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Endocrinology Vol. 148, No. 2 726-734
Copyright © 2007 by The Endocrine Society

Liver Receptor Homolog-1 and Steroidogenic Factor-1 Have Similar Actions on Rat Granulosa Cell Steroidogenesis

Deeksha Saxena, Rosalba Escamilla-Hernandez, Lynda Little-Ihrig and Anthony J. Zeleznik

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Anthony J. Zeleznik, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 830 Scaife Hall, 3500 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail: zeleznik{at}pitt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Granulosa cells express the closely related orphan nuclear receptors steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). To determine whether SF-1 and LRH-1 have differential effects on steroid production, we compared the effects of overexpressing LRH-1 and SF-1 on estrogen and progesterone production by undifferentiated rat granulosa cells. Adenovirus mediated overexpression of LRH-1 or SF-1 had qualitatively similar effects. Neither LRH-1 nor SF-1 alone stimulated estrogen or progesterone production, but when combined with FSH and testosterone, each significantly augmented progesterone production and mRNAs for cholesterol side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase above that observed with FSH alone, with SF-1 being more effective than LRH-1. LRH-1 did not augment FSH-stimulated estrogen production, whereas SF-1 produced only a slight (~30%) augmentation of FSH-stimulated estrogen production. The stimulatory actions of both were reduced by overexpression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1. Expression of either LRH-1 or SF-1 together with constitutively active protein kinase B in the absence of FSH stimulated progesterone production and mRNAs for 3ß-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme but did not stimulate estrogen production or mRNA for aromatase. These findings demonstrate that LRH-1 and SF-1 have qualitatively similar actions on FSH-stimulated estrogen and progesterone production, which would suggest that these factors may have overlapping actions in the regulation of steroidogenesis that accompanies granulosa cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH-MEDIATED GRANULOSA cell differentiation is associated with the induction of the LH receptor (LHr) as well as the induction of the estrogen and progesterone biosynthetic pathways (1, 2). Our previous studies demonstrated that whereas activation of the Gs{alpha}/cAMP/protein kinase A intracellular signaling system appears to be sufficient for the induction of the progesterone biosynthesis, additional signaling pathways seem to be necessary for the optimal induction of the LHr and aromatase (3). We previously explored the possible contribution of the orphan nuclear receptor liver receptor homolog-1 (LRH-1) on the induction of aromatase in granulosa cells because it has been shown by others that it synergizes with cAMP to stimulate the aromatase promoter II in transient transfection studies in granulosa cells and preadipocytes (4, 5). We demonstrated that adenovirus-directed overexpression of LRH-1 augmented the actions of FSH on progesterone biosynthesis and the induction of mRNAs for cholesterol side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) but had little or no effect on estrogen production or on the induction of mRNAs for aromatase (P450arom) and the LHr (6).

Granulosa cells also express the closely related orphan nuclear receptor steroidogenic factor-1 (SF-1) (4, 7, 8), and studies by others have shown that both SF-1 and LRH-1 bind to and regulate the same hexameric enhancer element in common target genes, including P450arom (5, 8). Moreover, the expression of LRH-1 and SF-1 in the ovary appears to be regulated in a development-specific manner because RNA for LRH-1, but not SF-1, is highly expressed in the corpus luteum, and mRNA for SF-1, but not LRH-1 is selectively expressed in theca cells (8). Granulosa cells appear to express both LRH-1 and SF-1 (8). The presence of both SF-1 and LRH-1 in granulosa cells raises the question as to whether there may be selective actions of each on the pattern of gene expression during granulosa cell differentiation. To address this question, we constructed an adenovirus vector that directs the expression of SF-1 and compared the effects of overexpression of LRH-1 with that of SF-1 on estrogen and progesterone biosynthesis and the induction of mRNAs for the LHr, P450scc, 3ß-HSD, and P450arom during FSH-stimulated differentiation of rat granulosa cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
Unless otherwise noted, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Human FSH (AFP-4161-B; 3205 IU second IRP FSH per milligram, 225 IU second IRP LH per milligram) was generously provided by Dr. A. F. Parlow [National Hormone and Pituitary Program, National Institute of Diabetes and Digestive Kidney Diseases, National Institutes of Health (NIH), Torrance, CA].

