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Hormone Research Center, Chonnam National University (K.-H.S., J.-I.P., J.S., K.L., H.-S.C.), Kwangju 500-757, and Department of Bioscience and Biotechnology, Sejong University (M.-O.L.), Seoul 143-747, Republic of Korea
Address all correspondence and requests for reprints to: Hueng-Sik Choi, Ph.D., Hormone Research Center, Chonnam National University, Kwangju 500-757, Republic of Korea. E-mail: hsc{at}chonnam.ac.kr
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase by
PGF2 (18). Using a genetic selection approach, Nur77 was found to recognize a specific nucleotide sequence called NGFI-B (Nur77)-responsive element (NBRE) (19, 20), which contains an E receptor half-site (AGGTCA) preceded by two adenines. Furthermore, Nur77 binds DNA as a monomer, and a region outside of the zinc finger domain (A box) was shown to play an important role in DNA binding specificity (19). Recently, a novel Nur77 target sequence called Nur response element (NurRE) was identified in the POMC promoter (21, 22), and Nur77 binds this element as a homodimer and heterodimer with other Nur family members (23). It has also been demonstrated that Nur 77 heterodimerizes with RXR and binds to a RAR element composed of direct repeats separated by five nucleotides (DR5) in the presence of 9-cis-retinoic acid (24). Although information regarding Nur77 function has been accumulated, the physiological role of this orphan nuclear receptor largely remains to be determined.
The present study demonstrates that LH induces Nur77 gene expression in mouse Leydig cells, and this LH-mediated induction of Nur77 gene expression is regulated via diverse cell signaling pathway. Furthermore, LH increased the Nur77 activity, and inhibition of Nur77 reduced LH-mediated progesterone biosynthesis in Leydig cells. Taken together, these results suggest that LH-induced Nur77 gene expression may play an important role in the steroidogenesis of Leydig cells in the testis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells
The K28 cell line was originally subcloned from the LK17 hybrid
clone, which was derived from the fusion between MA-10 mouse Leydig
tumor cells and freshly isolated mouse Leydig cells (25, 26). The K28 mouse Leydig tumor cell line has been characterized
as a suitable cell culture model for steroidogenesis (27, 28).
Plasmids
The mouse Nur77 cDNA, dominant negative Nur77 (DN-Nur77) cDNA,
and Nur77 promoter-luciferase reporter, NBRE-tk-Luc reporter construct
were described previously (14, 15, 18, 21, 29, 30), and
NurRE 3 copy-POMC-Luc reporter construct and Nurr-1 were obtained from
Dr. Jacques Drouin (Institut de Recherches Cliniques de Montréal,
Canada) and Dr. Thomas Perlmann (Institute for Cancer Research,
Sweden), respectively.
Northern blot analysis
K28 cells were grown in DMEM supplemented with 15% FBS. The
cells were serum-starved at 80% confluence in serum-free medium for
24 h. After the culture medium was changed to the serum-free
condition, cells were treated with LH (200 ng/ml) from 30 min to
24 h. Total RNA was isolated using Tri-Reagent
(Sigma). Twenty micrograms of total RNA were fractionated
by electrophoresis on 1.2% agarose gel containing formaldehyde and
were transferred to a nylon membrane (
-probe, Bio-Rad Laboratories, Inc., Richmond, CA) by capillary blotting with
10x SSC. After UV cross-linking and prehybridization, membranes were
hybridized 24 h at 42 C in solution containing 50% formamide,
10% dextran sulfate, 5x SSC, 1 mM EDTA, 10 mg/ml
denatured salmon sperm DNA, and a total of 2–4 x
106 cpm -32P-labeled
mouse Nur77 cDNA containing ligand-binding domain and Nurr-1 cDNA.
After hybridization, membranes were washed twice for 5 min at room
temperature in 2x SSC and 0.1% SDS, followed by 1 h at 65 C in
0.5x SSC and 0.1% SDS. Membranes were then exposed using
Kodak RX films (Eastman Kodak Co., Rochester,
NY) for 12–24 h at –70 C. The expression of
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an
internal control. The band intensities were subsequently measured using
a phosphorimager (Bio-Rad Laboratories, Inc., Hercules,
CA), and the signals were normalized to the GAPDH internal control.
