Reproductive Toxicology, Vol. 3, pp. 261-267. 1989 0890-6238/89 $3.00 + .00 Printed in the U.S.A. Copyright © 1989 Pergamon Press plc

E F F E C T S O F F L U O R I D E O N T H E H I S T O A R C H I T E C T U R E O F

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

NILOUFER J. CHINOY and EVELINE SEQUEIRA

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India

Abstract -- The effects of sodium fluoride (NaF) ingestion in two doses (10 and 20 mg/kg body weight) for 30 days on histology and histocytometry of reproductive organs of the adult male mouse were investigated. In order to study reversibility, treatment was withdrawn for one and two months. The testes, epididymides, vas deferens, prostate, and seminal vesicle were utilized for the study by standard hematoxylin-eosin staining and an ocular eye piece and micrometer scale. NaF treatment caused severe disorganization and denudation of germinal epithelial cells of seminiferous tubules with absence of sperm in the lumina. The Leydig cell and nucleus diameters were not affected. The caput epididymis showed fewer changes than the cauda. However, epithelial cell nuclear pyknosis and absence of luminal sperm were observed. A reduction in epithelial cell height, nuclear pyknosis, denudation of cells, and absence of sperm occurred in the cauda epididymis. The vas deferens epithelium showed nuclear pyknosis, clumped stereocilia, and cell debris but no sperm in the lumen and an increase in the lamina propria. The prostate and seminal vesicles were not affected by treatment. Withdrawal of treatment caused marked recovery in the histoarchitecture of these organs. The effects of NaF treatment are therefore transient and reversible.

Key Words: Histoarchitecture, Fluoride, Testes, Epididymides, Vas deferens, Prostate, Seminal vesicles, Mice.

INTRODUCTION

In response to reports by Schwarz and Milne (1) and Messer et al. (2) that fluoride is an essential nutrient, Tao and Suttie (3) undertook a set of experiments to deter- mine the essentiality of fluoride, especially as it relates to reproduction. These authors reported that fluoride does not have an essential role in reproduction of female mice. However, Messer et al. (4,5) reported that a low fluoride intake by female mice caused fluorine defi- ciency leading to impaired fertility and reproductive capacity of mice as well as a progressive decline in litter production over two successive generations; however, growth rate and litter size were not affected.

In view of the above conflicting data, it is evident that although fluoride may be essential for some physi- ologic processes, sound evidence for its exact role in reproduction is still lacking. Kour and Singh (6) reported that following fluoride ingestion (500 ppm and 1000 ppm) in mice for a period extending from 2 to 3 months, the testis showed lack of maturation and differentiation of spermatocytes. Cessation of spermatogenesis and necrosis of seminiferous tubules was also observed. Hence, a definite relationship between fluorosis and

Address correspondence to: Prof. Dr. Niloufer J. Chinoy, Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, Gujarat, India.

Received 1 December, 1988; Revised 20 February 1989; Ac- cepted 4 March 1989.

261

damage to the testis was established by these authors. Although some work has been carried out on the

effects of fluoride on the reproductive functions of female mice, there is a paucity of data on its effects on male reproduction. Hence, the present study was under- taken to investigate the effects of two doses of fluoride treatment on the histoarchitecture of reproductive organs of the male albino mouse and the reversibility of the induced effects, if any.

Method

Animals Healthy, adult male mice (Mus musculus) of Swiss

strain weighing between 20 and 30 g were used in the present study were maintained on a standard chow and water ad libitum. They were housed in an air-condi- tioned animal house at a temperature of 26 ___ 2 °C and exposed to 12 to 14 daylight hours. The animals were 6 to 8 weeks of age at the beginning of the treatment.

Exposures The experimental protocol is presented in Table 1.

The animals were divided into four weight-matched treatment groups. Two groups of animals received a dose of 10 mg/kg body weight/mouse/day and 20 mg/kg body weight/mouse/day orally for 30 days.

