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The dynamics of activation of steroidogenic pathways in cultured neonatal rat ovaries
0022-473l/X3iO30353-04$03.00~0 Copyright 0 1983 Pergamon Press Ltd
J. srcrod Bioc~/im. Vol. IX. No. 3. pp. 353-356, 1983 Printed in Great Britain. All rights reserved
THE DYNAMICS OF ACTIVATION OF STEROIDOGENIC PATHWAYS IN CULTURED NEONATAL RAT OVARIES B.
FUNKENSTEIN*
and A. NIMROD?
Department of Hormone Research, The Weizmann institute of Science, Rehovot 76100, Israel (Received 15 J&J: 1982) SUMMARY Ovaries of 4-day-old rats can be stimulated by cyclic AMP to produce steroids in culture. The timecourse of activation of the various steps in the biosynthetic pathway was studied in vitro by the conversion of various steroid precursors to their prospective products. Ovaries (6 per dish) cultured with (Bu)~ CAMP (1 mM) without exogenous precursors synthesized low amounts of progesterone (P), testosterone (Te) and estradiol-17/l (E,) during the first 8 h, and the production gradually increased thereafter. P production was not affected by exogenous cholesterol, but greatly increased by addition of pregnenolone already during the first 8 h even in the absence of (Bu& CAMP (11.0 + 1.9 ng/dish.S h with pregnenolone vs ~0.2 ng/dish. 8 h with (Bu)~ CAMP alone). Addition of P resulted in increased Te production only after 32 h (0.29 + 0.02 vs 0.10 rt 0.02 ng/dish 8 h, respectively), while exogenous 17ff-hydroxy-progesterone (17x-OH-P) significantly enhanced Te formation already during the first 8 h (0.38 + 0.09 vs 0.04 & 0.01 ng/dish’8 h, respectively). Aromatase activity, as measured by E, formation from exogenous Te, was stimulated by (Bu)~cAMP already during the first 8 h (105 ~5 6 pg/disb’8 h with (Bu)r CAMP + Te vs 47 $- 5 with Te alone), and reached a maximum (200 _+ 37 pg/dish.8 h) after 16 h of culture. The results indicate that (i) 38-01 dehydrogenase is relatively active and is not rate limiting in neonatal ovaries; (ii) the aromatase and 17:20 lyase can be activated relatively quickly (8 h) and (iii) cholesterol cleavage enzyme and 17a-hydroxylase are induced more slowly and thus appear to be rate-limiting.
INTRODUCTION At birth, the capability of the rat ovary to produce hormonal steroids is limited [l-7]. The ability to synthesize steroids develops later on, and during the second week of life the ovary begins to secrete appreciable amounts of estrogen El, 21. The development of steroidogenic capability was shown by various in vitro studies [3-71, and is also evident from histochemical demonstration of 3P-HSD activity at the age of 9 days [8,9]. While at 4 days of age the ovaries are not responsive to LH in terms of steroidogenesis, they can be stimulated in vitro by cyclic AMP to produce steroids [S-7]. The dynamics of this developmental process are not known, and the involvement of the various biosynthetic steps of the steroidogenic pathways is not clear. In this study, we have maint~ned ovaries from 4-day-old rats in culture with (Buf, CAMP in order to examine the dynamics of the activation of the steroidogenic apparatus in some detail.
MATERIALS AND METHODS
Animals Wistar-derived rats of the Hormone Research Departmental Colony were used. The day on which
* Present address: Department of Obstetrics and Gynecology, The University of Texas, Dallas, Texas 75235, U.S.A. t To whom all correspondence should be addressed.
pups were found with their mother Day 1 of life.
