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Ovary |
Department of Biological Sciences and Walther Cancer Center, 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) (monitored by Erk phosphorylation) blocked TGFß1-induced LH-R expression, and this effect was associated with the inhibition of Smad2 phosphorylation. We conclude that a primary differentiation-inducing action of activin A and TGFß1 on hen granulosa cells from prehierarchal follicles is directed toward LH-R expression. Enhanced LH-R levels subsequently sensitize granulosa cells to LH, which in turn promotes StAR plus P450scc expression and subsequently an increase in P4 production. Significantly, the finding that TGFß signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.
activin, follicle-stimulating hormone receptor, granulosa, granulosa cells, hen ovary, LH receptor, luteinizing hormone, ovary, StAR, steroidogenesis, TGFß
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transforming growth factor (TGF) ß and activin belong to a large superfamily of extracellular regulatory proteins. A number of such family members are expressed within the mammalian ovary, including TGFß (TGFß1, ß2, ß3), activins (activin A, AB, B), growth differentiation factor-9 (GDF-9), inhibins, anti-Mullerian hormone, and at least five bone morphogenic proteins (BMP-2, -4, -6, -7, and -15) [5– 8]. To date, various members of the TGFß superfamily have been found to act in the mammalian ovary either as positive or negative regulators of granulosa cell differentiation [9– 12].
Both activin and TGFß induce their biological effects by forming a heterodimeric complex with two type II and two type I serine and threonine kinase receptors. Activation of the type I receptor results in the phosphorylation of one or more receptor (r)-Smads (e.g., Smad2, -3) [8, 13]. Activated r-Smad subsequently associates with a co-Smad (e.g., Smad4) and is translocated to the nucleus to initiate gene transcription. On the other hand, negative modulation of activin and TGFß signaling can occur via inhibitory (i)-Smads (e.g., Smad6, -7), which act by inhibiting r-Smad phosphorylation and preventing r-Smad/co-Smad complexes from translocating to the nucleus [13].
Studies to date have documented the presence of TGFß expression in both the theca layer from prehierarchal and preovulatory hen follicles and within granulosa cells from the largest preovulatory follicle [14], and recent studies have identified a chicken TGFß4 hypothesized to represent the avian homolog to mammalian TGFß1 [15]. Immunoreactive activin A is localized mainly within the theca layer throughout follicle development, with lesser amounts detected within granulosa cells of preovulatory follicles. There is a significant increase in the theca content of activin A found within the most recently selected (9-mm-diameter) follicle [16], suggesting the potential for paracrine signaling at the time of follicle selection. Moreover, avian homologs to type I and type II TGFß and activin receptors as well as their associated cell signaling components (Smad2, 3, 4, and 7) have been identified [17, 18; GenBank accession AF230192], and several of these have been described within hen granulosa cells [19–21]. Such findings support the possibility of a paracrine and/or autocrine function for activin and TGFß within hen follicles.
Previously published data demonstrate that activin A treatment promotes Smad2 phosphorylation and nuclear translocation in hen granulosa cells from preovulatory follicles [21]. Additionally, both FSH and TGFß can upregulate levels of smad2 (but not smad3) mRNA expression [19, 21]. Combination treatment with FSH and activin A has been reported to stimulate or inhibit cell proliferation and to decrease LH-R mRNA expression in preovulatory follicle granulosa cells [22, 23]. To date, however, the actions of activin A and TGFß in prehierarchal follicles have yet to be studied, particularly with regard to their potential involvement in the initiation of granulosa cell differentiation.
Accordingly, the present studies were designed to evaluate the role and cellular mechanism(s) of action for activin- and TGFß-mediated differentiation of granulosa cells from prehierarchal follicles. In addition, in an attempt to explain the absence of granulosa cell differentiation in all but the single selected follicle per day, studies were initiated to identify factors that can negatively modulate activin and TGFß signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Single-comb white Leghorn hens (Creighton Bros., Warsaw, IN), 25– 35 wk of age and laying regular sequences of five or more 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 photoperiod of 15L:9D, with lights on at midnight. Laying cycles were monitored by the daily timing of oviposition. Hens were killed approximately 16–18 h prior to 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 (rh) TGFß1 and rh transforming growth factor (TGF) were purchased from PeproTech, Inc. (Rocky Hill, NJ) while rh activin A was from R&D Systems (Minneapolis, MN). The deduced amino acid sequence for the mature form of chicken TGFß4 is predicted to be 82% identical (89% similar) to human TGFß1 [15, 24], with all nine Cys residues and conserved domains maintained. The chicken processed form of activin A is predicted to be 98% similar to human activin A [25]. Ovine LH (lot 26) and rhFSH (lot AP8468A) were obtained from Dr. A.F. Parlow and the National Hormone and Pituitary Program. Maximally effective doses of TGF (50 ng/ml), FSH (100 ng/ml), and LH (100 ng/ml) used in the present studies were based on results from previous studies [1]. The MAP kinase inhibitor, U0126, was from BioMol (Plymouth Meeting, PA) and has previously been found to inhibit Erk (but not Akt) phosphorylation [1]. The Smad2 and phospho-specific Smad 2 antibodies, which selectively recognize activin and TGFß receptor-mediated phosphorylation sites, were from Zymed Laboratories and Upstate Biotechnology, respectively. The StAR antibody was generously provided by Dr. D.B. Hales (University of Illinois, Chicago, IL), while -tubulin monoclonal antibody used for standardization of StAR was from Sigma Chemical Co. (St. Louis, MO).