Sodium fluoride (NaF) was dissolved in doubly distilled water to make up the desired volume. The dose was decided on the basis of previous reports as well as LDso value of fluoride in male mice (7,8). The present

262 Reproductive Toxicology Volume 3, Number 4, 1989

Table 1. Experimental protocol.

Group Treatment

Number of Duration Day of animals

(days) autopsy used

I Control

II NaF treated

III NaF treated

*IV NaF treated

*V NaF treated

10 mg/kg body weight equivalent to 230 ppm/animal/ day.

20 mg/kg body 30 31 weight equivalent to 400 ppm/animal/ day.

Withdrawal of 30 31 treatment for 1 month

Withdrawal of 60 61 treatment for two months

- - Along with 40 treated

30 31 40

*The withdrawal treatments were carried out on

dose is equivalent to 230 ppm and 400 ppm/animal/day

respectively (6). The treatment was administered orally using a feeding tube attached to a hypodermic syringe. The treatment of 10 mg/kg body weight was withdrawn

for a period of 30 and 60 days, respectively, and the animals were utilized for recovery studies.

Data collection After the respective treatments, the males were

40

40

40

mice treated as in group II.

sacrificed by cervical dislocation and the different or- gans, namely, testis, caput and cauda epididymides, vas

deferens, prostate, and seminal vesicles from control, treated, and withdrawal groups of mice were fixed in

Bouin 's fixative, embedded in paraffin wax, and sec- tioned at 5 ~zm. The sections were stained by standard hematoxylin-eosin staining technique. The stained tissue sections were subsequently utilized for histocytometric measurements, using an ocular eyepiece and micrometer

scale.

(a) (b) (c) Fig. 1 (a). Control mouse testis (Hemotoxylin-eosin stain) showing different stages of spermatogenesis and sperm in the lumen, x 460. (b). Testis of NaF (10 mg/kg body weight) treated mouse. Note the disarrayed germinal epithelium, denudation of germinal cells in the lumen, arrest of spermatogenesis, and absence of sperms, x 450. (c). Testis of mouse two months after withdrawal of treatment, x 600.

Male reproductive organs and fluoride • N. J. CHINOY and E. SEQUEIRA 263

RESULTS

(a)

(b)

(c)

Fig. 2 (a). Caput epididymis of control mouse showing tubules with pseudostratified epithelium with stereocilia and sperm bundles, x 450. (b). Caput epididymis after NaF treatment (20 mg) for 30 days. Note the absence of sperm in the lumen. × 450. (C). Caput epididymis two months after withdrawal of treatment showing recovery, x 450.

Statistics A minimum of 30 replicates of each histocytometric

parameter was obtained and subjected to statistical analysis using the Student's t test. The level of signifi- cance was determined using Fisher and Yates table.

Testis The normal testis consists of highly coiled semin-

iferous tubules with healthy germinal epithelium. Be- tween the tubules are interstitial cells (Leydig cells) which synthesize androgens (Figure l(a)).

The histoarchitecture of the testis was altered after treatment as compared to control. The seminiferous tubule diameter decreased with NaF treatment at 20 mg/kg body weight. The germinal epithelium was disorganized with denudation of cells in the lumen, which was devoid of sperm. The germinal epithelial cell height was significantly reduced (p<0.001). However, diameters of Leydig cells and their nuclei remained unaltered (Table 2). The effects were almost the same at both doses (Figure l(b)). Withdrawal of treatment re- suited in recovery in the histology of the testis as compared to the treated group (Fig. l(c); Table 2).

Epididymis The caput epididymis of control animals consisted

of many tubules with pseudostratified epithelium having stereocilia. The intertubular interstitium was observed between the tubules. The lumen of the tubules contained sperm bundles (Figure 2(a)). In the treated animals, the tubular diameter insignificantly increased (Table 2). The secretory epithelium showed nuclear pyknosis, and no sperm were observed in the lumen (Figure 2(b)).

