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Effect of prolactin, testosterone and estrogen on prolactin binding in the rat testis, prostate, seminal vesicle and liver
Molecular and Cellular Endocrinology, 30 ( 1983) 119- 187 Elsevier Scientific Publishers Ireland, Ltd.
179
EFFECT OF PROLACTIN, TESTOSTERONE AND ESTROGEN PROLACTIN BINDING IN THE RAT TESTIS, PROSTATE, SEMINAL VESICLE AND LIVER
ON
T. AMIT, R.J. BARKEY * AND M.B.H. YOUDIM Rappaport Family Institute for Research in the Medical Sciences, Department of Phannacology, Faculty of Medicine, Technion- Israel Institute of Technology, P.O. Box 9649, Haifa 31096 (Israel) Received 19 July 198:!; revisions January 1983
received
27 October
1982 and 3 January
1983; accepted
6
We have studied the hormonal control of prolactin (PRL) binding in the male rat sex glands and liver, subsequent to the recent demonstration and characterization of specific PRL binding sites in rat testis, prostate and seminal vesicle. Ovine PRL (200 jtg/rat/day, 7 days) caused a time-dependent reduction in testicular binding of ‘2SI-labelled PRL (measured 2 days after last injection) to 58% of control. Testosterone alone (1 mg/rat/day, 7 days) or PRL caused similar reductions in binding, while their coadministration further lowered PRL binding to 10% of control. The synergism of PRL and testosterone suggests that either these doses are submaximal, or that they are acting on different systems. Estradiol was administered as a single dose of 2 mg/rat and the PRL binding determined on day 10 and day 19 was reduced to 37% of control, as after testosterone. Addition of PRL whether from day 1 to day 7 or from day 11 to day 17 of estradiol injection had no effect, suggesting that the EB site of action is closer to the PRL receptor than that for PRL or testosterone. Bstradiol resulted in a 72% reduction of PRL binding in the prostate, after 10 days, which subsequent PRL completely restored. PRL also partially restored the estradiolinduced time-dependent weight reduction of the prostate, but PRL coadministered from day 1 of estradiol did not inhibit the estradiol effects, suggesting a competitive mechanism for the two. While testosterone more than doubled PRL binding in the seminal vesicle, estradiol reduced it by 32% and organ weight by 21%. PRL given after estradiol restored the weight loss, but not the binding, suggesting that two different mechanisms of action are involved. In the liver, coadministration of testosterone with PRL could not inhibit the induction by PRL of its own hepatic sites, in keeping with a more direct site of action for PRL than for testosterone. These results demonstrate the profound effects of PRL, and of the sex steroids testosterone and estrogen, on PRL binding in the male sex glands and liver. The physiological implication of these findings on the role of PRL in male sexual function is currently being investigated. Keywords:
prolactin receptors; icle; liver.
* To whom all correspondence
0303-7207/83/0000-0000/$03.00
hormonal
regulation;
testis;
prostate;
seminal
should be addressed.
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
ves-
T. Amit et al.
180
We have previously reported the presence of specific binding sites for prolactin (PRL) in the rat testis, prostate, seminal vesicle and liver (Barkey et al., 1977). The involvement of PRL in the regulation of male reproduction and fertility is becoming increasingly clear (for review see Horrobin, 1979). The recent demonstration that administration of PRL dissolved in 10% polyvinylpyrrolidone (PVP) in saline greatly enhances the effect of PRL on its own receptors in the male rat liver (Barkey et al., 1981) probably through the slow release achieved with PVP (Morishige and Rothchild, 1974), prompted the study of the effects of PRL and the sex steroids testosterone and estradiol, alone and in various combinations, on the PRL binding site and on organ weights of the sex glands, in order to gain a better understanding of the role and mechanism of action of PRL in these glands.
MATERIALS Chemicals
AND
METHODS
and animals
Ovine PRL (oPRL; NIH-P-S12, 35 IU/mg) was generously supplied by the Hormone Distribution Program of NIAMDD, NIH. Lactoperoxidase (from milk), bovine serum albumin (fraction V), testosterone propionate and estradiol benzoate were purchased from Sigma Chemical Co. (U.S.A.); Sephadex G-100 was purchased from Pharmacia (Sweden), PVP (average molecular weight 360000; practical grade) from Fluka AG (Switzerland) and carrier-free Na’251 from the Radiochemical Centre, Amersham (Great Britain). All reagents and chemicals were of analytical grade. The rats were of the ‘Sabra’ strain of the Hebrew University (descendants of the Wistar strain) and the mice of the ICR strain. The animals were housed 5 to a cage, under natural lighting and temperature and receiving food and water ad libitum. Preparation
of tissue homogenates
The methods for the preparation of the tissue homogenates, iodination and purification of PRL and testing of the binding activity of the ‘251-labelled oPRL have been reported previously (Barkey et al., 1977, 1979a) and will be only briefly reviewed to report minor modifications. Adult male rats weighing 200-250 g were killed by decapitation. The testes, seminal vesicles, ventral prostate and liver were immediately removed, weighed, homogenized in the cold (4’C) in 5 ~01s. (only 2 ~01s. for the testes) of Tris-sucrose buffer, pH 7.6, aliquotted and frozen at -20°C until used. The protein concentration was determined by a modification (Miller, 1959) of the method of Lowry et al. (1951).
