Demonstration of acrosomal membranes using the hypoosmotic swelling test
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ANDROLOGIA 25, 1-2 (1993) ACCEPTED: MAY11, 1992 Demonstration of acrosomal membranes using the hypo- osmotic swelling test R. Sanchez,' M. Concha,2 E. Topfer-Petersen3and W.-B. Schil14 'Department of Preclinical Sciences, University of La Frontera, Chile 'Institute of Histology and Pathology, Austral University of Chile, Chile 3Department of Dermatology, Andrology Unit, Ludwig Maximilians University, Munich, Germany 4Department of Dermatology and Andrology, Justus Liebig University, Giessen, Germany Key words. Spermatozoa - acrosomal membranes - hypo-osmotic swelling test - indirect immunofluorescence - human. Summary. The state of the acrosomal mem- branes in human spermatozoa was studied by means of the hypo-osmotic swelling test and indirect immunofluorescence using anti-boar outer acrosomal membrane antibodies. The swell- ing phenomenon observed in the acrosomal region was characterized by expansion of the plasma membrane without modification of the outer acrosomal membrane. Introduction One property of the cell membrane is its ability to permit the selective transport of molecules. Human spermatozoa swell under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. The sperm tail appears to be particularly susceptible to hypo- osmotic treatment (Drevius & Eriksson, 1966). These osmotic changes induce typical morpho- logical alterations characterized by the presence of a swollen area. The occurrence of these mor- phological changes has been considered an indi- cation of the membrane integrity and normal functional activity of human spermatozoa (Jeyen- dran et al., 1984). We report the visualization of a swollen region in the head of spermatozoa after hypo-osmotic treatment and subsequent immuno- fluorescence. This phenomenon was confirmed by transmission electron microscopy. Correspondence: Professor Dr W.-B. Schill, Zentrum fir Dermatolo- gie und Andrologie, Gaflkystrasse 14, D-6300 Giessen, Germany. Material and methods Sperm suspension obtained by 'swim-up' was centrifuged at 250 x g for 10 min and the sperm pellet was resuspended in 1 ml of hypo-osmotic medium (150 mOsm) which contained 7.35 g sodium citrate (Merck, Darmstadt, Germany) and 15.3 1 g fructose (Merck, Germany) dissolved in 1000 ml of distilled water according to Jeyend- ran et al. (1984). After 60 min of incubation at 37 "C the sperm suspension was centrifuged at 250 x g for 10 min. The supernatant was dis- carded and the remaining sperm pellet was pro- cessed according to one of the following procedures: (1) ten microlitres were smeared on two slides, air dried for 15 min, and fixed in absolute ethanol (Merck, Germany) at 4 "C for 30min. After air drying, the smears were pre- incubated with 0.5% BSA-PBS (30min) and then allowed to react with purified rabbit anti- boar outer acrosomal membrane antibodies (1 : 100 in PBS) for 2 h at 37 "C in a moisture chamber (Topfer-Petersen et al., 1986). Controls were incubated with normal rabbit serum (1 : 100 in PBS). After the washing procedure (3 x for 10 min in PBS), the second antibody conjugated to FITC (anti-rabbit IgG) (Miles, Is Scientific, Munich, Germany) was added to the smears at a dilution of 1 : 16 in PBS and incubated for 60 min at 37 "C in a moisture chamber, followed again by washing steps (3 x for 10 rnin). The smears were embedded in PBS glycerol (1 +9) and examined under oil immersion at 1000 x magnification, using a Leitz fluorescence micro- scope, and (2) the pellets for the preparation of free cells by electron microscopy were processed as described by Sanchez et al. (1989).
Transcript of Demonstration of acrosomal membranes using the hypoosmotic swelling test
ANDROLOGIA 25, 1-2 (1993) ACCEPTED: MAY 11, 1992
Demonstration of acrosomal membranes using the hypo- osmotic swelling test
R. Sanchez,' M. Concha,2 E. Topfer-Petersen3 and W.-B. Schil14 'Department of Preclinical Sciences, University of La Frontera, Chile 'Institute of Histology and Pathology, Austral University of Chile, Chile 3Department of Dermatology, Andrology Unit, Ludwig Maximilians University, Munich, Germany 4Department of Dermatology and Andrology, Justus Liebig University, Giessen, Germany
Key words. Spermatozoa - acrosomal membranes - hypo-osmotic swelling test - indirect immunofluorescence - human.
Summary. The state of the acrosomal mem- branes in human spermatozoa was studied by means of the hypo-osmotic swelling test and indirect immunofluorescence using anti-boar outer acrosomal membrane antibodies. The swell- ing phenomenon observed in the acrosomal region was characterized by expansion of the plasma membrane without modification of the outer acrosomal membrane.
