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a Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF) treatment blocks FSH-induced differentiation and StAR expression in cultured hen granulosa cells, whereas inhibitors of MAP kinase signaling enhance FSH-induced differentiation. The present in vitro studies demonstrate that in addition to MAP kinase signaling, activation of protein kinase C (PKC) blocks both FSH-induced StAR expression and the initiation of progesterone production in prehierarchal follicle granulosa cells, whereas the pharmacologic inhibitor of PKC, GF109203X, potentiates FSH-induced StAR expression and, as a consequence, the initiation of progesterone synthesis. Moreover, we demonstrate in granulosa cells collected from preovulatory follicles that although an acute increase in progesterone production in response to LH treatment requires rapid transcription and translation of StAR, the magnitude of progesterone production is rate-limited by one or more factors other than StAR (e.g., the P450 cholesterol side-chain enzyme). Finally, the rapid turnover of StAR protein, such as occurs following the withdrawal of LH, provides an additional mechanism for the tight regulation of progesterone production that occurs during the hen ovulatory cycle, and explains the rapid loss of steroidogenesis in the postovulatory follicle. In summary, data reported herein support the proposal that paracrine/autocrine factors (including but not necessarily limited to TGF) prevent premature expression of StAR in prehierarchal follicle granulosa cells by more than one receptor-mediated signaling pathway. Furthermore, subsequent to follicle selection into the preovulatory hierarchy, StAR transcription and translation is necessary but not sufficient for the full potentiation of the preovulatory surge of serum progesterone.
follicular development, granulosa cells, growth factors, ovary, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cloning and characterization of the chicken StAR cDNA [5] has enabled us to document the induction of StAR mRNA and protein expression as a critical component of FSH-mediated differentiation in hen granulosa cells derived from immature, prehierarchal follicles [12]. This up-regulation of StAR is a prerequisite for the induction of granulosa cell progesterone production, and is associated with the selection of a follicle into the preovulatory hierarchy. Moreover, the full potentiation of FSH-induced StAR expression and progesterone production at this stage of development appears to be dependent on the inhibition of paracrine or autocrine factor-mediated mitogen-activated protein (MAP) kinase signaling (e.g., via transforming growth factor ). Once a follicle reaches the preovulatory stage of development, granulosa cells become LH receptor-dominant [13], and LH-mediated progesterone production occurs in association with the rapid induction of StAR mRNA (within 1 h) and protein (within 3 h) expression [12]. It is well established that a fully potentiated preovulatory surge of serum progesterone is obligatory for ovulation of the largest preovulatory follicle [14, 15].
Accordingly, the present studies were conducted to extend our understanding of mechanisms regulating StAR expression and function in hen granulosa cells both at approximately the time of follicle selection, as well as during the period leading up to and following an LH-induced progesterone surge in preovulatory follicles. A primary difference between these two stages of follicle development rests in the fact that undifferentiated cells from prehierarchal follicles fail to express StAR to any significant extent (despite FSH receptor expression), whereas differentiated granulosa cells from preovulatory follicles readily up-regulate the expression of StAR and produce large amounts of progesterone in response to gonadotropin (largely LH) treatment. Moreover, the working hypothesis tested herein is that the full potentiation of FSH-mediated differentiation in FSH receptor-dominant granulosa cells from prehierarchal follicles requires a concomitant removal of tonic inhibitory paracrine signaling. Specifically, experiments were designed to address 1) the role of PKC signaling in modulating FSH-induced differentiation in granulosa cells from prehierarchal follicles, and 2) the relationship between LH-induced StAR expression and the preovulatory progesterone surge in granulosa cells from hierarchal follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Single-comb white Leghorn hens (Creighton Brothers, Warsaw, IN), 25–35 wk of age and laying regular sequences of more than six eggs, were used in all studies described. Birds were housed individually in laying batteries, provided with free access to feed (Purina Layena Mash; Purina Mills, St. Louis, MO) and water, and were exposed to a 15L:9D photoperiod, with lights-on at midnight. Ovulatory cycles were monitored by recording oviposition on a daily basis. Hens were killed approximately 16–18 h before a midsequence ovulation by cervical dislocation. All procedures described herein were reviewed and approved by the University of Notre Dame Institutional Animal Care and Use Committee, and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Recombinant human transforming growth factor (TGF) was obtained from PeproTech, Inc (Rocky Hill, NJ), whereas ovine LH (AFP-5551B) and recombinant human FSH (lot AFP-8468A) were provided by Dr. A.F. Parlow through the National Hormone and Peptide Program. The MAP kinase inhibitor, U0126, and PKC inhibitor, GF109203X, were from Biomol (Plymouth Meeting, PA), and phorbol 12-myristate 13-acetate (PMA; an activator of PKC), actinomycin D (an inhibitor of RNA transcription), and cycloheximide (a protein synthesis inhibitor) were from Sigma Chemical Co. (St. Louis, MO).
