BIOLOGY OF REPRODUCTION 14.665-669(1976) Effect of Testosterone Implants on erum Gonadotropin Concentrations in the Male Rat W. H. MOGER Department of Physiology and Biophysics, Daihousie University, Halifax, Nova cotia, Canada B3H 4H7 ABTRACT ubcutaneous implantation of testosterone in ilasticcapsules in castrated male rats resultsin sustained elevations of serum testosterone. In rats castrated and implanted with capsules 2cm long at 30 days of age, serum testosterone concentrations were elevated within 24 h and then gradually declined during the following nine days. This decline appears related to the animals growth. In rats castrated and implanted at 30 days of age with capsules ranging in length from 0.5 to 2cm, serum testosterone concentrations at autopsy 5 days after implantation were comparable to those of intact control animals of the same age. The capsules suppressed the castration induced risein serum gonadotropin concentrations. imilar results were obtained with rats castrated and implanted with either one or two capsules 4cm long at 80 days of age. Although these results suggest that serum testosterone concentrations in either sexually immature or mature male rats can regulate serum gonadotropin concentrations, the question arises whether the sustained release of testosterone from ilastic capsules mimics the pattern of endogenous testosterone secretion. INTRODUCTION Extensive evidence suggests that testicular secretions regulate the secretion of pituitary gonadotropins (see Recommended Reviews). However two recent attempts to document the relationship between the concentrations of circulating testosterone and luteinizing hormone in male rats have indicated that serum testosterone concentrations must be supraphysiologic to suppress the castration induced rise in serum LH concentrations (Moger, 1975a; Verjans et al., 1975). In both of these experiments testosterone was administered by daily or twice daily subcutaneous injections in oil. This method of testosterone administration is believed to result in the rapid absorption of testosterone from the site of injection (James et al., 1969). For this reason the relationship between serum testosterone and gonadotropin concentrations was reassessed using an alternative method of administration. MATERIAL AND METHOD Male prague-dawley rats at 24 or 27 days of age were purchased from Bio-Breeding Laboratories, Ottawa and housed under conditions of controlled Accepted February 27, 1976. Received January 15, 1976. lighting (12 h light, 12 h dark) and temperature (18-21#{176}C). Castrations were performed under halothane anesthesia at either 30 or 85 days of age via the scrotal route. At castration, implants were placed subcutaneously via a small skin incision. Implants were prepared by cutting ilastic Medical Grade tubing, Dow Corning (0.062 in ID X 0.125 in OD) to the desired length and sealing one end with ilastic Medical Adhesive, Type A. After 24 h the tubing was packed with testosterone (igma Chemical Co.) and the open end sealed with adhesive which was allowed to set at least 24 h before implantation. The implants were placed in absolute ethanol for 15 mm before implantation to remove testosterone that might have adhered to the outside of the implant. Castrate control animals were implanted with capsules 2cm in length containing cholesterol. Intact control animals were not implanted. Animals were killed at different times (see Results) after castration by decapitation and trunk blood was collected. erum was collected by allowing coagulation at room temperature for 2 h followed by standing overnight at 4#{176}C. Following centrifugation, serum was stored at -18#{176} C until assayed for testosterone, LII and FH. erum testosterone was assayed as previously described (Moger, 1975a; Moger and Armstrong, 1974). This method measures both testosterone and dihydrotestosterone. erum LH and FH were assayed with materials generously supplied by the Rat Pituitary Hormone Distribution Program of the National Institute of Arthritis, Metabolism and Digestive Diseases as previously described (Moger, 1975b). The results are expressed in terms of the RP-1 standards supplied by this agency. tatistical analysis was by one way analysis of variance and Duncan s new multiple range test (teel and Tone, 1960). ignificance is defined as [ <0.05. 665
