Response of midpiece vesicles on human sperm to osmotic stress

Joanna V. Abraham-Peskir1,4, Eric Chantler2, Erik Uggerhøj1 and Jens Fedder3

1 ISA, Institute for Storage Ring Facilities, University of Aarhus, 8000 Aarhus C, Denmark, 2 Academic Unit of Obstetrics and Gynaecology and Reproductive Health Care, St Mary's Hospital, University of Manchester, Manchester M13 0JH, UK and 3 Fertility Clinic, Brædstrup Hospital, 8740 Brædstrup, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: We investigated the osmotic response of midpiece vesicles (MPV) on human sperm. METHODS: Light microscopy, transmission X-ray microscopy and computer-aided semen analysis was used to investigate sperm in normozoospermic semen from healthy donors, separated from semen and suspended in hyper- or hypo-osmotic solutions. RESULTS: The mean incidence of MPV-bearing sperm in semen was 31% (range 8–71; n = 47). MPV were morphologically different from cytoplasmic droplets. The incidence of MPV-bearing sperm in separated populations increased reversibly after washing in Sperm Preparation Medium but not after washing in seminal plasma. There was an inverse relationship between medium osmolality and both MPV-bearing sperm incidence and MPV diameter. However, initial osmolality in semen from different donors did not correlate with incidence of MPV-bearing sperm. Furthermore, a direct relationship was observed in semen as osmolality increased with time. No correlation existed between progressive motility and semen osmolality. Progressive motility and the amplitude of lateral head displacement were significantly reduced in sperm with an MPV (three out of four semen samples, 26–32 sperm). The incidence of MPV-bearing sperm in those that had penetrated cervical mucus (75, 46, and 40%) was increased compared with the adjacent semen (24, 35, and 24%). CONCLUSIONS: MPV are ubiquitous and distinct from cytoplasmic droplets. They respond to osmolality changes of the surrounding medium. The presence of an MPV can reduce motility but not survival in cervical mucus. Therefore, they should not be considered detrimental to sperm function.

Key words: midpiece vesicles/osmolality/sperm/X-ray microscopy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The spermatozoon is a highly regionalized cell with localized membrane domains that have specific functions (Martinez and Morros, 1996Go). The domains have heterogeneous fluidity, shape, diffusion coefficients and composition of phospholipids, glycolipids and steroids (see review by Ladha, 1998Go). There are five specialized regions: the acrosomal, equatorial, postacrosomal, midpiece and tail region (Bedford and Hoskins, 1990Go). The spermatozoon membrane is a dynamic system undergoing many changes, often domain specific, as the spermatozoon passes through the reproductive tract. These maturational changes are thought to be essential for eventual fertilization of the oocyte.

Sperm undergo membrane changes as they mature in the epididymis. It is during this period that the spermatozoon surface is modified by integration of proteins, glycoproteins and lipids such as phosphatidylcholine (Haidl and Opper, 1997Go). Changes in lipid concentration during epididymal transportation have been reported in several animal species (Hall et al., 1991Go; Rana et al., 1993Go). These lipids are significant for induction of progressive motility as well as for subsequent functions and processes such as capacitation and the acrosome reaction. Water loss from sperm also occurs as they pass through the epididymis, consequently, sperm are good osmometers (Drevius, 1972Go). Furthermore, the packed cell volume of sperm is sensitive to osmotic pressure (Liu and Foote, 1998Go). The osmotic water permeability coefficient of human sperm membranes is very high whilst the associated activation energy is low (Noiles et al., 1993Go), suggesting the presence of a porous membrane.

In humans, cytoplasm necessary for spermatogenesis is normally eliminated from spermatids while they are still in contact with the Sertoli cell (Smith and Lacy, 1959Go) as a structure commonly called the residual body, which forms on severance of the cytoplasmic stalk. Any residual cytoplasm is eliminated from the spermatozoon flagellum at a later time, during the period within the epididymis and before the release of sperm (Russell, 1979Go; Sprando and Russell, 1987Go). Cytoplasm retained abnormally as a sac or droplet at the spermatozoon midpiece is termed the cytoplasmic droplet. Its presence on sperm in semen indicates aberrant spermatogenesis and is associated with sub-fertility (Jouannet et al., 1988Go; Keating et al., 1997Go). Thus, in freshly ejaculated semen, the incidence of sperm with a cytoplasmic droplet is low (Keating et al., 1997Go; Laudat et al., 1998Go). We have demonstrated previously, using light and X-ray microscopy (XM), that another type of vesicular body that is caused by swelling of the midpiece region is also present in freshly ejaculated sperm (Abraham-Peskir et al., 1998Go).