Transient transfection and ß-galactosidase assay
Twenty-four hours before transfection, K28 cells were plated in
24-well culture dishes at a density of 2.5 x
104 cells/well. Transfection was performed using
LipofectAMINE Plus reagent (Life Technologies, Inc.,
Gaithersburg, MD) with Nur77 promoter-Luc, NBRE-Luc,
NurRE-Luc, Nur77, DN-Nur77, and internal control
promoter CMV (pCMV)-ß-galactosidase as recommended by the
manufacturer. Twenty-four hours after transfection, the cells were
treated with various concentrations of LH. Thirty-six hours after LH
treatment, cells were lysed with 100 µl 1% Triton X-100, 25
mM GLY-GLY (pH 7.5), 15 mM
MgSO4, and 2 mM EGTA for 15 min.
Twenty microliters of the cell lysates were assayed for luciferase
activity with a dual luciferase reporter assay system (Promega Corp., Madison, WI) and determined with an MLX microtiter
luminometer (Dynex Technologies, Inc., Chantilly,
VA). The lysates were transferred into 96-well microtiter plates
for ß-galactosidase assay by using the
o-nitrophenyl-ß-D-galactopyranoside
(Sigma) as a substrate as described previously
(31). The luciferase activities were normalized to the
ß-galactosidase activity expressed from the cotransfected
pCMV-ß-gal plasmid and reported as the mean ±
SE in relative light units. All transfection
experiments were performed at least five times in duplicate.
Western blot analysis
LH-treated K28 cells were resuspended in lysis buffer [50
mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EGTA, 1
mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1
µg/ml leupeptin, and 1 mM sodium fluoride] and incubated
on ice for 10 min and then lysed by homogenizer. The lysates were
centrifuged to remove cell debris, and the supernatant was collected
and frozen at -80 C until further use. Protein concentrations were
estimated using bicinchoninic acids protein assays (Pierce Chemical Co., Rockford, IL). Protein lysates (50 µg) were
boiled for 5 min in denaturing sample buffer and loaded onto a 12%
continuous gradient SDS-polyacrylamide gel, and proteins were
transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was blocked with
TBST buffer [10 mM Tris-buffered isotonic saline (pH 7.0),
0.1% merthiolate, and 0.1% Tween-20] containing 5% nonfat dry milk
for 30 min at room temperature with shaking, followed by incubation
with primary antibody (anti-Nur77 at 0.5 µg/ml dilution;
PharMingen, San Diego, CA) in TBST buffer for 24 h at
4 C with gentle shaking. Membrane was washed twice with TBST for 10 min
each time and incubated with antimouse IgG conjugated to alkaline
phosphatase (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBST for 1 h at room temperature.
Finally, membrane was washed twice with TBST, and Nur77-specific bands
were visualized using a Western-Star chemiluminescent detection system
(Tropix, Inc., Bedford, MA) according to the manufacturer’s
guidelines.
EMSA
Briefly, cultured K-28 cells were washed twice with cold PBS and
pelleted by centrifugation at 3,000 rpm for 5 min at 4 C. The pellets
were gently resuspended in buffer (20 mM HEPES, 10
mM EDTA, 0.1% Nonidet P-40, 100 mM NaCl, 0.15
M phenylmethylsulfonylfluoride, leupeptin, and pepstatin)
and broken by passing 20 times through a 25-gauge needle. The cells
were kept on ice for 1 h at 4 C and centrifuged at 14,000 rpm at 4
C for 30 min. The supernatants containing the whole cell extracts were
aliquoted and stored at -80 C. Probes used for EMSA experiments were
prepared by labeling 10 pmol double stranded oligonucleotides with T4
polynucleotide kinase (Promega Corp.) at 37 C for 30 min.
The labeled probes were purified by Sephadex G-50 column
chromatography. A sample containing 40,000–50,000 cpm of the purified
double stranded oligonucleotides was used for each reaction. EMSA was
performed with 1 µg poly(dI/dC)/sample as a nonspecific competitor.
The DNA-protein complexes were separated from the unbound DNA probe via
6% nondenaturing gel electrophoresis at 4 C in Tris base glacial
acetic EDTA buffer, and the binding reaction was carried out at 25 C
for 30 min. The sequences of oligonucleotides used as probes for NBRE
and mutated SF-1RE were 5'-GGAGTTTAAAAGGTCATGCTC-3' and
5'-CCCATCAATTATATAAAT-3', respectively.
