Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany
1 To whom correspondence should be addressed. e-mail: yeung{at}uni-muenster.de
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Abstract |
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Key words: organic osmolytes/quinine/regulatory volume decrease/sperm function/sperm motility
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Introduction |
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Epididymal fluid is characterized by progressive decreases in Na+ and increases in K+ concentrations along the duct. Osmolality is made up by high amounts of small organic molecules including amino acids such as glutamate and taurine, carnitine, glycerophosphocholine (GPC) and myo-inositol (see Cooper and Yeung, 2003). These organic molecules, as well as K+, are commonly used by somatic cells as osmolytes for volume regulation (see Lang et al., 1998
; Fürst et al., 2002
). The increase in osmolality from testicular to epididymal fluid should induce uptake of osmolytes by epididymal sperm to counteract cell shrinkage. These compounds could then be utilized by sperm upon ejaculation into a relatively hypotonic environment in the female tract, by mechanisms of regulatory volume decrease (RVD) resulting in efflux of osmolytes and cellular water (Cooper and Yeung, 2003
). In our preliminary study of murine sperm, it was revealed that the presence of such molecules in the incubation medium, with the exception of GPC, could prevent sperm RVD, thus supporting the hypothesis that these are sperm osmolytes. In the present study, these putative osmolytes were tested on human ejaculated sperm.
When challenged with physiological changes in osmolality, murine epididymal sperm swell considerably upon inhibition of RVD with induction of tail angulation (Yeung et al., 1999, 2002a). However, human ejaculated sperm increase in cell volume only to a minimal extent with little morphological changes in the tail (Yeung and Cooper, 2001
). This contrasts with the conventional hypo-osmotic swelling (HOS) test for viability where sperm are subjected to a non-physiological osmolality of 150 mosmol/kg (Jeyendran et al., 1992
). In the HOS test, sperm with intact plasma membranes form coils in the tails because of the rapid and drastic swelling, and lose their motility within 2 min (Hossain et al., 1998
). On the other hand, human sperm swollen in physiological osmolalities display greater changes in kinematic parameters than in cell volume (Yeung and Cooper, 2001
). Such kinematic changes can be detected with more sensitivity using computer-aided sperm analysis (CASA) than changes in cell volume measured by flow cytometry, which are readily detectable in the mouse (Yeung et al., 2002a
). Therefore, both cell volume and kinematics were used as end-points in the present study in the search for putative human sperm osmolytes.
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Materials and methods |
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Evaluation of sperm volume by flow cytometry
The laser forward scatter signal of viable sperm as a reflection of sperm cell volume (Yeung et al., 2002a) was analysed by a flow cytometer (Coulter Epics XL, version 3.0; Germany) as described previously (Yeung and Cooper, 2001
). The same flow cytometer settings were used throughout the study. For each sample,
10 000 particles were analysed and the mean forward scatter intensity was calculated after gating out the cell debris and aggregates using the forward and side-scatter dot plot and eliminating the non-viable sperm based on PI fluorescence.
It should be noted that the forward scatter signals obtained in terms of channel numbers are relative values that depend on the voltage settings of the photo-multiplier tube, which were chosen to be sensitive enough to detect changes and were kept constant for all experiments in the study. Signals so detected for human sperm are therefore not comparable with those obtained for mouse studies which used different voltage settings (Yeung et al., 2002a,b).
Simultaneous measurement of sperm volume by flow cytometry and electronic sizing using the Coulter counter
Aliquots of sperm suspensions diluted from eight ejaculates into BWW medium were measured simultaneously by the flow cytometer as described above and by a Coulter counter (model Z; Beckman Coulter, Germany) as previously described for mouse sperm (Yeung et al., 2002a). Volume was measured in femtolitres by the Coulter counter, and the size distribution of the 10 00030 000 particles measured in each sample was analysed using the Accucomp software provided by the manufacturer. The mean sperm volume was calculated after gating out debris and cell aggregates. The mean forward scatter signal of all the sperm from an aliquot of the same sample analysed by the flow cytometer was also calculated for comparison. To remove the sperm plasma membrane and cytoplasm in order to reduce sperm size to the minimum, Triton X-100 was added to the sperm suspension at a final concentration of 1 % (v/v) for 3 min before reading by both machines.
