Department of Chemistry I, Karolinska Institutet. Stockholm
INFLUENCE OF PROLACTIN ON THE METABOLISM OF STEROID HORMONES IN RAT LIVER AND ADRENALS
By
Jan-\l=A%o\keGustafsson
and
\l=A%o\keStenberg
ABSTRACT The influence of prolactin treatment on the hepatic metabolism of 4-[4-14C]androstene-3,17-dione (in the microsomal and 105 000 \m=x\gsupernatant fractions) and 5\g=a\-[4-14C]androstane-3\g=a\,17\g=b\-diol(in the microsomal fraction) and on the adrenal metabolism of 4-[4-14C]androstene\x=req-\
3,17-dione
was studied in intact and castrated male and female rats with and without concomitant treatment with testosterone propionate. Whereas prolactin gave a significant and specific decrease in the activity of adrenal 5\g=a\-reductase by about 20\p=n-\30% in both male and female rats no specific effects were noted in the metabolism of steroids in the liver. Neither did prolactin compensate for the relative androgen unresponsiveness characteristic of neonatally castrated male rats. These results suggest that prolactin does not play any significant role in mediating the recently discovered hypophyseal control of sexual differentiation of hepatic steroid metabolism in the rat whereas it may have a function in maintaining sexual differences in alrenal 5\g=a\-reductase activity.
Recent findings give strong indications that the well-known sexual differen¬ tiation of hepatic metabolism of steroid hormones and drugs in the rat occurs at birth and is under hypothalamo-hypophyseal control (Gustafsson 8c Stenberg 1974c; Gustafsson et al, in press). This control seems to be at least partly mediated via a hypophyseal feminizing factor that feminizes the basic masculine pattern of rat liver enzyme activities. Certain results also point to the existence of a hypophyseal masculinizing tactor necessary for the action of androgens upon the liver. At the present time nothing is known about the natures of these postulated feminizing and masculinizing factors. It seems essential to
investigate the possibility that some of the already characterized hypophyseal hormones are involved in the regulation of liver enzyme activities. Prolactin has been suggested as a regulator of steroid hormone metabolizing enzymes in certain tissues (Wiest Sc Kidwell 1969; Witorsch Sc Kitay 1972) and it has also been proposed that prolactin could play a role in the response of prostatic tissue to androgens (Grayhack Sc Lebowitz 1967). Neonatal treatment of female rats with testosterone propionate has been shown to result in a very high concentration of prolactin in the adult rats (Mallampati Se Johnson 1973). In view of these results we have investigated the effects of prolactin upon the activities of hepatic steroid metabolizing enzymes, also in combination with treatment with testosterone propionate. The effects of prolactin treatment have also been studied on some adrenal enzyme activities.
MATERIALS AND METHODS
Reference compounds The
given
sources
in
of the reference steroids used in the present investigation have been Se Gustafsson 1973; Einarsson et al. 1973).
previous publications (Berg
Radioactive steroids 4-[4-14C] Androstene-3,17 dione
was
obtained from The Radiochemical Centre
(Amersham, England), purified by thin-layer chromatography and diluted with un¬ labelled 4-androstene-3,17-dione to 29 /(Ci/mg (for incubations with adrenals) or to 0.49 ,aCi/mg (for incubations with hepatic subfractions). 5a-[4-14C] Androstane-3a,17/¿diol (specific radioactivity, 3.0 //Ci/mg) was prepared from 4-[4-14C]androstene-3,17dione as described previously (Berg 8c Gustafsson 1973). -
Animal
experiments
In experiment I male and female rats of the Sprague-Dawley strain were castrated at the age of 56 days and treated for 7 days (between 63 and 69 days of age) by subcutaneous injections of 0.2 mg of ovine prolactin (Lot No. 17024. National Pituitary Agency, National Institute of Health, Baltimore, Maryland, USA) in 13 µ\ of 1 °/o sodium carboxymethyl cellulose. Castrated control rats received vehicle only. The rats were killed at 70 days of age. In experiment II 63-day-old intact rats of both sexes were treated for 7 days with the same amount of prolactin. In experiment III male rats were chilled on ice and castrated on their first day of life or at the age of 14 days. From the age of 56 days one group of neonatally castrated rats and one group of rats castrated at 14 days of age received daily intra¬ muscular injections of 100 «g of testosterone propionate in 0.5 ml of propylene glycol for 14 days. Another group of neonatally castrated rats received the same treatment but was, in addition, given daily subcutaneous injections of 0.2 mg of ovine prolactin in 13 µ\ of sodium carboxymethyl cellulose during the same period. The rats were killed at 70 days of age. In all experiments each group of rats contained 4 animals.