PRL site regulation in male sex glanak and liver
181
oPRL was iodinated with lactoperoxidase (Rogol and Chrambach, 1975) and chromatographed on Sephadex G-100. The specific activity of the ‘251-labelled oPRL was 70-80 Ci/g. Incubation with 0.1 ml of freshly thawed homogenate, 1 ng of ‘251-labelled oPRL, and either phosphate buffer (to determine the total binding) or 1 pg of unlabelled oPRL (to determine non-specific binding), was carried out in triplicate at 20°C, or at 4°C for the liver, with constant shaking and for 44-48 h. The reaction was stopped by dilution and centrifugation at 8000 x g for 40 min at 5’C, followed by aspiration of the supernatant. Specific binding was the difference between total and non-specific binding and was expressed as a percentage of the total cpm incubated and corrected for the protein concentration of the homogenate. Hormonal treatments The injection volume of 0.1 ml/100 g body weight was administered S.C. oPRL was dissolved in 10% PVP in saline as previously described (Barkey et al., 1981) and 200 pg/rat injected once daily for 7 days. The rats were killed 48 h after the last PRL injection. Testosterone propionate was dissolved in olive oil and 1 mg/rat administered once daily for 7 days. Where indicated, oPRL and testosterone were combined into the one treatment group. Estradiol benzoate was dissolved in olive oil and administered as a single dose of 2 mg/rat on day 1, the rats being killed and binding measured on day 10, since this dose regimen was found to be optimal when a similar long-acting estrogen preparation was used and PRL hepatic binding determined (Kelly et al., 1975). Other treatment groups received estradiol on day 1 + oPRL daily from days 1-7, or estradiol on day 1 + oPRL daily from days 11-17, those rats being killed on day 19, in order to study possible interactions of PRL with estradiol, whether initially prior to attaining maximal estradiol effects at around day 10 (Kelly et al., 1975) or subsequent to attaining such an effect. The vehicle control groups received either PVP 10% in saline or olive oil, as shown.
RESULTS Administration of oPRL in 10% PVP at a dose of 200 pg/rat/day induced a time-dependent and well-correlated reduction in 1251-labelled oPRL binding to the testis, binding falling to 58% of the vehicle-injected controls after 7 days of injection (Fig. 1). Testosterone similarly reduced ‘ZSI-labelled oPRL binding in the testis (Fig. 2, panel A). However, coadministration of testosterone and PRL
182
T. Amii er al.
DURATION OF oPRL ADMINISTRATION
(DAYS1
Fig. 1. Effect of duration of oPRL administration on ‘251-labelled oPRL binding in the rat testis. oPRL in 10% PVP in saline was injected S.C. at a dose of 200 pg/rat/day for 2, 5 or 7 days and binding measured 2 days afier the last injection. The line drawn was obtained by least squares linear regression for the points shown as mean f SEM. The number of rats * P < 0.05 vs. 7 days and P < 0.01 vs. per experimental group is shown in parentheses. control; ** P -C 0.001 vs. control, Student’s two-tailed t-test.
further and significantly reduced ‘251-labelled oPRL binding to 10% of control or to 25% of the group administered oPRL only, suggesting additive effects of testosterone and PRL. PRL specific binding measured 10 or 19 days after a single injection of estradiol was reduced to 37% or 39%, respectively, of the level measured in the vehicle-injected controls (Fig. 2, panel B), comparable to the effect of testosterone. However, in contrast to administration with testosterone, coadministration of PRL in PVP had no further effect. In contrast to its effect on the PRL binding sites, estradiol had no effect on testicular weight (panel B), PRL caused no effect on PRL binding in the rat ventral prostate and testosterone caused a slight (11%) but significant increase (panel A). Estradiol administration on day 1 resulted in a 72% loss in PRL binding sites measured after 10 days, which was completely restored by subsequent administration of PRL. Estradiol also resulted in a time-dependent weight reduction of the prostate, which was only partially restored by PRL. It is interesting to note that PRL coadministered for 7 days from day 1 of the estradiol injection was not able to inhibit the estradiol-induced reduction in prostate weight or of PRL binding. PRL also had no effect on PRL binding in the seminal vesicles, but testosterone more than doubled it, confirming the androgen dependence of this gland. Estradiol caused a slight reduction in both PRL binding
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Fig. 2. Effect of various hormonal treatments on PRL binding and organ weight of male rat sex glands and liver. 5 rats per experimental group were treated with the doses shown of PRL and/or testosterone in panel A or PRL and/or estradiol in panel B, as described in Materials and Methods. The statistical analysis was based on the two-tailed Student’s t-test, with 8 degrees of freedom. (a) NS, (b) P c:0.05, (c) P < 0.01, (d) P < 0.001 vs. vehicle control in the same panel (A or B); (e) NS, (f) P < 0.02, (g) P < 0.01, (h) P < 0.001VS. testosterone in panel A or vs. estradiol in panel B; (i) NS, (i) P < 0.01, (k) P < 0.001 vs. PRL only in panel A or PRL days 1-7 in panel B; (1) NS, (m) P < 0.05, (n) P < 0.001 vs. estradiol+ PVP in panel B.