Tissue Collection and Granulosa Cell Cultures
Granulosa layers from the three largest preovulatory (F1, F2, and F3) follicles and undifferentiated prehierarchal (6–8 mm) follicles were collected, combined within their respective group, and dispersed in 0.3% collagenase (type 2; Worthington, Freehold, NJ) before treatment. An aliquot of cells from each group was frozen immediately at -70°C (T0 controls), whereas the remaining cells were plated at 40°C in an atmosphere containing 5% CO2/95% O2 in six-well polystyrene culture plates (Falcon 3046; Fisher Scientific, Pittsburgh, PA) at a density of approximately 106/well in 1 ml of M199-Hepes supplemented with Hanks salts (Gibco-BRL, Gaithersburg, MD) plus 1 ml of Dulbecco modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS; Gibco-BRL). In some instances, granulosa cells from the F1 follicle, or from the combined F2 plus F3 follicle layers, or both, were studied separately because it had previously been established that levels of StAR mRNA were dramatically higher in F1 follicles than in F2 or F3 follicles [5]. Morphologically normal and atretic prehierarchal follicles (3–8 mm) were collected and frozen without separating the granulosa and theca layers, as previously described [16].
In the first set of experiments, levels of StAR mRNA were compared in normal versus atretic prehierarchal follicles. In addition, we evaluated the involvement of MAP kinase signaling versus PKC signaling for initiation of StAR expression and progesterone production in functionally undifferentiated prehierarchal follicle granulosa cells. Granulosa cells were precultured for 1 h in the absence or presence of U0126 (50 µM), then cultured an additional 20 h with FSH (100 ng/ml), or TGF (50 ng/ml) plus FSH. Doses used were based on previous experiments in hen granulosa cells [12, 17]. Following culture, media and plated cells were collected, cells were pelleted by centrifugation at 500 x g for 5 min, then media and cells were frozen separately at -70°C until media were assayed for progesterone and cells were prepared for total cellular mRNA or protein.
To explore alternative signaling pathways by which TGF can affect StAR expression, prehierarchal follicle granulosa cells were cultured in the absence or presence of FSH, PMA [18] or FSH plus 162 nM PMA for 20 h. In addition, cells were precultured for 1 h in the absence or presence of GF109203X (10 µM), then cultured an additional 20 h with FSH or TGF plus FSH. The dose and preculture time for GF109203X was derived from previous studies of this pharmacologic inhibitor in granulosa/luteal cells [19, 20], and importantly, neither U0126 nor GF109203X induce cell death in hen granulosa cells after 24 h of culture ([12] and unpublished data). Media and cells were collected and processed as described above. In a related experiment, the effectiveness of each inhibitor to block MAP kinase signaling through extracellular signal-regulated kinases 1 and 2 (Erk1/2) was tested by pretreating freshly cultured granulosa cells for 1 h in the absence or presence of U0126 or GF109203X, then treating cells for an additional 20 min without or with PMA or TGF. Samples were processed for levels of phosphorylated Erk (Erk-P). Treatment with this dose of TGF has previously been demonstrated to be an effective inducer of Erk-P in hen granulosa cells [17].
