0013-7227/91/1293-1452$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
Regulation of Cytochrome P450 Aromatase Messenger Ribonucleic Acid and Activity by Steroids and Gonadotropins in Rat Granulosa Cells* SUSAN L. FITZPATRICK AND J O A N N E S. RICHARDS Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT. Estradiol (E) biosynthesis by the cytochrome P450 aromatase (P450arom) enzyme system increases as preovulatory follicles develop and is subsequently reduced by the ovulatory LH surge. To determine the specific effects of gonadotropins and steroids on expression of P450arom in rat granulosa cells, steady state levels of messenger (m) RNA were examined in vivo and in vitro, with the latter also being related to aromatase enzyme activity and cAMP production. P450arom mRNA and activity were induced in granulosa cells by FSH alone in a dose-, time-, and stage-dependent manner. E enhanced the effects of FSH in vivo and in vitro. The synergistic effect of E with FSH (50 ng/ml) was observed in the absence/presence of serum and was mimicked by a similar concentration (20 nM) of testos-
A
romatase cytochrome P450 (P450arom) is required for the conversion of testosterone (T) to estradiol (E). In the mammalian ovary this enzyme has been localized to the granulosa cells of antral follicles (1, 2). Synthesis of E has been characterized extensively in vivo (3) and in vitro (4) by measuring either steroid concentrations or enzymatic activity using androgens as substrates. Factors known to increase aromatase activity include the gonadotropins, forskolin, and 8-bromo cAMP (1-4); factors known to inhibit activity include serum (5), epidermal growth factor (6), transforming growth factor-a (7), and PRL (8,9). In addition, various steroids, including E (4), T (10), and dihydrotestosterone (DHT, (10, 11) have been shown to augment FSH-stimulated aromatase activity, whereas others, such as dexamethasone (Dex, 12, 13) and progesterone (P; 14, 15), appear inhibitory. Receptors for estrogens (3), androgens (16), glucocorticoids (17), and progestins (18) have been identified in rat granulosa cells. Thus, hormonal control of this key enzyme which catalyzes estrogen biosynthesis is complex. Received April 30,1991. Address all correspondence and requests for reprints to: Dr. JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. * This work was supported by NIH Grants HD-16272 and ACSBC627.
terone, dihydrotestosterone, or dexamethasone. In contrast, ovulatory doses of LH (500 ng/ml) or forskolin (10 nu) but not concentrations of progesterone reached in preovulatory follicles (100-1000 nM) acted on differentiated (FSH -I- E) granulosa cells to cause a rapid loss of P450arom mRNA. Whereas cycloheximide prevented the LH/cAMP-mediated decrease in P450arom mRNA in the differentiated cells, enzyme activity remained unaltered during the same 6-h period. Thus, expression of aromatase mRNA in rat granulosa cells is induced primarily by low FSH/cAMP, enhanced by physiological doses of several steroids (except progesterone), and, once induced, can be rapidly inhibited by elevated gonadotropin/cAMP via a pathway requiring protein synthesis. {Endocrinology 129: 1452-1462,1991)
The availability of complementary DNA clones encoding rat (19-22) and human (23-25) aromatase have permitted investigators to begin relating enzyme activity with intracellular levels of steady state messenger (m) RNA. Antibodies have further provided a means to relate mRNA to enzyme content (19, 20). Although parallel increases in P450arom mRNA and protein have been observed in rat preovulatory (PO) follicles (2, 19), the direct effects of peptides, serum, and steroids on granulosa cell expression of P450arom mRNA have not been studied and related to aromatase activity. Such analyses were deemed critical based on two observations: 1) that the LH surge causes aromatase mRNA and protein to decrease in luteinizing follicles; and 2) that in the rat corpus luteum, aromatase mRNA and protein are elevated at midgestation when luteal cell aromatase activity is reduced (2, 19, 20). Conversely, enzyme activity in the corpus luteum increases at parturition when mRNA and enzyme content have declined. Reasons for the divergent relationship among aromatase activity, protein, and mRNA in the corpus luteum remain unclear. We wished to determine if similar phenomena occurred in granulosa cells in response to specific peptides/steroids. Thus, the following study was undertaken to determine the effects of FSH, LH, steroids, and serum on aromatase mRNA and activity in granulosa cells in vivo and in vitro.
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Materials and Methods Animals Intact immature Sprague-Dawley female rats (days 24-26 of age) were obtained from Holtzman (Madison, WI) and kept under a 16-8 h light-dark regimen with food and water ad libitum. Hypophysectomized (H) rats (surgery done on day 26 of age) were obtained from Johnson Laboratory (Chicago, IL) 1 day after surgery. Treatments in vivo
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sodium dodecyl sulfate at room temperature, followed by two 15-min washes in O.lx SSC, 0.1% sodium dodecyl sulfate at 50 C. In some cases blots were stripped of radioactivity with 50% formamide in 10 mM sodium phosphate, pH 6.5, for 3 h at 68 C and reprobed with other labeled cDNAs: 1.5-kb RII/3 (29), the regulatory subunit of A-kinase type II, 2.2-kb C« (30), catalytic subunit of A-kinase, 1.2-kb P450scc, cytochrome P450 cholesterol side-chain cleavage (31). Aromatase assay
In some experiments, intact rats were primed by sc injections of 1.5 mg E 170 (Sigma Chemical Co., St. Louis, MO) in 0.2 ml propylene glycol administered daily for 3 days beginning on day 26 of age (2, 26). H rats were left untreated or were injected with E (1.5 mg/day) alone for 3 days, FSH (NIAMDD oFSH16,1 ng twice daily) alone for 2 days, or E followed by FSH (3). Cells were isolated from all rats the morning after the last day of hormone treatment.
The [3H]Androstenedione assay was performed as described elsewhere (32). [3H]Androstenedione (Androst-4-ene-3,17dione, 25.4 Ci/mmol, NEN, Boston, MA) was added to the media of each dish (0.8 /nCi, 106 nM). Three hours later, half the medium from each group (2 ml) was removed and extracted with 1 ml cold 30% trichloroacetic acid. The supernatant was extracted with 5 ml chloroform, and 2 ml of the supernatant was counted in a liquid scintillation counter. The results were expressed as total picomoles per ml.
Granulosa cell cultures
P and E RIA
Granulosa cells from ovaries of immature rats (26-29 days of age) were isolated as described previously (26). Trypsin (20 jig/ml), soybean trypsin inhibitor (300 ng/m\), and DNase I (100 ng/m\) were used to remove dead cells before plating (26). The cells were harvested and cultured in Dulbecco's modified Eagle's medium:Ham's F12 Nutrient Mixture (1:1, DMEM:F12) supplemented with 20 mM HEPES (pH 7.4), 400 mM glutamine, 100 IU penicillin, and 100 iig/m\ streptomycin, without fetal bovine serum (FBS, Hyclone, Logan, UT) unless noted, as described previously (26). Cells were plated in 35-mm cell culture dishes at 2 X 106 cells per dish (four dishes per treatment) and cultured for up to 48 h at 37 C in 5% CO2 and 95% air. T, P, Dex, and DHT were added at 10 or 20 nM final concentration or as indicated for each experiment. All steroids except E used for injections were purchased from Steraloids, Inc. (Wilton, NH). Ovine FSH was added at 50 ng/ml.
Progesterone (P) and E concentrations in the culture media were determined by RIA as described previously (33) using antisera kindly provided by Dr. Gordon D. Niswender (Department of Physiology and Biophysics, Colorado State University, Fort Collins, CO). [2,4,6,7-3H]E and [1,2,6,7-3H]P were obtained from Amersham Corp. (Arlington Heights, IL).