Adenovirus vectors
The preparation of an adenovirus vector that directs the expression of human LRH-1 was described previously (6). An adenovirus vector that directs the expression of SF-1 was constructed using a cDNA for mouse SF-1 provided by Dr. Keith Parker (University of Texas Southwestern Medical School, Dallas, TX) as previously described (9). An adenovirus vector that directs the expression of human dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1(DAX-1) was constructed using a cDNA (pCMX.AHC-wt) provided by Dr. Larry Jameson (Northwestern University, Chicago, IL) as described previously (9). An adenovirus vector that directs the expression of ß-galactosidase (Ad-ß-gal) was provided by Dr. Joseph Alcorn (University of Texas Medical School, Dallas, TX). An adenovirus vector that directs the expression of constitutively active protein kinase B (PKB) was provided by Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA). All viral vectors used a cytomegalovirus promoter. Virus concentrations were determined by measuring the OD at 260 nm using a ratio of 1 x 1012 viral particles per 1 OD unit (10).

Granulosa cell culture
All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Immature female rats (24 d old) were purchased from Hilltop Lab Animals (Scottdale, PA). Granulosa cells were collected from the ovaries by puncturing follicles with a 25-gauge hypodermic needle and cells were expressed into Medium 199 (M199; Life Technologies, Inc.-Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA). Granulosa cells were seeded into 6-well (~106 cells/well) or 24-well (~2 x 105 cells/well) tissue culture plates and allowed to attach overnight. The next morning, medium and unattached cells were removed and the granulosa cell monolayers were exposed to adenoviruses and stimulatory agents and maintained in serum-free M199 as described in the figure legends. At the end of the experiment, tissue culture medium was collected and stored at –20 C for subsequent RIAs. Where indicated, total RNA was prepared from the cell monolayers using RNA-Bee (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s directions.

For descriptive purposes, granulosa cells from sexually immature 24-d-old rats are referred to as undifferentiated because they lack the presence of functional LHrs and do not produce estrogen or progesterone under basal conditions (3). However, these cells respond to FSH with respect to the production of cAMP and induction of LH receptors and the induction of the estrogen and progesterone biosynthetic pathways (3).

mRNA analysis
Samples of total RNA (1–5 µg) were analyzed for mRNAs for cytochrome P450arom, 3ß-HSD, P450scc, and the LHr by RNase protection assay according to the instructions provided by the supplier (Ambion, Inc., Austin, TX). Antisense RNA probes were prepared using [32P]CTP (PerkinElmer Life Sciences, Boston, MA) from the following cDNA inserts: P450arom (bp 1034–1295) (11); rat LHr (bp 1–622) (12); P450scc (bp 18–816) (13); 3ß-HSD (bp 453–932) (14); and cyclophylin (bp 34–142) (15). After electrophoresis (5% acrylamide containing 8 M urea), gels were dried and exposed to x-ray film for 16–96 h. Densitometric analysis of protected RNA fragments was performed using NIH Image (version 1.61).

RT-PCR analysis
Total RNA was isolated as above and was reverse transcribed using random hexamer primers (I.D.T., Coralville, IA). PCRs were performed with the ABI Prism 7700 sequence detector system (PerkinElmer Applied Biosystems, Foster City, CA). The PCRs were performed in a volume of 25 µl containing 1 x Absolute SYBR green mix (ABgene House, Epsom, Surrey, UK), 70 nM of each forward and reverse primer (as described below), and 2 µl template. The templates included cDNA obtained from the reverse-transcribed RNA isolated from granulosa cells as well as serial dilutions (100 pg/µl to 1 fg/µl) of plasmids (pDC316) that contained the complete cDNA sequence for either human LRH-1 or mouse SF-1 to construct a standard curve. The PCRs were carried out at 50 C for 2 min, 95 C for 5 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. After completion of the PCR, a melting curve program (60–95 C with a heating rate of 1.75 C/min) was performed to confirm that specific PCR amplification product was generated. The quantification of copy number in the cDNA samples derived from granulosa cell RNA was done by interpolating the threshold cycle as a function of numbers of copies of input plasmid cDNA for SF-1 or LRH-1 (16).