Granulosa Cell Cultures
Granulosa cells from prehierarchal (6- to 8-mm-diameter) follicles were collected and prepared for culture as previously described [1, 2]. Cells were plated in six-well polystyrene culture plates (Falcon 3046; Fisher Scientific, Hanover Park, IL) at a density of approximately 106/well in 2 ml Dulbecco modified Eagle medium (DMEM) containing 2.5% fetal bovine serum (FBS; Gibco-BRL). Where appropriate, an aliquot of dispersed cells was immediately frozen at –70°C to serve as an uncultured (T0) control.
In the first experiment, prehierarchal follicle granulosa cells were plated in the absence or presence of FSH, activin A (1–50 ng/ml) or TGFß1 (1–25 ng/ml), then cultured for 20 h. Cells were subsequently collected and frozen at –70°C until processed and probed for LH-R mRNA. In a related experiment, cells were cultured for 20 h in the absence or presence of FSH, activin A (25 ng/ml), TGFß1 (10 ng/ml), FSH plus activin A (1– 50 ng/ml), or FSH plus TGFß1 (1–25 ng/ml). Media and cells were collected separately and frozen at –70°C until assayed for progesterone (media), and LH-R, P450scc, and StAR mRNA (cell pellet). Prehierarchal follicle granulosa cells were also treated without or with FSH, TGFß1 (10 ng/ml), and/or activin A (25 ng/ml) for 20 h to determine effects on FSH-receptor (FSH-R) mRNA expression. In a fourth experiment, granulosa cells were preplated for 20 h in the absence or presence of TGFß1 (10 ng/ml) or activin A (25 ng/ml), then challenged for an additional 3 h without or with FSH or LH. Media and cells were collected and frozen at –70°C for subsequent analysis of LH-R mRNA, StAR protein, and progesterone.
To evaluate the potential for interaction between MAP kinase signaling and TGFß1-mediated signaling, granulosa cells were precultured for 30 min without or with TGF, then treated with FSH or TGFß1 (10 ng/ml) for 20 h. Cells were collected and subsequently probed for LH-R mRNA. Signaling via Smad2 was evaluated by preculturing cells without or with TGF for 30 min, then treating for an additional 2 h with TGFß1 or activin A. A recently published study reported that the stimulatory effects of activin A on Smad2 mRNA levels in hen granulosa cells requires a minimum of 6 h [21]. Finally, to specifically assess MAP kinase involvement in Smad2 signaling, cells were precultured for 1 h with U0126, then treated for 2 h with TGF and TGFß1. Cellular proteins were collected and analyzed for phosphorylated Smad2 and total Smad2 protein by Western blot analysis.
Northern Blot Analysis
The chicken LH-R, FSH-R, and StAR cDNAs used for probing Northern blots have been previously described [3, 26, 27]. Although both gonadotropin receptors have been reported to express multiple transcripts, only the predominant transcript for LH-R (3.0 kilobases [kb]) and FSH-R (2.5 kb) were quantitated in the present studies. A 522-nucleotide chicken P450scc probe corresponding to base pairs 269–790 of the predicted coding region was generated from the chicken P450scc cDNA sequence (GenBank accession D49803) by reverse transcriptase-polymerase chain reaction, and the cloned sequence was subsequently confirmed by sequence analysis. All Northern blots were reprobed with a chicken 18S ribosomal RNA cDNA to standardize for equal loading of RNA samples. Conditions for conducting Northern blot analysis have been previously detailed [28], and all blots were visualized using a Storm 840 Phosphorimager and ImageQuant analysis (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis
Western blot analyses were conducted essentially as described [1, 28]. Briefly, tissues were homogenized in a protein lysis buffer containing a cocktail of enzyme (including phosphatase) inhibitors (Sigma). Proteins were separated on a 10% polyacrylamide gel under denaturing conditions, then transferred to a polyvinylidene fluoride membrane (Millipore Immobilon-P; Fisher Scientific). Incubations with the StAR, phospho-specific Erk, or phospho-specific Smad2 primary antibodies were conducted overnight at 4°C, while those for horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Pierce, Rockford, IL) were for 1 h. Blots were incubated with enhanced chemiluminescence Western blotting agent (Amersham Corp., Arlington Heights, IL) for 1 min and exposed to x-ray film for 1–5 min. Membranes were subsequently reblotted for levels of -tubulin, Erk2, or total Smad2 protein to enable standardization. Western data were quantitated by densitometry using an UltraScan XL laser densitometer (Pharmacia LKB, Piscataway, NJ).