RNA isolation Total RNA was extracted from granulosa cells in 1% Nonidet P-40-containing buffer (27) phenol-chloroform extracted, ethanol precipitated, and quantified by absorbance at 260 nm. A 1.2-kilobase (kb) rat P450arom cDNA (19) was used to identify and quantify mRNA transcripts. Labeling was performed according to the procedure of Feinberg and Vogelstein (28) using random hexanucleotides as primers and [a-32P] deoxycytidine triphosphate (3000 Ci/mol, ICN, Irvine, CA), to label to a specific activity of more than 1 x 108 cpm/Vg DNA. Northern blot analysis Total RNA (20 ng) was denatured in 45% formamide-5.4% formaldehyde and run at 20 C in a 1.5% agarose gel containing 1.0% formaldehyde and 20 mM Na phosphate, pH 7.0. RNA was transferred to Nylon filters (0.2 or 1.2 ^m; BioDyne, ICN), prehybridized, hybridized to a random primed 1.2-kb P450arom cDNA probe (1 X 106 cpm/ml), and washed according to the specifications of ICN. Blots were washed four times for 5 min each in 2x SSC (0.3 M NaCl, 30 mM sodium citrate), 0.1%
cAMP RIA cAMP was measured by RIA as previously described (33) using 2'-O-succinyl-3-[125I]iodotyrosine cAMP (2000 Ci/mmol, 20,000 cpm/100 /ul, ICN) as the labeled ligand and an anticAMP serum generously provided by Dr. Judith Vaitukaitis (NIH, Bethesda, MD).
Results Effects of E and FSH in vivo on induction of aromatase mRNA To determine the effects of specific hormones on the expression of aromatase in granulosa cells of developing follicles, H rats were injected in vivo with E (1.5 mg/day X 3), FSH (1 /tig twice daily for 2 days), or sequentially with both hormones. Previous studies have documented that these two hormones are required in the rat for the development of preovulatory follicles and differentiation of granulosa cells characterized by increased aromatase activity (1, 3) and the acquisition of receptors for LH (3, 34). Northern blot analyses using a rat P450arom cDNA (19) identified three aromatase transcripts in RNA prepared from granulosa cells isolated from ovaries of H rats treated with both FSH and E (Fig. 1). No P450arom mRNA was detected in samples prepared from granulosa cells of untreated H animals or from rats treated with either hormone alone. These results suggest that both FSH and E are required in vivo for stimulating and
HORMONAL REGULATION OF RAT P450 AROMATASE
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FIG. 1. Induction of aromatase mRNA in granulosa cells of H rats. Granulosa cells were isolated from H immature female rats that were untreated (H) or treated with E (HE, 1.5 mg daily) for 3 days, FSH (HF, 1 fig twice daily) for 2 days, or E followed by F (HEF). RNA was
extracted and 20 fig total RNA analyzed by Northern blot using a rat aromatase cDNA probe as described in Materials and Methods. An RNA ladder was used to determine the size of the aromatase mRNA transcripts by staining the gels with acridine orange before transfer.
FSH (ng/injection) 0
1
5 10 5 0
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few medium and no large antral follicles. In contrast, ovaries of animals given 5 or 10 /ug FSH contained many healthy, large antral follicles. At 50 \ig FSH, the ovaries contained many follicles that appeared in the early stages of luteinization, and the RNA prepared from these ovaries exhibited an intense signal when hybridized with a cDNA to cytochrome P450 side-chain cleavage (P450scc), the enzyme which converts cholesterol to pregnenolone (data not shown). Uterine wts were also increased in these H rats treated with 1, 5,10, and 50 \x% FSH (67 ± 14, 151 ± 15, 157 ± 16, and 160 ± 27 mg), indicating further that these doses of hormone stimulated endogenous production of steroids. This is similar to the increase in uterine wt seen in previous studies (35) when H rats were treated with a different preparation of FSH (103 ± 3.5 mg) compared to untreated rats (21 ± 2.1 mg). Although aromatase was stimulated by FSH, results of these in vivo experiments did not permit us to distinguish unequivocally between the effects of FSH alone and those of FSH combined with steroids (E) produced endogenously. Therefore, the following series of experiments were designed to analyze the effects of specific peptide and steroid hormones on aromatase mRNA and activity using serum-free cultures of granulosa cells harvested either from ovaries of H rats or from ovaries of intact immature rats. Induction of aromatase mRNA in vitro
In Vivo FIG. 2. Dose-dependent effects of FSH on aromatase mRNA in granulosa cells of H immature female rats. H rats were treated in vivo with 0,1, 5, 10, or 50 Mg FSH twice daily for 2 days. RNA was isolated and analyzed as described in Fig. 1.
maintaining aromatase mRNA and that increased mRNA is associated with increased aromatase activity. Dose response of FSH in vivo To determine if higher doses of FSH given alone would be able to stimulate aromatase expression in granulosa cells in the absence of endogenous hormones or exogenous E, H rats were injected with 1, 5, 10, or 50 ng FSH twice daily for 2 days. As shown in Fig. 1, aromatase mRNA was negligible in granulosa cells of untreated H rats and was low in animals injected with 1 ng FSH (Figs. 1 and 2). However, when rats were treated with 5 or 10 Hg FSH, elevated amounts of P450arom mRNA were observed (Fig. 2). At a higher dose (50 fig) of FSH, the level of aromatase expression decreased. Histological sections of ovaries from H rats treated with 1 /xg FSH revealed many atretic small antral follicles. There were
To analyze the effects of FSH alone on granulosa cells isolated from a peptide- and steroid-depleted environment, we first harvested granulosa cells from ovaries of H rats 2 days after surgery and cultured them in serumfree conditions. Aromatase transcripts, negligible in RNA isolated from H rats (Fig. 2), were increased in cells cultured for 48 h with FSH (Fig. 3). This effect was augmented by the addition of E or DHT to the culture media (Fig. 3). These data indicate that FSH is able to induce aromatase expression in granulosa cells in the absence of steroids, but steroids can facilitate this induction. However, because the amount of aromatase mRNA in these granulosa cells of H rats in culture (+ FSH + steroid) did not reach the levels observed in response to FSH in vivo (Figs. 1 and 2), other factors also may be involved. Therefore, we next examined the response to FSH of granulosa cells isolated from ovaries of intact immature rats. Granulosa cells were harvested from ovaries of 26-dayold intact immature rats that were either untreated or had been injected for 3 days with E (1.5 mg/day). Cells were then cultured in serum-free media alone (control), with FSH (50 ng/ml), or E and FSH (10 nM) for 48 h. In cells isolated from untreated animals and cultured with FSH alone, all three aromatase mRNA transcripts were increased over the negligible amount observed in
HORMONAL REGULATION OF RAT P450 AROMATASE
1455
isolated from ovaries of untreated intact rats on day 26 of age exhibited a greater response to FSH or FSH and E in culture than did granulosa cells isolated from ovaries of rats at day 24 of age (data not shown) or from H rats (Fig. 3). These age- and hormone-dependent differences may be associated with differences in the stage of follicular development, levels of endogenous follicular production of steroids, or the presence of other regulators of granulosa cell differentiation. Time course of aromatase induction by FSH in vitro
In
Vitro
FlG. 3. In vitro effects of FSH, E, and DHT on aromatase mRNA in granulosa cells of H rats. Granulosa cells were isolated from ovaries of H rats and cultured in serum-free medium (DMEM:F-12, 1:1) with FSH (50 ng/ml) alone or in combination with either E (10 nM) or DHT (20 nM). RNA was isolated after 48 h and analyzed as described in Fig. 1.
