Effect of cadmium chloride on the serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and androgens in the adult male rat

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1 Proc. Indian Acad. Sci., Vol. 87 B, (Experimental Biology-3), No.7, July 1978, pp. printed in India Effect of cadmium chloride on the serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and androgens in the adult male rat N H GOPAL DUTI*, K WAKABAYASHI and S INOUE Institute of Endocrinology, Gunma University, Maebashi, Japan *Post-graduate Department of Zoology, University of Mysore, Manasagangotri, Mysore MS received 6 February 1978; revised 31 May 1978 Abstract. Adult male rats injected with cadmium chloride were compared with controls with respect to serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH). androgens and testicular histology. A single injection of cadmium chloride (9 mg/kg) was found to bring about no consistent short term changes in the plasma levels of FSH or LH, but after a long period the levels of both these hormones were elevated. In contrast, the levels of androgen showed a sharp increase at 6 h which declined by 12 h. In accordance with the elevated levels of gonadotropins found at 9-28 days after cadmium chloride injection, the androgen levels showed a drastic reduction. Histological aspect of the testis revealed acute necrotic changes of which the vacuolation of spermatid nuclei and fibrosis of Leydig cells are noteworthy. Keywords. Cadmium chloride; follicle stimulating hormone: luteinizing hormone; androgens; rat. 1. Introduction The effects of cadmium chloride (CdCI 2 ) on the histology and biochemical composition of testicular tissue in rats have been studied by many investigators (Parizek and Zahor 1956; Allanson and Deanesly 1962; Mason et a11964; Web 1972). A few workers have also explained the mechanism of action of CdCl 2 on the rat testis (Parizek 1957, 1960). However, there is no information available on hormone levels of serum after the administration of cadmium. The present communication pertains to changes in the levels of FSH, LH and androgens after the administration of CdCI 2 to adult male rats. These results are correlated to parallel histological changes in the testis. 2. Materials and methods Male rats of Wistar strain weighing around 300 g were used in this study. Cadmium chloride was dissolved in distilled water at a concentration of 0 04 M (9'12 gmjlitre). One mljkg body weight was injected subcutaneously as recemmended by Parizek (1957). Distilled water injected rats served as controls. Both the experimental and 161

2 162 NH Gopal Dutt, K Wakabayashi and S Inoue control rats were fed ad lib on stock diet and water. Blood was collected at intervals of 0, 6, 12, 24, 48 h, and 9 and 28 days after the injection of CdCl a under light anaesthesia. Blood samples of 0 h group were collected between 9 a.m, and a.m, Time schedule for blood collection varied 30 to 45 minutes between the experimental groups. The gonadotropins and androgens were estimated in the same serum samples at all these periods. All the animals were killed by decapitation. Testes were fixed in Hollande Bouins fluid. For a histological study 8 microns paraffin sections of the testes were stained with iron haematoxylin and Mallory's triple stains. Rat FSH and LH were assayed with radioimmunoassay (RIA) kit obtained from Rat Pituitary Hormone Distribution Programme, National Institute of Arthritis, Metabolic and Digestive Deseases, National Institute of Health, Bathesda, Md., USA, according to the method of Monroe et al (1968). The assay results were expressed in terms of ngjml equivalents of the standard preparations of NIH-LH-SI and NIH-FSH-SI. To check for the consistency ofresults, assays for FSH and LH were repeated. The antiserum to testosterone used in the androgen assays was a gift from Teikoku Hormone Mfg. Co. Tokyo, Japan. Since this antiserum cross-reacted equally with testosterone and dihydrotestosterone (Makino 1973), the values reported represent total androgen equivalents. 3. Results While the testes of control rats revealed intact seminiferous tubules (figure I), those of CdCI:a-treated rats showed a marked degeneration of the tubules and acute necrosis was observed 48 h after CdCl:a injection. Degenerative changes included vacuolation of the seminiferous tubules (figure 2), pycnosis of nuclei, karyorrhexis, degeneration of tunica propria, peritubular fibrosis, etc. In addition it was also observed that many spermatids Were vacuolated (figure 3). At 6 h the Leydig cells were slightly oedematous. Later particularly at 48 h these cells appeared to be acutely degenerated. At this time, however, serum contained still detectable quantities of androgen. By 9th day the seminiferous tubules were seen severely damaged. These observations in general are in excellent agreement with those described earlier by Parizek and Zahor (1956). A marked decrease in the levels of FSH and LH was observed after 6 h ofinjection of CdC1 2 (experiment 1: tables 1 and 2). However, this acute effect observed at 6 h could perhaps bea spurious result, since it could not be reproduced in a second experiment; no variation could be found in the serum levels of FSH and LH up to 48 h in CdCl z administered rats as compared to those controls (tables 1 and 2). However, by 9th day, both FSH and LH levels were found to be significantly elevated in the CdCl 2 injected rats and the levels continued to remain high even after 28 days. Excepting for a sharp rise at 6 h after CdCl t injection, the androgen levels tended to show a gradual drop which remained low even at 28 days after CdCI! (table 3). It can be noted that the serum levels of androgen of the controls at 6 (1530 h) and 12 h (2130 h) were low compared to 0 h sample taken at 0930 h. By 28 days new Leydig cells were seen proliferating from the fibroblasts.

