| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
-Hydroxylase-17/20 Lyase (CYP17) Messenger Ribonucleic Acid in Hamster Primary Interstitial Cells In Vitro: Differential Regulation of Steroidogenesis by Cyclic Adenosine Monophosphate1
a Leland J. and Dorothy H. Olson Center for Women's Health, Department of Obstetrics and Gynecology and Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
ABSTRACT
Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17
-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYP11A1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.
cAMP, ovary, theca cells
INTRODUCTION
It is axiomatic that LH stimulates thecal and interstitial cells of adult mammals for progesterone and androgen biosynthesis, and the effect is mediated via a cell surface LH receptor coupled to the adenylate-cyclase-cyclic AMP second messenger system [1]. However, limited data are available regarding the initiation of steroidogenesis in developing interstitial cells of the mammalian ovary. Ovaries of 10-day-old hamsters contain only primary and early secondary (2–3 layers of granulosa cells) follicles without any evidence of thecal cell formation [2, 3]; however, by Day 13–14 of postnatal life, follicles with 6 layers of granulosa cells with a developing thecal layer are visible [3]. The appearance of large preantral follicles with theca coincides with the rise in serum LH [3, 4], suggesting, therefore, that the differentiation of thecal cells occurs between Days 10–14 of postnatal life and may be attributed, at least in part, to LH. Differentiated thecal cells express P450CYP11A1 and CYP17 enzyme protein [1; Schwartz and Roy, unpublished observations] and produce progesterone and androstenedione in vitro [5, 6].
Studies using in vitro culture of thecal-interstitial cells (TICs) [7] from immature rats have shown that LH as well as insulin-like growth factor I (IGF-I) [8–10] induce the expression of steroidogenic enzyme mRNA and influence their activities. However, because of the presence of thecal and secondary interstitial cells in the preparation, addressing the role of hormones in the differentiation of primary interstitial cells, especially under in vitro culture conditions, becomes limited. Therefore, we have selected the model of hamster primary interstitial cell culture [11] to study the regulation of interstitial cell differentiation. Although the regulatory mechanisms of steroidogenesis in rat TICs in vitro have been addressed [12], the mechanisms and factors involving the differentiation of primary interstitial cells need further evaluation.
The objective of these studies was to determine whether LH induces the differentiation of primary interstitial cells in culture via the cAMP second messenger system. We selected CYP11A1 and CYP17 as markers of interstitial cell differentiation into thecal cells.
MATERIALS AND METHODS
Animals
Golden Syrian hamsters were obtained from Sasco (Kingston, NY) and housed in a climate-controlled facility in accordance with U.S. Department of Agriculture and institutional care committee guidelines for the care and handling of experimental animals. Females were mated on the afternoon of proestrus (Day 4) and the presence of sperm in the vagina the next morning was considered as Day 1 of pregnancy. Ten-day-old pups were anesthetized with a s.c. injection of pentobarbitol sodium (Sigma Chemical Company, St. Louis, MO; 8 mg/100 g body weight) and ovaries were placed in sterile Krebs-Ringer bicarbonate medium (pH 7.4) containing 0.1% glucose (KRBG).
Isolation and Culture of Primary Interstitial Cells
Primary interstitial cells were isolated from 10-day-old hamster ovaries as described previously [11]. Cells were cultured in 0.5 ml Dulbeccos modified Eagle's medium (DMEM) containing 1% TS+ (Stock Concentration: 625 µg/ml transferrin, 625 ng/ml selenium, and 535 µg/ml lenoleic acid; Collaborative Research, Waltham, MA), antibiotics (Antibiotic-Antimycotoic, Gibco BRL, Grand Island, NY), 0.5% BSA (Fraction V, Sigma) and hydrocortisone (40 ng/ml) at 37°C under 5% CO2 in air in a 24-well culture plate (Costar, Cambridge, MA). For all experiments described here, cells were cultured for 96 h in the presence of 5% heat-inactivated FBS (Gibco BRL) to establish sufficient attachment and confluency. Test factors were added in serum-free medium at 96 h and the cultures were terminated 24 h later. Twenty-four hours was selected on the basis of response of hamster ovarian cells to steroidogenic stimuli [13]. At the end of each experiment, cell number was determined using an aqueous nonradioactive cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's instructions [11].