The PCR primers for LRH-1 were designed to amplify both endogenous rat LRH-1 as well as adenovirus-directed human LRH-1: forward, 5'-TTCGGGCCAATGTACAAGAGA-3', reverse, 5'-TGGATCACCTGAGACATGGCT-3'.

The primers for SF-1 amplify both endogenous rat SF-1 and adenovirus-directed mouse SF-1: forward, 5'-GCAAAATCGACAAGACGCAG-3', reverse, 5'-CATTCGATCAGCACGCACAG-3'.

The efficiency of amplification and validation of the LRH-1 and SF-1 primers were determined using the standard curve data derived from the amplification of pDC316-hLRH-1 and pDC316-mSF-1. The efficiency of amplification for LRH-1 was 0.987 and 0.974 for SF-1. The predicted size of the PCR product was 101 bp for both, which was confirmed by gel electrophoresis.

RIA
Estradiol and progesterone content in the culture medium were determined by RIAs as described previously (17).

Statistics
Where indicated, results were assessed for statistical significance by ANOVA followed by comparison of group means with Fisher’s least significant difference analysis (StatView, version 4.5; Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of overexpression of SF-1 and LRH-1 on FSH-stimulated estrogen and progesterone production by undifferentiated granulosa cells
Figure 1Go illustrates the effects of infecting undifferentiated granulosa cells with increasing amounts of either Ad-LRH-1 or Ad-SF-1 in the presence of a fixed amount of human FSH (100 ng/ml). Testosterone (30 ng/ml) was also included in the culture medium in this and subsequent studies. As shown in Fig. 1Go (left panel, solid bars), FSH treatment alone resulted in a significant stimulation of progesterone production (P < 0.05). The magnitude of this stimulation was further increased (P < 0.05) by infecting the cells with adenoviral vectors for SF-1 and LRH-1 before FSH stimulation. However, the extent of the augmentation of FSH-stimulated progesterone production was significantly greater (P < 0.05) by Ad-SF-1(~4-fold) when compared with Ad-LRH-1 (~2-fold).


Figure 1
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  		FIG. 1. Effects of Ad-SF-1 and Ad-LRH-1 on progesterone and estradiol production by undifferentiated granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ß-gal, Ad-LRH-1, or Ad-SF-1 at concentrations ranging from 1 x 1010 particles/ml to 5 x 1011 particles/ml for 3 h, after which the virus-containing medium was removed and replaced with fresh serum-free M199. After 24 h, medium was removed and replaced with fresh medium containing 30 ng/ml testosterone with or without FSH (100 ng/ml). Forty-eight hours later, medium was collected and analyzed for progesterone and estradiol content by RIA. Results show mean ± 1 SEM of nine groups of granulosa cells. Values with different lower-case letters differ significantly (P < 0.05).

 
As shown in Fig. 1Go (right panel, solid bars), FSH treatment alone stimulated estrogen production by undifferentiated granulosa cells as expected. The extent of this stimulation was not increased by infection with Ad-LRH-1 (P > 0.05; FSH vs. FSH+LRH-1) and was only marginally increased (~1.7-fold) by infection with Ad-SF-1. Neither SF-1 nor LRH-1, when expressed in the presence of testosterone alone, stimulated estrogen or progesterone production (data not shown).

To determine whether SF-1 and LRH-1 are expressed at similar levels by the adenoviral vectors, we infected granulosa cells with increasing amounts of each vector and resultant mRNA levels of SF-1 and LRH-1 were determined by real-time RT-PCR analysis using SYBR green. Table 1Go illustrates that endogenous levels of SF-1 and LRH-1 were comparable in unstimulated granulosa cells, and the levels of both SF-1 and LRH-1 were increased 5- to 6-fold after stimulation of cells by FSH for 48 h. Infection of granulosa cells with Ad-LRH-1 or Ad-SF-1 resulted in further increases in each respective mRNA. Assuming that mRNA levels reflect those of the actual protein, these results indicated that comparable amounts of SF-1 and LRH-1 were achieved in response to infection with their respective adenoviral vectors.