Untreated
E-Primed
FlG. 4. Effects of FSH and E on aromatase mRNA in granulosa cells of untreated and E-primed immature intact rats. Granulosa cells were isolated from 26-day-old rats that were untreated (left panel) or primed with E (1.5 ng daily for 3 days, right panel). Cells were incubated in serum-free media in the absence (Control) or presence of FSH (50 ng/ ml) or FSH + E (10 nM) for 2 days. RNA was extracted from the cultured cells and from granulosa cells of PO follicles and analyzed as described in Fig. 1.
cells cultured in media alone (Fig. 4) or with E alone (data not shown). Greater expression was observed in cells cultured in the presence of both E and FSH. In contrast, FSH alone induced maximal expression of aromatase mRNA in granulosa cells harvested from ovaries of animals that had been primed with E in vivo (Fig. 4). No further increase was observed by the addition of E to the culture medium. The levels of aromatase mRNA induced by FSH and E in these cultured cells from intact rats on day 26 were similar to that observed in granulosa cells isolated from preovulatory follicles of proestrous rats. These results demonstrate that both hormones are required for elevated steady state levels of aromatase mRNA. It is important to note that granulosa cells
To determine the time course of aromatase induction by FSH, granulosa cells from E-primed rats were placed in serum-free culture with FSH (50 ng/ml) for 0, 12, 24, or 48 h. As seen in the Northern blot (Fig. 5), no detectable aromatase transcripts were observed in total RNA extracted from cells before culture [time (t) = 0] or after 12 h. However, by 24 h, FSH had induced detectable levels of aromatase mRNA; by 48 h an elevated response was observed. Thus, induction of aromatase by FSH in these granulosa cells from E-primed rats did not occur rapidly. Rather, the increase in aromatase was delayed until 24-48 h after exposure to FSH. Because FSH can rapidly stimulate cAMP production by these cells (3) and because cAMP is presumed to mediate the effects of FSH, we next determined if the time-dependent changes in aromatase mRNA were associated with changes in A-kinase subunits, one of which (RII/3) is known to be regulated in these cells by E and FSH (36). Specifically, the Northern blot shown in Fig. 5 was stripped and sequentially hybridized with a cDNA to the regulatory subunit RII/3 (29), and with a cDNA to Ca, the ubiquitous catalytic subunit (30). RII/3 mRNA was Hours 0 12 24 48 P450arom mRNA
RllfS — Ccx
-3.2 2.2
FIG. 5. Time course of aromatase mRNA induction by FSH in granulosa cells from E-primed rats. Granulosa cells, isolated from immature rats that had been primed with E (1.5 ng daily for 3 days), were incubated in serum-free media containing FSH (50 ng/ml). RNA was isolated at t = 0, 12, 24, or 48 h of culture. Aromatase mRNA was detected by Northern blot analysis using a rat aromatase cDNA probe. The blot was stripped and rehybridized sequentially with rat cDNA probes for the regulatory (RII/3) and catalytic (Ca) subunits of type II cAMP dependent protein kinase (A-kinase).
HORMONAL REGULATION OF RAT P450 AROMATASE
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present in granulosa cells at the initiation of culture (t = 0) and increased gradually by 48 h to levels approximately 5-fold above those at t = 0. Ca subunit expression increased by 12 h compared to t = 0 and remained at that level at t = 48. Thus, in contrast to P450arom, the A-kinase presumed to be mediating the actions of FSH, is present in granulosa cells isolated from 26-day-old rats, and its mRNA expression is enhanced by the addition of FSH to the culture media. Effects of LH, forskolin, and cycloheximide on aromatase induction by FSH and E Previous studies have indicated that aromatase mRNA and activity are elevated in granulosa cells of PO follicles but are rapidly turned off as a consequence of the LH surge and elevated amounts of cAMP. To determine if we could mimic this effect in culture, granulosa cells of E-primed rats were differentiated in the presence of FSH and E for 2 days. At that time the cells were incubated an additional 6 h in the presence of an ovulatory dose of LH (500 ng/ml), forskolin (Fo, 10 /iM), cycloheximide (CHX, 10 /ig/ml), or CHX + LH. LH and Fo caused a rapid decrease in the steady state levels of aromatase mRNA compared to the control at t = 0 (Fig. 6). In contrast, treatment with CHX alone or LH and CHX resulted in steady state levels of aromatase mRNA that far exceeded that observed in cells cultured with FSH and E (control; Fig. 6). The superinduction occurring in the presence of CHX (+ E + FSH) and the greater effect
kb -3.3 P450
arom mRNA
-2.6 -1.9
Rllfl
-3.2
of CHX in the presence of elevated LH suggest that steady state levels of aromatase mRNA in granulosa cells are submaximal. The results further suggest that the synthesis of a negative regulatory protein is an important factor. To determine if the effects of LH, Fo, and CHX were specific for P450arom mRNA, the blot was reprobed with other cDNAs. mRNA for RII|8 exhibited a pattern similar to that of aromatase mRNA, whereas P450scc was induced 2-fold by LH, Fo, or CHX + LH and 4-fold by CHX alone (Fig. 6). The presence of LH, Fo, CHX, or CHX + LH for only 6 h had no significant effect on aromatase activity or E synthesis (Table 1), suggesting that enzyme levels were not effected. In contrast, the high dose of LH and Fo superimposed on FSH and E for 6 h stimulated marked increases in both cAMP and P (Table 1), with cAMP reaching approximately 10- to 100-fold above that observed with FSH only (control). CHX alone added to cultures containing FSH + E also increased cAMP production but caused P biosynthesis to decrease. Thus granulosa cells isolated from ovaries of immature rats can differentiate in culture in the presence of FSH + E and achieve increased amounts of steady state levels of aromatase mRNA. These differentiated cells can then respond to LH or Fo and exhibit functional changes identical to granulosa cells of preovulatory follicles in vivo in response to the ovulatory LH surge. Effect of androgens on induction of aromatase expression Steroids in addition to E have been shown to alter granulosa cell function and aromatase activity. These include androgens (T, DHT) (10, 11), glucocorticoids (Dex) (12,13), and progestins (P) (14,15). To determine if these steroids modify enzyme activity directly or alter cellular levels of aromatase mRNA, the following experiments were done. Granulosa cells from untreated and E-primed immature rats were cultured with 20 nM androgens and 50 ng/ TABLE 1. Aromatase activity and production of cAMP, E, or P in cultured granulosa cells treated with LH, Fo, CHX, or CHX + LH Hormone treatment
P450 sec
-2.0
FIG. 6. Effects of LH and CHX on aromatase mRNA in differentiated granulosa cells cultured with FSH and E. Granulosa cells were isolated from day 26 immature rats and cultured in serum-free media with FSH (50 ng/ml) and E (10 nM) for 42 h. Cells were then incubated for 6 h (42-48 h) with FSH + E alone (Control) or in the presence of LH (500 ng/ml), forskolin (10 M M), CHX (10 jig/ml), or CHX + LH. RNA was isolated and aromatase mRNA detected by Northern blot analysis using a rat aromatase cDNA probe. The filter was stripped and rehybridized with a rat cDNA encoding the regulatory subunit of type II Akinase (RII/3) or cholesterol side-chain cleavage cytochrome P450 enzyme (P450scc).
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Control (F + E) + LH + Fo + CHX + CHX + LH
Aromatase (pmol/ml) 1.1 ± 1.2 ± 1.1 ± 0.8 ± 0.9 ±
0.2 0.2 0.03 0.01 0.07
cAMP (pmol/ml)
E
P
(ng/ml)
(ng/ml)
55 600 7000 200 1400
1.8 2.3 4.4 2.9 4.5
14 213 179 16 11
Media from granulosa cell cultures described in Fig. 6 were assayed by RIA for the presence of cAMP, E, or P after 6 h hormone treatment. Media from four dishes were pooled and the results expressed as concentration per sample group. Aromatase activity was determined by the conversion of [3H]androstenedione to 3H2O (picomoles of [3H] androstenedione converted to 3H2O per ml media) as described in Materials and Methods, in each dish of granulosa cells. Results of four dishes per group were expressed as average ± SE.