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(by 32%) and seminal vesicle weight (by 21%) after 10 days and both parameters were reduced even further after 19 days. PRL given after estradiol was able to restore the weight of the seminal vesicles, but not the PRL binding. In the liver, PRL caused a major (170-fold) increase in PRL specific binding, confirming our earlier findings (Barkey et al., 1981). Testosterone coadministered with PRL was not able to modulate this inductive process. Estradiol also induced a 24-fold increase in PRL binding measured 19 days after the estradiol injection, but administration of PRL from days 1 l- 17 increased the induction further to 32-fold over control. Coadministration of PRL from days l-7 resulted in a further stimulation of PRL binding, greater than achieved by PRL alone.
DISCUSSION This study describes the effects of the sex steroids on the regulation of PRL receptors and organ weight of the male rat sex glands and the liver as well as the interaction of PRL with these effects. In the testis the down-regulating effect of PRL on its own binding sites (Barkey et al., 1979b; Morris and Saxena, 1980) is confirmed and shown to be time-dependent for up to 9 days. While Chan et al. (1981) did not observe a down-regulatory effect of PRL on its own receptors, in that study PRL was administered without PVP and only for 3 days. Receptor down-regulation has been demonstrated for a number of peptide hormone receptors and appears to result from a change in either receptor synthesis or degradation, probably mediated by processing of the hormone-receptor complex (Catt et al., 1979). Testosterone propionate was administered at a dose of 1 mg/rat/day since this dose was previously shown to affect both PRL binding sites and accessory sex gland weights (Kledzik et al., 1976; Barkey et al., 1979b) and since Aragona and Friesen (1975) found no effect 10 days after a single 20 mg dose of testosterone cypionate on PRL binding to the rat prostate gland. This dose of testosterone propionate, like PRL, also exerted an inhibitory effect on testicular PRL sites. This effect was additive to that of PRL, suggesting two possibilities as to their mode of action: (1) PRL and testosterone, at the dose level used, may each be acting submaximally through a common mechanism; (2) they may be acting through separate mechanisms, eventually synergising with one another. Beyond its direct effect at the testicular level, testosterone may be affecting PRL binding sites in the testis, indirectly, through feedback inhibition of LH release. Chan et al. (198 1) observed a stimulatory effect
PRL site regulation in male sex glands and liver
185
of LH on PRL testicular binding, and it might therefore be expected that inhibition of LH secretion would result in a reduction in PRL binding. The down-regulating effect of estradiol on testicular binding was quantitatively similar to that of testosterone alone, but was not affected by added PRL. Estradiol is a potent releaser of PRL (Chen and Meites, 1970) and these results do not eliminate the possibility that part, at least, of the estradiol effect is achieved through the pituitary release of PRL. However, if the estradiol effect was solely mediated by its effect on endogenous PRL release, concomitant exogenous PRL would then be expected to lower PRL binding further, as it did with testosterone. Thus a direct effect of estradiol on PRL receptors at the testicular level would seem more likely to predominate, as indeed has already been shown in vitro in incubated liver and kidney slices (Moenkemeyer et al., 1974). In the prostate, the stimulatory effect of testosterone on PRL binding is confirmed, as is the lack of effect of PRL, either alone or with testosterone (Kledzik et al., 1976; Barkey et al., 1979b). In view of the demonstrated androgen dependence of the prostate ,PRL receptors, their down-regulation by estradiol is consistent with an anti-androgenic effect of the estrogen, and in keeping with earlier observations (Aragona and Friesen, 1975). Such an effect could be achieved either through feedback inhibition of pituitary gonadotropin secretion or through direct androgen antagonism at the testicular level (Kalla et al., 1980). PRL administered in PVP for 7 days from day 10 after the estradiol injection, but not from day 1, was able to restore PRL binding completely to control levels and partially restore the prostatic weight loss. These results also suggest a competitive antagonism between estradiol and PRL on the regulation of PRL sites: at the high initial concentrations of estradiol, PRL is not able to antagonize the estradiol-induced inhibition; after 10 days, when estradiol levels can be assumed to have been lowered by degradation, PRL is now able to antagonize the estradiol effects and restore PRL binding and organ weight. It is interesting to compare the effect of PRL on its own binding site in the prostate of untreated rats (no effect) and of estrogen-treated rats (stimulatory effect). It could be said that in face of a lowered number of sites (after estrogen), PRL is able to restore the number to the basal situation, but it is not able to increase this number further above the basal situation. An analogous situation is observed with the effect of testosterone treatments on PRL sites in intact and castrated rats. When PRL sites in the prostate are lowered, such as subsequent to castration, testosterone has a much more pronounced effect than in the normal situation of the intact rat (Aragona and Friesen, 1975; Kledzik et al., 1976; Barkey et al., 1979b).
