Astra Pharmaceutical Products Inc.1, Worcester, MA; Cecil H. & Ida Green Center for Reproductive Biology Sciences, Department of Biochemistry and Obstetrics-Gynaecology2, University of Texas Southwestern Medical School, Dallas, TX; Department of Obstetrics & Gynaecology3, The University of Texas Health Science Center, San Antonio, TX, USA
Suppression of pituitary and testicular function in rats by a transplanted adrenocortical carcinoma By M.E. Harris Rice1, J. I. Mason2 and A. Bartke3
Snell adrenocortical carcinoma was transplanted into immature 4-week-old male rats, and the animals were sacrificed 3 weeks afterward for study of adenohypophyseal and testicular function. The weight of the tumour was 11 ± 5 g; plasma corticosterone levels were elevated and plasma progesterone levels were massively increased compared to those in rats with no tumours. The weights of the adrenals, testes and androgen-dependent accessory reproductive glands were significantly decreased, as was the diameter of seminiferous tubules. The concentrations of testosterone, LH and FSH in the plasma were significantly reduced, whereas the levels of Prl and oestradiol were not affected. We suggest that steroid products of the transplanted tumour suppressed release of gonadotrophins from the pituitary, leading to a severe reduction of testosterone synthesis in the testes.
Key words: adrenocortical tumour - pituitary function - testes - rats - progesterone - seminal vesicles.
Activation of the pituitary-adrenal axis by stress or by experimental procedures can interfere with testicular function in many mammalian species (Matsumoto et al. 1970; Saez et al. 1977; Pratt & Longcope 1978; Welsh & Johnson 1981). Under
natural conditions, high population density can lead to delayed puberty and suppression of fertility, and these effects are believed to be caused by increased ACTH secretion (Christian 1971). In people with Cushing’s syndrome, congenital adrenal hyperplasia or cortisol-secreting testicular tumours, LH release may be suppressed (Penny et al. 1973; Luton et al. 1977; Radfar et al. 1977), and stress was reported to reduce peripheral LH levels in the bull (Welsh & Johnson 1981). It was of interest therefore to ascertain whether pituitary and testicular function in rats is affected by Snell adrenocortical carcinoma 494. It has been shown that this transplanted rat adrenal tumour produces corticosterone (Ney et al. 1969; Kimmel et al. 1974) which is the principal steroid product of normal adrenals in this species.
Materials and Methods
A Snell adrenocortical carcinoma 494 was transplanted into 12 immature (4-week- old, 60-70 g) male Sprague-Dawley (Charles River Breeding Laboratories, Wil- mington, MA) rats at a subcutaneous site near the base of the tail as described previously (Mason & Robidoux 1979). Untreated animals from the same shipment served as controls. The animals were housed in a room with controlled light (12 h of illumination per day) and temperature (22° ± 2℃) with constant access to food and water. Three weeks later, the animals were decapitated, trunk blood was saved for hormone measurements, and the tumour, adrenals, testes, seminal vesicles and ventral prostate were weighed. Testes of 6 animals from each group were fixed in Zenker-formol solution, embedded and sectioned at 5 um, stained with haematoxy- lin PAS for the evaluation of spermatogenesis (Clermont & Morgentaler 1955; Clermont & Harvey 1967) and the measurement of the diameter of seminiferous tubules and the size of Leydig cells. Plasma corticosterone was measured by a fluorometric technique as described previously (Silber et al. 1958). Other hormones were measured by radioimmunoassays by use of kits provided by NIAMDD for measurement of rat LH, rat FSH and rat Prl, NIAMDD reference preparations RP-1, and antisera to testosterone, oestradiol and progesterone (provided by Drs. B. Caldwell, J. McCracken, W. Stylos, J. Patrick, and K. Williams), respectively, as described in our earlier publications (Bartke et al. 1977a,b; Harris & Bartke 1975; Herbert et al. 1978).
Statistical significance of the results was calculated by Student’s t-test.
Results
At the time of autopsy, the average weight of the transplanted tumour was 11 g. There was a significant decrease in the rate of weight gain of the tumour-bearing rats compared to the controls. However, the tumour-bearing animals were not
| Control | Tumour | P | |
|---|---|---|---|
| Body weight (g) | 208 ± 8 | 167.0 ± 19 | < 0.001 |
| Tumour (g) | 11.0 ± 4.9 | ||
| Adrenal (mg) | 45.4 ± 3.17 | 16.8 ± 2.04 | < 0.001 |
| Ventral prostate (mg) | 99.2 ± 22.1 | 31.6 ± 17.0 | < 0.001 |
| Seminal vesicles (mg) | 106.5 ± 10.2 | 30.8 ± 11.8 | < 0.001 |
| Testes (g) | 2.35 ± 0.17 | 1.97 ± 0.16 | <0.1 |
| Tubule diameter (u) | 245 ± 42 | 186.0 ± 25 | < 0.01 |
| Leydig cell diameter (u) | 8.81 ± 1.61 | 9.03 ± 1.30 | N.S. |
acutely ill and did not appear malnourished at the time of sacrifice. The weights of the adrenals, the seminal vesicles and the ventral prostate in tumour-bearing rats were reduced by approximately 70%, whereas body and testicular weights were reduced by approximately 20% (Table 1). The diameter of the seminiferous tubules was significantly reduced, whereas the size of the Leydig cells was not affected. The number of different classes of primary spermatocytes was reduced by 7-11%, while the number of step 7 spermatids was reduced by 27% (data not shown).