All animals were killed by cervical dislocation, the adrenals and liver were excised and cooled in a modified Bucher medium, pH 7.4 (Bergström Se Gloor 1955). Liver homogenates, 20 °/o (w/v), were prepared with a Potter-Elvehjem homogenizer equipped with a loosely fitting teflon pestle. The microsomal and cytosol (105 000 x g super¬ natant) fractions were prepared using differential centrifugation (Berg Se Gustafsson 1973) and the microsomal fraction was resuspended.
Incubations of 4-[4-14C]androstene-3,17-dione and
5a-[4-14C]androstane-3a,17ß-diol with liver preparations Microsomal suspension from 0.10 g of liver of 105 000 x g supernatant from 0.60 g of liver was incubated in 3.0 ml of Bûcher medium with 500 pg of 4-[4-14C]androstene3,17-dione (added in 50 /d of acetone) at 37CC for 8 min in the presence of an NADPH-regcnerating system (Gustafsson Se Stenberg 1974a). Incubations with 5a[4-14C]androstane-3a,17/?-diol were carried out for 10 min using 200 pg of substrate (added in 50 µ of acetone) and microsomal suspension from 0.50 g of liver in 4.0 ml of Bûcher medium containing an NADPH-regenerating system. The incubation condi¬ tions were designed to give linear total conversion of substrate with time and enzyme concentration. Total conversion was not affected by small variations in substrate or cofactor amounts. The incubation mixtures were extracted with chloroform/methanol, 2:1 (v/v), as described previously (Gustafsson Se Stenberg 1974a).
Incubations of The adrenals
4-[4-,4C]androstene-3,17-elione
with adrenals
rendered free from adherent tissue and weighed. They were then transferred to a small vial and 4-[4-14C]androstene-3,17-dione was incubated with the intact adrenals using a micromethod (Goldman et al. 1974): the adrenals were suspended in 200 pi of Bûcher medium to which was added 2.5 pg of the steroid dissolved in 5 «1 of acetone and 10/d of a solution containing 3 //moles of NADH, 3 «moles of NADP, 12.5 /(moles of isocitrate, 10 «1 of isocitric dehydrogenase, 0.02 /(moles of MnCl2 and 400 µ of Bucher medium. NADP, NADH, DL-isocitric acid (trisodium salt) and isocitric dehydrogenase (type IV) were obtained from Sigma Chemical Co. The incubations were carried out for 75 min at 37CC. The conditions described gave conversions linear with respect to time and adrenal weight. The in¬ cubations were terminated by the addition of 400 pi of acetone/ethanol (1:1, v/v). After centrifugation the supernatant was taken off and the adrenals were extracted a second time with 400 /d of acetone/ethanol. This procedure gave a complete extrac¬ tion of all radioactivity in the adrenals. were
Analytical procedure The extracts from incubation with liver subfraction and adrenals were chromato¬ on precoated silica gel plates (250/(, Merck AG., Darmstadt. Germany) using chloroform/ethyl acetate, 4:1 (v/v), as the mobile phase. The radioactive zones were localized by radioautography for 7-10 days, scraped off separately and eluted with 5 ml of methanol. After removal of an aliquot for measurement of radioactivity (Packard Liquid Scintillation Spectrometer Model 4322) the rest was analyzed using radio-gas chromatography (Hewlett-Packard Gas Chromatograph Model 402, equipped with a Barber-Colman Radioactivity monitoring system Model 5190). Zones containing more than one metabolite were thus resolved and individual metabolites could be
graphed
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Table 3. metabolism of 4-[4-14C]androstene-3,17-dione and hepatic 5a-[4-14C]androstane-3a,17/?-diol in neonatally castrated male rats given testosterone propionate between 56 and 69 days of age. Abbreviations: NC ¿5 neonatally cas¬ trated male rats given testosterone propionate when adult; 14DC(5T male rats cas¬ trated at 14 days of age given testosterone propionate when adult; NC(3T, neo¬ natally castrated male rats given testosterone propionate and prolactin when adult. The conversions were calculated from the amounts of radioactivity in the different zones of the thin-layer chromatograms. The values listed at the means + gd. The effects of
prolactin
on
=
=
=
nmoles of metabolites
Enzymes
active
5/?-Reductase 5«-Reductase
17cc-Hydroxysteroid
reducíase
reductase Ratio StWSa-reduced metabolites Ratio 3/?-/5a-reduced metabolites
6/>-Hydroxylase 7«-Hydroxylase 16a-Hydroxylase
14DCc?T
6.64 1.15 17 96.8 ± 17.3