186
T. Amit et al.
The same pattern of androgen dependence and lack of PRL effect seen with the prostate is observed with regard to the seminal vesicle PRL binding site. Unlike the prostate, however, PRL could not restore the estradiol-induced loss in PRL binding, although it did restore completely the estradiol-induced loss in seminal vesicle weight, suggesting the involvement of two different mechanisms of action of PRL on these parameters. Coadministration of testosterone with PRL could not inhibit the induction by PRL of its own hepatic receptors, in keeping with a more distal or less direct site of action for the normal down-regulating effects of testosterone in the liver. The strong inductive effects of estradiol could be interpreted both as an anti-androgen effect as well as a result of increased PRL secretion (Chen and Meites, 1970; Tresguerres et al., 1981) in addition to the demonstrated direct effect on liver cells (Moenkemeyer et al., 1974). The fact that coadministration of PRL significantly enhanced the estradiol induction of hepatic PRL binding sites, regardless of the timing of the PRL administration, suggests that at least a major component of the estradiol effect involves increasing PRL secretion. Alternatively, PRL and estradiol could be acting through separate and additive mechanisms. Since the completion of this study, we have been preoccupied with the possibility that ovine PRL administration with PVP might evoke an immunological response in addition to its effect on its own receptor site. Indeed, Hughes et al. (1982) recently suggested that PRL administered in PVP for 10 days evoked an antibody response and that such an immunological artifact, rather than autoregulation of the PRL receptor, accounts for the induction observed. Our recent studies (submitted for publication) in fact suggest that, in parallel to affecting its own receptors, PRL indeed evokes an immunological response. In those studies, the receptor and antibody binding entities were separated and the up-regulating effects of PRL on its own receptors in the rat lung and liver were clearly demonstrated. Further, in the present study, PRL was administered for 7 days, and this treatment was clearly shown to cause both a down-regulation of testicular PRL sites as well as restoration of seminal vesicle weights, neither of which could be accounted for by an antibody to ovine PRL. In summary, these results demonstrate the complex and profound effects of PRL and the sex steroids on PRL binding sites in the male rat sex glands and liver. The profile of these hormones must therefore be carefully evaluated prior to determination of PRL receptors in those organs. The interaction of these hormones at the level of the target organ assumes even greater importance in view of the recent clinical findings of hyperprolactinaemic male impotence and infertility (Horrobin, 1979)
PRL site regulation in male sex glands and liver
and we are currently male sex glands.
evaluating
187
the role of PRL in the function
of the
ACKNOWLEDGEMENTS This work was supported of Sciences and Humanities
by grants from the Israel National and the Wellcome Trust (U.K.).
Academy
REFERENCES Aragona, C. and Friesen, H.G. (1975) Endocrinology 97, 677-684. Barkey, R.J., Shani, J., Amit, T. and Barzilai, D. (1977) J. Endocrinol. 74, 163-173. Barkey, R.J., Shani, J., Amit, T. and Barzilai, D. (1979a) J. Endocrinol. 80, 181- 189. Barkey, R.J., Shani, J. and Barzilai, D. (1979b) J. Endocrinol. 81, 1 l-18. Barkey, R.J., Shani, J., Lahav, M., Amit, T. and Youdim, M.B.H. (1981) Mol. Cell. Endocrinol. 21, 129-138. Catt, K.J., Hanwod, J.P., Aguilera, G. and Dufau, M.L. (1979) Nature (London) 280, 109-115. Chan, V., Katikineni, M., Davies, T.F. and Catt, K.J. (1981) Endocrinology 108, 1607-1612. Chen, C.L. and Meites, J. (1970) Endocrinology 86, 503-508. Horrobin, D.F. (1979) In: Prolactin, Vol. 7 (Eden Press Inc., Quebec) pp. 23-26. Hughes, J.P., Elsholtz, H.P. and Friesen, H.G. (1982) Endocrinology 111, 702-704. Kalla, N.R., Nisula, B.C., Menard, R. and Loriaux, D.L. (1980) Endocrinology 106, 35-39. Kelly, P.A., Posner, B.I. and Friesen, H.G. (1975) Endocrinology 97, 1408-1415. Kledzik, G.S., Marshall, S., Campbell, G.A., Gelato, M. and Meites, J. (1976) Endocrinology 98, 373-379. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Miller, G.L. (1959) Anal. Chem. 31, 964. Moenkemeyer, H., Kelly, P.A. and Friesen, H.G. (1974) Clin. Res. 22, 733A (abstract). Morisbige, W.K. and Rothchild, I. (1974) Endocrinology 95, 260-274. Morris, P.L. and Saxena, B.B. (1980) Endocrinology 107, 1639-1645. Rogol, A.D. and Chrambach, A. (1975) Endocrinology 97, 406-417. Tresguerres, J.A.F., Esquifino, AI., Perez-Mendez, L.F. and Lopez-Calderon, A. (1981) Endocrinology 108, 83-87.
