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From the * Institute of Reproductive Medicine and
the Department of Obstetrics and Gynaecology
of the University of Münster, Germany.
| Correspondence to: Dr T. G. Cooper, Institute of Reproductive Medicine of the University of Münster, Domagkstrasse 11, D-48129 Münster, Germany. |
| Received for publication July 18, 2005; accepted for publication September 27, 2005. |
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Abstract |
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Key words: Fathers, mucus penetration, sperm swelling, semen analysis, diagnosis
In mice, CDs located at the end of the midpiece are the site of water entry, so in cases of defective volume regulation, the resultant swelling initiates flagellar angulation leading to hairpin-bend morphology (Yeung et al, 1999). This is a characteristic that can be mimicked by blocking the cation and anion channels involved in volume regulation (Yeung et al, 2002, 2005; Barfield et al, 2005) and which leads to failure of spermatozoa to reach the oviduct (Yeung et al, 2000). Osmotically sensitive "mid-piece vesicles" (MPVs) were described by Abraham-Peskir et al (2002) and Chantler and Abraham-Peskir (2004), our interpretation of which is that they are grossly swollen CDs (Cooper et al, 2004) categorized by the former authors as MPVs only when they extend the length of the midpiece.
Human ejaculated spermatozoa swell when presented with a physiologically relevant hypo-osmotic challenge (Yeung and Cooper, 2001) of cervical mucus osmolality (286 mmol/kg: Rossato et al, 1996), unless they are able to perform regulatory volume decrease (RVD). As quinine blocks the channels employed for osmolyte efflux during volume regulation (Yeung and Cooper, 2001; Yeung et al, 2003, 2005), a simple test of the volume-regulating status of spermatozoa is to monitor their size in the presence and absence of quinine. This drug would prevent RVD, leading to a larger volume only for cells capable of undergoing RVD. The smaller the difference in size in the presence and absence of quinine, the lower potential of the cell for regulating volume.
In this work, experiments were performed to examine the relationship, if any, among CDs, volume regulation, and mucus migration of spermatozoa from patients, volunteers, and fathers.
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Materials and Methods |
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Measurement of Osmotic Pressure and Composition of Media
The osmolality of 10 µL of fluids was measured with a vapor pressure
osmometer (Wescor Vapro 5510, Kreienfeld Scientific Measuring Systems,
Langenfeld, Germany) using no delay for media or a 2-minute delay to ensure
chamber saturation and accurate results for liquefied semen. Various
modifications of Biggers, Whittem, Whittingham
(Biggers et al, 1971) media
(BWW) were prepared with osmolality altered by addition or removal of NaCl and
inclusion of 20 mM HEPES, pH 7.4, in addition to the bicarbonate buffer: BWW
of 320 mmol/kg (BWW320) was used to mimic the osmolality of freshly
liquefied semen and to dissolve quinine in experiment 1; BWW230 was
used in experiment 1 to reduce semen osmolality to that of mucus and in
experiment 2 to achieve with BWW390 the desired osmolality of
surrogate mucus; and BWW290 was used in experiment 3 to mimic the
osmolality of human cervical mucus (286 mmol/kg:
Rossato et al, 1996) in the
preparation of Percoll. The osmolalities of surrogate mucus of hyaluronic acid
(6 mg/mL: Tang et al, 1999;
from rooster comb, Sigma H5388) and 4 mg/mL bovine serum albumin were 311
± 3 mmol/kg and 267 ± 3 mmol/kg.
Experiment 1: Effect of Osmolality on Percentages of Cytoplasmic Droplets
These experiments were done in order to establish if CDs were more easily
detected on sperm in semen of lower osmolality or when volume regulation was
blocked by quinine.
Patients with sperm concentrations of more than 10 x 106/mL supplied semen for this study. For microscopic observations of spermatozoa in wet preparations, either quinine in BWW320 (10 µL stock) or BWW320 alone (10 µL as control) was added to 100 µL liquefied semen. After a 15-minute incubation at 37°C in 5% (vol/vol) CO2, a 3-µL aliquot was placed under a 22 x 22-mm cover slip and examined by phase contrast microscopy (Olympus BH-20, Tokyo, Japan) with a 40x objective and 10x eyepiece on a heated stage at 37°C (Mini-Tüb, Tiefenbach, Germany). The percent motility (WHO grades a+b+c) was determined, and immotile and motile spermatozoa were separately assessed for the presence of I) CDs (regular distensions at the neck or along the midpiece), II) residual cytoplasm (large, irregular material along the midpiece), III) coiled or looped tails, or IV) none of the above categories (Cooper et al, 2004).
