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Effect of testosterone propionate on ABP levels in rats hypophysectomised at different ages using individual sampling
Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
6 (1977) 203-209 Scientific Publishers Ltd.
EFFECT OF TESTOSTERONE PROPIONATE ON ABP LEVELS IN RATS HYPOPHYSECTOMISED AT DIFFERENT AGES USING INDIVIDUAL SAMPLING
J.S.H. ELKINGTON *, B.M. SANBORN **, M.W. MARTIN, A.K. CHOWDHURY and E. STEINBERGER Department of Reproductive Biology and Endocrinology, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77025, USA
Received 2 March 1976; accepted 25 June 1976
The effect of age at hypophysectomy on the response of the regressed rat testis to testosterone propionate (TP) and FSH with respect to androgen-binding protein (ABP) levels was studied in individual animals. All treatments were begun 30 days after surgery. Treatment of rats 35,45,5.5 and 75 days of age at surgery with TP (1 mg/260 g for 25 days) significantly increased the level of ABP in the testes of animals in all age groups except those hypophysectomized at 35 days of age. TP treatment did not significantly elevate epididymal levels of ABP above those found in untreated rats in any age group. In animals hypophysectomized at 100 days of age, acute treatment (3 days) with FSH (150 and 300 pg/day) significantly increased the ABP levels per testis and per epididymis. Similar treatment with 750 fig TP/day did not result in a statistically significant increase in testicular ABP. No synergism between the two hormones was noted under the conditions described. Significant restoration of testicular ABP levels per mg protein was achieved with 1 mg TP/day by 5 days of treatment. Treatment of hypophysectomized adult rats with FSH raised the epididymalltesticular ratio of ABP to about 40% of that found in intact rats while comparable treatment with TP (750 ng/ day for 3 days or 1 mg/day for 10 days) only slightly affected the ratio. It is postulated that FSH may facilitate ABP transport to the epididymis in addition to affecting its production by the testis. Keywords:
androgen-binding
protein; testis; FSH; testosterone
propionate.
It has long been recognized that the hormonal requirements for the initiation, maintenance and restoration of spermatogenesis in the rat may not be identical and * Present address: International Fertility Research Program, Research Triangle Park, NC 27709, USA. ** To whom reprint requests should be sent. Portions of this work were presented or appear as abstracts of the Annual Meeting of the Society for the Study of Reproduction, Ottawa, Canada, 1974 (Abst. 22) and the 57th Meeting of the Endocrine Society, New York, 1975 (Abst. 347). 203
204
J.S.H. Elkingtorl et al.
may include simultaneous or sequential requirements for FSH and testosterone (see Steinberger, 1971, for review). It is therefore conceivable that the hormonal control of the testicular and epididymal levels of androgen-binding protein (ABP) may also differ depending upon the condition of the testis at the time of study. The testicular level of ABP has been shown to be affected by FSH in both immature and mature rats (Hansson et al., 1973; Sanborn et al., 1974a, 1975; Vernon et al., 1974; Tindall et al., 1974). Testosterone propionate (TP) was shown to maintain or restore ABP levels in the testes and epididymides of rats hypophysectomized at 100 days of age (Sanborn et al., 1974b; Elkington et al., 1975). Using 60-day-old rats, Hansson et al. (1975) subsequently demonstrated maintenance but not restoration of epididymal ABP with TP. In 28-day-old animals Hansson et al. (1974b) reported no effect of TP alone but a marked enhancement of the FSH effect on epididymal ABP when the steroid was administered from the day after surgery. Weddington et al. (1975) subsequently reported that 0.5 mg/day TP would maintain epididymal ABP in these animals but would not restore ABP if treatment was initiated 10 days after surgery. This paper provides data on the responsiveness of chronically hypophysectomized rat testis to TP with respect to ABP levels as a function of age at hypophysectomy and compares the acute affects of FSH and TP on testicular and epididyma1 levels of ABP.