In a second set of experiments, granulosa cells from F2 plus F3 follicles were precultured for 1 h in the absence and presence of actinomycin D (1 µg/ml) or cycloheximide (0.5 µg/ml), then cultured an additional 3 h without or with LH (100 ng/ml) to evaluate the requirement of StAR transcription, translation for progesterone production, or both. In addition, equal numbers of F1 or F2 plus F3 granulosa cells were incubated in the absence or presence of LH for 30 to 180 min to correlate levels of induced StAR protein with progesterone production according to stage within the hierarchy. For both of these experiments, media and cells were collected separately and frozen at -70°C until assayed for progesterone or prepared for total cellular RNA, protein levels, or both. For comparison, levels of P450scc mRNA in freshly collected granulosa cells from the F1 follicle were compared with those in F2 follicles.
To evaluate the turnover of StAR protein in cultured cells, granulosa cells from F1 follicles were pretreated with LH for 3 h, then medium was removed and cells were rinsed once with medium before culturing an additional 1 to 6 h in the absence of LH. Control cells included those that were untreated or that remained in the presence of LH for the entire 9-h experimental period. All granulosa cells were collected, frozen, and subsequently processed for protein. Finally, levels of StAR mRNA in freshly collected tissue and progesterone production from incubated (for 3 h) granulosa cells derived from the largest (F1) preovulatory follicle were compared with those collected from the most recent and second-most recent postovulatory follicles (POF1 and POF2, respectively; collected approximately 10 to 12 h following the most recent preovulatory LH surge). Granulosa cell layers from postovulatory follicles were collected by gently scraping the granulosa layer away from the theca layer with a scalpel blade.
Northern Blot Analysis
The chicken StAR cDNA used for probing Northern blots has been previously described [5], while the chicken P450scc probe consisted of a 569-base pair polymerase chain reaction (PCR) product corresponding to nucleotides 383 to 952 from chicken P450scc (GenBank accession number D49803). Separate blots were probed for StAR mRNA or P450scc mRNA, then each was reprobed for chicken 18S ribosomal RNA to standardize for equal loading of RNA samples. Conditions for conducting Northern blot analysis have been previously detailed [21], and all blots were visualized on phosphor screens using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Analysis of blots was conducted using the ImageQuant data reduction system (Molecular Dynamics).
Western Blot Analysis
The StAR polyclonal antibody was generously provided by Dr. D.B. Hales (University of Illinois-Chicago; [22]). The phospho-specific Erk1/2 monoclonal antiserum was from Upstate Biotechnology (Lake Placid, NY), the Erk2 polyclonal antiserum was from Santa Cruz Biotechnology (Santa Cruz, CA), and the -tubulin monoclonal antibody used for standardization was from Sigma. Western blot analyses were conducted essentially as previously described [12, 17]. Incubations with primary antibodies (phospho-Erk at a 1:1000 dilution; Erk2 at a 1:1000 dilution; and StAR at a 1:5000 dilution) were conducted overnight at 4°C, whereas those for the horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (Ig) G secondary antibody (for phospho-Erk; Pierce, Rockford, IL) or HRP-conjugated anti-rabbit IgG secondary antibody (for Erk2 and StAR; Pierce) were incubated for 1 h at room temperature. Blots were incubated with enhanced chemiluminescence Western blotting agent (Amersham Corp., Arlington Heights, IL) for 1 min and exposed to x-ray film for 1–15 min. The extent of antibody binding was quantitated by densitometry (UltraScan XL laser densitometer, Pharmacia LKB, Piscataway, NJ).
Progesterone Assays
Progesterone levels in media samples were quantified without extraction by radioimmunoassay according to previously published methods [18].