was designated
Culture sysmn Ovaries from 4-day-old rats were taken for organ culture as described before [7]. Briefly, 6 ovaries were put onto a stainless steel grid in a Falcon organ-culture dish (No. 3037) containing 1 ml medium (Eagle’s Minimum Essential Medium with modified Earle’s salts supplemented with 1 mM HEPES, 10% fetal calf serum, 5 pg insulin and 1 pg/ml gentamycin) and cultured at 37°C under an oxygen/air mixture (1: 1). Wherever indicated, the medium included (Bu)~ CAMP (1 mM). In addition, some dishes contained the steroid precursors cholesterol, pregnenolone, I7cc-hydroxyprogesterone or testosterone (0.5 or 1 pg/ml; added in ethanol to 0.1% final corm., Ikapharm, Ramat Gan, Israel). Media were changed every 8 h, and culture was continued up to 48 h. Spent media were extracted with diethyl-ether and steroid content was measured by radioimmunoassay as described before [7, lo]. The antibody used for assay of Te (against Testosterone-7-BSA; [lo]) had a significant cross-reaction with DHT. Therefore, some samples were also purified by thin layer chromatography (benzene-acetone, 5: 1, v/v) before assay. The values obtained before and after chromatography did, not differ significantly (P < 0.9 by paired r-test; n = 30). Statistical significance of the resuits was examined t-test and by Student-N~umanby Student Keuhls[ 1 t] multiple-range test.
353
(ELI), CAMP
(Imhl)
+
(Bu I2 CAMP
Preg (I~glrnl)
(Bu j2 CAMP +
chol (Ipg/mJ)
Preg
4 4 2 Fig. 1. El%ecect of (13ufz cAMP. cholesterol (cholj and pregnenolone (preg) on progesterone production by ovaries of+day-old rats, Ovaries (6 per dish) were cultured for 48 h in the presence of added compounds as specified. Media were replaced every 8 h, and progesterone content was assayed. Values represent the mean 5 SEM for N replicate cultures (IV > 3) or mean + range for duplicates (N = 2). N=
2
RESULTS
As described before [73> steroid production by ovaries cultured without addition of the cyclic nucleotide was negligible (
production
The ability of ovaries to further metabolize P was examined by culturing them with (Bu)~ CAMP in combination with exogenous P or 17r-OH-P. In the presence of (Bu), CAMP alone, the ovaries accumulated low amounts of Te during the first 8 h of culture and the amounts produced gradually increased thereafter (Fig. 2), Addition of P together with (Bu), CAMP did not affect Te production during the first 32 h of
culture, but a significant increase in Te formation was found thereafter (P -C 0.001 for P I fB& CAMP vs (Bu), CAMP alone; Fig. 2). By contrast, exogenous 17cr-OH-P was converted into Te by ovaries stimulated with (Bu), CAMP already during the first 8 h of culture, and this activity increased only slightly afterwards (Fig. 2). Ovaries that have not been stimulated by (Buf, CAMP exhibited a low activity of conversion of 17r-OH-P to Te throughout the entire cuItute period. Estrogen production Estradiol 17@ (E,) secretion by ovaries cultured with (Bu), CAMP alone was very low during the first 8 h and gradually increased thereafter (from 23 + 12 pg/dish. 8 h during O-8 h to 77 f 16 during 4C-48 h ; P < 0.05 ; Fig. 3). In order to assay aromatase activity in these ovaries, exogenous Te was added as substrate. Ovaries that have not been stimulated by (Bu), CAMP exhibited significant aromatase activity during the first 24 h of culture, and this activity slightly decreased thereafter (Fig. 3). (BuIz CAMP markedly stimulated aromatase activity already durthe first 8h of culture (48 + 9 vs ing 106 i: 12pgjdish.8 h for Te alone vs (au), CAMP + Te, respectively; P -=z0.001; Fig. 3) to reach a plateau (approx. 200 pg/dish.8 h) during the subsequent culture periods. Ovaries that have not been stimulated by (Bu)~ CAMP were also capable of utilizing exogenous 17c(-OH-P for estrogen formation (presumably via androgen formation), resulting in a constant low rate
355
S~~roidogenicpathways in neonatal ovary
ClMW, tima (h):
0
16 32
46
0
16
32
48
0
16
32
46
e
16
)
48
+ 17a--OH-P
6
7
3
32
(Bu),eAMP
IO.5 or Ip p/ml
N=
0
ITa-OH-P
iBut,cAMP
Fig. 2. Effect of (Bu)~CAMP, progesterone (prog) and 17a-hydroxy-progesterone (17x-OH-P) on testosterone production by ovaries of 4-day-old rats. For details, see legend to Fig. 1.
