Molecular Human Reproduction vol.3 no.9 pp. 763–767, 1997
Autometallographic demonstration of zinc ions in rat sperm cells
Meredin Stoltenberg1, Michael Busk Sørensen, Gorm Danscher, Søren Juhl, Arne Andreasen and
Erik Ernst
Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
1To
whom correspondence should be addressed
An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated
sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat
spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and
a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in
the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal
AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was
seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate
(DEDTC) was found to block the AMG staining whereas calcium–EDTA, known not to pass through cell
membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located.
Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope
levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the
mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place
between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.
Key words: chelating agents/diethyldithiocarbamate/semen/silver sulphide/spermatozoa
Introduction
Materials and methods
The total zinc content of mammalian semen is high, 800–3000
µg/g dry weight (Bertrand and Vladesco, 1921; Mann, 1945;
Mawson and Fischer, 1953; Vallee and Falchuk, 1993), and it
has been demonstrated that zinc deficiency induces atrophy of
the seminiferous tubules and causes failure of spermatogenesis
in rats (Follis et al., 1941; Millar et al., 1958, 1960; Underwood,
1977; Endre et al., 1990). A handful of articles have used
histochemical methods based on Timm’s original sulphide
silver method (Timm, 1958) for light microscopical (Timm
and Schulz, 1966; Boström, 1971; Danscher and Rebbe, 1974)
and electron microscopical demonstration of zinc in human
sperm cells (Stoltenberg et al., 1997).
It has been suggested that zinc ions might be of significance
for the maturation process of rat sperm cells during their
passage through the epididymis (Schell and Hornstein, 1974;
Fujimori et al., 1988; Stoltenberg et al., 1996). In accordance
with this, treatment with zinc-chelating agents has been found
to affect the motility of rat spermatozoa (Saito et al., 1967).
However, it is still far from clear how zinc ions may affect
sperm cell motility and male fecundity.
This paper describes an autometallographic (AMG) method
suitable for histochemical detection of zinc ions in rat sperm
cells at light and electron microscopical levels. A description
of the localization of zinc ions in rat epididymal and ejaculated
spermatozoa is presented.
A total of 20 rats were used in this study. Five rats were electroejaculated and spermatozoa was collected from caput and cauda of
epididymides from 15 rats. The animals were housed in plastic cages
under the following conditions: 12 h light/dark cycle, 22 6 2°C, and
50 6 10% relative humidity. Food (Altromin No. 1314, Altromin
Spezialfutterwerke, Germany) and tap water were available ad libitum.
© European Society for Human Reproduction and Embryology
Procedure for electroejaculation of rats
Electroejaculation was performed using a bipolar probe electrode
connected to the output of an electrical generator according to Hundal
et al. (1978). The signal sequence leading to ejaculation consisted of
ten consecutive pulses, 3.5 mA and 8 V, each with a duration of 3 s.
One sequence liberates 0.084 J, an amount of energy which is
tolerable but should not be exceeded due to the risk of heat damage
to the rectum.
Procedure for epididymal sperm collection
The rats were killed with i.p. injections of 1.5 ml sodium pentobarbital
(50 mg/ml). The entire epididymis was removed, trimmed of adherent
tissue, separated into caput and cauda segments, and each placed in
individual Petri dishes. The epididymal tissue was cut into tiny pieces
with a pair of scissors, and the ‘tissue soup’ with sperm cells
was flushed with 1.0 ml of SpermWash (Ciconia Products ApS,
Copenhagen, Denmark). After adding a further 1 ml of SpermWash
the suspension was filtered through a 80 µm nylon filter (KE Fibertech,
Vejen, Denmark) and placed in a funnel (for details see Ernst, 1989).
The filtered samples were stored in Eppendorf vials (Teknunc, Aarhus,
Denmark).
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M.Stoltenberg et al.
Preparation for electron microscopy (EM) of epididymal
cells: The centrifugation method
1.0 ml semen collected as described above was mixed with 1.0 ml
0.5% sodium sulphide in phosphate buffer for 30 min at 37°C and
then with 4.5 ml 6% glutaraldehyde in 0.1 M Sørensen buffer for 30
min at room temperature.
