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
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Abstract |
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Key words: midpiece vesicles/osmolality/sperm/X-ray microscopy
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Introduction |
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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, 1997). Changes in lipid concentration during epididymal transportation have been reported in several animal species (Hall et al., 1991
; Rana et al., 1993
). 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, 1972
). Furthermore, the packed cell volume of sperm is sensitive to osmotic pressure (Liu and Foote, 1998
). The osmotic water permeability coefficient of human sperm membranes is very high whilst the associated activation energy is low (Noiles et al., 1993
), 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, 1959) 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, 1979
; Sprando and Russell, 1987
). 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., 1988
; Keating et al., 1997
). Thus, in freshly ejaculated semen, the incidence of sperm with a cytoplasmic droplet is low (Keating et al., 1997
; Laudat et al., 1998
). 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., 1998
).
X-ray microscopy is a relatively new imaging technique with a practical resolving power of 3050 nm. High-resolution ultrastructural studies of live cells in physiological solution with high contrast are possible with transmission XM (Kirz et al., 1995; Abraham-Peskir, 2000
). 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., 1991
; DaSilva et al., 1992
). 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., 1992
). XM has also revealed changes that occur in spermatozoon mitochondrial morphology after exposure to capacitating conditions (Vorup-Jensen et al., 1999
).
Immediately after ejaculation, sperm must migrate across the semenmucus 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, 1981). 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., 1988
; Katz et al., 1990
). 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., 1989
).
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.
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Materials and methods |
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Sperm preparation
Semen samples were collected by masturbation from men (age 2143) 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, 1999), 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 KurzrokMiller method (Mortimer, 1994) 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, 1998) 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 35 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 510 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.
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Results |
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Discussion |
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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., 1999). MPV were considered to be artefacts in two TEM studies: one that examined the effect of Percoll gradients on human sperm (Arcidiacono et al., 1983
) and another that reported damage incurred by sperm during purification protocols (Rodriguez-Martinez et al., 1997
). 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, 1972). 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, 1998
). The motor apparatus of sperm exposed to hypo-osmotic Ringer's solution coil up within the plasmalemma (Drevius and Eriksson, 1966
). 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 ~340375 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, 315340 mOsm/kg (Levine and Marsh, 1971); 312380 mOsm/kg (Hinton et al., 1981
) or uterine fluid, 280294 mOsm/kg (Casslen and Nillson, 1984
). 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., 1990
), 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., 1983) 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., 1983
) 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, 1984). The osmolality of semen increases progressively after liquefaction due to the breakdown of seminal proteins (Velazquez et al., 1977
). 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., 1997; Suzuki-Toyota et al., 1999
) 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.
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Acknowledgements |
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Notes |
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References |
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accepted on October 15, 2001.