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Endocrinology Vol. 141, No. 8 2735-2742
Copyright © 2000 by The Endocrine Society

ARTICLES

Interactive Stimulation by Luteinizing Hormone and Insulin of the Steroidogenic Acute Regulatory (StAR) Protein and 17{alpha}-Hydroxylase/17, 20-Lyase (CYP17) Genes in Porcine Theca Cells1

Gongqiao Zhang, James C. Garmey and Johannes D. Veldhuis

Division of Endocrinology, Department of Internal Medicine, Specialized Cooperative Center in Reproductive Research, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Department of Internal Medicine, P.O. Box 800202, University of Virginia School of Medicine, Charlottesville, Virginia 22908. E-mail: jdv{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14–18 vs. 3–5.5 pmol/µg DNA/12–48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH’s effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL CLINICAL studies have postulated that dual stimulatory actions of LH and insulin on theca-cell androstenedione and testosterone biosynthesis may contribute to the anovulatory hyperandrogenism characteristic of the polycystic ovarian syndrome (1, 2). In relation to LH, earlier analyses established a key role for this gonadotropin in enhancing theca-cell steroidgenesis, in major part through the second-messenger cAMP-protein kinase A (PKA) pathway (3). Other studies have documented the expression of functional insulin and insulin-like growth factor (IGF)-1 receptors on ovaries in different species; e.g. insulin receptors on pig granulosa cells can catalyze autophosphorylation of the insulin-receptor 41-kDa ß subunit (4). The molecular mechanisms by which insulin regulates steroidogenesis are less well understood, although substantial evidence indicates that insulin, IGFs and other intraovarian growth factors, such as epidermal growth factor and fibroblast growth factor, modulate basal and gonadotropin-stimulated steroidogenesis (5). For example, insulin and IGF-1 increase gonadotropin-receptor density by elevating LH receptor messenger RNA (mRNA) levels, and also enhance CYP11A and CYP17 mRNA accumulation.

Selected in vitro experiments have demonstrated the ability of combined LH and insulin (or IGF-I) to stimulate androgen biosynthesis by interstitial-theca cells in a synergistic (supraadditive) manner (6, 7). Because LH and insulin act physiologically via distinct intracellular signaling mechanisms, their synergistic enhancement of theca-cell steroidogenesis likely entails important interactions between these two effector pathways. For example, one could hypothesize that LH and insulin jointly up-regulate the expression of one or more specific genes that control the access and/or utilization of sterol substrate in the androgen-biosynthetic pathway, and thereby augment androgen biosynthesis. To explore this notion further and to test the specificity of possible LH-insulin synergy on selected molecular endpoints, we have investigated the single and dual actions of LH and insulin on the theca-cell expression of genes that govern intracellular sterol substrate delivery (steroidogenic acute regulatory protein, StAR), and subsequent utilization in steroidogenesis [17-{alpha} hydroxylase/C17–20 lyase (CYP17)] in serum-free primary cultures of normal porcine theca cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and oligonucleotides
Ovine LH (NIDDK-oLH-25, 2.3 U/mg, 1 U = activity of NIH-LH-S1) was provided by the National Hormone and Pituitary Program of NIDDK. Insulin (from porcine pancreas) and 8 Br-cAMP were obtained from Sigma (St. Louis, MO). Oligo (dT)15 primer and dNTP’s were purchased from Roche Molecular Biochemicals (Indianapolis, IN), RNase inhibitor, MuLV reverse transcriptase and Amplitaq Gold DNA polymerase from Perkin-Elmer Corp. (Brandsberg, NJ), and PicoGreen DNA assay reagents from Molecular Probes, Inc. (Eugene, OR). All oligonucleotides used for primers were obtained from Operon Technologies (Alameda, CA). The sequences selected for forward and reverse oligonucleotide primers are given in Table 1Go. Degenerate reverse primer sequences were selected from the fourth and third introns of the pig StAR and CYP17 genes, respectively, for RT-PCR of hnRNA (below). Porcine StAR hnRNA and 18S ribosomal RNA (18S rRNA) sequences were not available in GenBank. Thus, we identified fully conserved sequences as templates to design primers among at least four species, including human, rat, mouse, rabbit, and/or cow. For StAR, primers were first designed to clone part of exon 4 and exon 5 including intron 4 from porcine theca-cell genomic DNA. The PCR products were sequenced three times for confirmation. Another pair of primers was then designed directed to pig exon 4 and intron 4 to allow assay of theca-cell hnRNA accumulation. Tables 2Go and 3Go show the PCR product sequences for porcine StAR hnRNA and 18S rRNA, respectively.


