1 Institute of Reproductive Medicine of the University Clinic, Domagkstrasse 11, D-48129 Münster, Germany and 2 Department of Anatomy, Tehran University of Medical Sciences, Tehran, Iran
3 To whom correspondence should be addressed. Email: cooper{at}uni-muenster.de
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Abstract |
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Key words: artefacts/cytoplasmic droplets/human sperm/morphology/nomenclature
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Introduction |
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Observations on human ejaculated sperm that have been swollen by the channel blocker quinine and that display poor penetration of surrogate mucus suggest that volume regulation may play a role in human fertility (Yeung and Cooper, 2001; Yeung et al., 2003
). The vast majority of literature on cytoplasmic droplets of human sperm considers them to be indicative of abnormality with sperm being described as of diminished maturity (Gergely et al., 1999
) or immature sperm (Ollero et al., 2000). The different terminology used to describe the cytoplasmic structure of human sperm (cytoplasmic droplets, Rago et al., 2003), cytoplasmic residues (Keating et al., 1997
), residual sperm cytoplasm (Aitken et al., 1994
), abnormal retention of cytoplasmic droplets (Zini et al., 2000
), retention of cytoplasm (Mak et al., 2000
) attests to the confusion surrounding this organelle.
Such disagreement in terminology is not helped by the different descriptions given by semen analysis manuals. According to the World Health Organization (1999), cytoplasmic droplets observed in air-dried semen smears should only be considered morphological defects when large (greater than one-third or one-half the sperm head size), implying that smaller droplets are not abnormal. The ESHRE/NAFA handbook (2002)
confirms that sperm with retained cytoplasm less than one-third the sperm head size are normal but adds to the confusion by stating that residues larger than this are abnormal and classified as cytoplasmic droplets, associating that name with an abnormal structure.
Recent observations on osmotically sensitive midpiece vesicles have prompted the view that such vesicles may be a normal component of human sperm (Abraham-Peskir et al., 2002; Chantler and Abraham-Peskir, 2004
), although these authors consider them distinct from cytoplasmic droplets by accepting that such droplets survive air-drying and are characteristic of immature sperm.
The recommended clinical procedure for evaluating human seminal sperm (the production of air-dried smears before fixation: World Health Organization, 1999) can cause morphological artefacts. For instance, it produces severely swollen sperm heads when applied to immature epididymal sperm (Yeung et al., 1997
; Soler et al., 2000
), yet no swollen sperm heads are observed if the cells are fixed before making the smears. Such a drastic procedure as air-drying has been shown to disrupt fragile, osmotically sensitive midpiece vesicles (Abraham-Peskir et al., 2002
). Thus the apparent absence of droplets from a normal spermiogram may be an artefact of semen preparation for sperm morphological analysis.
This report describes experiments performed in order to assess the presence of true (not abnormal) cytoplasmic droplets on human sperm in living, in fixed and wet, and in air-dried preparations.
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Materials and methods |
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Ejaculates
Human ejaculates of widely differing quality were obtained with informed consent from 37 patients attending the Institute of Reproductive Medicine and from 12 student volunteers. Ten normozoospermic samples were included and the characteristics of the semen provided in this study, as analysed according to the World Health Organization manual (1999), are presented in Table I.
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Microscopical observations
After liquefaction, semen samples were incubated for 15 min at 37°C and 3 µl were examined under a 22 x 22 mm cover slip by phase contrast microscopy (Olympus BH-20, Japan) with a x40 objective and x10 ocular on a heated stage (Mini-Tüb, Germany) at 37°C. The percentage of motile sperm (WHO grades a + b + c) was determined and immotile and motile sperm were separately assessed for the presence of (I) cytoplasmic droplets (small, regular distensions at the neck or midpiece: Figure 1a, c, e), (II) abnormal residual cytoplasm (large, irregular material along the mid-piece: Figure 1d, h, iFigure 1d, h, i), (III) coiled or looped tails (Figure 1g, j, k) or (IV) none of the above categories. To 1020 µl of these samples was added an equal volume of 7% (v/v) glutaraldehyde and after 60 min at room temperature the fixed cells were washed by addition of 1 ml PBS, and centrifugation at 500 g for 5 min. The pellet was examined as a wet preparation at x400 magnification for the presence of categories IIV above. Similar preparations were examined in which semen was mixed with appropriate volumes of BWW230 to a final osmolality of 290 mmol/kg, the osmotic pressure of cervical mucus (Rossato et al., 1996).
