Pergamon

0890-6238(93)E0009-7

Reproductive Toxicology, Vol. 8, No. 2, pp. 155-159, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

0890-6238/94 $6.00 + .00

• Original Contribution

I N V I T R O F L U O R I D E T O X I C I T Y I N H U M A N S P E R M A T O Z O A

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

Previous investigations in fluoride intoxicated ex- perimental animals and humans afflicted with fluo- rosis reported the interrelationship of fluoride and reproductive function. Fluoride has been found to damage testicular seminiferous tubules, causing vac- uolization and denudation of spermatogenic ele- ments, 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 con- sequent reduction in fertility (6-8).

Preliminary studies in human subjects suffering from industrial fluorosis reported azoospermia and oligospermia, which may have been due to hypogo- nudism (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 In- dia. However, the exact effect of fluoride on human

Address correspondence to Prof. Dr. (Ms.) N. J. Chinoy, Head, Zoology Department, School of Sciences, Gujarat Univer- sity, Ahmedabad - - 380 009, Gujarat, India.

155

sperm structure and metabolism is hitherto unex- plored. The present investigation studied the meta- bolic and morphologic alterations induced by so- dium fluoride (NaF) treatment in vitro in human spermatozoa.

M E T H O D S

Semen 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 morn- ing. After liquefaction, the fresh semen was centri- fuged 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

156 Reproductive Toxicology Volume 8, Number 2, 1994

osmotic shock. The effects of NaF on sperm mor- phology 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 solu- tion to concentrations of 25, 50, and 250 raM.

pH. The extracellular pH of sperm was mea- sured 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 incu- bated for 5, I0, and 20 min. Sperm suspensions with- out NaF were similarly incubated and used as con- trols. 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 for- ward progression rating of the total motile popula- tion, only those cells meeting minimum tracking re- quirements 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 37°C for 30 min, followed by addition of 4 mL of 0.1 N sodium hydroxide. The colour in- tensity 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 sus- pension.

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 sus- pended in 2 mL of KORPB solution. Acrosome ex- traction 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

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 acrosome- less spermatozoa were discarded, and the superna- tant 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 mea- sured at 585 nm against a blank prepared as above with glass-distilled water substituted for the enzyme solution. The activity of hyaluronidase was ex- pressed 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 centri- fuged. 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 car- bonate-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 glutathi- one was expressed as /xmoles/100 mL sperm sus- pension.

Silver nitrate staining of sperm. Differential sil- ver 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 ammo- nia (16). The modified technique has greatly im- proved the differential staining patterns of acroso- real, subacrosomal, and postacrosomal regions of spermatozoa. Acrosomal intactness was evaluated

Fluoride toxicity in human sperm • N. J. CHINOY and M. V. NARAYANA 157

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 distrib- uted 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 differ- entiated 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-

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 pat- tern. 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%) progres- sion, with complete loss of good and excellent pro- gression 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 treat- ment with NaF produced a return of activity to con- trol values (Table 2).

Hyaluronidase. Sodium fluoride treatment sig- nificantly enhanced (P < 0.001) the acrosomal hyal- uronidase 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 treat- ment. 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 un- treated sperm stained with acidic alcoholic silver

Table 1. Sperm extracellular pH, motility, acid phosphatase (ACPase), hyaluronidase, and glutathione (GSH)

NaF treatment (duration in min)

Parameter Control 5 10 20

pH 7.1 ± 0.5 7.1 -+ 0.5 6.9 --- 0.28 6.4 -+ 0.3 Sperm motility (%) 71 ± 1.1 61 -+ 1.2 48 -+ 1.12 6.3 - 0.42* ACPase (U/100 m L sperm 61.3 -_+ 1.88 103 ± 1.71" 103 -+-+ 1.88" 81 --+ 1.02

suspension) Hyaluronidase 18.0 ± 1.02 29 - 0.89 33.6 ± 0.78* 8.5 - 0.89* /xmoles N-acetyl g lucosamine

liberated/h/106 sperma- tozoa.