activity is demonstrable at this stage in spite of the fact that the total cellular protein kinase activity was shown to be responsive to CAMP only during the second week of life [12,133. At least 3 biosynthetic steps were shown to be stimulatable by the cyclic nucleotide: (i) the conversion of cholesterol to C-21 steroids; (ii) the cleavage of the side-chain of 17a-OH-P to form androgens; and (iii) the aromatization of androgens to estrogens. By contrast, the activity of a key enzyme in steroid biosynthesis--the 3fl-HSD-was found to be relatively active in un-
of Ez accumulation throughout the culture period (Fig. 3). Addition of (Bu)~ CAMP to the medium resulted in an increase of Ez formation during the 24118 h culture period, (P < 0.05) but not during the first 24 h (Fig. 3). DISCUSSION
The results of the present study show that a cyclicAMP-responsive steroidogenic apparatus is present in the ovary even at the age of 4 days. This biochemical
300
250 i
cuitun ~~ timrfh): 0
16 3248
fBul,
CAMP
O-- 16 32 46
6
To
16 32 46
0
16 32 &
fi%&cAMP
17a-OH-P
l*
tlpglmlt
+ (IIMI N:
4
(lpglml) 2
5
2
0
IS Y
46
(%ut,cAMP + 17a-OH-P 2
Fig. 3. Effect of (Bu)~ CAMP, 17a-hydroxy-progesterone (17a-OH-P) and testosterone (Te) on estradioL17fl production by ovaries from 4-day-old rats. For details, see Fig. I.
356
B. FI \LI.USII.I\
stimulated 4-day ovaries, and did not appear to be rate-limiting. Its activity was further stimulated by CAMP only after 40 h of culture (Fig. I). This finding does not accord well with histochemical studies that have shown 3/CHSD activity in ovaries only 9 days post partum 18.91. This discrepancy is probably due to the relative insensitivity of the determination of the enzymatic activity by histochemical methods. The ineffectiveness of exogenous cholesterol in enhancing P production may indicate ample supply of endogenous substrate for steroidogenesis. However, it could be the result of improper penetration of the exogenous sterol into the tissue. Although the experiments reported here did not inelude detailed kinetic studies. they supply some information on the dynamics of the induction process. The first enzymatic steps that were activated in the neonatal tissue are the 17:20 lyase and aromatase; these enzymes reached half-maximal activities during the first 8 h of exposure to (Bu)* CAMP. The cleavage of cholesterol to give P was induced more slowly: the stimulation of this activity became significant only after 8 h. and it gradually increased thereafter (Fig. 1). An even more sustained response was seen for the 17~hydroxylase. which became active only after 32 h of culture. It can be argued that a prolonged use of exogenous CAMP for the stimulation of the steroidogenic apparatus may be accompanied by rapid degradation of the cyclic nucleotide by phosphodiesterase. thus causing a decrease in its cellular concentration during culture. Hence, the temporal relationship of the various enzymatic steps obtained in our culture system could be a reflection of different sensitivities of the various pathways to stimulation. However, the relative stability of the dibutyryl derivative compared with the native nucleotide and the fact that a high concentration (1 mM) of (Bu), CAMP was added every 8 h make this possibility unlikely. The prolonged lag periods found in our system for the stimulatory effect of CAMP are inconsistent with the classical rapid activation of protein kinase and phosphorylation. thus suggesting that &-non synthesis of some enzymes may be involved. In fact, activation by CAMP of the transcription of some P-450 enzymes in adrenal tissue has recently been demonstrated (Simpson and Funkenstein. unpublished). The differences in the response of the various enzymatic steps to the cyclic nucleotide may represent an inherent characteristic of the cellular steroidogenic mechanism. Alternatively. these may be a reflection of
and A. NIMKOI)
synthetic processes occurring in different cell types. The cellular localization of the various steroidogenic processes in the infantile ovary during development remains to be elucidated.
REFERElVCES
I. Diihler K. D. and Wuttke W.: Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. Endo~r;~ ology 97 (1975) 898~907. H. M. A., De Greef W. J. and Uilen2. Meijs-Roelofs
3,
4,
5.
6.
7.
8.