MATERIALS AND METHODS Male Sprague-Dawley rats were hypophysectomized by Hormone Assay Laboratories, Chicago, Illinois, at various ages and 30 days later were treated subcutaneously with TP or FSH (NIH-FSH-Pl) as indicated in the text and the legends to tables 1 and 2. At the end of the treatment period, supernatants (140,OOOg) were prepared from homogenates of paired testes or epididymides from individual animals and were extracted with charcoal as previously described (Elkington et al., 1975). ABP was estimated by steady-state polyacrylamide gel electrophoresis (Ritzen et al., 1974), using 7.5% gels labeled with 2 nM [3H]dihydrotestosterone (DHT; Elkington et al., 1975). Protein estimations were made on the charcoal-extracted supernatants (Layne, 1957). Results were analysed by the analysis of variance; Duncan’s multiple range test was used to make within-treatment group comparisons (Steel and Torrie, 1960).
RESULTS Effect of age at hypophysectomy on respodeness to TP Table 1 summarizes the testicular and epididymal levels of ABP measured in rats
3 2
35
3 2 2
3 6 5 .__-_..
5s
75
---
3 3 3
45
2
Pools (no.)
Age at surgery (days) .-
1 I 1
1 1 293
1 3 3
t. 4 3,4
Animals/ pool (no.)
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Treatment group
0.51 f 0.08 0.16 * 0.03 b 0.30 f. 0.03 bc ---~___---
0.47 + 0.04 NDb 0.26 f 0.01 bd
0.38 + 0.02 0.13 f 0.03 a 0.34 * 0.08 c
0.42 rt 0.05 NDb 0.17 a 0.05 a
pmol/mg P
Testicular ABP
14.9 f 3.0 2.1 i 0.03 b 5.8 t 0.7 bc
31.5 2 3.7 NDb 2.4 f 0.07 b
26.0 i 1.3 0.8 + 0.2 b 3.1 + 0.5 b
1.8 2 0.9 b
NDb
19.1 * 1.1
pmoljorgan
59.6 + 4.5 NDb 1.6 + 0.4 b 39.8 1: 10.0 0.3 * 0.06 b 5.2 f 1.5 b
3.7 r 0.6 0.10 f 0.02 b 0.49 r 0.13 b ~--
65.3 1 21.9 ND a 2.2 * 0.8 a 3.0 I: 0.5 NDb 0.39 4 0.07 b
3.0 t 0.3 NDb 0.43 c 0.07 b
1.5
33.2 f NDb NDb
2.0 It 0.2. NDb NDb
-l_-pmol/org~
__-
pmol/mg P
Epididymal ABP -~--.-
Table 1 Effect of testosterone propionate (TP; 1 mg/260 g body weight), administered beginning 30 days after hypophysectomy, to rats hypophysectomized at 35, 45, 55 and 7.5 days of age (hypox.). The figures represent mean ABP levels + S.E.M. Differences between treatment groups within an age group were tested by Duncan’s Multiple Range Test. The significance levels of differences between intact groups and others are denoted by ‘a’ (p < 0.051, and ‘b’ (P < 0.011. while differences between ABP Ievels in untreated and treated (TP hypophysectom~zed rats are denoted by ‘c’ (f’< 0.05) and ‘d’ (P.< O.Ol).‘ND = not determinable. --. ~.--.-._ -----~
4
2 : B 2.
3
-a.
: 2 5 2
2
8
(6) (8) (10) (9) (8) (11)
c 0.05 j: 0.05 I 0.05 t 0.05 t 0.03 $0.08 4 0.05
2.2 i 0.4 ND 1.2 + 0.4 0.9 + 0.2 0.02 + 0.01 1.0 + 0.4 0.9 * 0.1
8.1 0 4.1 2.1 0.08 2.8 3.0 .~__
17.2 2.3 6.3 7.7 3.7 7.5 7.3
* 4.5 f: 0.6 + 0.6 i 1.0 + 0.4 + 1.3 t 1.0
(5)
0.27 0.17 0.29 0.33 0.24 0.35 0.30
intact NT FSHl FSH2 TP FSHI + TP FSH2 + TP
.-._ . .._ ._
Epid./Testis Testis
._-._ ~..