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HORMONAL REGULATION OF RAT P450 AROMATASE
ml FSH for 48 h. [3H] Androstenedione was added for the last 3 h of culture to measure aromatase activity (32). In unprimed animals, FSH combined with E, T, or DHT was able to induce aromatase mRNA above that seen with FSH alone (Fig. 7A). No aromatase mRNA was seen in the presence of DHT alone (Fig. 7A, upper). In contrast, FSH alone induced a marked increase in mRNA in cells from E-primed rats, and this was not augmented by the presence of steroids (T, DHT, E) (Fig. 7A, lower). The ability of DHT, a nonaromatizable androgen, to induce aromatase expression in the presence of FSH as shown here and in granulosa cells from H rats (Fig. 2), suggests that androgens are not acting solely via conversion to E but by an androgen-specific pathway. Stimulatory effects of androgens on E production have also been reported using granulosa cell cultures established in 5% FBS (11). To determine if aromatase mRNA was also regulated under these culture conditions, granulosa cells were cultured with either FSH alone or FSH -I- T in the presence of 5% FBS. FBS inhibited the ability of FSH to increase aromatase mRNA in granulosa cells from both unprimed and E-primed rats (Fig. 7A). Addition of T, however, restored the ability of FSH to induce aromatase mRNA (Fig. 7A). In all treatments, changes in aromatase mRNA were associated with similar changes in aromatase activity (Fig. 7B, data for primed rats is not shown) and E production. T, DHT, and E all increased the FSH-induced aromatase activity compared to FSH alone, with T having the greatest effect, whereas serum inhibited FSH-induced aromatase activity and E synthesis. Because T could override the negative effect of serum, the results suggest that FBS contains an inhibitor(s) of FSH action that is (are) reversed by the addition of T. P secretion was also enhanced by the synergistic actions of FSH and T, DHT, or E but was lower in all cells cultured with serum (data not shown). Likewise, FSH stimulation of cAMP (Fig. 7C) was augmented by the presence of T, DHT, or E and inhibited by the addition of FBS to the culture media. Thus, one important role of steroids (androgens and estrogens) appears to be that of maintaining the FSH response system in the presence of inhibitory substances in serum. Effect of glucocorticoids and progestins on aromatase expression and activity Dex previously has been shown to decrease aromatase activity in cultured rat granulosa cells (12,13). To determine if Dex modified the ability of FSH to induce aromatase mRNA and activity in our system, granulosa cells from intact immature rats were isolated and cultured with FSH and either Dex or T. As shown in Fig. 8A, T and Dex augmented induction of aromatase mRNA and increased enzyme activity 4.4-fold and 3.6-fold, respec-
X
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Hormone Treatment FIG. 7. Effects of FSH and steroids on aromatase mRNA, activity, and cAMP levels in cultured granulosa cells. Granulosa cells were isolated from 26-day-old rats that had been untreated or primed with E (1.5 mg) daily for 3 days (panel A). Cells were incubated in serum-free media with DHT (20 nM), FSH (50 ng/ml), or FSH in combination with DHT, T (20 nM), or E (20 nM). Cells were also incubated in medium containing 5% FBS with and FSH alone (50 ng/ml) or in combination with T (20 nM). After 48 h in culture, RNA was extracted and aromatase mRNA detected by Northern blot analysis (20 Mg total RNA/lane). Aromatase activity (panel B) and cAMP levels (panel C) were measured as described in Table 1 for granulosa cells depicted in panel A (unprimed rats).
HORMONAL REGULATION OF RAT P450 AROMATASE
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kb 3.3 2.6
1.9 B 1.0-
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Endo • 1991 Voll29-No3
other experiments, indicating that Dex, as well as T and E, is able to enhance FSH stimulation of cAMP, aromatase mRNA and activity, and progesterone biosynthesis in these cultured granulosa cells. To determine if Dex may be acting via a P response pathway and to analyze the effects of P at elevated concentrations observed at the time of ovulation and in corpora lutea, P was added in increasing concentrations (20-1000 nM) to granulosa cell cultures. As in previous experiments, T enhanced FSH stimulation of aromatase mRNA (Fig. 9A) and enzyme activity (Fig. 9B). P had no effect on the response of these cells to FSH + T when added either at low (PL> 20 nM), medium (PM, 100 nM), or high (PH, 1000 nM) concentrations for 48 h of culture or at the high concentration added at 42 h (PH, lane 4). Low and medium concentrations of P also did not alter the ability of FSH alone to increase aromatase mRNA and activity (Fig. 9, A and B). In contrast, the high concentration of P (1000 nM) did augment induction of aromatase by FSH alone (Fig. 9A, lane 10), FSH + E (lanes 12 and 13), and forskolin (lanes 14 and 15). In these same cultures the high dose of P increased cAMP levels (Fig. 9C) 15-fold (F + P), 4-fold (F + E + P), and 4-fold (Fo) above FSH alone (Fig. 9C). P had no effect on cAMP in the other treatment groups.
Discussion 30-
20-
10-
F+T
F • Dex
Hormone Treatment
FIG. 8. Effects of FSH, T, and Dex on aromatase mRNA, activity, and cAMP levels in cultured granulosa cells. Granulosa cells were isolated from 26-day-old immature rats and incubated in serum-free media in the presence of FSH (F, 50 ng/ml) alone or in combination with T (20 nM) or Dex (20 nM). RNA was isolated after 48 h of culture and aromatase mRNA detected by Northern blot analysis using a rat aromatase cDNA probe {panel A). Aromatase activity {panel B) and cAMP levels {panel C) were measured as described in Table 1.
tively, above FSH alone (Fig. 8B). T and Dex in the presence of FSH also increased the concentrations of cAMP 4- and 10-fold, respectively (Fig. 8C), and P biosynthesis (data not shown) greater than 10-fold over that with FSH alone. Similar results were seen in two
Results of this study document that FSH alone can induce expression of aromatase mRNA in granulosa cells isolated either from untreated H rats or intact immature rats and cultured in serum-free medium. Induction of aromatase by FSH was influenced by the age of the rats from which the cells were obtained, the addition of specific steroids, and the presence of serum in the culture media. Under a variety of culture conditions tested, changes in aromatase mRNA were consistently associated with parallel changes in aromatase activity, suggesting that activity reflects enzyme levels. These results contrast with those observed previously in the pregnant rat corpus luteum in which elevated levels of mRNA and protein were associated with low enzyme activity (19, 20). Results from in vivo and in vitro studies also document that E enhances the ability of FSH to increase aromatase mRNA and activity in rat granulosa cells but has no effect alone. These results support previous studies in which E and FSH have been shown to synergistically increase aromatase enzyme activity and E biosynthesis (4) as well as expression of mRNA and protein for other genes such as LH receptor (34), P450scc (37), RII0 (36), inhibin (38, 39), and tissue plasminogen activator (40). Measurement of cAMP in cells cultured with FSH alone compared to FSH plus E showed that the presence
HORMONAL REGULATION OF RAT P450 AROMATASE
1 2
3
4
5
6
7
8
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10 7' 11 12 13 14
1.9
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3
4
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8
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I o.
Vol. 129, No. 3 Printed in U.S.A.