9.
broek J. Th. J.: Plasma progesterone and its relationship to serum follicle stimulating hormone in immature female rats. J. Endocr. 64 (1975) 329-336. Lamprecht S. A., Kohen F.. Ausher J.. Zor U. and Lindner H. R.: Hormonal stimulation of oestradiol 178 release from the rat ovary during early postnatal development. J. Endocr. 68 (1976) 343. 344. Levina S. E., Gyevai A. and Horvath E.: Responsiveness of the ovary to gonadotropins in pre- and perinatal life: oestrogen secretion in tissue organ culture. J. Endocr. 65 (1975) 219-233. Lamprecht S. A., Funkenstein B. and Nimrod A.: Ontogeny of responsiveness to gonadotrophins and prostaglandin E in the neonatal rat ovary. Alan. Biol. A/1im. Biochem. Bioph~,s. 19 (1979) 1351-l 361, Nimrod A. and Funkenstein B.: Control of resposiveness to gonatropins and steroids in the neonatal rat ovary. Adc. Biosci. 25 (1979) 343%359. Funkenstem B.. Nimrod A. and Lindner H. R.: The development of steroidogenic capability and responsiveness to gonadotropins in cultured neonatal rat ovaries. Endouinoloqy 106 (I 980) 9% 106. Presl J., Jirasek J., Horsky J. and Henzl M.: Observations on steroid-3/(-ol dehydrogenase activity in the ovary during early postnatal development in the rat. J. Endocr. 31 ( 1965) 293-294. Schlegel R. J.. Farias E.. Russo N. C.. Moore J. R. and Gardner L. I.: Structural changes in the fetal gonads and gonaducts during maturation of an enzyme. steroid 3/j-ol-dehydrogenase, in the gonads. adrenal cortex and placenta of fetal rats. Endoc~rinohy 81 (1967)
565- 572.
10. Bauminger
S.. Kohen F. and Lindner H. R. Steroids as haptens: Optimal design of antigens for the formation of antibodies to steroid hormones. d. steroid Biochn. 5, (1974)
739- 797.
I I. Sokal R. S. and Rohlf F. J. Bionlefr~~ Freeman. Franctsco ( 1969). 12. Lamprecht
San
S. A., Zor U., Tsafriri A. and Lindner H. R.: Action of prostaglandin EZ and of luteinizing hormone on ovarian adenylate cyclase. protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J. Endocr. 57 (1973) 217m 233. R. A., 13. De Angelo A. B., Schweppe J. S.. Jungmann Huber P. and Eppenberger U.: Ovarian cyclic adenosine monophosphate-dependent protein kinase activity: Ontogeny and effect of gonadotropins. Endocrinology 97 (I 975) 1509% 1520.
J. srcrod Bioc~/im. Vol. IX. No. 3. pp. 353-356, 1983 Printed in Great Britain. All rights reserved
THE DYNAMICS OF ACTIVATION OF STEROIDOGENIC PATHWAYS IN CULTURED NEONATAL RAT OVARIES B.
FUNKENSTEIN*
and A. NIMROD?
Department of Hormone Research, The Weizmann institute of Science, Rehovot 76100, Israel (Received 15 J&J: 1982) SUMMARY Ovaries of 4-day-old rats can be stimulated by cyclic AMP to produce steroids in culture. The timecourse of activation of the various steps in the biosynthetic pathway was studied in vitro by the conversion of various steroid precursors to their prospective products. Ovaries (6 per dish) cultured with (Bu)~ CAMP (1 mM) without exogenous precursors synthesized low amounts of progesterone (P), testosterone (Te) and estradiol-17/l (E,) during the first 8 h, and the production gradually increased thereafter. P production was not affected by exogenous cholesterol, but greatly increased by addition of pregnenolone already during the first 8 h even in the absence of (Bu& CAMP (11.0 + 1.9 ng/dish.S h with pregnenolone vs ~0.2 ng/dish. 8 h with (Bu)~ CAMP alone). Addition of P resulted in increased Te production only after 32 h (0.29 + 0.02 vs 0.10 rt 0.02 ng/dish 8 h, respectively), while exogenous 17ff-hydroxy-progesterone (17x-OH-P) significantly enhanced Te formation already during the first 8 h (0.38 + 0.09 vs 0.04 & 0.01 ng/dish’8 h, respectively). Aromatase activity, as measured by E, formation from exogenous Te, was stimulated by (Bu)~cAMP already during the first 8 h (105 ~5 6 pg/disb’8 h with (Bu)r CAMP + Te vs 47 $- 5 with Te alone), and reached a maximum (200 _+ 37 pg/dish.8 h) after 16 h of culture. The results indicate that (i) 38-01 dehydrogenase is relatively active and is not rate limiting in neonatal ovaries; (ii) the aromatase and 17:20 lyase can be activated relatively quickly (8 h) and (iii) cholesterol cleavage enzyme and 17a-hydroxylase are induced more slowly and thus appear to be rate-limiting.
