0013-7227/79/l()51-0135$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 105, No. 4 Printed in U.S.A.

Alterations in Steroidogenesis and Human Chorionic Gonadotropin Binding in the Cryptorchid Rat Testis* D. M. DE KRETSER, R. M. SHARPE, AND I. A. SWANSTON MRC Unit of Reproductive Biology, Edinburgh, Scotland; and the Department of Anatomy, Monash University, Melbourne, Australia

ABSTRACT. One month after the induction of cryptorchidism in adult rats, serum levels of LH and FSH were significantly elevated in comparison with sham-operated controls, whereas serum levels of testosterone remained low to normal. Testis weight in cryptorchid rats was reduced by over 66%, and once the extratubular fluid was removed by decapsulation, the reduction in weight was 78%. The basal production of testosterone, pregnenolone, and estradiol in vitro by testes from cryptorchid rats was similar to

controls, whereas significantly less androstenedione was produced. Testicular stimulation in vitro with a high dose of hCG (360 pM) resulted in significantly greater production of testosterone, pregnenolone, and estradiol by cryptorchid than by control rat tissue. The in vitro binding of [l25I]hCG per testis was decreased in the cryptorchid state to 40% of control values, probably as a result of down-regulation of LH receptors due to the 4-fold elevation of serum LH levels in the cryptorchid rats. (Endocrinology 105: 135, 1979)


Four weeks after the induction of cryptorchidism, the animals were anesthetized with ether and decapitated, and trunk blood was collected for subsequent RIA of LH, FSH, and testosterone. The paired testes were removed, cleaned of extraneous material, placed into preweighed 5-ml polystyrene serum vials, weighed, and decapsulated; in the case of cryptorchid rat testes, decapsulation was performed over a serum vial to enable collection of the copious testicular (extratubular) fluid. Decapsulated testes were then used immediately to assess in vitro steroid production, as described below, while testes used for the measurement of in vitro binding of [125I]hCG were stored overnight at -20 C before use.

T HAS been established that surgically induced intraabdorninal translocation of the rat testes results in disruption of spermatogenesis and infertility (1, 2). Our recent studies have demonstrated that a state of compensated interstitial cell failure exists, characterized by elevated LH levels and low or normal testosterone levels together with a subnormal testosterone response to hCG in vivo (3). Paradoxically, morphometric studies have shown that in cryptorchid rats, the interstitial cells are larger in size and contain increased quantities of the organelles involved in steroid biosynthesis (3). On the basis of these findings, it was postulated that the failure of these hypertrophic interstitial cells to secrete testosterone was related to the influence of factors produced by the damaged seminiferous tubules (3). In this paper, we have examined whether changes in LH (hCG) binding and steroid production in vitro by cryptorchid rat testes can explain the previous in vivo findings.

In vitro binding of [l2r>IJhCG

Testes from individual rats were thawed and homogenized in Krebs-Ringer bicarbonate solution (KRB; 1 ml/100 mg tissue), as described previously (4); 0.2-ml aliquots of this suspension were then incubated in 63 X 11-mm polystyrene tubes together with 25 ng/ml [125I]hCG in KRB containing 0.2% bovine serum albumin (fraction V; Sigma Chemical Co., St. Louis, MO) for 3 h at 37 C in a shaking water bath. Estimation of nonspecific binding and separation procedures were as described previously (4). Labeling of hCG (hCG CR119; 11,600 IU/mg) with I26I was performed by the enzymic method of Miyachi et al. (5) to a specific activity of 63 jiiCi/jtig.

Materials and Methods Inbred PVG (Piebald Viral Glaxo strain) male rats, aged 6580 days, were anesthetized with ether and their testes were translocated into the abdominal cavity, after which the inguinal canal was closed by suture to prevent redescent of the testes into the scrotum. Sham-operated animals were used as controls.

In vitro steroid production

Received October 11, 1978. Address requests for reprints to: Prof. D. M. de Kretser, Department of Anatomy, Monash University, Clayton Victoria 3168, Australia. * Presented in part at the 60th Annual Meeting of The American Endocrine Society (Abstract 594). This work was supported in part by the National Health and Medical Research Council of Australia.

