ARCHIVES OF ANDROLOGY 48:209 ±219 (2002)

Copyright # 2002 Taylor & Francis

0148-5016 /02 $12.00+.00

HEPARIN±GLUTATHIONE III: STUDY WITH

FLUORESCENT PROBES AS INDICATORS
OF MEMBRANE STATUS OF BULL SPERM
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R. REYES
J. C. MARTINEZ
N. M. DELGADO
Biology of Reproduction Division, East Biomedical Research Center,
General Zone Hospital, #5, Mexican Institute of Social Security (IMSS),
Puebla, Mexico

H. MERCHANT-LARIOS
Biomedical Research Institute, National Autonomy of Mexico,
University City, Mexico, DF, Mexico
For personal use only.

Sperm obtained from bull epididymes were used to validate in vitro the e€ ect of heparin and re-
duced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and
Triton X-100 in the presence of propidium iodide (IP) and diacetate ¯uorescein (FDA). The meta-
bolic activities of treated sperm were qualitatively monitored using an alamarBlue Redox ¯uores-
cence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at
26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time
zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage.
Results validated by electron microscopy of thin sections of treated sperm showed complete lack
of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100.
Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus,
leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic
activity was supported over 52 h of incubation in any of the incubation systems tested, including
Triton X-100, which showed a spermaticide e€ ect. The authors propose that membrane damage
does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining
can be used as a ¯uorescent marker of sperm plasmatic membrane permeabilization and nuclear
swelling.

Keywords decondensation, ¯uorescein diacetate, GSH, heparin, iodium propidium, redox, sodium dodecyl
sulfate, Triton X-100

We thank M.V.Z. Jose GonzaÂlez Cruz and M.V.Z. Eduardo Cabrera of the Rastro Municipal de Atlixco,
Puebla, MeÂxico, for bovine epididymis.
Address correspondence to Rosalina Reyes, DivisioÂn de BiologõÂ a de la ReproduccioÂn, Centro de InvestigacioÂn
BiomeÂdica de Oriente, 34 Norte No. 1816, Col. Humboldt, Puebla, Pue. 72330, Mexico.
E-mail: ndelgado@prodigy.net.mx

209
 210 R. Reyes et al.

Membrane permeabilization seems to be a necessary condition for the internalization of

key compounds that might induce sperm nucleus decondensation [1, 9]. Over the past
decade, we have been able to show that heparin±GSH acts as the physiological decon-
densing agents that induce the decondensation of mammalian sperm nuclei [24]. Heparin
binds to the sperm plasma membrane in a process that seems to be analogous to the in-
teraction between hormones and the cell surface receptors [6, 13]. The mechanism that has
been suggested to occur in the rat liver cells may be explained by the fact that when sperm
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cells are treated with heparin, it might release phospholipase A1 and it binds at the site of
phospholipas e A1 [11, 20, 28, 29]. Binding to membrane and nuclear decondensation
might be interrelated, with heparin ®rst binding and then exerting a destabilization e€ ect
on the cell membrane. Another interesting possibility would be the action that heparin
and=or reduced glutathione might have on the reactive oxygen species (ROS).
Assessing the permeabilization and=or the loss of the sperm plasma membrane integrity
with the usual stain procedures would mislead us regarding some of the reproduction
events. Therefore, the need for accurate assessment to monitored membrane permeability,
sperm nuclei swelling, sperm±egg fusion, chromatin condensation, cell viability, embryo-
nic viability, metabolism, etc., makes us search for ¯uorescent dyes that might solve such a
requirement in a quick and easy way [10, 12, 15, 27].
Reduced glutathione (GSH) may play important roles in protecting the cell from oxi-
For personal use only.

dative damage and in sperm nuclear decondensation. There is an increase in the synthesis
and levels of GSH during oocyte maturation for pig, hamster, mouse, and bovine.
However, GSH at these levels in vitro does not induce sperm nuclear decondensation, even
when combined with other thiol reagents [2, 8, 19, 23, 24, 30]. Furthermore, GSH alone at
the concentration found in each one of the animal species environment in which fertili-
zation take place is incapable of provoking nuclear decondensation of the sperm nuclei of
all the animal species tested. Nevertheless, when heparin±GSH are tested together, they
were able to induced nuclei decondensation [3±5, 7, 23±25, 28].
Therefore, it is important to evaluate if the sperm plasma membrane destabilization
might be induced by the presence of heparin and=or GSH and if this process can be
measured by a ¯uorescent test designed to determine cell viability. This test is an adap-
tation of standard staining methods with propidium iodide and ¯uorescein diacetate for
use in a simultaneous double-staining procedure, in which viable cells stained bright green
(intact membrane) and nonviable cells stained bright red (damaged membrane) [16].