The cauda epididymis of control animals consisted of tubules with a larger diameter in comparison to the caput, with a pseudostratified epithelium with stereo- cilia. The intertubular interstitium was observed. Sperm bundles were present within the lumen of the tubule (Figure 3(a)). The cauda epididymis of treated animals revealed larger tubules with extensive denudation of cells in the lumen, which was devoid of sperm (Figure 3(b), (c)). The epithelial cell height was significantly decreased in comparison to control by the 10 mg dose. A marked recovery was observed in the histology of the epididymides after withdrawal of treatment (Figures 2(c); 3(d); Table 2).

Vas deferens The vas deferens of control animals consisted of

three muscle layers, namely the outer longitudinal, middle circular, and inner longitudinal layers. The lam- ina propria was present between the inner longitudinal muscle layer and the pseudostratified epithelium, which contained stereocilia. The epithelial cell layer was folded so as to form a stellate lumen in which the sperm bundles were present (Figure 4(a), (b)). The epithelial cell height was not affected much in treated animals but the epithe-

264 Reproductive Toxicology Volume 3, Number 4, 1989

Table 2. Histocytometry (values in ixm)

Tissues Parameter Control

NaF treated

10 mg/kg 20 mg/kg body weight body weight

NaF Withdrawal

One month Two months

Testis

Caput epididymis

Cauda epididymis

Was deferens (distal)

Prostate

Seminal vesicle

Diameter 195 --- 3.5 193 ± 2.8 183 _+ 3.5 143 ± 6.2 168 --- 5.3 Germinal

epithelium cell height 68.1 ± 3.8 25.6 --- 4.3 a 42.3 _+ 2.6 a 37.8 - 3.2 a 66.9 ~ 4.2

Leydig cell diameter 7.6 --- 0.5 7.3 --- 0.4 7.8 -+ 0.5 7.5 -+ 0.6 7.9 - 0.6

Leydig cell nuclear diameter 3.1 _+ 0.1 3.2 ± 0.3 3.4 _+ 0.2 3.9 + 0.3 3.7 ± 0.3

Tubular diameter 109 _+ 3 113 _+ 2 113 ~ 3 94 _+ 6 114 -- 4

Epithelial cell height 38.4 _+ 1.8 28.5 ± 0.8 b 30.2 ± 0.7 b 35.5 --- 1.2 41.4 - 1.5

Tubular diameter 254 --- 10 257 +-- 9 262 _+ 6 249 _+ 6 252 _+ 10

Epithelial cell height 42.5 _+ 2.4 14.2 ± 0.9 a 19.8 + 1.4 a 18.9 +-- 1.1 a 40.8 ± 2.3 c

Epithelial cell height 52.9 ± 0.6 52.2 +- 0.6 51.2 +-- 0.6 48.6 -_+ 1.6 52.8 _+ 1.4

Muscle Coat 252.9 +-- 1.8 124.0 - - - - - 18.8 a 214.3 ± 8.4 177.6 ± 7.8 198.2 ----- 9.0 c

Lamina Propria 11.3 ± 0.8 35.6 +-- 4.2 23.8 _+_ 3.2 31.9 _+ 2.9 19.7 -+ 1.3

Epithelial cell height 16.7 - 0.5 17.1 _ 0.5 18.5 --- 0.9 15.9 --- 1.1 14.9 _+ 1.0

Epithelial cell height 13.1 - 0.7 13.8 --- 0.8 14.7 --+ 0.8 15.7 _+ 0.9 15.6 -+- 0.8

Values are mean ___ S.E. aDifferent from control, p<0.001. bDifferent from control, p<0.2. CDifferent from 10 mg/kg group, p<0.001.

l i um s h o w e d nuc lea r pyknos i s e spec ia l ly in the r eg ion of

the folds . The s te reoci l ia were c l u m p e d and the l u m e n

c o n t a i n e d cel l debr i s and no sperm. T he l a m i n a p ropr i a

was inc reased in th ickness . T h e d i a m e t e r o f the vas

de fe rens and the th i ckness o f the m u s c l e coat were

s ign i f i can t ly less than in the con t ro l (F igure 4(c) , (d)).