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  		Table 1. Primer sequences used for amplification of porcine genes

 

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  		Table 2. Sequence of the porcine StAR hnRNA PCR product (253 bp)

 

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  		Table 3. Sequence of the porcine 18S rRNA PCR product (315 bp)

 
Porcine theca-cell culture
Theca cells were isolated as described previously (14, 15). Briefly, combined theca-granulosa membranes were removed from immature follicles (diameter < 5 mm) using forceps, and granulosa cells were detached by mechanical agitation for 3 min. Residual theca-basal lamina linings were allowed to settle at unit gravity, and the supernatant containing granulosa cells was discarded. Follicle linings were then digested with collagenase (1 mg/ml) and DNase (10 µg/ml), and filtered 0.2 µm Nylon screen using a Millipore Corp. filter unit. Theca cells were washed 3 times in F12/DMEM Media (1:1) by centrifugation for 5 min at 1,000 RPM.

Theca cells were purified further using Percoll density gradient, as described previously (14). Cells were then suspended in F-12/DMEM (1:1) media and plated in 96-well plates (Costar, Cambridge, MA) at a density of 105 cells/well in a humidified 95% air, 5% CO2 incubator at 37 C. After an initial 40 h of culture in F-12/DMEM to allow anchorage, theca cells were exposed to LH, insulin alone, or their combination. Culture media were collected at the indicated times (0, 6, 12, 24, and 48 h) for later analysis of androstenedione, progesterone, and cAMP concentrations. Androstenedione was analyzed by commercially available RIA (ICN Pharmaceuticals, Inc. Costa Mesa, CA), which has a sensitivity of 0.17 ng/ml and exhibits low cross-reactivity with progesterone (0.02%) and testosterone (0.87%). Progesterone, cAMP and DNA were measured as described previously (16). Data were normalized per unit cellular DNA in the cultures.

RNA isolation
Total RNA was isolated from cells pooled from eight wells using the Tri-Reagent total RNA isolation kit (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacture’s instruction, and quantified spectrophotometrically by absorbance at 260/280 nm (Spectronics Model 601; Milton Roy, Rochester NY). Total RNA was used to examine the expression of mRNA and hnRNA.

RT-PCR
Semiquantitive RT-PCR was used to assess mRNA levels by first reverse transcribing 4 µl (1 µg) of total RNA/sample in a final volume of 40 microliters. Each reaction contained 1 mM of each dNTP, 2.5 µM of oligo (dT)15, 40 U of RNase inhibitor, 5 mM MgCl2, 4 µl of 10x RT-PCR buffer, 50 U of murine leukemia virus reverse transcriptase (MuLV-RT). RT was performed at 42 C for 15 min followed by 99 C for 5 min.

PCR reaction mixtures of 100 microliters final volume contained 2.5 mM MgCl2, 100 pmol each of forward and reverse gene specific primers, 0.2 mM of each dNTP, 10 microliters of 10x RT-PCR buffer, one fifth of the total RT volume for gene specific products, and 2.5 U of AmpliTaq Gold DNA polymerase. PCR amplification was optimized for each gene as follows: 95 C for 12 min, then 28 cycles at 94 C for 30 sec, 62 C for 2 min, followed by 62 C for 10 min.

To amplify hnRNA for StAR, CYP17, and 18S rRNA, a different RT-PCR protocol was used. In the RT step, 2 pmol reverse gene-specific primer instead of oligo (dT)15 was used to synthesize complementary DNA that was primed by intron-specific reverse primers. In the PCR step, a different thermal-cycling protocol was used: 30 sec at 95 C, 35 sec at 62 C, and 1 min 30 sec at 67 C.

To control for potential DNA contamination, RT-PCR reactions were also conducted using total RNA without reverse transcriptase. To control for errors in input of complementary DNA used in PCR reactions, amplification of porcine endogenous 18S ribosomal RNA was performed in parallel and results were expressed as a ratio of specific gene product amplified to that of 18S ribosomal RNA.