X-ray microscopy is a relatively new imaging technique with a practical resolving power of 30–50 nm. High-resolution ultrastructural studies of live cells in physiological solution with high contrast are possible with transmission XM (Kirz et al., 1995Go; Abraham-Peskir, 2000Go). Although electron microscopy has revealed structural details of the spermatozoon cell, contributing much to our understanding of the mechanisms involved in the transformation of the spermatozoon membrane during maturation, artefacts can occur during specimen preparation and the cell has to be removed from the physiological environment. Thus, the examination of fragile or delicate structures is compromised, which is especially pertinent to studies of the plasma membrane. X-ray microscopy eliminates artefact-inducing preparation techniques because the live specimen is loaded into the microscope at atmospheric pressure and does not require chemical fixation, staining or drying. The first XM images of sperm were obtained using laser-plasma X-ray sources (Tomie et al., 1991Go; DaSilva et al., 1992Go). More recently, using a synchrotron radiation source, mammalian sperm were analysed by combining scanning XM with X-ray absorption measurements to determine the DNA to protein ratios of sperm (Balhorn et al., 1992Go). XM has also revealed changes that occur in spermatozoon mitochondrial morphology after exposure to capacitating conditions (Vorup-Jensen et al., 1999Go).

Immediately after ejaculation, sperm must migrate across the semen–mucus interface. Cervical mucus receptivity to sperm is cyclic; maximal penetration occurs about the time of the luteinizing hormone peak. Cervical mucus can also provide a physical barrier to sperm that have abnormal morphology (Hanson and Overstreet, 1981Go). This process may result from the different motility pattern exhibited by sperm with abnormal morphology. Therefore, cervical mucus selects for morphologically normal sperm, based on the differential motility of normal versus abnormal sperm (Morales et al., 1988Go; Katz et al., 1990Go). Once within the cervical mucus, spermatozoon motility alters dramatically and the movement of the sperm causes an alteration in the microstructure of the mucus (Katz et al., 1989Go).

In the present study, the incidence of midpiece vesicle (MPV)-bearing sperm among healthy men with normozoospermia was examined, and the morphology and incidence of MPVs and cytoplasmic droplets compared. The relationship between the size and prevalence of MPVs and changes in osmotic pressure was evaluated. Finally, the effect of an MPV on motility characteristics of sperm in both semen and mid-cycle cervical mucus was examined.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials
All chemicals and media were analytical grade (Sigma-Aldrich, Poole, UK) unless otherwise stated. Semen samples were generously supplied by Cryos International Sperm Bank Ltd, Aarhus, Denmark.

Sperm preparation
Semen samples were collected by masturbation from men (age 21–43) after >3 days abstinence. The semen was allowed to liquefy at 37°C for at least 30 min and thereafter kept at 37°C until use. Normozoospermia was verified within 1 h of ejaculation (WHO, 1999).

The concentration of sperm in the semen was assessed using a Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) and osmolality measured with a Camlab automatic micro-osmometer. Sperm motility was determined using computer aided semen analysis (CASA) (see below). After liquefaction, sperm were separated by centrifugation through a 40/80% discontinuous density gradient of Percoll or standard swim-up method (WHO, 1999) followed by two washes in Sperm Preparation Medium (Medicult, Copenhagen, Denmark). Seminal plasma was collected by centrifugation (300 g, 20 min) of ~1ml semen. The incidence of MPV-bearing sperm was determined using a Leica DMR microscope with differential interference contrast, x100 oil immersion lens and a x10 magnification eyepiece.

Papanicolaou stain
Cytoplasmic droplets on sperm in fresh ejaculates were investigated. A 10 µl smear was prepared on a clean glass microscope slide and allowed to air dry. The smear was fixed in equal parts of ethanol (95%, v/v) and diethylether for 10 min. The fixed smears were stained according to the World Health Organization Laboratory Manual (World Health Organization, 1999Go), using Haematoxylin, Orange G solution and Papanicolaou EA-50. Cytoplasmic droplets located around the midpiece stained green and their presence was scored using bright field light microscopy (LM) with a dry x100 lens.

Cervical mucus penetration
Mid-cycle cervical mucus was collected using a catheter from women attending Brædstrup or Maigaard Fertility Clinics. Three days sexual abstinence was required to ensure absence of sperm. Mucus used in experiments had a spinnbarkeit of >10 cm and absence of leukocytes. The Kurzrok–Miller method (Mortimer, 1994Go) was used to assess spermatozoon penetration and to score for MPV-bearing sperm in the mucus.