RIA
For LH treatment, the exponentially growing K28 cells in the
100-mm dish were split into an appropriate number of 60-mm dishes
(3.5 x 105 cells/dish) and cultured for 24 h in
DMEM supplemented with 15% FBS. Transfection was performed using
LipofectAMINE Plus reagent (Life Technologies, Inc.) with
DN-Nur77 expression vector (1 µg) or Nur77 expression vector (1 µg)
and internal control pCMV-ß-gal. After 24-h transfection,
cells were washed twice with Dulbecco’s PBS, and medium was replaced
with 2 ml DMEM containing 15% FBS and incubated for 24 h. Cells
were washed twice with Dulbecco’s PBS and 4 ml serum-free DMEM
containing low density lipoprotein (5 µg/ml) was added to the
cells. After cells were incubated with or without LH (200 ng/ml) for
the designated period, the cell culture medium was obtained for RIA.
Culture media were assayed directly without further purification. The
general assay procedure was used as described previously
(32). The progesterone concentration was calculated with
SecuRIA program (Packard, Downers Grove, IL). Coefficients of variation
for progesterone between and within assay were 9.4% and 9.2%,
respectively. The lower limit of assay sensitivity for progesterone was
6.5 pg. Transfection efficiency was normalized by ß-galactosidase
activity. Each treatment group contained duplicate cultures, and each
experiment was repeated at least twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). The levels of testis Nur77
expression were 3-fold (n = 2) higher in 30-d-old mice than in
20-d-old mice. This result suggests that Nur77 gene expression is
regulated during the postnatal stage of testis development.
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).
Quantitative analysis showed that hCG treatment resulted in a 4.7-fold
increase in Nur77 mRNA at 12 h (Fig. 1D), indicating that LH/hCG
could regulate Nur77 gene expression in testis.
LH induces Nur77 gene expression in a time- and dose-dependent
manner
As the secretion of LH is increased during the puberty in testis,
and a large number of studies have shown that LH is the main regulator
of adult Leydig cells (33, 34), we investigated the effect
of LH on the induction of Nur77 gene expression in the mouse testis
Leydig cell line, K28. Northern blot analysis showed that LH treatment
(200 ng/ml) caused a transient increase in Nur77 mRNA expression,
reaching a maximum level, 63-fold higher than the basal level, within
1 h and returning to the basal level after 3 h (Fig. 2, A and B). Moreover, LH induced
Nur77-related member Nurr-1 mRNA expression with a similar time-course
pattern of Nur77 mRNA expression (Fig. 2A), and LH induced Nur77 mRNA
in a dose-dependent manner, reaching a saturation level at 100 ng/ml
(Fig. 2C). To investigate whether LH could also increase Nur77 protein
expression, Western blot analysis with the Nur77-specific antibody was
performed. LH retained its ability to induce Nur77 synthesis (Fig. 2D),
and the induction of Nur77 protein was dramatically increased within 30
min of LH treatment and remained at its highest level for 2 h
after LH treatment. A broad range of Nur77 protein sizes (70 up to 90
kDa) was observed, suggesting that phosphorylation of Nur77 might be
involved in the response to LH. Taken together, these results suggest
that one of the important roles of LH in Leydig cells might be the
induction of orphan nuclear receptor Nur77 gene expression.
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, A and B).
Moreover, adenylate cyclase activator, forskolin (10 µM),
cell-permeable cAMP analog, 8-bromo-cAMP (1 mM), or TPA
(200 nM) alone induced Nur77 gene expression, confirming
that PKA and PKC pathways are involved in Nur77 gene induction.
Finally, wortmannin (10 nM), an inhibitor of PI3 kinase,
inhibited 70% of LH-mediated Nur77 gene induction, suggesting that the
PI3K signaling pathway might also be involved in this induction. Taken
together, these results suggest that the Nur77 gene induction by LH
stimulation is mediated through diverse signal transduction
pathways.
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). Forskolin and TPA also significantly
induced Nur77 promoter activity (Fig. 4B), suggesting that Nur77 gene
transcription is regulated by both PKA and PKC cell signaling. These
results indicate that the transcriptional regulation of Nur77 gene
expression is associated with LH-mediated Nurr77 gene induction via a
specific cell signaling pathway.