Computerized analysis of sperm kinematics (using CASA)
Washed sperm incubated in various media for 20 min were recorded at 37°C in a 20 µm deep chamber (2X-Cel; HamiltonThorne Research, USA) on video tape as described in Yeung and Cooper (2001). For each sample, >200 motile sperm were tracked for 1 s at 50 frame/s, with an average path velocity threshold value of 3 µm/s and a minimum of 26 track points for each motile cell, using a HamiltonThorne CASA system (IVOS version 10.8; HamiltonThorne Research). To circumvent any non-Gaussian distribution of individual track data, the median values of all sperm tracks in each sample were taken to represent the kinematic parameters of that sample. Data for each treatment group were calculated and presented as mean ± SEM of the n median values from n samples.
Statistics
In each experiment, the effects of osmolality and quinine treatment on sperm volume were analysed by comparison with the reference value initially obtained with non-washed sperm in BWWsemen (see Figure 1) and expressed as ratios to overcome basal differences between ejaculates. Data from osmolyte incubation were expressed as ratios of the control incubation (BWW290) in the absence of osmolytes. Analysis of variance was performed using SigmaStat computer software (version 2.03, SPSS Inc., Germany). For each group of osmolyte or quinine treatment, statistical comparisons with controls were done using the StudentNewmanKeuls method. In cases where the normality test failed, one way analysis of variance on ranks was performed followed by Dunns comparison.
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Results |
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Discussion |
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Semen osmolality measured in the present study after 30 min of liquefaction varied from 289 to 351 with an average of 318 mosmol/kg, as found by Rossato et al. (2002). In our previous study, a mean ± SD value of 342 ± 21 mosmol/kg from 66 normozoospermic ejaculates was obtained 6090 min after collection (Yeung and Cooper, 2001
). There are reports of similar (Makler et al., 1981
) or higher values (Velazquez et al., 1977
; Polak and Daunter, 1984
; Gopalkrishnan et al., 1989
). However, it is well known that semen osmolality increases with time after ejaculation owing to proteolysis and other chemical changes (see Mann and Lutwak-Mann, 1981
), e.g. increasing from
330 mosmol/kg at 50 min to 380 mosmol/kg at 150 min (Abraham-Peskir et al., 2002
).
The measured seminal osmolality was lower than that reported in the vas deferens and higher than that of female tract fluids. Therefore during normal copulation sperm experience a stepwise decrease in surrounding osmolality from the cauda epididymidis and vas deferens (342 mosmol/kg; Hinton et al., 1981) via the fresh ejaculate (318 mosmol/kg) to cervical mucus (287 mosmol/kg; Casslén and Nilsson, 1984
) and uterine fluid (284 mosmol/kg; Rossato et al., 1996
). The gradient of change (
60 mosmol/kg) is less than that experienced by murine ejaculated sperm (
80 mosmol/kg). In the present study, dilution of ejaculated sperm into medium with cervical mucus osmolality in the presence of quinine, the wide spectrum blocker of ion channels involved in RVD, also induced both volume increases and kinematic changes. In the absence of quinine, sperm size and kinematics remained unchanged either in BWWsemen or BWW290, indicating that RVD mechanisms were functioning within the physiological range of osmolality.
In the search for potential sperm osmolytes, K+ and small organic molecules found in high concentrations in epididymal fluid of laboratory mammals were tested in the present work. Because of the paucity of data on human (or other primate) epididymal fluid composition, these substances were included in the incubation medium at concentrations found to be effective in inhibiting RVD of murine sperm, presumably by preventing efflux down concentration gradients through opened channels, resulting in the removal of water from the cell (C.H.Yeung, M.Anapolski and T.G.Cooper, unpublished data). However, none was found to be effective in inducing a volume increase in human sperm. When the related, but more sensitive, kinematic parameters were examined, glutamate and K+ were found to have slight effects. In vas deferens fluid (Hinton et al., 1981), K+ is at far higher concentration (111 mmol/l) than myo-inositol (6 mmol/l) and carnitine (6 mmol/l in both vas deferens and cauda epididymidal fluid from one man) (Turner, 1979
). This differs from the rat (there are no data from mouse) where all three osmolytes are
50 mmol/l in caudal fluid (Levine and Marsh, 1971
; Hinton and Palladino, 1995
). Therefore, K+ could be the major osmolyte utilized by human sperm for RVD.
It is worth noting that uterine (Casslén and Nilsson, 1984) and oviductal fluids are generally rich in K+, with mean values ranging from 7 to 26 mmol/l in women (see Borland et al., 1980
) and as high as 30 mmol/l in rabbits oviducts (Burkman et al., 1984
). Nevertheless, these levels are well below the intracellular concentrations reported to be 75 mmol/l in human sperm (Patrat et al., 2002
) and as high as 120 mmol/l in bovine and murine sperm (Babcock, 1983
; Chou et al., 1989
), creating large diffusion gradients which would enable efficient efflux of K+ as an osmolyte for volume regulation. Evidence for this is provided by the reversal of the quinine-induced changes in volume and kinematics by the K+ ionophores valinomycin and gramicidin (Yeung and Cooper, 2001
). The present finding of an effective concentration of 100 mmol/l but not of 30 mmol/l is in agreement with this hypothesis. Rabbit sperm motility is also inhibited by 50 mmol/l K+ (Burkman et al., 1984
), which led the authors to suggest a role of K+ in the regulation of sperm motility in the female tract.