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  		TABLE 1. Quantitative real-time RT-PCR analysis for LRH-1 mRNA and SF-1 mRNA in rat granulosa cells infected with Ad-LRH-1 and Ad-SF-1

 
Effects of overexpression of SF-1 and LRH-1 on the time course of estrogen and progesterone production
Undifferentiated granulosa cells undergo a characteristic 24-h lag period after FSH stimulation before maximal production of estrogen and progesterone commences. To determine whether SF-1 or LRH-1 differentially affect the timing of the induction of the estrogen and/or progesterone biosynthetic pathways, undifferentiated granulosa cells were infected with 5 x 1010 particles/ml of Ad-SF-1 or Ad-LRH-1, and estrogen and progesterone levels were measured after 24 and 48 h stimulation by FSH. Results shown in Fig. 2Go indicate that although both SF-1 and LRH-1 augmented FSH-stimulated progesterone production, neither dramatically altered the time course of either estrogen or progesterone production when compared with FSH stimulation alone.


Figure 2
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  		FIG. 2. Effects of Ad-SF-1 and Ad-LRH-1 on the time course of progesterone and estradiol production by undifferentiated granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ß-gal, Ad-LRH-1, or Ad-SF-1 at concentrations of 5 x 1010 particles/ml for 3 h, after which the virus-containing medium was removed and replaced with fresh serum-free M199. After 24 h, medium was removed and replaced with fresh medium containing 30 ng/ml testosterone with or without FSH (100 ng/ml). Medium was collected 24 and 48 h after the addition of FSH and analyzed for progesterone and estradiol content by RIA. Results show mean ± 1 SEM of three groups of granulosa cells.

 
The steroidogenic actions of both SF-1 and LRH-1 are amplified by PKB
We previously demonstrated that LRH-1 synergizes with PKB to augment FSH-stimulated progesterone production but not estrogen production and that in the absence of FSH, costimulation of granulosa cells by LRH-1 and PKB induced progesterone but not estrogen synthesis (18). The current study now shows that the overexpression of SF-1 produced comparable results to that previously reported for LRH-1. As illustrated in Fig. 3AGo, LRH-1 amplified FSH-stimulated progesterone production (FSH+Ad-LRH-1 vs. FSH; P < 0.05), whereas there was only a modest nonsignificant augmentation of FSH-stimulated progesterone production by adenovirus-mediated expression of constitutively active PKB (Ad-myr-PKB+FSH vs. FSH, P = 0.115), The combination of Ad-LRH-1 and Ad-myrPKB dramatically increased FSH-stimulated progesterone secretion (approximately 8-fold) over FSH alone (FSH+Ad-myrPKB+Ad-LRH-1 vs. FSH; P < 0.0001) Also, as reported previously (17), coinfection of undifferentiated granulosa cells with Ad-LRH-and Ad-myrPKB stimulated progesterone production in the absence of FSH (Ad-LRH1+Ad-myrPKB vs. control; P < 0.001).


Figure 3
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  		FIG. 3. LRH-1 and SF-1 increase progesterone production in combination with constitutively active PKB by undifferentiated rat granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ß-gal, Ad-LRH-1, or Ad-SF-1 with or without Ad-myrPKB at concentration of 5 x 1010 particles/ml for 3 h, after which the virus-containing medium was removed and replaced with fresh serum-free M199. After 24 h, medium was removed and replaced with fresh medium containing 30 ng/ml testosterone with or without FSH (100 ng/ml). Forty-eight hours later, medium was collected and analyzed for progesterone and estradiol content by RIA. Results show mean ± 1 SEM of six groups of granulosa cells.

 
As shown in Fig. 3BGo, the responses of granulosa cells to overexpression of SF-1 were qualitatively similar to those observed for LRH-1. As was observed for LRH-1, SF-1 overexpression augmented FSH-stimulated progesterone production (FSH+Ad-SF-1 vs. FSH; P < 0.001) and synergized with constitutively active PKB to augment progesterone production greater than 10-fold over FSH alone (FSH+Ad-myrPKB+Ad-SF-1 vs. FSH; P < 0.0001). In combination with constitutively active PKB, SF-1 stimulated progesterone production in the absence of FSH (Ad-SF-1+Ad-myrPKB vs. control; P < 0.005).