Effect of LH on Interstitial Cell Steroidogenesis
In the first experiment, interstitial cells were cultured in the presence of 0, 50, or 100 ng/ml of ovine LH (NIADDK-oLH-26) for 24 h. The medium was collected and stored at -20°C until assayed for androstenedione and progesterone using radioimmunoassay (RIA; [13]).
Effect of Forskolin on Interstitial Cell cAMP Production
Interstitial cells were cultured for 24 h in the presence of 1, 5, 10, or 50 µm of forskolin (Calbiochem, La Jolla, CA) and 0.5 mM isobutylmethylxanthine (IBMX). Upon termination, the medium was collected, boiled for 5 min (to inactivate any remaining phosphodiesterase activity), and then stored frozen at -20°C until assayed for cAMP using an RIA kit (Biomedical Technologies, Stoughton, MA) with a sensitivity of detection of 1 pmol/ml of acetylated cAMP.
Effect of Forskolin and 8-Br-cAMP on Interstitial Cell Steroidogenesis
Interstitial cells were cultured in the presence of 1 or 10 µM forskolin or 1 mM 8-Br-cAMP (Sigma) for 24 h. The medium was stored frozen at -20°C until assayed for progesterone and androstenedione by specific RIA [13].
Semiquantitiative Analysis of P450CYP11A1 and CYP17 mRNA Levels in Hamster Interstitial Cells
Total RNA was extracted using 500 µl of TRI reagent (MRC, Cincinnati, OH) according to the manufacturer's instructions and stored at -80°C until used for reverse transcription-polymerase chain reaction (RT-PCR) evaluation of CYP11A1 and CYP17 mRNA. CYP11A1 and CYP17 mRNA levels in cultured interstitial cells were determined using an RT-PCR protocol similar to that described by Paria et al. [14] with modifications. The sequences for the forward and reverse primers for CYP11A1 were 5'-CTCGATCCTTCAATGAGATC-3' and 5'-TACAACACTGGTGATGGACT-3', respectively [15]. The sequences for the forward and reverse primers for CYP17 were 5'-ACTCTAGGCCTCTTGTCGGACCAA-3' and 5'-CAACCACGGGAATATGTCCACCAG-3', respectively [16, 17]. For normalization and to determine the specificity of enzyme mRNA expression following exposure to various stimuli, levels of hamster ribosomal protein S4 mRNA was also determined using 5'-GGCGATGAAGTCAAGAAG-3' and 5'-CCCAAGTTAGCACCTCC-3' as forward and reverse primers, respectively [18]. Five hundred nanograms of total RNA was reverse-transcribed in a final volume of 7 µl using a gene-specific antisense primer. The RT reaction mixture consisted of RT buffer (250 mM Tris, 375 mM KCl, 15 mM MgCl2, pH 8.3), 10-picomole reverse primer, 10 mM dNTP, 1 mg/ml BSA, dithiothreotol, RNasin (Promega), and 50 U M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD). The cDNA generated by RT was amplified in a PCR reaction containing gene-specific forward and reverse primers in a final volume of 25 µl, using a PTC 200 thermocycler (MJ Research, Inc., Watertown, MA). The conditions for the PCR for S4 and CYP11A1 reaction were 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C. The conditions for the PCR for CYP17 were 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C. The cDNA for S4 and CYP17 was amplified for 30 cycles, whereas cDNA for CYP11A1 was amplified for 34 cycles. The relationship between PCR cycle number and product formation was linear up to 50 cycles. A final 10-min incubation at 72°C was carried out to complete the extension reaction. The reaction mixture containing no RNA was used to verify the specificity of the PCR product, whereas the RT reaction without M-MLV reverse transcriptase was used in the PCR to rule out any DNA contamination.
PCR products were resolved in a 1% agarose gel containing ethidium bromide in Tris-Borate-EDTA (TBE) buffer, transferred to a Zeta Probe nylon membrane (Bio-Rad, Hercules, CA), UV cross-linked, and hybridized to a 32P-labeled sequence-specific antisense oligonucleotide. The radioactive signal was quantified using a Cyclone phosphorimager (Packard Instruments, Meridan, CT). The digital light units (DLUs) for CYP11A1 and CYP17 were normalized against the S4 DLU and expressed as enzyme-specific DLU relative to S4 signal.