The weak, or lack of, response of human sperm to incubation with potential osmolytes compared with murine sperm was reflected in the difference in their extents of swelling at physiological osmolality induced by quinine. Whereas a 30% increase in laser forward scatter (as indicator of sperm size) is demonstrated in murine sperm (Yeung et al., 2002b), the increase in human sperm is very small, amounting to only a few per cent. One explanation of a lower swelling response to quinine in human sperm is a smaller drop in osmolality from the native fluid of the sperm sample (seminal plasma in man and cauda epididymidal fluid in mouse) to the test medium (an average of 318290 = 28 mosmol/kg in the human and 415330 = 85 mosmol/kg in the mouse studies). Another explanation could be the difference in the size of the cytoplasmic droplets which hold most of the sperm cytoplasm. These are relatively large and present in
80% of mature murine sperm (Cooper and Yeung, 2003
). The presence of cytoplasmic droplets in dried smears is mostly considered an abnormal morphology of human sperm, but the disruptive forces during smearing and air drying may well disrupt osmotically sensitive organelles. Recently, vesicles around the human sperm mid-piece (MPV) have been reported in wet preparations using differential interference contrast or X-ray microscopy (Abraham-Peskir et al., 2002
). The incidence, as well as the size, of these MPV increases with decreasing osmolality of the medium ranging from 450 to 200 mosmol/kg. These authors reported that the association of MPV with reduced motility, in agreement with our previous observation of inhibition of mucus penetration and migration upon inhibition of RVD (Yeung and Cooper, 2001
). Therefore visible MPV may be a manifestation of insufficient cell volume regulation.
The present flow cytometric method could offer a rapid analysis of sperm cell volume in semen analysis. It has been reported that in normozoospermic ejaculates, 30% of sperm, separated by density gradients, have enlarged mitochondrial diameter and low creatine kinase isoform ratios (Gergely et al., 1999
) which is an indication of cytoplasmic retention during spermiogenesis (Huszar et al., 1998
). The presence of such defective, mis-matured sperm would increase the mean cell volume of the ejaculated sperm. This cause of poor sperm quality may partly contribute to the present correlations of poor kinematics with higher laser scatter signals. Nevertheless, the correlation with the changes in laser scatter signal were stronger than that with the absolute values, reflecting more the functional deficiency in cell volume regulation than the abnormal morphology.
The reversal of both sperm size increase and alterations in kinematics in the presence of quinine by the ionophores valinomycin and gramicidin (Yeung and Cooper, 2001) suggests that the dynamics of flagellation are influenced by the physical dimension/structure of sperm components. In the present study, a linear correlation was established between changes in sperm volume as reflected by laser forward scatter and simultaneous changes in various kinematic parameters, particularly the straightness of the sperm track, which decreases with increases in sperm volume. Although the low values of the correlation coefficients for the other parameters warrant a note of caution, the finding suggests that an increase in cell volume may lead to a decreased efficiency of forward progression (decreases in straight line velocity and linearity) without compromising vigour of flagellar beating (reflected in increases in curvilinear velocity and lateral head displacement).
That there was no correlation between semen osmolality and sperm size indicates that normal sperm can regulate their volume in semen. Nevertheless, weak but significant correlations were demonstrated between sperm size and kinematic parameters, in particular the association of faster forward progression with smaller sperm. Upon cell swelling, the signal transduction leading to channel opening for osmolyte effluxes (if there are concentration gradients) is known to involve diverse pathways including protein kinase A (PKA) and PKC (see Hoffmann, 2000; Fürst et al., 2002
), and in some cells increases in intracellular pH and Ca2+ (MacLeod and Hamilton, 1999
). Since such factors could be the direct modulators of motility, it is not clear whether the motility changes found in the present study were effected by any of these factors, besides the direct mechanical effect that volume has on the kinematics. Volume regulation as a component of the complex signal transduction pathways and interplay among various aspects of sperm function warrants further investigation. It also offers a new lead in the development of post-testicular contraceptive.
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Acknowledgements |
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Submitted on July 26, 2002; resubmitted on November 26, 2002; accepted on January 15, 2003.