Image acquisition and analysis
Differential interference contrast images of sperm were collected using a Leica DMR microscope and x100 oil immersion lens. X-ray microscopy images were collected at 2.4 nm wavelength using the Aarhus transmission XM (Medenwaldt and Uggerhøj, 1998Go) at the ASTRID storage ring, University of Aarhus, Denmark. The microscope was equipped with Fresnal zone plates (Department of Physics, Göttingen, Germany) as optical elements, giving 30nm resolution. Synchrotron radiation was focused by a condenser zone plate onto the target. A micro zone plate acting as an objective lens magnified the image onto a back-illuminated, Peltier-cooled charge-coupled device camera (Photometrics, Tectronix CCD array, USA). A 3 µl aliquot of the wet sample was mounted between two thin silicon wafers etched to <150 nm in the central part and sealed in a specially constructed chamber kept at ambient temperature and atmosphere throughout imaging. The depth of the liquid layer was maintained at 3–5 µm, the lower limit set by the addition of washed 5 µm Dynospheres (Plano, Marburg-Cappel, Germany), which ensured high X-ray transmission and facilitated sperm motility. Sperm were motile in this environment for several hours. Exposure times were 5–10 s, during which time the spermatozoon had to be stationary. The target spermatozoon was immobilized with a short exposure to the radiation beam immediately prior to imaging. All other sperm remained motile and were not exposed to the radiation beam. Images were processed with PMIS software (Photometrics, Tucson, AZ, USA).

Motility changes
Semen in a 20 µm depth microslide (Conception Technologies, San Diego, CA, USA) at 35°C was examined using a Hamilton Thorne IVOS version 10 CASA apparatus (Berkley, CA, USA) and motility data analysed remotely as Excel files. The presence of an MPV increases spermatozoon volume, which could significantly affect their motility. This possibility was examined using CASA of individual tracks of video-recorded sperm both with and without an MPV. Measurement of the MPV-bearing sperm was done using video-recorded images selected using the EDIT function; CASA gate functions were modified to maximize the capture of LM images.

Osmolality experiments
In preliminary investigations, trisaccharide raffinose or sodium chloride (data not shown) was used to change the osmolality of Sperm Preparation Medium and raffinose was chosen for use in the following experiments. Semen from four donors was divided and sperm separated by swim-up method into medium of varying osmolality, 200, 280, 350, 450 mOsm/kg (if the osmolality of the medium was >400 mOsm/kg, it was placed below the semen). After 2 h, the medium fraction was aspirated and for each treatment, the incidence of MPV-bearing sperm was scored using LM (n = 100).

Sperm were separated from semen by standard swim-up method into Sperm Preparation Medium (four semen samples). After 2 h, the medium fraction was aspirated and centrifuged. The pellet of sperm was divided into six 100 µl aliquots of solutions with osmolalities adjusted to 280, 315, 340, 380, 415 and 450 mOsm/kg. For each treatment, the incidence of MPV-bearing sperm was scored using LM (n = 100). For two samples, at least 50 cells from each treatment were recorded onto videotape via a CCD camera attached to a Leica LM using differential interference contrast (DIC) x100 oil immersion lens. The diameter of the MPV was measured in single frames directly from the screen image. Motility parameters were also measured at each osmotic pressure using CASA.

Sperm were transferred to hypo-osmotic (200 mOsm/kg) and hyper-osmotic (400 mOsm/kg) solutions, and examined by XM.

Separated samples
Sperm were separated by swim-up or on a Percoll gradient and washed twice in seminal plasma or Sperm Preparation Medium. The incidence of MPVs was determined on 100 sperm (counted in duplicate and the mean taken) by LM for untreated sperm in fresh ejaculate, separated sperm, and each wash in seminal plasma or medium. Sperm were separated on a Percoll gradient and washed twice. The pellet was halved and re-suspended in either Sperm Preparation Medium or seminal plasma. The incidence of MPVs on 100 sperm in the semen, after separation and two washes and after the third wash was recorded using LM.

Semen samples
The incidence of MPV-bearing sperm was measured in 47 semen samples from different donors within 60 min of liquefaction. Seventeen of these donors provided samples on different dates; the data from these samples were used for the investigation of intra-donor variation. A further 19 normozoospermic samples were collected from different donors. The osmolality of the semen, incidence of MPV-bearing sperm and sperm motility parameters were measured for each semen sample within 1 h of liquefaction. Four of the samples were incubated for a further 7 h and the osmolality measured and incidence of MPV-bearing sperm recorded.