666 MOGER TABLE 1. Effect of 2 cm testosterone implants on body and ventral prostate weights in rats castrated and implanted at 30 days of age. Ventral prost ate weight* Days post Body (mg/100 g implantation N weight (g) (mg) body weight) 0 4 82 ± 3 51.2 ± 3.6a** 0.62 ± o.o3 1 5 93 ± 3 71.6 ± 6.6a 0.76 ± 007ab 3 5 113 ± 3 113.9 60b ± 1.00 ± 0.05 5 5 125 ± 2 127.8 ± 76b 1.02 ± o.o7c 10 5 107 ± 4 158.8 ± 12.1C 0.93 ± 006cb #{149}Mean ±.E.M. Within a column, different letters signify significant differences between means. ([ <0.05). REULT In a preliminary experiment, rats were castrated at 30 days of age and implanted with capsules 2cm long. Groups of animals were killed 0, 1, 3, 5 and 10 days following implantation. Body and ventral prostate weights are reported in Table 1. Growth rate was similar to that of intact rats of the same age and strain (Moger, unpublished observation). Ventral prostate weight increased throughout the implantation period. When expressed as a percentage of body weight, ventral prostate weight did not change significantly after 3 days postimplantation. erum testosterone, LH and FH concentrations are reported for these animals in Table 2. erum testosterone concentrations were elevated by 24 h after implantation. The concentration at this time was significantly greater than 3, 5 or 10 days postimplantation and the trend was for a continuous decline during the course of the experiment. Expressing the serum testosterone concentrations on a body weight basis (Table 2) eliminates this change in concentrations. erum LH concentration did not change significantly during the postimplantation period and were not significantly different from intact 30 day old animals. erum FH concentrations during the postimplantation period were significantly lower than at implantation. In a subsequent experiment, animals were castrated at 30 days of age and implanted with capsules 0.5, 1.0, 1.5 or 2.0cm in length. The rats were autopsied 5 days following implantation. Results are shown in Fig. 1. Castrated cholesterol implanted control animals had barely detectable testosterone concentrations. Ventral prostate weight was significantly reduced, and serum LH and FH concentrations were significantly increased compared to those for intact animals. Testosterone implants increased serum testosterone levels and ventral prostate weights in a dose dependent manner. Although the groups receiving 0.5 or 1.0 cm implants have serum testosterone concentrations signifi- TABLE 2. Effect of 2 cm testosterone implants on serum testosterone, LH and FH concentrations in rats castrated and implanted at 30 days of age. erum Days post (ng/mlx 100 g) erum LH erum FH implantation N (ng/ml) body weight) (ng/ml) (ng/ml) 0 4 0.43 ga**... 23.9± 9.1 700±96a 1 5 3.96 ± 043b 3.4k ± 0.41 26.4 ± 4.8a 264 ± 3 5 2.47 ± 0.36c 2.77 ± 0.34a 32.0 ± 5.1 158 ± 35bc 5 5 1.92 ± o.18 2.39 ± o.18 27.6 ± lo.la 253 ± 54b 10 5 1.52 ± 0.22c 2.71 ± o.36 32.0 ± 49a 107 ± 26c Mean ±.E.M. * *Within a column different letters signify significant differences between means, [ <0.05.
TETOTERONE IMPLANT, ERUM LH, FH LEVEL 667 (a) Ill lit- = it- EEl lb.) & 551 8 & 8 8 it - B (b( Its) (b( a IEI let 1.2 II 101 (Oct I.d1 0-0 5 0.2 8 cat 05 5.0 1.5 GO INTACT CAT 0.5 5,0 I.E 2.0 INTACT a 8 (6 3 C, LENGTH OF IIOTTROM CAPUCI c,, LENGTH OF TETOTEROHCAPUIO I c* P c0 FIG. 1. erum testosterone, LH and FH concentrations, and ventral prostate weights in rats castrated and implanted at 30 days of age with testosterone containing capsules. The animals were killed five days after implantation. Vertical bars represent the standard error of mean. There are six animals per group. Within each graph, columns with different letters are significantly(p<0.05) different from each other. cantly lower than those implanted with either 1.5 or 2.0cm capsules none of the testosterone implanted groups had serum testosterone concentrations significantly different from those of control animals. None of the testosterone implanted groups had serum LH or FH concentrations significantly different from those of the intact control group. Although there were no significant differences in serum LH among experimental groups, those implanted with 1.5 or 2.0cm capsules had serum FH concentrations significantly lower than the groups implanted with 0.5 or 1.0cm capsules. An experiment similar to the above was performed with animals castrated at 85 days of age and implanted with either one or two, 4cm capsules. The animals were autopsied at 90 days of age and the results are shown in Fig. 2. erum testosterone concentrations, ventral prostate weights and serum LH and FH concentrations did not differ significantly among the testosterone implanted groups and intact control group. However, these parameters were all significantly different for the castrate control group. DICUI ON everal reports indicate that testosterone (werdloff et al., 1973; Eldridge and Mahesh, OF CAT 4 8 INTACT LENGTH OF IETOItROFI CAPULE (cml CAT 4 8 INTACT LENGTH OF IETO1TROt CAPULE I cm FIG. 2. erum testosterone, LH and FH concentrations, and ventral prostate weights in rats castrated and implanted at 85 days of age with testosterone containing capsules. The animals were killed 5 days after implantation. Vertical bars represent the standard error of the mean. There are 5 animals per group. Within each graph, columns with different letters are significantly ([ <0.05) different from each other. 