Similar preparations were examined in which semen was mixed with appropriate volumes of BWW230 to reach a final osmolality of 290 mmol/kg before the incubation with or without a final concentration 0.27 mM quinine. An equal volume of 7% (vol/vol) glutaraldehyde was added to 10 to 20 µL of these samples, and after 60 minutes at room temperature, the fixed cells were washed by addition of 1 mL phosphate-buffered saline and centrifuged at 500 x g for 5 minutes. The fixed pellet was examined as a wet preparation at 400x magnification for the presence of categories I through IV on the previous page. Centrifugation of fixed cells did not influence the percentages of CDs observed.
To assess if CDs were related to the regulation of cell volume, cell size (given in channel number) was measured by flow cytometry on other aliquots after dilution with the same medium.
Experiment 2: Sperm Migration Through Surrogate Mucus
These experiments were done to ascertain if the presence of CDs would
hinder the migration of spermatozoa through fluid of cervical mucus viscosity
and osmolality.
Twenty-one patients with sperm concentrations of more than 10 x 106/mL, 3 healthy volunteers, and 7 fathers provided semen for this study. The osmolality of the ejaculates after about 30 minutes' liquefaction was 321 plus or minus 4 mmol/kg.
Flat capillary tubes (10 cm long, 3 mm wide, and 0.3 mm deep; Camlab, Cambridge, United Kingdom) were filled with the mucus. One end was sealed with plasticine and the other end was inserted into an Eppendorf tube containing 75 µL liquefied ejaculate and incubated horizontally in a moist chamber at 37°C and 5% (vol/vol) CO2 in air for 90 minutes. The number of sperm at 1 cm (representing penetrated sperm) and at 4 cm (representing migrated sperm) was counted, and the migration distance of vanguard sperm (the most distal field containing the most advanced 10 cells) in a low-power microscopic field (10x objective and 10x eyepiece) was measured.
In order to examine the presence of droplets on these spermatozoa, the capillary was scored with a diamond knife and snapped at 1 and 4 cm, a drop of the mucus was expressed by blowing the contents onto a slide, and 2 x 100 spermatozoa were evaluated with a 40x phase contrast objective. To test for the effect of mucus alone on the presence of CDs, 4-µL aliquots of the ejaculates were mixed with 20 µL mucus by stirring with the pipette tip and incubated in parallel with the capillary and analyzed at the same time point for the presence of CDs. For determination of percentages, 100 cells were counted in duplicate, except in cases in which fewer than 100 cells were seen, in which case all were counted.
Experiment 3: Volume Regulatory Capacity of Spermatozoa in Physiologically Hypotonic Medium
These experiments were done to determine if the regulatory response of
spermatozoa from recent fathers to a physiologically relevant osmotic
challenge differed from that of patients.
Aliquots of 200 µL freshly liquefied semen from patients or fathers were diluted in 200 µL BWW290 with or without a final concentration of 0.27 mM quinine and were incubated for 5 minutes at 37°C. To remove non-sperm particles, the semen was then washed through Percoll 30% (vol/vol)/60% (vol/vol) made up in modified BWW290 with or without 0.27 mM quinine; we gently layered the sperm sample over 1.4-mL Percoll in a 2-mL Eppendorf tube and centrifuged at 700 x g for 20 minutes. In this way, the osmotic challenge with and without quinine was already present during centrifugation.
Preliminary experiments revealed that this washing procedure did not select for quinine-responding or nonresponding spermatozoa. After removal of the supernatant the sperm pellet (about 50 µL) was then resuspended and the cell volume measured by flow cytometry after diluting a 5-10-µL aliquot in 200 µL of the same incubation medium. Measurements were also made on these washed samples after 5-minute demembranation by addition of 20-µL sample to 5 µL 10% (vol/vol) Triton X-100.
Flow Cytometry
Laser forward scatter by flow cytometry, as previously described and
validated, was used as an index of cell volume
(Yeung et al, 2003). The DNA
stain propidium iodide (PI) was added to the sample (final concentration 6
µg/mL) before flow cytometric analysis to discriminate nonviable from
viable sperm in the control and quinine-treated samples and to distinguish
demembranated sperm from cell debris in the Triton-treated samples. The
difference in cell volume (b - a) between nontreated viable sperm performing
volume regulation (a) and quinine-treated sperm in which volume regulation was
blocked (b) indicates the extent of volume regulation. This capacity was
standardized against the cytoplasmic volume (a - c), in which c is the volume
given by the demembranated sperm devoid of cytoplasm. The sperm volume
regulation index was calculated as (b - a)/(a - c).