MATERIALS AND METHODS

Bovine testes were obtained at the slaughterhouse of Atlixco, Puebla and trans-
ported on ice to the laboratory. The entire epididymis was removed and repeatedly
washed in salt medium. The isolated epididymides were perfused via the spermatic
artery with the same solution. Sperm were obtained from the cauda region by pro-
cedures previously described [3]. To diminish interdonor variability, pools of sperm
cells obtained from 5 di€ erent animals were used for individual experiments [14].
Samples were made up to 10 mL with the salt medium proposed by Keyhani and
Storey [18]: 113 mM KCl, 12.5 mM KH 2 PO4 , 2.5 mM MgCl2 , and 20 mM Tris, all
adjusted to pH 7.4. These samples were washed by centrifugation, resuspended,
 Heparin ~ Glutat hione in Sperm 211

counted, and diluted if necessary, to obtain a ®nal concentration of 108

spermatozoa=0.1 mL.
Bioassay Procedure
Separate aliquots of 5£10 7 sperm cells were added the salt medium to give a total
volume of 1 mL. Suspensions were incubated at 37 ¯ C and aliquots were evaluated at
preestablished times, under the following conditions:
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1. When the incubation was performed in the absence of heparin=GSH.

2. When the incubation was performed in the presence of 80 m M heparin alone.
3. When the incubation was performed in the presence of 15 mM GSH alone.
4. When the incubation was performed in the presence of both compounds (80 mM
heparin=15 mM GSH).
5. When incubation was performed in the presence of 0.01% Triton X-100.
6. When incubation was performed in the presence of 0.01% SDS.
Sperm Viability
Cell viability and plasma membrane integrity of sperm cells under di€ erent experi-
mental conditions tested was determined by simultaneous staining with 100 m L (2 m g)
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¯uorescein diacetate and 30 m L (0.6 m g) propidium iodide [16]. Stained cells were examined
with a microscope (Nikon E-600) equipped with epi¯uorescence attachment . Evaluation of
the percentage of sperm cells that ¯uoresce bright green (intact membrane) or red
(damaged membrane) was performed by counting 20 random high-magni®cation ®elds
(400 £ [200±400 sperm cells]).
Sperm Redox State
The alamarBlue assay incorporates a ¯uorometric=colorimetric indicator based on
detection of metabolic activity. The system incorporates an oxidation±reduction (redox)
indicator that both ¯uoresces and changes color in responses to chemical reduction of
culture medium resulting from cell activity [21]. The speci®c (¯uorometric=colorimetric)
redox indicator incorporated into alamarBlue has been carefully selected based on several
properties: (1) This indicator exhibits both ¯uorescence and colorimetric change in the
appropriate oxidation±reduction range relating to cellular metabolic reduction. (2) This
indicator is minimally toxic to living cells. (3) The redox indicator produces a clear, stable,
distinct change, which is easy to interpret, from oxidized (non¯uorescent, blue) to reduced
(¯uorescent, red) form.
A total of 5£107 sperm cells in 1 mL of the incubation mixture were stained by adding
10 m L alamarBlue and left at 37 ¯ C. Aliquots were extracted at preestablished times and
evaluated for metabolic activity.
Electron Microscopy
At the selected times, aliquots of the treated spermatozoa were ®xed for 1 h at room
temperature by adding 9 volumes of 0.1 M cacodylate bu€ er, pH 7.3, containing 1%
paraformaldehyd e plus 2.5% glutaraldehyde in 0.1 M sodium cacodylate bu€ er (pH 7.3)
[17]. The samples were then washed by centrifugation and resuspended with 2 changes of
 212 R. Reyes et al.
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Figures 1 and 2. Fluorescence images of untreated cauda epididymal bull sperm, stained with IP,
which ¯uoresces bright red (damaged membrane), and FDA, which ¯uoresces bright green (intact
membrane). £400.
For personal use only.