Regulation of Cytochrome P450 Aromatase Messenger Ribonucleic Acid and Activity by Steroids and Gonadotropins in Rat Granulosa Cells* SUSAN L. FITZPATRICK AND J O A N N E S. RICHARDS Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT. Estradiol (E) biosynthesis by the cytochrome P450 aromatase (P450arom) enzyme system increases as preovulatory follicles develop and is subsequently reduced by the ovulatory LH surge. To determine the specific effects of gonadotropins and steroids on expression of P450arom in rat granulosa cells, steady state levels of messenger (m) RNA were examined in vivo and in vitro, with the latter also being related to aromatase enzyme activity and cAMP production. P450arom mRNA and activity were induced in granulosa cells by FSH alone in a dose-, time-, and stage-dependent manner. E enhanced the effects of FSH in vivo and in vitro. The synergistic effect of E with FSH (50 ng/ml) was observed in the absence/presence of serum and was mimicked by a similar concentration (20 nM) of testos-
A
romatase cytochrome P450 (P450arom) is required for the conversion of testosterone (T) to estradiol (E). In the mammalian ovary this enzyme has been localized to the granulosa cells of antral follicles (1, 2). Synthesis of E has been characterized extensively in vivo (3) and in vitro (4) by measuring either steroid concentrations or enzymatic activity using androgens as substrates. Factors known to increase aromatase activity include the gonadotropins, forskolin, and 8-bromo cAMP (1-4); factors known to inhibit activity include serum (5), epidermal growth factor (6), transforming growth factor-a (7), and PRL (8,9). In addition, various steroids, including E (4), T (10), and dihydrotestosterone (DHT, (10, 11) have been shown to augment FSH-stimulated aromatase activity, whereas others, such as dexamethasone (Dex, 12, 13) and progesterone (P; 14, 15), appear inhibitory. Receptors for estrogens (3), androgens (16), glucocorticoids (17), and progestins (18) have been identified in rat granulosa cells. Thus, hormonal control of this key enzyme which catalyzes estrogen biosynthesis is complex. Received April 30,1991. Address all correspondence and requests for reprints to: Dr. JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. * This work was supported by NIH Grants HD-16272 and ACSBC627.
terone, dihydrotestosterone, or dexamethasone. In contrast, ovulatory doses of LH (500 ng/ml) or forskolin (10 nu) but not concentrations of progesterone reached in preovulatory follicles (100-1000 nM) acted on differentiated (FSH -I- E) granulosa cells to cause a rapid loss of P450arom mRNA. Whereas cycloheximide prevented the LH/cAMP-mediated decrease in P450arom mRNA in the differentiated cells, enzyme activity remained unaltered during the same 6-h period. Thus, expression of aromatase mRNA in rat granulosa cells is induced primarily by low FSH/cAMP, enhanced by physiological doses of several steroids (except progesterone), and, once induced, can be rapidly inhibited by elevated gonadotropin/cAMP via a pathway requiring protein synthesis. {Endocrinology 129: 1452-1462,1991)
The availability of complementary DNA clones encoding rat (19-22) and human (23-25) aromatase have permitted investigators to begin relating enzyme activity with intracellular levels of steady state messenger (m) RNA. Antibodies have further provided a means to relate mRNA to enzyme content (19, 20). Although parallel increases in P450arom mRNA and protein have been observed in rat preovulatory (PO) follicles (2, 19), the direct effects of peptides, serum, and steroids on granulosa cell expression of P450arom mRNA have not been studied and related to aromatase activity. Such analyses were deemed critical based on two observations: 1) that the LH surge causes aromatase mRNA and protein to decrease in luteinizing follicles; and 2) that in the rat corpus luteum, aromatase mRNA and protein are elevated at midgestation when luteal cell aromatase activity is reduced (2, 19, 20). Conversely, enzyme activity in the corpus luteum increases at parturition when mRNA and enzyme content have declined. Reasons for the divergent relationship among aromatase activity, protein, and mRNA in the corpus luteum remain unclear. We wished to determine if similar phenomena occurred in granulosa cells in response to specific peptides/steroids. Thus, the following study was undertaken to determine the effects of FSH, LH, steroids, and serum on aromatase mRNA and activity in granulosa cells in vivo and in vitro.
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HORMONAL REGULATION OF RAT P450 AROMATASE
Materials and Methods Animals Intact immature Sprague-Dawley female rats (days 24-26 of age) were obtained from Holtzman (Madison, WI) and kept under a 16-8 h light-dark regimen with food and water ad libitum. Hypophysectomized (H) rats (surgery done on day 26 of age) were obtained from Johnson Laboratory (Chicago, IL) 1 day after surgery. Treatments in vivo
1453
sodium dodecyl sulfate at room temperature, followed by two 15-min washes in O.lx SSC, 0.1% sodium dodecyl sulfate at 50 C. In some cases blots were stripped of radioactivity with 50% formamide in 10 mM sodium phosphate, pH 6.5, for 3 h at 68 C and reprobed with other labeled cDNAs: 1.5-kb RII/3 (29), the regulatory subunit of A-kinase type II, 2.2-kb C« (30), catalytic subunit of A-kinase, 1.2-kb P450scc, cytochrome P450 cholesterol side-chain cleavage (31). Aromatase assay
In some experiments, intact rats were primed by sc injections of 1.5 mg E 170 (Sigma Chemical Co., St. Louis, MO) in 0.2 ml propylene glycol administered daily for 3 days beginning on day 26 of age (2, 26). H rats were left untreated or were injected with E (1.5 mg/day) alone for 3 days, FSH (NIAMDD oFSH16,1 ng twice daily) alone for 2 days, or E followed by FSH (3). Cells were isolated from all rats the morning after the last day of hormone treatment.
The [3H]Androstenedione assay was performed as described elsewhere (32). [3H]Androstenedione (Androst-4-ene-3,17dione, 25.4 Ci/mmol, NEN, Boston, MA) was added to the media of each dish (0.8 /nCi, 106 nM). Three hours later, half the medium from each group (2 ml) was removed and extracted with 1 ml cold 30% trichloroacetic acid. The supernatant was extracted with 5 ml chloroform, and 2 ml of the supernatant was counted in a liquid scintillation counter. The results were expressed as total picomoles per ml.
Granulosa cell cultures
P and E RIA
Granulosa cells from ovaries of immature rats (26-29 days of age) were isolated as described previously (26). Trypsin (20 jig/ml), soybean trypsin inhibitor (300 ng/m\), and DNase I (100 ng/m\) were used to remove dead cells before plating (26). The cells were harvested and cultured in Dulbecco's modified Eagle's medium:Ham's F12 Nutrient Mixture (1:1, DMEM:F12) supplemented with 20 mM HEPES (pH 7.4), 400 mM glutamine, 100 IU penicillin, and 100 iig/m\ streptomycin, without fetal bovine serum (FBS, Hyclone, Logan, UT) unless noted, as described previously (26). Cells were plated in 35-mm cell culture dishes at 2 X 106 cells per dish (four dishes per treatment) and cultured for up to 48 h at 37 C in 5% CO2 and 95% air. T, P, Dex, and DHT were added at 10 or 20 nM final concentration or as indicated for each experiment. All steroids except E used for injections were purchased from Steraloids, Inc. (Wilton, NH). Ovine FSH was added at 50 ng/ml.
Progesterone (P) and E concentrations in the culture media were determined by RIA as described previously (33) using antisera kindly provided by Dr. Gordon D. Niswender (Department of Physiology and Biophysics, Colorado State University, Fort Collins, CO). [2,4,6,7-3H]E and [1,2,6,7-3H]P were obtained from Amersham Corp. (Arlington Heights, IL).
