[ The Medicine and Surgery (1986): (XXVI), 2-3, 33-35]

Protection of Mouse Testes, Epididymis and Adrenals with Speman against Cadmium Intoxication

Rathore, H.S., M. Sc., Ph.D., M. Phil., F.A.Z., F.A.E.B.,

Professor and Head Faculty of Life Sciences and

Varsha Saraswat, M.Sc., Research Scholar School of Studies in Zoology, Vikram University, Ujjain, Madhya Pradesh, India.

INTRODUCTION Cadmium is a heavy metal pollutant which is toxic to plants, animals and human beings (Nriagu, 1980). In mammals, cadmium damages the testes, accessory sex organs, sperms and adrenals (Gunn and Gould, 1970). Fortunately sterility and sexual disorders in man have not been reported following cadmium intoxication but osteomalacia, polynephritis, cancer of the nasopharynx and prostate have been reported in men professionally exposed to cadmium (Kjellstrom et al., 1979; Lemen et al., 1976; Yosumura et al., 1980). In the present investigation an attempt has been made to prevent destruction of mice testes, epididymis and adrenals with Speman (an indigenous drug), which is known to promote spermatogenesis (Parikh, 1971; Subbarao, et al., 1973; Khaleeluddin et al., 1973: Talaulikar and Nagarsekar, 1976: Jayatilak et al., 1976 (a) and (b), Pardanani et al., 1976; Nath, 1982) and is used to cure male sterility (oligozoospermia). The aim of this investigation was to test the ability of Speman to nullify the toxic effects of cadmium so that in future it can be recommended to cure testicular dysfunction in man if it results following cadmium poisoning. MATERIALS AND METHODS Three and half month old male albino Swiss mice were obtained from the Biological Products Division, Veterinary College, Mhow (M.P.) and were used in the present experiments. Ten mice were placed in each propylene cage (290 x 220 x 140 mm) and given standard mice food and tap water ad libitum. CdCl2 made by BDH was dissolved in distilled water to prepare a solution of 1 mg/ml. Each Speman tablet (The Himalaya Drug Company, Bombay) contains the following constituents: Orchis mascula 65 mg Lactuca scariola 16 mg Hygrophila spinosa 32 mg Mucuna pruriens 16 mg

Exts. Parmelia perlata 16 mg Argyreia speciosa 32 mg Tribulus terrestris 32 mg Leptadenia reticulata 32 mg Suvarnavang (Mosaic Gold) 16 mg

Speman is not soluble in water, hence The Himalaya Drug Company supplied a suspension having 10 mg Speman per ml, which was administered orally through injection using a blunt needle. The mice were divided into four groups: I. Group A: All mice were injected with 1 mg CdCI2 intramuscularly only once + placebo

given orally daily. II. Group B: All mice received a single injection of 1 mg CdCl2 + 0.5 ml suspension

containing 5 mg Speman given orally daily. III. Group C: All mice received a single injection of 1 mg CdCl2 + 1 ml suspension

containing 10 mg Speman given orally daily. IV. Group D: Controls no treatment at all. The contents of Speman suspension were as follows: Speman powder (200 mesh) 100 gm Nipagin Sodium (0.25%) 2.5 gm Nipasol Sodium (0.15%) 10 gm Distilled water to make 1000 ml Placebo contained the same constituents as cited above except Speman. Speman administration was continued for 60 days. The mice were killed on the 15th, 30th and 60th days. Their testes, epididymis and adrenals were fixed in alcoholic Bouin’s fluid and routine microtome sections of 6 to 8 microns were cut and stained in Delafield’s haematoxylin and eosin. The experiments were done thrice. Observations of permanent slides, camera lucida drawings and photomicrographs have formed the basis of the present results. RESULTS The results, which are presented in the form of Tables 1 to 5 and photomicrographs, reveal the following facts: a) Pattern of Speman plus CdCl2 alone, induced changes A careful persual of Tables 1 to 4 and all photomicrographs shows the effects of CdCl2

alone, and in combination with Speman, on the testes, epididymis and adrenals. Typical responses are evident after the 15th 30th and 60th days.