The sperm suspension was centrifuged at 1300 g for 3 min and
resuspended in 0.1 M Sørensen buffer. This procedure was repeated
three times. The pellets were mixed with the autometallographic
developer and placed in a dark box for 60 min. After AMG the sperm
cells were centrifuged once and washed three times in phosphate
buffer, before 0.5% osmium (1 ml 0.2 M buffer 1 0.5 ml 2% osmium
1 0.5 ml distilled water) was added for 30 min. The sperm cells
were washed three times in buffer and centrifuged once. The resulting
sperm pellets were transferred in a cryotube to a 50°C warm water
bath, and 1 ml 2% agar solution (60°C) was added and quickly mixed
with the sperm cells. The agar–sperm solution was centrifuged once
before the sperm cell pellets were placed in 3% glutaraldehyde until
embedding in epon.
Semithin (3 µm) sections were cut and placed on glass slides.
After light microscopic analysis, selected sections were re-embedded
in epon, and ultrathin sections were made and counterstained with
lead citrate and uranyl acetate before examination in a JEOL 100S
electron microscope.
Figure 1. Autometallographic (AMG)-developed smears,
counterstained with Toluidine Blue. (a) Sperm cells from the caput
of epididymis. Zinc ions are seen in the tail parts as a double line
of AMG staining. Small zinc grains are also seen in the heads
(original magnification 3600). (b) Sperm from the cauda of
epididymis. AMG grains completely cover the acrosomes. No zinc
grains are located in other parts of the sperm cells (original
magnification 3600). (c) Electroejaculated semen. The acrosome
staining appears identical to that of sperm cells from the caudal
part of the epididymis. In the tail large heavily stained areas
interrupted by non-stained bands are seen (original magnification
3375).
Autometallographic development
The semen was mixed with 0.5% sodium sulphide in phosphate
buffer (1:1 v/v) for 30 min. Smears were made, air-dried and fixed
in 96, 70, and 50% alcohol for 15, 2, and 2 min respectively, and
then finally washed for 10 min with distilled water before development.
Autometallographic development was performed according to
Danscher (1981, 1996). Briefly, the semen smears were developed at
26°C for 60 min in a dark-box and the sections were post-fixed with
70% ethanol for 30 min. Counterstaining was performed with a 0.1%
aqueous Toluidine Blue solution, pH 4.0.
The developer consisted of 60 ml filtered gum arabic solution (1 kg
dissolved in 2 l distilled water), 10 ml sodium citrate buffer (1.21 M
citric acid, 0.8 M sodium citrate) and 0.85 g hydroquinone dissolved
in 15 ml distilled water. Immediately before use, 0.12 g silver lactate
in 15 ml distilled water was added and the solution was mixed
thoroughly (for further details see Stoltenberg et al., 1997).
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Preparations of semen smears for EM: The smear epon
embedding method
Semen was mixed with an equal volume amounts of a 0.5% phosphate
buffered sodium sulphide solution for 30 min at 37°C. Smears were
made on one part 10% potassium ferricyanide and nine parts 10%
sodium thiosulphate (Farmer)-rinsed glass slides, air-dried for 30 s,
transferred to 3% glutaraldehyde for 30 min, and washed three
times in phosphate buffer and once in distilled water. After AMG
development for 60 min the smears were washed once in distilled
water and three times in phosphate buffer. Thereafter 0.5% osmium
(1 ml 0.2M buffer 1 0.5 ml 2% osmium 1 0.5 ml distilled water)
was added for 30 min, and a final wash with buffer and distilled
water was performed. The osmium-contrasted smears were studied
in the light microscope, and areas of interest for further electron
microscopical analysis were marked with a diamond.
The selected smears were dehydrated in graded alcohol solutions
and embedded in epon. Epon-blocks placed on top of the previously
marked areas of interest were kept at 60°C for 24 h and then removed
by fracture from the glass smears. These epon blocks, now containing
the sperm cells previously found in the smears, were placed in a
microtome, and ultrathin sections were made and counterstained
with lead citrate and uranyl acetate before electron microscopical
examination.