Introduction
One property of the cell membrane is its ability to permit the selective transport of molecules. Human spermatozoa swell under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. The sperm tail appears to be particularly susceptible to hypo- osmotic treatment (Drevius & Eriksson, 1966). These osmotic changes induce typical morpho- logical alterations characterized by the presence of a swollen area. The occurrence of these mor- phological changes has been considered an indi- cation of the membrane integrity and normal functional activity of human spermatozoa (Jeyen- dran et al., 1984). We report the visualization of a swollen region in the head of spermatozoa after hypo-osmotic treatment and subsequent immuno- fluorescence. This phenomenon was confirmed by transmission electron microscopy.
Correspondence: Professor Dr W.-B. Schill, Zentrum f i r Dermatolo- gie und Andrologie, Gaflkystrasse 14, D-6300 Giessen, Germany.
Material and methods
Sperm suspension obtained by 'swim-up' was centrifuged at 250 x g for 10 min and the sperm pellet was resuspended in 1 ml of hypo-osmotic medium (150 mOsm) which contained 7.35 g sodium citrate (Merck, Darmstadt, Germany) and 15.3 1 g fructose (Merck, Germany) dissolved in 1000 ml of distilled water according to Jeyend- ran et al. (1984). After 60 min of incubation at 37 "C the sperm suspension was centrifuged at 250 x g for 10 min. The supernatant was dis- carded and the remaining sperm pellet was pro- cessed according to one of the following procedures: (1) ten microlitres were smeared on two slides, air dried for 15 min, and fixed in absolute ethanol (Merck, Germany) at 4 "C for 30min. After air drying, the smears were pre- incubated with 0.5% BSA-PBS (30min) and then allowed to react with purified rabbit anti- boar outer acrosomal membrane antibodies (1 : 100 in PBS) for 2 h at 37 "C in a moisture chamber (Topfer-Petersen et al., 1986). Controls were incubated with normal rabbit serum (1 : 100 in PBS). After the washing procedure (3 x for 10 min in PBS), the second antibody conjugated to FITC (anti-rabbit IgG) (Miles, Is Scientific, Munich, Germany) was added to the smears at a dilution of 1 : 16 in PBS and incubated for 60 min at 37 "C in a moisture chamber, followed again by washing steps (3 x for 10 rnin). The smears were embedded in PBS glycerol (1 +9) and examined under oil immersion at 1000 x magnification, using a Leitz fluorescence micro- scope, and (2) the pellets for the preparation of free cells by electron microscopy were processed as described by Sanchez et al. (1989).
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Results Discussion
Figure 1 a illustrates human spermatozoa treated by exposure to hypo-osmotic medium and indirect immunofluorescence. These show an intense acro- somal fluorescence obtained by an excellent cross reactivity of anti-boar outer acrosomal membrane antibodies with the outer acrosomal membrane of human spermatozoa (human spermatozoa with acrosomal reaction presented no fluorescence in the acrosomal region). The swelling phenomenon of the sperm head is observed as an expansion of the plasma membrane, whereas the outer acro- somal membrane is persisting without any modifi- cation. Figure l b shows this process which is visualized at the electron microscopic level as a partial separation of the plasma membrane in the acrosomal region (arrow), whereas the remaining acrosomal structures are well preserved.
Figure 1. (a) Human spermatozoa treated by exposure to hypo- osmotic medium and indirect immunofluorescence. The swelling phenomenon in the sperm head is observed as an expansion of the plasma membrane (arrow). The outer acrosomal membrane is unaltered and shows an intense immunofluorescence reaction. (b) This process is confirmed at the electron microscopic level as a separation of the plasma membrane from the outer acrosomal membrane only in the acrosomal region (arrow). The acrosomal membranes are not altered.
Morphological changes induced by hypo-osmotic solutions in the human spermatozoon occur pref- erentially in the tail, as described by Jeyendran et al. (1984). These changes were clearly visible by phase-contrast microscopy. By means of indirect immunofluorescence with anti-boar outer acrosomal membrane antibodies and exposure of human spermatozoa to hypo-osmotic medium it was possible, for the first time, to visualize the swelling phenomenon in the sperm head. This shows the capacity for expansion of the plasma membrane in the acrosomal region and the prin- cipal specificity of this antiserum for the outer acrosomal membrane. The slight fluorescence of the plasma membrane is due to small contami- nations of the outer acrosomal membrane prep- aration used for immunization (Topfer-Petersen & Schill, 1983; Hinrichsen et al., 1985).
The indirect immunofluorescence technique in conjunction with the exposure to hypo-osmotic solution provides a new method for the visualiz- ation of the state of the acrosomal membranes in viable human spermatozoa.
Acknowledgements
This study was supported by Deutsche For- schungsgemeinschaft Sch 86/7-6 and TO/ 1 14- 1. R. Sanchez is a recipient of a fellowship of the Alexander von Humboldt Foundation, Bonn-Bad Godesberg, Germany.
References
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Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJD (1984) Development of an assay to assess the functional integrity of the human sperm mem- brane and its relationship to other semen characteristics. J Reprod Fertil 70:219-228.
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