Statistical analysis
Statistical analyses were performed using SPSS version 8 (Chicago, IL, USA) and Excel version 7.0a (Microsoft Corporation, USA). For the healthy donor investigations and the removal of seminal plasma experiments, results were analysed using Student's paired t-test or Student's t-tests adjusted for unequal variance: P < 0.02 was considered significant. Data are presented as means and range.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
MPV on sperm were large with high transparency (Figure 1aGo) and were seen to disappear during drying. At high resolution they were usually homogeneously smooth, with no indication of Golgi apparatus or membranous inclusions (Figure 1bGo). In 47 normozoospermic semen samples, the mean incidence of MPV-bearing sperm was 31% (range 8–71). The incidence was donor specific, with low intra-donor variation (Figure 2Go). The incidence of MPV-bearing sperm in wet samples determined using DIC LM (mean 41%, range 9–73, n = 21) was significantly different from that of cytoplasmic droplets (Papanicolaou stained) on sperm in the same sample (mean 6.6%, range 1–13, n = 21) (P < 0.001).



View larger version (127K):
[in this window]
[in a new window]
 
Figure 1. Vesicular bodies surrounding the midpiece of human sperm. (a) Spermatozoon in semen imaged using differential interference contrast light microscopy with x100 lens, (b) spermatozoon separated by swim-up method and imaged usingX-ray microscopy. Scale bars represent 2 µm.

 


View larger version (23K):
[in this window]
[in a new window]
 
Figure 2. Incidence of MPV-bearing sperm in semen samples from 17 donors (a-q), number of samples investigated per donor, a-m = 2, n-p = 3, q = 7. Error bars are means ± SEM.

 
Osmolality experiments
There was an inverse relationship between the incidence of MPV-bearing sperm and osmolality when sperm were transferred from seminal plasma to medium of a different osmolality (Figures 3a, bGo). This inverse proportionality was of similar magnitude if either raffinose or sodium chloride was used to make the hyper-osmolality solution. However, a similar response was not seen in the motion kinetics of the sperm for the two compounds. There appeared to be a dual action of low ionic strength and hypo-osmolality. Therefore, raffinose was used to change the osmolality of Sperm Preparation Medium.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 3. Incidence of MPV-bearing sperm after transfer from seminal plasma to solutions of varying osmolalities.(a) Swim-up method into four different media in the range of200–450 mOsm/kg, n = 6, (b) swim-up method into SpermPrep medium (280 mOsm/kg), centrifugation and resuspension into~100 µl of solution with osmolalities adjusted to 280, 315, 340, 380, 415 and 450 mOsm/kg, n = 4.

 
The mean diameter of the MPVs decreased as the osmolality of the medium was increased over the range 280–375 mOsm/kg (Figure 4Go). The baseline osmolality of semen was taken as the value after liquefaction. In the XM, at hypo-osmolality the vesicles were large and translucent (Figure 5a,bGo) and at hyper-osmolality they were absent or if present small (Figure 5c,dGo). Motility did not change significantly when sperm were placed in media that corresponded to the range of physiological osmolality (Figure 6Go). Outside this range of osmolality there was a marked decline in progressive motility. The size of MPVs measured using LM corresponded to that seen in the XM.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 4. Size of MPVs (µm) compared with the osmolality of the external medium, for two different donors. Values are expressed as the means ± SEM from 50 MPVs.

 


View larger version (140K):
[in this window]
[in a new window]
 
Figure 5. X-ray microscopy images of human sperm washed in medium. (a, b) Hypo-osmotic (200 mOsm/kg), or (c, d) hyper-osmotic (450 mOsm/kg) to semen. In hypo-osmotic medium all sperm had an MPV, but in hyper-osmotic medium there were no MPVs present. Scale bar = 2 µm.

 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 6. Progressive motility of separated sperm at various osmolalities of medium. n = 2–7 for each range of osmolality. Thirty-two points used to derive trend line R2 = 0.79.

 
Separated samples
After Percoll separation and washing in medium there was always an increase in the incidence of MPV-bearing sperm (mean 47%, range 24–88, n = 36). We therefore attempted to determine the processing stage at which MPV formation was stimulated. Centrifugation per se did not cause an increase in MPV-bearing sperm; neither did the initial separation through Percoll (Figure 7Go). The incidence of MPV-bearing sperm increased after the first wash in medium, but not if washed instead in seminal plasma. The measured osmolalities for Sperm Preparation Medium, 80% Percoll and 40% Percoll were 276, 382, and 347 mOsm/kg respectively. Using the swim-up method, a significant increase in MPV-bearing sperm occurred when sperm swam out of seminal plasma and into medium (Figure 8Go), which could be increased further by washing in medium. The increase in incidence of MPV-bearing sperm was reversible by returning separated washed sperm to seminal plasma (Figure 9Go).