Estrogen production by undifferentiated granulosa cells in response to overexpression of SF-1 was also comparable with that of LRH-1. As shown in Fig. 3Go, C and D, overexpression of PKB alone significantly enhanced FSH-stimulated estrogen production approximately 3-fold over FSH alone (P < 0.05; FSH vs. FSH+PKB). However, unlike that seen for progesterone production, neither LRH-1 nor SF-1 in combination with constitutively active PKB and FSH stimulated estrogen production above that seen with FSH+PKB alone (P > 0.1; FSH+PKB vs. FSH+PKB+LRH-1 or SF-1). Likewise and again in contrast to progesterone production, constitutively active PKB in combination with either LRH-1 or SF-1 did not stimulate estrogen production in the absence of FSH (Ad-LRH-1+Ad-myrPKB vs. control; P = 0.66; Ad-SF-1+Ad-myrPKB vs. control; P = 0.62). Expression of constitutively active PKB alone did not stimulate either estrogen or progesterone production (6).

Interactions among FSH, PKB, LRH-1, and SF-1 in stimulating granulosa cell mRNAs associated with granulosa cell differentiation
We performed RNase protection assays on total RNA harvested from granulosa cells stimulated by various combinations of FSH, Ad-myrPKB, and either Ad-LRH-1 or Ad-SF-1 as shown in Fig. 4AGo. Figure 4Go, B and C, display the comparison of the relative intensities, obtained by densitometric scanning, of P450scc, 3ß-HSD, LHr, and P450arom mRNAs in response to the individual treatments. Each hybridization band was normalized to the respective intensity of the corresponding cyclophilin band. To standardize each of the treatments to one another, the response seen in the presence of FSH was set to 1.0.


Figure 4
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  		FIG. 4. Effect of expression of LRH-1 or SF-1 and constitutively active PKB on differentiation-associated mRNA level in undifferentiated granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ß-gal, Ad-LRH-1, AD-SF-1, or Ad-myrPKB at concentrations of 5.0 x 1010 particles/ml for 3 h, after which the virus-containing medium was removed and replaced with fresh serum-free M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without human FSH (100 ng/ml). Forty-eight hours later, total RNA was extracted from monolayers and analyzed for mRNAs by ribonuclease protection assay. A, Representative autoradiograph of hybridization signals for each of the treatments. B and C, Means ± 1 SEM (n = 3) of the densitometric analysis of the hybridization signals for cells infected with Ad-LRH-1 (B) and Ad-SF-1 (C). Results show the relative intensities of each mRNA normalized to its respective cyclophilin signal and standardized to intensity of the signal seen in response to FSH alone, which was set to 1.0.

 
Quantitative comparison of the overall profile of mRNA expression in cells revealed a remarkable similarity of the mRNA expression pattern of cells stimulated by LRH-1 (Fig. 4BGo) and SF-1 (Fig. 4CGo). FSH-stimulated mRNA levels for enzymes involved in progesterone production (P450scc and 3ß-HSD) exhibited the greatest response to overexpression of both LRH-1 and SF-1. Specifically, FSH-stimulated mRNA levels for P450scc exhibited the greatest response to overexpression of either LRH-1 or SF-1 in the absence of PKB (~5-fold) (P < 0.05 FSH+LRH-1 vs. FSH; P < 0.005 FSH+SF-1 vs. FSH). The levels of P450scc mRNA in FSH+LRH-1- and FSH+SF-1-treated cells were further stimulated to approximately 12-fold in the presence of constitutively active PKB (P < 0.0001 FSH+LRH-1+PKB vs. FSH; P < 0.0001 FSH+SF-1+PKB vs. FSH). Similarly, FSH-stimulated mRNA levels for 3ß-HSD were augmented by overexpression of either LRH-1 or SF-1 in the absence of PKB (3- to 5-fold) (P < 0.001 FSH+LRH-1 vs. FSH; P < 0.0001 FSH+SF-1 vs. FSH) and were further stimulated (~10-fold) in the presence of PKB (P < 0.0001 FSH+LRH-1+PKB vs. FSH; P < 0.0001 FSH+SF-1+PKB vs. FSH).