The identities of the mRNA- and hnRNA-derived RT-PCR products were confirmed in multiple (at least 3 to 5) independent samples from different batches of ovaries by automated gene sequencing (University of Virginia Molecular Core Lab).

PicoGreen quantification of PCR products
The amount of amplified PCR product was quantified for each sample using the PicoGreen ultrasensitive fluorometric assay (Molecular Probes, Inc., Eugene, OR). Twenty microliters of PCR product/sample were prepared in 1 x TE buffer in a final volume 100 µl, and added in a 96-well microplate. One hundred microliters of 1:200 PicoGreen in 1 x TE was then mixed with each PCR sample. After incubation at room temperature for 2–5 min, microtiter plates were scanned at excitation = 488 nm and emission = 530 nm using a FluorImager 595 Optical Scanner, version 5.01. The image was analyzed using Imagequant version 5.0 (Molecular Dynamics, Inc., Sunnyvale, CA). The amount of PCR product was quantitated by extrapolation from DNA standards as previously reported (17). The assay has a sensitivity of 25 pg/ml with a linear range from 250 pg/ml to 1000 ng/ml. Lambda dsDNA was used as standard. Intra and interassay coefficients of variation were 5.1% and 8.3%, respectively.

Statistical analyses
Data are presented in the figures as the mean ± SEM of five independent experiments using separate batches of ovaries to confirm reproducibility of results. Data were subjected to the Student’s two-tailed t test or one-way ANOVA to determine significant (P < 0.05) treatment effects. A synergistic effect was assessed by testing for supraadditive incremental (above control) effects using factorial ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LH and insulin synergy on theca-cell androstenedione and progesterone biosynthesis
Initial studies were performed to optimize hormone dose and time course effects on androstenedione and progesterone production. These analyses showed that 100 ng/ml LH and 100 ng/ml insulin were maximally effective (Fig. 1Go, A and B). Thus, further studies were done using these hormone concentrations. As shown in the time course data of Fig. 1Go, C and D, cotreatment with LH and insulin synergistically increased both androstenedione and progesterone accumulation normalized against DNA (P < 0.001). Synergism was evident statistically as early as 12 and 6 h for androstenedione and progesterone, respectively. Maximal effects of LH or insulin singly were apparent at 24 h. Androstenedione and progesterone concentrations driven by LH or insulin alone decreased slightly at 48 h, but continued to rise in response to the combined actions of LH and insulin.



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  		Figure 1. A and B, C and D, Dose-response and time course of LH and insulin’s individual and combined stimulation of porcine theca-cell androstenedione and progesterone accumulation. Serum-free primary monolayer cultures of theca cells (105 cells/0.25 ml medium/well) were stimulated with vehicle, LH (100 ng/ml), insulin (100 ng/ml) or their combination. At 0, 6, 12, 24, and 48 h, culture media were collected and the concentrations of androstenedione and progesterone determined by specific RIAs. Values are normalized for DNA content of the cultures. Data are the mean ± SEM of five different experiments each conducted with triplicates. Each treatment effect was significant at P < 0.05 (see text).

 
Joint LH and insulin actions on theca-cell StAR and CYP17 mRNA accumulation
Figure 2AGo shows that LH alone effectively stimulated StAR mRNA accumulation within 6 h (P < 0.01). StAR message levels peaked at 12 h, and decreased thereafter. Insulin alone also enhanced StAR mRNA accumulation, but less rapidly than LH, and then maintained an approximately constant amount of StAR message at 12 and 24 h (P < 0.01). After 24–48 h, combined LH and insulin stimulated StAR mRNA expression significantly (P < 0.01). A generally similar time-course was observed for CYP17 mRNA expression in response to these agonists alone and combined (Fig. 2BGo).



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  		Figure 2. A and B, Time course of accumulation of porcine theca-cell StAR and CYP17 mRNA basally and when stimulated by LH (100 ng/ml), insulin (100 ng/ml) or both. Total RNA was extracted from theca cells and specific mRNA for StAR, CYP17, and 18S ribosomal RNA quantitated by RT-PCR-PicoGreen assay (see Materials and Methods). Data are normalized to 18S rRNA and given as the mean ± SEM of triplicate samples from five independent experiments. Each overall treatment effect was significant at P < 0.05 (see text).

