Reproductive Toxicology, Vol. 8, No. 2, pp.

155-159, 1994
Copyright 1994 Elsevier Science Ltd
Printed in the USA. All rights reserved
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Pergamon
0890-6238(93)E0009-7

Original Contribution

IN VITRO

FLUORIDE

TOXICITY

IN HUMAN

SPERMATOZOA

NILOUFER J. CHINOY and M U R A K O N D A V . NARAYANA

Reproductive Endocrinology & Toxicology Unit, Department of Zoology, School of Sciences, Gujarat
University, Ahmedabad, India
Abstract m Effects of sodium fluoride (NaF) on washed, ejaculated human spermatozoa at doses of 25, 50,
and 250 mM were investigated in vitro at intervals of 5, 10, and 20 min. Sodium fluoride (NaF) did not affect
the extracellular pH of sperm, except that a slight acidification was caused by the 250 mM dose only. The
treatment caused a significant enhancement in acid phosphatase (ACPase) and hyaluronidase activities after
5 and 10 min. However, the decrease in the lysosomal enzyme activity after 20 min treatment could have been
due to the gradual increase in fluoride accumulation by spermatozoa leading to membrane damage. Silver
nitrate staining of sperm revealed elongated heads, deflagellation, and loss of the acrosome together with
coiling of the tail. Sperm glutathione levels also showed a time-dependent decrease with complete depletion
after 20 min indicating rapid glutathione oxidation in detoxification of the NaF. The altered lysosomal enzyme
activity and glutathione levels together with morphologic anomalies resulted in a significant decline in sperm
motility with an effective dose of 250 mM.
Key Words: NaF; human sperm; in vitro; pH; forward progression; ACPase; hyaluronidase; GSH; morphology.

INTRODUCTION

sperm structure and metabolism is hitherto unexplored. The present investigation studied the metabolic and morphologic alterations induced by sodium fluoride (NaF) treatment in vitro in human
spermatozoa.

Previous investigations in fluoride intoxicated experimental animals and humans afflicted with fluorosis reported the interrelationship of fluoride and
reproductive function. Fluoride has been found to
damage testicular seminiferous tubules, causing vacuolization and denudation of spermatogenic elements, which hampered spermatogenesis in several
species (I-5). Similarly, fluoride treatment rendered
the epididymal internal milieu hostile to the residual
spermatozoa, resulting in loss of motility and a consequent reduction in fertility (6-8).
Preliminary studies in human subjects suffering
from industrial fluorosis reported azoospermia and
oligospermia, which may have been due to hypogonudism (9). Further studies have reported reduced
testosterone and elevated concentrations of FSH
and LH in patients with fluorosis (10). Recently,
Neelam and colleagues (11) found infertility among
young married men in fluoride endemic areas in India. However, the exact effect of fluoride on human

METHODS
S e m e n collection

Semen samples were collected separately from

8 individuals of age 28 to 30 years referred to our
departmental clinic. The donors were normal and
healthy and had no infections. They did not smoke
or use alcohol. The semen samples were collected
by masturbation in clean, sterilized glass-stoppered
vials at our laboratory in the early hours of the morning. After liquefaction, the fresh semen was centrifuged at 1500 rpm and spermatozoa were isolated;
this was followed by two cycles of resuspension in
2 mL of Kreb's Original Ringer Phosphate Buffer
(KORPB) (NaCI, 0.154 M; KCI, 0.154 M; KH2PO 4,
0.154 M; MgSO4 7H20, 0.154 M; 0.1 M phosphate
buffer, pH 7.4). The spermatozoa were resuspended
in 3 mL of KORPB at 75 to 80 x 106 sperm/mL.
To this sperm suspension, 2.2% polyethylene glycol
(PEG) was added to prevent water absorption and

Address correspondence to Prof. Dr. (Ms.) N. J. Chinoy,

Head, Zoology Department, School of Sciences, Gujarat University, Ahmedabad - - 380 009, Gujarat, India.
155

156

Reproductive Toxicology

osmotic shock. The effects of NaF on sperm morphology and metabolism at a dose of 5, 10, and
250 mM were investigated after 5-, 10-, and 20-rain
intervals.