GSH (/z moles/100 m L sperm suspension) 9.0 - 0.61 7.1 ±- 0.76 4.5 --- 0.63 2.24 -+ 0.29*

Values are mean -+ S.E. *P < 0.001 compared to control.

158 Reproductive Toxicology Volume 8, Number 2, 1994

Table 2. Compute r Au toma ted Semen Analyser (CASA) data on sperm forward progress ion rating after 20 min incubation

Group

Sperm density/ ejaculate

(million/mL Cells

analysed FWR ~ % FWR (0-4) °

Control 85.2 225 0-1 9% 2.5 1-2 46% 2-3 40% 3-4 5%

NaF 85,2 210 0-1 50% 1.8 250 mM 1-2 50%

2-3 0% 3 -4 0%

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, mid- piece, and tail regions. However, following their incubation with NaF, loss of the acrosome and de- capitation occurred, especially after 20 minutes.

Fluoride-treated sperm exhibited a high percent of morphologic abnormalities, including a large num- ber (10.59%) of elongated heads and 2.1% amor- phous 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 extra- cellular pH of sperm at these concentrations (25, 50

Table 3. Percent sperm with abnormal morphology, us- ing silver nitrate staining technique (17)

% Morphologic abnormalities

Control NaF (250 mM)

1. Head a. amorphous head 0.87 2.10 b. pointed/elongated 1.81 10.59

head 2. Tail

a. split tail 0.47 2.19 b. coiled tail 1.04 11.60 c. deflagellated/ 1.29 22.43

missing tail 1.08 3.12 3. bent neck 1.54 16.75 4. diminutive acrosome/

abnormal acrosome

% Total abnormalities 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 immobi- lized by the 20-min exposure. The alterations in pH alone could not account for the inhibition of sperm motility, although low sperm pH generally corre- sponds to reduced motility (17).

In the present study treatment of spermatozoa with fluoride for 5 and 10 min showed a time-depen- dent increase in the activities of both acid phospha- tase 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 attrib- uted 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 modi- fied acidic alcoholic silver nitrate staining of sperm enhanced the differential staining pattern, facilitat- ing scoring of various acrosomal anomalies. Fluo- ride treatment revealed a high proportion of abnor- mal sperm with elongated and amorphous heads in addition to bent necks and diminished acrosome size. The tails exhibited splitting, coiling, and defla- gellation. 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 signifi- cant enhancement in poor to fair forward progres- sion and failure of good and excellent forward pro- gression, leading to a significant decline in sperm

Fluoride toxicity in human sperm • N. J. CHINOY and M. V. NARAYANA 159

motility. In support of these findings, several experi- mental studies performed in the rat, rabbit, mouse, and guinea pig also revealed disintegration of sperm acrosome and decapitation, which resulted in sig- nificant 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 com- pounds. Bruggeman and colleagues (20) reported that depletion of GSH in cells enhances the suscepti- bility to toxicity. In the present study, sperm GSH showed a time-dependent decrease. The signifi- cantly lower GSH levels after 20 min of fluoride treatment suggest a rapid oxidation of GSH to detox- ify the toxicant; the extremely suppressed GSH lev- els might render the sperm more susceptible to fluo- ride toxicity. The depleted sperm GSH in the present investigation strongly suggests that, like several ex- ogenous compounds, fluoride is largely dependent upon glutathione for detoxification.

These results demonstrate alterations in lyso- somal 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 chro- matid exchanges (SCE) as compared to the control population (22). Hence, it is concluded that detailed investigation in this area in humans exposed to ex- tremely high concentrations of fluoride should be given top priority.

A c k n o w l e d g m e n t - - The financial support provided by the Coun- cil of Scientific and Industrial Research (CSIR), New Delhi, to one of the authors (MVN) is gratefully acknowledged.

