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From the * Institute of Reproductive Medicine of
the University, Münster, Germany; and the
Institute of Physiology, University of
Tübingen, Tübingen, Germany.
| Correspondence to: Dr C. H. Yeung, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany (e-mail: yeung{at}uni-muenster.de). |
| Received for publication August 25, 2003; accepted for publication November 21, 2003. |
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Abstract |
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Key words: Sperm swelling, regulatory volume decrease, quinine, organic osmolytes, infertility
A characteristic of epididymal fluid of all mammalian species is the distally increasing osmolality, which should induce osmotic changes in sperm cells. It has recently been proposed that these conditions, in conjunction with the long time (days) it takes sperm to pass through the epididymis, favor isovolumetric regulation of sperm osmolality (Cooper and Yeung, 2003). In this process in general, major changes in cell volume are avoided during imposition of small incremental changes in extracellular OP that result in influx of osmolytes (Pasantes-Morales et al, 2000; Souza et al, 2000). In the epididymis, the increasing tonicity along the tubule would encourage similar osmolyte uptake by maturing sperm, and it is suggested that these osmolytes, provided by the epididymis, are used by sperm in the female tract in response to its relative hypo-osmolality (Cooper and Yeung, 2003).
A number of low–molecular weight, water-soluble organic components are present in extremely high (mM) concentrations in epididymal fluid (see Cooper, 1998), and several of them (glutamate, taurine, myo-inositol, carnitine [a betaine derivative], and glycerophosphocholine) are employed in somatic cells as nonperturbing solutes for volume regulation (Strange et al, 1996; Lang et al, 1998; Furst et al, 2002) and could be relevant for the volume regulatory properties of spermatozoa.
One way to determine the nature of the osmolytes used by spermatozoa in volume regulation is to monitor volume changes in response to inhibitors of channels mediating osmolyte efflux. In this way, evidence for a role of ion channels in sperm volume regulation was provided by the effect of quinine on bovine sperm volume (Kulkarni et al, 1997; Petrunkina et al, 2001). Quinine (a wide-spectrum, though conventional, K+ channel blocker), BaCl2 (a K+ channel blocker), and 5-nitro-2-(3-phenypropylamine)-benzoic acid (NPPB; a Cl– channel blocker) all promote the angulation of murine sperm, reflecting a swollen status (Yeung et al, 1998, 1999, 2002a). This suggests that osmolytes that use these channels are involved in sperm volume regulation.
In this study, the identities of potential osmolytes were elucidated by compromising their concentration gradients across the sperm membrane and examining the effect on RVD. The amounts of these effective osmolytes in caudal epididymal luminal contents were also compared between the fertile heterozygous and infertile homozygous c-ros knockout mice because the heterozygous males are identical to the wild type in phenotype, whereas the homozygous mice lack the initial segment of the epididymis that normally differentiates from the proximal caput during puberty (Sonnenberg-Riethmacher et al, 1996). Although sperm production in the testis and deposition in the uterus are normal in the infertile male, spermatozoa recovered from the uterus after mating or released from the cauda epididymis into medium show angulation of the tail, as exhibited by normal sperm swollen by ion channel blockers (Yeung et al, 1999, 2000). Increases in cell volume of these sperm have been confirmed (Yeung et al, 2002a,b). Therefore, these transgenic animals are a useful model for the study of the role of the epididymis in sperm volume regulation.
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Materials and Methods |
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Incubation Media
The basal (control) medium used was BWW
(Biggers et al, 1971),
containing bovine serum albumin (BSA) at 4 mg/mL, with the osmolality made to
330 mmol/kg with NaCl, which is identical to that of the uterine contents of
wild-type mice (Yeung et al,
2000). All chemicals were obtained from Sigma (Taufenkirchen,
Germany) unless stated otherwise. Osmolytes tested at various concentrations
(see Results section) were first made in individual stock solutions (1.0 M for
KCl, 250 mM for the organic osmolytes, including sodium glutamate, taurine,
L-carnitine [inner salt] and myo-inositol), with pH adjusted to 7.0
when necessary. Cadmium-free glycerophosphocholine (GPC) was dissolved in
methanol, and the desired amount was blown dry in glass tubes at 37°C and
taken up by BWW medium. The osmolality of all media used for sperm incubation
was adjusted to 330 mmol/kg by omission and further adjustment of NaCl
content. Quinine was made up in a 100 mM aqueous stock solution and diluted
into BWW medium to give 200, 400, or 800 µM just before use.
