variations in time of sample processing.
Ejaculated spermatozoa
Male monkeys (n=15) were anaesthetized as above and prepared for electro-ejaculation using a rectal probe. The samples were brought to the laboratory in an incubator at 37°C within 30 min and the exudate was separated from the coagulum. A portion of the exudate was taken to assess sperm concentration and osmotic pressure and 210 µl aliquots were dispensed into 100 µl relevant media (see below) to a concentration of 220 x 106/ml.
Osmotic pressure measurements
The osmotic pressure of semen was measured in a vapour pressure device (Wescor Vapro model 5520, Kreienbaum Messsystem, Langenfeld, Germany) on 10 µl undiluted fluid, as dew point depression is not affected by sample viscosity or the presence of suspended particles (Sweeney and Beuchat, 1993). A delay of 2 min was employed to ensure chamber saturation and reproducible results.
Flow cytometry
Immediately after sperm dispersion (12 min) and incubation at 37°C in 5% (v/v) CO2 for 20 and 40 min (epididymal spermatozoa) or for 5 and 30 min (ejaculated spermatozoa), 10 µl sperm suspension was added to 200 µl incubation medium containing the vital dye propidium iodide (PI, 3 µl 0.5 mg/ml). A minimum of 6000 cells was read in an Epics XL flow cytometer (Coulter version 3.0, Krefeld, Germany) for PI (emission at 605635 nm) and for forward and side scatter of laser with an excitation at 488 nm. Forward scatter signals were analysed after gating for viable spermatozoa. All flow cytometer settings (voltage, gain, signal trigger threshold, analysis windows, etc.) in the flow cytometer protocol used for each experiment were maintained throughout the whole study. With each individual monkey and each ejaculate used, a non-treated sample was always included, which served as a control value for comparison for each replicate treatment sample.
Incubation media
All chemicals were from Sigma (Taufkirchen, Germany) unless otherwise stated. Modified BiggersWhittenWhittingham (BWW) medium (pH 7.4, containing 20 mM Hepes in addition to 25 mM HCO3 and 4 mg/ml albumin) was made to osmolality of 290 mmol/kg (BWW290) by adjusting the amount of NaCl. BWW290 is medium of similar osmolality to that of the female tract, and is anticipated to be hypotonic to both epididymal and ejaculated spermatozoa. To this was added extra KCl (40 mM, 80 mM, replacing an equivalent amount of NaCl) or channel blockers involved in volume regulation at concentrations effective for somatic and sperm cells; the broad spectrum cationic blockers quinine HCl (QUI, 0.25 mM for epididymal and 0.3 mM for ejaculated sperm), tetraethylammonium (TEA, 10 mM, 50 mM: Mathie et al., 1998) and BaCl2 (Ba, 1 mM, 5 mM: Furlong and Spring, 1990
; Cho, 2002
), the voltage-gated potassium channel blocker 4-aminopyridine (4-AP, 1 mM, 4 mM: Krasznai et al., 1995
; Mathie et al., 1998
), the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 µM: Okada, 1997
); the Kv1.3-specific voltage-dependent K+ channel blocker margatoxin (MTX, 10 nM, 200 nM: Garcia-Calvo et al., 1993
); the Ca2+ -activated K+ channel blocker charybdotoxin (CTX 10 nM, 100 nM: Hanner et al., 1998
); the outwardly rectifying K channel blocker dendrotoxin-
(DTX, 150 nM, 400 nM; Calbiochem, Schwalbach, Germany: Gasparini et al., 1998
); the ATP-type, Ca2+ -activated K+ channel inhibitor apamin (APM, 5 nM, 200 nM: Tanabe et al., 1999
), the ATP-sensitive K+ channel blocker glybenclamide (GLY, 10 µM, 100 µM: Lindstrom et al., 1986
; Macho et al., 2001
; Cho, 2002
), the acid-sensitive K+ channel inhibitor clofilium tosylate (CLO, 10 µM, 100 µM; Alexis, Grünberg, Germany: Niemeyer et al., 2001a
,b
) and the transient channel receptor protein inhibitor GdCl3 (Gd, 10 µM, 100 µM: Taouil and Hannaert, 1999
; Chen and Barritt, 2003
).
Computer-assisted analysis (CASA) of sperm kinematics
Percentages of motile sperm were estimated manually. For CASA measurement, video-recordings of 515 microscopic fields of the sperm sample in the 20 µm slide chamber (2X-CEL; Hamilton-Thorne Research, Beverly, MA) was made at 37°C using a negative phase contrast x10 objective and x3.3 photo-eyepiece. For each sample, 200 motile spermatozoa were tracked for 1 s and analysed with the HamiltonThorne CASA system (Beverly, MA). Kinematic parameters measured included curvilinear (VCL), straight-line (VSL) and averaged path (VAP) velocities, amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and the derived parameters of linearity (LIN = VSL/VCL x100%) and straightness (STR = VAP/VCL x100%). Epididymal spermatozoa were analysed at 25 Hz frame rate for 25 frames (version 10.8) and ejaculated sperm at 50 Hz for 50 frames, as described in Yeung et al. (2003)
.
Statistics
Differences in sperm kinematic data were compared by one way ANOVA for repeated measures, followed by Dunnett's post-hoc test. Volume changes by various treatments were expressed as a ratio of each control value by one way ANOVA followed by a Dunnett's post-hoc test, or for non-normal distributions by the non-parametric KruskalWallis one way ANOVA on ranks followed by Dunn's test.