Treatment. Sodium fluoride (Loba Chemie,

Bombay, 99% purity) was dissolved in KORPB solution to concentrations of 25, 50, and 250 raM.
pH. The extracellular pH of sperm was measured using pH indicators at different time intervals
(5, 10, and 20 rain). Sperm suspension (1 mL) in
KORPB was diluted to 2 mL with the same buffer
and pH was measured; then 1 mL of the sperm
suspension with 1 mL of KORPB containing NaF
were mixed, and pH was determined after intervals
of 5, I0, and 20 min.
Forward progression. To 0.5 mL of the sperm
suspension in KORPB, an equal volume (0.5 mL)
of NaF (dissolved in KORPB) was added and incubated for 5, I0, and 20 min. Sperm suspensions without NaF were similarly incubated and used as controls. A 0.1-mL aliquot of the sperm suspension was
placed on a clean, dry glass slide and sperm forward
progression and motility were evaluated on a Cell
Soft Computerized Automated Semen Analyser
2000 (CASA). A maximum of 15 fields of about 250
to 300 cells were analyzed. Fields with deflagellated
spermatozoa on the monitor were avoided). For forward progression rating of the total motile population, only those cells meeting minimum tracking requirements are rated by CASA and expressed as a
percentage.
Acid phosphatase (ACPase). ACPase activity
was assayed by the method of Bessey and colleagues
(12). To 0.2 mL of sperm suspension (20 to 22 x
10 6 sperm/mL) at incubation intervals of 5, 10, and
20 rain, 0.6 mL of substrate buffer was added and
incubated at 37C for 30 min, followed by addition
of 4 mL of 0.1 N sodium hydroxide. The colour intensity was measured at 420 nm on a Bausch and
Lomb Spectronic 88 colorimeter. The activity of the
enzyme was expressed as U/100 mL sperm suspension.
Hyaluronidase.

Hyaluronidase activity in
sperm before and after sodium fluoride treatment
was determined by using the method of Linker (13).
Spermatozoa were isolated by centrifugation at 1500
rpm for 15 rain, and the spermatozoa were suspended in 2 mL of KORPB solution. Acrosome extraction and disruption of spermatozoa were carried
out by suspending the sperm in an equal volume (2.0
mL) of 10% glycerol. The pH was adjusted to 3.0

Volume8, Number 2, 1994

with 4% acetic acid, and the sperm were incubated
overnight at 4 C at a concentration of 35 +_ 2 106
sperm/mL. Thereafter, the sample was centrifuged
at 2000 rpm for 15 min. The precipitated acrosomeless spermatozoa were discarded, and the supernatant containing the acrosomal enzymes was assayed
as follows: To 0.3 mL of the supernatant on ice, 0.3
mL of the substrate solution (0.8 mg/mL hyaluronic
acid in 0.1 M sodium acetate buffer, pH 3.8, and
0.15 M NaC1) was added, mixed, and incubated at
37 C for 1 h. The reaction was terminated by adding
0.1 mL potassium tetraborate buffer (0.8 M; pH 9.1)
and 0.25 mL 1 M NaOH. This mixture was heated
at 100 C in a water bath for precisely 3 min and
cooled under tap water. To this was added 3 mL
p-dimethyl aminobenzaldehyde (DMAB) reagent
(10 g DMAB dissolved in 100 mL glacial acetic acid
containing 12.5% (w/v) 10 N HCI). After 20 min
incubation at 37 C, the colour intensity was measured at 585 nm against a blank prepared as above
with glass-distilled water substituted for the enzyme
solution. The activity of hyaluronidase was expressed as/xmoles N-acetyl glucosamine liberated/
h/106 spermatozoa.