Data Analysis
All experiments were repeated a minimum of three times, unless otherwise stated. Summarized levels of StAR and P450scc mRNA and progesterone were expressed as fold-difference (mean ± SEM) versus a designated reference treatment such as freshly collected (T0) or cultured control cells (the value for the reference treatment was arbitrarily set at 1). Data were analyzed by t-test or by one-way ANOVA (analysis of fold-difference data did not include the reference treatment) followed by the Fisher protected least significant difference multiple range test. Post hoc analysis of selected data was conducted by paired t-test using original (nontransformed) data (e.g., Fig. 2, progesterone data).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A single 2.8-kilobase transcript for StAR mRNA was detected in whole, morphologically normal prehierarchal follicles, but it was virtually undetectable in atretic follicles (Fig. 1). As recently reported [12], the MAP kinase inhibitor, U0126, potentiates FSH-induced StAR mRNA and protein expression plus progesterone production in cultured prehierarchal follicle granulosa cells. The novel finding is that TGF treatment attenuates this stimulatory effect on StAR expression and progesterone production (Fig. 2) even though U0126 completely prevents TGF-induced Erk-P (Fig. 3).
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Similar to the effects of TGF, cotreatment with PMA, a pharmacologic activator of PKC, effectively blocks the induction of FSH-induced StAR mRNA and progesterone production after 20 h in culture (Fig. 4), and this inhibitory effect is not mediated via Erk-P (Fig. 3). Furthermore, pretreatment of granulosa cells with the PKC inhibitor GF109203X potentiates FSH-induced StAR mRNA and protein expression plus progesterone production. However, under these conditions, cotreatment with TGF completely reverses the potentiating effect of GF109203X (Fig. 5) and, importantly, GF109203X fails to attenuate TGF-mediated Erk-P (Fig. 3).
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Relationship Between StAR Expression and Progesterone Production in Preovulatory Follicles
Preculture of F2 plus F3 follicle granulosa cells with actinomycin D effectively blocks LH-induced StAR mRNA transcription and protein translation, and greatly attenuates LH-induced progesterone production after a 3-h culture (Fig. 6). By comparison, preculture with cycloheximide fails to alter LH-induced StAR mRNA, but completely inhibits any increase in StAR protein and, moreover, effectively blocks LH-induced progesterone production.
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Although granulosa cells from the F1 follicle and F2 plus F3 follicles show comparable LH-induced responses after 90 to 180 min of treatment in terms of StAR protein expression, there is significantly greater progesterone production from F1 follicle granulosa cells at each interval (Fig. 7). To address this apparent discrepancy, levels of P450scc mRNA were compared in F1 versus F2 follicles, and were found to be significantly higher in granulosa cells from F1 follicles (Fig. 8).
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Finally, levels of StAR protein are increased by 17.5 ± 4.4-fold after a 3-h pretreatment of cultured F1 follicle granulosa cells with LH (compared to control). Following the withdrawal of LH, StAR protein rapidly decreases to nondetectable levels within 6 h (Fig. 9). In an effort to relate this rapid loss of StAR expression to the interval following the preovulatory LH surge and early postovulatory period, in vivo, StAR mRNA (a more sensitive measure of detection than protein levels) was evaluated in freshly collected granulosa layers from the F1 and two most recent postovulatory follicles. Basal expression of the StAR transcript was readily detected in granulosa cells from the F1 follicle, but was decreased by more than 50% in granulosa cells from POF1 (collected 6 to 8 h following ovulation) and was virtually nondetectable in POF2. Similarly, basal progesterone produced after a 3-h incubation (expressed as nanograms of progesterone per micrograms of RNA) decreased from 4.9 ± 0.6 to 0.6 ± 0.1, respectively (P < 0.01; Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability to detect StAR mRNA in whole, nonatretic prehierarchal follicles likely represents expression almost entirely within the theca layer, as granulosa cells at this stage of development express little to no StAR [5] and produce essentially no progesterone [24]. Not unexpectedly, follicle atresia is associated with a significant decrease in LH and FSH receptor mRNA expression [13, 25] and, presumably due to the withdrawal of gonadotropin support, the loss of StAR mRNA expression. Although the theca layer from viable prehierarchal follicles is known to be steroidogenically active [26], it has not yet been established whether the loss of StAR expression, and as a result steroidogenic potential, is a contributing factor to the death of the granulosa layer or simply a result of the atretic process.
