1 Department of Medical Microbiology and Immunology and 2 Institute for Storage Ring Facilities, University of Aarhus,DK-8000 Aarhus C, Denmark, and 3 Forschungseinrichtung Röntgenphysik, Georg-August-Universität Göttingen,Geiststr. 11, D-37073 Göttingen, and Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung m.b.H. (BESSY), Lentzeallee 100, D-14195 Berlin, Germany
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Abstract |
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Key words: capacitation/mitochondria/spermatozoa/X-ray microscopy
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Introduction |
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Hyperactivation, a vigorous but poorly progressive state of motility, is associated with capacitation. A formal characterization of this mode of sperm motility has been difficult and no consensus has so far been reached (for reviews see de Lamirande et al., 1997; Mortimer, 1997
). However, advanced mathematical modelling of trajectories collected in computer-assisted sperm analysis (CASA) has improved classification of hyperactivation (Mortimer et al., 1996
).
There is evidence of change in the membrane density of cholesterol during the progression of sperm maturation, and studies have linked a cholesterol efflux from the spermatozoon with capacitation (Ravnik et al., 1990). Also, it has been suggested that membrane bound proteins such as lectins appear on the sperm membrane after capacitation which thus may serve to expose putative zona pellucida binding proteins (Ahuja, 1985
; Singer et al., 1985
; Cross and Overstreet, 1987
; Benoff et al., 1993
).
The intracellular calcium concentration has been demonstrated to increase stepwise during sperm maturation (Baldi et al., 1991), and other divalent metal ions such as zinc may play a role in capacitation (Fraser, 1995
). Studies point to Ca2+-ATPase activity as an important regulator of the intracellular calcium concentration during capacitation (DasGupta et al., 1994
) and this activity has been shown to be localized in head membranes (Adeoya-Osiguwa and Fraser, 1996
). Voltage-dependent calcium channels appear to be involved in Ca2+ regulation in the terminal stage of the maturation process (Florman et al., 1992
) which ultimately include the loss of the acrosome.
The large number of studies on the capacitation process, apart from highlighting the multifarious biochemistry involved, also emphasize the difficulty in delineating capacitation as a distinct stage on the path to maturation of the spermatozoon. Although staining techniques are available which can distinguish between non-capacitated, capacitated and acrosome-reacted spermatozoa (Lee et al., 1987), these are indirect means of evaluating the state of maturity when compared to the morphological change, the loss of the acrosomal cap, of acrosome-reacted spermatozoa.
Electron microscopy (EM) has revealed fine structural details of the spermatozoon and has contributed much to our understanding of the processes transforming the immature spermatozoon into a potentially fertilizing gamete (Hafez, 1971). In the 1960s it was concluded from EM studies that capacitation did not include a morphological characteristic with a distinctness similar to the loss of the acrosome (Adams and Chang, 1962
; Bedford, 1969
). Subsequent reviews have thus stated that `no major morphological changes are induced by capacitation' (Langlais and Roberts, 1985
) or emphasized events at the molecular level as important (de Lamirande et al., 1997
). Sample treatment required for conventional EM, e.g. dehydration, staining, and metal coating, can introduce artefacts and certainly removes the cell from physiological conditions supporting viability. This treatment thus compromises the interpretation of physiological changes which involves fragile structures of the specimen. X-ray microscopy offers the opportunity of obtaining high resolution images of cells in a liquid medium with the simple preparation techniques used for light microscopy (for a detailed overview of the biological applications of X-ray microscopy see Kirz et al., 1995
). In previous studies, spermatozoa from mammalian species have been examined by combining X-ray absorption measurements with X-ray microscopy with 50 nm resolution in experiments aimed at analysing the DNA to protein ratios (Zhang et al., 1996
). Recent developments in X-ray microscopy allow the visualization of structures down to 30 nm and have improved sample contrast in a liquid medium. By use of this equipment ultrastructural studies on human spermatozoa have been carried out showing details of the surface membranes (Abraham-Peskir et al., 1998
).
In the present study we report on two morphological states of human sperm mitochondria, one apparently associated with fresh spermatozoa and the other with a treatment for in-vitro capacitation.
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Materials and methods |
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X-ray microscopy
After preliminary studies on human spermatozoa carried out with the Göttingen transmission X-ray microscope at BESSY (Berlin, Germany) (Schmahl et al., 1995, 1996
), the full field imaging X-ray microscope located at ISA (Aarhus, Denmark) (Medenwaldt and Uggerhøj, 1998
), was used to obtain the images presented in this study. Both microscopes are equipped with circular diffraction gratings, so called zone plates, as optical elements, providing a resolution of around 30 nm, which is an order of magnitude better than achieved by visible light microscopy. The high resolution was combined with high contrast in the micrographs by choosing an X-ray wavelength of 2.4 nm, where the absorption in organic material is much higher than in water. The optical set-up of the X-ray microscope (Figure 1
) is equivalent to visible light transmission microscopes. Radiation from a source, in our case the synchrotron, is focused by a zone plate corresponding to a condenser lens onto the sample. A microzone plate (MZP) acting as an objective lens behind the sample forms a transmission image on a charge-coupled device (CCD) detector. The condenser has a large central stop which makes the transmitted X-rays form a hollow cone. Thereby, the too intense direct radiation from the storage ring is prevented from reaching the object and the detector.