View larger version (33K):
[in this window]
[in a new window]
 
Figure 7. The incidence of MPV-bearing sperm in fresh ejaculate, after separation through a Percoll gradient, and subsequent washing in medium (light grey shading) or seminal plasma (dark grey shading). *P < 0.02; **P < 0.005 (Student's t-test paired two sample for means adjusted for unequal variance) when comparing medium wash with seminal plasma wash or fresh ejaculate.

 


View larger version (35K):
[in this window]
[in a new window]
 
Figure 8. The incidence of MPV-bearing sperm in fresh ejaculate, after standard swim-up (top), and subsequent washing in medium (dark grey shading) or seminal plasma (light grey shading).*P < 0.02; **P < 0.01; ***P < 0.001 (Student's t-test paired two sample for means assuming unequal variance) when comparing medium wash with seminal plasma wash or fresh ejaculate.

 


View larger version (50K):
[in this window]
[in a new window]
 
Figure 9. The incidence of MPV-bearing sperm in semen (treatment 1), after Percoll separation and two washes (treatment 2), a subsequent wash in seminal plasma (treatment 3) or medium (treatment 4). *P < 0.0015 when comparing after Percoll and two washes with the fresh ejaculate. **P < 0.001 when comparing a third wash in Sperm Preparation Medium with a wash in seminal plasma.

 
Semen samples
The mean osmolality of semen was 346 mOsm/kg, range 307–371, n = 25. The incidence of MPV-bearing sperm and progressive motility was not correlated with semen osmolality, measured within 30 min of liquefaction. Nor was there a correlation between incidence of MPV-bearing sperm and sample volume (mean 2.4 ml, range 1.0–4.0) or spermatozoon concentration (mean 102x106/ml, range 44–376). There was an increase in osmolality (P < 0.001) (Figure 10aGo) and incidence of MPV-bearing sperm (P = 0.01) (Figure 10bGo) with time, in semen incubated for up to 7 h at 37°C.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 10. Effect of time on seminal plasma osmolality and MPV incidence. Semen from four different donors was incubatedfor up to 7 h at 37°C, (a) semen osmolality plotted against time, (b) incidence of MPV-bearing sperm (n = 200) with time.

 
Motility changes
Both progressive velocity (VAP) and the amplitude of lateral head displacement (ALH) were significantly reduced in the presence of an MPV on the sperm of three out of four semen samples examined (n = 26–32, P <= 0.02). This observation was extended to a physiological system by observing the movement of sperm after they had penetrated human mid-cycle cervical mucus (n = 3) (Figure 11Go). In each case the incidence of MPV-bearing sperm in mucus (75, 46, and 40%) increased compared with that in the semen (24, 35, and 24%).



View larger version (109K):
[in this window]
[in a new window]
 
Figure 11. Light microscopy image of MPV-bearing sperm in mid-cycle cervical mucus. Scale bar = 2 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
There were two populations of MPV found on sperm in seminal plasma: cytoplasmic droplets of low incidence and another vesicle that did not contain cytoplasm and was formed by plasmalemma swelling of the midpiece region. The incidence of MPV-bearing sperm for healthy men was 31%, which did not correspond to that of cytoplasmic droplets reported in the literature, 0–21.5% (Keating et al., 1997Go) and 3–28% (Laudat et al., 1998Go). That the MPV were clearly visible by LM on freshly ejaculated sperm was unquestionable evidence of their existence in the normal population. Additionally, MPV-bearing sperm were ubiquitous, showing a substantial incidence in fresh ejaculate from all donors studied. Incidence was donor specific and did not vary considerably between ejaculates from the same man, indicating that the occurrence of vesicles was a specific characteristic of the subject and not a random process. It is intriguing that MPVs have not been widely commented on in the literature. MPVs described in the present study were seen to disappear during drying; consequently they would not be seen during routine morphological analysis where the semen smear is often dried. The XM images were crucial to this study, as visualization of detail not seen by LM was possible. Using XM, it was clear that MPVs did not contain Golgi apparatus or other membranous structures that are often found in cytoplasmic droplets (Hermo et al., 1988Go). Further evidence against the structures described in the present study being cytoplasmic droplets was that transfer from seminal plasma to medium induced a doubling in the incidence of MPV-bearing sperm, whilst return to seminal plasma reversed this.