Sperm Preparation for Cell Volume Estimation
Mice were killed by cervical dislocation after CO2 asphyxiation.
The epididymis was dissected out and cleaned of blood. For the study of mature
sperm, the cauda region was decapsulated at around the flexure. From each
side, 3 short tubule segments (6 from 1 mouse) were excised and processed in
sequence. Each segment was transferred to a spatula into a drop of control or
test medium, and more incisions were further made in the tubule to release the
sperm. After removing the empty tubule, the released sperm were dispersed in
200 µL of the same prewarmed medium and incubated at 37°C with 5%
CO2 in air. For the comparison of mature and immature sperm,
samples were taken from 3 regions of the epididymis: the caput from the lobule
just distal to the initial segment (the equivalent gross anatomical site of
the epididymal head in the c-ros knockout mice, which do not have an
initial segment), the corpus region proximal to the narrowest midsegment, and
the cauda at the flexure. These sampling sites correspond respectively to
region II, proximal region IV, and midregion V described by Abe et al
(1983). The order of sampling
and the sequence of test media used were alternated between experiments to
randomize any possible effect of sperm preparation time.
Measurement of Sperm Volume by Flow Cytometry
Changes in individual cell volume were estimated by comparing the laser
forward and side scatter signals of control and treated sperm samples from the
same mouse, using the flow cytometry method established and validated
previously (Yeung et al,
2002a). After 1 minute of incubation for dispersion and at 10, 40,
and 60 minutes of incubation, a 50 µL aliquot of the incubated sperm
suspension was added to 200 µL of the same medium, but without BSA and
containing 3 µL of a propidium iodide solution (PI, 500 µg/mL, final
concentration 6 µg/mL). The sample was analyzed in a flow cytometer
(Coulter Epics XL, version 3.0, Krefeld, Germany) with laser excitation at 488
nm. With cellular debris and aggregates gated out, laser emissions from 10 000
particles were collected. With the use of PI fluorescence signals, sperm were
gated as viable (PI-negative) and nonviable (PI-positive), and the forward and
side scatter signals from viable sperm were analyzed.
Quantification of Sperm Cytoplasmic Droplets
The possibility that sperm cytoplasmic droplets were associated with the
different responses to osmolyte incubation by sperm from the heterozygous and
knockout mice was investigated. Sperm from the caudal region released into
phosphate-buffered saline (PBS), with osmolality adjusted to 420 mmol/kg (that
of cauda epididymal fluid) (Yeung et al,
1999) were immediately fixed with 3% (vol/vol) glutaraldehyde and
examined at 200x magnification for the presence of cytoplasmic
droplets.
Collection of Cauda Epididymal Fluid and Sperm for Assay of Organic Osmolytes
Mice were killed by cervical dislocation after CO2 asphyxiation,
and the cauda epididymis with the proximal vas deferens were isolated. The vas
deferens was cannulated with a drawn-out polyvinyl chloride catheter, and the
epididymal luminal contents were flushed out by retrograde perfusion through a
cut end of the tubule in the proximal cauda region. The perfusion solution was
PBS (Gibco, Berlin, Germany) adjusted to 420 mmol/kg to mimic the osmolality
of caudal fluid. The exudate was taken up into a positive displacement
pipette, and the collections from both sides of the animal were dispersed in
100 µL of medium. Sperm cells were separated by centrifugation at 2000
x g for 2 minutes at 4°C, and the supernatant was
centrifuged again at 2000 x g for 5 minutes before storing the
diluted cauda epididymal fluid at –20°C for use in assays. The sperm
pellet was washed twice with the perfusion medium by centrifugation at 600
x g for 5 minutes, and the number of spermatozoa collected was
estimated by nephelometry (Bone et al,
2000). Sperm pellets were stored at –20°C. To extract
sperm for the assay of organic osmolytes, 120 µL of assay buffer was added
to each freeze-thawed pellet, and the sample was vortexed and sonicated
(1.5-mm tip; Vibra-Cell-Sonicator, Sonics & Materials Inc, Danby, Conn)
with 4- by 1-second ultrasound burst. The supernatant was obtained by
centrifugation at 20 000 x g for 10 minutes at 4°C.