Progesterone Assays
Levels of media progesterone were quantitated by radioimmunoassay as previously described [29] and expressed as mean nanogram per well ± SEM for the replicate experiments.
Data Analysis
Experiments were independently replicated a minimum total of three times unless otherwise noted. Northern blot data were standardized to 18S ribosomal (r) RNA while StAR, phospho-Erk, and phospho-Smad2 protein levels were standardized to -tubulin, Erk, and Smad2, respectively. Standardized data were expressed as a fold-change compared with the appropriate control (freshly collected [T0] or FBS-cultured cells). Data were analyzed by one-way analysis of variance (without including the reference value, which was set to 1.0) and the Fisher protected least significant difference multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Levels of LH-R mRNA were increased above FBS control levels following treatment with 10 and 50 ng/ml activin A and 10 and 25 ng/ml TGFß1 (Fig. 1). There was no significant additive effect found for LH-R mRNA expression when cells were cultured for 20 h with FSH plus activin A or TGFß1 (Fig. 2). Interestingly, the highest (50 ng/ml) dose of activin plus FSH failed to increase LH-R expression above control levels. A related set of experiments was conducted to evaluate the ability of activin A and TGFß1 to influence cell differentiation at sites other than the LH-R. Culture for 20 h with activin A or TGFß1 alone failed to increase P450scc or StAR mRNA above levels in control cultured cells, and FSH combined with activin A or TGFß1 resulted in no greater increase in P450scc or StAR mRNA expression compared with FSH treatment alone after 20 h of culture (Fig. 3). TGFß1 or activin A alone also had no direct stimulatory effect on progesterone production (Fig. 4). However, in combination with FSH, activin A (10 and 50 ng/ml) and TGFß1 (10 and 25 ng/ml) stimulated significantly greater progesterone production compared with the FSH treatment. Levels of FSH-R mRNA were also significantly increased by TGFß1, but not by activin A or FSH, when compared with FBS control cells following 20 h of culture (Fig. 5). Furthermore, there was a stimulatory effect of activin A and an additive effect of TGFß1 on FSH-R mRNA levels when combined with FSH treatment.
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Subsequent experiments were designed to determine whether increased levels of gonadotropin receptor mRNA induced by activin A and TGFß1 resulted in the expression of functional gonadotropin receptors. To accomplish this, cells were precultured for 20 h in the absence or presence of TGFß1 or activin A, and then challenged an additional 3 h without or with FSH or LH. Consistent with results from the first experiment, TGFß1 and activin A preculture increased LH-R mRNA expression (by 1.6 ± 0.1- and 2.8 ± 0.3-fold, respectively) compared with control cultured cells (P < 0.05; data not shown). Evaluation of StAR protein and progesterone production determined that preculture with TGFß1 or activin A followed by gonadotropin challenge produced significantly higher StAR protein levels and progesterone production compared with comparable treatments in cells precultured with media alone (Fig. 6).
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Influence of the MAP Kinase Pathway on TGFß1- and Activin A-Induced Signaling
The next set of experiments evaluated the potential for TGF acting via MAP kinase signaling to modulate TGFß1 and activin A signaling. Consistent with results from a previous study [1], a 20-min treatment with TGF induced a significant increase in levels of phosphorylated Erk (Erk-P). In the present studies, TGFß1 and activin A neither stimulated an increase in Erk-P nor inhibited TGF-induced Erk-P (P > 0.5 for TGFß1 and activin A versus TGF alone; data not shown). However, with regard to effects on granulosa cell differentiation, TGF treatment completely blocked TGFß1-induced LH-R expression after 20 h of culture (Fig. 7). Finally, both TGFß1 and activin A induced Smad2 phosphorylation (Smad2-P) following a 2-h culture, while levels of total Smad2 protein were unchanged (Fig. 8, top panel). Significantly, pretreatment for 15 min with TGF completely blocked TGFß1- and activin A-induced Smad-P. This inhibitory effect could be prevented by a prior treatment with the MAP kinase inhibitor, U0126 (Fig. 8, bottom panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced MAP kinase/Erk signaling is also shown to prevent TGFß1- and activin A-induced LH-R expression. A summary of events describing activin A- and TGFß1-mediated signaling and their proposed relationship to the initiation of steroidogenesis in undifferentiated granulosa cells from prehierarchal (6- to 8-mm-diameter) follicles is depicted in Figure 9.