RNA isolation Total RNA was extracted from granulosa cells in 1% Nonidet P-40-containing buffer (27) phenol-chloroform extracted, ethanol precipitated, and quantified by absorbance at 260 nm. A 1.2-kilobase (kb) rat P450arom cDNA (19) was used to identify and quantify mRNA transcripts. Labeling was performed according to the procedure of Feinberg and Vogelstein (28) using random hexanucleotides as primers and [a-32P] deoxycytidine triphosphate (3000 Ci/mol, ICN, Irvine, CA), to label to a specific activity of more than 1 x 108 cpm/Vg DNA. Northern blot analysis Total RNA (20 ng) was denatured in 45% formamide-5.4% formaldehyde and run at 20 C in a 1.5% agarose gel containing 1.0% formaldehyde and 20 mM Na phosphate, pH 7.0. RNA was transferred to Nylon filters (0.2 or 1.2 ^m; BioDyne, ICN), prehybridized, hybridized to a random primed 1.2-kb P450arom cDNA probe (1 X 106 cpm/ml), and washed according to the specifications of ICN. Blots were washed four times for 5 min each in 2x SSC (0.3 M NaCl, 30 mM sodium citrate), 0.1%
cAMP RIA cAMP was measured by RIA as previously described (33) using 2'-O-succinyl-3-[125I]iodotyrosine cAMP (2000 Ci/mmol, 20,000 cpm/100 /ul, ICN) as the labeled ligand and an anticAMP serum generously provided by Dr. Judith Vaitukaitis (NIH, Bethesda, MD).
Results Effects of E and FSH in vivo on induction of aromatase mRNA To determine the effects of specific hormones on the expression of aromatase in granulosa cells of developing follicles, H rats were injected in vivo with E (1.5 mg/day X 3), FSH (1 /tig twice daily for 2 days), or sequentially with both hormones. Previous studies have documented that these two hormones are required in the rat for the development of preovulatory follicles and differentiation of granulosa cells characterized by increased aromatase activity (1, 3) and the acquisition of receptors for LH (3, 34). Northern blot analyses using a rat P450arom cDNA (19) identified three aromatase transcripts in RNA prepared from granulosa cells isolated from ovaries of H rats treated with both FSH and E (Fig. 1). No P450arom mRNA was detected in samples prepared from granulosa cells of untreated H animals or from rats treated with either hormone alone. These results suggest that both FSH and E are required in vivo for stimulating and
HORMONAL REGULATION OF RAT P450 AROMATASE
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FIG. 1. Induction of aromatase mRNA in granulosa cells of H rats. Granulosa cells were isolated from H immature female rats that were untreated (H) or treated with E (HE, 1.5 mg daily) for 3 days, FSH (HF, 1 fig twice daily) for 2 days, or E followed by F (HEF). RNA was
extracted and 20 fig total RNA analyzed by Northern blot using a rat aromatase cDNA probe as described in Materials and Methods. An RNA ladder was used to determine the size of the aromatase mRNA transcripts by staining the gels with acridine orange before transfer.
FSH (ng/injection) 0
1
5 10 5 0
Endo • 1991 Voll29«No3
few medium and no large antral follicles. In contrast, ovaries of animals given 5 or 10 /ug FSH contained many healthy, large antral follicles. At 50 \ig FSH, the ovaries contained many follicles that appeared in the early stages of luteinization, and the RNA prepared from these ovaries exhibited an intense signal when hybridized with a cDNA to cytochrome P450 side-chain cleavage (P450scc), the enzyme which converts cholesterol to pregnenolone (data not shown). Uterine wts were also increased in these H rats treated with 1, 5,10, and 50 \x% FSH (67 ± 14, 151 ± 15, 157 ± 16, and 160 ± 27 mg), indicating further that these doses of hormone stimulated endogenous production of steroids. This is similar to the increase in uterine wt seen in previous studies (35) when H rats were treated with a different preparation of FSH (103 ± 3.5 mg) compared to untreated rats (21 ± 2.1 mg). Although aromatase was stimulated by FSH, results of these in vivo experiments did not permit us to distinguish unequivocally between the effects of FSH alone and those of FSH combined with steroids (E) produced endogenously. Therefore, the following series of experiments were designed to analyze the effects of specific peptide and steroid hormones on aromatase mRNA and activity using serum-free cultures of granulosa cells harvested either from ovaries of H rats or from ovaries of intact immature rats. Induction of aromatase mRNA in vitro
In Vivo FIG. 2. Dose-dependent effects of FSH on aromatase mRNA in granulosa cells of H immature female rats. H rats were treated in vivo with 0,1, 5, 10, or 50 Mg FSH twice daily for 2 days. RNA was isolated and analyzed as described in Fig. 1.
maintaining aromatase mRNA and that increased mRNA is associated with increased aromatase activity. Dose response of FSH in vivo To determine if higher doses of FSH given alone would be able to stimulate aromatase expression in granulosa cells in the absence of endogenous hormones or exogenous E, H rats were injected with 1, 5, 10, or 50 ng FSH twice daily for 2 days. As shown in Fig. 1, aromatase mRNA was negligible in granulosa cells of untreated H rats and was low in animals injected with 1 ng FSH (Figs. 1 and 2). However, when rats were treated with 5 or 10 Hg FSH, elevated amounts of P450arom mRNA were observed (Fig. 2). At a higher dose (50 fig) of FSH, the level of aromatase expression decreased. Histological sections of ovaries from H rats treated with 1 /xg FSH revealed many atretic small antral follicles. There were
To analyze the effects of FSH alone on granulosa cells isolated from a peptide- and steroid-depleted environment, we first harvested granulosa cells from ovaries of H rats 2 days after surgery and cultured them in serumfree conditions. Aromatase transcripts, negligible in RNA isolated from H rats (Fig. 2), were increased in cells cultured for 48 h with FSH (Fig. 3). This effect was augmented by the addition of E or DHT to the culture media (Fig. 3). These data indicate that FSH is able to induce aromatase expression in granulosa cells in the absence of steroids, but steroids can facilitate this induction. However, because the amount of aromatase mRNA in these granulosa cells of H rats in culture (+ FSH + steroid) did not reach the levels observed in response to FSH in vivo (Figs. 1 and 2), other factors also may be involved. Therefore, we next examined the response to FSH of granulosa cells isolated from ovaries of intact immature rats. Granulosa cells were harvested from ovaries of 26-dayold intact immature rats that were either untreated or had been injected for 3 days with E (1.5 mg/day). Cells were then cultured in serum-free media alone (control), with FSH (50 ng/ml), or E and FSH (10 nM) for 48 h. In cells isolated from untreated animals and cultured with FSH alone, all three aromatase mRNA transcripts were increased over the negligible amount observed in
HORMONAL REGULATION OF RAT P450 AROMATASE
1455
isolated from ovaries of untreated intact rats on day 26 of age exhibited a greater response to FSH or FSH and E in culture than did granulosa cells isolated from ovaries of rats at day 24 of age (data not shown) or from H rats (Fig. 3). These age- and hormone-dependent differences may be associated with differences in the stage of follicular development, levels of endogenous follicular production of steroids, or the presence of other regulators of granulosa cell differentiation. Time course of aromatase induction by FSH in vitro
In
Vitro
FlG. 3. In vitro effects of FSH, E, and DHT on aromatase mRNA in granulosa cells of H rats. Granulosa cells were isolated from ovaries of H rats and cultured in serum-free medium (DMEM:F-12, 1:1) with FSH (50 ng/ml) alone or in combination with either E (10 nM) or DHT (20 nM). RNA was isolated after 48 h and analyzed as described in Fig. 1.