Statistical Analysis
Cellular production of cAMP, progesterone, and androstenedione was normalized for 105 cells. Each data point represents the mean of at least three separate experimental analysis with three replicates for each. The data were analyzed using two-way ANOVA with Scheffe's F-test and the level of significance was 5%.
RESULTS
Effect of LH on Interstitial Cell cAMP and Steroid Production
Administration of LH to interstitial cell cultures did not induce either progesterone or androstenedione production regardless of the dosage used (data not shown). In fact, steroidogenesis appeared to have decreased in the presence of LH, although not significantly.
Effect of Forskolin on cAMP Production
In this experiment, interstitial cells were exposed to increasing doses of forskolin in the presence of IBMX. Forskolin stimulated cAMP production in a dose-dependent manner and a significant increase was noted with a dose level as little as 1 µM (Fig. 1). Although the increase in cAMP production was more or less linear up to 10 µM of forskolin, a marked increase in cAMP production was evident when cells were exposed to 50 µM of forskolin (Fig. 1).
|
Effect of Forskolin and 8-Br-cAMP on Progesterone and Androstenedione Production
Interstitial cells maintained a baseline production of progesterone, indicating the presence of active CYP11A1 (Fig. 2). Forskolin at a dose of 1 µm stimulated progesterone production by 2.5-fold; a further increase was noted when the forskolin dose was increased to 10 µM (Fig. 2). However, progesterone production increased almost sevenfold in response to 1 mM 8-Br-cAMP (Fig. 2). Despite a significant increase in progesterone production, no increase in androstenedione production was noted in response to either forskolin or 8-Br-cAMP (Fig. 3). Interstitial cells, however, were capable of producing detectable amounts of androstenedione regardless of the treatment condition (Fig. 3), thus indicating that some CYP17 activity was present in cultured primary interstitial cells.
|
|
Effect of Forskolin and cAMP on Interstitial Cell CYP11A1 and CYP17 mRNA Levels
The basal expression of CYP11A1 mRNA was very low in primary interstitial cells following 5 days of culture in vitro. However, a significant (P < 0.05) induction of CYP11A1 mRNA was noted with 1 µM forskolin and the induction further increased with higher doses (Fig. 4). In fact, the levels of CYP11A1 mRNA increased by as much as 28-fold with 10 µM of forskolin (Fig. 4). Administration of 8-Br-cAMP stimulated the induction of CYP11A1 mRNA 40-fold over that observed for untreated interstitial cells alone (Fig. 4).
|
In contrast, basal levels of CYP17 mRNA were very low compared with those of CYP11A1 gene transcripts (Fig. 5). Nevertheless, CYP17 mRNA levels increased more than 2.3-fold when cells were exposed to forskolin or cAMP (Fig. 5). Contrary to the effect of forskolin on the induction of CYP11A1 mRNA expression, a higher dose of forskolin appeared to suppress CYP17 mRNA induction (Fig. 5). Similarly, 8-Br-cAMP significantly (P < 0.05) stimulated CYP17 mRNA expression; however, the effect was significantly lower than that stimulated by the 1 µM dose of forskolin (Fig. 5).
|
DISCUSSION
The results of these studies clearly indicate that neonatal hamster primary interstitial cells have a functional adenylate cyclase system, which is involved in regulating progesterone biosynthesis. Moreover, cAMP can induce differentiation of hamster primary interstitial cells in vitro. The failure of LH to induce steroidogenesis indicates that primary interstitial cells of the neonatal ovary either do not express LH receptors or the cAMP second messenger system is not functionally coupled to the LH receptor. We could not with consistency detect LH receptors in cultured neonatal cells (data not shown). Shaha and Greenwald [5] have shown that specific binding of labeled hCG (125I-hCG) does not occur until postnatal Day 15 in hamster interstitial cells, which corresponds to the rise in serum LH in vivo [3, 4]. Despite a lack in cellular response to LH, the results of these studies demonstrate that, in vitro, the cAMP-generating system is operational in neonatal interstitial cells.