One testis from each rat was incubated in 1 ml KRB containing glucose (1.5 mg/ml) and bovine serum albumin (0.25%) for 4 h at 37 C in a shaking water bath using glass scintillation vials. The other testis from each rat was incubated under identical conditions in the presence of 166 mlU/ml hCG (Preg-


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nyl, Organon, Pharmaceuticals, West Orange, NJ; 360 pM, assuming a molecular weight of 38,000). In another experiment, testes from control and cryptorchid rats were incubated, as above, with concentrations of hCG ranging from 0.5«fi500 pM to obtain dose-response characteristics for testosterone production. The incubation medium was aspirated and centrifuged for 5 min at 1500 X g to remove cellular debris, and the resultant supernatant was stored at —20 C for subsequent measurement of steroid concentrations. Hormone measurements Serum levels of LH and FSH were determined by RIA, as described by Fraser and Sandow (6) and de Jong and Sharpe (7), respectively; results are expressed in terms of the appropriate NIAMDD reference preparation (RP-1). The sensitivity for LH assay was 5 ng/ml and for FSH was 50 ng/ml. Testosterone in serum, incubation medium, and testicular fluid was measured by RIA, as described by Corker and Davidson (8) using the method of Furuyama, Mayes, and Nugent (9). Serum was extracted with 10 vol of hexane-diethyl ether (4:1, vol/vol) before assay, while incubation medium and testicular fluid was assayed directly, as extracted and unextracted samples gave similar results (10, 11). The sensitivity of the testosterone assay was 120 pg/ml. For the measurement of androstenedione and pregnenolone, aliquots (0.3 ml) of incubation media were extracted with 2 ml hexane-diethyl ether mixture (4:1). The extracts were then chromatographed on aluminium oxide columns (3 X 0.5 cm) and eluted sequentially with 6 ml 0.2% hexane-ethanol, which was discarded, then with 4 ml 0.2% hexane-ethanol (androstenedione fraction), and then with 3 ml 2.0% hexane-ethanol, which yielded the pregnenolone fraction. The fractions were dried down, redissolved in phosphate-buffered saline (0.1% gelatin, pH 7.0), and assayed by RIA. [3H]androstenedione and [3H]pregnenolone were added before extraction and chromatography to assess recovery. Androstenedione was measured by the RIA described previously (12) and pregnenolone by RIA using an antiserum kindly provided by Dr. H. Van der Molen with specificities previously published (13). The sensitivity of the assay for androstenedione was 800 pg/ml and for pregnenolone was 120 pg/ml. For the measurement of estradiol, aliquots of incubation media were extracted with diethyl ether, dried down, and reconstituted, as described above, before inclusion in a RIA for estradiol routinely performed in our laboratory, whose characteristics have been previously described (14). The sensitivity of the estradiol assay was 20 pg/ml.

Results There was a 66% decrease in the weight of the cryptorchid testis compared to control animals; this difference was even larger (78%) after decapsulation, as this released a large amount of fluid (range, 120-670 mg) from cryptorchid rat testes (Table 1). After the induction of cryptorchidism, serum levels of FSH and LH were both significantly elevated, but despite the 4-fold rise in serum

Kndo Vol 105

1979 Nol

TABLE 1. Testis weight and serum levels of LH, FSH, and testosterone in control and cryptorchid rats (mean ± SD) No. of animals Paired testes wt (mg) Testis fluid wt (mg) Corrected testis wt (mg)* Serum FSH (ng/ml) Serum LH (ng/ml) Serum T (ng/ml)

22 14 22 14 14 22



2573 ± 110

849 ± 172" 272 ± 164 577 ± 78" 1505 ± 82" 234.8 ± 66.4" 2.28 ± 1.23

2573 ± 722 ± 55.3 ± 3.25 ±

110 75 20.8 2.05

° P < 0.001. * After the removal of fluid.