Table 1: Diameter of seminiferous tubules of mice (in microns) at different intervals after cadmium chloride and Speman treatment (Mean ± SE)

Duration of Experiment Groups Treatment

15 days 30 days 60 days A Single injection of CdCl2 + Placebo 16.70 ± 0.74 16.96 ± 0.52 8.32 ± 0.32* B Single injection of CdCl2 + Speman 5 mg daily 18.51 ± 0.93 21.09 ± 1.82 18.05 ± 1.81 C Single injection of CdCl2 + Speman 10 mg daily 15.87 ± 1.52 15.61 ± 0.21* 15.74 ± 0.21* D Control 19.64 ± 1.19 – –

*Statistically significant, based on ‘t’ test at 5% level of significance.

Table 2: Diameter of tubules of epididymis and height of cells lining tubules

at different intervals (in microns) after CdCl2 and Speman treatment (Mean ± SE) Diameter after Height of cells after

Group Treatment 15 days 30 days 60 days 15 days 30 days 60 days

A Single injection of CdCl2 + Placebo

26.90* ± 3.02

32.51* ± 2.04

6.00* ± 0.04

2.19* ± 0.26

2.00* ± 0.22

0.90* ± 0.05

B Single injection of CdCl2 + Speman 5 mg daily

27.80* ± 0.22

23.80* ± 2.90

34.45 ± 3.53

2.90* ± 0.22

2.70* ± 0.17

3.80* ± 0.44

C Single injection of CdCl2 + Speman 10 mg daily

19.87* ± 0.66

34.58 ± 0.44

27.35* ± 1.67

2.25* ± 0.29

5.48* ± 1.49

4.96* ± 1.11

D Control 40.38 ± 0.63 – – 10.06

± 0.03 – –

*Statistically significant, based on student’s ‘t’ test at 5% level of significance.

Table 3: Number of germinal and other cells present in the testes of mice at various intervals following

CdCl2 and Speman treatment (Mean ± SE)

Group Treatment Days Primary

Spermato-cytes

Secondary Spermato-

cytes

Sperma-tids

Sperms + =Present – =Absent

Sertoli cells

Leydig cells

15 58.75* ±10.67

80.37* ±11.22

40.64* ±6.54 + – –

30 47.00* ±1.76

75.45* ±7.34

28.88* ±1.21 + 5.25*

±0.12 10.00*±0.91 A

Single injection of CdCl2 + Placebo

60 53.33* ±3.34

55.00* ±8.68

12.50* ±1.77 – 4.66*

±0.33 31.66*±3.82

15 66.56* ±3.68

83.50* ±6.71

60.16* ±2.24 + 6.55*

±0.84 19.82*±1.26

30 79.85* ±1.65

96.50* ±4.25

73.12* ±1.81 + 25.60*

±0.85 26.00*±1.58 B

Single injection of CdCl2 + Speman 5 mg daily 60 83.33

±13.36 93.33

±13.36 36.66* ±4.08 + 5.33

±1.35 18.33*±1.80

15 60.00* ±7.09

76.66* ±6.68

30.00* ±2.89 + – –

30 75.78* ±4.89

92.50* ±6.49

54.03* ±6.95 + 29.16

±7.65 17.16*±0.58 C

Single injection of CdCl2 + Speman 10 mg daily 60 69.29*

±3.02 91.10* ±9.24

54.03* ±6.95 + 30.00*

±1.90 25.72*±1.20

D Control 97.32 ±4.15

148.20 ±15.02

99.68 ±5.61 + 41.56

±1.01 35.38 ±1.43

*Statistically significant, based on ‘t’ test at 5% level of significance.

Table 4: Diameter of cortex and medulla of adrenals in mice at different intervals following CdCl2 and

Speman treatment (Values in microns; Mean ± SE) Group Treatment Duration in days Cortex Medulla

15 108.12 ± 3.05* 60.83 ±3.36* 30 93.87 ± 7.30 50.83 ± 4.89 A Single injection of CdCl2 + Placebo 60 92.45 ± 0.96* 42.12 ± 0.17* 15 66.12 ± 1.44* 33.87 ± 1.55* 30 85.16 ±2.14* 43.54 ± 1.82* B Single injection of CdCl2 + Speman

5 mg daily 60 96.64 ± 5.07* 48.06 ± 6.14 15 86.90 ± 10.42 48.70 ± 6.07 30 79.03 ± 10.70 45.41 ±5.75 C Single injection of CdCl2 + Speman

10 mg daily 60 79.35 ± 5.52 37.16 ± 7.73

D Control – 79.09 ± 0.31 44.96 ± 1.49 *Statistically significant, based on ‘t’ test at 5% level of significance.