Untreated
E-Primed
FlG. 4. Effects of FSH and E on aromatase mRNA in granulosa cells of untreated and E-primed immature intact rats. Granulosa cells were isolated from 26-day-old rats that were untreated (left panel) or primed with E (1.5 ng daily for 3 days, right panel). Cells were incubated in serum-free media in the absence (Control) or presence of FSH (50 ng/ ml) or FSH + E (10 nM) for 2 days. RNA was extracted from the cultured cells and from granulosa cells of PO follicles and analyzed as described in Fig. 1.
cells cultured in media alone (Fig. 4) or with E alone (data not shown). Greater expression was observed in cells cultured in the presence of both E and FSH. In contrast, FSH alone induced maximal expression of aromatase mRNA in granulosa cells harvested from ovaries of animals that had been primed with E in vivo (Fig. 4). No further increase was observed by the addition of E to the culture medium. The levels of aromatase mRNA induced by FSH and E in these cultured cells from intact rats on day 26 were similar to that observed in granulosa cells isolated from preovulatory follicles of proestrous rats. These results demonstrate that both hormones are required for elevated steady state levels of aromatase mRNA. It is important to note that granulosa cells
To determine the time course of aromatase induction by FSH, granulosa cells from E-primed rats were placed in serum-free culture with FSH (50 ng/ml) for 0, 12, 24, or 48 h. As seen in the Northern blot (Fig. 5), no detectable aromatase transcripts were observed in total RNA extracted from cells before culture [time (t) = 0] or after 12 h. However, by 24 h, FSH had induced detectable levels of aromatase mRNA; by 48 h an elevated response was observed. Thus, induction of aromatase by FSH in these granulosa cells from E-primed rats did not occur rapidly. Rather, the increase in aromatase was delayed until 24-48 h after exposure to FSH. Because FSH can rapidly stimulate cAMP production by these cells (3) and because cAMP is presumed to mediate the effects of FSH, we next determined if the time-dependent changes in aromatase mRNA were associated with changes in A-kinase subunits, one of which (RII/3) is known to be regulated in these cells by E and FSH (36). Specifically, the Northern blot shown in Fig. 5 was stripped and sequentially hybridized with a cDNA to the regulatory subunit RII/3 (29), and with a cDNA to Ca, the ubiquitous catalytic subunit (30). RII/3 mRNA was Hours 0 12 24 48 P450arom mRNA
RllfS — Ccx
-3.2 2.2
FIG. 5. Time course of aromatase mRNA induction by FSH in granulosa cells from E-primed rats. Granulosa cells, isolated from immature rats that had been primed with E (1.5 ng daily for 3 days), were incubated in serum-free media containing FSH (50 ng/ml). RNA was isolated at t = 0, 12, 24, or 48 h of culture. Aromatase mRNA was detected by Northern blot analysis using a rat aromatase cDNA probe. The blot was stripped and rehybridized sequentially with rat cDNA probes for the regulatory (RII/3) and catalytic (Ca) subunits of type II cAMP dependent protein kinase (A-kinase).
HORMONAL REGULATION OF RAT P450 AROMATASE
1456
present in granulosa cells at the initiation of culture (t = 0) and increased gradually by 48 h to levels approximately 5-fold above those at t = 0. Ca subunit expression increased by 12 h compared to t = 0 and remained at that level at t = 48. Thus, in contrast to P450arom, the A-kinase presumed to be mediating the actions of FSH, is present in granulosa cells isolated from 26-day-old rats, and its mRNA expression is enhanced by the addition of FSH to the culture media. Effects of LH, forskolin, and cycloheximide on aromatase induction by FSH and E Previous studies have indicated that aromatase mRNA and activity are elevated in granulosa cells of PO follicles but are rapidly turned off as a consequence of the LH surge and elevated amounts of cAMP. To determine if we could mimic this effect in culture, granulosa cells of E-primed rats were differentiated in the presence of FSH and E for 2 days. At that time the cells were incubated an additional 6 h in the presence of an ovulatory dose of LH (500 ng/ml), forskolin (Fo, 10 /iM), cycloheximide (CHX, 10 /ig/ml), or CHX + LH. LH and Fo caused a rapid decrease in the steady state levels of aromatase mRNA compared to the control at t = 0 (Fig. 6). In contrast, treatment with CHX alone or LH and CHX resulted in steady state levels of aromatase mRNA that far exceeded that observed in cells cultured with FSH and E (control; Fig. 6). The superinduction occurring in the presence of CHX (+ E + FSH) and the greater effect
kb -3.3 P450
arom mRNA
-2.6 -1.9
Rllfl
-3.2
of CHX in the presence of elevated LH suggest that steady state levels of aromatase mRNA in granulosa cells are submaximal. The results further suggest that the synthesis of a negative regulatory protein is an important factor. To determine if the effects of LH, Fo, and CHX were specific for P450arom mRNA, the blot was reprobed with other cDNAs. mRNA for RII|8 exhibited a pattern similar to that of aromatase mRNA, whereas P450scc was induced 2-fold by LH, Fo, or CHX + LH and 4-fold by CHX alone (Fig. 6). The presence of LH, Fo, CHX, or CHX + LH for only 6 h had no significant effect on aromatase activity or E synthesis (Table 1), suggesting that enzyme levels were not effected. In contrast, the high dose of LH and Fo superimposed on FSH and E for 6 h stimulated marked increases in both cAMP and P (Table 1), with cAMP reaching approximately 10- to 100-fold above that observed with FSH only (control). CHX alone added to cultures containing FSH + E also increased cAMP production but caused P biosynthesis to decrease. Thus granulosa cells isolated from ovaries of immature rats can differentiate in culture in the presence of FSH + E and achieve increased amounts of steady state levels of aromatase mRNA. These differentiated cells can then respond to LH or Fo and exhibit functional changes identical to granulosa cells of preovulatory follicles in vivo in response to the ovulatory LH surge. Effect of androgens on induction of aromatase expression Steroids in addition to E have been shown to alter granulosa cell function and aromatase activity. These include androgens (T, DHT) (10, 11), glucocorticoids (Dex) (12,13), and progestins (P) (14,15). To determine if these steroids modify enzyme activity directly or alter cellular levels of aromatase mRNA, the following experiments were done. Granulosa cells from untreated and E-primed immature rats were cultured with 20 nM androgens and 50 ng/ TABLE 1. Aromatase activity and production of cAMP, E, or P in cultured granulosa cells treated with LH, Fo, CHX, or CHX + LH Hormone treatment
P450 sec
-2.0
FIG. 6. Effects of LH and CHX on aromatase mRNA in differentiated granulosa cells cultured with FSH and E. Granulosa cells were isolated from day 26 immature rats and cultured in serum-free media with FSH (50 ng/ml) and E (10 nM) for 42 h. Cells were then incubated for 6 h (42-48 h) with FSH + E alone (Control) or in the presence of LH (500 ng/ml), forskolin (10 M M), CHX (10 jig/ml), or CHX + LH. RNA was isolated and aromatase mRNA detected by Northern blot analysis using a rat aromatase cDNA probe. The filter was stripped and rehybridized with a rat cDNA encoding the regulatory subunit of type II Akinase (RII/3) or cholesterol side-chain cleavage cytochrome P450 enzyme (P450scc).
Endo• 1991 Voll29«No3
Control (F + E) + LH + Fo + CHX + CHX + LH
Aromatase (pmol/ml) 1.1 ± 1.2 ± 1.1 ± 0.8 ± 0.9 ±
0.2 0.2 0.03 0.01 0.07
cAMP (pmol/ml)
E
P
(ng/ml)
(ng/ml)
55 600 7000 200 1400
1.8 2.3 4.4 2.9 4.5
14 213 179 16 11
Media from granulosa cell cultures described in Fig. 6 were assayed by RIA for the presence of cAMP, E, or P after 6 h hormone treatment. Media from four dishes were pooled and the results expressed as concentration per sample group. Aromatase activity was determined by the conversion of [3H]androstenedione to 3H2O (picomoles of [3H] androstenedione converted to 3H2O per ml media) as described in Materials and Methods, in each dish of granulosa cells. Results of four dishes per group were expressed as average ± SE.