XM provided a distinct advantage in the holistic study of membranes on intact cells, making it possible to study whole, hydrated cells in physiological medium. Vesicles remained a constant size even after repeated exposure to X-rays, providing evidence for their existence prior to exposure. Corresponding structures have unwittingly been presented in studies using EM. In a comparative study of normal human germ cells, an LM image of an MPV-bearing spermatozoon was shown but not described (Johnson et al., 1999Go). MPV were considered to be artefacts in two TEM studies: one that examined the effect of Percoll gradients on human sperm (Arcidiacono et al., 1983Go) and another that reported damage incurred by sperm during purification protocols (Rodriguez-Martinez et al., 1997Go). The structures described were wrinkled, damaged or crenate rather than smooth inflated vesicles described here, and were interpreted as detached plasmalemma. It is likely that the previously published EM images represented vesicular bodies that had been smooth and intact but had collapsed during preparation.

The increase in MPV-bearing sperm following washing or swim-up into Sperm Preparation Medium was indicative of their sensitivity to osmolality changes, as Sperm Preparation Medium (276 mOsm/kg) had a lower osmolality than semen (>300 mOsm/kg). The combined osmolality of 100% Percoll and buffer/medium was closer to that of semen than the buffer/medium alone. Therefore, it was not surprising that centrifugation through Percoll did not cause an increase in MPV-bearing sperm. The reversal of MPV occurrence on return of sperm to seminal plasma was evidence for an osmosis-governed process. The mean MPV size decreased as the osmolality of the external medium increased and explains why the incidence of MPV-bearing sperm decreased in hyper-osmotic conditions. That both the size and prevalence of MPVs were responsive to changes in osmotic pressure in the present study supports the observation that sperm are good osmometers (Drevius, 1972Go). Furthermore, changes in MPV response to osmotic pressure could account for the alteration in sperm volume previously reported under varying external osmolalities (Liu and Foote, 1998Go). The motor apparatus of sperm exposed to hypo-osmotic Ringer's solution coil up within the plasmalemma (Drevius and Eriksson, 1966Go). As the spermatozoon cell swells it is thought to take on a more spherical shape. However, other constituents required for spermatozoon vitality are not present in salt solutions, which could account for swelling. When Sperm Preparation Medium was used in the present study, swelling was only observed in the midpiece domain. Even at the lowest osmolality, the motor apparatus did not curl up nor did the cells burst. One could speculate that the dynamic nature of MPV generation allows spermatozoon structure to adapt to water uptake, preventing lysis as they travel through microenvironments of varying osmolality. However, at ~340–375 mOsm/kg there was no further decrease in MPV size. The vesicles were not responsive to external osmotic changes above physiological levels, supporting the suggestion of a regulatory mechanism.

Motility did not change significantly when sperm were placed in media that corresponded to the range of osmolality reported for human testicular tubular fluid, 315–340 mOsm/kg (Levine and Marsh, 1971Go); 312–380 mOsm/kg (Hinton et al., 1981Go) or uterine fluid, 280–294 mOsm/kg (Casslen and Nillson, 1984Go). Outside this range of osmolality, there was a marked decline in progressive motility. Motility was reduced in MPV-bearing sperm. Therefore, the observation that the incidence of MPV-bearing sperm in mucus increased compared with that in the semen was unexpected, as the more vigorously moving sperm (those without an MPV) would be expected to penetrate mucus. Although mucus is thought to select against abnormal sperm (Katz et al., 1990Go), MPV-bearing sperm were not affected by this selective barrier.

Our findings support previous studies that report membrane changes induced by preparation techniques, however, we also show that membrane alterations are a normal feature on live motile cells. EM images were presented (Arcidiacono et al., 1983Go) of fully intact swollen membranes (as seen in the present study) but attributed to the effect of Percoll. In the present study, we did not see an increase in MPV incidence after Percoll separation, only after the subsequent washing stages. So the membrane changes can only be attributed to exposure to medium not Percoll. It was significant that the same vesicles were visible by LM before separation. It was reported (Arcidiacono et al., 1983Go) that no such structures were seen at the light microscopic level. This is not surprising, as they used a 10-fold lower magnification (x100) than in the present study (x1000). MPVs are not easily visible at low magnification partly due to their low contrast. Also in their study of EM images, it is only stated how many sections were examined, not how many sperm. Therefore, the claim that Percoll induced extensive damage was not substantiated. Our study does give supporting evidence for an increase in morphological changes after Percoll separation; however, the cause was osmolality changes during washes in medium and not exposure to Percoll per se.

The inverse relationship between osmolality and incidence of MPV-bearing sperm in physiological medium did not hold true for sperm in seminal plasma. In the general population, there was no correlation between osmolality and the incidence of MPV-bearing sperm in normozoospermic semen. The semen osmolality measured in the present study agreed with that of the previously reported value (Polak and Daunter, 1984Go). The osmolality of semen increases progressively after liquefaction due to the breakdown of seminal proteins (Velazquez et al., 1977Go). However, we did not find an inverse relationship between the incidence of MPV-bearing sperm and increasing osmolality of seminal plasma with time; surprisingly, there was a direct relationship. This indicates a seminal plasma factor other than osmolality that inhibited MPV reduction or stimulated MPV production.

