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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.
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Abstract |
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Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active Gs Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and Gs signaling pathways are required. |
Introduction |
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We have adopted the use of replication-defective adenovirus vectors to explore the signaling pathways involved in granulosa cell function. Because these vectors infect granulosa cells and direct the expression of proteins with high efficiency (10), it is possible to express constitutively activated as well as dominant-negative signaling proteins and directly assess their influences on granulosa cell steroidogenesis and cAMP production as well as the expression of mRNAs that encode for differentiation and proliferation-associated proteins. Previously, with this approach we expressed both the wild-type and a constitutively active human LH receptor in undifferentiated granulosa cells (i.e. no prior exposure to FSH) and found that although activation of FSH receptors and LH receptors produced comparable responses in stimulating progesterone production and increasing mRNA levels for -inhibin and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), activation of FSH receptors was more effective than both the wild-type and the constitutively active LH receptor in stimulating mRNA levels for aromatase and the endogenous granulosa cell LH receptor (11). Collectively, these observations suggest that the expression of aromatase and the LH receptor during FSH-induced granulosa cell differentiation may be controlled by signaling pathways preferentially used by the FSH receptor. In the present study we continue our use of adenovirus vectors to further elucidate the signaling pathways involved in FSH-stimulated granulosa cell differentiation and proliferation.
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Materials and Methods |
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Adenovirus vectors
Preparation of the constitutively activated human LH receptor adenovirus vector Ad-RSV-LHrD578H was described previously (11). Adenovirus vectors that direct the expression of a constitutively active PKB (Ad-myrAKT) and a dominant negative PKB (Ad-dnAKT) under the control of a cytomegalovirus (CMV) promoter were obtained from Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA) (12). A constitutively active MAPK/ERK [MAPK kinase (MEK)] adenoviral vector (Ad-caMEK) was provided by Dr. N. Mitsuda (Osaka University, Osaka, Japan) (13). An adenovirus that directs the expression of a ß-galactosidase (Ad-ßgal) was provided by Dr. Joseph Alcorn (University of Texas Medical School, Dallas, TX). An adenovirus vector that directs the expression of a constitutively activated Gs (Ad-GsQ227L) was constructed using the AdMax system (Microbix Biosystems, Inc., Toronto, Canada). A 1195-bp fragment from plasmid GNAOSLOOCO (Guthrie Research Institute, Sayre, PA) corresponding to the coding sequence of human GsQ227L was excised and subcloned into the adenovirus shuttle vector pDC316. Five micrograms of the pDC316 GsQ227L shuttle vector were cotransfected with 5 µg of the shuttle vector pBHGlox
E,3Cre into the human embryonic kidney cell line 293 in 60-mm culture dishes using the Superfect reagent according to the manufacturer’s directions (QIAGEN, Inc., Valencia, CA). Transfected cells were maintained in DMEM containing 4.5 g/liter glucose (Life Technologies, Inc., Gaithersburg, MD) and 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcorss, GA) at 37 C in 5% CO2. Approximately 14 d after transfection when the viral cytopathic effect was observed, the cells were collected and frozen on dry ice, thawed three times, then further propagated in 293 cells. When the cells exhibited a complete viral cytopathic effect, the cells were collected, resuspended in PBS, frozen and thawed on dry ice three times, and then centrifuged (1000 x g, 4 C, 10 min) to remove cellular debris. Aliquots of virus stocks were diluted 50- and 100-fold in lysis solution [0.1% sodium dodecyl sulfate, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA] and incubated for 10 min at 56 C in a shaking water bath. The OD of the samples was measured at 260 nm, and the value obtained was used to calculate virus content using the relationship 1012 virus particles/ml/OD 260 U (14).
Granulosa cell culture
All procedures were approved by the University of Pittsburgh institutional animal care and use committee. Immature female rats (23–25 d old) were purchased from Hilltop Lab Animals (Scottsdale, PA). Granulosa cells were collected from the ovaries by puncturing follicles with a 25-gauge hypodermic needle, and cells were expressed into medium 199 (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum. 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 as described in the figure legends. At the end of the experiment, tissue culture medium was collected, boiled for 10 min to inactivate phosphodiesterases, 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.
mRNA analysis
Samples of total RNA (1–5 µg) were analyzed for mRNAs for cytochrome P450 aromatase, 3ß-HSD, the -subunit of inhibin, and the LH receptor by ribonuclease 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: P450 aromatase (bp 1034–1295) (15), rat LH receptor (bp 1–622) (16), subunit of inhibin (bp 694-1095) (17), 3ß-hydroxysteroid dehydrogenase (bp 453–932) (18), proliferating cell nuclear antigen (PCNA; bp 204–456) (19), and cyclophylin (bp 34–142) (20). After electrophoresis (5% acrylamide containing 8 M urea), gels were dried and exposed to x-ray film for 16–96 h.