Measurement of Organic Osmolytes in Epididymal Fluid and Sperm
These measurements were taken by fluorometric assays modified for 96-well
plate format. L-Glutamate was measured by estimating the
H2O2 liberated by the action of glutamate oxidase with
Amplex Red reagent (Kit A-12216; Molecular Probes, Leiden, The Netherlands;
Ex = 530 nm, Em = 590 nm). L-Carnitine was
quantified by measuring, as a fluorescent
N-[4-(2-benzimidazolyl)phenyl]maleimide (BIPM) adjunct, the free
coenzyme A liberated by the action of L-carnitine acetyl
transferase (Maehara et al,
1988; Ex = 365 nm, Em = 460 nm). The
myo-inositol assay was developed from that of O'Neill et al
(1998) in a linked enzyme
reaction, in which the NADH+ liberated by the action of inositol
dehydrogenase was quantified after conversion by NADH oxidase to
H2O2 and detection of the latter with Amplex Red
(Ex = 530 nm, Em = 590 nm). To 5-µL (epididymal
fluid) or 50-µL (sperm extract) samples was added 140 µL reaction
mixture containing (final concentrations) 3.5 mM NAD, 0.18 U/mL inositol
dehydrogenase, 0.21 mM flavin adenine dinucleotide, 14.3 mU/mL NADH oxidase,
0.18 µg/mL Amplex Red, and 1.42 U/mL horseradish peroxidase). The samples
were incubated for 60 minutes at 30 °C.
Measurement of K+ Concentration in Epididymal Fluid Using Ion-Selective Electrodes
The measurement of potential differences in drops of samples against
calibration standards with the use of glass capillary microelectrodes was made
as previously described for other ions (Xu
et al, 2003). Potassium ionophore I cocktail A (Fluka Chemicals,
Deisenhofen, Germany) was used to fill the ion-selective electrodes. The
perfusion solution for flushing out epididymal luminal contents contained
trypan blue (12 mg/mL) to ensure collection of uncontaminated samples.
Statistics
Data were analyzed by SigmaStat software (version 2.03; SPSS Inc, Erkrath,
Germany) and presented as mean ± SEM. Differences between the
transgenic and control mice within the same epididymal regions and differences
between regions within each genotype and over the 60-minute incubation time
were tested by 3-way analysis of variance with the Student-Newman-Keuls method
for comparison. The effect of different osmolytes and quinine on aliquots of
the same source of sperm in each experiment was tested statistically against
the controls (expressed as a ratio of control values) by 1-way repeated
measures analysis of variance with the Dunnett method. Differences between
genotypes in osmolyte contents were tested by the Student's t test.
Differences were considered statistically significant at P <
.05.
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Results |
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All of the osmolytes tested, except glycerophosphocholine, caused an increase in the laser forward scatter (Figure 1) during the 60-minute incubation. Dose responses were demonstrated, particularly with K+, taurine, and glutamate, although statistically significant effects by carnitine and myo-inositol were obtained only at the highest dose tested (50 mM). Among these osmolytes, K+ induced the most immediate and the largest effect and was effective already at 10 mM, which was double the concentration in the control medium mimicking serum K+ concentration.
Lack of Effect of Extracellular Putative Osmolytes on Volume of Sperm From c-ros Knockout Mice
At concentrations causing volume increases in sperm from the c-ros
heterozygous fertile mice, none of the osmolytes affected the volume of sperm
released from the cauda epididymis of the infertile c-ros knockout
mice (Figure 2).
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This lack of response to the osmolyte incubation by knockout mouse sperm was not due to the absence of cytoplasmic droplets, where the bulk of sperm cytoplasm is located, because there was no difference from the heterozygous mice in the percentage of sperm bearing cytoplasmic droplets (83% ± 2% vs 77% ± 2%; n = 7).
Contents of Osmolytes in Luminal Fluid and Sperm From the Cauda Epididymidis of Heterozygous and Knockout Mice
The concentrations of K+ in cauda epididymal fluid measured by
ion-selective microelectrodes were significantly higher in the knockout than
the heterozygous mice. The content of organic osmolytes measured by
spectrofluorometric methods showed no difference between the 2 genotypes in
the luminal fluid, but glutamate and inositol contents were lower in the sperm
recovered from the knockout compared with the heterozygous mice as normal
controls (Table).
Comparison of Laser Scatter by Cauda and Caput Spermatozoa From c-ros Heterozygous and Knockout Mice in Basal Medium
Mature sperm from the cauda epididymis of the fertile c-ros
heterozygous mice showed a tendency toward volume decrease after the initial
increase, unlike immature sperm from the caput region, which showed a
continuous volume increase for up to 40 minutes of incubation in basal medium
(Figure 3). By comparison,
cauda sperm from the knockout mice exhibited larger volumes, especially during
the first 10 minutes of incubation, but also showed a decline with time,
whereas caput sperm were smaller than those from the heterozygous mice but
swelled to the same extent with time. Sperm from the corpus epididymis from
both genotypes responded identically, exhibiting larger forward scatter than
the caput and cauda sperm initially, with a slight tendency of decrease over
time (Figure
3).