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Statistics
Differences between populations were assessed by paired or unpaired t-tests, and relationships by linear regression. P<0.05 was accepted as statistically significant.
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Results |
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Sperm morphology and motility
The majority of live sperm viewed in wet preparations in semen (mean 336 mmol/kg at the time of processing) and in media of female tract tonicity (290 mmol/kg) were observed to have cytoplasmic droplets at the neck regions, sometimes extending along the length of the midpiece (Figure 1b, f). The percentage of motile sperm with droplets significantly exceeded that of immotile cells in both semen and at the osmolality of cervical mucus (290 mmol/kg) and there was no difference in motility of sperm in these fluids (Table II). The same sperm suspensions fixed in glutaraldehyde also revealed cytoplasmic droplets on the majority of them. In order to compare this value with that obtained from the live wet preparations, a weighted mean was obtained by multiplying the percentage of motile and immotile sperm by their respective percentages of droplets. This percentage (40.5±1.5, n=50, mean±SEM) was not different (paired t-test) from that of the glutaraldehyde-fixed sperm (38.4±1.7).
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Discussion |
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A related and important observation for the morphological assessment of human ejaculates was the far lower percentage of droplets on sperm observed in air-dried, Papanicolaou-stained smears. Indeed, the same observer counted far fewer droplets in stained smears than in the live or fixed wet preparations of the same ejaculates. This attests to the general inadequacy of the routine method for preserving structures sensitive to the stresses accompanying air-drying before fixing and staining. As even human sperm heads may expand under these conditions (Yeung et al., 1997; Soler et al., 2000
), it should not be surprising that far less rigid and osmotically sensitive vesicles would collapse during preparation.
These findings confirm and extend the observations of osmotically sensitive midpiece vesicles (MPV) extending along the length of the midpiece of human sperm in semen and cervical mucus (Abraham-Peskir et al., 2002; Chantler and Abraham-Peskir, 2004
), which were also considered not detrimental to sperm and were not found in air-dried preparations. These authors considered the so-called MPV to be distinguishable from cytoplasmic droplets by the absence of visible content and their higher incidence than conventional cytoplasmic droplets. MPV were found to have no content as rendered visible by X-ray and differential contrast microscopy of wet preparations, whereas cytoplasmic droplets assessed in air-dried preparations were expected to contain a proteinaceous content which is stained green with Papanicolaou dye. Others have considered swollen cytoplasmic droplets to be artefacts of Percoll preparations (Arcidiacono et al., 1983
), although they clearly can be observed in the absence of Percoll, and their detection by confocal microscopy (Sofikitis et al., 1994
) was also considered abnormal, presumably also because they resembled large cytoplasmic remnants observed in air-dried preparations.
We consider that MPV are true cytoplasmic droplets as observed here (see Figure 1b, f), as they are present on living gametes and neither survive air-drying well. Thus we disagree with Chantler and Abraham-Peskir (2004) that the two organelles are distinct and believe that this difference reflects one of terminology: the abnormal (large, protein-rich, green-stained) cytoplasmic droplets that survive the air-drying procedure are considered by many to be the equivalent of cytoplasmic droplets found on well-fixed sperm, whereas we argue that they are excess residual cytoplasm retained by abnormal sperm produced by imperfect spermiogenesis. This view is supported by electron micrographs of well-fixed semen that reveal the membranous components typical of a true cytoplasmic droplet within a vesicle extending the entire length of the human sperm midpiece (see Figure 3 in Smith et al., 1988). The presence of droplets on living sperm within cervical mucus (Abraham-Peskir et al., 2002
) and on fixed sperm recovered from the Fallopian tube after artificial insemination by husband (Mortimer et al., 1982
) suggest that they may be markers for functionally superior cells. Whether the presence of true cytoplasmic droplets observed in semen smears are related to fertility deserves examination.