Glutathione. The concentration of glutathione

in sperm suspensions was estimated by the modified
procedure of Grunert and Phillips (14). To 1 mL of
sperm suspension at a concentration of 35 to 40 x
106/mL was added 3 mL of 3% metaphosphoric acid
and 1 mL of glass-distilled water. The mixture was
saturated with sodium chloride (NaC|) and centrifuged. To 2 mL of supernatant, 6 mL of saturated
NaCI was added. After equilibration at 20 C for 5
to 10 min, l mL of sodium nitroprusside solution
(0.67 M) was added, followed by 1 mL sodium carbonate-sodium cyanide mixture (1.5 M and
0.067 M, respectively). The intensity of the resulting
colour was measured on a Spectronic 20 Bausch
and Lomb colorimeter at 520 nm within 1 min. The
reagent blank was 2 mL of 2% metaphosphoric acid
saturated with NaCI. The concentration of glutathione was expressed as /xmoles/100 mL sperm suspension.

Silver nitrate staining of sperm. Differential silver staining patterns were demonstrated by Bongso
(15) in mammalian spermatozoa using an aqueous
silver nitrate reagent. This method was modified by
the use of an alcoholic, acidic silver nitrate reagent
with subsequent differentiation in alcoholic ammonia (16). The modified technique has greatly improved the differential staining patterns of acrosoreal, subacrosomal, and postacrosomal regions of
spermatozoa. Acrosomal intactness was evaluated

Fluoride toxicity in h u m a n sperm N. J. CHINOY and M. V. NARAYANA

by this modified alcoholic acidic silver nitrate stain

procedure (16). The silver staining properties of the
sperm are attributed to the presence of protein
bound sulphydryl moieties, which are richly distributed in the sperm membranes, particularly those of
the postacrosomal region. A 0.2-mL aliquot of the
freshly obtained semen sample was suspended in
0.2 mL of Hank's balanced salt solution (Ca +z and
Mg +2 free). The suspension was smeared uniformly
on clean glass slides, air-dried, and fixed in 70% and
90% ethyl alcohol for 2 min each. The slides were
then stained with 1 or 2 drops of 5% alcoholic acidic
silver nitrate (5 g AgNO 3 in 34 mL of distilled water
+ 60 mL 100% alcohol + 5 mL glacial acetic acid).
To each slide 1 drop of 1% gelatin containing 10
drops of formic acid was added. The slides were
covered with a coverslip and placed at 4 C overnight
in a moist, airtight chamber. The slides were differentiated in 5% alcoholic ammonia, dehydrated in
90% and 100% ethyl alcohol, and cleared in xylene.
The spermatozoa were observed under 1000 x (oil
immersion) magnification and photographs were
taken on a Nikon microscope with a photographic
attachment.

Statistics. For all biochemical parameters, a

minimum of 10 replicates were used and the data
subjected to statistical analysis by Student's t test.
RESULTS
NaF at 25 and 50 mM did not show an inhibitory
effect on sperm motility at any time interval (data
not presented). NaF at 250 mM inhibited motility
and metabolism after 5, 10, and especially after 20
min. Hence, the results of the 250-mM concentration
are presented and discussed.

pH. Sperm suspended in KORPB showed an

extracellular pH of 7.1 - 0.5 at 30 C. A slight acidi-

157

fication to a pH of 6.4 - 0.3 was observed in sperm

after the addition of fluoride (Table 1).