5.28 ± 1.12 63.8 ± 7.9
5.42 ± 0.81 101 ± 40
-18 +4
0.62 ± 0.39
1.05 ± 0.40
0.44
± 0.18
-29
4.05 ± 1.28
-20
1.04 ± 121
2/?-Hydroxylase 7a-Hydroxylase 7/?-Hydroxylase 18-Hydroxylase
4.43
± 0.82
NCcST,
0.153
±
0.045
0.174 ± 0.017
0.131 ± 0.025
-14
0.084 4.94 4.34 3.68
± ± ± ±
0.064 1.30 0.61 1.33
0.150 ± 0.041 11.44 ± 2.60 6.22 ± 2.18 6.82 ± 1.68
0.065 4.53 4.89 3.26
± ± ± ±
-23 -8 +13
on
nmoles of metabolites
5a-A-3a,17^-diol 2a-Hydroxylase
"lo Change with
NCr^T
17/?-Hydroxysteroid
active
10 min
on
A4-3,l 7-dione
Enzymes
formed/mg protein
3.52 0.84 2.52 0.68 1.27
± ± ± ± ±
1.96 0.43 0.21 0.19 1.05
0.017 0.29 0.92 0.64
formed/mg protein
4.94 ± 1.4S 1.53 ± 0.18
2.24 ± 0.24 0.83 ± 0.13 2.56 ± 0.46
2.61 0.68 2.45 0.62 1.08
± ± ± ± ±
0.92 0.11 0.12 0.08 0.26
-11
8 min
-26 -19 -3 -9 -15
The strongest effect of prolactin treatment on adrenal enzyme levels (Table 4) was noted with the 5a-reductase activity. This decreased 20-30 %> in both male and female rats (P < 0.05). The other adrenal enzyme activities were not significantly affected.
Table 4. The effects of prolactin on adrenal metabolism of 4-[4-14C]androstene-3.17-dione in 63-day-old male and female rats treated with prolactin for 7 days. Abbreviations: (5= control males; c5P males given prolactin; Ç control females; Ç females given prolactin. The conversions were calculated from the amounts of radioactivity in the different zones of the thin-layer chromatograms. The values listed are the means ± sd. =
=
=
Change
ÇP
with
¿VA. ?,%> 5a-Reductase
177 ± 13
128 ± 19*
214 ± 33
118 ± 19 18.8 ± 3.8 44.3 ± 7.7
139 ± 12 21.6 ± 3.5 49.7 ± 12.5
98 ± 1 21.5 ± 7.6 33.1 ± 6.3
17/?-Hydroxysteroid reductase
6/5-Hydroxylasc ll^-Hydroxylase *
149
± 29*
87 ± 8 18.8 ± 3.8 34.2 ± 4.6
-22*
-30*
+12 +15 +12
-11 -13 +3
< 0.05.
DISCUSSION
Besides its mammotrophic actions, prolactin may be involved in the control of steroid hormone production, transport and metabolism in several peripheral target organs. Thus, prolactin exerts an effect on the rat corpus luteum by inhibiting the reduction of the 20-oxo group of progesterone resulting in a net increase in progesterone secretion (Wiest 8c Kidwell 1969). Furthermore, Witorsch 8c Kitay (1972) have demonstrated that prolactin reduces the adrenal 5a-reductase activity and have suggested that prolactin plays a physiological role in regulating corticosterone secretion by preventing the intra-adrenal conversion of corticosterone to reduced metabolites. Finally, prolactin has been ascribed a role in regulating the uptake of testosterone into different organs (Grayhack 8c Lebowitz 1967). In the present investigation it was possible to confirm the finding of Witorsch 8c Kitay (1972) that prolactin reduces the activity of the adrenal 5a-reductase. This effect seemed to be quite specific since adrenal 17/j-hydroxysteroid re¬ ductase and 6ß- and 11/5-hydroxylase activities were not significantly affected following treatment with prolactin. In a previous study we have suggested that during the neonatal period testicular androgens irreversibly program the adrenal 5a-reductase activity to a lower, masculine level (Goldman et al. 1974). Kitay et al. (1971) have shown that hypophysectomy results in higher 5areductase levels in the adrenals. In consideration of the results of Mallampati
Johnson (1973) who found high concentrations of prolactin in neonatally androgenized female rats it may be speculated that prolactin could play a role in mediating the hypophyseal regulation of sexual differentiation of adrenal 5a-reductase activity. On the other hand, prolactin seemed to have less significant and unspecific effects on the hepatic enzyme activities. This was true both in experiments where prolactin was administered to intact and to castrated male and female rats. It was also shown that prolactin administration did not increase the androgen responsiveness in neonatally castrated male rats. These experiments would seem to make it less probable that prolactin is identical to either the feminizing or the masculinizing factor postulated to be secreted from the rat hypophysis even if it cannot be excluded that prolactin may be more effective when administered in other doses or together with other hormones. Sc
ACKNOWLEDGMENTS This work
was
(13X-2819)
supported by grants from the Swedish Medical Research Council
and WHO.