Statistics
Differences between populations were assessed by paired t test (%
sperm with CDs with or without quinine), unpaired t tests (comparison
of patients and fathers), or 3-way analysis of variance (% sperm with CDs for
3 sources of variation: patients or fathers; mucus of 267 or 311 mmol/kg;
distance 0 (semen and mucus mixed in tube), 1, or 4 cm in capillaries) and
relationships by linear regression. The statistical program SigmaStat
(Erkrath, Germany) prompted the use of nonparametric tests when conditions of
normality or equal variances were not met. Significance was accepted when
P < .05.
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Results |
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Flow cytometric determination of forward scatter revealed that there was no relationship between the size of sperm and the osmolality of seminal plasma (Figure 2). In the presence of quinine, live spermatozoa were significantly larger than in its absence both at the osmolality of native semen (mean forward scatter [FS, channel number] 569.7 ± 31.0 vs 543.6 ± 3.2) and of cervical mucus (290 mmol/kg) (FS: 560.5 ± 4.1 vs 536.4 ± 3.4), whereas nonviable cells permitting PI entry did not increase in size (FS: 531.1-537.1; differences between nontreated and quinine-treated were not significantly different from zero). As expected, Triton-demembranated sperm were smaller (FS 474.5 ± 3.4; n = 40), as membranes and their contained cytoplasm were lost.
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Experiment 2: The Percentage of Spermatozoa With CDs After Migration Through Viscous Surrogate Mucus
The ejaculates used for this study came from 7 fathers, 3 student donors,
and 21 patients. Since there was no difference between the groups in terms of
how their spermatozoa behaved in mucus
(Table 2), the results given
are from all 31 subjects. As a group, the percentage of motile spermatozoa
bearing CDs in the native semen (63% ± 3%, range: 40%-86%) was similar
to the percentage of spermatozoa with CDs recovered from the 1-cm and 4-cm
marks of capillaries containing mucus at high and low osmolalities
(Table 2). The percentages of
spermatozoa with CDs found within surrogate mucus at the 1-cm and 4-cm marks
were similar at both osmolalities (Table
2), as were the vanguard distances (5.6 ± 0.3 cm [267
mmol/kg] and 5.7 ± 0.3 cm [311 mmol/kg]). The CDs on sperm in the mucus
were of various sizes, ranging from small but distinguishable droplets at the
neck region to a vesicle surrounding the whole of the midpiece
(Figure 5); smaller ones were
more frequently encountered. There was no correlation between the percentage
of CDs and any values of the penetration score or the osmolality of the
semen.
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There was no significant difference between the osmolality of semen from fathers (317 ± 9 mmol/kg) and semen from patients (323 ± 4 mmol/kg). The percentage of spermatozoa with droplets within semen of fathers was not significantly higher than that of patients (Table 2). However, when mixed with surrogate mucus of either osmolality, or after penetrating 1 cm and 4 cm through mucus, there were consistently higher percentages of spermatozoa with droplets from fathers than with droplets from patients (Table 2). Three-way analysis of variance on these percentages revealed a significantly higher, albeit small, percentage of spermatozoa with droplets within the surrogate mucus when obtained from fathers rather than from patients (mean 7.5%).
Experiment 3: Volume Regulatory Capacity of Spermatozoa From Fathers and Patients to Physiologically Hypotonic Medium
There was no difference between fathers and patients in the uptake of the
vital dye PI by spermatozoa from fathers (F: 74.3% ± 4.2% PI-negative,
n = 13) or patients (P: 65.6% ± 4.5% PI-negative, n = 14) in the
absence of quinine or in its presence (F: 74.1% ± 4.6%; P: 64.9%
± 4.1%), in their size (FS, channel number) in medium with the
osmolality of human cervical mucus in the absence (F: 502.8 ± 7.7; P:
505.4 ± 5.9) or presence (F: 520.9 ± 7.5; P: 517.5 ± 6.3)
of quinine, or in the size of demembranated spermatozoa (F: 441.2 ±
5.0; P: 442.0 ± 4.1) and, hence, their surmised cytoplasmic volume.
However, there was a significant difference between fathers and patients in the response of their spermatozoa to quinine at the osmolality of mucus, with a significantly greater quinine/control ratio for fathers (3.6% ± 0.5% increase in the presence of quinine) than for patients (2.4% ± 0.4% increase) (Table 3). When expressed as a percentage of the cytoplasmic volume, this value was significantly more marked in fathers (32.7% ± 4.7% increase) than in patients (18.7% ± 3.5%; Figure 6).