Testis ~_ .---
__-..
-~-~
Epididymis
pmol/organ
pmol/mg protein
Treatment group
.._
49.6 i 8-4 ND 4.8 * 1.9 4.3 Yk0.7 0.12 i- 0.06 10.4 f 5.3 5.0 f 0.6
Epididymis _-.__ _-.
_._~
2.8 0 0.76 0.56 0.03 1.4 0.68
EpidJTestis --...-.-
.-.. -.
The effect of treatment of chronically hypophysectomized adult rats for 3 days with FSII (FSHr = 150 &day; FSII2 = 300 pg/day) and testosterone propionate (TP = 750 &day) alone and in combination on testicular and epididymal levels of ABP. Rats were 100 days of age at surgery and treatment was begun after an additional 30 days. Figures represent mean responses * SE. and the number of animals is indicated in parentheses. NT = no treatment; ND = not determinable. -_... _~_.. .___-.. _.._.
Table 2
2 ir: b 3 Z 8 Z a?
Hormonal effects on ABP in rat testis
207
35, 4.5, 55 and 75 days of age at hypophysectomy after treatment for 25 days with TP, beginning 30 days after surgery. In all age groups the ABP levels in both organs in untreated animals were not detectable or very low compared to intact animals of comparable age. The testicular response to TP was in part dependent on the manner of expressing the results. TP significantly elevated testicular ABP levels expressed as pmoles per mg protein above those in untreated rats at 45, 55 or 75 days of age but was ineffective in 35-day-old animals. However, TP significantly elevated ABP levels per organ only in the rats hypophysectomized at 75 days of age. Epididymal ABP levels were not significantly elevated by TP treatment in any age group. Acute effects of FSH and TP on ABP in chronically hypophysectomized adult rats Table 2 summarizes the acute effects of the administration of TP and FSH for 3 days on testicular and epididymal ABP, determined in individual chronically hypophysectomized adult animals, 100 days of age at surgery. The small but detectable androgen-binding activity found in supernatants from untreated hypophysectomized animals was demonstrated to be ABP by competition with 100 nM unlabeled DHT included in both incubates and gels. Because of this activity, the differences in testicular ABP levels between untreated and hormone-treated groups were not statistically significant when expressed as pmoles per mg protein, although the FSH effect was significant when expressed per organ. FSH also significantly elevated ABP levels per mg protein and per organ in the epididymis, but TP had no effect. FSH and TP did not show any synergism under the conditions of this experiment. Additional experiments with chronically hypophysectomized animals (100 days old at surgery) revealed restoration of testicular ABP per mg protein by 5 days of treatment with 1 mg TP/day (0.34 f 0.03 and 0.27 rt 0.05 pmoles/mg protein in TP-treated and intact animals, respectively) but no statistically significant elevation in epididymal levels with up to 10 days of treatment. Table 2 also shows that the ratio of epididymal to testicular ABP levels was increased above that of untreated animals by FSH to about 40% of that in the intact rat but was not greatly increased by TP after the 3-day treatment period. The epididymal/testicular ABP level was found to be 0.4-0.9 after 5- 10 days of treatment with 1 mg TP, the dose resulting in a statistically significant restoration of testicular levels.