1974; Dafy-Barbe and Frachimont, 1972) or testosterone propionate (Block et al., 1974; Negro-Vilar et al., 1973a; Kalra et al., 1973) administered by subcutaneous injection in oil can suppress the castration induced rise in LH or LH and FH. The general conclusion from these reports is that physiologic doses of testosterone or testosterone propionate are probably adequate to inhibit the post castration rise in serum LH concentrations particularly if treatment is begun immediately after castration. On the other hand, larger doses are required to suppress FH concentrations. This conclusion has been more directly investigated in two reports in which both serum testosterone and gonadotropin concentrations were determined. Moger (1975a) reported that supraphysiological serum concentrations of testosterone were required to inhibit the post castration rise in serum LH concentrations in immature rats, and Verjans et al. (1975) reported similar findings for both LH and FH in mature rats. Our results fail to support the earlier findings in that maintenance of serum testosterone concentrations within the normal range of either sexually immature or mature animals
668 MOGER prevented the castration induced rise of both serum LH and FH. The major difference between our report and previous ones involves the method of testosterone administration. ilastic capsules have been shown to release testosterone (tratton et al., 1973) and other steroids at uniform rates (Dziuk and Cook, 1966) and although this is supported by our results in Tables 1 and 2, the serum testosterone concentrations are influenced by animal growth during the implantation period. This pattern of release is believed to be quite different from the fluctuating concentration that results when testosterone is administered in oil as testosterone is rapidly absorbed from the injection site (James et al., 1969). It appears, therefore, that a more uniform concentration of testosterone is more effective than are fluctuating levels in suppressing the castration induced rise in serum gonadotropin concentrations. Hutchison and Goldman (1975) have recently confirmed this by comparing the ability of testosterone administered as a single injection in oil with the same quantity administered by constant infusion to inhibit the castration induced rise in serum LH concentrations. Their results indicate that administration by constant infusion is considerably more effective. It is not clear, however, whether the pattern of release obtained with implants or that obtained by injection in oil most closely mimics the endogenous pattern of testosterone release. erum testosterone concentrations have been reported to show periods of elevation during a 24 h period in a number of species including man (Rowe et al., 1975a), bull (Katongole et al., 1971) rabbit (Rowe et al., 1975b) and rat (Bartke et al., 1973). This is reflected in the present report where the average coefficient of variation of serum testosterone concentrations was 57.3 percent for the intact animals. This value is comparable to that reported by Bartke et al. (1973) for adult rats. The coefficient of variation for the testosterone implanted groups averaged only 22.5 percent. Many of the reports cited above on the effect of testosterone injections on serum gonadotropin concentrations were investigations of the possibility that the hypothalamic-pituitary axis becomes less sensitive to feedback inhibition during sexual development. The present study was not designed to investigate this point, and no inferences regarding changes in the sensitivity of LH suppression can be made as no dose-response relationship was observed between serum testosterone and LH concentrations. In the immature animals a dose-response relationship was observed between serum testosterone and FH concentrations (Fig. 1). This relationship is observed only at serum testosterone concentrations below approximately one ng/ml. At concentrations of testosterone above this level mean serum FH concentrations ranged from approximately 100 to 260 ng/ml (Table 2 and Fig. 1). Although these levels are generally below FH concentrations in intact immature animals, they are not greatly different from serum FH concentrations in the 90 day old adult rats (252 ± 17 ng/ml, Fig. 2). It is therefore possible that the decrease in serum FH concentrations that occurs during sexual development (werdloff et al., 1972; Negro- Vilar et al., 1973b; Gupta et al., 1975) may be accounted for largely by the increases in serum testosterone concentrations that occur during puberty. Gupta et al. (1975) have suggested that the changes in serum FH concentrations during puberty correlate