INTRODUCTION At birth, the capability of the rat ovary to produce hormonal steroids is limited [l-7]. The ability to synthesize steroids develops later on, and during the second week of life the ovary begins to secrete appreciable amounts of estrogen El, 21. The development of steroidogenic capability was shown by various in vitro studies [3-71, and is also evident from histochemical demonstration of 3P-HSD activity at the age of 9 days [8,9]. While at 4 days of age the ovaries are not responsive to LH in terms of steroidogenesis, they can be stimulated in vitro by cyclic AMP to produce steroids [S-7]. The dynamics of this developmental process are not known, and the involvement of the various biosynthetic steps of the steroidogenic pathways is not clear. In this study, we have maint~ned ovaries from 4-day-old rats in culture with (Buf, CAMP in order to examine the dynamics of the activation of the steroidogenic apparatus in some detail.
MATERIALS AND METHODS
Animals Wistar-derived rats of the Hormone Research Departmental Colony were used. The day on which
* Present address: Department of Obstetrics and Gynecology, The University of Texas, Dallas, Texas 75235, U.S.A. t To whom all correspondence should be addressed.
pups were found with their mother Day 1 of life.
was designated
Culture sysmn Ovaries from 4-day-old rats were taken for organ culture as described before [7]. Briefly, 6 ovaries were put onto a stainless steel grid in a Falcon organ-culture dish (No. 3037) containing 1 ml medium (Eagle’s Minimum Essential Medium with modified Earle’s salts supplemented with 1 mM HEPES, 10% fetal calf serum, 5 pg insulin and 1 pg/ml gentamycin) and cultured at 37°C under an oxygen/air mixture (1: 1). Wherever indicated, the medium included (Bu)~ CAMP (1 mM). In addition, some dishes contained the steroid precursors cholesterol, pregnenolone, I7cc-hydroxyprogesterone or testosterone (0.5 or 1 pg/ml; added in ethanol to 0.1% final corm., Ikapharm, Ramat Gan, Israel). Media were changed every 8 h, and culture was continued up to 48 h. Spent media were extracted with diethyl-ether and steroid content was measured by radioimmunoassay as described before [7, lo]. The antibody used for assay of Te (against Testosterone-7-BSA; [lo]) had a significant cross-reaction with DHT. Therefore, some samples were also purified by thin layer chromatography (benzene-acetone, 5: 1, v/v) before assay. The values obtained before and after chromatography did, not differ significantly (P < 0.9 by paired r-test; n = 30). Statistical significance of the resuits was examined t-test and by Student-N~umanby Student Keuhls[ 1 t] multiple-range test.
353
(ELI), CAMP
(Imhl)
+
(Bu I2 CAMP
Preg (I~glrnl)
(Bu j2 CAMP +
chol (Ipg/mJ)
Preg
4 4 2 Fig. 1. El%ecect of (13ufz cAMP. cholesterol (cholj and pregnenolone (preg) on progesterone production by ovaries of+day-old rats, Ovaries (6 per dish) were cultured for 48 h in the presence of added compounds as specified. Media were replaced every 8 h, and progesterone content was assayed. Values represent the mean 5 SEM for N replicate cultures (IV > 3) or mean + range for duplicates (N = 2). N=
2
RESULTS
As described before [73> steroid production by ovaries cultured without addition of the cyclic nucleotide was negligible (
production