3 Effect of CdCl 2-serum FSH, LH and androgens 163 Figures Intact seminiferous tubules from a control rat. Bouins fluid, Mallory's triple stain, x Vacuolated seminiferous tubules from the testis of CdCl 2 injected rat. Bouins fluid, Mallory's triple stain, x Degenerating spermatocytes and vacuolated sperrnatids in a seminiferous tubule from a Cd Cl, injected rat. Bouins fluid, Mallory's triple stain, x 860.

4 Effect ofcdcl 2 - serum FSH, LH and androgens 165 Tablen. Changes in serum level offsh* after a single injection ofcadmiumchloride. Time of blood sampling Experiment I Control (10) Experimental (9) Experiment II Control (5) Experimental (5) Oh 6h 12 h 24h 48 h 9 days 28 days 1'07 ± 0' ± ± '04 ± ± 0' ± ± '10 ± 0'05 0'96 ± 0'05* 0 95 ± ± ± 0' ± 0'09** 2'04 ± 0'18** 0 79 ± 0' ± ± '78 ± 0'06 0'91 ± ± ± 0'07 0'96 ± '94 ± 0 17 Serum FSH levels (mean ± SE) are expressed as NIH-FSHwS 1jmI. paranthesis indicates the number of animals used for group. *p<0'05, **p<o'ool, compared to corresponding control. Number in the Table 2. Changes in serum level of LH after single injection of cadmium chloride. Time of blood Experiment I Experiment II sampling Control (l0) Experimental (9) Control (5) Experimental (5) Oh 0 80 ± 0' ± 0'14 1'86 ± 0'38 6h ],06 ± 0'10 0'44 ± 0'08 ]'25 ± 0' ± h 0 98 ± '70 ± ± 0'55 1'03 ± h 1-98 ± 0' ± ± 0'15 0'94 ± h 1 47 ± ± ± ± 0'40 9 days 0'64 ± ± 0'45 28 days 0 56 ± ± 0 74 Serum LH levels (mean ± SE) are expressed as ng NIH-LH-Slfml. paranthesis indicates the number ofrats per group. p<o OOl, compared to the corresponding control. Number in Table 3. Changes in serum testosterone level after single injection of cadmium chloride.* oht 6h 12 h 24 h 48 h 28 days Control 1756 ± ± ± ± ± ± 285 (10) (5) (5) (5) (5) (9) Experimental 5782 ± ± ± 6~ 104 ± ± 17 (5) (5) (5) (5) (9) Statistical significance P <0'05 N.S. N.S <0 01 <0 001 "'Serum testosterone levels are expressed as pg testosterone/ml. toh sample was collected at 0930 h. N.S. Not significant. Mean ±SE