We have conclusively demonstrated that human sperm can have two distinct types of vesicle associated with the midpiece but only the cytoplasmic droplet is associated with impaired fertility. Midpiece vesicles have been recently detected but not fully described previously. They may act as an osmotic buffer, allowing cells to adapt to the varying osmotic environment that they encounter in the male and female reproductive tracts. In support of this, aquaporin water channels have been located in the midpiece region of human sperm but not the head or tail regions (Ishibashi et al., 1997Go; Suzuki-Toyota et al., 1999Go) and water movement through these channels is bi-directional. However, there are unknown factors in seminal plasma that can override the osmotic affects. The presence of an MPV reduced both progressive velocity and lateral head movement of a spermatozoon but there was a predominance of MPV-bearing sperm amongst sperm that had penetrated cervical mucus. Therefore, MPVs should not be considered detrimental to either motility or male fertility.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Semen samples generously supplied by Cryos International Sperm Bank Ltd. Aarhus. Thanks to Svend Maigaard, Maigaard Fertility Clinic, Aarhus, for collection of cervical mucus. Financial support was provided by ISA and Manchester University. Approval for collection of cervical mucus was given by the Local Ethical Committee (protocol no. 20000201).


    Notes
 
4 To whom correspondence should be addressed. E-mail: jabraham{at}ifa.au.dk Back

Submitted in June 25, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Abraham-Peskir, J.V. (2000) X-ray microscopy with synchrotron radiation: applications to cellular biology. Cell. Mol. Biol., 46, 1045–1052.[ISI]

Abraham-Peskir, J., Chantler, E., McCann, C. et al. (1998) Ultrastructure of human sperm using X-ray microscopy. Med. Sci. Res., 26, 663–667.[ISI]

Arcidiacono, A., Walt, H., Campana, A. et al. (1983) The use of Percoll gradients for the preparation of subpopulations of human spermatozoa. Int. J. Androl., 6, 433–445.[ISI][Medline]

Balhorn, R., Corzett, M., Allen, M.J. et al. (1992) Application of X-rays to the analysis of DNA packaging in mammalian sperm. Soft x-ray Microscopy SPIE, 1741, 374–385.

Bedford, J.M. and Hoskins, D.D. (1990) The mammalian spermatozoon: morphology, biochemistry and physiology. In Lamming G.E. (ed.), Marshall's Physiology of Reproduction. Churchill Livingstone, Edinburgh, pp. 379–568.

Casslen, B. and Nillson, B. (1984) Human uterine fluid examined in undiluted samples for osmolarity, and the concentrations of inorganic ions, albumin, glucose, and urea. Am. J. Obstet. Gynecol., 150, 877–881.[ISI][Medline]

DaSilva, L.B., Trebes, J.E., Balhorn, R. et al. (1992) X-ray laser microscopy of rat sperm nuclei. Science, 258, 269–271.[ISI][Medline]

Drevius, L.O. (1972) Bull spermatozoa as osmometers. J. Reprod. Fertil., 28, 29–39.[Medline]

Drevius, L.O. and Eriksson, H. (1966) Osmotic swelling of mammalian spermatozoa. Exp. Cell. Res., 42, 136–156.[ISI][Medline]

Haidl, G. and Opper, C. (1997) Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation. Hum. Reprod., 12, 2720–2723.[Abstract]

Hall, J.C., Hadley, J. and Doman, T. (1991) Correlation between changes in rat sperm membrane lipids, protein, and the membrane physical state during epididymal maturation. J. Androl., 12, 76–87.[Abstract/Free Full Text]

Hanson, F.W. and Overstreet, J.W. (1981) The interaction of human spermatozoa with cervical mucus in vivo. Am. J. Obstet. Gynecol., 140, 173–178.[ISI][Medline]

Hermo, L., Dworkin, J. and Richard, O. (1988) Role of epithelia clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa. Am. J. Anat., 183, 107–124.[ISI][Medline]

Hinton, B.T., Pryor, J.P., Hirsh, A.V. et al. (1981) The concentration of some inorganic ions and organic compounds in the luminal fluid of the human ductus deferens. Int. J. Androl., 4, 457–461.[ISI][Medline]

Ishibashi, K., Kuwahara, M., Kageyama, Y. et al. (1997) Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem. Biophys. Res. Commun., 237, 714–718.[ISI][Medline]

Johnson, L., Neaves, W.B., Barnard, J.J. et al. (1999) Comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules. Biol. Reprod., 61, 927–934.[Abstract/Free Full Text]

