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Regular Article |
a Institute of Reproductive Medicine of the University, D-48129 Münster, Germany
b Department of Physiology, Institute of Biomedicine, University of Turku, 20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gamete biology, male reproductive tract, sperm maturation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another transgenic mouse model with a sperm phenotype similar to that of c-ros deficient mice is the GPX5-Tag2 mouse [6]. GPX5-Tag2 transgenic mice are generated by directing expression of the simian virus 40 (SV40) large T-antigen (Tag) into the epididymis using the promoter of murine glutathione peroxidase 5 (GPX5), a protein expressed specifically in the caput epididymidis [7]. Males grow to healthy adults and express SV40 Tag along the epididymis. They have normal reproductive hormones; apparently normal testicular histology, sperm production, motile epididymal sperm; and normal mating behavior, but they are infertile [6]. Angulation of the sperm tail, as described in infertile c-ros knockout mice, appears in these transgenic mice within the epididymal canal, first in the proximal corpus and more frequently in the distal regions. More than 90% of sperm in the cauda epididymidis show this tail defect, and upon release and dilution, >80% display extreme tail angulation into a hairpin shape. In addition, osmolality of the cauda epididymidal fluid is around 40 mmol/kg lower in GPX5-Tag2 mice than in wild-type mice [6].
A quantitative, objective, flow cytometric method has been validated using electronic sizing analysis for the direct measurement of changes in the volume of viable murine spermatozoa [8]. Sperm volume is reflected by the intensity of forward scattered laser signals, as occurs with somatic cells. Induction of swelling by quinine, the ion-channel blocker, leads to an increase in mean forward scatter and the appearance of a subpopulation of sperm exhibiting a large side scatter signal (LSS). The size of the LSS population, as well as the mean forward scatter, are both correlated to the percentage of sperm with angulated tails.
The first aim of the present study was to use cell volume measurements to provide evidence for the involvement of sperm swelling in the angulation of the sperm tail in both transgenic mouse models mentioned above. The novel aspect of sperm maturation in the regulation of cell volume was also investigated. In transgenic mice, failure in sperm transport from the uterus to the oviduct is caused by angulated tails of presumably swollen sperm. In this respect, hindrance of transport of swollen human sperm through surrogate cervical mucus has recently been demonstrated [9]. In that study, the increase in sperm volume induced by quinine was associated with both coiling of the tail and changes in motility pattern. Furthermore, sperm kinematics, as quantified by computerized analysis, was much more sensitive to quinine than the manifestation of coiled tails. For this reason, both sperm kinematics and tail angulation were investigated in the present study, together with sperm volume increases in response to quinine, in order to understand the relationship of these 3 factors in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transgenic c-ros mice born from C57BL6 and Ola129 parents [1] were bred and genotyped as previously described [8]. Adult heterozygous and homozygous knockout mice of about 4 mo of age were used. Sperm from the heterozygous animals, which are normal in phenotype and fertility, were used as controls. GPX5-Tag2 transgenic mice were generated using FVB/N mice as genetic background and were genotyped using polymerase chain reaction [6]. Adult mice of 3–7 mo of age were used, and wild-type FVB/N males served as controls. Present experiments using these animals were conducted according to German federal law on the care and use of laboratory animals (license 41/98).
Incubation Media
We used Biggers Whitten Whittingham (BWW) media [10] containing BSA at 4 mg/ml with the osmolality adjusted with NaCl. In the study of sperm maturation in c-ros mice, BWW had the same osmolality as the uterine contents of wild-type mice (330 mmol/kg; BWW330) in which sperm tail angulation of the knockout mice was observed [2]. Because such measurements in FVB/N females are not available, and sperm tail angulation in GPX5-Tag2 transgenic mice already exists in the epididymis, the medium we used in the maturation study of GPX5-Tag2 mice was cauda epididymidal fluid of GPX5-Tag2 mice (360 mmol/kg), in order to eliminate in vitro volume changes of cauda sperm due to osmotic differences. To study the effects of quinine using c-ros control mice, BWW330 and BWW290 were used. The latter osmolality is the same as conventional isotonic medium and the same as that used for conventional mouse in vitro fertilization (IVF). Quinine (Sigma, Taufkirchen, Germany) was made up in a 100 mM aqueous stock solution and diluted into BWW medium to give 50, 200, 400, or 800 µM just before use.