LH levels, the mean serum level of testosterone was not significantly altered (Table 1). The testicular fluid collected from cryptorchid rat testes had a high concentration of testosterone [161 ± 77 ng/ml, (mean ± SD)] and there was a strong positive correlation (r = 0.87; P < 0.001) between the concentration of testosterone in serum and that in testicular fluid from individual animals. Testicular fluid in control rats was not collected, as it requires 3-4 h to obtain adequate amounts (15); however, the concentration observed in cryptorchid rats was within the range observed for normal rats of different ages (15). In vitro, the basal production of testosterone, pregnenolone, and 17/?-estradiol by decapsulated testes from cryptorchid rats did not differ significantly from the controls, but the production of androstenedione was significantly lower (Table 2). Stimulation with hCG produced a significant increase above basal in both control and cryptorchid rats for all of the steroids measured; however, the increment in the concentrations of testosterone, pregnenolone, and estradiol caused by hCG stimulation was significantly greater in the testes from cryptorchid animals (Table 2). During in vitro incubation, a dose-dependent increase in testosterone production in response to hCG stimulation could be shown for the testes from both control and cryptorchid animals (Fig. 1). At the lower concentrations (0.5-11 pM) of hCG used, the testosterone responses were not significantly (P > 0.05) different, but at higher concentrations (450-4500 pM), the capacity of the cryptorchid testes to produce testosterone was significantly (P < 0.001) greater than controls. The binding of [125I]hCG per mg testicular homogenate was significantly greater in the cryptorchid state than in controls but, when considered on a per testis basis, binding of [125I]hCG to the cryptorchid rat testis was significantly decreased to 40% of control levels (Table 3).

Discussion This study demonstrates that the testes of cryptorchid rats show significant differences in their in vitro steroid-

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STEROIDOGENESIS IN CRYPTORCHIDISM TABLE 2. Basal and hCG-stimulated steroid production in vitro by testes from control and cryptorchid rats

Basal Control Cryptorchid hCG-stimulated Control Cryptorchid.

Testis wt (mg)

Testosterone (ng)

Pregnenolone (ng)

Androstenedione (ng)

Estradiol (pg)

1340 ± 53 414 ± 45"

341.7 ± 54.3 309.6 ± 90.9

1.64 ± 0.31 1.54 ± 0.82

25.3 ± 12.0 5.7 ± 1.8*

84.9 ± 13.8 97.1 ± 14.5

1363 ± 81 477 ± 61"

820.8 ± 115.6 1824.0 ± 582*

2.50 ± 0.38 4.92 ± 1.93C

140.1 ± 49.5 108.3 ± 71.1

126.5 ± 19.1 168.5 ± 13.2"

Results are the mean ± SD for five testes. " P < 0.001 compared with the corresponding control value. h P < 0.01 compared with the corresponding control value. '' P < 0.05 compared with the corresponding control value. TABLE 3. The in vitro binding of [l25I]hCG to testis tissue from control and cryptorchid rats


o —




[l2r>I]hCG bound (pg) -


Control Homogenate (20 mg wet wt decaps, testis) Whole testis




1000 (pM)


FIG. 1. Dose-response relationship between the concentration of added hCG and testosterone production in vitro by testes from control ( J ) and cryptorchid (D) rats. Each point represents the mean ± SD for three testes.

ogenic secretory capacity compared to testes from control rats. The increased capacity for steroidogenesis in the cryptorchid testes is reflected by the significantly greater production of pregnenolone, testosterone, and estradiol. This increased steroidogenic capacity is consistent with the cytological features of Leydig cells in the cryptorchid testis, which have been shown to be hypertrophic, with all of the organelles associated with steroid biosynthesis being present in greater quantities than in normal Leydig cells (3). However, the increased capacity of the cryptorchid testis to produce testosterone during in vitro stimulation with hCG contrasts remarkably with the results of our previous study in vivo, which demonstrated a subnormal testosterone response in cryptorchid rats to an iv injection of 50 IU hCG (3). The reason for this paradox remains unknown. The in vivo study was performed at a single dose, and it is possible that with higher doses of hCG an increased capacity for * testosterone production in vivo by cryptorchid testes may be observed. In this respect, it is interesting to note that the increased capacity of the cryptorchid rat testis to secrete testosterone was demonstrated only at the higher doses of hCG (see Fig. 1). Alternatively, the subnormal steroidogenic response of the cryptorchid rat testis to hCG in vivo may be a consequence of the reported reduction in



± 37.1



± 4500


± 49.7 ±


Binding to cryptorchid (% of control) 170.0"


Values are the mean ± SD for five testes. " P < 0.001 compared to control.