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Differences in Response to Quinine by Cauda and Caput Spermatozoa From Heterozygous and Knockout Mice
In the presence of the ion channel blocker quinine, mature (cauda) sperm
from the c-ros heterozygous mice manifested marked, immediate, and
persistent dose-dependent increases in laser forward scatter
(Figure 4), whereas cauda sperm
from the knockout mice failed to show any significant response. On the other
hand, immature (caput) sperm from the fertile genotype failed to respond to
quinine with volume increases characteristic of mature sperm. Surprisingly,
the knockout caput sperm responded with a transient increase at 10 minutes of
incubation (Figure 5).
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Discussion |
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When subjected to the osmolality faced in the uterus, caudal sperm from heterozygous males initially swelled and then reduced their volume, as previously demonstrated (Yeung et al, 2002b). That caudal sperm from knockout mice were larger in initial volume but also reduced their volume with time is consistent with the view that they contain a reduced complement of osmolytes available for volume regulation, although defective ion channels in an abnormal plasma membrane cannot be ruled out. Corpus sperm from both genotypes behaved similarly; namely, they were unable to reduce their larger volume over 1 hour, but their volumes did not increase with time, indicating a minor ability to regulate volume. The larger volume might reflect a higher intracellular osmolality compared with cauda sperm. Caput sperm from both genotypes swelled continuously during incubation—even faster for the knockout sperm, which were initially smaller—demonstrating a complete lack of ability to regulate volume as they entered the epididymis from the testis.
The amino acid content of whole epididymal tissue from the mouse is known to be high, with taurine and glutamate among the highest in caput tissue, with glutamate content decreasing and taurine increasing toward the cauda (Kochakian, 1975). Little is known of the nature of luminal osmotic components in the murine epididymis, and most knowledge has come from the rat (see Cooper, 1998), in which L-carnitine (60 mM), myo-inositol (50 mM), GPC (40 mM), and taurine (3 mM) are major osmolytes in caudal fluid: corpus fluid contains 20 mM glutamate and 6 mM taurine, and caput fluid contains 50 mM glutamate and 2 mM taurine. Each of these components has a distinct profile, such that sperm entering the epididymis are bathed consecutively in high concentrations of GPC followed by glutamate and taurine, K+, carnitine, and then myo-inositol (Hinton and Palladino, 1995; Cooper and Yeung, 2003). Even less is known of intrasperm concentrations of epididymal osmolytes: the carnitine content of sperm from many species increases distally (Cooper, 1986), whereas intracellular potassium in the mouse is reported to be 90–120 mM in mature sperm (Babcock, 1983; Chou et al, 1989; Zeng et al, 1995).
From the data presented here, assuming an approximate dilution of 50- to 100-fold when luminal contents were flushed out and dispersed in 100 µL of medium, the corresponding neat concentrations would be 35–70 mM for myo-inositol, 60–120 mM for carnitine, and 0.23–0.45 mM for glutamate, which are values similar to those measured in rats. A difference in provision of osmolytes in this fluid in the infertile c-ros knockout males was not evident because no differences between genotypes were detected for the organic osmolytes expressed per unit protein of fluid. No differences in taurine content of epididymal fluid between genotypes were previously reported by Xu et al (2003), and neither is there any detectable differences in the expression of the epithelial carnitine transporter genes OCT1, OCT2, OCT3, and OCTN2 (Cooper et al, 2003). By contrast, higher K+ concentrations in cauda epididymal fluid were detected in the infertile knockout mice. Thus, the only detectable change in caudal fluid from the mutant males with compromised fertility was an increase, rather than decrease, in a potential osmolyte, which nevertheless indicates abnormal epithelial function in the c-ros knockout male. It is tempting to speculate that the increased extracellular K+ concentration leads to cellular K+ uptake and swelling, as shown in other cells (Lang et al, 1998). This in situ swelling might then inhibit the cellular accumulation of organic osmolytes, such as glutamate and myo-inositol (see the Table). It could be the lack of these osmolytes that leads to deranged cell volume regulation and function of sperm from the knockout mice.