Chelating agents
The effects on the staining pattern of prior treatment with the chelating
agents diethyldithiocarbamate (DEDTC) and EDTA were evaluated.
Concentrations of 5 and 10 mM of the two chelating agents were
mixed with the sperm samples for 30 min. Then 0.5% sodium
sulphide (Na2S) was added and the procedure described above was
strictly followed.
Controls
Semen smears not exposed to sulphide were AMG developed, and
smears exposed to sulphide only served as negative controls.
Results
In sperm cells from the caput region of the epididymis the tail
was visible as a double line and the head was discretely dotted
AMG demonstration of sperm zinc
Figure 2. Electron micrographs of the ‘centrifugation method’. (a) Sperm cell from the caput of epididymis. Autometallographic (AMG)
grains are mainly found in the helecine mitochondria of the midpiece. Few scattered grains are seen in the acrosome (original magnification
314 000). (b) Sperm cells from the cauda of epididymis. Zinc grains are exclusively located to the acrosome, no zinc was seen in the
postacrosomal region or tail of the sperm cells (original magnification 311100).
Figure 3. Electron micrographs of the ‘smear epon embedding method’ and the ‘centrifugation method’. (a,b) Micrographs of sperm cells
from the caput and cauda of epididymis after the ‘centrifugation method’ (original magnification 317 400 and 329 900). The lower part of
(a) shows a cross section of a sperm tail in which some mitochondrial grains can be seen. (c,d) A single sperm cell from a smear is
embedded in epon. The sperm cells are from the caput and cauda of the epididymis (original magnification 319 400 and 329 900).
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M.Stoltenberg et al.
Figure 4. (a) Sperm cells from cauda epididymidis treated with diethyldithiocarbamate (DEDTC) (10 mM) before autometallographic
(AMG) and counterstaining with Toluidine Blue (original magnification 31500). (b) Sperm cells from cauda epididymides treated with
EDTA (10 mM) before AMG and counterstaining with Toluidine Blue (original magnification 3600). DEDTC at a concentration of 10 mM
resulted in a complete bleaching of the zinc staining in all regions of the sperm cells. EDTA treatment did not affect the intensity of the
sperm cell zinc staining.
with AMG grains (Figure 1a). In contrast, sperm cells from
cauda epididymis showed a heavy staining with AMG grains
completely covering the acrosome in a confluent pattern (Figure
1b). In these spermatozoa no staining was seen in the midpiece,
tail or post-acrosomal part of the sperm head. In sperm cells
obtained by electroejaculation the acrosomal staining was
identical to that seen in caudal epididymal spermatozoa. The
staining of the tail parts was however quite different, with a
segmentation of large heavily stained bands interrupted by
non-stained bands (Figure 1c).
Sperm cells from caput epididymis showed AMG grains in
the helecine mitochondria of the midpiece. A few grains were
seen in the lower parts of the sperm tail and in the acrosomal
cap (Figures 2a, 3a,c). In sperm cells from cauda epididymis
the AMG grains were exclusively located in the acrosome
(Figure 2b).
Treatment with the chelating agent DEDTC at a concentration of 10 mM resulted in a complete bleaching of the zinc
staining in all regions of epididymal and electroejaculated
sperm cells. An example of caudal sperm cell bleaching is
shown in Figure 4a. Calcium EDTA (10 mM) did not affect
the acrosomal and midpiece staining, as is shown for caudal
spermatozoa (Figure 4b). In the negative control smears, i.e.
smears from semen not exposed to sulphide ions which were
developed by AMG, or smears exposed to disulphide only, no
staining appeared.
Discussion
Two methods of demonstrating ultrastructurally the presence
of zinc ions in rat sperm cells are given. Both methods provide
satisfactory results; the ‘smear epon embedding method’ has
the clear advantage that it is possible to analyse a single
spermatozoon at both light and electron microscopical levels.