Figures 3 and 4. Microphotographs of cauda epididymal bull sperm nucleus swelling (*). Left:
Aspect of a swollen nucleus under phase-contrast microscopy where it is atmost impossible to ob-
serve the nucleus. £400 Right: Same microscopy ®eld but the swollen sperm nucleus was stained with
IP to ¯uoresce bright red. £400.

0.1 M cacodylate bu€ er alone and post®xed in 1% OsO4 for 2 h [31]. This was followed
by dehydration in ascending ethanol concentrations and embedding in Epon [22]. Thin
sections were stained with uranyl acetate and lead citrate [26] and observed using an
electron microscope Joel 1010.

RESULTS=DISCUSSION
Sperm from 150 bull epididymides was obtained to perform normal spermbioassay with
the aid of FDA-IP. The study was done utilizing only sperm cells from the cauda epidi-
 Heparin ~ Glutat hione in Sperm 213
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Figure 4b. Fluorescence images of a representative sperm when the metabolic activity was quali-
tatively measured with the almarBlue redox indicator. No matter that the sperm cells were incubated
for 72 h in the presences of heparin, GSH, SDS, or Triton X-100 the metabolic activity was sus-
tained, producing a ¯uorescent pink-red color (alamarBlue, reduced form). £400.
For personal use only.

Table 1. E€ ect of Physiological Spermatozoa Nuclei Decondensing Agents and Detergents on Viability

Incubation Time at 37 ¯ C (h)

Additions T0 1 2 3 22 24 26

Non 83a 78a 69 a 65a 45a 41 a 29 a

Heparin 80 mM 84 81 71 65 24a 24 a 15 a
GSH 15 mM 75a 66a 57 a 50a 36a 24 a 17 a
HEP=GSH 78 64a 60 55 30a 22.4a 14.3a
SDS 0.01% 65a 63a 57.3 a 54.6 a 39 35.7 24.8
Triton X-100 0.01% 25¤ 6¤ 2¤ 1¤ nil¤ nil¤ nil¤

Note. Simultaneous double-staining procedure (FDA-IP) was used to determine sperm viability. Viable sper-
matozoa ¯uoresce bright green (intact membrane) and nonviable spermatozoa are bright red (damaged mem-
brane). Data show the mean expressed as a percentage of 10 determinations of sperm viable cells.
a
Non-addition data versus all other means in the same column were statistically di€ erent (unpaired t-test),
p<.05.
* Triton X-100 data versus all other means in the same column were statistically di€ erent (unpaired t-test),
p<.05.

dymis and the sperm average per bull cauda epididymis was of 1617£106 spermatozoa.
Twenty percent of this sperm had double tails or heads with anomalous shape, or were
without motility. However, from this 20%, half showed membrane damage, since they
¯uoresced bright red, while the spermatozoa with intact membrane ¯uoresced bright green
(Figures 1, 2). On the other hand, a notable observation is the involuntary error that is
consistently made in evaluating the sperm quality or nuclei sperm swelling with the use of
vital stains and light or phase-contras t microscopy, instead of using a ¯uorescence probe
 214 R. Reyes et al.
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Figure 5. Electron microphotograph of a thin section of bull sperm after being incubated 26 h in the
presence of 80 m M heparin. Note the insertion point of the plasma membrane at the basal plate level in the
implantation fossa (arrow); the sperm nucleus appears highly condensed (n) and the plasma membrane
completely loose (p) as well as the postacrosomal membrane system (e) and the acrosome (a). Bar = 1 m M.
 Heparin ~ Glutat hione in Sperm 215
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Figure 6. Electron microphotograph of a thin section of bull sperm after being incubated 26 h in the
presence of 15 mM GSH. Note the complete lack of the acrosome, the insertion point of plasma
membrane at the basal plate level in the implantation fossa (arrow) and completely loose in the
middle piece (p); the sperm nucleus appears highly condensed (n); postacrosomal membrane system
appears completely loose but apparently without damage (e). Bar = 1 m M.