On the other hand, granulosa cells collected from normal prehierarchal follicles respond to FSH treatment with a modest but significant increase in StAR and P450scc expression plus progesterone production after 24 h of culture, yet coculture with TGF blocks each of these effects [12, 27]. The MAP kinase inhibitors U0126 and PD98059 completely inhibit TGF-induced Erk-P, and, importantly, they potentiate FSH-induced StAR expression and progesterone production (Fig. 2; [12]). These findings support the working hypothesis that tonic MAP kinase activity prevents premature differentiation of granulosa cells and, moreover, that the inhibition of such signaling may be a prerequisite for the full potentiation of steroidogenesis following follicle selection. It is worthy of note that the ability of the MAP kinase signaling via Erk to inhibit gonadotropin-stimulated steroidogenesis in human granulosa-derived cells has recently been reported [28, 29].
However, the present data also demonstrate that even in the absence of MAP kinase signaling via Erk-P, TGF remains capable of attenuating FSH-induced StAR expression and progesterone production (Figs. 2 and 3). Previously published data have implicated PKC signaling as a negative modulator of steroid production in hen granulosa cells [18]. The present results confirm that this negative regulation is at least in part attributed to PKC-mediated inhibition of StAR transcription (Fig. 4), and this is further supported by the finding that preculture with the pharmacologic PKC inhibitor GF109203X, greatly potentiates FSH-induced StAR expression and progesterone induction (Fig. 5). Furthermore, this effect is not mediated via MAP kinase/Erk signaling (Fig. 3). Although neither the specific PKC isoforms involved nor the precise cellular mechanisms effecting negative regulation of StAR transcription are currently known, there is evidence from mammalian model systems for the involvement of the transcription factors DAX-1 and Yin Yang 1 [1, 30].
In contrast to the inability of TGF to completely reverse the potentiating effects of U0126 on differentiation, TGF effectively blocks GF109203X-potentiated differentiation, presumably through the alternative MAP kinase signaling pathway. This indicates that TGF can independently signal via both the MAP kinase/Erk-P and PKC pathways in prehierarchal follicle granulosa cells, yet suggest that MAP kinase signaling may represent the more potent differentiation-inhibiting pathway. Furthermore, unlike gonadotropin-releasing hormone in human granulosa-luteal cells [19], TGF-induced Erk-P is not dependent on PKC signaling (Fig. 3). The findings reported herein are similar to those using cultured bovine granulosa cells from which it was concluded that TGF inhibits PKA-mediated progesterone and estradiol production via both MAP kinase and PKC signaling [31]. We found it interesting that in the same studies, inhibition of MAP kinase signaling (using genistein) was also found to potentiate FSH-induced estradiol production. We also note that both epidermal growth factor and PMA have previously been found to reverse the phenotypic remodeling that occurs in porcine granulosa cells during FSH-induced differentiation and spontaneous luteinization in vitro [32, 33]. These results collectively indicate that the MAP kinase and PKC signaling pathways represent a conserved mechanism by which granulosa cell differentiation is tonically inhibited.