In contrast, whereas mRNA levels for P450arom and LHr in response to FSH were both further augmented (~4-fold) by expression of constitutively active PKB (P < 0.005, FSH+PKB-1 vs. FSH; P < 0.001, FSH+PKB vs. FSH), unlike that seen for P450scc and 3ß-HSD, this was not further increased by overexpression of either LRH-1 or SF-1 (P > 0.1 FSH+PKB-1 vs. FSH+PKB+LRH-1; P > 0.1 FSH+PKB vs. FSH+PKB+SF-1). In addition, in the absence of FSH, both SF-1 and LRH-1 in combination with PKB stimulated mRNAs for P450scc and 3ß-HSD (control vs. PKB+LRH-1 or SF-1; P < 0.05) but not mRNAs for P450arom or the LH receptor (control vs. PKB+LRH-1 or SF-1; P > 0.05).

SF-1 reverses DAX-1-mediated suppression of FSH-stimulated estrogen and progesterone production
Our inability to observe dramatic effects of overexpression of either SF-1 or LRH-1 on FSH-stimulated estrogen production is surprising in light of previously published observations demonstrating that both LRH-1 and SF-1 stimulate aromatase promoter activity in transient transfection assays (4, 5). This raised the possibility that the stimulation of estrogen biosynthesis (aromatase) in our cell system is refractory to overexpression of both SF-1 and LRH-1 because endogenous levels of these transcription factors (see Table 1Go) may be sufficient to optimally stimulate aromatase expression. DAX-1 has been shown to block steroidogenesis by interfering with transcriptional activity of SF-1 and LRH-1 (19, 20, 21). In agreement with these findings, Fig. 5Go illustrates that adenovirus-mediated overexpression of DAX-1 suppressed both FSH-stimulated progesterone (Fig. 5Go, A and C) and estrogen (Fig. 5Go, B and D) production (P < 0.05 FSH vs. FSH+DAX-1). Moreover, overexpression of SF-1 reversed DAX-1-mediated suppression of both FSH-stimulated progesterone (Fig. 5AGo) and estrogen (Fig. 5BGo) production. The effect of LRH-1 was less dramatic (Fig. 5Go, C and D) and did not result in a restoration of either FSH-stimulated estrogen or progesterone production to the extent seen in the absence of DAX-1.


Figure 5
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  		FIG. 5. DAX-1 inhibition of FSH-induced estrogen and progesterone production is reversed by SF-1. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ß-gal or Ad-DAX-1 (1 x 1010 particles/ml) in the absence or presence of increasing amounts of Ad-LRH-1 or Ad-SF-1 at concentrations ranging from 0.5 x 1010 to 1 x 1011 particles/ml for 3 h, after which the virus-containing medium was removed and replaced with fresh serum-free M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without human FSH (100 ng/ml). Forty-eight hours later, medium was collected and analyzed for estradiol and progesterone content by RIA. Result shows the mean ± 1 SEM of three groups of granulosa cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously demonstrated that overexpression of LRH-1 in undifferentiated granulosa cells augmented FSH-stimulated progesterone production and mRNAs for P450scc and 3ß-HSD but did not enhance FSH-stimulated estrogen production or expression of mRNA for P450arom (6). This was an unexpected finding because it has been shown by others that LRH-1 synergizes with forskolin to stimulate P450arom promoter activity in granulosa cells and preadipocytes (4, 5). Granulosa cells also express the closely related orphan nuclear receptor SF-1, which also has been shown to stimulate P450arom promoter activity in granulosa cells. LRH-1 and SF-1 share their greatest degree of homology in their DNA binding domains but differ in other regions, which could result in differential recruitment of coactivators or corepressors (22). This raises the possibility that LRH-1 and SF-1 could have different functional roles in granulosa cells with respect to the regulation of the estrogen and progesterone biosynthetic pathways. To test this hypothesis, the current study was undertaken to compare the effects of adenovirus-mediated overexpression of SF-1 and LRH-1 on estrogen and progesterone biosynthesis and the induction of the LH receptor that accompanies FSH-induced granulosa cell differentiation.