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). Nur77 DNA-binding activity was
completely abolished by 25- and 50-fold excesses of unlabeled specific
probe. However, a 50-fold excess of oligomer containing the mutated
SF-1RE motif could not competitively inhibit protein binding. To
determine whether Nur77 is present in this complex, whole cell extracts
were preincubated with a monoclonal antibody against Nur77 protein.
Nur77-specific antibody reduced the formation of the LH-induced
Nur77-DNA complex, whereas nonspecific antibody had no significant
effect on Nur77-DNA complex formation (Fig. 5B). These results clearly
demonstrate that LH induces Nur77 DNA-binding activity.
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, A and B), suggesting that the monomer
or dimer formation of Nur77 was differentially regulated by LH. These
results indicate that LH regulates Nur77 activity via direct induction
of its gene expression.
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, DN-Nur77 significantly inhibited Nur77 trans-activation in a
dose-dependent manner. To correlate the LH-mediated induction of Nur77
gene expression with the regulation of steroidogenesis, DN-Nur77 was
transiently expressed in K28 cells, the cell culture media were
collected, and the amount of progesterone was determined by RIA.
Progesterone synthesis was significantly induced after 1 h and
gradually declined up to 6 h in LH-treated (200 ng/ml) K28 cells.
In contrast, overexpression of DN-Nur77 in LH-treated cells was
accompanied by a significant reduction in progesterone (Fig. 7B).
DN-Nur77 maintained an average transfection efficiency of about 40%
(data not shown). Moreover, overexpression of the active form of Nur77
in K28 cells significantly increased progesterone production in the
absence of LH (Fig. 7C). These results strongly suggest that
LH-mediated Nur77 expression may be involved in steroidogenesis in
Leydig cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that ACTH, an anterior pituitary peptide hormone, rapidly induces Nur77 mRNA synthesis in the mouse adrenocortical tumor cell, Y1 (10), and more recently that PTH also induces Nur77 mRNA in primary mouse osteoblasts (37), and LH induces NGFI-B mRNA in rat ovarian follicle (38). Our results demonstrated that LH rapidly increased the expression of Nur77 mRNA in the Leydig cell line, K28. Unlike ACTH, which induced Nur77 mRNA up to 16 h, LH-mediated induction of Nur77 mRNA returned to basal levels after 6 h, suggesting that LH-mediated induction of Nur77 gene expression is more transient than that of ACTH-mediated induction. Moreover, LH induced the subfamily member of Nur77, Nurr-1, suggesting that the normal phenotype in Nur77 null mice (11) might be due to functional redundancy among the Nur77 family. Western blot analysis showed that LH induces Nur77 protein ranging from 70–90 kDa. It has been well documented that Nur77 is phosphorylated at multiple sites (7, 8, 9, 39) related to its trans-activation, and a recent study demonstrates that NGF induces phosphorylation of Nur77 on Ser105 in PC12 pheochromocytoma cells, which results in the translocation of Nur77 from nucleus to cytoplasm (40), and the phosphorylation on Ser354 decreases Nur77 DNA-binding activity (41, 42). Therefore, LH-mediated hyperphosphorylation of Nur77 protein may modulate both its DNA-binding and trans-activation activities, and the potential amino acid residues of phosphorylation of Nur77 by LH still remain to be determined.
To investigate the intracellular signaling pathways controlling Nur77 gene expression, we examined several inhibitors and activators of signaling pathway. In addition to LH, forskolin, TPA, and 8-bromo-cAMP also induced Nur77 expression in K28 cells. Although it has been well documented that cAMP is the major second message for LH, there is considerable evidence to suggest that other intracellular signaling systems may also be involved in LH action (43). These include IP3, diacylglycerol, calcium, arachidonic acid, and various free radicals. This idea is strongly supported by the observation that LH-mediated Nur77 gene expression was abolished by H-89, an inhibitor of PKA; bisindolylmaleimide I (GF109203X), an inhibitor of PKC; and wortmannin, an inhibitor of PI3 kinase, whereas there was no significant effect of PD98059, an inhibitor of MAPK, on Nur77 gene expression (data not shown). In contrast to a previous report on ACTH- and angiotensin II-mediated Nur77 induction in adrenal cortical cells (44), LH-mediated Nur77 induction in K28 was strongly inhibited by H-89, indicating that Nur77 expression is differentially regulated in a stimulus- and cell type-specific-manner. Therefore, it seems reasonable to speculate that LH induces Nur77 gene expression through diverse cell signaling pathways in Leydig cells.