The w i t h d r a w a l o f t r e a tmen t resu l ted in r ecove ry in the

h i s toa rch i t ec tu re o f the vas de fe rens (F igure 4(e) ;

T a b l e 2).

Seminal vesicle The s emina l ves ic le cons i s t s o f m ucos a l fo lds w h i c h

e x t e n d in to the l u m e n wi th secre t ion p r o d u c e d by the

g l andu la r ep i the l ium. T h e r e were no s ign i f ican t differ-

ences in the s emina l ves ic les b e t w e e n con t ro l and t rea ted

g roups (Tab le 2).

Prostate gland T h e pros ta te g l and cons i s t s o f severa l a lveol i con-

t a in ing sec re t ions and l ined wi th s imple as wel l as

pseudos t ra t i f i ed c o l u m n a r ep i the l ium. T h e r e is an inter-

v e n i n g f i b r o m u s c u l a r s t roma. No s ign i f ican t c h a n g e s

were o b s e r v e d in the pros ta te of t rea ted an ima l s as

c o m p a r e d to con t ro l s (Tab le 2).

D I S C U S S I O N

The p re sen t s tudy r evea l ed that N a F t r ea tmen t o f

ma le mice resu l ted in a l te ra t ions in the h i s toa rch i t ec tu re

of the testis. The g e r m i n a l ep i the l ia l cel l h e i g h t o f the

s emin i f e rous tubu le was s ign i f ican t ly r educed in t rea ted

an ima l s in c o m p a r i s o n to cont ro ls . Kour and S i n g h (6)

have also r epor ted m a r k e d degene ra t i ve c h a n g e s such as

necros i s o f the s emin i f e rous tubu les and lack of differ-

en t i a t ion as wel l as m a t u r a t i o n o f spe rma tocy t e s in mice

t rea ted w i th N a F at doses o f 500 and 1000 p p m in

d r ink ing water . Hence , it is ev iden t tha t f luor ide in tox-

ica t ion affects tes t icu lar h i s toa rch i t ec tu re , and it fo l lows

tha t spe rma togenes i s wil l be h a m p e r e d . The dec rease in

total spe rm c o u n t in the ep id idymis in NaF- t r ea t ed

mouse , as repor ted e l s e w h e r e by us (9), suppor ts the

a b o v e data.

In the test is o f NaF- t r ea t ed an imal s , the Leyd ig cel l

d i a m e t e r and its nuc l ea r d i a m e t e r were not af fec ted ,

ind ica t ing tha t Leyd ig cell func t ions may not be c h a n g e d

Male reproductive organs and fluoride • N. J. CHn~oY and E. SEQtrEIRA 265

(a) (b)

(c) (d) Fig. 3 (a). Cauda epididymis of control mouse showing tubules with pseudostratified epithelium having stereocilia and sperm in the lumen. × 480. (b). Cauda epididymis after NaF (10 mg/kg body weight) treatment for 30 days. Note pyknosis of epithelial nuclei and disorganization of the epithelium, x 480. (c). Treatment with 20 mg/kg body weight NaF. × 480. (d). Cauda epididymis two months after withdrawal of treatment. Observe recovery of the epithelium and sperm in the lumen. × 480.

by treatment. Moreover, Chinoy and Sequeira (10) have reported elsewhere that testis cholesterol and serum testosterone levels in NaF-treated mouse were not sig- nificantly altered compared to control. Hence, androgen biosynthesis may not be altered in these animals. How- ever, since a number of androgen-sensitive parameters in target organs were found to be altered by NaF, as reported by Chinoy and Sequeira (10), it is probable that the structure and functions of the epididymides and other accessory sex organs are changed.