Aromatase assay
Granulosa cells were plated in 12-well dishes and stimulated with FSH, 8-bromo-cAMP (8Br-cAMP), and adenovirus vectors as described above. After stimulation, cells were washed twice with PBS and incubated for 2 h in 1 ml medium 199 containing 30 pmol 1ß-[3H]androstenedione (PerkinElmer Life Sciences) and 120 pmol unlabeled androstenedione for 2 h at 37 C in 5% CO2. After incubation, the medium was transferred to an extraction tube containing 0.5 ml 30% trichloroacetic acid, and 2 ml chloroform were added. The tubes were mixed vigorously for 15 sec and centrifuged for 5 min at 3000 rpm, after which 1.0 ml of the aqueous upper phase were transferred to a 12 x 75-mm glass tube. At 4 C, 1 ml dextran/charcoal (5.0 g Norit plus 0.5 g dextran in 100 ml H2O) was added to each tube and incubated for 20 min. The samples were centrifuged at 3500 rpm for 30 min, and 1.0 ml of the supernatants was transferred and counted for 3H radioactivity in a liquid scintillation counter. Resultant counts were corrected for background and extraction efficiency, and cell monolayers were analyzed for protein content by Bradford assay.
RIA
Estradiol and progesterone concentrations in culture medium were determined by RIAs as described previously (21). cAMP concentrations in culture medium were analyzed by RIA using [125I]cAMP-2-0' monosuccinlyl cAMP tyrosine methyl ester (22) and anti-cAMP in accordance with the instructions provided by the National Hormone and Pituitary Program.
Western immunoblotting
Granulosa cells were scraped into cold PBS, centrifuged at 14,000 x g for 10 min, and resuspended in TE buffer [50 mM Tris-HCl (pH 7.4) and 1.0 mM EDTA] supplemented with 20 µg/ml phenylmethylsulfonylfluoride and 0.5 µg/ml leupeptin, 0.2 mM sodium vanadate, and 100 nM microcystin (10). Whole cell lysates were separated on 12% SDS-discontinuous polyacrylamide gels, and the resolved proteins were electrophoretically transferred to nitrocellulose membranes. Anti-PKB immunoblotting was performed using a polyclonal Anti-PKB (no. 9272, Cell Signaling, Beverly, MA) at a final concentration of 1 µg/ml. Chemiluminescent detection was accomplished using the BM Chemiluminescence Western Blotting Kit (Roche, Indianapolis, IN) with the appropriate horseradish peroxidase-conjugated secondary antibody diluted to 1:2000 according to the manufacturer’s directions. Membranes were stripped and probed with an anti-CREB antibody (SC-186, Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 1 µg/ml to verify equivalent sample loading and analyzed as described above.
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).
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Results |
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SQ227L on estrogen, progesterone, and cAMP production by undifferentiated granulosa cellsQ227L (Ad-GSQ227L). Samples of culture medium were collected 48 h after FSH stimulation and 72 h after virus infection and were analyzed for estrogen, progesterone, and cAMP content by RIA. Figure 1
illustrates that each of the stimuli resulted in dose-dependent increases in cAMP, progesterone, and estrogen production. The absolute amounts of progesterone produced were comparable among the cells stimulated by FSH, Ad-LHrD578H, and Ad-GSQ227L, whereas the production of estrogen, an index of aromatase activity, was greater in cells stimulated by FSH than in cells stimulated by Ad-LHrD578H and Ad-GSQ227L (P < 0.01). It can be seen in Fig. 1 that cAMP production rates were similar in response to 100 ng/ml FSH, Ad-LHrD578H at 5 x 1010 particles/ml, and Ad-GSQ227L at 1.3 x 1010 particles/ml. These concentrations of FSH and adenoviral vectors were used in subsequent studies to achieve comparable levels of cAMP with the different treatments to diminish the likelihood that any differences in granulosa cell responses would be due to differences in cAMP production.