DISCUSSION Evaluation of the roles of FSH and TP in regulating testicular and epididymal levels of ABP has been the subject of some controversy. The original reports using immature rats indicated that FSH would both maintain and restore testicular and epididymal ABP (Hansson et al., 1973). TP had no direct effect but enhanced the
208
J.S.H. tlkington
et al.
responsiveness of FSH (Hansson et al., 1974b). However, in the hypophysectomized adult rat both FSH and TP maintained and restored testicular and epididyma1 ABP (Sanborn et al., 1974b, 1975; Vernon et al., 1974; Elkington et al., 1975). The data in table 1 support the contention that chronic hypophysectomy and the resulting testicular regression may be reflected in a progressive loss of responsiveness to TP and that this loss is more marked the less mature the animal at the time of surgery. This concept may help to explain why TP was effective in restoring ABP levels in rats hypophysectomized at 100 days of age and treated after a subsequent 30 days for lo-60 days (Elkington et al., 1975) but was ineffective when administered to rats hypophysectomised at 60 days and treated after a subsequent 60 days (Hansson et al., 1975). The data in table 2 show an effect of FSH on ABP levels in the testes and epididymides of mature rats after 3 days of treatment but no effect of TP nor any synergism between the two hormones under these conditions. The concentrations of hormone chosen had been previously show to be submaximal (Elkington et al., 1975; Sanborn et al., 1975), and therefore should have been suitable to detect synergism. Treatment for 5 days with a higher does of TP (1 mg/day) did result in a statistically significant restoration of testicular but not epididymal levels of ABP expressed per mg protein. FSH raised the epididymal/testicular ratio of ABP per mg protein to almost 40% of the level in intact rats (table 2). A dose of TP which resulted in restoration of the testicular levels of ABP (1 mg, 5-10 days) only raised this ratio to 5-10% of the level in intact rats. These data are consistent with the hypothesis that, in addition to having a direct effect on ABP production, FSH may facilitate the movement of ABP from the testis to the epididymis and may do so in part via an increase in testicular fluid production. The effect of prolonged TP treatment on epididymal levels of ABP may depend in part on other factors relating to testicular function which may in turn be affected by the age of the animal at hypophysectomy and the duration of regression in the absence of pituitary hormones prior to administration of the hormone. Note ad&d in proof
Means (1976) has recently reported that TP will restore testicular ABP levels in rats hypophysectomized at 28 days of age when treatment is begun 3 days after sur-
gery. ACKNOWLEDGEMENTS The excellent technical assistance of Ms. Hsu Kuo and Ms. C. Williamson is greatly acknowledged. This work was supported by Contract NOl-HD3-2782, NICHHD (BMS), Program Project NIH-1POl-HD08338 (E.S.) and Grant 7200075A, Ford Foundation (E.S.). The FSH was a gift from the NIAMD Pituitary Distribution Program.
Hormottul effects on ABP dn rat testis
209