 
Impact of LH and insulin on StAR and CYP17 hnRNA accumulation
To monitor gene transcriptional activity indirectly, we used gene intron-specific reverse primers for PCR-based quantitation of (incompletely processed) heterogeneous nuclear (hn) RNA (see Table 1Go). The accumulation of hnRNA at a given time reflects a function of the rate of transcription of the target gene and the rate of processing of the immature hnRNA into mRNA (18). The differences in the hnRNA levels among samples can be attributed to transcription.

Figure 3Go (A and B) depicts the time-course of StAR and CYP17 hnRNA accumulation basally and in response to stimulation with LH, insulin and their combination. LH maximally enhanced StAR and CYP17 hnRNA accumulation at 6 and 12 h, respectively. Then, hnRNA levels decreased gradually. In contrast, insulin significantly increased StAR and P450 hnRNA after 12 to 24 h. Combined LH and insulin treatment consistently elevated concentrations of hnRNA’s for both StAR and CYP17 hnRNA expression more than the corresponding values stimulated by either LH or insulin alone (P < 0.05).



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  		Figure 3. A and B, Time course of accumulation of intron-specific heterogeneous nuclear (hn) RNA for StAR and CYP17 basally and in response to stimulation by LH and/or insulin in porcine theca-cell monolayer cultures. Theca cells were pretreated for the indicated times with vehicle, LH (100 ng/ml), insulin (100 ng/ml) or their combination. Heterogeneous nuclear RNA levels were quantified by RT-PCR-PicoGreen assay using gene-specific primers complementary to introns 4 and 3 of the swine StAR and CYP17 genes, respectively. Data are normalized to 18S rRNA and presented as the mean ± SEM of triplicate samples from five independent experiments. Each overall treatment effect was significant at P < 0.05 (see text).

 
Effects of insulin on basal and LH-driven cAMP accumulation
Insulin alone did not alter cAMP accumulation but significantly enhanced LH-stimulated cAMP concentrations (Fig. 4Go). LH-stimulated cAMP concentrations were maximal at 6 h, and then declined slowly. In contrast, LH plus insulin increased cAMP accumulation during the entire time course of 48 h.



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  		Figure 4. Insulin significantly increases LH-driven cAMP accumulation in porcine theca cells cultured as described in Fig. 1Go. Media were collected, diluted with HCl to 0.1 N, and cAMP concentrations quantified by RIA. Data are presented as the mean ± SEM for five separate experiments each performed in triplicate. Each overall treatment effect was significant at P < 0.05 (see text).

 
Actions of a stable cAMP analog, 8 Br-cAMP, on theca-cell progesterone and androstenedione biosynthesis
Initial studies were performed to optimize 8 Br-cAMP dose and time course effects on androstenedione and progesterone production. These analyses showed that 1 mM 8 Br-cAMP and 100 ng/ml insulin were maximally effective (Fig. 5Go, A and B). Thus, further studies were done using these concentrations. 8 Br-cAMP (1 mM) increased theca-cell androstenedione and progesterone biosynthesis in a time-dependent manner (Fig. 5Go, C and D). However, in contrast to LH and insulin, coadministration of 8 Br-cAMP and insulin synergized only in stimulating progesterone biosynthesis (20- to 31-fold within 6 to 48 h, P < 0.001).



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  		Figure 5. A and B, C and D, Dose response and time course of 8 Br-cAMP and insulin’s individual and combined stimulation of porcine theca-cell androstenedione and progesterone accumulation. Monolayer cultures of porcine theca cells were stimulated with control solvent, 8 Br-cAMP (1 mM), insulin (100 ng/ml) or their combination for 0, 6, 12, 24, and 48 h. Media were collected for the later determination of androstenedione and progesterone concentrations by specific RIAs. Data are normalized for DNA content of the cultures, and reported as the mean ± SEM of five independent experiments each done in triplicate. Each overall treatment effect was significant at P < 0.05 (see text).