Once a follicle has been recruited into the preovulatory hierarchy, granulosa cells become competent to continuously secrete low levels of progesterone throughout the ovulatory cycle, plus respond to LH with a transient preovulatory surge of progesterone during the period 6 h through 2 h before ovulation [34]. Studies have previously demonstrated that the granulosa cell layer is the primary source of progesterone production during the preovulatory LH surge, and both in vivo and in vitro data demonstrate that LH-induced progesterone production is significantly greater within F1 follicles than it is in F2 and smaller preovulatory follicles [35]. The latter observation is confirmed in the present in vitro experiment (Fig. 7). Significantly, however, the enhanced potential for progesterone synthesis is not associated with a detectable difference in LH receptor mRNA levels [13] or LH-induced StAR expression between these two stages of development, but is associated with a higher level of P450scc expression in F1 follicle granulosa cells (Fig. 8). We conclude that the increase in StAR expression in F2 follicles is necessary for rapid, LH-induced progesterone production, but is not sufficient for producing surge levels of progesterone. Instead, the data support the longstanding proposal that levels of P450scc activity [36] represent the rate-limiting step in producing the preovulatory surge of progesterone primarily by the F1 follicle granulosa layer.
In contrast to StAR expression, which increases within 90 to 180 min following gonadotropin treatment (Fig. 7, [12]), significant increases in P450scc expression occur after 8 to 16 h of treatment [27]. It is interesting to speculate why the transient preovulatory surge of progesterone induced by LH might be regulated by the product of a gene that is not rapidly regulated (e.g., P450scc) rather than one that can be acutely up-regulated (StAR). In this regard, there is evidence that progesterone, in the absence of gonadotropins, may directly affect the follicle to promote the rupture of the stigma at ovulation [37, 38]. These effects may be mediated via progesterone receptors that are known to be expressed in both the granulosa and theca layers of preovulatory follicles [39]. Therefore, from a physiologic perspective, the inability of the F2 follicle to produce sufficiently large amounts of progesterone in response to a preovulatory LH surge (due to limiting amounts of P450scc) may preclude its ability to initiate the ovulatory process, and thus ensure ovulation of only the F1 follicle.
Similar to findings derived from porcine luteinized granulosa cells [11] and mouse Leydig tumor cells [40], PKA-induced StAR expression, and as a consequence enhanced progesterone production, is largely dependent on new transcription and translation (Fig. 6). In the presence of actinomycin D, there is a marked (but not complete) reduction in LH-induced progesterone production from hen granulosa cells. This suggests that StAR protein synthesis continues in the presence of actinomycin D, and that the low but detectable levels of StAR protein are capable of facilitating limited progesterone synthesis after a 3-h culture. On the other hand, although cycloheximide does not block LH-induced StAR mRNA expression (an indication that StAR transcription does not require new protein synthesis), granulosa cells treated with cycloheximide plus LH fail to respond with either increased StAR protein expression or progesterone synthesis compared with those treated with cycloheximide alone. This latter result demonstrates the virtual requirement for new protein synthesis to mediate agonist-induced steroid production.
Following the preovulatory release of LH in vivo, serum progesterone levels decline to presurge levels by approximately 2 h before ovulation [34]. Results from the present studies suggest that this decrease in circulating progesterone is associated with a rapid loss of StAR protein, as is observed following the withdrawal of LH in cultured granulosa cells (Fig. 9). The ability of StAR protein to be rapidly turned over as described here is consistent with the previously estimated half-life of StAR mRNA (3–3.5 h, [40]). Mechanisms responsible for terminating the transient preovulatory LH and progesterone surges have yet to be adequately defined.
Unlike mammalian species in which a steroidogenically active corpus luteum forms from the remaining follicle tissues following ovulation, the postovulatory follicle of the hen rapidly becomes steroidogenically inactive and is completely resorbed within a few days to a week [41, 42]. Not unexpectedly, this decline in basal progesterone production in hen postovulatory follicles is associated with the loss of StAR mRNA expression (Fig. 10).