Table 5: Recovery pattern of some parameters in mice after a single injection of CdCl2 by Speman treatment for 60 days

Gro

up

com

paris

on

Dia

met

er o

f se

min

ifero

us

tubu

le

Dia

met

er o

f tu

bule

of t

he

epid

idym

is

Hei

ght o

f tub

ular

lin

ing

of th

e ep

idid

ymis

Dia

met

er o

f the

co

rtex

Dia

met

er o

f the

m

edul

la

No.

of p

rimar

y sp

erm

atoc

ytes

No.

of s

econ

dary

sp

erm

atoc

ytes

No.

of s

perm

atid

s

No.

of L

eydi

g ce

lls

No.

of S

erto

li ce

lls

Sper

ms

+ =

Pres

ent

– =

Abs

ent

A (Speman 5 mg

daily with placebo)

+ + + – + – – + – – +

B (Speman 10 mg

daily with placebo)

+ + + + + + + + – + +

Only significant results at 5% level of significance are presented. Recovery means significant betterment towards normal values with the drug when results are compared with those obtained after CdCl2 treatment.

b) Pattern of maximum recovery with Speman therapy following CdCl2 intoxication. Here experiments were conducted for 60 days in order to find out the actual recovery

i.e. whether Speman checks and /or reverses the toxic effects of CdCl2. The values of various parameters obtained following 60 days after CdCl2 alone were compared with the values of the same parameters obtained following Speman therapy for 60 days. And ‘t’ test at 5% level of significance was applied to the data for this comparison. The results are presented in Table 5, which shows that Speman at 5 mg/daily dose for 60 days shows a beneficial role by abolishing the toxic effects of CdCl2 on the seminiferous tubules, epididymis, spermatide and medulla of the adrenals. Speman at

10 mg/daily dose is more effective as it removes the toxic effects of CdCl2 on all the parameters studied. Speman at both doses shows typical relationship for Leydig cells.

PLATE-I Photomicrographs of mice testes with Bouin’s haematoxylin and eosin preparation

Fig. 1:

Control. Normal relationship of intertubular structure to tubules . 100X

Fig

15 days after CdCl2 injecti

Fig. 3: 30 days after CdCl2 injection. Few interstitial cells. 100X

2 . Few interstitial cells. Ext mely few sperms. 100X

n daily.

is shown. 2:

on complete absence of interstitial cells resulted in the intertubular spaces. 100X

Fig. 4:

60 days after CdCl injection. Seminiferous tubules shrinkre

Fig. 5:

15 days after CdCl2 injection + 5 mg SpemaInterstitial cells are present. 100X

Fig. 6:

30 days after CdCl2 injection + 5 mg Speman daily. No changes are visible. 100X

PLATE-II

Fi

60 days after CdCl2 injection + 5 mg Speman daily.

Fig

15 days after CdCl2 injection + 10 mg Speman daily.

Fig. 9:

30 days after CdCl2 injection + 10 mg Speman daily. Few Fig. 10:

60 days after CdCl2 injection + 10 mg Speman daily. Sh ls

Fig. 1: Magnified view niferous tubule

sh .

g. 7:

No change. 100X

. 8:

Interstitial cells are absent. 100X

interstitial cells are seen in between shrunken seminiferous tubules. 100X.

rinkage of seminiferous tubules and few interstitial celwhen compared with Fig. 1 but situation is better when

compared with Fig. 4. 100X

1

of a semiowing spermatogenesis and sperms. 400X

PLATE-III

Photomicrographs of mice caput epididymis, with Bouin’s haematoxylin and eosin. All slides photographed at 50X.

Figs. 12 and 13

Epididymis, control. Note the height of the lining epithelium and degree of intervening tissue between tubules.

Fig. 14:

15 days after CdCl2 injection. Marked widening of the intertubular spaces. Spermatozoa are present. The height of

the lining is low.

30 days after CdCl2 injection. Diameter of tubules and height of the lining epit e reduced. Sperms are

2

height of the lining epithelium are reduced. Sperms present. 2Diameter of tubules and height of the lining epithelium are

reduced. S e present.

