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ABSTRACT: Volume regulation by spermatozoa has been demonstrated to be crucial in both mice and men for transport in the female tract. In order to determine the nature of osmolytes used by spermatozoa, they were released from the cauda epididymis of fertile c-ros heterozygous mice into incubation medium of uterine osmolality (representing an osmotic challenge), containing increasing concentrations of compounds that are major epididymal fluid components and known osmolytes in somatic cells. This should nullify the concentration gradients for osmolytes that mediate volume regulation, prevent osmolyte efflux, and lead to swelling. Of the osmolytes tested, K+ caused the most rapid and extensive volume increases; glutamate, taurine, l-carnitine, and myo-inositol also were effective, but glycerophosphocholine was not. Such effects were not observed in cauda sperm from the infertile knockout mice, demonstrating a defect in normal volume regulation. K+ concentrations in cauda epididymal fluid were 21 mM higher in the knockout than the heterozygous mice, but no differences were found in caudal fluid glutamate, carnitine, or myo-inositol. The carnitine content of cauda sperm from knockout males was not different from that of fertile males, but lower amounts of glutamate and inositol were found that could explain the poor volume regulation. In heterozygous mice, cauda but not caput sperm responded to the K+ channel blocker quinine by swelling, demonstrating development of volume regulation during epididymal transit, whereas knockout cauda sperm showed no response, as with the osmolytes. Major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge in the normal fertile mice; the reduced sperm content of inositol and glutamate in the c-ros knockout mice might reflect maturational abnormalities in volume regulation.
Anovel cause of male infertility in 2 transgenic mouse models is the angulated tails of spermatozoa that fail to negotiate the uterotubal junction and hence reach the site of fertilization (Yeung et al, 2000; Sipilä et al, 2002). Tail angulation reflects a volume increase in the spermatozoa (Yeung et al, 2002a,b) caused by an inadequate regulatory volume decrease (RVD), which is normally initiated after osmotic entry of water in hypotonic environments. Although somatic cells rarely experience this phenomenon (O'Neill, 1999), it is a normal occurrence for sperm upon ejaculation when they are rapidly expelled into the uterus (osmotic pressure [OP] around 330 mmol/kg) (Yeung et al, 2000) from the relatively hypertonic environment of the cauda epididymis (OP around 420 mmol/kg) (Yeung et al, 1999). In RVD, the response is to lose cell water in parallel with efflux of osmolytes through pertinent membrane channels. In the early 1970s, studies of bovine ejaculated sperm incubated in medium with osmolality identical to serum showed that Na+ and K+ close to serum levels, Ca2+, and serum albumin as well as metabolic factors all contributed to the maintenance of sperm volume stability (Bredderman and Foote, 1971a,b,c). More recently, potassium has been associated with sperm volume regulation in bulls (Kulkarni et al, 1997; Petrunkina et al, 2001), boars (Petrunkina et al, 2001), mice (Yeung et al, 2002a), and humans (Yeung and Cooper, 2001). Besides these reports, little is known about other inorganic or organic osmolytes or osmolyte channels for spermatozoa.
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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Although sperm volume regulation and its association with fertility has been highlighted in the infertile c-ros knockout mice, it can be envisaged that multiple factors should be involved because the lack of a differentiated epididymal initial segment would render the luminal milieu abnormal and, hence, affect various aspects of maturational changes in sperm. This study concentrated on sperm volume regulation. The working hypothesis was that sperm migrating through the epididymis are gradually confronted with increases in osmolality and consequently take up osmolytes that subsequently would be lost in RVD in the female tract (see the Introduction). These osmolytes should be present at high concentrations in the epididymal lumen, and their identities would be revealed if spermatozoa were to swell in media containing these molecules at concentrations approaching intrasperm levels that prevent their diffusional efflux. In the c-ros knockout males, sperm osmolytes are anticipated to be limiting.
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).