179
EFFECT OF PROLACTIN, TESTOSTERONE AND ESTROGEN PROLACTIN BINDING IN THE RAT TESTIS, PROSTATE, SEMINAL VESICLE AND LIVER
ON
T. AMIT, R.J. BARKEY * AND M.B.H. YOUDIM Rappaport Family Institute for Research in the Medical Sciences, Department of Phannacology, Faculty of Medicine, Technion- Israel Institute of Technology, P.O. Box 9649, Haifa 31096 (Israel) Received 19 July 198:!; revisions January 1983
received
27 October
1982 and 3 January
1983; accepted
6
We have studied the hormonal control of prolactin (PRL) binding in the male rat sex glands and liver, subsequent to the recent demonstration and characterization of specific PRL binding sites in rat testis, prostate and seminal vesicle. Ovine PRL (200 jtg/rat/day, 7 days) caused a time-dependent reduction in testicular binding of ‘2SI-labelled PRL (measured 2 days after last injection) to 58% of control. Testosterone alone (1 mg/rat/day, 7 days) or PRL caused similar reductions in binding, while their coadministration further lowered PRL binding to 10% of control. The synergism of PRL and testosterone suggests that either these doses are submaximal, or that they are acting on different systems. Estradiol was administered as a single dose of 2 mg/rat and the PRL binding determined on day 10 and day 19 was reduced to 37% of control, as after testosterone. Addition of PRL whether from day 1 to day 7 or from day 11 to day 17 of estradiol injection had no effect, suggesting that the EB site of action is closer to the PRL receptor than that for PRL or testosterone. Bstradiol resulted in a 72% reduction of PRL binding in the prostate, after 10 days, which subsequent PRL completely restored. PRL also partially restored the estradiolinduced time-dependent weight reduction of the prostate, but PRL coadministered from day 1 of estradiol did not inhibit the estradiol effects, suggesting a competitive mechanism for the two. While testosterone more than doubled PRL binding in the seminal vesicle, estradiol reduced it by 32% and organ weight by 21%. PRL given after estradiol restored the weight loss, but not the binding, suggesting that two different mechanisms of action are involved. In the liver, coadministration of testosterone with PRL could not inhibit the induction by PRL of its own hepatic sites, in keeping with a more direct site of action for PRL than for testosterone. These results demonstrate the profound effects of PRL, and of the sex steroids testosterone and estrogen, on PRL binding in the male sex glands and liver. The physiological implication of these findings on the role of PRL in male sexual function is currently being investigated. Keywords:
prolactin receptors; icle; liver.
* To whom all correspondence
0303-7207/83/0000-0000/$03.00
hormonal
regulation;
testis;
prostate;
seminal
should be addressed.
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
ves-
T. Amit et al.
180
We have previously reported the presence of specific binding sites for prolactin (PRL) in the rat testis, prostate, seminal vesicle and liver (Barkey et al., 1977). The involvement of PRL in the regulation of male reproduction and fertility is becoming increasingly clear (for review see Horrobin, 1979). The recent demonstration that administration of PRL dissolved in 10% polyvinylpyrrolidone (PVP) in saline greatly enhances the effect of PRL on its own receptors in the male rat liver (Barkey et al., 1981) probably through the slow release achieved with PVP (Morishige and Rothchild, 1974), prompted the study of the effects of PRL and the sex steroids testosterone and estradiol, alone and in various combinations, on the PRL binding site and on organ weights of the sex glands, in order to gain a better understanding of the role and mechanism of action of PRL in these glands.
MATERIALS Chemicals
AND
METHODS
and animals
Ovine PRL (oPRL; NIH-P-S12, 35 IU/mg) was generously supplied by the Hormone Distribution Program of NIAMDD, NIH. Lactoperoxidase (from milk), bovine serum albumin (fraction V), testosterone propionate and estradiol benzoate were purchased from Sigma Chemical Co. (U.S.A.); Sephadex G-100 was purchased from Pharmacia (Sweden), PVP (average molecular weight 360000; practical grade) from Fluka AG (Switzerland) and carrier-free Na’251 from the Radiochemical Centre, Amersham (Great Britain). All reagents and chemicals were of analytical grade. The rats were of the ‘Sabra’ strain of the Hebrew University (descendants of the Wistar strain) and the mice of the ICR strain. The animals were housed 5 to a cage, under natural lighting and temperature and receiving food and water ad libitum. Preparation
of tissue homogenates
The methods for the preparation of the tissue homogenates, iodination and purification of PRL and testing of the binding activity of the ‘251-labelled oPRL have been reported previously (Barkey et al., 1977, 1979a) and will be only briefly reviewed to report minor modifications. Adult male rats weighing 200-250 g were killed by decapitation. The testes, seminal vesicles, ventral prostate and liver were immediately removed, weighed, homogenized in the cold (4’C) in 5 ~01s. (only 2 ~01s. for the testes) of Tris-sucrose buffer, pH 7.6, aliquotted and frozen at -20°C until used. The protein concentration was determined by a modification (Miller, 1959) of the method of Lowry et al. (1951).