 
Effect of 8 Br-cAMP on theca-cell StAR and CYP17 mRNA and hnRNA expression
8 Br-cAMP alone significantly increased StAR at 24 h (Fig. 6AGo) and CYP17 mRNA accumulation at 12 and 24 h (Fig. 6CGo) as well as StAR hnRNA (Fig. 6BGo) and CYP17 hnRNA expression (Fig. 6DGo). This analog combined with insulin also mimicked the synergistic effect of LH and insulin on progesterone, and StAR mRNA (12 h) accumulation. Combined 8 Br-cAMP and insulin treatment consistently elevated concentrations of hnRNA for StAR expression more than the corresponding values stimulated by either 8 Br-cAMP or insulin alone (P < 0.05). The supraadditive effects on CYP17 mRNA (Fig. 6CGo) or hnRNA (Fig. 6DGo) concentrations over the full range of culture time explored were not observed.



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  		Figure 6. A and B, C and D, The stable cAMP analog, 8 Br-cAMP, combined with insulin mimics the joint effect of LH and insulin on StAR mRNA (A) and hnRNA (B), but not on CYP17 mRNA (C) and hnRNA (D), accumulation. Theca cells were cultured as described in the legend of Fig. 5Go. At 0, 6, 12, 24, and 48 h, total cellular RNA was extracted and the amounts of mRNA and hnRNA for StAR and CYP17 quantitated by the RT-PCR and PicoGreen assay (see Materials and Methods). Data are the mean ± SEM of five separate experiments each done in triplicate. Each overall treatment effect was significant at P < 0.05 (see text).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies implement an in vitro serum-free monolayer culture system of Percoll-purified porcine theca cells that are responsive to LH and insulin singly and jointly. Thereby, we explore the molecular mechanisms of the putative steroidogenic interaction between these two pivotal but distinct signaling pathways. Given the key role of StAR and CYP17 genes in controlling the intracellular transport, and enzymatic processing of sterol substrate (19, 20, 21), we investigated the regulation of these primary loci of steroidogenic control by LH and insulin individually and in concert. Time-course experiments first corroborated that LH and insulin individually and synergistically (at a maximally effectively concentration of each) drive theca-cell androstenedione biosynthesis above control levels. The action of LH was more rapid in onset but less sustained than that of insulin, whereas bihormonal stimulation evoked both a rapid and a sustained increase in steroidogenesis. Based on the consistent responses evident in primary theca-cell cultures, we could examine LH and insulin’s interactive control of the foregoing critical genes that govern cellular sterol economy and steroidogenic activity. We found that bihormonal synergy is explicable by selective supraadditive stimulation of mRNA’s encoding CYP17 and StAR, which points to novel LH and insulin-dependent effects on these genes and/or their regulatory cofactors.

Detailed time-course analyses of CYP17 and StAR expression under LH and/or insulin drive revealed several distinctions, which were agonist specific. For example, the onset of LH’s stimulation of StAR and CYP17 mRNA synthesis were more rapid than that for insulin. Conversely, insulin action was more sustained. Combined LH and insulin augmented CYP17 and StAR intron-specific transcript synthesis (hnRNA) and accumulation (mRNA), more rapidly in a more persistent manner than LH or insulin individually. The molecular bases for these hormone-specific time-course distinctions are not known but likely reflect differences in the proximal signaling elements and distal effector pathways used by the two (LH and insulin) agonists, and/or unequal effects on protein or mRNA turnover. For example, gonadotropins rapidly (within minutes) activate G protein-linked receptor-mediated cAMP and PKA-dependent signaling (22), whereas insulin initiates a complex cascade of tyrosine phosphorylation-dependent reactions leading to altered gene transcription (23). In this regard, the CYP17 and StAR genes are both cAMP-PKA (24, 25, 26) and insulin/IGF-I (27, 28) responsive in various endocrine cells. In the present studies, CYP17 and StAR gene expression were increased by LH and insulin in theca cells. Bihormonal synergy also is achieved by FSH (or cAMP) and IGF-I (or insulin) in stimulating CYP11A gene expression in (porcine) granulosa cells (29, 30).

The 17{alpha}-hydroxylase and lyase activities of cytochrome CYP17 are essential for male and female sex-hormone production (31). This enzyme represents a single protein that catalyzes two biochemical reactions, the 17{alpha}-hydroxylation of progesterone (and pregnenolone) and the subsequent cleavage of the C17–20 bond to yield androstenedione (and DHEA). These precursors can be converted to potent androgens, such as testosterone and 5{alpha} DHT (32), or undergo aromatization to estrogens. Here, we show in vitro that LH and insulin can jointly induce marked theca-cell androgen production via molecular synergy at the level of steady-state expression of specific steroidogenic genes.