Our findings demonstrated that neither SF-1 nor LRH-1, in the absence of FSH, stimulated estrogen or progesterone production. However, both SF-1 and LRH-1 significantly amplified the stimulatory effects of FSH on progesterone production and the expression of mRNAs for P450scc and 3ß-HSD. When combined with constitutively active PKB, both SF-1 and LRH-1 stimulated progesterone production in the absence of FSH. The stimulation of the progesterone biosynthetic pathway by SF-1 and LRH-1 seen in the current studies is consistent with those of others that demonstrated that LRH-1 and/or SF-1 stimulate the expression of P450scc, steroidogenic acute regulatory protein, and 3ß-HSD in ovarian cells (21, 23, 24). Collectively, these results demonstrate that adenovirus-mediated overexpression of SF-1 or LRH-1 in undifferentiated granulosa cells produced qualitatively similar responses on FSH-stimulated progesterone production and mRNA levels for P450scc and 3ß-HSD. These observations are in agreement with recently reported results of Weck and Mayo (25), which demonstrated by transfection assays that SF-1 and LRH-1 acted similarly to increase inhibin {alpha}-subunit promoter activity in transformed mouse granulosa cells. Their studies also demonstrated by chromatin immunoprecipitation analysis that both SF-1 and LRH-1 occupy the {alpha}-subunit promoter in granulosa cells after treatment of rats with pregnant mare serum gonadotropin in vivo.

In contrast to the marked effects on progesterone production, neither FSH-stimulated estrogen production nor the expression of mRNA for P450arom nor the expression of mRNA for the LH receptor was augmented by overexpression of LRH-1 and was only slightly increased (0.6-fold) by overexpression of SF-1 (Figs. 1Go and 4Go). As noted above, the lack of a strong effect of either LRH-1 or SF-1 on estrogen production and mRNA for P450arom is surprising because previous studies using transient transfection approaches have shown that both SF-1 and LRH-1 augment cAMP-stimulation of the type II aromatase promoter. For example, Hinshelwood et al. (5) demonstrated in bovine granulosa cells that SF-1 and LRH-1 stimulated basal aromatase promoter activity 3-fold and forskolin stimulated promoter activity approximately 6-fold. Interestingly, Parakh et al. (26) recently demonstrated that the same adenoviral vector for SF-1 as we used in our current studies resulted in a strong augmentation of FSH-stimulated P450arom mRNA in undifferentiated rat granulosa cells. However, in these studies (26), granulosa cells were cultured in the absence of testosterone, a condition that results in only a minimal induction of aromatase by FSH (27). It is likely that the relatively small increases in aromatase expression expected to be seen in the absence of androgens would have been dwarfed in our studies by inclusion of testosterone in the culture medium.

One explanation for our current findings could be that in rodents, neither LRH-1 nor SF-1 is involved in the FSH-mediated stimulation of the estrogen biosynthetic pathway during granulosa cell differentiation. However, this must also be tempered with caution because of the recent findings that mice with a targeted disruption of SF-1 in granulosa cells exhibited hypoplastic uteri that, by inference, would suggest a defect in the expression of aromatase (28).