Sperm forward progression. CASA revealed a

significant decline in the forward progression pattern. There was a high percentage of good (40%)
and 5% excellent forward progression in control
samples. With fluoride treatment a high percent of
sperm revealed poor (50%) and fair (50%) progression, with complete loss of good and excellent progression ratings only after 20 min exposure
(Table 2).
Sperm motility. The sperm treated with NaF
showed no significant change in motility pattern after
5 and 10 min incubation. However, 20 minutes NaF
treatment significantly (P < 0.001) suppressed sperm
motility (Table I).
Acid phosphatase (ACPase). Exposure of
sperm to NaF for 5 and I0 rain increased the activity
of ACPase significantly (P < 0.001), but 20 min treatment with NaF produced a return of activity to control values (Table 2).
Hyaluronidase. Sodium fluoride treatment significantly enhanced (P < 0.001) the acrosomal hyaluronidase activity after 10 min incubation. Further
incubation of sperm with NaF for 20 min caused a
significant decrease (P < 0.001) in enzyme activity
as compared to both the 5- and 10-min treatment
(Table 1).
Glutathione (GSH). The levels of GSH were
depleted in a time-dependent manner after treatment. Prolonged exposure (20 min) of sperm to NaF
resulted in a significant (P < 0.001) depletion in GSH
levels revealing rapid oxidation (Table 1).
Morphology- silver nitrate stain. The untreated sperm stained with acidic alcoholic silver

Table 1. Sperm extracellular pH, motility, acid phosphatase (ACPase),

hyaluronidase, and glutathione (GSH)
N a F treatment (duration in min)
Parameter
pH
Sperm motility (%)
A C P a s e (U/100 m L sperm
suspension)
Hyaluronidase
/xmoles N-acetyl glucosamine
liberated/h/106 spermatozoa.
G S H (/z moles/100 m L sperm
suspension)
Values are m e a n -+ S.E.
*P < 0.001 compared to control.

Control
7.1 0.5
71 1.1
61.3 -_+ 1.88
18.0 1.02

9.0 - 0.61

10

7.1 -+ 0.5
61 -+ 1.2
103 1.71"

6.9 --- 0.28

48 -+ 1.12
103 -+-+1.88"

6.4
6.3
81

-+ 0.3
- 0.42*
--+ 1.02

33.6 0.78*

8.5

- 0.89*

29

- 0.89

7.1 - 0.76

4.5 --- 0.63

20

2.24 -+ 0.29*

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Reproductive Toxicology

Volume 8, Number 2, 1994

Table 2. C o m p u t e r A u t o m a t e d S e m e n A n a l y s e r (CASA) data on

s p e r m f o r w a r d p r o g r e s s i o n rating after 20 min incubation
Sperm density/
ejaculate
(million/mL

Cells
analysed

Control

85.2

225

NaF
250 mM

85,2

210

Group

FWR ~

FWR (0-4)

0-1
1-2
2-3
3-4
0-1
1-2
2-3
3-4

9%
46%
40%
5%
50%
50%
0%
0%

2.5

1.8

aFWR = forward progression rating: 0-1 = poor; 1-2 = fair; 2-3 = good;
3-4 = excellent.
bMedian values for FWR from column 5 (%), where the maximum percentage
of cells are scored by CASA to be within the 1-2, 2-3 forward progression
range (column 4).

nitrate revealed heads with intact acrosome, midpiece, and tail regions. However, following their
incubation with NaF, loss of the acrosome and decapitation occurred, especially after 20 minutes.
Fluoride-treated sperm exhibited a high percent
of morphologic abnormalities, including a large number (10.59%) of elongated heads and 2.1% amorphous heads. The tail also revealed splitting (2.19%),
coiling (11.6%), and deflagellation (22.43%). A few
sperm had bent necks, and 16.75% of spermatozoa
showed a diminutive acrosome (Table 3).
DISCUSSION
Fluoride at a concentration of 25 mM did not
inhibit sperm motility even after 20 min exposure.
Sperm incubated with 50 mM fluoride for 20 minutes
showed only a 10% inhibition of motility. The extracellular pH of sperm at these concentrations (25, 50

Table 3. Percent sperm with abnormal morphology, using silver nitrate staining technique (17)
% Morphologic
abnormalities

1. Head
a. amorphous head
b. pointed/elongated
head
2. Tail
a. split tail
b. coiled tail
c. deflagellated/
missing tail
3. bent neck
4. diminutive acrosome/
abnormal acrosome
% Total abnormalities