PRL site regulation in male sex glanak and liver
181
oPRL was iodinated with lactoperoxidase (Rogol and Chrambach, 1975) and chromatographed on Sephadex G-100. The specific activity of the ‘251-labelled oPRL was 70-80 Ci/g. Incubation with 0.1 ml of freshly thawed homogenate, 1 ng of ‘251-labelled oPRL, and either phosphate buffer (to determine the total binding) or 1 pg of unlabelled oPRL (to determine non-specific binding), was carried out in triplicate at 20°C, or at 4°C for the liver, with constant shaking and for 44-48 h. The reaction was stopped by dilution and centrifugation at 8000 x g for 40 min at 5’C, followed by aspiration of the supernatant. Specific binding was the difference between total and non-specific binding and was expressed as a percentage of the total cpm incubated and corrected for the protein concentration of the homogenate. Hormonal treatments The injection volume of 0.1 ml/100 g body weight was administered S.C. oPRL was dissolved in 10% PVP in saline as previously described (Barkey et al., 1981) and 200 pg/rat injected once daily for 7 days. The rats were killed 48 h after the last PRL injection. Testosterone propionate was dissolved in olive oil and 1 mg/rat administered once daily for 7 days. Where indicated, oPRL and testosterone were combined into the one treatment group. Estradiol benzoate was dissolved in olive oil and administered as a single dose of 2 mg/rat on day 1, the rats being killed and binding measured on day 10, since this dose regimen was found to be optimal when a similar long-acting estrogen preparation was used and PRL hepatic binding determined (Kelly et al., 1975). Other treatment groups received estradiol on day 1 + oPRL daily from days 1-7, or estradiol on day 1 + oPRL daily from days 11-17, those rats being killed on day 19, in order to study possible interactions of PRL with estradiol, whether initially prior to attaining maximal estradiol effects at around day 10 (Kelly et al., 1975) or subsequent to attaining such an effect. The vehicle control groups received either PVP 10% in saline or olive oil, as shown.
RESULTS Administration of oPRL in 10% PVP at a dose of 200 pg/rat/day induced a time-dependent and well-correlated reduction in 1251-labelled oPRL binding to the testis, binding falling to 58% of the vehicle-injected controls after 7 days of injection (Fig. 1). Testosterone similarly reduced ‘ZSI-labelled oPRL binding in the testis (Fig. 2, panel A). However, coadministration of testosterone and PRL
182
T. Amii er al.
DURATION OF oPRL ADMINISTRATION
(DAYS1
Fig. 1. Effect of duration of oPRL administration on ‘251-labelled oPRL binding in the rat testis. oPRL in 10% PVP in saline was injected S.C. at a dose of 200 pg/rat/day for 2, 5 or 7 days and binding measured 2 days afier the last injection. The line drawn was obtained by least squares linear regression for the points shown as mean f SEM. The number of rats * P < 0.05 vs. 7 days and P < 0.01 vs. per experimental group is shown in parentheses. control; ** P -C 0.001 vs. control, Student’s two-tailed t-test.
further and significantly reduced ‘251-labelled oPRL binding to 10% of control or to 25% of the group administered oPRL only, suggesting additive effects of testosterone and PRL. PRL specific binding measured 10 or 19 days after a single injection of estradiol was reduced to 37% or 39%, respectively, of the level measured in the vehicle-injected controls (Fig. 2, panel B), comparable to the effect of testosterone. However, in contrast to administration with testosterone, coadministration of PRL in PVP had no further effect. In contrast to its effect on the PRL binding sites, estradiol had no effect on testicular weight (panel B), PRL caused no effect on PRL binding in the rat ventral prostate and testosterone caused a slight (11%) but significant increase (panel A). Estradiol administration on day 1 resulted in a 72% loss in PRL binding sites measured after 10 days, which was completely restored by subsequent administration of PRL. Estradiol also resulted in a time-dependent weight reduction of the prostate, which was only partially restored by PRL. It is interesting to note that PRL coadministered for 7 days from day 1 of the estradiol injection was not able to inhibit the estradiol-induced reduction in prostate weight or of PRL binding. PRL also had no effect on PRL binding in the seminal vesicles, but testosterone more than doubled it, confirming the androgen dependence of this gland. Estradiol caused a slight reduction in both PRL binding
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Fig. 2. Effect of various hormonal treatments on PRL binding and organ weight of male rat sex glands and liver. 5 rats per experimental group were treated with the doses shown of PRL and/or testosterone in panel A or PRL and/or estradiol in panel B, as described in Materials and Methods. The statistical analysis was based on the two-tailed Student’s t-test, with 8 degrees of freedom. (a) NS, (b) P c:0.05, (c) P < 0.01, (d) P < 0.001 vs. vehicle control in the same panel (A or B); (e) NS, (f) P < 0.02, (g) P < 0.01, (h) P < 0.001VS. testosterone in panel A or vs. estradiol in panel B; (i) NS, (i) P < 0.01, (k) P < 0.001 vs. PRL only in panel A or PRL days 1-7 in panel B; (1) NS, (m) P < 0.05, (n) P < 0.001 vs. estradiol+ PVP in panel B.