Transient transfection experiments with Nur77 promoter (bp -336 to +67)-Luc reporter indicate that LH induces Nur77 promoter activity. Although we have not ruled out the possible implication of the further upstream region of Nur77 promoter, this promoter region was sufficient to confer LH-mediated Nur77 gene induction in K28 cells. Multiple cis-acting elements, such as GC-rich/SP-1 site, activating protein-1-like elements, and RSRF elements in this promoter region, were characterized previously (15, 41, 45, 46, 47, 48). Therefore, multiple transcription factors may also be involved in the LH-mediated Nur77 induction in Leydig cells.
It has been reported that Nur77 recognizes a specific sequence called the NBRE, AAAGGTCA (19). We showed that Nur77 constitutes NBRE-binding activity in LH-stimulated Leydig cells. Interestingly, Nur77 DNA-binding activity was observed from 2–24 h, indicating that the Nur77-DNA complex is continuously maintained in LH-treated testicular Leydig cells. This pattern of Nur77-binding activity is reminiscent of a previous report (49) of the induction of immediate-early response gene c-fos/c-jun gene expression by antioxidant. This immediate-early response gene expression is transient, but the activating protein-1 binding activity is continuously maintained, and this delayed-type DNA-binding activity does not correlate with transient induction of c-fos/c-jun. Therefore, we concluded that the delayed and continuous DNA-binding activity of Nur77 by LH treatment might be due to the intrinsic characteristics of the orphan nuclear receptor Nur77.
To identify the Nur77 target gene in Leydig cells, we examined the cholesterol side-chain cleavage enzyme and steroidogenic acute regulatory protein gene promoter activity by Nur77 in K28 cells. Consistent with previous report (50), Nur77 did not show any specific effect on steroidogenic acute regulatory protein or cholesterol side-chain cleavage enzyme promoter activity (data not shown). Although we could not determine the putative Nur77 target gene that is involved in response to LH, identification of specific genes regulated by Nur77 in Leydig cells is currently under investigation in our laboratory.
A recent study has demonstrated that Nur77 is the sole transcription
factor that mediates PGF2 stimulation of
20-hydroxysteroid dehydrogenase (20HSD), which converts
progesterone into biologically inactive steroid (18). In
contrast to this report, there was no significant change in 20HSD
mRNA after LH treatment in K28 cells (data not shown), indicating that
LH-mediated Nur77 induction may not be implicated in the regulation of
20HSD gene expression. However, the existence of NBRE sequence in
several steroidogenic enzyme gene promoters (51, 52)
suggests that Nur77 may participate in the regulation of
steroidogenesis in Leydig cells. Interestingly, we observed that
overexpression of DN-Nur77 suppressed LH-induced progesterone
biosynthesis. Moreover, overexpression of the active form of Nur77
significantly increased progesterone biosynthesis, suggesting that
LH-mediated Nur77 may play an important role in the regulation of
steroidogenesis in Leydig cells. However DN-Nur77 could not completely
abolish LH-mediated progesterone biosynthesis, suggesting that the
induction of steroidogenic enzymes by other transcription factors may
also be implicated in LH-mediated steroidogenesis. To our knowledge,
this is the first report that Nur77 is directly linked to
steroidogenesis in testicular Leydig cells.
In summary, we have shown that orphan nuclear receptor Nur77 gene expression is regulated by LH in a testis Leydig cell line. LH treatment induces Nur77 gene expression via diverse signaling pathway, and this induction is regulated at a transcriptional level. Furthermore, LH-mediated Nur77 gene expression is involved in steroidogenesis in testicular Leydig cells. Identification of the target genes regulated by Nur77 will be necessary to understand the detailed function of Nur77 responding to LH in testis development.