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illustrates the time course of progesterone production and aromatase activity as assessed by the conversion of [3H]androstenedione to 3H2O in response to FSH, Ad-LHrD578H, and Ad-GSQ227L. At selected time intervals, progesterone production in response to each stimulus was comparable. However, similar to the estrogen production rates shown in Fig. 1
, aromatase activity was significantly elevated by FSH at 48 and 72 h compared with activity in response to both Ad-LHrD578H and GsQ227L (P < 0.01).
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illustrates the expression of selected mRNAs by granulosa cells stimulated by FSH, Ad-LHrD578H, and Ad-GSQ227L. As reported previously (11), activation of the FSH receptor and the LH receptor produced similar increases in mRNAs for 3ß-HSD and -inhibin, whereas activation of the FSH receptor was more effective in inducing mRNA for the LH receptor and aromatase. In the current study the effects of Ad-GSQ227L were similar to those of Ad-LHrD5788H, in that both stimulated 3ß-HSD and -inhibin, but were not as effective as FSH in stimulating mRNAs for aromatase and the LH receptor. Collectively, the results presented in Figs. 1–3 confirm our earlier results that activation of the FSH receptor is more effective than activation of the LH receptor with respect to the induction of aromatase and the induction of mRNA for the LH receptor. In addition, the current results show that Ad-GSQ227L exerted effects similar to those of Ad-LHrD578H on aromatase and LH receptor mRNA, suggesting that the optimal induction of aromatase and the LH receptor by FSH relies on other intracellular signaling pathways in addition to Gs.
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signaling pathway in the induction of aromatase and the LH receptor presents data from a pilot study to explore this possibility. As expected, FSH plus testosterone stimulated the production of estrogen and progesterone. Although neither Ad-myrPKB nor Ad-caMEK in the presence of testosterone stimulated estrogen or progesterone production, Ad-myrPKB amplified the stimulatory effects of FSH plus testosterone on both estrogen and progesterone production. Ad-caMEK appeared to suppress FSH-stimulated estrogen and progesterone production; however, this observation was not pursued further in the current study. Figure 5 illustrates an immunoblot analysis to document that infection of granulosa cells with Ad-myrPKB leads to enhanced expression of PKB.
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SQ227L, and 8Br-cAMP in the presence and absence of the constitutively activated PKB (Ad-myrPKB). As shown in Fig. 6, Ad-myrPKB amplified the stimulatory effects of FSH on both estrogen and progesterone production. In addition, neither Ad-LHrD578H, Ad-GSQ227L, nor 8Br-cAMP alone stimulated estrogen production to the extent seen with FSH, although each, in combination with Ad-myrPKB, stimulated estrogen production comparable to that seen in cells stimulated with FSH. An obligatory role for PKB in FSH-stimulated granulosa cell differentiation was revealed by cells stimulated with FSH after being infected with an adenovirus vector that expresses a dominant-negative mutant of PKB (Ad-dnPKB). In these cells, FSH-stimulated production of estrogen and progesterone was inhibited without any reduction in FSH-stimulated cAMP production. The lack of effect of Ad-dnPKB on FSH-stimulated cAMP production indicates that its inhibition of steroidogenesis was probably not due to a toxic effect of viral infection. Assessment of aromatase activity using the tritiated water production assay yielded results identical to those obtained by measurement of estradiol by RIA (data not shown). Figure 6 also illustrates that Ad-myrPKB enhanced FSH-stimulated cAMP production, but did not appear to elevate cAMP production in response to Ad-LHrD578H or Ad-GSQ227L. Table 1 shows that Ad-myrPKB also slightly elevated basal cAMP production by undifferentiated granulosa cells.
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shows that the expression of constitutively activated PKB amplified the expression of the differentiation-associated mRNAs for 3ß-HSD, -inhibin, P450 aromatase, and the LH receptor in response to FSH, Ad-LHrD578H, 8Br-cAMP, and Ad-GSQ227L. However, neither mRNA for cyclin D2 nor PCNA was amplified by the expression of the constitutively activated PKB. Furthermore, whereas the dominant-negative mutant of PKB (Ad-dnPKB) abrogated the FSH stimulation of differentiation-associated mRNAs, it did not reduce the basal expression of mRNAs for cyclin D2 or PCNA.