In summary, the data reported herein support the proposal that paracrine/autocrine factors (including but not limited to TGF) prevent premature expression of StAR in prehierarchal follicle granulosa cells by more than one receptor-mediated signaling pathway. Accordingly, in addition to ongoing efforts directed toward identifying the mechanisms by which trophic hormones promote StAR gene transcription, and by implication enhanced steroidogenic potential, emphasis should be also focused on downstream target regulatory proteins that can actively prevent StAR transcription. Subsequent to follicle selection into the preovulatory hierarchy, StAR transcription and translation can be rapidly increased within the granulosa layer in response to a preovulatory LH surge. Although greater StAR expression is clearly required to produce the preovulatory surge of circulating progesterone, it is evident that additional factors, such as levels of P450scc expression, ultimately determine the stage-dependent quantity of progesterone synthesis. Finally, in combination with the ability of StAR expression to be rapidly up-regulated by PKA signaling, the subsequent rapid loss of StAR protein following LH withdrawal provides for an ongoing, tightly controlled regulation of granulosa cell steroidogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A.L. Johnson, Department of Biological Sciences, P.O. Box 369, The University of Notre Dame, Notre Dame, IN 46556. FAX: 574 631 7413; johnson.128{at}nd.edu
Received: 25 February 2002.
First decision: 17 March 2002.
Accepted: 27 May 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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action in human granulosa-luteal cells. J Clin Endocrinol Metab 2001 86:375-380This article has been cited by other articles:
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  P. Olivero, E. Leiva-Salcedo, L. Devoto, and A. Stutzin Activation of Cl- Channels by Human Chorionic Gonadotropin in Luteinized Granulosa Cells of the Human Ovary Modulates Progesterone Biosynthesis Endocrinology, September 1, 2008; 149(9): 4680 - 4687. [Abstract] [Full Text] [PDF]
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A. L. Johnson, M. J. Haugen, and D. C. Woods Role for Inhibitor of Differentiation/Deoxyribonucleic Acid-Binding (Id) Proteins in Granulosa Cell Differentiation Endocrinology, June 1, 2008; 149(6): 3187 - 3195. [Abstract] [Full Text] [PDF]
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N. Nakao, S. Yasuo, A. Nishimura, T. Yamamura, T. Watanabe, T. Anraku, T. Okano, Y. Fukada, P. J. Sharp, S. Ebihara, et al. Circadian Clock Gene Regulation of Steroidogenic Acute Regulatory Protein Gene Expression in Preovulatory Ovarian Follicles Endocrinology, July 1, 2007; 148(7): 3031 - 3038. [Abstract] [Full Text] [PDF]
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 D. C. Woods, M. J. Haugen, and A.L. Johnson Actions of Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase and Protein Kinase C Signaling in Granulosa Cells from Gallus gallus Are Dependent upon Stage of Differentiation Biol Reprod, July 1, 2007; 77(1): 61 - 70. [Abstract] [Full Text] [PDF]
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D. C. Woods and A. L. Johnson Phosphatase Activation by Epidermal Growth Factor Family Ligands Regulates Extracellular Regulated Kinase Signaling in Undifferentiated Hen Granulosa Cells Endocrinology, October 1, 2006; 147(10): 4931 - 4940. [Abstract] [Full Text] [PDF]
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 L. Tosca, S. Crochet, P. Ferre, F. Foufelle, S. Tesseraud, and J. Dupont AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles. J. Endocrinol., July 1, 2006; 190(1): 85 - 97. [Abstract] [Full Text] [PDF]
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D. C. Woods and A.L. Johnson Regulation of Follicle-Stimulating Hormone-Receptor Messenger RNA in Hen Granulosa Cells Relative to Follicle Selection Biol Reprod, March 1, 2005; 72(3): 643 - 650. [Abstract] [Full Text] [PDF]
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J.T. Bridgham and A.L. Johnson Alternatively Spliced Variants of Gallus gallus TNFRSF23 Are Expressed in the Ovary and Differentially Regulated by Cell Signaling Pathways Biol Reprod, April 1, 2004; 70(4): 972 - 979. [Abstract] [Full Text] [PDF]
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