Sperm Preparation
Mice were anesthetized with 0.6 ml of 2.5% (v/v) Avertin (Sigma). The epididymis was dissected out and cleaned of blood. Sperm were sampled 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 c-ros knockout mice, which do not have an initial segment), the corpus region proximal to the narrowest mid segment, and the cauda at the flexure. From each site, a small segment of the tubule was excised and transferred on a spatula into a drop of test medium. One or more further incisions were made in the tubule to release the sperm, which were dispersed in 200 µl of the same, prewarmed medium and incubated at 37°C with 5% v/v CO2 in air.
For each parameter, one analysis was made with each treatment of sperm prepared from each of the 3 epididymal regions of 1 mouse. Different doses of quinine (see below) were applied on different aliquots of the sperm obtained from the same epididymal segment of the same animal. Similar experiments using wild-type and transgenic mice were done on the same days (for all GPX5-Tag2 mice and some c-ros mice) or on different days (for some c-ros mice). The number of mice used for each experiment is given in the Results section and in the figure legends (see Statistics for data analysis).
Measurement of Sperm by Laser Scatter Using Flow Cytometry
This method has been validated using electronic sizing for direct volume measurement [8]. A good linear correlation is obtained for the 2 methods. The flow cytometric method is preferable because it can distinguish between live and dead sperm, and it is superior in sensitivity and stability, is quicker and easier to operate, and allows more efficient use of sperm samples. However, the voltage of the photomultiplier tube necessary for detecting the minute volume changes under investigation did not allow construction of a standard curve using standard size beads. Therefore, results are given in units of forward laser scatter (channel number) as measured, instead of units of volume.
After 1 min of incubation for dispersion and at 10, 40, and 60 min 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 (PI) solution (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, the forward and side scatter signals from 10 000 particles were collected. Using the fluorescence signals from PI, sperm were gated as viable (PI negative) or nonviable (PI positive), and their forward and side scatter signals were analyzed.
Evaluation of Sperm Tail Morphology
Aliquots (13 µl) of incubated sperm were examined with phase contrast microscopy at 200x magnification at 20 and 50 min. The shape of the tail from 2 x 100 spermatozoa was examined as an indication of cell swelling. When the 2 counts gave unacceptably different results (as recommended by the World Health Organization [11]), a third count was made before calculating the mean. Sperm were classified into 3 different categories based on the extent of angulation of the flagellum: straight form (no angulation), hairpin form, and any degree of angulation in between (see Fig. 1 for GPX5-Tag2 mice; see micrographs in [3] for c-ros mice). Each spermatozoon was also noted as motile or immotile.
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Analysis of Sperm Kinematics
At 20 and 50 min of incubation, 13 µl of sperm was placed on a siliconized slide with a 18 ;ts 18 mm coverslip to give a chamber depth of 40 µm. Motility of the sperm was examined at 37°C using pseudodarkfield optics and recorded for 1 min with a 4x objective, a 40x condenser ring, and 3.3x photo-ocular, covering about 10 microscopic fields. Kinematic parameters of >200 motile sperm from each sample were analyzed using a computer-assisted sperm analysis system (IVOS, version 10.8, Hamilton Thorne Research, Beverly, MA) that tracked motile sperm for 60 frames at a frame rate of 50 Hz.
Statistics
Data were analyzed using SigmaStat computer software (version 2.03, SPSS Inc., Erkrath, Germany). Differences between GPX5-Tag2 transgenic and control mice within the same epididymal regions, and differences between regions within each genotype, were tested using two-way ANOVA with the Student-Newman-Keuls method. Differences between c-ros knockout and heterozygous controls were analyzed similarly. The effect of different doses of quinine on aliquots of the same source of sperm in each experiment were tested statistically against the controls using one-way repeated measure ANOVA with the Dunnett method. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clear changes in the distribution of output signals from the forward and side scatter of laser light were observed among sperm sampled from different regions of the epididymis and were examined immediately after dispersion (Fig. 2). As reflected in the forward scatter signals, the cell volume of corpus sperm from wild-type mice was greater than those of caput and cauda sperm. In GPX5-Tag2 mice, immature caput sperm had the same cell volume as wild-type mice. However, the more-mature sperm from the corpus and cauda were both larger in size in infertile transgenic animals than in wild-type mice, showing no shrinkage during complete maturation and storage (Figs. 2 and 3, left panels). These changes in forward scatter signal were associated with changes in the side scatter signal, producing a conspicuous subpopulation of sperm with a large side scatter signal (LSS) in the corpus region of wild-type mice (80%–90% of GPX5-Tag2 corpus and cauda sperm exhibited LSS; Figs. 2 and 3, left panels). Caput sperm from some GPX5-Tag2 mice showed a small but discernible LSS population (e.g., Fig. 2), but statistically, there was no difference from wild-type controls (Fig. 3).