The present observations on the cytoplasmic droplet on the midpiece of the majority of living human sperm highlights the inconsistent and confusing nomenclature whereby cytoplasmic droplets are considered normal components of healthy mammalian sperm by basic scientists, but to be a structure associated with abnormal sperm by clinicians. This difference may well have to do with the preparation methods employed by each; generally well-fixed epididymal sperm versus air-dried smears of ejaculated sperm, respectively. Midpiece structures surviving the latter, drastic treatment would include abnormally large amounts of excess cytoplasm not removed at spermiation, adhering to the midpiece and staining green with Papanicolaou. As such excess residual cytoplasm has been associated with sperm from smokers (Mak et al., 2000) and men with varicocele (Zini et al., 2000
) and with deficiencies in sperm DNA (Fischer et al., 2003
) and phospholipid-bound docosahexanoic acid (Zini et al., 2000
), its presence is indeed indicative of abnormal spermiogenesis. Such sperm should not be described as of diminished maturity (Gergely et al., 1999
) or as immature (Ollero et al., 2000
), but merely abnormal, since they cannot and do not undergo maturation in the epididymis.
A plea is made to adhere to a common nomenclature and reserve the term cytoplasmic droplet of human sperm to that defined for sperm from all other Eutherian species, namely an anatomically normal, osmotically sensitive component of a well-formed spermatozoon, produced by a functional testicular tubule, and which does not survive well the air-drying of semen smears. Conversely, the large, irregular abnormal cytoplasmic droplet (World Health Organization, 1999), that does survive air-drying and is stained in routine semen smears, should not be termed a cytoplasmic droplet; the term excess residual cytoplasm (Aitken et al., 1994
) is suggested to indicate the nature of this organelle, found on abnormally formed sperm liberated from a testis displaying damage to the seminiferous epithelium. As both true cytoplasmic droplets and residual cytoplasm could be said to be forms of midpiece vesicles, this term is rejected in favour of the long-existing term cytoplasmic droplet. Acceptance of this terminology should obviate the confusion that has surrounded this topic in the past and reduce confusion arising when the same term is used for two completely different sperm structures.
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Acknowledgements |
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References |
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---|
Aitken R, Krausz C and Buckingham D (1994) Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod Dev 39, 268279.[ISI][Medline]
Ånberg Å. (1957) The ultrastructure of the spermatozoon. An electronmicroscopic study of the spermatozoa from sperm samples and the epididymis including some observations of the spermatid. Acta Obstet Gynecol Scand 36 (Suppl 2), 1133.
Arcidiacono A, Walt H, Campana A and Balerna M (1983) The use of Percoll gradients for the preparation of subpopulations of human spermatozoa. Int J Androl 6, 433445.[ISI][Medline]
Biggers JD, Whittem WK and Whittingham D (1971) The culture of mouse embryos in vitro. In Daniel Jr, JC (ed.) Methods in Mammalian Embryology. Freeman, San Francisco, pp. 86116.
Branton C and Salisbury GW (1947) Morphology of spermatozoa from different levels of the reproductive tract of the bull. J Anim Sci 6, 154160.[ISI]
Chantler E and Abraham-Peskir JV (2004) Significance of midpiece vesicles and functional integrity of the membranes of human spermatozoa after osmotic stress. Andrologia 36, 8793.[CrossRef][ISI][Medline]
Cooper TG and Yeung CH (2003) Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. In Toshimori K (ed.) The Biology of Spermatozoa Maturation. Wiley-Liss Inc, New York, pp. 2838.
Cooper TG, Yeung C-H, Wagenfeld A, Nieschlag E, Poutanen M, Huhtaniemi I and Sipilä P (2004) Mouse models of infertility due to swollen spermatozoa. Mol Cell Endocrinol 216, 5563.[CrossRef][ISI][Medline]
ESHRE/NAFA (2002) Manual on basic semen analysis. http://www.ki.se/org/nafa/manual.
Fischer MA, Willis J and Zini A (2003) Human sperm DNA integrity: correlation with sperm cytoplasmic droplets. Urology 61, 207211.[CrossRef][ISI][Medline]
Gergely A, Kovanci E, Senturk L, Cosmi E, Vigue L and Huszar G (1999) Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity. Hum Reprod 14, 20072014.