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demonstrates that the phosphoinositol 3-kinase (PI-3 kinase) inhibitor wortmannin (100 nM) blocked FSH-stimulated increases in estrogen and progesterone production by granulosa cells. Infection with Ad-myrPKB overcame the wortmannin-induced inhibition, indicating that the enhancement of FSH-stimulated granulosa cell differentiation by PKB is probably mediated through PI-3 kinase.
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indicate that this Epac-selective cAMP analog failed to increase estrogen production alone or in combination with either FSH or Ad-GSQ227L.
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illustrates that inhibition of protein kinase A by 10 µM H-89 abolished FSH-stimulated induction of aromatase in undifferentiated granulosa cells, as assessed by measurement of estrogen production. Although Ad-myrPKB augmented FSH-stimulated estrogen production, it did not overcome the inhibition imposed by H-89. These results indicate that PKB is not downstream of PKA and that optimal induction of aromatase requires both PKA and PKB.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Q227L can stimulate cAMP production to an extent equivalent to or greater than that of FSH. This vector was effective in stimulating progesterone production and increasing mRNA levels for -inhibin and 3ß-HSD comparable to that of FSH, but was less effective than FSH in stimulating aromatase activity and the expression of mRNA for the LH receptor. In this regard, the cellular responses to Ad-GSQ227L were similar to those of Ad-LHrD578H and those resulting from the direct stimulation of granulosa cells by 8Br-cAMP. These observations therefore indicate that activation of the Gs signaling pathway and its resultant stimulation of cAMP production alone are not sufficient for the optimal induction of aromatase and the LH receptor by FSH. Interestingly, Laven et al. (27) recently investigated ovarian function in a women with McCune-Albright syndrome, a sporadic disease caused by an activating mutation of the GNAS1 gene, which in this patient encodes for a constitutively active GsR201H. As a result of this mutation, numerous follicular cysts were present in the affected ovary, and analysis of follicular fluid estrogen and progesterone concentrations in smaller follicles of the affected ovary revealed a disproportionate increase in the progesterone to estrogen ratio, similar to the steroidogenic profile seen in our current study of granulosa cells infected with the adenoviral vector which directs the expression of the constitutively active GsQ227L. To identify other interacting pathways, we infected undifferentiated granulosa cells with adenoviral vectors that direct the expression of constitutively active mutants of PKB and MEK. Our findings indicated that neither PKB nor MEK alone was sufficient to stimulate estrogen or progesterone production. However, when constitutively activated PKB was expressed in combination with FSH treatment, we observed a dramatic amplification of estrogen production and, to a lesser extent, progesterone production compared with cells that were stimulated by FSH alone. Moreover, the expression of a dominant-negative mutant of PKB blocked FSH-stimulated estrogen production and aromatase activity without interfering with FSH-stimulated cAMP production. By contrast, the constitutively activated MEK did not synergize with FSH to stimulate estrogen or progesterone production. In fact, this preliminary study suggests that MEK may be antagonistic to granulosa cell steroidogenesis, as was recently reported by others (28).
The constitutively activated PKB also amplified progesterone production by granulosa cells either stimulated by FSH or 8Br-cAMP or infected with an adenoviral vectors that directs the expression of constitutively activated LH receptors and Gs. More importantly, however, although neither the constitutively active LH receptor, the constitutively active Gs, nor 8Br-cAMP was effective in stimulating estrogen production, aromatase activity, or mRNAs for aromatase and the LH receptor, each in combination with activated PKB, became effective in stimulating these hallmarks of FSH-stimulated granulosa cell differentiation. The finding that neither 8Br-cAMP nor GsQ227L optimally stimulated the expression of aromatase is somewhat surprising in view of fact that the aromatase promoter contains a cAMP response element (29). However, the results shown in Fig. 10 demonstrate that inhibition of PKA by H-89 completely blocks the stimulation of estrogen production by FSH, indicating that PKA (and presumably cAMP response element-binding protein activation) is also required for induction of aromatase. Moreover, the finding that Ad-myrPKB cannot overcome the inhibition of aromatase imposed by H-89 indicates that both the PKA and PKB signaling pathways are required. Elucidation of the targets downstream of PKB and PKB that underlie this synergism is probably the key in understanding granulosa cell differentiation.