REFERENCES

1. Kour K, Singh J. Histological findings of mice testes follow- ing fluoride ingestion. Fluoride. 1980;13:160-2.

2. Chinoy N J, Sequeira E. Effects of fluoride on the histoarchi- tecture of reproductive organs of the male mouse. Reprod Toxicol. 1989;3:261-7.

3. Shashi A. Histopathological changes in rabbit testes during experimental fluorosis. Folia Morphol. 1990;38:63-5.

4. Chinoy N J, Rao MV, Narayana MV, Neelakanta E. Micro- dose vasal injection of sodium fluoride in the rat. Reprod Toxicol. 1991 ;5:505-12.

5. Susheela AK, Kumar A. A study of the effect of high concen- trations of fluoride on the reproductive organs of male rabbits using light and scanning electron microscopy. J Reprod Fer- til. 1991;92:353-60.

6. Chinoy N J, Sequeira E. Fluoride induced biochemical changes in reproductive organs of male mice. Fluoride. 1989;22:78-85.

7. Chinoy N J, Sequeira E. Reversible fluoride induced fertility impairment in male mice. Fluoride. 1992;25:71-6.

8. Chinoy NJ, Sequeira E, Narayana MV. Effects of vitamin C and calcium on the reversibility of fluoride induced alter- ations in spermatozoa of rabbit. Fluoride. 1991;24:29-39.

9. Tarinsky AP. The influence of some industrial factors of aluminium industry on generative function of men. Ph.D. Thesis, Sverdlovsk, 1972.

10. Tokar VI, Savchenko ON. The influence of inorganic fluorine compounds on functional condition of the hypophysis-testes system. Probl Endocrinol. 1977;23:104-7.

11. Neelam K, Suhasini RV, Sudhakar RY. Incidence of preva- lence of infertility among married male members of endemic fluorosis district of Andhra Pradesh. Proceedings of a Confer- ence of the International Society for Fluoride Research. Swit- zerland (Nyon) (Abstract) 1987.

12. Bessey OA, Lowry OH~ Brock MJ. A method for the rapid determination of acid and alkaline phosphatase with 5 cubic mm of serum. J Biol Chem. 1946;164:321-9.

13. Linker A. Hyaluronidase. In Bergmeyer HU, ed. vol 4. Meth- ods of enzymatic analysis; Weinheim: Verlag Chemie; 1984:156-62.

14. Grunert RR, Phillips PH. A modification of the nitroprusside method of analysis for glutathione. Arch Biochem. 1951 ;217:25.

15. Bongso TA. Comparative silver staining patterns of water buffalo, goat and pig spermatozoa. Arch Androl. 1983; 11:13-17.

16. Schoff PK, Lardy HA. Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermato- zoa. Biol Reprod. 1987;37:1037-46.

17. Chinoy NJ, Ranga GM, Highland HN, D'Souza KJ, Sequeira E. A modified method for the differential staining of sperma- tozoa using acidic alcoholic silver nitrate stain. Int J Fertil. 1992;37:232-6.

18. Reddy PN, Dural Raj G, Dhar SC. Toxic effects of different concentrations of dimethoate on lysosomal enzymes of fe- male albino rats. Ind J Expt Biol. 1992;30:394-8.

19. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711-60.

20. Bruggeman IM, Spenkelink A, Temmink JHM, Van Bladeren PJ. Differential effects of raising and lowering intracellular glutathione levels on the cytotoxicity of allyl isothiocyanate, tert-butyl hydroperoxide and chlorodinitrobenzene. Toxicol. In Vitro. 1988;2:31-5.

21. Li Y, Dunipace AJ, Stookey GK. Effects of fluoride on the mouse sperm morphology test. J Dent Res. 1987;66:1509-11.

22. Sheth F J, Multani AS, Chinoy NJ. Increased frequency of SCEs in fluorotic individuals of North Gujarat. XVI All India Cell Biology Conference, held at Varanasi, India, 1-3 Janu- ary, 1993 (Abstract).