REFERENCES
Gustafsson J.-A.: J. biol. Chem. 248 (1973) 6559. S. 8- Gloor U.: Acta chem. scand. 9 (1955) 34. Einarsson K., Gustafsson J.-A. 8c Stenberg .: J. biol. Chem. 248 (1973) 4787. Goldman A. S., Gustafsson J.-A. & Stenberg .: Acta endocr. (Kbh.) 76 (1974) 719. Grayhack J. T. Se Lebowitz J. M.: Invest. Urol. 5 (1967) 87. Gustafsson J.-A. 8- Stenberg .: J. biol. Chem. 249 (1974«) 711. Gustafsson J.-A. Sc Stenberg .: J. biol. Chem. 249 (1974Ô) 719.
Berg
A. &
Bergström
Gustafsson J.-Ä. & Stenberg .: Endocrinology 95 (1974c) 891. Kitay J. I., Coyne M. D. & Swygert . H.: Endocrinology 89 (1971) 432. Lowry 0. H., Rosebrough N. J., Fair A. L. & Randall R. J.: J. biol. Chem.
(1951) 265. Mallampati R.
193
S. Se Johnson D. C: Neuroendocrinology // (1973) 46. Wiest W. G. Se Kidwell W. R. In: McKerns K. W., Ed. The Gonads, Appleton-CenturyCrofts, New York (1969) p. 295. Witorsch R. J. 8- Kitay J. L: Endocrinology 91 (1972) 764. Received
on
April 29th,
1974.
INFLUENCE OF PROLACTIN ON THE METABOLISM OF STEROID HORMONES IN RAT LIVER AND ADRENALS
By
Jan-\l=A%o\keGustafsson
and
\l=A%o\keStenberg
ABSTRACT The influence of prolactin treatment on the hepatic metabolism of 4-[4-14C]androstene-3,17-dione (in the microsomal and 105 000 \m=x\gsupernatant fractions) and 5\g=a\-[4-14C]androstane-3\g=a\,17\g=b\-diol(in the microsomal fraction) and on the adrenal metabolism of 4-[4-14C]androstene\x=req-\
3,17-dione
was studied in intact and castrated male and female rats with and without concomitant treatment with testosterone propionate. Whereas prolactin gave a significant and specific decrease in the activity of adrenal 5\g=a\-reductase by about 20\p=n-\30% in both male and female rats no specific effects were noted in the metabolism of steroids in the liver. Neither did prolactin compensate for the relative androgen unresponsiveness characteristic of neonatally castrated male rats. These results suggest that prolactin does not play any significant role in mediating the recently discovered hypophyseal control of sexual differentiation of hepatic steroid metabolism in the rat whereas it may have a function in maintaining sexual differences in alrenal 5\g=a\-reductase activity.