Surprisingly, infection of granulosa cells with Ad-myrPKB amplified FSH-stimulated cAMP production, but did not appear to augment cAMP production in response to Ad-LHrD578H or Ad-GSQ227L. At the present time, the mechanism by which this occurred is not known. Whether PKB regulates the activity of G proteins and/or phosphodiesterases in granulosa cells remains a question for future investigation. It is also possible that there is cellular compartmentalization of the FSH signaling system (3), and that local changes in phosphodiesterase activity might selectively affect FSH-stimulated cAMP production, but not that stimulated by Ad-LHrD578H or Ad-GaSQ227L.
The synergism between the PKB and the Gs signaling pathways in regulating granulosa cell differentiation is not confined to aromatase and the LH receptor, as mRNAs for -inhibin and 3ß-HSD were also amplified in the presence of PKB. Likewise, FSH-stimulated progesterone production was amplified by PKB. This raises the question of whether PKB exerts a global effect on granulosa cell function such as modifications of cellular metabolism that could indirectly amplify gene expression. This does not appear to be the case because, as shown in Fig. 7, PKB did not amplify the expression of the proliferation-associated mRNAs PCNA and cyclin D2, and the dominant-negative PKB did not reduce the levels of expression of these mRNAs. In this regard, we demonstrated previously that activin synergizes with both FSH and forskolin to regulate the expression of PCNA and cyclin D2 in granulosa cells (19). It would therefore appear that a number of individual signaling factors, including PKB, Smad, and androgens (30), may converge upon the Gs pathway to regulate granulosa cell differentiation and proliferation.
The activation of PKB is complex and can occur in a PI-3 kinase-dependent and independent manner (31). Although we have not examined directly the activation of PKB in the current studies, the finding that the PI-3 kinase inhibitor wortmannin blocked FSH-stimulated estrogen and progesterone production and that this inhibition was completely reversed by the constitutively active PKB (Fig. 8) provides indirect evidence for a link between PI-3 kinase and PKB in FSH-stimulated granulosa cell differentiation. In support of this, Gonzalez-Robayna et al. (5) demonstrated that FSH stimulates the phosphorylation and activation of PKB which is blocked by wortmannin but not the PKA inhibitor H-89. These authors suggest that phosphorylation of PKB could be mediated by activation of PI-3 kinase through cAMP-dependent guanine nucleotide exchange factors (cAMP-GEF). However, our preliminary observation that 8CPT-2Me-cAMP, a cAMP analog that selectively activates the cAMP-GEF Epac1 (26), does not stimulate estrogen and progesterone production (Fig. 9) alone or in combination with constitutively active Gs indicates that the cAMP-Epac-Rap1/Rap2 pathway is not a principal participant in granulosa cell differentiation, at least as assessed by estrogen and progesterone production under the tissue culture conditions in which these studies were conducted. Alternate mechanisms for PKB activation could also include either stimulation of PI-3 kinase by IGF-I (32), which, in turn, could augment the responsiveness of granulosa cells to FSH (33) or direct stimulation of PI-3 kinase by a G protein ß
heterodimer (34). However, a caveat to this hypothesis is that Gonzalez-Robayna et al. (5) also demonstrated that 8Br-cAMP stimulates PKB phosphorylation in granulosa cells. This would indicate that both the cAMP and PKB pathways would be concurrently activated by 8Br-cAMP, yet in our studies 8Br-cAMP did not optimally induce aromatase or mRNA for the LH receptor (Figs. 5–7). At the present time, we have no explanation for this other than the possibility that the extent to which PKB is activated by FSH and 8Br-cAMP could differ either as a function of time or as absolute activity.
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mimics the effect of FSH on aromatase and LH receptor mRNA, it appears that both the PKB and Gs pathways are required for the expression of these hallmarks of granulosa cell differentiation. However, because the constitutively active PKB did not amplify mRNA levels for PCNA or cyclin D2, and the dominant-negative PKB did not reduce the expression of these mRNAs, it appears that PKB may not be required for the proliferative effects of FSH.
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Acknowledgments |
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cDNA into the adenovirus shuttle vector; Drs. J. Alcorn, Kenneth Walsh, and N. Mitsuda for providing us with adenovirus vectors; and Dr. Carole Mendelson for providing us with a protocol for the aromatase assay. |
Footnotes |
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Abbreviations: 8Br-cAMP, 8-Bromo-cAMP; CMV, cytomegalovirus; 8CTP-2Me-cAMP, 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP; Epac, guanine exchange factor directly activated by cAMP; FBS, fetal bovine serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; MEK, MAPK kinase; PCNA, proliferating cell nuclear antigen; PI-3 kinase, phosphoinositol 3-kinase; PKB, protein kinase B.