Holstein AF and Roosen-Runge EC (1981) Atlas of Human Spermatogenesis. Grosse Verlag, Berlin.
Johnson L (1982) A re-evaluation of daily sperm output of men. Fertil Steril 37, 811816.[ISI][Medline]
Keating J, Grundy CE, Fivey PS, Elliott M and Robinson J (1997) Investigation of the association between the presence of cytoplasmic residues on the human sperm midpiece and defective sperm function. J Reprod Fertil 110, 7177.[ISI][Medline]
Lasley JF and Bogart RA (1944a) Comparative study of epididymal and ejaculated spermatozoa of the boar. J Anim Sci 3, 360370.
Lasley JF and Bogart R (1944b) Some factors affecting the resistance of ejaculated and epididymal spermatozoa of the boar to different environmental conditions. Am J Physiol 141, 619624.
Mak V, Jarvi K, Buckspan M, Freeman M, Hechter S and Zini A (2000) Smoking is associated with the retention of cytoplasm by human spermatozoa. Urology 56, 463466.[CrossRef][ISI][Medline]
Mortimer D, Leslie EE, Kelly RW and Templeton AA (1982) Morphological selection of human spermatozoa in vivo and in vitro. J Reprod Fertil 64, 391399.[ISI][Medline]
Neugebauer DC, Neuwinger J, Jockenhoevel F and Nieschlag E (1990) 9 + 0 axoneme in spermatozoa and some nasal cilia of a patient with totally immotile spermatozoa associated with thickened sheath and short mid-piece. Hum Reprod 5, 981986.[Abstract]
O'Donnell JM (1969) Electrical counting and sizing of mammalian spermatozoa and cytoplasmic droplets. J Reprod Fertil 19, 263272.[ISI][Medline]
Ollero M, Powers RD and Alvarez JG (2000) Variation of docosahexanoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage. Mol Reprod Dev 55, 326334.[CrossRef][ISI][Medline]
Rao CK and Hart GH (1948) Morphology of bovine spermatozoa. Am J Vet Res 9, 117124.[ISI]
Rossato M, Di Virgillio F and Foresta C (1996) Involvement of osmo-sensitive calcium influx in human sperm activation. Mol Hum Reprod 2, 903909.[Abstract]
Smith AC, Anderson CW, Barratt CLR and Williams MA (1988) Ultrastructural morphometric data on human spermatozoa. Andrologia 20, 396403.[ISI][Medline]
Sofikitis NV, Miyagawa I, Zavos PM, Toda T, Iino A and Terakawa N (1994) Confocal scanning laser microscopy of morphometric human sperm parameters: correlation with acrosin profiles and fertilising capacity. Fertil Steril 62, 376386.[ISI][Medline]
Soler C, Perez-Sanchez F, Schulze H, Bergmann M, Oberpenning F, Yeung C and Cooper TG (2000) Objective evaluation of the morphology of human epididymal sperm heads. Int J Androl 23, 7784.[Medline]
White IG, Larsen LH and Wales RG (1959) Method for the in vivo collection of epididymal spermatozoa and for their comparison with ejaculated cells. Fertil Steril 10, 571577.[ISI]
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th edn., Cambridge University Press, Cambridge.
Yeung CH and Cooper TG (2001) Effects of the ion-channel blocker quinine on human sperm volume, kinematics and mucus penetration, and the involvement of potassium channels. Mol Hum Reprod 7, 819828.
Yeung CH, Perez-Sanchez F, Soler C, Poser D, Kliesch S and Cooper TG (1997) Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction. Hum Reprod Update 3, 205213.
Yeung CH, Anapolski M, Depenbusch M, Zitzmann M and Cooper TG (2003) Human sperm volume regulation. Response to physiological changes in osmolality, potential sperm osmolytes and channel blockers. Hum Reprod 18, 10291036.
Zini A, Defreitas G, Freeman M, Hechter S and Jarvi K (2000) Varicoele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa. Fertil Steril 74, 461464.[CrossRef][ISI][Medline]
Submitted on May 5, 2004; accepted on June 16, 2004.