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Volume Changes on Maturation in c-ros Transgenic Mice
As was found for wild-type FVB/N mice, the initial volume of corpus sperm in both fertile heterozygous (+/-) and infertile c-ros knockout (-/-) mice was larger than that of caput sperm, whereas fully matured sperm, during storage in the cauda region, were smaller in initial volume (Fig. 3, right panel). Corpus sperm from both c-ros genotypes (+/- and -/-) did not differ in cell volume. However, caput sperm from knockout (-/-) mice were smaller, and cauda sperm were larger, than sperm from the same regions in control (+/-) mice. Differences in cell volume were associated with similar changes in LSS population size (Fig. 3).
Changes in Volume on Incubation and Maturational Status
In control mice (c-ros +/-), caput sperm steadily increased in volume when incubated until a plateau was reached at 40 min, such that they were larger than more-mature sperm after 1 h (Fig. 4a). Cauda sperm, which showed the smallest initial volume, showed a slight increase in size during 10 min, then showed a tendency to decrease slightly when incubated further. Corpus sperm manifested the largest volume initially and also showed a tendency toward a slight decrease with time, but remained larger than the cauda sperm throughout. These changes during 1 h of incubation were mirrored by changes in the size of the LSS subpopulation, except at the end of incubation (Fig. 4b). By 60 min, forward scatter signals were higher for caput sperm than for corpus sperm, which in turn were higher than for cauda sperm, whereas both caput and corpus sperm displayed a similarly high percentage of LSS cells, which was higher than that of cauda sperm.
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Different Responses of Control (c-ros +/-) Mature and Immature Sperm to the Channel Blocker Quinine
Quinine in the incubation medium (osmolality at 330 mmol/kg) at 200–800 µM caused a dose-dependent increase in the volume of control mature sperm as reflected by the laser forward scatter signal (cauda sperm in Fig. 5). The effect was maximal by 10 min and was maintained over 60 min. Over this period, the size of the LSS subpopulation intensified with time. Unlike mature sperm, which largely maintained their laser scatter characteristics during incubation, sperm from the caput epididymidis showed a gradual increase in forward scatter as well as side scatter. These parameters were not affected by the presence of quinine throughout incubation.
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Effect of Quinine on Tail Morphology of Mature Sperm
During 50 min of incubation in the control media at both osmolalities of 290 and 330 mmol/kg, the majority of spermatozoa maintained a straight tail, which always existed in higher percentages among motile cells than in the entire sperm population (Fig. 6). Quinine at 50–800 µM showed a dose-dependent effect on the shape of the sperm tail, causing it to bend at the site of the cytoplasmic droplet to angles of various magnitudes. The maximum angulation was expressed as a hairpin shape with the looping mid piece/principal piece junction forming the leading end when spermatozoa swam, and this tail form predominated among the affected sperm. Tail angulation was detectable at 20 min and increased with the time of incubation in media at both osmolalities of 290 and 330 mmol/kg, indicating greater effects with lower extracellular osmolality.
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Effect of Quinine on Kinematics of Mature Sperm
Aliquots of the same preparations used for the morphology study described above were examined. Quinine at 50 and 200 µM did not affect percentage motility of sperm during 50 min of incubation. At 800 µM, there were decreases of around 10 percentage points in medium of 290 mmol/kg at 20 min (from 52% ± 1% to 43% ± 4%) and 50 min (from 50% ± 1% to 41% ± 2%). Similar decreases were also found in medium at 330 mmol/kg at 20 min (from 53% ± 4% to 41% ± 3%) and 50 min (from 50% ± 3% to 33% ± 5%).