Fig. 15:

helium arpresent.

Fig. 16:

60 days after CdCl injection. Diameter of tubules and

Fig. 17:

15 days after CdCl injection + 5 mg Speman daily.

perms ar

PLATE-IV

Fig.

30 days after CdCl2 injection + 5 mg Speman daily. Dia re

Fig. 9:

60 days after CdCl2 inject g Speman daily. Tubular

Fig. 15 days after CdCl2 injection + 10 mg Speman daily.

T e

Fig. 1: 30 days after CdCl2 injection + 10 mg Speman daily.

Tub m

60 days after CdCl2 injection + 10 mg Speman daily. Tubular

(H ).

18:

meter of tubules and height of the lining epithelium areduced.

1ion + 5 m

diameter is normal. Height of the lining epithelium is less as compared to controls but more when compared with Fig. 15.

20:

ubular diameter and height of the lining epithelium arreduced.

2

ular diameter normal but height of the lining epitheliuis low.

Fig. 2: 2

diameter and height of the lining epithelium are reduced owever the situation is better when compared with Fig. 15

PLATE-V

Photomicrographs of mice adrenal gland, with C.S. Bouin’s haematoxylin and eosin preparation. All slides were photographed at 50X.

Fig.

Control adrenals, medulla normal.

Fig. 4:

15 days after CdCl2 injection. Hypertrophy of both zones.

Fig. 25: 30 days after CdCl2 injection. Cortex swells but medulla is

normal. Fig. 26: 60 days after CdCl2. Cortex ells but medulla is normal.

23:cortex and

2

sw

Fig. 27:

15 days after CdCl2 injection + 5 mg Speman daily. Cortex and medulla shrink.

Fig. 28:

30 days after CdCl2 injection + 5 mg Speman daily. Cortex hypertrophies but medulla shrinks.

PLATE-VI

Fig. 29:

Fig. 31:

30 days after CdCl2 injection + 10 mg Speman daily. No change.

Fig. 32: 60 days after CdCl2 injection + 10 mg Speman daily.

No change.

DISCUSSION Cadmiun-induced changes in the testes, epididymis and adrenals as observed here are in good conformity with the findings of earlier workers (Gunn and Gould, 1970). The presence of androgens is a must for the maintenance of spermatogenesis (Steinberger and Duckett, 1965) and normal functioning of the epididymis (Prasad et al., 1973; Tuohima and Niema, 1974; Allen, 1958). In the present investigation, when 60 days after Speman administration cadmium-induced changes were nullified significantly, it is logical to think that Speman must have exerted an “androgen-like” activity as earlier reported by Jayatilak et al., (1976), because it is known that exogenous androgens can maintain spermatogenesis in rats and guinea pigs and upto a certain extent in man and monkeys (Steinberger, 1971). Speman’s ability to reverse and/or check cadmium-induced changes further proved its “androgen-like” activity, as CdCl2 lowers testosterone synthesis in mammals (Saxena et al., 1977) and in fish (Sangland and O’Halloran, 1973).

Fig. 30: 15 days after CdCl2 injection + 10 mg Speman daily. No

change.60 days after CdCl2 injection + 5 mg Speman daily. Cortex

swells, medulla normal.

CdCl2 lowers oxygen uptake (Rao and Patnaik, 1977) and Speman has been recently reported to enhance oxygen uptake in the testes of rats following irradiation (Sethi et al., 1980). This property of Speman can also be cited for its beneficial role following cadmium intoxication. Speman (10 mg daily) fully nullified the effects of CdCl2 on the adrenals and the normal functioning of adrenals can be held responsible for protecting the testes and adrenals at 60 days, as the adrenals also produce androgens (Turner and Bagnara, 1976), and some steroids which influence cell renewal in accessory sex glands (Tullner, 1963). CdCl2 also causes stress (Singhal and Merali, 1979) and the ability of Speman to maintain the normal structure of the adrenals is reflected in its “antistress” property, as the cortical steroids act against stressors (Selye, 1959). AThe authors thank Prof. G.N. Johri, D.Sc. r giving departmental facilities and to The Himalaya Drug Company, Bombay for the research grants. REFERENCES 1. Allen, J.M., Exp. Cell. Res. (1958): 14, 142.

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