blood supply to the Leydig cells of such rats (16). The low basal androstenedione concentration and the failure of hCG to stimulate its secretion in vitro may be related to the decrease in 3/?-hydroxysteroid dehydrogenase and 17-desmolase activity seen in the cryptorchid rat testis (17, 18). Testes from cryptorchid rats contain a greater concentration of Leydig cell tissue than the normal testis and this is due to both the reduction in the amount of tubular tissue and the increased number and size of the Leydig cells (3, 16). Presumably, this change accounts for the increased binding of [125I]hCG per U testis weight in cryptorchid rats. In the same animals, the marked decrease in binding of [125I]hCG per testis probably represents a pathophysiological demonstration of the phenomenon of down-regulation of LH receptors (4, 19) as a result of the 4-fold elevation of endogenous LH levels (11, 20, 21). It should be emphasized that the binding of [125I]hCG in vitro measures only unoccupied receptors, but since the degree of receptor occupancy is usually very low (22), this technique should provide a good estimate of the total receptor sites present. Again, it has been shown that steroid biosynthesis may be disrupted in the down-regulated testis when studies of steroidogenesis are performed 1-3 days after the injection of hCG (22, 23) or after experimental elevation of endogenous LH levels (11). It is also relevant that treatment of rats with high doses of hCG results in the accumulation of

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testicular fluid (10) similar to that observed in cryptorchid rat testes in the present investigation. To date, no data are available concerning the in vitro steroid biosynthetic potential of Leydig cells from rats subjected to a chronic (>1 week) elevation of LH levels, though Zipf et at. (24) showed that the in vivo response of the Leydig cells was increased after LH stimulation for 10 days. In considering the steroidogenic secretory potential of such cells, two factors must be assessed, since, on one hand, acutely down-regulated cells have a diminished biosynthetic potential yet in chronically stimulated Leydig cells the trophic action of the elevated LH levels results in cellular hypertrophy, as seen in the cryptorchid testis (3). The summation of these factors may result in the effect demonstrated in this study: namely, testes with only 40% of the hCG-binding capacity of controls which at low doses of hCG stimulation produce steroids in amounts equal to the controls, but at higher doses, the hypertrophic cellular apparatus produces significantly larger quantities of all of the steroids measured. The higher doses of hCG appear to be able to overcome any receptor deficiency, enabling the true steroidogenic potential of the cells to emerge. The relationship of the loss of receptors to the evolvement of Leydig cell hyperresponsivity remains unclear, since data are now available to indicate that the loss of receptors does not directly affect testicular responsiveness (19, 24) or the sensitivity of the testis to LH/hCG (11). The recent studies by Aoki and Fawcett (25) have demonstrated that local feedback between the compartments of the testis may be the reason for the hypertrophy of Leydig cells surrounding tubules with spermatogenic disruption, and a similar mechanism may be responsible in the cryptorchid testis. Detailed studies of parameters of Leydig cell function are required at weekly intervals after the induction of cryptorchidism to obtain a clear understanding of the temporal relationship between the elevation of serum LH levels and the changes in steroidogenesis and hCG binding. Such studies are of importance not only to the cryptorchid state, since similar disturbances of Leydig cell function have been shown with other types of testicular damage induced by fetal irradiation, hydroxyurea treatment, or chronic vitamin A deficiency (26).

References 1. Moore, C. R., Properties of gonads as controllers of somatic and physical characteristics: testicular reactions in experimental cryptorchidism, Am JAnat 34: 269,. 1924. 2. Nelson, W. 0., Mammalian spermatogenesis: effects of experimental cryptorchidism in the rat and non-descent of the testis in man, Recent Prog Horm Res 6: 29, 1951. 3. Kerr, J. B., K. A. Rich, and D. M. de Kretser, Alterations of the fine structure and androgen secretion of the interstitial cells in the experimentally cryptorchid rat testis, Biol Reprod, in press.