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Statistical analysis
The proportions of tightly wrapped mitochondria and mitochondrial sheaths with circular areas of high X-ray transmission were compared by a one-tailed Fisher's exact test for 2x2 tables, calculated by use of the SOLO statistical software package (BMDP Statistical Software, Los Angeles, CA, USA).
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Results |
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A vigorous motility pattern of in-vitro capacitated spermatozoa was observed although no formal characterization of hyperactivation was carried out with computer-assisted semen analysis (CASA). Addition of calcium ionophore induced acrosome-reaction in 8090% of the spermatozoa (acrosome loss was observed in ~3% of the cells in control without ionophore added). Spermatozoa treated only by washing, i.e. not harvested by the swim-up procedure, showed no significant change in the staining pattern after incubation with ionophore.
Head dimensions corresponded in general with the morphological classification of human spermatozoa given in the WHO laboratory handbook (4.05.5 µm long, 2.53.5 µm wide), and the mitochondrial sheath contained 1115 turns consistent with previous reports on human spermatozoa (Curry and Watson, 1995). The dimensions measured on the X-ray microscopic images are shown as a model of the loosely wrapped state (Figure 3
) of a simplified mid-piece region, depicting a single helix.
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Discussion |
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Evaluation of the maturation stage of spermatozoa by the ability to undergo calcium ionophore-induced acrosome reaction suffers from involving an agent which is unlikely to have any chemical counterpart in the intrauterine environment and consequently suffers from lack of good reference conditions. In the present study, the great majority of spermatozoa treated according to the in-vitro capacitation protocol were able to undergo acrosome reaction whereas washed spermatozoa lacked this ability. This indicates that the ability to undergo acrosome reaction is caused by in-vitro capacitation and not by adverse effects such as toxicity of the ionophore. The high percentage of spermatozoa which underwent ionophore-induced acrosome reaction may be associated with the likewise high percentage of in-vitro capacitated spermatozoa with morphologically transformed mitochondria, thereby suggesting that the altered mitochondrial morphology is part of the process of capacitation.
Change in mitochondrial morphology is in line with the general concept that mitochondria are dynamic organelles which can change their size and shape depending on the metabolic state of a cell (Sadava, 1993) and during spermiogenesis (Woolley 1970
; Otani et al., 1988
). The model in Figure 3
depicts a single mitochondrial helix; however, it has been considered that mammalian sperm mitochondria wrap around the axonemal complex as a double (Otani et al., 1988
), triple or quadruple helix (Phillips, 1977
). In the present study we did not determine the number of helical strands from our X-ray micrographs. Based on studies of spermatozoa from over 200 mammalian species Cardullo and Baltz (1991) calculated the tail beat frequency to be increasing with the mitochondrial volume. If the morphological change seen after capacitation is interpreted as a distension of the mitochondria then our study does support an increase in mitochondrial volume as accompanying the capacitation process. It may thus be speculated that the observed change in mitochondrial morphology is associated with the change of motility of capacitated spermatozoa into the state of hyperactivitation.
The focus on changes in the sperm head and acrosomal regions as a part of the maturation process seems a natural choice when considering that the ultimate aim of the capacitation process is to allow for fusion between the spermatozoon and the oocyte. Our study, however, suggests that the changes in the mitochondrial region may also be of importance in the understanding of the complex phenomenon of capacitation.
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Acknowledgments |
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Notes |
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References |
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Adams, C.E. and Chang, M.C. (1962) Capacitation of rabbit spermatozoa in the Fallopian tubes and in the uterus. J. Exp. Zool., 151, 159166.[ISI]
Adeoya-Osiguwa, S.A. and Fraser, L.R. (1996) Evidence for Ca(2+)-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm. Mol. Reprod. Dev., 44, 111120.[ISI][Medline]
Ahuja, K.K. (1985) Carbohydrate determinants involved in mammalian fertilization. Am. J. Anat., 174, 207223.[ISI][Medline]
Austin, C.R. (1951) Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res. B, 4, 581596.
Baldi, E., Casana, R., Falsetti, C. et al. (1991) Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J. Androl., 12, 323330.[Abstract]
Bedford, J.M. (1969) Morphological aspects of sperm capacitation in mammals. Adv. Biosci., 4, 3550.