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Results |
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Effects of channel inhibitors on volume and kinematics of ejaculated spermatozoa
Osmolality of the exudate of ejaculates from 15 monkeys was 333±6 mmol/kg (mean±SEM). The size of ejaculated spermatozoa at 5 min incubation in BWW290 was no different from that examined after 30 min (1.0045±0.0047, n=26; expressed as ratios of the 5 min values). Of the range of channel inhibitors tested at both time points, only quinine (0.3 mM) and 4-aminopyridine (0.4 mM) increased the sperm size examined at 30 min (Figure 3). None of the specific K+ channel inhibitors (margatoxin, charybdotoxin, dendrotoxin or clofilium) nor the less specific TEA or BaCl2 affected volume. Gadolinium also had no effect.
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Table II shows that quinine and 4-AP had no significant effect on sperm vigour (VCL) but both depressed STR, LIN and BCF and increased ALH, indicative of reduced forward progression. Quinine additionally depressed VAP and VSL. With the other blockers tested, no effects on sperm kinematics can be detected (data not shown).
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Discussion |
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These findings suggest that the size of the spermatozoa within the epididymis would be similar to that exhibited by the caput sperm. As caput spermatozoa move along the epididymal duct, they are anticipated to encounter increasing osmolality, as in all mammals studied (see Cooper and Yeung, 2003), and act against the tendency of cell shrinkage by taking up the osmolytes plentiful in epididymal fluid, increasing their intracellular osmolality as they mature. When released in the medium of 290 mmol/kg, both the maturing and mature sperm from the corpus and cauda epididymidis would be challenged by the relative hypotonicity and increase in cell volume initially, as measured at 1 min after dispersion in medium. This would trigger the volume regulatory mechanism and cause the opening of the relevant ion channels to allow efflux of osmolytes, resulting in a decrease in cell volume to counteract the swelling. Such mechanisms would be fully developed in the mature cauda sperm, as shown in the mouse (Yeung et al., 1999
, 2002b
), and allow the cell to return to the original volume. Corpus sperm may have only partially acquired these mechanisms and therefore failed to recover their volume fully. This interpretation is supported by the finding that in the presence of quinine, which is a wide spectrum blocker of channels involved in volume regulation (Lang et al., 1998
), volume decrease was inhibited so that both corpus and cauda sperm retained their initially swollen status, with cell volume significantly larger than in the absence of quinine, which allowed volume regulatory mechanisms to take effect fully in cauda sperm and partially in corpus sperm.
The differences between mature and immature sperm in their volume regulatory ability could partly be attributed to the differences in their plasma membrane lipid components, which are known to change during maturation in the epididymis (see Jones, 2002), as composition of membrane lipid rafts is known to modulate ion channel activities (see Brown and London, 1998
). It has been shown that sperm capacitation condition, which alters lipid cholesterol contents and destabilizes plasma membranes (Harrison et al., 1996
; Flesch et al., 2001
) can also alter volume regulatory behaviour of bovine and porcine mature sperm (Kulkarni et al., 1997
; Petrunkina and Töpfer-Petersen, 2000; Petrunkina et al., 2004a
).
In the present study, monkey ejaculated spermatozoa were used as mature sperm for further study because of their greater availability than epididymal sperm. As quinine also blocks volume regulation of ejaculated spermatozoa from men (Yeung and Cooper, 2001; Yeung et al., 2003
), bulls (Kulkarni et al., 1997
; Petrunkina et al., 2001
), boars (Petrunkina et al., 2001
) and dogs (Petrunkina et al., 2004b
), the quinine-sensitive channel seems to be conserved between species. Employing a wide range of inhibitors of channels involved in the volume regulation of somatic cells failed to indicate any specific channels involved in the volume response in hypotonic media. At the concentrations used (effective in somatic cells) there was no evidence that the Kv1.3-specific voltage-dependent K channel (inhibited by margatoxin), Ca2+ -activated K channels (inhibited by charybdotoxin), outwardly rectifying K channels (inhibited by dendrotoxin), ATP-type Ca2+ -activated K channels (inhibited by apamin), ATP-sensitive K channels (inhibited by glybenclamide), the acid-sensitive K channel TASK2 (inhibited by clofilium) or the transient channel protein (inhibited by gadolinium) were involved in volume regulation, as they are in some somatic cells.
The ability of the chloride channel blocker NPPB to cause swelling of monkey spermatozoa may indicate that anion channels are involved in the regulation of volume, as also found in the mouse (Yeung et al., 1999). Such channels in somatic cells also permit the efflux of organic osmolytes (Strange and Jackson, 1995
), which are present at high concentration in the epididymis and could be provided to spermatozoa during their maturation. The effect of NPPB and other chloride channel blockers involved in cell volume regulation on primate ejaculated spermatozoa warrants further study.
Finally, the factors (quinine, NPPB and 4-Ap) also affecting monkey sperm volume, affected motility parameters. Quinine and 4-AP also reduce VSL and linearity of human ejaculated spermatozoa, whereas TEA, charybdotoxin and margatoxin have no effect (Yeung and Cooper, 2001). Thus, changes in cell volume were associated with changes in the kinematics of both the epididymal and ejaculated monkey sperm, as was demonstrated in human ejaculated sperm (Yeung et al., 2003
). That volume regulation in the non-human primate resembles that demonstrated in man and mouse may reflect the presence of primitive channels that evolved to prevent stresses in spermatozoa, encountered during maturation in the epididymis and upon ejaculation into the female tract. The monkey may be a suitable model that could be used in contraceptive studies designed to mimic the infertility of transgenic mice caused by inadequate volume regulation (Cooper et al., 2004
).
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Acknowledgements |
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Notes |
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References |
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Submitted on May 7, 2004; resubmitted on June 30, 2004; accepted on July 20, 2004.