Recent findings give strong indications that the well-known sexual differen¬ tiation of hepatic metabolism of steroid hormones and drugs in the rat occurs at birth and is under hypothalamo-hypophyseal control (Gustafsson 8c Stenberg 1974c; Gustafsson et al, in press). This control seems to be at least partly mediated via a hypophyseal feminizing factor that feminizes the basic masculine pattern of rat liver enzyme activities. Certain results also point to the existence of a hypophyseal masculinizing tactor necessary for the action of androgens upon the liver. At the present time nothing is known about the natures of these postulated feminizing and masculinizing factors. It seems essential to
investigate the possibility that some of the already characterized hypophyseal hormones are involved in the regulation of liver enzyme activities. Prolactin has been suggested as a regulator of steroid hormone metabolizing enzymes in certain tissues (Wiest Sc Kidwell 1969; Witorsch Sc Kitay 1972) and it has also been proposed that prolactin could play a role in the response of prostatic tissue to androgens (Grayhack Sc Lebowitz 1967). Neonatal treatment of female rats with testosterone propionate has been shown to result in a very high concentration of prolactin in the adult rats (Mallampati Se Johnson 1973). In view of these results we have investigated the effects of prolactin upon the activities of hepatic steroid metabolizing enzymes, also in combination with treatment with testosterone propionate. The effects of prolactin treatment have also been studied on some adrenal enzyme activities.
MATERIALS AND METHODS
Reference compounds The
given
sources
in
of the reference steroids used in the present investigation have been Se Gustafsson 1973; Einarsson et al. 1973).
previous publications (Berg
Radioactive steroids 4-[4-14C] Androstene-3,17 dione
was
obtained from The Radiochemical Centre
(Amersham, England), purified by thin-layer chromatography and diluted with un¬ labelled 4-androstene-3,17-dione to 29 /(Ci/mg (for incubations with adrenals) or to 0.49 ,aCi/mg (for incubations with hepatic subfractions). 5a-[4-14C] Androstane-3a,17/¿diol (specific radioactivity, 3.0 //Ci/mg) was prepared from 4-[4-14C]androstene-3,17dione as described previously (Berg 8c Gustafsson 1973). -
Animal
experiments
In experiment I male and female rats of the Sprague-Dawley strain were castrated at the age of 56 days and treated for 7 days (between 63 and 69 days of age) by subcutaneous injections of 0.2 mg of ovine prolactin (Lot No. 17024. National Pituitary Agency, National Institute of Health, Baltimore, Maryland, USA) in 13 µ\ of 1 °/o sodium carboxymethyl cellulose. Castrated control rats received vehicle only. The rats were killed at 70 days of age. In experiment II 63-day-old intact rats of both sexes were treated for 7 days with the same amount of prolactin. In experiment III male rats were chilled on ice and castrated on their first day of life or at the age of 14 days. From the age of 56 days one group of neonatally castrated rats and one group of rats castrated at 14 days of age received daily intra¬ muscular injections of 100 «g of testosterone propionate in 0.5 ml of propylene glycol for 14 days. Another group of neonatally castrated rats received the same treatment but was, in addition, given daily subcutaneous injections of 0.2 mg of ovine prolactin in 13 µ\ of sodium carboxymethyl cellulose during the same period. The rats were killed at 70 days of age. In all experiments each group of rats contained 4 animals.
All animals were killed by cervical dislocation, the adrenals and liver were excised and cooled in a modified Bucher medium, pH 7.4 (Bergström Se Gloor 1955). Liver homogenates, 20 °/o (w/v), were prepared with a Potter-Elvehjem homogenizer equipped with a loosely fitting teflon pestle. The microsomal and cytosol (105 000 x g super¬ natant) fractions were prepared using differential centrifugation (Berg Se Gustafsson 1973) and the microsomal fraction was resuspended.
Incubations of 4-[4-14C]androstene-3,17-dione and
5a-[4-14C]androstane-3a,17ß-diol with liver preparations Microsomal suspension from 0.10 g of liver of 105 000 x g supernatant from 0.60 g of liver was incubated in 3.0 ml of Bûcher medium with 500 pg of 4-[4-14C]androstene3,17-dione (added in 50 /d of acetone) at 37CC for 8 min in the presence of an NADPH-regcnerating system (Gustafsson Se Stenberg 1974a). Incubations with 5a[4-14C]androstane-3a,17/?-diol were carried out for 10 min using 200 pg of substrate (added in 50 µ of acetone) and microsomal suspension from 0.50 g of liver in 4.0 ml of Bûcher medium containing an NADPH-regenerating system. The incubation condi¬ tions were designed to give linear total conversion of substrate with time and enzyme concentration. Total conversion was not affected by small variations in substrate or cofactor amounts. The incubation mixtures were extracted with chloroform/methanol, 2:1 (v/v), as described previously (Gustafsson Se Stenberg 1974a).