Plasma corticosterone levels were significantly increased and the concentration of progesterone in the plasma was increased over 50-fold in the tumour-bearing rats compared to those in rats with no tumour. The levels of testosterone, LH and FSH were markedly depressed, while the levels of oestradiol and Prl were not affected in the tumour-bearing rats compared to controls (Table 2).
| Control | Tumour | P | |
|---|---|---|---|
| Corticosterone (µg/ml) | 0.26 ± 0.01 | 0.44 ± 0.03 | < 0.001 |
| Progesterone (ng/ml) | 0.23 ± 0.06 | 14.5 ± 4.6 | < 0.001 |
| Testosterone (ng/ml) | 2.18 ± 1.47 | 0.42 ± 0.07 | < 0.001 |
| Oestradiol (pg/ml) | 24.9 ± 11.1 | 35.4 ± 13.2 | N.S. |
| LH (ng/ml) | 48.8 ± 37.6 | 10.4 ± 3.3 | < 0.1 |
| FSH (ng/ml) | 577 ± 65 | 382 ± 57 | < 0.001 |
| Prl (ng/ml) | 52.4 ± 25.8 | 40.2 ± 24.4 | N.S. |
Discussion
The results of the present study are indicative that transplanted adrenocortical carcinoma 494 markedly suppresses the secretion of gonadotrophins and testoste- rone in male rats. The concomitant reduction in the levels of LH and testosterone in peripheral plasma are suggestive that the primary effect of the tumour was at the hypothalamic and/or pituitary level, but the possibility that steroids secreted by the tumour also acted directly on the testes cannot be eliminated on the basis of these data. The decline in the weight of the testes, the decrease in the diameter of the seminiferous tubules and the number of germinal cells in tumour-bearing rats were relatively modest. This may have been due to maintenance of peripheral FSH at approximately 60% of pre-treatment levels and possibly also to intratesticular testosterone distribution mechanisms which favour transport to seminiferous tubules during periods of reduced androgen biosynthesis (Setchell 1980). In support of the latter possibility, spermatogenesis and tubular testosterone levels can be reasonably well maintained in hypophysectomized, pregnenolone-injected rats in which peripheral testosterone levels are extremely low (Harris & Bartke 1975). Recently it was reported that during treatment of rats with testosterone propionate, peripheral FSH levels were only slightly depressed and spermatogenesis was maintained although testicular testosterone levels were reduced significantly (Cunningham & Huckins 1979).
Suppression of pituitary gonadotrophin release in tumour-bearing rats was almost certainly due to the secretion of hormonal steroids, including corticosterone and progesterone, by the tumour. In the present study the concentration of corticosterone in the plasma was increased by Snell tumour 494 in agreement with earlier observations (Ney et al. 1969; Kimmel et al. 1974); and the concentration of progesterone was elevated enormously. Corticosteroids apparently can suppress LH release (Penny et al. 1973; Radfar et al. 1977; Luton et al. 1977; Welsh & Johnson 1981) and progesterone can inhibit the release of both LH and FSH, at least in the presence of oestrogen (Savoy-Moore & Schwartz 1980). However, at present, we cannot completely exclude the possibility that the growth of the neoplasm may have a direct effect on the development of the testis and accessory sex tissues.
In additional experiments (Mason & Harris, unpublished observations) we have attempted to examine the effects of the adrenal tumour in mature male rats (10-11 weeks old at the time of implantation). Interestingly, this study was made difficult by a low rate of successful implantation and tumour development obtained in mature rats compared to that in young animals. In 2 mature male rats with demonstrable tumours 3 weeks after implantation, the adrenal weights were suppressed compared to the corresponding control animals. However, the weights of testes, seminal vesicles and prostates overlapped with those of the controls. These findings are suggestive that the effects of the tumour on testicular-pituitary function may be transient in nature and might represent a delay of puberty.
Acknowledgments
We thank Ms. Carol Roberson, Ms. Penny Bonin and Mr. William Robidoux for hormone determinations, NIAMDD and Drs. B. Caldwell, J. McCracken, W. Stylos, J. Patrick and K. Williams for materials used in radioimmunoassays and Ms. Lydia Morris for excellent editorial assistance.
Supported by grants CA 30253, HD 12641 and HD 12642, DHHS.
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Author’s address:
Dr. J. I. Mason, Departments of Biochemistry and Obstetrics-Gynecology,
The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA.