The effect of chronically administered cannabis extract on the testicular function of mice

The effect of chronically administered cannabis extract on the testicular function of mice

EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 111-114. NORTH-HOLLAND PUBLISHING COMPANY Short c o m m u n i c a t i o n THE EFFECT OF CHRONICALLY ADMINI...

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EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 111-114. NORTH-HOLLAND PUBLISHING COMPANY

Short c o m m u n i c a t i o n THE EFFECT OF CHRONICALLY ADMINISTERED CANNABIS EXTRACT ON THE TESTICULAR FUNCTION OF MICE V.P. DIXIT, V.N. SHARMA and N.K. LOHIYA Reproduction Physiology Section, Department of Zoology, University of Rajasthan, Jaipur, and Department of Pharmacology, S.M.S. Medical College, Jaipur-302004, India

Received 21 December 1973

Accepted 21 January 1974

V.P. DIXIT, V.N. SHARMA and N.K. LOHIYA, The effect of chromically administered cannabis extract on the testicularfunction o f mice, European J. Pharmacol. 26 (1974) 111-114. Daily administration of cannabis extract (2 mg/day for 45 days: Total dose 90 mg) produced a complete arrest of spermatogenesis in mice. Distinct effects were produced upon various cellular stages in the seminiferous epithelium. Regression of Leydig celi tissue and of accessory sex glands was conspicuous. These effects were revertible. Cannabis extract

Testicular epithelium

Spermatogenetis inhibition

1. Introduction

2. Materials and methods

Marihuana is a crude preparation of flowering tops, leaves, seeds, and stems of female plants of Indian hemp Cannabis sativa L. There is as yet little known about its biological effects. Kew et al. (1969) performed liver function tests on 12 regular marihuana smokers and suggested hepatotoxicity. Martin (1969) studied possible chromosome aberrations on human leucocytes and rat embryo cells in culture. Acute toxicity of cannabis resin was also reported in rats and mice (Phillips et al., 1971). Reproductive studies have indicated that chronic oral administration of cannabis resin significantly reduces fertility in the rat (Miras, 1965). As there is so far no information available on the effect o f marihuana on spermatogenic processes we have studied testicular function of young adult male mice after long term administration of low doses of cannabis extract.

Pharmacological studies of cannabis have indicated that the tetrahydrocannabinol (THC) fraction of the resin is the most active portion. Since THC has however not yet been established to be the only fraction of marihuana with biological activity, we felt that it was important to use whole plant extracts. Therefore, we extracted the authenticated samples of Cannabis indica with 98% ethanol in a Soxhlet apparatus for 4 hr. The extract was evaporated to dryness and the residue weighed and redissolved in a mixture of alcohol, Tween 80 and distilled water (10 : 1 : 89) to make a final concentration of 10 mg/ml cannabis extract. 40 young adult male mice of the Swiss albino strain weighing 30 + 3 g were divided into groups o f 10 each and were treated as outlined in table 1. The extract was injected i.p. The controls received vehicle alone. 10 animals were allowed to recover for a per-

Control (10) i Cannabis extract (Total dose o f 50 m g in 25 days (10) Cannabis extract (Total dose of 90 m g in 45 days (10) Recovery (63 days after last d o s e ) ( 1 0 )

1 2

35-+ 1

8.6-+ 1.2

8.2 +- 1.2

9.0-+ 1.0

31 -+ 1.7

32 -+ 2.5

9.1 -+ 1.2

Thyroid wt. (mg]100 g b o d y wt.)

30 -+ 1.9

Body wt. (g)

593-+51

534 +- 64*

515 -+ 1 0 " * *

697 ± 28

Testes wt. (mg/100 g b o d y wt.)

456-+56

274 -+ 2 9 * * *

293 ± 2 0 * * *

520 +- 38

Seminal vesicle (mg/100 g b o d y wt.)

14.2-+ 1.8

25.1 -+ 2.1"

20.7 -+ 0.4

16.8 +- 2.4

Adrenal wt. (mg/100 g b o d y wt.)

135-+8.0

64.7 -+ 2.0***

97 ± 2.7***

123 +- 3

Levator ani (mg/100 g b o d y wt.)

***p < 0.001 compared with group 1; *p < 0.05 compared with group 1; **p < 0.01 compared with group 1.

4

3

Tratment

Group

57.1-+

7

32.6 -+ 5.0

25.2 +- 2.1"

59.2 -+ 12

T h y m u s wt. (mg/100 g b o d y wt.)

198-+ 3

148 -+ 3***

155 ± 6 * * *

196 -+ 2

Seminiferous tubule diameter (/~)

Figures in parentheses: n u m b e r of animals RNA: m e a n s o f 6 determinations. All figures m e a n values _+ S.E.M.

2.173 +- 0.35**

--

3.485 ± 0.19

R N A (#g/mg testis)

Table 1 Organ weights, diameters of seminiferous tubules and testicular R N A concentrations o f control mice and mice treated with cannabis extract (2 m g / m o u s e / d a y , i.p.).