A second possible explanation for our current findings that the expression of aromatase is refractory to LRH-1 and SF-1 in granulosa cells is that the endogenous level of one or both of these transcription factors in undifferentiated granulosa is sufficient for optimal induction of aromatase by FSH. If so, overexpression of either (or both) would not be expected to further augment the level of aromatase, which is what we observed in this study. In accordance with this hypothesis, we would expect that inhibition of either LRH-1 or SF-1 signaling would curtail the expression of aromatase, which appears to be the case in the aforementioned SF-1 knockout mouse (28). To test this hypothesis, granulosa cells were infected with an adenoviral vector that directs the expression of DAX-1. The rationale for this study was that because one effect of DAX-1 is to interfere with the transcriptional activity of both SF-1 and LRH-1, the overexpression of DAX-1 should inhibit their endogenous actions in granulosa cells. Consistent with this notion, as shown in Fig. 5Go, overexpression of DAX-1 suppressed FSH stimulation of both estrogen and progesterone production. Moreover, DAX-1 inhibition of both estrogen and progesterone production was reversed by coinfection of granulosa cells with Ad-SF-1 and to a lesser extent by LRH-1. The ability of DAX-1 to suppress FSH stimulation of both estrogen and progesterone production raises the possibility that regulation of endogenous levels of DAX-1 could play a role in FSH-stimulated granulosa cell differentiation as it has been shown by others that stimulation of rat granulosa cells by FSH rapidly reduces DAX-1 expression at both the mRNA and protein levels (29). However, it must be noted that in addition to repressing SF-1 and LRH-1 stimulated transcription, DAX-1 has also been shown to interfere with the transcriptional activities of other transcription factors including the estrogen, progesterone and androgen receptors and Nur77 (30, 31, 32, 33). Thus, it cannot be concluded that the inhibitory actions of DAX-1 on steroidogenesis are due solely to the inhibition of the actions of SF-1 and/or LRH-1. Studies are currently underway in our laboratory to test this hypothesis further by constructing adenovirus vectors to deliver small interfering RNAs to reduce endogenous DAX-and SF-1 levels in granulosa cells.

Although the effects of overexpression of SF-1 and LRH-1 produced qualitatively similar responses in granulosa cells, it appeared that, on a quantitative level, SF-1 overexpression resulted in a greater augmentation of progesterone production than did LRH-1 (Fig. 1Go). In addition, whereas overexpression of LRH-1 did not augment estrogen production, there was a very slight, but consistent, augmentation of FSH-stimulated estrogen production by SF-1 (Fig. 1Go). One possible explanation for these quantitative differences is that the efficacy of the individual adenoviral vectors for SF-1 and LRH-1 differ such that greater levels of SF-1 may have been achieved than LRH-1. To the best of our knowledge, there is no way to directly compare the exact levels of LRH-1 and SF-1 proteins after viral infection. However, if these quantitative differences between SF-1 and LRH-1 were due simply to the extent of their levels of expression, this should have been revealed by dose-response studies in which granulosa cells were infected with progressively increasing amounts of each adenoviral vector. As can be seen in Fig. 1Go, both progesterone and estradiol responses to increasing amounts of Ad-LRH-1 plateaued at maximal levels that were less than the responses to increasing amounts of Ad-SF-1, which would argue against these quantitative differences between SF-1 and LRH-1 being due to different levels of their expression. Moreover, real-time quantitative RT-PCR analysis revealed that comparable levels of mRNA for LRH-1 and SF-1 were achieved as a result of adenoviral infection. A final caveat to our current study is that we used human LRH-1 and mouse SF-1 in these studies. However, it is unlikely that the use of proteins from these different species compromised the validity of our findings because both human LRH-1 and mouse SF-1 require the binding of endogenous phospholipid ligands for maximal activity (34).

In conclusion, our current findings indicate that overexpression of LRH-1 and SF-1 have qualitatively similar actions on FSH-stimulated estrogen and progesterone production. However, on a quantitative basis, SF-1 appeared to be more effective than LRH-1 in stimulating estrogen production, which could explain the apparent lack of estrogen production in mice with a targeted disruption of SF-1 in granulosa cells (28).


   Footnotes
 
This work was supported by NIH Grant HD 47260 (to A.J.Z.) and NIH Training Grant HD 07332 (to D.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 9, 2006

Abbreviations: Ad-ß-gal, Adenovirus vector directing expression of ß-galactosidase; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1; FBS, fetal bovine serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; LHr, LH receptor; LRH-1, liver receptor homolog-1; M199, Medium 199; P450arom, P450 aromatase; PKB, protein kinase B; P450scc, cholesterol side-chain cleavage enzyme; SF-1, steroidogenic factor-1.

Received January 25, 2006.

Accepted for publication October 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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