In somatic cells, the predominant osmolyte and the mechanism of volume regulation can vary under different conditions and is dependent on cell type. In cardiomyocytes, RVD induced by drastic hypo-osmotic change is achieved mainly by taurine efflux, whereas in isovolumetric regulation (IVR) with gradual osmolality decrease, K+ loss is predominant (Souza et al, 2000). However, in hippocampal tissue, IVR does not involve K+ but mainly taurine efflux, whereas RVD is associated with loss of glutamate, taurine, and K+ (Franco et al, 2000). The nature of osmolytes used by sperm is unknown, but hyperosmotic stress in chimpanzee sperm can be alleviated by 2 mM taurine (Ozasa and Gould, 1982), suggesting that it might be a physiological osmolyte taken up by sperm during the sojourn of increasing osmolalities in the epididymis.
In this study, when cauda sperm were subjected to a physiological 90 mmol/kg decrease in extracellular osmolality, K+ was the most effective extracellular osmolyte tested that caused swelling of murine mature sperm and induced the fastest response. Myo-inositol and L-carnitine at assumed physiological concentrations were able to sustain high cell volumes over 40 minutes, whereas cell volumes began to decline in the presence of supraphysiological concentrations of glutamate and taurine, suggesting that other osmolytes were operating to maintain volume. GPC had no effect on sperm volume, probably because it is impermeant, as it is for renal cells (Zablocki et al, 1991). These positive responses in the induction of swelling from almost all the osmolytes tested suggest that murine sperm can use a number of different molecules for volume regulation.
The failure of knockout cauda sperm to respond to the osmolytes tested could be because the sperm are already swollen (Yeung et al, 2002a). The same argument would explain the resistance of c-ros knockout sperm to swelling induced by quinine. Normal immature sperm from the caput swelled in the basal medium and did not respond to quinine because the volume regulation mechanism is largely undeveloped. Although quinine enhanced the swelling of caput sperm from the knockout mice at 10 minutes, this effect was not sustained at later time points. This could mean that because the knockout sperm had been in the caput environment longer than the normal sperm because of the missing initial segment, they might have started the early stages of the development of volume regulation mechanism but failed to complete it normally because of epididymal malfunction. The swollen status of the knockout cauda sperm in the basal medium could be a consequence of abnormal osmolyte uptake in the mutant epididymis, and indeed, glutamate and myo-inositol were decreased in sperm from the knockout males, although the carnitine and taurine levels (Xu et al, 2003) within spermatozoa were not different between genotypes.
This study demonstrates that major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge. This capacity of volume regulation is developed during the sojourn in the epididymis and is important for normal sperm function in the female tract. The infertile c-ros knockout mouse sperm were found to have less sperm glutamate and myo-inositol, despite normal concentrations in epididymal fluid. Insofar as this animal model can be useful for investigating the relationship between epithelial and sperm function, for purposes of developing a contraceptive for males, these observations suggest that attacking the sperm channels to limit the uptake of epididymal osmolytes might be more effective than targeting the epithelial transporters in order to limit the provision of luminal secretions to the sperm. This reiterates findings in rats and hamsters that reducing epididymal carnitine by increasing excretion of pivalolyl carnitine does not lead to infertility or reduce sperm motility because sperm carnitine was unaltered (Cooper et al, 1997; Lewin et al, 1997).
Because K+ and quinine consistently provided the most rapid and extensive swelling responses, K+ could be a major regulator of sperm volume. Again, the nature of the channels used by sperm in mediating osmolyte influx and efflux in the male and female tracts requires investigation and could be controlled by secretions of the initial segment. Such elucidation of sperm volume regulation mechanisms contributes to the understanding of infertility and the development of new male contraceptives because volume regulation has been demonstrated in human sperm and swollen sperm have altered motility pattern that hinders mucus penetration (Yeung and Cooper, 2001; Yeung et al, 2003).
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Acknowledgments |
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Footnotes |
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A M Petrunkina, D Waberski, A R Gunzel-Apel, and E Topfer-Petersen Determinants of sperm quality and fertility in domestic species Reproduction, July 1, 2007; 134(1): 3 - 17. [Abstract] [Full Text] [PDF]
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A M Petrunkina, R A P Harrison, M Tsolova, E Jebe, and E Topfer-Petersen Signalling pathways involved in the control of sperm cell volume Reproduction, January 1, 2007; 133(1): 61 - 73. [Abstract] [Full Text] [PDF]
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 Di Zhang and M. Gopalakrishnan Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines? J Androl, November 1, 2005; 26(6): 643 - 653. [Full Text] [PDF]
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J.P. Barfield, C.H. Yeung, and T.G. Cooper The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa Biol Reprod, May 1, 2005; 72(5): 1275 - 1281. [Abstract] [Full Text] [PDF]
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) obtained at the same incubation time
point.