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Acknowledgments |
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Footnotes |
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Abbreviations: CMV-ß-gal,
Cytomegalovirus-ß-galactosidase; DN-Nur77, dominant
negative Nur77; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase;
hCG, human CG; 20HSD, 20-hydroxysteroid dehydrogenase; NBRE,
NGFI-B (Nur77)-responsive element; NGF, nerve growth factor;
NurRE, Nur response element; TBST, 10 mM Tris-buffered
isotonic saline (pH 7.0), 0.1% merthiolate, and 0.1% Tween-20; TPA,
12-O-tetradecanoyl-phorbol-13-acetate.
Received April 6, 2001.
Accepted for publication August 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced
expression of 20-hydroxysteroid dehydrogenase involves the
transcription factor NUR77. J Biol Chem 275:37202–37211
5–4-isomerase and
17-hydroxylase/C17–20lyase cytochrome P-450 messenger ribonucleic
acid levels in the K9 mouse Leydig cell line. Mol Cell Endocrinol 106:31–39[CrossRef][Medline]
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W. Li, H. Amri, H. Huang, C. Wu, and V. Papadopoulos Gene and Protein Profiling of the Response of MA-10 Leydig Tumor Cells to Human Chorionic Gonadotropin J Androl, November 1, 2004; 25(6): 900 - 913. [Abstract] [Full Text] [PDF]
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K.-H. Song, Y.-Y. Park, K. C. Park, C. Y. Hong, J. H. Park, M. Shong, K. Lee, and H.-S. Choi The Atypical Orphan Nuclear Receptor DAX-1 Interacts with Orphan Nuclear Receptor Nur77 and Represses Its Transactivation Mol. Endocrinol., August 1, 2004; 18(8): 1929 - 1940. [Abstract] [Full Text] [PDF]
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 H.-J. Shin, B.-H. Lee, M. G. Yeo, S.-H. Oh, J.-D. Park, K.-K. Park, J.-H. Chung, C.-K. Moon, and M.-O. Lee Induction of orphan nuclear receptor Nur77 gene expression and its role in cadmium-induced apoptosis in lung Carcinogenesis, August 1, 2004; 25(8): 1467 - 1475. [Abstract] [Full Text] [PDF]
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 C. Y. Hong, J. H. Park, R. S. Ahn, S. Y. Im, H.-S. Choi, J. Soh, S. H. Mellon, and K. Lee Molecular Mechanism of Suppression of Testicular Steroidogenesis by Proinflammatory Cytokine Tumor Necrosis Factor Alpha Mol. Cell. Biol., April 1, 2004; 24(7): 2593 - 2604. [Abstract] [Full Text] [PDF]
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J. J. Tremblay and R. S. Viger Transcription Factor GATA-4 Is Activated by Phosphorylation of Serine 261 via the cAMP/Protein Kinase A Signaling Pathway in Gonadal Cells J. Biol. Chem., June 6, 2003; 278(24): 22128 - 22135. [Abstract] [Full Text] [PDF]
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D. J. McLean, P. J. Friel, D. Pouchnik, and M. D. Griswold Oligonucleotide Microarray Analysis of Gene Expression in Follicle-Stimulating Hormone-Treated Rat Sertoli Cells Mol. Endocrinol., December 1, 2002; 16(12): 2780 - 2792. [Abstract] [Full Text] [PDF]
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K.-H. Song, K. Lee, and H.-S. Choi Endocrine Disrupter Bisphenol A Induces Orphan Nuclear Receptor Nur77 Gene Expression and Steroidogenesis in Mouse Testicular Leydig Cells Endocrinology, June 1, 2002; 143(6): 2208 - 2215. [Abstract] [Full Text] [PDF]
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) and a 50-fold excess of
nonspecific competitor (N. S.), mutated SF-1 binding sequence, and
1 µg Nur77-specific antibody (Nur77 Ab) or nonspecific antibody,
progesterone antibody (PR Ab), were added to the reaction mixture
before the addition of labeled probe (B). Free, Running of labeled
probe only; arrows, Nur77-DNA complex;
arrowheads, nonspecific binding (N.B.).
) or presence of LH (200 ng/ml;
) and DN-Nur77 (
) at the designated time points; the cultured
medium was collected; and the concentration of progesterone in the
medium was measured by RIA as described in Materials and
Methods. Each point represents the average
amount of progesterone per 1 ml. C, K28 cells were transfected with
empty vector (1 µg;