Benoff, S., Hurley, I., Cooper, G.W. et al. (1993) Head-specific mannose-ligand receptor expression in human spermatozoa is dependent on capacitation-associated membrane cholesterol loss. Hum. Reprod., 8, 21412154.[Abstract]
Cardullo, R.A. and Baltz, J.M. (1991) Metabolic regulation in mammalian sperm: mitochondrial volume determines sperm length and flagellar beat frequency. Cell Motil. Cytoskeleton, 19, 180188.[ISI][Medline]
Chang, M.C. (1951) Fertilizing capacity of spermatozoa deposited in fallopian tubes. Nature, 168, 997998.[ISI]
Cross, N.L. and Overstreet, J.W. (1987) Glycoconjugates of the human sperm surface: distribution and alterations that accompany capacitation in vitro. Gamete Res., 16, 2335.[ISI][Medline]
Curry, M.R. and Watson, P.F. (1995) Sperm structure and function. In Grudzinskas, J.G. and Yovich, J.L. (eds), Gametes The Spermatozoa. Cambridge University Press, Cambridge, UK, p. 52.
DasGupta, S., Mills, C.L. and Fraser L.R. (1994) A possible role for Ca(2+)-ATPase in human sperm capacitation. J. Reprod. Fertil., 102, 10716.[Abstract]
Florman, H.M., Corron, M.E., Kim, T.D.H. et al. (1992) Activation of voltage-dependent calcium channels of mammalian sperm is required for zona-induced acrosomal exocytosis. Dev. Biol., 152, 304314.[ISI][Medline]
Fraser, L.R. (1995) Cellular biology of capacitation and the acrosome reaction. Hum. Reprod., 10 (Suppl. l), 2230.
Hafez, E.S.A. (1971) Atlas of Mammalian Reproduction. G.Thieme Publishers, Stuttgart, Germany.
Jacobsen, C., Medenwaldt, R. and Williams, S. (1998) A perspective on biological X-ray and electron microscopy. In Thieme, J., Schmahl, G., Umbach, E. et al. (eds), X-ray Microscopy and Spectromicroscopy. Springer Verlag, Heidelberg, Germany.
Kirz, J., Jacobsen, C. and Howells, M. (1995) Soft X-ray microscopes and their biological applications. Q. Rev. Biophys., 28, 33130.[ISI][Medline]
de Lamirande, E., Leclerc, P. and Gagnon, C. (1997) Capacitation as regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol. Hum. Reprod., 3, 175194.[Abstract]
Langlais, J. and Roberts, K.D. (1985) A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res., 12, 183224.[ISI]
Lee, M.A., Trucco, G.S., Bechtol, K.B. et al. (1987) Capacitation and acrosome reaction of human spermatozoa monitored by a chlortetracycline fluorescence assay. Fertil. Steril., 48, 649658.[ISI][Medline]
Medenwaldt, R. and Uggerhøj, E. (1998) Description of an X-ray microscope with 30 nm resolution. Rev. Sci. Instrum., 69, 29742977.[ISI]
Mortimer, S.T. (1997) A critical review of the physiological importance and analysis of sperm movement in mammals. Hum. Reprod. Update, 3, 40339.
Mortimer, S.T., Swan, M.A. and Mortimer, D. (1996) Fractal analysis of capacitating human spermatozoa. Hum. Reprod., 11, 10491054.[Abstract]
Otani, H., Tanaka, O., Kasai, K. and Yoshioka, T. (1988) Development of mitochondrial helical sheath in the middle piece of the mouse spermatid tail: Regular dispositions and synchronized changes. Anat. Rec., 222, 2633.[ISI][Medline]
Phillips, D.M. (1977) Mitochondrial disposition in mammalian spermatozoa. J. Ultrastruct. Res., 58, 144154.[ISI]
Ravnik, S.E., Zarutskie, P.W. and Muller, C.H. (1990) Lipid transfer activity in human follicular fluid: relation to human sperm capacitation. J. Androl., 11, 216226.
Sadava, D.E. (1993) Cell Biology Organelle Structure and Function. Jones and Bartlett Publishers, Boston, MA, USA.
Schmahl, G., Rudolph, D., Guttmann, P. et al. (1995) Phase contrast studies of biological specimens with the X-ray microscope at BESSY. Rev. Sci. Instrum., 66, 12821286.[ISI]
Schmahl, G., Rudolph, D., Niemann, B. et al. (1996) Naturwissenschaften, 83, 6170.[ISI][Medline]
Schneider, G. (1994) Investigation of soft X-radiation induced structural changes in wet biological objects. In Aristov, V.V. and Erko, A.I. (eds), X-Ray Microscopy IV. Bogorodskii Pechatnik Publishing Company, Chernogolovka, Russia, pp.181195.
Singer, S.L., Lambert, H., Overstreet, J.W. et al. (1985) The kinetics of human sperm binding to the human zona pellucida and zona-free hamster oocyte in vitro. Gamete Res., 12, 2940.[ISI]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 3rd edn. Cambridge University Press, Cambridge, UK.
Woolley, D.M. (1970) The midpiece of the mouse spermatozoon: Its form and development as seen by surface replication. J. Cell Sci., 6, 865879.[ISI][Medline]
Zhang, X., Balhorn, R., Mazrimas, J. et al. (1996) Mapping and measuring DNA to protein ratios in mammalian sperm head by XANES imaging. J. Struct. Biol., 116, 335344.[ISI][Medline]
Submitted on August 20, 1998; accepted on January 11, 1999.