more with changes in dihydrotestosterone concentrations than with testosterone concentrations. As our assay measures both testosterone and dihydrotestosterone we cannot dispute this suggestion. ACKNOWLEDGMENT The technical assistance of Ms. V. Masland and the editorial assistance of Miss E. hapter are gratefully acknowledged. Acknowledgment is also extended to Dr. T. Kennedy for advice on the preparation of the testosterone capsules. This work was supported by Grant MA-5401 from the Medical Research Council of Canada. REFERENCE Bartke, A., teele, R. E., Musto, N. and Caldwell, B. V. (1973). Fluctuations in plasma testosterone levels in adult male rats and mice. Endocrinology 92, 1223-1228. Bloch, G. J., Masken, J., Kragt, C. L. and Ganong, W. F. (1974). Effect of testosterone on plasma LH in male rats of various ages. Endocrinology 94, 947-95 1. Dufy-Barbe, L. and Franchimont, P. (1972). Influence des differents steroides gonadiques sur Ic taux de la FH et de Ia LB chez Ic rat castr#{233}cr. oc. Biol. 166, 960-964. Dziuk, P. J. and Cook, B. (1966). Passage of steroids through silicone rubber. Endocrinology 78, 208-211. Eldridge, J. C. and Mahesh, V. B. (1974). Pituitarygonadal axis before puberty: evaluation of testicular steroids in the male rat. Biol. Reprod. 11, 385-397. Gupta, D., Roger, K., Attanasio, A., Klemm, W. and Eichnez, M. (1975). ex steroid hormones during multiphase pubertal developments. J. teroid Bio-
TETOTERONE IMPLANT, ERUM LB. FH LEVEL 669 chem. 6, 8 59-868. Hutchison, J.. and Goldman, B. D. (1975). The relationship between the rate of testosterone infusion and gonadotropin secretion. Endocrinology 97, 725-730. James, K. C., Nicholls, P. J. and Roberts, (Mrs.) M. (1969). Biological half-lives of (4-4C) testosterone and some of its esters after injection into the rat. J. Pharm. Pharmacol. 21, 24-27. Kalra, P. 5., Fawcett, C. P., Krulich, L. and McCann,. M. (1973). The effects of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrinology 92, 1256-1 268. Katongole, C. B., Naftolin, F. and hort, R. V. (1971). Relationship between blood levels of luteinizing hormone and testosterone in bulls, and the effects of sexual stimulation. J. Endocrinol. 50, 457-466. Moger, W. H. (1975a). erum testosterone and luteinizing hormone concentrations in castrated immature rats treated with testosterone. J. Endocrinol. 67, 135-136. Moger, W. H. (1975b). Effects of testosterone metabolites on serum gonadotropin concentrations in immature male rats. Can. J. Physiol. Pharmacol. 53, 839-844. Moger, W. H. and Armstrong, D. T. (1974). Changes in serum testosterone levels following acute LH treatment in immature and mature rats. Biol. Reprod. 11, 1-6. Negro-Vilar, A., Ojeda,. R. and McCann,. M. (1973a). Evidence for changes in sensitivity to testosterone negative feedback on gonadotropin release during sexual development in the male rat. Endocrinology 93, 729-735. Negro- Vilar, A., Krulich, L. and McCann,. M. (1973b). Changes in serum prolactin and gonadotropins during sexual development of the male rat. Endocrinology 93, 660-664. Rowe, P. H., Racey, P. A., Lincoln, G. A., Ellwood, M., Lehane, J. and henton, J. C. (1975). The temporal relationship between the secretion of luteinizing hormone and testosterone in man. J. Endocrinol. 64, 17-26. Rowe, P. H., Hopkinson, C. R. N., henton, J. C. and Glover, T. D. (1975). The secretion of LH and testosterone in the rabbit. teroids 25, 313-321. teel, R. G. D. and Tone, J. J. (1960). Principles and Procedures of tatistics, McGraw-Hill, New York. tratton, L. G., Ewing, L. L. and Desjardins, C. (1973). Efficacy of testosterone-filled polydimethylsiloxane implants in maintaining plasma testosterone in rabbits. J. Reprod. Fertil. 35, 235-244. werdloff, R.., Jacobs, H. J. and Odell, W. D. (1972). Hypothalamic-pituitary-gonadal interrelationships in the rat during sexual maturation. in Gonadotropins, B. B. axena, C. G. Beling and H. M. Grandy, Eds., pp. 546-561, Wiley, New York. werdloff, R.., Grover, P. K., Jacobs, H.. and Bain, J. (1973). earch for a substance which selectively inhibits FH-Effects of steroids and prostaglandins on serum FH and LH levels. teroids 21, 703-722. Verjans, H. L., van der Molen, H. J. and Eik-Nes, K. B. (1975). Relation between circulating levels of testosterone, LH and FH in intact and castrated, adult, male rats after testosterone administration. Acta Endocrinol. 79, 380-386. RECOMMENDED REVIEW McCann,. M. and Ramirez, V. D. (1964). The neuroendocrine regulation of hypophyseal luteinizing hormone secretion. Rec. Progr. in Hormone Res. 20, 131-181. Odell, W. D., werdloff, R.., Abraham, C. E., Jacobs, H.. and Walsh, P. C. (1971). Pituitary- Gonadal Interrelations, in Control of Gonadal teroid ecretion, D. T. Baird and J. A. trong, Eds., pp. 3 1-62, Edinburgh University Press, Edinburgh.