REFERENCES Elkington, J.S.H., Sanborn, B.M. and Steinberger, E. (1975) Mol. Cell. Endocrinol. 2, 157. Hansson, V., Reusch, E., Trygstad, O., Torgerson, O., Ritzen, E.M. and French, F.S. (1973) Nature New Biol. 246,56. Hansson, V., Trygstad, O., French, F.S., McLean, W.S., Smith, A.A., Tindail, D.J., ~~eddington, SC., Petrusz, P. Nayfeh, S.N. and Ritz&r, E.M. (1974a) Nature 250,389. Hansson, V., French, F.S., Weddington, S.C., Nayfeh, S.N. and Ritzen, E.M. (1974b) In: Hormone Binding and Target Cell Activation in the Testis, Eds.: M.L. Dufau and A.R. Means (Plenum Press, New York) p. 287. Hansson, V., Weddington, S.C., Naess, 0. Attramadal, A., French, F.S., Kotite, N., Nayfeh. S.N. Ritz&n, E.M., and Hagenas, L. (1975) in: Hormonal Regulation of Spermatogenesis, Eds.: F.S. French, V. Hansson, E.M. Ritzen and S.N. Nayfeh (Plenum Press, New York) p. 323. Layne, D. (1957) In: Methods in Enzymology, Vol. III, Eds.: S.P. Colowick and N.O. Kaplan (Academic Press, New York and London) p. 454. Means, A.R. (1976) In: Regulatory Mechanisms of Male Reproductive Physiology. Eds.: C.H. Spilman, T.J. Lob1 and K.T. Kirton. (Excerpta Medica, Amsterdam) p. 87. Ritzen, E.M., French, F.S., Weddington, S.C., Nayfeh, S.N. and Hansson, V. (1974) J. Biol. Chem. 249,6597. Sanborn, B.M., Elkington, J.S.H., and Steinberger, E. (1974a) In: Hormone Binding and Target Cell Activation in the Testis, Eds.: M.L. Dufau and A.R. Means (Plenum Press, New York) p. 291. Sanborn, B.M., Elkington, J.S.H., and Steinberger, E. (1974b) Endocrine Sot., 56th, Annu. Meet., Atlanta, Ga., Abstr. 288. Sanborn, B.M., Elkington, J.S.H., Chowdhury, M., Tcholakian, R.K. and Steinberger, E. (1975) Endocrinology 96,304. Steinberger, E. (1971) Physiol. Rev. 51,L. Steel, R.G.D. and Torrie, J.H. (1960) Principles and Procedures of Statistics, (McGraw-Hill Book Company, New York). Tindall, D.J., Schrader, W.T. and Means, A.R. (1974) In: Hormone Binding and Target Cell Activation in the Testis, Eds.: M.L. Dufau and A.R. Means (Plenum Press, New York) p. 167. Vernon, R.G., Kopec, B. and Fritz, LB. (1974) Mol. Cell. Endocrinol. 1,167. Weddington, SC., Hansson, V., Ritzin, EM., Hagenas, L., French, F.S. and Nayfeh, S.N. (1975) Nature 254,145.
0 Elsevier/North-Holland
6 (1977) 203-209 Scientific Publishers Ltd.
EFFECT OF TESTOSTERONE PROPIONATE ON ABP LEVELS IN RATS HYPOPHYSECTOMISED AT DIFFERENT AGES USING INDIVIDUAL SAMPLING
J.S.H. ELKINGTON *, B.M. SANBORN **, M.W. MARTIN, A.K. CHOWDHURY and E. STEINBERGER Department of Reproductive Biology and Endocrinology, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77025, USA
Received 2 March 1976; accepted 25 June 1976
The effect of age at hypophysectomy on the response of the regressed rat testis to testosterone propionate (TP) and FSH with respect to androgen-binding protein (ABP) levels was studied in individual animals. All treatments were begun 30 days after surgery. Treatment of rats 35,45,5.5 and 75 days of age at surgery with TP (1 mg/260 g for 25 days) significantly increased the level of ABP in the testes of animals in all age groups except those hypophysectomized at 35 days of age. TP treatment did not significantly elevate epididymal levels of ABP above those found in untreated rats in any age group. In animals hypophysectomized at 100 days of age, acute treatment (3 days) with FSH (150 and 300 pg/day) significantly increased the ABP levels per testis and per epididymis. Similar treatment with 750 fig TP/day did not result in a statistically significant increase in testicular ABP. No synergism between the two hormones was noted under the conditions described. Significant restoration of testicular ABP levels per mg protein was achieved with 1 mg TP/day by 5 days of treatment. Treatment of hypophysectomized adult rats with FSH raised the epididymalltesticular ratio of ABP to about 40% of that found in intact rats while comparable treatment with TP (750 ng/ day for 3 days or 1 mg/day for 10 days) only slightly affected the ratio. It is postulated that FSH may facilitate ABP transport to the epididymis in addition to affecting its production by the testis. Keywords:
androgen-binding
protein; testis; FSH; testosterone
propionate.