Jouannet, P., Ducot, B., Feneux, D. et al. (1988) Male factors and the likelihood of pregnancy in infertile couples. I. Study of sperm characteristics. Int. J. Androl., 11, 379–394.[ISI][Medline]

Katz, D.F., Drobnis, E.Z. and Overstreet, J.W. (1989) Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res., 22, 443–469.[ISI][Medline]

Katz, D.F., Morales, P., Samuels, S.J. et al. (1990) Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus. Fertil. Steril., 54, 513–526.[ISI][Medline]

Keating, J., Grundy, C.E., Fivey, P.S. et al. (1997) Investigation of the association between the presence of cytoplasmic residues on the human sperm midpiece and defective sperm function. J. Reprod. Fertil., 110, 71–77.[Abstract]

Kirz, J., Jacobsen, C. and Howells, M. (1995) Soft X-ray microscopes and their biological applications. Q. Rev. Biophys., 28, 33–130.[ISI][Medline]

Ladha, S. (1998) Lipid heterogeneity and membrane fluidity in a highly polarized cell, the mammalian spermatozoon. J. Membrane Biol., 165, 1–10.[ISI][Medline]

Laudat, A., Guechot, J. and Palluel, A.M. (1998) Seminal androgen concentrations and residual sperm cytoplasm. Clin. Chim. Acta, 276, 11–18.

Levine, N. and Marsh, D.J. (1971) Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens in rats. J. Physiol., 213, 557–570.[ISI][Medline]

Liu, Z. and Foote, R.H. (1998) Bull sperm motility and membrane integrity in media varying in osmolality. J. Dairy Sci., 81, 1868–1873.[Abstract/Free Full Text]

Martinez, P. and Morros, A. (1996) Membrane lipid dynamics during human sperm capacitation. Frontiers in Bioscience, 1, d103–117.[Medline]

Medenwaldt, R. and Uggerhøj, E. (1998) Description of an X-ray microscope with 30 nm resolution. Rev. Sci. Instrum., 69, 2974–2977.[ISI]

Morales, P., Overstreet, J.W., Katz, D.F. (1988) Changes in human sperm motion during capacitation in vitro. J. Reprod. Fertil., 83, 119–128.[Abstract]

Mortimer, D. (1994) Practical Laboratory Andrology. Oxford University Press.

Noiles, E.E., Mazur, P., Watson, P.F. et al. (1993) Determination of water permeability coefficient for human spermatozoa and its activation energy. Biol. Reprod., 48, 99–109.[Abstract]

Polak, B. and Daunter, B. (1984) Osmolarity of human seminal plasma. Andrology, 16, 224–227.

Rana, A.P., Misra, S., Majumder, G.C. et al. (1993) Phospholipid asymmetry of goat sperm plasma membrane during epididymal maturation. Biochim. Biophys. Acta, 1210, 1–7.[ISI][Medline]

Rodriguez-Martinez, H., Larsson, B. and Pertoft, H. (1997) Evaluation of sperm damage and techniques for sperm clean-up. Reprod. Fertil. Dev., 9, 297–308.[ISI][Medline]

Russell, L.D. (1979) Spermatid-sertoli tubulobulbar complexes as devices for elimination of cytoplasm from the head region of late spermatids of the rat. Anat. Rec., 194, 233–246.[ISI][Medline]

Smith, K.B.V. and Lacy, D. (1959) Residual bodies in the seminiferous tubules of the rat. Nature, 184, 249–251.[ISI]

Sprando, R.L. and Russell, L.D. (1987) Comparative study of cytoplasmic elimination in spermatids of selected mammalian species. Am. J. Anat., 178, 72–80.[ISI][Medline]

Suzuki-Toyota, F., Ishibashi, K. and Yuasa, S. (1999) Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis. Cell. Tissue Res., 295, 279–285.[ISI][Medline]

Tomie, T., Shimizu, H., Majima, T. et al. (1991) Three-dimensional readout of flash X-ray images of living sperm in water by atomic-force microscopy. Science, 252, 691–693.[ISI][Medline]

Velazquez, A., Pedron, N., Delgado, N.M. et al. (1977) Osmolality and conductance of normal and abnormal human seminal plasma. Int. J. Fertil., 22, 92–97.[ISI][Medline]

Vorup-Jensen, T., Hjort, T., Abraham-Peskir, J.V. et al. (1999) X-ray microscopy of human spermatozoa shows change of mitochondrial morphology after capacitation. Hum. Reprod., 14, 880–884.[Abstract/Free Full Text]

World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm–cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, UK.

accepted on October 15, 2001.