1457
HORMONAL REGULATION OF RAT P450 AROMATASE
ml FSH for 48 h. [3H] Androstenedione was added for the last 3 h of culture to measure aromatase activity (32). In unprimed animals, FSH combined with E, T, or DHT was able to induce aromatase mRNA above that seen with FSH alone (Fig. 7A). No aromatase mRNA was seen in the presence of DHT alone (Fig. 7A, upper). In contrast, FSH alone induced a marked increase in mRNA in cells from E-primed rats, and this was not augmented by the presence of steroids (T, DHT, E) (Fig. 7A, lower). The ability of DHT, a nonaromatizable androgen, to induce aromatase expression in the presence of FSH as shown here and in granulosa cells from H rats (Fig. 2), suggests that androgens are not acting solely via conversion to E but by an androgen-specific pathway. Stimulatory effects of androgens on E production have also been reported using granulosa cell cultures established in 5% FBS (11). To determine if aromatase mRNA was also regulated under these culture conditions, granulosa cells were cultured with either FSH alone or FSH -I- T in the presence of 5% FBS. FBS inhibited the ability of FSH to increase aromatase mRNA in granulosa cells from both unprimed and E-primed rats (Fig. 7A). Addition of T, however, restored the ability of FSH to induce aromatase mRNA (Fig. 7A). In all treatments, changes in aromatase mRNA were associated with similar changes in aromatase activity (Fig. 7B, data for primed rats is not shown) and E production. T, DHT, and E all increased the FSH-induced aromatase activity compared to FSH alone, with T having the greatest effect, whereas serum inhibited FSH-induced aromatase activity and E synthesis. Because T could override the negative effect of serum, the results suggest that FBS contains an inhibitor(s) of FSH action that is (are) reversed by the addition of T. P secretion was also enhanced by the synergistic actions of FSH and T, DHT, or E but was lower in all cells cultured with serum (data not shown). Likewise, FSH stimulation of cAMP (Fig. 7C) was augmented by the presence of T, DHT, or E and inhibited by the addition of FBS to the culture media. Thus, one important role of steroids (androgens and estrogens) appears to be that of maintaining the FSH response system in the presence of inhibitory substances in serum. Effect of glucocorticoids and progestins on aromatase expression and activity Dex previously has been shown to decrease aromatase activity in cultured rat granulosa cells (12,13). To determine if Dex modified the ability of FSH to induce aromatase mRNA and activity in our system, granulosa cells from intact immature rats were isolated and cultured with FSH and either Dex or T. As shown in Fig. 8A, T and Dex augmented induction of aromatase mRNA and increased enzyme activity 4.4-fold and 3.6-fold, respec-
X
kb •3.3
-2.6 •1.9
Unprimed kb •3.3
-2.6 -1.9 E-Primed
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1.5-
Q.
T
1.0-
T 0.5-
o
:-:-:-:•
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Hormone Treatment FIG. 7. Effects of FSH and steroids on aromatase mRNA, activity, and cAMP levels in cultured granulosa cells. Granulosa cells were isolated from 26-day-old rats that had been untreated or primed with E (1.5 mg) daily for 3 days (panel A). Cells were incubated in serum-free media with DHT (20 nM), FSH (50 ng/ml), or FSH in combination with DHT, T (20 nM), or E (20 nM). Cells were also incubated in medium containing 5% FBS with and FSH alone (50 ng/ml) or in combination with T (20 nM). After 48 h in culture, RNA was extracted and aromatase mRNA detected by Northern blot analysis (20 Mg total RNA/lane). Aromatase activity (panel B) and cAMP levels (panel C) were measured as described in Table 1 for granulosa cells depicted in panel A (unprimed rats).
HORMONAL REGULATION OF RAT P450 AROMATASE
1458
kb 3.3 2.6
1.9 B 1.0-
T y / / / / *
V/V. xxxx /// V/V
0.5
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xxxx
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F + Dex
Hormone Treatment
Endo • 1991 Voll29-No3
other experiments, indicating that Dex, as well as T and E, is able to enhance FSH stimulation of cAMP, aromatase mRNA and activity, and progesterone biosynthesis in these cultured granulosa cells. To determine if Dex may be acting via a P response pathway and to analyze the effects of P at elevated concentrations observed at the time of ovulation and in corpora lutea, P was added in increasing concentrations (20-1000 nM) to granulosa cell cultures. As in previous experiments, T enhanced FSH stimulation of aromatase mRNA (Fig. 9A) and enzyme activity (Fig. 9B). P had no effect on the response of these cells to FSH + T when added either at low (PL> 20 nM), medium (PM, 100 nM), or high (PH, 1000 nM) concentrations for 48 h of culture or at the high concentration added at 42 h (PH, lane 4). Low and medium concentrations of P also did not alter the ability of FSH alone to increase aromatase mRNA and activity (Fig. 9, A and B). In contrast, the high concentration of P (1000 nM) did augment induction of aromatase by FSH alone (Fig. 9A, lane 10), FSH + E (lanes 12 and 13), and forskolin (lanes 14 and 15). In these same cultures the high dose of P increased cAMP levels (Fig. 9C) 15-fold (F + P), 4-fold (F + E + P), and 4-fold (Fo) above FSH alone (Fig. 9C). P had no effect on cAMP in the other treatment groups.
Discussion 30-
20-
10-
F+T
F • Dex
Hormone Treatment
FIG. 8. Effects of FSH, T, and Dex on aromatase mRNA, activity, and cAMP levels in cultured granulosa cells. Granulosa cells were isolated from 26-day-old immature rats and incubated in serum-free media in the presence of FSH (F, 50 ng/ml) alone or in combination with T (20 nM) or Dex (20 nM). RNA was isolated after 48 h of culture and aromatase mRNA detected by Northern blot analysis using a rat aromatase cDNA probe {panel A). Aromatase activity {panel B) and cAMP levels {panel C) were measured as described in Table 1.
tively, above FSH alone (Fig. 8B). T and Dex in the presence of FSH also increased the concentrations of cAMP 4- and 10-fold, respectively (Fig. 8C), and P biosynthesis (data not shown) greater than 10-fold over that with FSH alone. Similar results were seen in two
Results of this study document that FSH alone can induce expression of aromatase mRNA in granulosa cells isolated either from untreated H rats or intact immature rats and cultured in serum-free medium. Induction of aromatase by FSH was influenced by the age of the rats from which the cells were obtained, the addition of specific steroids, and the presence of serum in the culture media. Under a variety of culture conditions tested, changes in aromatase mRNA were consistently associated with parallel changes in aromatase activity, suggesting that activity reflects enzyme levels. These results contrast with those observed previously in the pregnant rat corpus luteum in which elevated levels of mRNA and protein were associated with low enzyme activity (19, 20). Results from in vivo and in vitro studies also document that E enhances the ability of FSH to increase aromatase mRNA and activity in rat granulosa cells but has no effect alone. These results support previous studies in which E and FSH have been shown to synergistically increase aromatase enzyme activity and E biosynthesis (4) as well as expression of mRNA and protein for other genes such as LH receptor (34), P450scc (37), RII0 (36), inhibin (38, 39), and tissue plasminogen activator (40). Measurement of cAMP in cells cultured with FSH alone compared to FSH plus E showed that the presence
HORMONAL REGULATION OF RAT P450 AROMATASE
1 2
3
4
5
6
7
8
9
10 7' 11 12 13 14
1.9
FSH
1
2
3
4
5
6
7
Fo
8
9
10 1 1 1 2 13 14
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