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(by 32%) and seminal vesicle weight (by 21%) after 10 days and both parameters were reduced even further after 19 days. PRL given after estradiol was able to restore the weight of the seminal vesicles, but not the PRL binding. In the liver, PRL caused a major (170-fold) increase in PRL specific binding, confirming our earlier findings (Barkey et al., 1981). Testosterone coadministered with PRL was not able to modulate this inductive process. Estradiol also induced a 24-fold increase in PRL binding measured 19 days after the estradiol injection, but administration of PRL from days 1 l- 17 increased the induction further to 32-fold over control. Coadministration of PRL from days l-7 resulted in a further stimulation of PRL binding, greater than achieved by PRL alone.
DISCUSSION This study describes the effects of the sex steroids on the regulation of PRL receptors and organ weight of the male rat sex glands and the liver as well as the interaction of PRL with these effects. In the testis the down-regulating effect of PRL on its own binding sites (Barkey et al., 1979b; Morris and Saxena, 1980) is confirmed and shown to be time-dependent for up to 9 days. While Chan et al. (1981) did not observe a down-regulatory effect of PRL on its own receptors, in that study PRL was administered without PVP and only for 3 days. Receptor down-regulation has been demonstrated for a number of peptide hormone receptors and appears to result from a change in either receptor synthesis or degradation, probably mediated by processing of the hormone-receptor complex (Catt et al., 1979). Testosterone propionate was administered at a dose of 1 mg/rat/day since this dose was previously shown to affect both PRL binding sites and accessory sex gland weights (Kledzik et al., 1976; Barkey et al., 1979b) and since Aragona and Friesen (1975) found no effect 10 days after a single 20 mg dose of testosterone cypionate on PRL binding to the rat prostate gland. This dose of testosterone propionate, like PRL, also exerted an inhibitory effect on testicular PRL sites. This effect was additive to that of PRL, suggesting two possibilities as to their mode of action: (1) PRL and testosterone, at the dose level used, may each be acting submaximally through a common mechanism; (2) they may be acting through separate mechanisms, eventually synergising with one another. Beyond its direct effect at the testicular level, testosterone may be affecting PRL binding sites in the testis, indirectly, through feedback inhibition of LH release. Chan et al. (198 1) observed a stimulatory effect
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of LH on PRL testicular binding, and it might therefore be expected that inhibition of LH secretion would result in a reduction in PRL binding. The down-regulating effect of estradiol on testicular binding was quantitatively similar to that of testosterone alone, but was not affected by added PRL. Estradiol is a potent releaser of PRL (Chen and Meites, 1970) and these results do not eliminate the possibility that part, at least, of the estradiol effect is achieved through the pituitary release of PRL. However, if the estradiol effect was solely mediated by its effect on endogenous PRL release, concomitant exogenous PRL would then be expected to lower PRL binding further, as it did with testosterone. Thus a direct effect of estradiol on PRL receptors at the testicular level would seem more likely to predominate, as indeed has already been shown in vitro in incubated liver and kidney slices (Moenkemeyer et al., 1974). In the prostate, the stimulatory effect of testosterone on PRL binding is confirmed, as is the lack of effect of PRL, either alone or with testosterone (Kledzik et al., 1976; Barkey et al., 1979b). In view of the demonstrated androgen dependence of the prostate ,PRL receptors, their down-regulation by estradiol is consistent with an anti-androgenic effect of the estrogen, and in keeping with earlier observations (Aragona and Friesen, 1975). Such an effect could be achieved either through feedback inhibition of pituitary gonadotropin secretion or through direct androgen antagonism at the testicular level (Kalla et al., 1980). PRL administered in PVP for 7 days from day 10 after the estradiol injection, but not from day 1, was able to restore PRL binding completely to control levels and partially restore the prostatic weight loss. These results also suggest a competitive antagonism between estradiol and PRL on the regulation of PRL sites: at the high initial concentrations of estradiol, PRL is not able to antagonize the estradiol-induced inhibition; after 10 days, when estradiol levels can be assumed to have been lowered by degradation, PRL is now able to antagonize the estradiol effects and restore PRL binding and organ weight. It is interesting to compare the effect of PRL on its own binding site in the prostate of untreated rats (no effect) and of estrogen-treated rats (stimulatory effect). It could be said that in face of a lowered number of sites (after estrogen), PRL is able to restore the number to the basal situation, but it is not able to increase this number further above the basal situation. An analogous situation is observed with the effect of testosterone treatments on PRL sites in intact and castrated rats. When PRL sites in the prostate are lowered, such as subsequent to castration, testosterone has a much more pronounced effect than in the normal situation of the intact rat (Aragona and Friesen, 1975; Kledzik et al., 1976; Barkey et al., 1979b).