Thecal cells are characterized by their ability to express CYP17 enzyme and to produce androstenedione [1]. In the hamster, LH-induced androgen synthesis occurs around Day 15 of postnatal development; however, 5- and 10-day-old hamster ovaries are capable of converting various steroid precursors in vitro when exposed to LH and cAMP [6]. Our data support this finding by demonstrating that cultured interstitial cells from 10-day-old hamsters respond to forskolin and cAMP with increased levels of CYP11A1 and CYP17 mRNA. The low level of CYP17 mRNA relative to that of CYP11A1, even after cAMP stimulation, may be inadequate to increase the net amount of CYP17 enzyme protein in cultured cells; thus resulting in no apparent increase in androstenedione output. The production of progesterone rules out any possible lack of substrate in vitro; however, whether one or more other factors that are crucial for the induction of androstenedione production are absent from these cells in culture remains to be determined.
Contrary to the finding of Shaha and Greenwald [6], androstenedione production by cultured interstitial cells did not increase in response to forskolin or cAMP. This discrepancy may be the result of a difference in culturing primary interstitial cells versus whole ovaries, in which preantral follicles at different stages of development are present. The increase in CYP17 mRNA levels without any associated increase in androstenedione production suggests that, despite the development of the CYP17-androgen producing system in interstitial cells from 10-day-old neonatal hamsters, androgen producing mechanisms remain immature, whereby increased presence of progesterone substrate or cAMP stimulation fails to induce androstenedione production. Differentiation of interstitial cells into thecal cells may involve the maturation of the steroidogenic cascade (i.e., a sequential activation of CYP11A1 followed by 3ß-hydroxysteroid dehydrogenase and CYP17 during ovarian development; [19, 20]). It is possible that the translation of CYP17 mRNA into 17-hydroxylase enzyme protein may not occur efficiently in cells derived from 10-day-old hamsters or the amount of enzyme protein synthesized from the small amount of mRNA does not adequately increase the existing enzyme level. Synthesis of androstenedione from progesterone requires other factors such as cytochrome b5 [21] in addition to hydroxylase enzymes, which suggests that other levels of control within interstitial cells may not be fully mature for androgen biosynthesis.
Because the development of thecal cells coincides with the development of large preantral follicles [3], a possible role of follicles in thecal cell differentiation is likely. Follicles secrete a variety of growth factors that modulate the steroidogenic functions of theca-interstitial cells [8–10, 22, 23]. IGF-I enhances LH-stimulated CYP17 mRNA expression and androgen production in cultured rat TICs [8–10, 24]. Stem cell factor has also been shown to increase production of androstenedione from bovine theca cells grown under confluent conditions [24]; however, transforming growth factor-ß [25] and hepatocyte growth factor [26] produced in vivo by ovarian thecal and interstitial cells attenuate androgen production in rat cultured thecal interstitial cells in vitro, perhaps to prevent detrimental effects on folliculogenesis [27, 28]. This line of evidence suggests that the control of androgen production may include negative regulatory mechanisms, which may be important during early ovarian development.
In summary, the results of these studies demonstrate that cultured interstitial cells from neonatal hamsters are capable of producing and responding to cAMP for the induction of CYP11A1 and CYP17 enzyme mRNA. Increase in progesterone production without a concurrent increase in the synthesis of androgen suggests that the control of androstenedione synthesis may be different from that of progesterone and may require either the maturation of interstitial cells, the presence of some paracrine factor or factors produced by intact follicles, or both.
ACKNOWLEDGMENTS
We thank the National Pituitary Program and Dr. A.V. Parlow for generously providing the gonadotropins.
FOOTNOTES
First decision: 2 February 2000.
1 This work was supported by a grant from NIHCHD (HD28165) and the Olson Foundation (Omaha, NE). 
2 Correspondence: Shyamal K. Roy, Departments of Obstetrics and Gynecology and Physiology and Biophysics, 984515 University of Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Accepted: March 23, 2000.
Received: November 12, 1999.
REFERENCES
-hydroxylase messenger ribonucleic acid expression by luteinizing hormone and insulin-like growth factor-I. Endocrinology 1993; 132:1945–1951.-hydroxylase/C17–20 lyase. Genomics 1991; 10:270–275.[CrossRef][Medline]-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse. J Mol Endocrinol 1993; 11:77–82.This article has been cited by other articles:
|
|
 |
|
  B.-M. Huang, K.-Y. Hsiao, P.-C. Chuang, M.-H. Wu, H.-A. Pan, and S.-J. Tsai Upregulation of Steroidogenic Enzymes and Ovarian 17{beta}-Estradiol in Human Granulosa-Lutein Cells by Cordyceps sinensis Mycelium Biol Reprod, May 1, 2004; 70(5): 1358 - 1364. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