The ability of ovaries to further metabolize P was examined by culturing them with (Bu)~ CAMP in combination with exogenous P or 17r-OH-P. In the presence of (Bu), CAMP alone, the ovaries accumulated low amounts of Te during the first 8 h of culture and the amounts produced gradually increased thereafter (Fig. 2), Addition of P together with (Bu), CAMP did not affect Te production during the first 32 h of
culture, but a significant increase in Te formation was found thereafter (P -C 0.001 for P I fB& CAMP vs (Bu), CAMP alone; Fig. 2). By contrast, exogenous 17cr-OH-P was converted into Te by ovaries stimulated with (Bu), CAMP already during the first 8 h of culture, and this activity increased only slightly afterwards (Fig. 2). Ovaries that have not been stimulated by (Buf, CAMP exhibited a low activity of conversion of 17r-OH-P to Te throughout the entire cuItute period. Estrogen production Estradiol 17@ (E,) secretion by ovaries cultured with (Bu), CAMP alone was very low during the first 8 h and gradually increased thereafter (from 23 + 12 pg/dish. 8 h during O-8 h to 77 f 16 during 4C-48 h ; P < 0.05 ; Fig. 3). In order to assay aromatase activity in these ovaries, exogenous Te was added as substrate. Ovaries that have not been stimulated by (Bu), CAMP exhibited significant aromatase activity during the first 24 h of culture, and this activity slightly decreased thereafter (Fig. 3). (BuIz CAMP markedly stimulated aromatase activity already durthe first 8h of culture (48 + 9 vs ing 106 i: 12pgjdish.8 h for Te alone vs (au), CAMP + Te, respectively; P -=z0.001; Fig. 3) to reach a plateau (approx. 200 pg/dish.8 h) during the subsequent culture periods. Ovaries that have not been stimulated by (Bu)~ CAMP were also capable of utilizing exogenous 17c(-OH-P for estrogen formation (presumably via androgen formation), resulting in a constant low rate
355
S~~roidogenicpathways in neonatal ovary
ClMW, tima (h):
0
16 32
46
0
16
32
48
0
16
32
46
e
16
)
48
+ 17a--OH-P
6
7
3
32
(Bu),eAMP
IO.5 or Ip p/ml
N=
0
ITa-OH-P
iBut,cAMP
Fig. 2. Effect of (Bu)~CAMP, progesterone (prog) and 17a-hydroxy-progesterone (17x-OH-P) on testosterone production by ovaries of 4-day-old rats. For details, see legend to Fig. 1.
activity is demonstrable at this stage in spite of the fact that the total cellular protein kinase activity was shown to be responsive to CAMP only during the second week of life [12,133. At least 3 biosynthetic steps were shown to be stimulatable by the cyclic nucleotide: (i) the conversion of cholesterol to C-21 steroids; (ii) the cleavage of the side-chain of 17a-OH-P to form androgens; and (iii) the aromatization of androgens to estrogens. By contrast, the activity of a key enzyme in steroid biosynthesis--the 3fl-HSD-was found to be relatively active in un-
of Ez accumulation throughout the culture period (Fig. 3). Addition of (Bu)~ CAMP to the medium resulted in an increase of Ez formation during the 24118 h culture period, (P < 0.05) but not during the first 24 h (Fig. 3). DISCUSSION
The results of the present study show that a cyclicAMP-responsive steroidogenic apparatus is present in the ovary even at the age of 4 days. This biochemical
300
250 i
cuitun ~~ timrfh): 0
16 3248
fBul,
CAMP
O-- 16 32 46
6
To
16 32 46
0
16 32 &
fi%&cAMP
17a-OH-P
l*
tlpglmlt
+ (IIMI N:
4
(lpglml) 2
5
2
0
IS Y
46
(%ut,cAMP + 17a-OH-P 2
Fig. 3. Effect of (Bu)~ CAMP, 17a-hydroxy-progesterone (17a-OH-P) and testosterone (Te) on estradioL17fl production by ovaries from 4-day-old rats. For details, see Fig. I.