5 166 N H Gopal Dutt, K Wakabayashi and S Inoue 4. Discussion Regressive changes in the testes, as evidenced by histology are detectable 6 h after CdC}2 injection. However, no consistent variation is noticed in the serum gonadotropins of these animals even 48 h after injection. By this time, the seminiferous tubules, particularly, the spermatids are considerably damaged, but the severity of damage is greater by 9 days after administration of the drug. Steinberger (1971) has reported that FSH is involved in the formation of spermatids and that the hypothalamo-hypophyseal feed-back mechanism comes into play only after this step of spermatogenesis. It is of considerable interest to note that there is no concordance in the pattern of change in serum FSH and LH levels after CdCl 2 injection. The rise in FSH is much faster than that of LH, (peak FSH levels being reached at 9 days as against 28 days in case of LH). Amatayakul et al (1971) have reported increase in the serum concentration of FSH and LH within two days following castration. Similarly serum levels of gonadotropins are also known to be elevated following surgical cryptorchidism, but the magnitude of elevation is less marked than that following castration (Amatayakul et al 1971) or testicular X-radiation (Wakabayashi et a/1971). The increase in the levels of gonadotropins especially after testicular necrosis due to CdCl 2 confirms the earlier reports on the existence of dynamic relationship between testis and pituitary. Immunohistochemical study of pituitary gonadotrophins in the hypophysis of CdCl 2 injected rats by Dutt et al (1976) further supports this view. The variation in the serum gonadotropin levels in CdCl2 treated rats between experiments 1 and 2, especially at 6 h may be due to some difference in the time schedule of the collection of blood samples. According to Nankin and Troen (1971) in human males, major differences in the levels of gonadotropins can occur within minutes during the day. The dramatic rise in plasma testosterone after 6 h may be due to a sudden discharge of hormone as a consequence of impairment in testes metabolism due to cadmium. That at least part of this testosterone is released from adrenal source also cannot be ruled out. In support of this possibility is the observation that detectable levels of testosterone are still found in serum at 48 h despite heavy damage to the interstitial cells. Kniewald et al (1971) have demonstrated that the adrenal gland of male rats is capable ofproducing testosterone and that this increased after castration. Further, the elevation in the level of plasma testosterone is consistent with the observations of Kar and Das (1960) and Maekawa et al (1966) who reported increase in the weights of accessory sex glands and in the concentration of fructose in the coagulating gland respectivelysoon after administration ofcdci 2 The acute drop in the levelsof androgen at longer intervals is parallel to acute necrosis seen in the Leydig cells. Restorative changes in the Leydig cells were noticed, however, after 28 days. It is possible that the decrease in serum testosterone levels at 6 hand 12 h in the controls is due to diurnal variations since the blood samples for these groups were collected for one group in the afternoon and in the night for the other. Alternatively, as reported by Eik-Nes (1962) and Bardin and Peterson (1967), that the decrease is due to frequent exposure to anaesthesia on the first day of injection of CdCl 2 cannot be excluded.

6 Effect of CdC/a-serum FSH, LH and androgens 167 References Allanson M and Deanesly R 1962 J. Endacrinol Amatayakul K, Ryan R, Uozomi T and Albert A 1971 Endocrinology Bardin C Wand Peterson R E 1967 Endocrinology Dutt N H G, Wakabayashi K and Inoue S 1976 Curro Sci Eik-Nes K B 1962 Endocrinology Kar A Band Das R P 1960 Acta Bioi. Med. Ger Kniewald Z, Zanisi M and Martini L 1971 Acta Endocrinol, Maekawa K, Tsunenari Y, Nobuki K and Okada J 1966 Dobutsugaku zasshi Makino T 1973 Folia Endocrinol. Jpn, Mason K E, Brown J A, Younge J 0 and Nesbit R R 1964 Anat, Rec Monroe S E, Parlow A F and Midley A R Jr 1968 Endocrinology Nankin H Rand Troen P 1971 J. CUn. Endocrtnol Parizek J 1957 J. Endocrinol, 1556 Parizek J 1960 J. Reprod, Fertil Parizek J and Zahor Z 1956 Nature (London) Steinberger E 1971 Physiol. Rev Wakabayashi K, Date Y and Tamaoki B 1971 Nat. Inst, Radiol. Sci. Ann. Rep Webb M 1972 J. Reprod. Fertil P.(B)-2