5

V.P. Dixit et al., Effect of cannabis on testicular function iod of 63 days. The animals were killed 24 hr after the administration of the final dose and various organs were removed and weighed. Immediately after weighing the right testis of each mouse, it was fixed in Bouin's fluid for histology. The left testis was frozen and total RNA determined later (Munro and Fleck, 1966). 50 seminiferous tubules, appearing circular in section, were traced, with a camera lucida at 80X magnification. 2 diameters, at right angles to each other, were measured of each tubule, and the mean values and standard errors calculated. Student's t-test was applied to compare means.

113

tion (table 1). The weights of the seminal vesicles and the levator ani muscles was drastically decreased. Involution of the thymus gland was observed (p < 0.05). The weight of the adrenal gland increased significantly (49%) whereas the weight of the thyroid gland did not change. 3.2. Histology o f testis

The relative testicular weight of mice treated with cannabis extract was significantly decreased, indicating testicular damage. A shrinkage of the seminiferous tubules occurred after cannabis extract administra-

After treatment with cannabis extract for 25 days (2 mg/day/animal: total dose 50 mg) the number of spermatogonia was decreased. No resting or leptotene spermatocytes were seen; the pachytene spermatocytes decreased in number. Cytolysis and chromatolysis were common and spermatozoa were retained in the basal layers of the germinal epithelium. After treatment for 45 days (2 mg/day/animal: Total dose 90 mg) a complete arrest of spermatogenesis occurred (figs. 1 and 2). Distinct effects were produced upon spermatogonia, spermatocytes and spematids. Occasional degenerating forms of spermatids and fragmented sperms were seen. Leydig cells were

Fig. 1. Section of a testicular tubule of a control mouse. Note the normal spermatogenesis. H.E. X320.

Fig. 2. Testis of a mouse treated with cannabis extract (90 rag) showing degenerative changes and complete absence

3. R e s u l t s

3.1. Organ weights

of spermatozoa. H.E. × 80.

114

v.P. Dixit et al., Effect of cannabis on testicula~ function

weakly eosinophillic. The cytoplasm was scanty and the nuclei shrunken. 3.3. Epididymis

Degenerating cells, spermatids and fibrous material from the testis appeared in the epididymal lumen. 3.4. Ribonucleic acid

Cannabis extract administration caused a significant decrease in the total amount of RNA in the testis (table 1).

The decreased synthesis of RNA in the testis of mice and the decrease in weight of the seminal vesicles indicate the possibility that cannabis extract administration inhibits androgen production. The findings (table 1) of a significantly higher adrenal weight might suggest that adrenal secretion is a contributing factor. The results of the present investigation further revealed that cannabis extract caused a regression of Leydig cell tissue and of accessory sex organs (e.g. seminal vesicle and levator ani muscles). These effects are reversible. Repopulation of testicular epithelial cells and normal spermatogenesis occurred following a recovery period of 63 days.

3.5. Recovery

Complete recovery of spermatogenesis and Leydig cell function was found in mice 63 days after medication. The organ weights and the dimensions of the seminiferous tubules had returned to normal.

Acknowledgement One of us (V.P.D.) is greatly indebted to Prof. P.N. Srivastava for providing facilities.

4. Discussion Treatment with cannabis extract caused cytolytic and chromatolytic changes in young spermatides of mice. The nuclei of spermatids appeared ring-shaped due to marginal accumulatiori of chromatin. The degenerative changes in the spermatocytes were followed by pyknosis, chromatolysis and gradual cytolysis. Cannabis extract seems to affect mouse testis cells in two different ways: (1) it causes degeneration of spermatids and the disappearance of spermatozoa; (2) it produces latent changes in spermatocytes which still allow some of them to develop into spermatozoa. Steinberger and Dixon (1959) and Chowdhury and Steinberger (1964) also concluded that not all stages of the development of primary spermatocytes are damaged when the testicular tissue is exposed to temperatures of 43°C for 15 min, which is similar to the occasional presence of spermatozoa in cannabis.treated mice. The sequence of events in the present investigations is that spermatids degenerate first and the other germinal cells undergo degenerative changes with time (Asdell and Salisbury, 1941).

References AsdeU, S.A, and G.W. Salisbury, 1941, The rate at which spermatogenesis occurs in the rabbit, Anat. Rec. 80, 145. Chowdhury, A.K. and E. Steinberger, 1964, A quantitative study of the effect of heat on the germinal epithelium of the rat testis, Amer. J. Anat. 115,509. Kew, M.C., I. Bersohn and S. Siew, 1969, Possible hepatotoxicity of cannabis, Lancet 1,578. Martin, P.A., 1969, Cannabis and chromosomes, Lancet 1, 370. Miras, C.J., 1965, Some aspects of cannabis action, in: Hashish: Its chemistry and Pharmacology,ed. G.E.W. Wolstenholme (Little Brown, Boston, Mass.) p. 37. Munro, H.N. and Fleck, 1966, The determination of nucleic acids, in: Methods of BiochemicalAnalysis, Vol. 14, ed. D. Glick (Interscience, New York) p. 113. Phillips, R.N., R.F. Turk and R.B. Fomey, 1971, Acute toxicity of Ag-tetrahydrocannabinol in rats and mice, Proc. Soc. Exptl. Biol. Med. 136,260. Steinberger, E. and W.J. Dixon, 1959, Some observations on the effect of heat on the testicular germinal epithelium, Fertil. Steril. 10,578.