It has long been recognized that the hormonal requirements for the initiation, maintenance and restoration of spermatogenesis in the rat may not be identical and * Present address: International Fertility Research Program, Research Triangle Park, NC 27709, USA. ** To whom reprint requests should be sent. Portions of this work were presented or appear as abstracts of the Annual Meeting of the Society for the Study of Reproduction, Ottawa, Canada, 1974 (Abst. 22) and the 57th Meeting of the Endocrine Society, New York, 1975 (Abst. 347). 203
204
J.S.H. Elkingtorl et al.
may include simultaneous or sequential requirements for FSH and testosterone (see Steinberger, 1971, for review). It is therefore conceivable that the hormonal control of the testicular and epididymal levels of androgen-binding protein (ABP) may also differ depending upon the condition of the testis at the time of study. The testicular level of ABP has been shown to be affected by FSH in both immature and mature rats (Hansson et al., 1973; Sanborn et al., 1974a, 1975; Vernon et al., 1974; Tindall et al., 1974). Testosterone propionate (TP) was shown to maintain or restore ABP levels in the testes and epididymides of rats hypophysectomized at 100 days of age (Sanborn et al., 1974b; Elkington et al., 1975). Using 60-day-old rats, Hansson et al. (1975) subsequently demonstrated maintenance but not restoration of epididymal ABP with TP. In 28-day-old animals Hansson et al. (1974b) reported no effect of TP alone but a marked enhancement of the FSH effect on epididymal ABP when the steroid was administered from the day after surgery. Weddington et al. (1975) subsequently reported that 0.5 mg/day TP would maintain epididymal ABP in these animals but would not restore ABP if treatment was initiated 10 days after surgery. This paper provides data on the responsiveness of chronically hypophysectomized rat testis to TP with respect to ABP levels as a function of age at hypophysectomy and compares the acute affects of FSH and TP on testicular and epididyma1 levels of ABP.
MATERIALS AND METHODS Male Sprague-Dawley rats were hypophysectomized by Hormone Assay Laboratories, Chicago, Illinois, at various ages and 30 days later were treated subcutaneously with TP or FSH (NIH-FSH-Pl) as indicated in the text and the legends to tables 1 and 2. At the end of the treatment period, supernatants (140,OOOg) were prepared from homogenates of paired testes or epididymides from individual animals and were extracted with charcoal as previously described (Elkington et al., 1975). ABP was estimated by steady-state polyacrylamide gel electrophoresis (Ritzen et al., 1974), using 7.5% gels labeled with 2 nM [3H]dihydrotestosterone (DHT; Elkington et al., 1975). Protein estimations were made on the charcoal-extracted supernatants (Layne, 1957). Results were analysed by the analysis of variance; Duncan’s multiple range test was used to make within-treatment group comparisons (Steel and Torrie, 1960).
RESULTS Effect of age at hypophysectomy on respodeness to TP Table 1 summarizes the testicular and epididymal levels of ABP measured in rats
3 2
35
3 2 2
3 6 5 .__-_..
5s
75
---
3 3 3
45
2
Pools (no.)
Age at surgery (days) .-
1 I 1
1 1 293
1 3 3
t. 4 3,4
Animals/ pool (no.)
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Intact Hypox. TP-treated
Treatment group
0.51 f 0.08 0.16 * 0.03 b 0.30 f. 0.03 bc ---~___---
0.47 + 0.04 NDb 0.26 f 0.01 bd
0.38 + 0.02 0.13 f 0.03 a 0.34 * 0.08 c
0.42 rt 0.05 NDb 0.17 a 0.05 a
pmol/mg P
Testicular ABP
14.9 f 3.0 2.1 i 0.03 b 5.8 t 0.7 bc
31.5 2 3.7 NDb 2.4 f 0.07 b
26.0 i 1.3 0.8 + 0.2 b 3.1 + 0.5 b
1.8 2 0.9 b
NDb
19.1 * 1.1
pmoljorgan
59.6 + 4.5 NDb 1.6 + 0.4 b 39.8 1: 10.0 0.3 * 0.06 b 5.2 f 1.5 b
3.7 r 0.6 0.10 f 0.02 b 0.49 r 0.13 b ~--
65.3 1 21.9 ND a 2.2 * 0.8 a 3.0 I: 0.5 NDb 0.39 4 0.07 b
3.0 t 0.3 NDb 0.43 c 0.07 b
1.5
33.2 f NDb NDb
2.0 It 0.2. NDb NDb
-l_-pmol/org~
__-
pmol/mg P
Epididymal ABP -~--.-
Table 1 Effect of testosterone propionate (TP; 1 mg/260 g body weight), administered beginning 30 days after hypophysectomy, to rats hypophysectomized at 35, 45, 55 and 7.5 days of age (hypox.). The figures represent mean ABP levels + S.E.M. Differences between treatment groups within an age group were tested by Duncan’s Multiple Range Test. The significance levels of differences between intact groups and others are denoted by ‘a’ (p < 0.051, and ‘b’ (P < 0.011. while differences between ABP Ievels in untreated and treated (TP hypophysectom~zed rats are denoted by ‘c’ (f’< 0.05) and ‘d’ (P.< O.Ol).‘ND = not determinable. --. ~.--.-._ -----~
4
2 : B 2.