Kndo • 1979 Vol 105 • No 1

4. Sharpe, R. M., hCG-induced decrease in rat testis receptors, Nature 264: 644, 1976. 5. Miyachi, Y., J. L. Vaitukaitis, E. Nieschlag, and M. B. Lipsett, Enzymatic radioiodination of gonadotropins, J Clin Endocrinol Metab 34: 23, 1972. 6. Fraser, H. M., and J. Sandow, Gonadotrophin release by a highly active analogue of luteinizing hormone releasing hormone in rats immunized against luteinizing hormone releasing hormone, J EndocrinollA: 291, 1977. 7. de Jong, F. H., and R. M. Sharpe, The onset and establishment of spermatogenesis in rats in relation to gonadotrophin and testosterone levels, J Endocrinol 75: 197, 1977. 8. Corker, C. S., and D. W. Davidson, Radioimmunoassay of testosterone in various biological fluids without chromatography, J Steroid Biochem 9: 373, 1978. 9. Furuyama, S., D. M. Mayes, and C. A. Nugent, A radioimmunoassay for testosterone, Steroids 16: 415, 1970. 10. Sharpe, R. M., Relationship between testosterone, fluid content and luteinizing hormone receptors in the rat testis, Biochem Biophys Res Commun 75: 711, 1977. 11. Sharpe, R. M., H. M. Fraser, and J. Sandow, Effect of treatment with an agonist of luteinizing hormone releasing hormone on early maturational changes in pituitary and testicular function in the rat, J Endocrinol, in press. 12. Baird, D. T., P. E. Burger, G. D. Hearon-Jones, and R. J. Scaramuzzi, The site of secretion of androstenedione in non-pregnant women, J Endocrinol 63: 201, 1974. 13. Van der Vusse, G. J., M. L. Kalkman, and H. J. Van der Molen, Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins, Biochim Biophys Ada 380: 473, 1975. 14. Van Look, P. F. A., W. M. Hunter, C. S. Corker, and D. T. Baird, Failure of positive feedback in normal men and subjects with testicular feminization, Clin Endocrinol (Oxf) 7: 353, 1977. 15. Sharpe, R. M., Gonadotrophin-induced accumulation of'interstitial fluid' in the rat testis, J Reprod Fertil, in press. 16. Damber, J. E., A Bergh, and P. O. Janson, Testicular blood flow and testosterone concentrations in the spermatic venous blood in rats with experimental cryptorchidism, Acta Endocrinol (Kbh) 88: 611, 1978. 17. Le Vier, R. R., and E. Spaziani, The influence of temperature on steroidogenesis in the rat testis, J Exp Zool 169: 113, 1968. 18. Inano, H., and B. Tamaoki, Effects of experimental bilateral cryptorchidism on testicular enzymes related to androgen formation, Endocrinology 83: 1074, 1968. 19. Hsueh, A. J. W., M. L. Dufau, and K. J. Catt, Regulation of luteinizing hormone receptors in testicular interstitial cells by gonadotropin, Biochem Biophys Res Commun 72: 1145, 1976. 20. Auclair, C, P. A. Kelly, D. H. Coy, A. V. Schally, and F. Labrie, Potent inhibitory activity of (D-LEU 6 , DES-GLY-NH210) LHRH ethylamide on LH/hCG and PRL testicular receptor levels in the rat, Endocrinology 101: 1890, 1978. 21. Huhtaniemi, I., and H. Martikainen, Rat testis LH/hCG receptors and testosterone production after treatment with GnRH, Mol Cell Endocrinol 11: 199, 1978. 22. Tsuruhara, T., M. L. Dufau, S. Cigorraga, and K. J. Catt, Hormonal regulation of testicular luteinizing hormone receptors. Effects of cyclic AMP and testosterone responses in isolated Leydig cells, J Biol Chem 252: 9002, 1977. 23. Sharpe, R. M., Gonadotrophin-induced reduction in the steroidogenic responsiveness of the immature rat testis, Biochem Biophys Res Commun 76: 957, 1977. 24. Zipf, W. B., A. H. Payne, and R. P. Kelch, Dissociation of lutropininduced loss of testicular lutropin receptors and lutropin-induced desensitization of testosterone synthesis, Biochim Biophys Acta 540: 330, 1978. 25. Aoki, A., and D. W. Fawcett, Is there a local feedback from the seminiferous tubules affecting activity of the Leydig cells? Biol Reprod 19: 144, 1978. 26. Rich, K. A., J. B. Kerr, and D. M. de Kretser, Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage, Mol Cell Endocrinol 13: 123, 1979.

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Alterations in steroidogenesis and human chorionic gonadotropin binding in the cryptorchid rat testis.

0013-7227/79/l()51-0135$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society Vol. 105, No. 4 Printed in U.S.A. Alterations in Steroidogen...
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