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The same pattern of androgen dependence and lack of PRL effect seen with the prostate is observed with regard to the seminal vesicle PRL binding site. Unlike the prostate, however, PRL could not restore the estradiol-induced loss in PRL binding, although it did restore completely the estradiol-induced loss in seminal vesicle weight, suggesting the involvement of two different mechanisms of action of PRL on these parameters. Coadministration of testosterone with PRL could not inhibit the induction by PRL of its own hepatic receptors, in keeping with a more distal or less direct site of action for the normal down-regulating effects of testosterone in the liver. The strong inductive effects of estradiol could be interpreted both as an anti-androgen effect as well as a result of increased PRL secretion (Chen and Meites, 1970; Tresguerres et al., 1981) in addition to the demonstrated direct effect on liver cells (Moenkemeyer et al., 1974). The fact that coadministration of PRL significantly enhanced the estradiol induction of hepatic PRL binding sites, regardless of the timing of the PRL administration, suggests that at least a major component of the estradiol effect involves increasing PRL secretion. Alternatively, PRL and estradiol could be acting through separate and additive mechanisms. Since the completion of this study, we have been preoccupied with the possibility that ovine PRL administration with PVP might evoke an immunological response in addition to its effect on its own receptor site. Indeed, Hughes et al. (1982) recently suggested that PRL administered in PVP for 10 days evoked an antibody response and that such an immunological artifact, rather than autoregulation of the PRL receptor, accounts for the induction observed. Our recent studies (submitted for publication) in fact suggest that, in parallel to affecting its own receptors, PRL indeed evokes an immunological response. In those studies, the receptor and antibody binding entities were separated and the up-regulating effects of PRL on its own receptors in the rat lung and liver were clearly demonstrated. Further, in the present study, PRL was administered for 7 days, and this treatment was clearly shown to cause both a down-regulation of testicular PRL sites as well as restoration of seminal vesicle weights, neither of which could be accounted for by an antibody to ovine PRL. In summary, these results demonstrate the complex and profound effects of PRL and the sex steroids on PRL binding sites in the male rat sex glands and liver. The profile of these hormones must therefore be carefully evaluated prior to determination of PRL receptors in those organs. The interaction of these hormones at the level of the target organ assumes even greater importance in view of the recent clinical findings of hyperprolactinaemic male impotence and infertility (Horrobin, 1979)
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and we are currently male sex glands.
evaluating
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the role of PRL in the function
of the
ACKNOWLEDGEMENTS This work was supported of Sciences and Humanities
by grants from the Israel National and the Wellcome Trust (U.K.).
Academy
REFERENCES Aragona, C. and Friesen, H.G. (1975) Endocrinology 97, 677-684. Barkey, R.J., Shani, J., Amit, T. and Barzilai, D. (1977) J. Endocrinol. 74, 163-173. Barkey, R.J., Shani, J., Amit, T. and Barzilai, D. (1979a) J. Endocrinol. 80, 181- 189. Barkey, R.J., Shani, J. and Barzilai, D. (1979b) J. Endocrinol. 81, 1 l-18. Barkey, R.J., Shani, J., Lahav, M., Amit, T. and Youdim, M.B.H. (1981) Mol. Cell. Endocrinol. 21, 129-138. Catt, K.J., Hanwod, J.P., Aguilera, G. and Dufau, M.L. (1979) Nature (London) 280, 109-115. Chan, V., Katikineni, M., Davies, T.F. and Catt, K.J. (1981) Endocrinology 108, 1607-1612. Chen, C.L. and Meites, J. (1970) Endocrinology 86, 503-508. Horrobin, D.F. (1979) In: Prolactin, Vol. 7 (Eden Press Inc., Quebec) pp. 23-26. Hughes, J.P., Elsholtz, H.P. and Friesen, H.G. (1982) Endocrinology 111, 702-704. Kalla, N.R., Nisula, B.C., Menard, R. and Loriaux, D.L. (1980) Endocrinology 106, 35-39. Kelly, P.A., Posner, B.I. and Friesen, H.G. (1975) Endocrinology 97, 1408-1415. Kledzik, G.S., Marshall, S., Campbell, G.A., Gelato, M. and Meites, J. (1976) Endocrinology 98, 373-379. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Miller, G.L. (1959) Anal. Chem. 31, 964. Moenkemeyer, H., Kelly, P.A. and Friesen, H.G. (1974) Clin. Res. 22, 733A (abstract). Morisbige, W.K. and Rothchild, I. (1974) Endocrinology 95, 260-274. Morris, P.L. and Saxena, B.B. (1980) Endocrinology 107, 1639-1645. Rogol, A.D. and Chrambach, A. (1975) Endocrinology 97, 406-417. Tresguerres, J.A.F., Esquifino, AI., Perez-Mendez, L.F. and Lopez-Calderon, A. (1981) Endocrinology 108, 83-87.