3
-a.
: 2 5 2
2
8
(6) (8) (10) (9) (8) (11)
c 0.05 j: 0.05 I 0.05 t 0.05 t 0.03 $0.08 4 0.05
2.2 i 0.4 ND 1.2 + 0.4 0.9 + 0.2 0.02 + 0.01 1.0 + 0.4 0.9 * 0.1
8.1 0 4.1 2.1 0.08 2.8 3.0 .~__
17.2 2.3 6.3 7.7 3.7 7.5 7.3
* 4.5 f: 0.6 + 0.6 i 1.0 + 0.4 + 1.3 t 1.0
(5)
0.27 0.17 0.29 0.33 0.24 0.35 0.30
intact NT FSHl FSH2 TP FSHI + TP FSH2 + TP
.-._ . .._ ._
Epid./Testis Testis
._-._ ~..
Testis ~_ .---
__-..
-~-~
Epididymis
pmol/organ
pmol/mg protein
Treatment group
.._
49.6 i 8-4 ND 4.8 * 1.9 4.3 Yk0.7 0.12 i- 0.06 10.4 f 5.3 5.0 f 0.6
Epididymis _-.__ _-.
_._~
2.8 0 0.76 0.56 0.03 1.4 0.68
EpidJTestis --...-.-
.-.. -.
The effect of treatment of chronically hypophysectomized adult rats for 3 days with FSII (FSHr = 150 &day; FSII2 = 300 pg/day) and testosterone propionate (TP = 750 &day) alone and in combination on testicular and epididymal levels of ABP. Rats were 100 days of age at surgery and treatment was begun after an additional 30 days. Figures represent mean responses * SE. and the number of animals is indicated in parentheses. NT = no treatment; ND = not determinable. -_... _~_.. .___-.. _.._.
Table 2
2 ir: b 3 Z 8 Z a?
Hormonal effects on ABP in rat testis
207
35, 4.5, 55 and 75 days of age at hypophysectomy after treatment for 25 days with TP, beginning 30 days after surgery. In all age groups the ABP levels in both organs in untreated animals were not detectable or very low compared to intact animals of comparable age. The testicular response to TP was in part dependent on the manner of expressing the results. TP significantly elevated testicular ABP levels expressed as pmoles per mg protein above those in untreated rats at 45, 55 or 75 days of age but was ineffective in 35-day-old animals. However, TP significantly elevated ABP levels per organ only in the rats hypophysectomized at 75 days of age. Epididymal ABP levels were not significantly elevated by TP treatment in any age group. Acute effects of FSH and TP on ABP in chronically hypophysectomized adult rats Table 2 summarizes the acute effects of the administration of TP and FSH for 3 days on testicular and epididymal ABP, determined in individual chronically hypophysectomized adult animals, 100 days of age at surgery. The small but detectable androgen-binding activity found in supernatants from untreated hypophysectomized animals was demonstrated to be ABP by competition with 100 nM unlabeled DHT included in both incubates and gels. Because of this activity, the differences in testicular ABP levels between untreated and hormone-treated groups were not statistically significant when expressed as pmoles per mg protein, although the FSH effect was significant when expressed per organ. FSH also significantly elevated ABP levels per mg protein and per organ in the epididymis, but TP had no effect. FSH and TP did not show any synergism under the conditions of this experiment. Additional experiments with chronically hypophysectomized animals (100 days old at surgery) revealed restoration of testicular ABP per mg protein by 5 days of treatment with 1 mg TP/day (0.34 f 0.03 and 0.27 rt 0.05 pmoles/mg protein in TP-treated and intact animals, respectively) but no statistically significant elevation in epididymal levels with up to 10 days of treatment. Table 2 also shows that the ratio of epididymal to testicular ABP levels was increased above that of untreated animals by FSH to about 40% of that in the intact rat but was not greatly increased by TP after the 3-day treatment period. The epididymal/testicular ABP level was found to be 0.4-0.9 after 5- 10 days of treatment with 1 mg TP, the dose resulting in a statistically significant restoration of testicular levels.