On the other hand, kinematics of motile sperm were more sensitive to treatment with quinine. At 50–800 µM, quinine showed dose-dependent decreases in straight-line velocity (VSL) of motile sperm without affecting their curvilinear velocity (VCL), resulting in dose-dependent decreases in the linearity (LIN) of the swim path. The maximal responses achieved at these concentrations were a decrease of 43% in both VSL and LIN (Fig. 7). Such effects were already discernible at 50 µM in the medium of 290 mmol/kg, but were only significantly expressed at 800 µM in the medium of 330 mmol/kg, amounting to decreases of around 25%. However, the effects manifested at 20 min of incubation were no longer obvious at 50 min except with the highest dose of quinine in low-osmotic medium. No effects were detected on the other kinematic parameters, including amplitude of the lateral head displacement and beat cross-frequency.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In control mice from both transgenic colonies (wild-type FVB/N and c-ros +/-), the main profile of maturational changes was the same. Sperm from the corpus were larger than those from the caput and cauda epididymidis when examined immediately after dispersion into medium. Cauda epididymidal fluid osmolality is much higher than that of serum (see [4]), and regional changes in luminal fluid osmolality have been reported in rats and hamsters [12, 13], in which the corpus has the highest value. As sperm are transported along the epididymis slowly enough to allow osmotic equilibrium between them and the surrounding fluid, corpus sperm are anticipated to have the highest intracellular osmolality. Because it took a minimum of about 3 min for sperm release, dispersion, and final dilution for flow cytometric measurement, it was impossible to measure sperm volume in their in situ status. Nevertheless, sperm volume measured in the physiologically hypotonic incubation medium employed here should reflect the net result of water influx, driven by the osmotic gradient, and subsequent water removal that accompanies the efflux of intracellular osmolytes by the mechanism of RVD induced by initial swelling. The initial volume measurements should therefore mainly reflect intracellular osmolality, which determines the rate and extent of water influx. This implies that the epididymis of the mouse also has the highest luminal and sperm osmolality in the corpus region, as in rats and hamsters.
During incubation, the volumes of both corpus and cauda sperm were first maintained or slightly increased, then decreased gradually after 10 min. The subsequent gradual volume decrease in corpus and cauda sperm is consistent with the initial triggering of RVD by swelling, which then dampened further volume increase. By contrast, the continuous steep increase in caput sperm volume during incubation demonstrates the lack of RVD to counteract the osmotic swelling. As a result, after 1 h of incubation, caput sperm were larger than corpus sperm, which were larger than cauda sperm. This is the same order as the extent of tail angulation during incubation observed in the previous study [3].
There is almost no information in the literature on comparative sizes of mature and immature sperm. The most direct data have been obtained in rats by electronic sizing using the Coulter counter, which show that cauda sperm dispersed in a medium of around 300 mmol/kg are 10% smaller than caput sperm [14]. This is close to the present findings of an 8% difference between the initial volumes of cauda and caput sperm from fertile control c-ros mice. There are no data on electronic sizing of corpus sperm. Hamster sperm yield 2 bands of distribution after 30 min of centrifugation through continuous Percoll density gradients, presumably depending on the cell volume [15]. Sperm originating from the head, body, and tail regions of hamster epididymides show an increasing prominence of the denser of the 2 bands. Such differences in specific gravity of mature and immature sperm are consistent with the present finding of a decrease in volume from caput, corpus, to cauda sperm after incubation.
The common features of the c-ros knockout and the GPX5-Tag2 transgenic male mice are their infertility and the manifestation of tail angulation by dispersed cauda epididymidal sperm. The present data have proved for both models that infertile cauda sperm are larger than those of fertile controls, and exhibit larger then normal LSS subpopulations, which are correlated with tail angulation and forward scatter, reflecting cell size [8]. The greater extent of abnormalities in GPX5-Tag2 mice substantiates previous observations of their more severe tail angulation than c-ros knockout mice when diluted [3, 6]. GPX5-Tag2 sperm also demonstrate a much more obvious swelling within the epididymis: whereas 80% of GPX5-Tag2 sperm within the cauda epididymidis exhibit some form of tail angulation (versus 40% in the c-ros knockout), those showing acute or extreme angulation are 80% vs. 20%, respectively. Additional differences between infertile c-ros and GPX5-Tag2 mice include 1) the absence of the initial segment in the c-ros knockout but not in the GPX5-Tag2 mice, and 2) the normal osmolality of the cauda luminal fluid in the former, but lower osmolality in the latter [6]. Therefore, there are probably different causes for their common defects in sperm volume regulation. It is likely that in c-ros knockout mice, epididymal dysfunction starts more proximally, because caput sperm were smaller than normal controls. The reduced osmolality within the cauda epididymidis of GPX5-Tag2 mice may explain the greater extent of swelling in this genotype.