Incubations of The adrenals
4-[4-,4C]androstene-3,17-elione
with adrenals
rendered free from adherent tissue and weighed. They were then transferred to a small vial and 4-[4-14C]androstene-3,17-dione was incubated with the intact adrenals using a micromethod (Goldman et al. 1974): the adrenals were suspended in 200 pi of Bûcher medium to which was added 2.5 pg of the steroid dissolved in 5 «1 of acetone and 10/d of a solution containing 3 //moles of NADH, 3 «moles of NADP, 12.5 /(moles of isocitrate, 10 «1 of isocitric dehydrogenase, 0.02 /(moles of MnCl2 and 400 µ of Bucher medium. NADP, NADH, DL-isocitric acid (trisodium salt) and isocitric dehydrogenase (type IV) were obtained from Sigma Chemical Co. The incubations were carried out for 75 min at 37CC. The conditions described gave conversions linear with respect to time and adrenal weight. The in¬ cubations were terminated by the addition of 400 pi of acetone/ethanol (1:1, v/v). After centrifugation the supernatant was taken off and the adrenals were extracted a second time with 400 /d of acetone/ethanol. This procedure gave a complete extrac¬ tion of all radioactivity in the adrenals. were
Analytical procedure The extracts from incubation with liver subfraction and adrenals were chromato¬ on precoated silica gel plates (250/(, Merck AG., Darmstadt. Germany) using chloroform/ethyl acetate, 4:1 (v/v), as the mobile phase. The radioactive zones were localized by radioautography for 7-10 days, scraped off separately and eluted with 5 ml of methanol. After removal of an aliquot for measurement of radioactivity (Packard Liquid Scintillation Spectrometer Model 4322) the rest was analyzed using radio-gas chromatography (Hewlett-Packard Gas Chromatograph Model 402, equipped with a Barber-Colman Radioactivity monitoring system Model 5190). Zones containing more than one metabolite were thus resolved and individual metabolites could be
graphed
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Table 3. metabolism of 4-[4-14C]androstene-3,17-dione and hepatic 5a-[4-14C]androstane-3a,17/?-diol in neonatally castrated male rats given testosterone propionate between 56 and 69 days of age. Abbreviations: NC ¿5 neonatally cas¬ trated male rats given testosterone propionate when adult; 14DC(5T male rats cas¬ trated at 14 days of age given testosterone propionate when adult; NC(3T, neo¬ natally castrated male rats given testosterone propionate and prolactin when adult. The conversions were calculated from the amounts of radioactivity in the different zones of the thin-layer chromatograms. The values listed at the means + gd. The effects of
prolactin
on
=
=
=
nmoles of metabolites
Enzymes
active
5/?-Reductase 5«-Reductase
17cc-Hydroxysteroid
reducíase
reductase Ratio StWSa-reduced metabolites Ratio 3/?-/5a-reduced metabolites
6/>-Hydroxylase 7«-Hydroxylase 16a-Hydroxylase
14DCc?T
6.64 1.15 17 96.8 ± 17.3
5.28 ± 1.12 63.8 ± 7.9
5.42 ± 0.81 101 ± 40
-18 +4
0.62 ± 0.39
1.05 ± 0.40
0.44
± 0.18
-29
4.05 ± 1.28
-20
1.04 ± 121
2/?-Hydroxylase 7a-Hydroxylase 7/?-Hydroxylase 18-Hydroxylase
4.43
± 0.82
NCcST,
0.153
±
0.045
0.174 ± 0.017
0.131 ± 0.025
-14
0.084 4.94 4.34 3.68
± ± ± ±
0.064 1.30 0.61 1.33
0.150 ± 0.041 11.44 ± 2.60 6.22 ± 2.18 6.82 ± 1.68
0.065 4.53 4.89 3.26
± ± ± ±
-23 -8 +13
on
nmoles of metabolites
5a-A-3a,17^-diol 2a-Hydroxylase
"lo Change with
NCr^T
17/?-Hydroxysteroid
active
10 min
on
A4-3,l 7-dione
Enzymes
formed/mg protein
3.52 0.84 2.52 0.68 1.27
± ± ± ± ±
1.96 0.43 0.21 0.19 1.05
0.017 0.29 0.92 0.64
formed/mg protein
4.94 ± 1.4S 1.53 ± 0.18
2.24 ± 0.24 0.83 ± 0.13 2.56 ± 0.46
2.61 0.68 2.45 0.62 1.08
± ± ± ± ±
0.92 0.11 0.12 0.08 0.26
-11
8 min
-26 -19 -3 -9 -15
The strongest effect of prolactin treatment on adrenal enzyme levels (Table 4) was noted with the 5a-reductase activity. This decreased 20-30 %> in both male and female rats (P < 0.05). The other adrenal enzyme activities were not significantly affected.