for sperm ARN=DNA, which makes it quite easy to score abnormal sperm (Figures 3, 4).
Therefore, it seems reasonable enough to use a ¯uorescent probe that is used to evaluate
cell viability, but it might also be used to evaluate the status of the sperm plasmatic
membrane.
At zero time, the results obtained between untreated cauda epididymal sperm (non-
addition) and the sperm incubated in presence of 80 m M of heparin shows that 15 to 20%
¯uoresce bright red (Table 1), meaning that these sperm have membrane damage. This
pattern was also observed when sperm were incubated in the presence of 80 m M heparin=15
mM reduced glutathione. On the contrary, a signi®cant 25% increase of sperm membrane
destabilization was obtained when sperm were incubated with 15 mM GSH alone (Table 1).
It would appear that there is a protecting role of heparin against a deleterious e€ ect of
reduced glutathione (GSH).
Moreover, when at zero time the incubation was carried out in the presence of sodium
dodecyl sulfate (SDS), which causes desegregation of cell wall bacteria, 35% of sperm
¯uoresced red. When instead of SDS we used under the same incubation and time con-
ditions 0.01% Triton X-100, a non-ionic detergent known for its spermaticide e€ ect, the
rate of cell membrane disarrangement reached 75%. This last striking e€ ect increased
 216 R. Reyes et al.
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Figure 7. Electron microphotograph of a thin section of bull sperm after being incubated 26 h in the
presence of 0.01% SDS. Note the loss of the acrosomal content remaining only the inner acrosomal
membrane (i); the insertion point of plasma membrane at the basal plate level in the implantation
fossa (arrow); the postacrosomal membrane system (e); and the highly condensed sperm nucleus (n).
Bar = 1 mM.

when the incubation time increase up to 22±26 h and reaches 100% sperm membrane
damage (Table 1). In contrast, when incubation time reaches 22, 24, and 26 h, the results
between untreated sperm and the sperm incubated with 80 m M heparin or with the mixture
of Hep=GSH were statistically signi®cant, contrary to what happens in the ®rst hours of
the incubation time (T0 = 3 h) (Table 1).
Electron microphotographs validate all the data presented in Table 1. In the presence of
heparin the plasmatic membrane is lost all over the sperm head and postacrosomal
membrane. Another feature is the acrosomal swelling and the maintenance of the highly
chromatin structure (Figure 5). The chromatin structure remains highly structured
(Figures 6, 7). The behavior of GSH and SDS are practically the same, in spite of the fact
that they are supposedly chemically and functionally di€ erent.
In contrast, the performanc e of Triton X-100 is quite di€ erent than heparin, SDS, or
GSH, at a concentration 10-fold less than normally used. In this case, the plasmatic, ex-
ternal and inner acrosomal, and nuclear membranes as well as the acrosomal content are
dissagregated in electron-dense spots. The nuclear chromatin structure remains without
morphological modi®cation (Figure 8). A relevant and striking result was obtained when
the metabolic activity was qualitatively measured with the almarBlue redox indicator
 Heparin ~ Glutat hione in Sperm 217
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Figure 8. Electron microphotograph of a thin section of bull sperm after being incubated 26 h in the
presence of 0. 01% Triton X-100. Note the complete lack of plasma membrane, acrosome structure,
and nuclear membrane; the insertion point of plasma membrane at the basal plate level in the im-
plantation fossa (arrow); the sperm nucleus appears highly condensed (n); and dot clusters of
electron-dense material (s). Bar = 1 mM.
 218 R. Reyes et al.

(Figure 4b). No matter that the sperm cells were incubated for 72 h in the presence of
heparin, GSH, SDS, or Triton X-100 the metabolic activity was sustained, producing a
¯uorescent pink-red color (alamarBlue, reduced form).
It would appear that membrane damage does not indicate cell death, irrespective of the
vital stain employed. We also suggest that FDA-IP can be used as a ¯uorescent marker of
sperm plasma membrane permeabilization and nuclear swelling.
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