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Discussion |
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In mice, osmotic swelling occurs at the cytoplasmic droplet at the end of the midpiece and prevents passage through the uterotubal junction (Yeung et al, 2000). In man the droplet is proximal (Cooper et al, 2004), and its role in volume regulation and migration through cervical mucus is obscure. If such droplets are the major site of water influx in hypo-osmotic solutions and if volume regulation cannot occur, their size may physically hinder penetration through viscous media encountered in the female tract as mucus penetration is inhibited by quinine (Yeung and Cooper, 2001). Were this to be the case, assessing the presence of droplet-bearing spermatozoa may be of diagnostic importance for human infertility. This study examined whether the extent of swelling of human spermatozoa and their capacity for regulating volume was reflected in their CDs and whether the presence of CDs influenced sperm function (motility or penetration of a surrogate mucus).
The greater percentage of motile spermatozoa with droplets compared to immotile cells in semen has been noted before (Cooper et al, 2004) and extended here to semen diluted in medium of 290 mmol/kg. As judged from wet preparations, there was no clear relationship between the percentage of spermatozoa bearing droplets and the osmolality of seminal plasma. Although statistically significant, the increased percentage of spermatozoa bearing droplets in the presence of inhibitory concentrations of the channel blocker quinine, as recorded in fixed preparations, amounted only to 2.3%. As droplets were recorded regardless of their size (and not just those extending the length of the midpiece), the slight increase presumably reflects the slightly greater size of droplets in the presence of quinine that made them more obvious and thus caught the subjective attention of the observer. Objective measurements of sperm size by flow cytometry, indeed, indicated a significant increase in sperm size in the presence of quinine. These quinine-induced increases in size of spermatozoa at female tract osmolality reflect the extent of regulatory volume decrease that would otherwise occur in the absence of the drug. The observations demonstrate that with physiological challenges in the range of osmolalities found within liquefied semen and cervical mucus, the majority of spermatozoa were functioning to control their volume using osmolytes passing through quinine-sensitive channels. The response of spermatozoa to quinine was not related to the percentage bearing CDs, most likely because the majority of spermatozoa bore these structures. The geometry of the droplet at the neck and not the annulus (Cooper et al, 2004) may prevent accumulation of cellular water in the droplet, as in mice, but may permit its incorporation along the entire flagellar length, which could account for the effect of quinine on human sperm kinematics (Yeung and Cooper, 2001).
As human mucus is difficult to obtain and variable in quality, use of a surrogate was deemed pertinent to answer questions relating to sperm migration through viscous media. In addition to the differences in rheological properties of hyaluronate (stemming from a polysaccharide) and those of human mucus (stemming from a range of mucin glycoproteins), soluble factors in human mucus may affect sperm progression and confound the interpretation of results. These components are lacking from the mucus substitute, which permits examination solely of viscosity effects at different osmolalities. In experiments with mucus of similar viscosity to that of mid-cycle cervical mucus, CDs neither hindered nor facilitated the penetration and migration of the spermatozoon into this matrix. These results are consistent with observations that spermatozoa within natural mucus (ie, selected for motility) are more likely to have droplets (midpiece vesicles) than (both motile and immotile) spermatozoa in semen (Abraham-Peskir et al, 2002), because mucus selects for motile sperm, which bear more droplets than immotile spermatozoa (data presented here; Cooper et al, 2004). The ability of droplet-bearing spermatozoa to penetrate mucus would also explain the presence of such spermatozoa in the Fallopian tube after artificial insemination (Mortimer et al, 1982).
The lack of differences in the migration of droplet-bearing spermatozoa within mucus implies efficient volume regulation by motile spermatozoa during exposure to a natural hypo-osmotic challenge of about 40 mmol/kg. Such efficient volume regulation may be required for mucus penetration, since quinine-treated spermatozoa were unable to penetrate surrogate mucus effectively (Yeung and Cooper, 2001), and thus, disturbances in volume regulation may be a cause of some cases of male infertility. That volume regulation has such an important role in fertility was indicated by the significantly greater swelling of spermatozoa from fathers compared to patients in response to quinine at female tract osmolality. This implies a greater RVD activity of spermatozoa from fathers and warrants a larger study to ascertain whether assessing volume regulation routinely in the Andrology laboratory would be a useful complement to the spermiogramme, especially for men with idiopathic infertility. The lack of relationship between RVD response and presence of CDs indicates that there is nothing to be gained by assessing the latter routinely for diagnostic purposes.
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Acknowledgments |
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Footnotes |
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and immotile (
)
human ejaculated spermatozoa bearing cytoplasmic droplets (ordinate, mean +
SEM, n = 50) within 30 minutes of liquefaction, undiluted in seminal plasma
(SP) or diluted in medium of 290 mmol/kg (290) in the absence (SP, 290) or
presence (SPQ, 290Q) of 0.27 mM quinine. *, for each medium,
significant difference between motile and immotile fractions (P < .05).
) quinine, in the presence of Triton X-100
(
), and the calculated cytoplasmic volume (
). Volume regulation
within semen is indicated by the constant sperm size over the entire range of
osmolality.