In the present study, NaF treatment caused alter- ations in the histology of both caput and cauda epi- didymides. On the whole, the cauda epididymis was affected more than the caput. The changes included the reduction in epithelial cell height, which was very significant in the cauda, disorganization of the secretory epithelium, nuclear pyknosis, and complete absence of spermatozoa in the tubular lumen, which corroborates the significantly reduced sperm count in treated animals reported by us elsewhere (9). It is known that the maintenance of the structure and function of accessory

reproductive organs, including the epididymis, is depen- dent on circulating levels of androgens (11-14). More- over, the different accessory sex organs manifest a differential sensitivity to androgens so that the cauda epididymis requires a higher threshold of the hormone for maintenance of its normal structure and secretory activity than does the caput, and the epididymis has a higher threshold requirement than seminal vesicle or prostate (11-14). Hence, the cauda epididymis was found to be affected more than the caput. In the vas deferens, an increase in the lamina propria but a reduc- tion in the muscle layers was observed while prostate and seminal vesicle histology was not affected by NaF treatment.

Since normal epididymal structure and internal milieu is important for sperm maturation and for main- taining sperm in a viable, motile state (12,13), the observed structural alterations in the epididymis would be expected to affect the contained spermatozoa. Chinoy and Sequeira (9) have reported that motility and mor- phology of epididymal spermatozoa were altered in the

266 Reproductive Toxicology Volume 3, Number 4 1989

(a) (b)

(c) (d) (e)

Fig. 4 (a). Distal vas deferens of control mouse showing pseudostratified folded epithelial layer with stereocilia and stellate lumen. The muscle layers and lamina propria are also seen. x 120; (b). Magnified view of the fold x 640.: (c). Distal vas deferens of treated mouse (10 mg/kg body weight) showing nuclear pyknosis and clumping of stereocilia, x 150. (d). Magnified view. x 480. (e). T.S. of a fold of distal vas deferens of mouse two months after withdrawal showing recovery, x 480.

NaF-treated mouse. Therefore, changes in epididymal structure lead to nonviable sperm and subsequently to infertility (9). This is in concurrence with the report of Kour and Singh (6). Similarly, Messer et al. (4,5) reported progressive development of infertility in female mice on a low fluoride intake.

The data of our studies, present and previous, reveal that fluoride has a role in male reproduction and affects fertility (9). However, the induced effects were transient and reversible by withdrawal of the treatment for two months, since the histoarchitecture of the testis, cauda epididymis, and vas deferens recovered almost to

the normal state. Hence, NaF treatment in the mouse does not cause any permanent structural alterations in the reproductive organs. Mohamed and Chandler (15) re- ported that chromosomal abnormalities occurred in bone marrow cells and spermatocytes in mice treated with 1 to 200 ppm fluoride for 3 to 6 weeks; this treatment also induced chromosomal changes in a dose-dependent man- ner. Based on these data, it follows that the exposed sperms might also possess chromosomal abnormalities. Smith reported (16) that fluoride might be responsible for causing genetic damage, birth defects, cancer, and allergic responses. This has important implications in

Male reproductive organs and fluoride • N. J. CtflNOY and E. SEQUEIRA 267

Ind ia whe re F luoros i s is e n d e m i c in 13 states. There fo re ,

ex t ens ive s tudies on f luor ide ef fec ts in l abora to ry ani-

ma l s as wel l as in a f fec ted popu la t ions shou ld be g iven

the top pr ior i ty in the Na t iona l T e c h n o l o g y M i s s i o n s and

Task Forces .

A c k n o w l e d g e m e n t s - - One of the authors (E.S.) is grateful for finan- cial assistance from R.J. Cama Research Project for carrying out this research work and to St. Theresa's College for granting the necessary permission to carry out doctoral work.

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3. Tao S, Suttie JW. Evidence for alack of an effect of dietary fluoride level on reproduction in mice. J Nutr. 1976; 106: 1115-22.

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