356
B. FI \LI.USII.I\
stimulated 4-day ovaries, and did not appear to be rate-limiting. Its activity was further stimulated by CAMP only after 40 h of culture (Fig. I). This finding does not accord well with histochemical studies that have shown 3/CHSD activity in ovaries only 9 days post partum 18.91. This discrepancy is probably due to the relative insensitivity of the determination of the enzymatic activity by histochemical methods. The ineffectiveness of exogenous cholesterol in enhancing P production may indicate ample supply of endogenous substrate for steroidogenesis. However, it could be the result of improper penetration of the exogenous sterol into the tissue. Although the experiments reported here did not inelude detailed kinetic studies. they supply some information on the dynamics of the induction process. The first enzymatic steps that were activated in the neonatal tissue are the 17:20 lyase and aromatase; these enzymes reached half-maximal activities during the first 8 h of exposure to (Bu)* CAMP. The cleavage of cholesterol to give P was induced more slowly: the stimulation of this activity became significant only after 8 h. and it gradually increased thereafter (Fig. 1). An even more sustained response was seen for the 17~hydroxylase. which became active only after 32 h of culture. It can be argued that a prolonged use of exogenous CAMP for the stimulation of the steroidogenic apparatus may be accompanied by rapid degradation of the cyclic nucleotide by phosphodiesterase. thus causing a decrease in its cellular concentration during culture. Hence, the temporal relationship of the various enzymatic steps obtained in our culture system could be a reflection of different sensitivities of the various pathways to stimulation. However, the relative stability of the dibutyryl derivative compared with the native nucleotide and the fact that a high concentration (1 mM) of (Bu), CAMP was added every 8 h make this possibility unlikely. The prolonged lag periods found in our system for the stimulatory effect of CAMP are inconsistent with the classical rapid activation of protein kinase and phosphorylation. thus suggesting that &-non synthesis of some enzymes may be involved. In fact, activation by CAMP of the transcription of some P-450 enzymes in adrenal tissue has recently been demonstrated (Simpson and Funkenstein. unpublished). The differences in the response of the various enzymatic steps to the cyclic nucleotide may represent an inherent characteristic of the cellular steroidogenic mechanism. Alternatively. these may be a reflection of
and A. NIMKOI)
synthetic processes occurring in different cell types. The cellular localization of the various steroidogenic processes in the infantile ovary during development remains to be elucidated.
REFERElVCES
I. Diihler K. D. and Wuttke W.: Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. Endo~r;~ ology 97 (1975) 898~907. H. M. A., De Greef W. J. and Uilen2. Meijs-Roelofs
3,
4,
5.
6.
7.
8.
9.
broek J. Th. J.: Plasma progesterone and its relationship to serum follicle stimulating hormone in immature female rats. J. Endocr. 64 (1975) 329-336. Lamprecht S. A., Kohen F.. Ausher J.. Zor U. and Lindner H. R.: Hormonal stimulation of oestradiol 178 release from the rat ovary during early postnatal development. J. Endocr. 68 (1976) 343. 344. Levina S. E., Gyevai A. and Horvath E.: Responsiveness of the ovary to gonadotropins in pre- and perinatal life: oestrogen secretion in tissue organ culture. J. Endocr. 65 (1975) 219-233. Lamprecht S. A., Funkenstein B. and Nimrod A.: Ontogeny of responsiveness to gonadotrophins and prostaglandin E in the neonatal rat ovary. Alan. Biol. A/1im. Biochem. Bioph~,s. 19 (1979) 1351-l 361, Nimrod A. and Funkenstein B.: Control of resposiveness to gonatropins and steroids in the neonatal rat ovary. Adc. Biosci. 25 (1979) 343%359. Funkenstem B.. Nimrod A. and Lindner H. R.: The development of steroidogenic capability and responsiveness to gonadotropins in cultured neonatal rat ovaries. Endouinoloqy 106 (I 980) 9% 106. Presl J., Jirasek J., Horsky J. and Henzl M.: Observations on steroid-3/(-ol dehydrogenase activity in the ovary during early postnatal development in the rat. J. Endocr. 31 ( 1965) 293-294. Schlegel R. J.. Farias E.. Russo N. C.. Moore J. R. and Gardner L. I.: Structural changes in the fetal gonads and gonaducts during maturation of an enzyme. steroid 3/j-ol-dehydrogenase, in the gonads. adrenal cortex and placenta of fetal rats. Endoc~rinohy 81 (1967)
565- 572.
10. Bauminger
S.. Kohen F. and Lindner H. R. Steroids as haptens: Optimal design of antigens for the formation of antibodies to steroid hormones. d. steroid Biochn. 5, (1974)
739- 797.
I I. Sokal R. S. and Rohlf F. J. Bionlefr~~ Freeman. Franctsco ( 1969). 12. Lamprecht
San
S. A., Zor U., Tsafriri A. and Lindner H. R.: Action of prostaglandin EZ and of luteinizing hormone on ovarian adenylate cyclase. protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J. Endocr. 57 (1973) 217m 233. R. A., 13. De Angelo A. B., Schweppe J. S.. Jungmann Huber P. and Eppenberger U.: Ovarian cyclic adenosine monophosphate-dependent protein kinase activity: Ontogeny and effect of gonadotropins. Endocrinology 97 (I 975) 1509% 1520.