Control

NaF (250 mM)

0.87
1.81

2.10
10.59

0.47
1.04
1.29
1.08
1.54

2.19
11.60
22.43
3.12
16.75

8.10

68.78

mM) was not altered; there was a slight acidification

at the 250-mM concentration. Spermatozoa treated
with 250 mM fluoride were nearly totally immobilized by the 20-min exposure. The alterations in pH
alone could not account for the inhibition of sperm
motility, although low sperm pH generally corresponds to reduced motility (17).
In the present study treatment of spermatozoa
with fluoride for 5 and 10 min showed a time-dependent increase in the activities of both acid phosphatase and hyaluronidase, probably to overcome the
toxicity, since lysosomal enzymes are liberated in
excess under pathologic and toxic conditions and
play a critical role in overcoming the ill-effects of
the toxic substance (18). However, 20-min fluoride
treatment resulted in a significant decline in both
enzyme activities. This discrepancy could be attributed to time variation of fluoride retention by sperm,
causing membrane damage and loss of permeability,
leading to impaired metabolism. These observations
were further corroborated with the time-dependent
morphologic observations by silver nitrate staining
of spermatozoa for acrosomal integrity. The modified acidic alcoholic silver nitrate staining of sperm
enhanced the differential staining pattern, facilitating scoring of various acrosomal anomalies. Fluoride treatment revealed a high proportion of abnormal sperm with elongated and amorphous heads in
addition to bent necks and diminished acrosome
size. The tails exhibited splitting, coiling, and deflagellation. These changes may have caused loss of
membrane integrity and reduced metabolic activity,
which ultimately resulted in deterioration of forward
progression rating. The treatment caused a significant enhancement in poor to fair forward progression and failure of good and excellent forward progression, leading to a significant decline in sperm

Fluoride toxicity in human sperm N. J. CHINOY and M. V. NARAYANA

motility. In support of these findings, several experimental studies performed in the rat, rabbit, mouse,
and guinea pig also revealed disintegration of sperm
acrosome and decapitation, which resulted in significant inhibition of sperm motility and ultimately
low fertility (2,7,8).
GSH is involved in the detoxification of various
xenobiotics. Meister and Anderson (19) noticed a
primary cellular defense mechanism in cells against
the lethal effects of toxic chemicals by GSH. Thus,
the intracellular GSH level is a very important factor
in the cytotoxic effect of a large number of compounds. Bruggeman and colleagues (20) reported
that depletion of GSH in cells enhances the susceptibility to toxicity. In the present study, sperm GSH
showed a time-dependent decrease. The significantly lower GSH levels after 20 min of fluoride
treatment suggest a rapid oxidation of GSH to detoxify the toxicant; the extremely suppressed GSH levels might render the sperm more susceptible to fluoride toxicity. The depleted sperm GSH in the present
investigation strongly suggests that, like several exogenous compounds, fluoride is largely dependent
upon glutathione for detoxification.
These results demonstrate alterations in lysosomal enzyme activities and glutathione levels along
with morphologic abnormalities of sperm by fluoride
treatment, ultimately suppressing sperm motility.
Thus, prolonged exposure of humans in endemic
areas to fluoride may have serious implications for
fertility, supporting earlier reports.
Genotoxic effects of fluoride cannot be ruled
out due to the sperm abnormalities after fluoride
exposure, as has been explored extensively by Li
and colleagues (21). Investigations of genotoxicity
of fluoride in fluorotic individuals of the Mehsana
and Banaskantha Districts of North Gujarat, India,
have revealed an increased incidence of sister chromatid exchanges (SCE) as compared to the control
population (22). Hence, it is concluded that detailed
investigation in this area in humans exposed to extremely high concentrations of fluoride should be
given top priority.
- - The financial support provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, to
one of the authors (MVN) is gratefully acknowledged.

Acknowledgment

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