DISCUSSION Evaluation of the roles of FSH and TP in regulating testicular and epididymal levels of ABP has been the subject of some controversy. The original reports using immature rats indicated that FSH would both maintain and restore testicular and epididymal ABP (Hansson et al., 1973). TP had no direct effect but enhanced the
208
J.S.H. tlkington
et al.
responsiveness of FSH (Hansson et al., 1974b). However, in the hypophysectomized adult rat both FSH and TP maintained and restored testicular and epididyma1 ABP (Sanborn et al., 1974b, 1975; Vernon et al., 1974; Elkington et al., 1975). The data in table 1 support the contention that chronic hypophysectomy and the resulting testicular regression may be reflected in a progressive loss of responsiveness to TP and that this loss is more marked the less mature the animal at the time of surgery. This concept may help to explain why TP was effective in restoring ABP levels in rats hypophysectomized at 100 days of age and treated after a subsequent 30 days for lo-60 days (Elkington et al., 1975) but was ineffective when administered to rats hypophysectomised at 60 days and treated after a subsequent 60 days (Hansson et al., 1975). The data in table 2 show an effect of FSH on ABP levels in the testes and epididymides of mature rats after 3 days of treatment but no effect of TP nor any synergism between the two hormones under these conditions. The concentrations of hormone chosen had been previously show to be submaximal (Elkington et al., 1975; Sanborn et al., 1975), and therefore should have been suitable to detect synergism. Treatment for 5 days with a higher does of TP (1 mg/day) did result in a statistically significant restoration of testicular but not epididymal levels of ABP expressed per mg protein. FSH raised the epididymal/testicular ratio of ABP per mg protein to almost 40% of the level in intact rats (table 2). A dose of TP which resulted in restoration of the testicular levels of ABP (1 mg, 5-10 days) only raised this ratio to 5-10% of the level in intact rats. These data are consistent with the hypothesis that, in addition to having a direct effect on ABP production, FSH may facilitate the movement of ABP from the testis to the epididymis and may do so in part via an increase in testicular fluid production. The effect of prolonged TP treatment on epididymal levels of ABP may depend in part on other factors relating to testicular function which may in turn be affected by the age of the animal at hypophysectomy and the duration of regression in the absence of pituitary hormones prior to administration of the hormone. Note ad&d in proof
Means (1976) has recently reported that TP will restore testicular ABP levels in rats hypophysectomized at 28 days of age when treatment is begun 3 days after sur-
gery. ACKNOWLEDGEMENTS The excellent technical assistance of Ms. Hsu Kuo and Ms. C. Williamson is greatly acknowledged. This work was supported by Contract NOl-HD3-2782, NICHHD (BMS), Program Project NIH-1POl-HD08338 (E.S.) and Grant 7200075A, Ford Foundation (E.S.). The FSH was a gift from the NIAMD Pituitary Distribution Program.
Hormottul effects on ABP dn rat testis
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