Table 4. The effects of prolactin on adrenal metabolism of 4-[4-14C]androstene-3.17-dione in 63-day-old male and female rats treated with prolactin for 7 days. Abbreviations: (5= control males; c5P males given prolactin; Ç control females; Ç females given prolactin. The conversions were calculated from the amounts of radioactivity in the different zones of the thin-layer chromatograms. The values listed are the means ± sd. =
=
=
Change
ÇP
with
¿VA. ?,%> 5a-Reductase
177 ± 13
128 ± 19*
214 ± 33
118 ± 19 18.8 ± 3.8 44.3 ± 7.7
139 ± 12 21.6 ± 3.5 49.7 ± 12.5
98 ± 1 21.5 ± 7.6 33.1 ± 6.3
17/?-Hydroxysteroid reductase
6/5-Hydroxylasc ll^-Hydroxylase *
149
± 29*
87 ± 8 18.8 ± 3.8 34.2 ± 4.6
-22*
-30*
+12 +15 +12
-11 -13 +3
< 0.05.
DISCUSSION
Besides its mammotrophic actions, prolactin may be involved in the control of steroid hormone production, transport and metabolism in several peripheral target organs. Thus, prolactin exerts an effect on the rat corpus luteum by inhibiting the reduction of the 20-oxo group of progesterone resulting in a net increase in progesterone secretion (Wiest 8c Kidwell 1969). Furthermore, Witorsch 8c Kitay (1972) have demonstrated that prolactin reduces the adrenal 5a-reductase activity and have suggested that prolactin plays a physiological role in regulating corticosterone secretion by preventing the intra-adrenal conversion of corticosterone to reduced metabolites. Finally, prolactin has been ascribed a role in regulating the uptake of testosterone into different organs (Grayhack 8c Lebowitz 1967). In the present investigation it was possible to confirm the finding of Witorsch 8c Kitay (1972) that prolactin reduces the activity of the adrenal 5a-reductase. This effect seemed to be quite specific since adrenal 17/j-hydroxysteroid re¬ ductase and 6ß- and 11/5-hydroxylase activities were not significantly affected following treatment with prolactin. In a previous study we have suggested that during the neonatal period testicular androgens irreversibly program the adrenal 5a-reductase activity to a lower, masculine level (Goldman et al. 1974). Kitay et al. (1971) have shown that hypophysectomy results in higher 5areductase levels in the adrenals. In consideration of the results of Mallampati
Johnson (1973) who found high concentrations of prolactin in neonatally androgenized female rats it may be speculated that prolactin could play a role in mediating the hypophyseal regulation of sexual differentiation of adrenal 5a-reductase activity. On the other hand, prolactin seemed to have less significant and unspecific effects on the hepatic enzyme activities. This was true both in experiments where prolactin was administered to intact and to castrated male and female rats. It was also shown that prolactin administration did not increase the androgen responsiveness in neonatally castrated male rats. These experiments would seem to make it less probable that prolactin is identical to either the feminizing or the masculinizing factor postulated to be secreted from the rat hypophysis even if it cannot be excluded that prolactin may be more effective when administered in other doses or together with other hormones. Sc
ACKNOWLEDGMENTS This work
was
(13X-2819)
supported by grants from the Swedish Medical Research Council
and WHO.
REFERENCES
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Berg
A. &
Bergström
Gustafsson J.-Ä. & Stenberg .: Endocrinology 95 (1974c) 891. Kitay J. I., Coyne M. D. & Swygert . H.: Endocrinology 89 (1971) 432. Lowry 0. H., Rosebrough N. J., Fair A. L. & Randall R. J.: J. biol. Chem.
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193
S. Se Johnson D. C: Neuroendocrinology // (1973) 46. Wiest W. G. Se Kidwell W. R. In: McKerns K. W., Ed. The Gonads, Appleton-CenturyCrofts, New York (1969) p. 295. Witorsch R. J. 8- Kitay J. L: Endocrinology 91 (1972) 764. Received
on
April 29th,
1974.