Received March 5, 2003.
Accepted for publication May 29, 2003.
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L. Tosca, P. Froment, P. Solnais, P. Ferre, F. Foufelle, and J. Dupont Adenosine 5'-Monophosphate-Activated Protein Kinase Regulates Progesterone Secretion in Rat Granulosa Cells Endocrinology, October 1, 2005; 146(10): 4500 - 4513. [Abstract] [Full Text] [PDF]
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F. X. Donadeu and M. Ascoli The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade Endocrinology, September 1, 2005; 146(9): 3907 - 3916. [Abstract] [Full Text] [PDF]
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F.-Q. Yu, C.-S. Han, W. Yang, X. Jin, Z.-Y. Hu, and Y.-X. Liu Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially J. Endocrinol., July 1, 2005; 186(1): 85 - 96. [Abstract] [Full Text] [PDF]
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E. M. Ongeri, M. F. Verderame, and J. M. Hammond Follicle-Stimulating Hormone Induction of Ovarian Insulin-Like Growth Factor-Binding Protein-3 Transcription Requires a TATA Box-Binding Protein and the Protein Kinase A and Phosphatidylinositol-3 Kinase Pathways Mol. Endocrinol., July 1, 2005; 19(7): 1837 - 1848. [Abstract] [Full Text] [PDF]
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C. Gillio-Meina, Y. Y. Hui, and H. A. LaVoie Expression of CCAAT/Enhancer Binding Proteins Alpha and Beta in the Porcine Ovary and Regulation in Primary Cultures of Granulosa Cells Biol Reprod, May 1, 2005; 72(5): 1194 - 1204. [Abstract] [Full Text] [PDF]
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 Y. Park, E. T. Maizels, Z. J. Feiger, H. Alam, C. A. Peters, T. K. Woodruff, T. G. Unterman, E. J. Lee, J. L. Jameson, and M. Hunzicker-Dunn Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad J. Biol. Chem., March 11, 2005; 280(10): 9135 - 9148. [Abstract] [Full Text] [PDF]
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 C. Daelemans, G. Smits, V. de Maertelaer, S. Costagliola, Y. Englert, G. Vassart, and A. Delbaere Prediction of Severity of Symptoms in Iatrogenic Ovarian Hyperstimulation Syndrome by Follicle-Stimulating Hormone Receptor Ser680Asn Polymorphism J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6310 - 6315. [Abstract] [Full Text] [PDF]
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E C Chin and D R E Abayasekara Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation J. Endocrinol., October 1, 2004; 183(1): 51 - 60. [Abstract] [Full Text] [PDF]
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D. Saxena, R. Safi, L. Little-Ihrig, and A. J. Zeleznik Liver Receptor Homolog-1 Stimulates the Progesterone Biosynthetic Pathway during Follicle-Stimulating Hormone-Induced Granulosa Cell Differentiation Endocrinology, August 1, 2004; 145(8): 3821 - 3829. [Abstract] [Full Text] [PDF]
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C. A. Nechamen, R. M. Thomas, B. D. Cohen, G. Acevedo, P. I. Poulikakos, J. R. Testa, and J. A. Dias Human Follicle-Stimulating Hormone (FSH) Receptor Interacts with the Adaptor Protein APPL1 in HEK 293 Cells: Potential Involvement of the PI3K Pathway in FSH Signaling Biol Reprod, August 1, 2004; 71(2): 629 - 636. [Abstract] [Full Text] [PDF]
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H. Alam, E. T. Maizels, Y. Park, S. Ghaey, Z. J. Feiger, N. S. Chandel, and M. Hunzicker-Dunn Follicle-stimulating Hormone Activation of Hypoxia-inducible Factor-1 by the Phosphatidylinositol 3-Kinase/AKT/Ras Homolog Enriched in Brain (Rheb)/Mammalian Target of Rapamycin (mTOR) Pathway Is Necessary for Induction of Select Protein Markers of Follicular Differentiation J. Biol. Chem., May 7, 2004; 279(19): 19431 - 19440. [Abstract] [Full Text] [PDF]
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