On the other hand, the ‘centrifugation method’ is faster and
gives a cleaner background staining since the sperm cells are
washed during the centrifugation procedure thus removing the
zinc ions contained in plasma (Figures 2 and 3).
DEDTC is a chelator of divalent cations and the active
766
metabolite of antabuse. In rats that have received a doses of
1000 mg DEDTC/kg body weight all free zinc ions and loosely
bound zinc ions in the body will be chelated, and the subsequent
treatment with sulphide ions will not result in the creation of
zinc sulpide crystal lattices. AMG development of e.g. brain
or testis sections from such animals will therefore result in
tissue sections without AMG silver grains (Danscher et al.,
1973; Danscher and Montagnese, 1994; Danscher, 1996).
In this study DEDTC was added to the sperm cells 30 min
before the sodium sulphide solution in order to evaluate
whether the AMG staining seen in spermatozoa was caused
by zinc ions. Treatment with DEDTC in concentrations of
10 mM or higher resulted in a complete bleaching of the zinc
staining from all regions of the sperm cells. On the other hand,
calcium EDTA did not affect the zinc staining. EDTA cannot
penetrate cell membranes, and it can therefore be concluded
that the AMG staining seen in the spermatozoa is intracellular
which is in accordance with the ultrastructural findings.
The differences in zinc staining pattern in the rat sperm
cells are dependent on the location in the epididymal duct and
therefore most likely on the maturity of the spermatozoa. The
heavy zinc staining in the acrosome of both epididymal and
ejaculated sperm cells supports this view. An explanation for
the tail zinc staining in the ejaculated sperm cells, as compared
to the cauda–epididymal spermatozoa, could be that some zinc
ions from the prostate are taken up by the sperm cells as they
swim in the prostatic liquid.
In a previous study it has been suggested that the variation
in amount of chelatable zinc in the epididymal epithelium
reveals a role for zinc ions in the maturation of sperm cells
(Stoltenberg et al., 1996). This theory is supported by the
present findings. The results of this study indicate that zinc
ions change their location from the tail region to the head of
the sperm as the cells transverse the epididymal duct.
Total zinc measured with atomic absorption spectrophotometry (AAS) has been analysed in relation to sperm motility
concentration (Lewis-Jones et al., 1996). The authors found
no correlation and concluded that seminal plasma zinc is
an unreliable marker of spermatogenic activity. The AMG
AMG demonstration of sperm zinc
technique demonstrates chelatable zinc ions, not total zinc. To
date, no studies have examined the relationship between zinc
ion staining of sperm cells and their motility, and the possible
significance of our own results remains to be elucidated.
Acknowledgements
The technical assistance of Ms H.Brandstrup, Ms D.Jensen, Ms
K.Wiedemann, Mr B.Krunderup, and Mr A.Meier, is gratefully
acknowledged. We thank Mr T.Madsen and Mr O.Sørensen from
Aalborg Sygehus Nord. Managing Director, M.B.A. Ole Schou from
the Cryos Sperm bank is thanked for delivering SpermWash. We
thank KE Fibertech, Vejen, for the 80 µm nylon filter. The study was
supported by grants from ‘Fonden til Lægevidenskabens Fremme’
and ‘Ciconia Foundation’.
Stoltenberg, M., Ernst, E., Andreasen, A. and Danscher, G. (1996)
Histochemical localization of zinc ions in the epididymis of the rat.
Histochem. J., 28, 173–185.
Stoltenberg, M., Sørensen, M.B. and Danscher, G. (1997) Histochemical
demonstration of zinc ions in ejaculated human semen. Int. J. Androl.,
in press.
Timm, F. (1958) Zur Histochemie des Zinks. Deutsche Z. gesamte ger.
Medizin, 47, 428–431.
Timm, F. and Schulz, G. (1966) Hoden und Schwermetalle. Histochemie, 7,
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Underwood, E.J. (1977) Zinc. In Trace Elements in Human and Animal
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Vallee, B.L. and Falchuk, K.H. (1993) The biochemical basis of zinc
physiology. Physiol. Rev., 73, 79–118.
Received on April 16, 1997; accepted on July 15, 1997
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