The channel blocker quinine caused consistent swelling of cauda sperm as reflected in both forward scatter and LSS population sizes in a clearly dose-dependent manner, and with effective dosages that were comparable to those that act on somatic cells (see [16]). Exhibition of a maximal effect within 10 min suggests a normally rapid onset of the RVD mechanism in mature sperm. On the other hand, caput sperm volume did not respond to quinine, suggesting the lack of RVD in normal immature cells.
In addition to the effect on sperm volume, quinine in the incubation medium resulted in dose-dependent increases in the number of sperm with tail angulation. The minimum effective concentration for this was 200 µM. The extent of the disruption of tail morphology increased with time and was intensified by lower osmolality of medium. A third effect of quinine, at a minimal dose of 50 µM, was on the pattern of movement, but it did not affect the percentage of motile sperm. The decreases in VSL and LIN of progression were not accompanied by any changes in VCL, which suggests that energy metabolism was not affected, but that translation of axonemal microtubule sliding into flagellar bending may be modified. According to the geometric clutch theory [17, 18], this translation of force is dependent on the resistance elements of the structures peripheral to the axoneme, which include the arrangement of outer dense fibers and the plasma membrane. It can readily be envisaged that an increase in cytosolic volume would alter the geometry of flagellar bending and the efficiency of cellular forward propulsion.
Whereas the effect of quinine on sperm volume was maintained throughout incubation, there was a time-related increase in quinine effect on tail morphology but a decrease in the effect on kinematics. These observations are consistent with the hypothesis that as the spermatozoon starts to swell, water moves in along the length of the cell increasing the flagellar diameter, and affects the kinematics. With time, the increase in volume accumulates in the cytoplasmic droplet, which is the main site of water influx, because it holds the most cytoplasm. This would cause a bending of the flagellum into an angle to prevent overstretching of the droplet membrane while accommodating the higher volume. The faster onset and lower minimum quinine concentration for the kinematic effects were also borne out in a previous study of human ejaculated sperm [9].
Despite much research on the mechanisms of volume regulation in somatic cells, which involve both K+ and Cl- as well as organic anion channels and amino acid transporters (see [5, 19, 20]), investigations on sperm cells are rare. Early studies on bovine ejaculated sperm suggested a role of Na+/K+-ATPase and modulation by Ca2+ [21, 22], and more recent reports identified quinine-sensitive K+ channels in bull [23], boar [24], and human sperm [9]. Quinine is effective in blocking a variety of channels, including cation, anion, and organic ion channels [16, 25]. Epididymal secretions such as myo-inositol, L-carnitine, and amino acids are common organic osmolytes used by renal and neural cells ([26, 27]). It is likely that the increasing osmolality of the luminal fluid from the caput to the corpus region would induce uptake of these osmolytes by the maturing sperm (to prevent dehydration), which could use them for RVD upon ejaculation.
In conclusion, the present study has demonstrated that sperm at different stages of maturation in the epididymis attain different cell volumes when released and diluted. This volume reflects 1) the in situ intracellular osmolality, which depends on the luminal milieu and osmolyte loading; and 2) the ability to undergo regulatory volume decrease, which is acquired during epididymal maturation. Disruption of sperm volume regulation would alter movement patterns, compromising forward progression, and lead to angulation of the sperm tail, which would hinder the transport of sperm in the female tract and may be of contraceptive significance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Deutsche Forschungsgemeinschaft, grant FOR197/3-1 for "The Male Gamete: Production, Maturation, Function"; and by the AMPPA Project, sponsored by the Rockefeller and Ernst Schering Research Foundations. 
2 Correspondence: C.H. Yeung, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany. FAX: 49 251 835 6449; yeung{at}uni-muenster.de
Accepted: February 8, 2002.
Received: December 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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