Using RT-PCR, we could show that LH and insulin augment accumulation of CYP17 and StAR hnRNAs in theca cells in an agonist- and time-specific manner. Assuming the rate of processing of the hnRNA is constant under the conditions of studies, then the actions of these two agonists points to enhanced CYP17 and StAR gene transcription. As importantly, combined stimulation with LH and insulin achieved a consistently greater effect on the accumulation of both hnRNAs than either stimulus alone. Heightened transcription of these genes by combined actions of LH and insulin, if confirmed by transcriptional run-on assays, could explicate the synergistic rise in StAR and CYP17 mRNA content and steroidgenesis in theca cells.

An unexpected finding in the case of the CYP17 gene was that an exogenous cAMP analog failed to mimic fully LHs enhancement of insulin action under our specific culture conditions. This allows for the possibility of greater regulatory complexity of the LH-insulin interaction. For example, some of the effects of combined LH and insulin to stimulate CYP17 gene expression synergistically may be exerted in part independently of cAMP accumulation. Alternatively, endogenous and exogenous cAMP may activate nonidentical cellular reactions. The latter consideration was also raised in Sertoli cell studies, whereas the former notion is consistent with the well established ability of LH to stimulate not only the cAMP/PKA but also the Ca2+/phospholipase C signaling pathway (33). In addition, LH may modify one or more critical steps in insulin’s signaling pathway. For example, the interaction between LH and insulin in mediating the synergistic stimulation of androstenedione production might be exerted via non-cAMP-dependent actions of LH via MAP-kinase dependent pathways (34).

Theca-cell cytodifferentiation and steroidogenesis appear to depend on LH’s trophic actions via its cell membrane receptor (24, 35). The theca cells used here were harvested from developing Grafian follicles (diameter < 5 mm), which express LH receptors (36). In addition to LH, insulin and various intragonadal insulin-like growth factors, such as IGF-I and IGF-II, can participate in the regulation of steroidogenesis by granulosa or theca cells (5, 37). Thus, one cellular mechanism of synergy between LH and insulin or its analog IGF-I could involve heightened gonadotropin-receptor expression in response to insulinomimetic peptides, as well as increased expression of specific mRNAs for CYP17 and StAR. Insulin’s trophic effects on ovarian steroidogenesis likewise are exerted via IGF-I/and or insulin receptors (4), and possibly in part via the inositalglycan pathway (38). For example, functional insulin receptors are expressed on granulosa cells in the rat, fish, pig, and human (4, 5), and insulin induces receptor autophosphorylation in pig granulosa cells (4). Here, we show that stimulation with insulin and LH synergistically increases ovarian theca-cell cAMP accumulation, which would be consistent with enhanced LH receptor expression, facilitated LH-receptor G protein coupling to adenylyl cyclase, and/or suppression of cAMP degradation. The last-mentioned consideration is suggested (but not proven) by insulin’s ability to oppose the decline in theca-cell cAMP accumulation over the 48 h time course of LH action. Independently of the mechanism involved, these loci of LH-insulin synergy could be bypassed experimentally by coadministration of a stable (phosphodiesterase-resistant) analog of cAMP, 8 Br-cAMP.

In summary, the present experiments delineate specific and time-dependently synergistic interactions between LH and insulin on theca-cell androstenedione biosynthesis and StAR and CYP17 gene expression. These actions in primary cultures of normal theca cells point to a possible physiological interplay of gonadotropic and insulinotropic stimuli to govern sterol economy and steroidogenesis in vivo. Interactions of LH and insulin on CYP17 and StAR hnRNA accumulation would further suggest that hormone-specific mechanisms of transcriptional control may mediate these bihormonal effects. The detailed nature of such molecular mechanisms will be important to clarify in future studies.


   Footnotes
 
1 This work was supported in part by NIH Grants HD-16393 and HD-16806 (to J.D.V.), NIH P30-HD-28934 (Center for Cellular and Molecular Reproduction), and the NIH U-54 Specialized Cooperative Centers Program in Reproductive Research (NICHD HD-96–008; U54–2HD 28934). Back

Received December 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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