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Collectively, the data reported herein demonstrate that activin A and TGFß1 directly induce LH-R expression and indirectly promote P450scc and StAR expression plus progesterone production following LH-R induction. This conclusion is supported by the apparent inability of activin A or TGFß1 to induce P450scc mRNA expression or StAR mRNA or protein expression and the absence of any additive effect on either parameter in combination with FSH (Fig. 3). The inability of activin A and TGFß1 to directly promote progesterone production or enhance production when combined with gonadotropin treatment is also consistent with previous reports using granulosa cells from preovulatory follicles [23, 30, 31], a stage of development where comparatively higher levels of P450scc and StAR expression are not limiting factors to progesterone production. On the other hand, these data contrast with those reported using undifferentiated granulosa cells from the rat in which activin A failed to increase LH-R mRNA expression and exhibited an additive effect on both LH-R and P450scc mRNA expression when combined with FSH pretreatment [32]. These differences may reflect a difference in the stage of differentiation for cultured granulosa cells used and/or species-related responses between the two studies.
Constitutive expression of FSH-R mRNA during follicle development is highest in granulosa cells from 6- to 8-mm-diameter follicles [27]. It is interesting to note that FSH-R mRNA levels significantly decrease (by greater than 50%) in control cultured cells following 20 h of culture (Fig. 5). This suggests that the steady-state level of FSH-R mRNA expression in vivo requires tonic stimulation by endocrine or paracrine factors. While neither FSH nor activin A alone prevented this decline in FSH-R expression, treatment with TGFß1 maintained FSH-R expression compared with T0 levels. The variability and comparative ineffectiveness of activin A to promote gonadotropin receptor expression is likely related to granulosa cell production of follistatin [16]. In support of this proposal, we have recently determined that the addition of an antifollistatin serum effectively enables activin to promote FSH-R expression in vitro (Woods and Johnson, unpublished data). By comparison, both TGFß1 and activin A produced a greater stimulatory effect on FSH-R mRNA levels when combined with FSH; thus, it appears that activin treatment still exerts some level of sensitization. In any event, it is likely that some combination of FSH, TGFß, and activin are among the factors normally required to maintain FSH-R expression in granulosa cells at this stage of development in vivo.
Additional evidence for the ability of activin and TGFß to induce not only increased levels of receptor mRNA but also translated and functionally coupled FSH-R and LH-R is provided by the finding that preculture for 20 h with activin A or TGFß1 significantly enhanced StAR expression and progesterone production following a 3-h challenge with FSH or LH compared with control precultured cells (Fig. 6). Similar results for StAR have been reported after a 24-h TGFß preculture in rat granulosa cells [33].
The only other report to date regarding effects on gonadotropin receptors in the hen ovary determined that activin A either had no effect on LH-R and FSH-R mRNA expression or a slight inhibitory effect [23]. However, these studies were conducted in granulosa cells collected from preovulatory follicles. The difference between such results and those reported herein indicate a role of activin and TGFß in the process of early granulosa cell differentiation rather than the facilitation of short-term secretion of progesterone (as occurs during the preovulatory surge of progesterone) at a stage of development when cells already express comparatively high levels of LH-R [3]. Interestingly, FSH plus the highest level of activin A utilized (50 ng/ ml) inhibited LH-R mRNA levels (Fig. 2) but stimulated progesterone production (Fig. 4). As speculated above, this result may represent a sensitizing effect of activin A at the level of postreceptor signaling.
While neither activin A nor TGFß1 directly induces progesterone production, the higher doses of each factor in combination with FSH treatment did produce an additive effect on progesterone secretion compared with the FSH treatment alone (Fig. 4). This response may be related to the additive effect of FSH combined with activin A and TGFß1 observed specifically for FSH-R expression (Fig. 5) because no additive effects of these treatments were found for P450scc and StAR expression (Fig. 3). It is possible that the increase in functional FSH-R expression may provide for somewhat higher and sustained levels of cAMP-mediated steroidogenesis without a requirement for a further increase in P450scc or StAR expression.
Significantly, TGF-induced MAP kinase/Erk signaling was found to block TGFß1-induced LH-R mRNA expression (Fig. 7). The inhibitory effect of TGF was associated with a prevention of Smad2 phosphorylation and, importantly, was reversed by pretreatment with the MAP kinase inhibitor, U0126 (Fig. 8, lower panels). Previous reports from the rat have determined that EGF treatment can induce expression of i-Smad7 mRNA [13, 34] and that the Smad7 promoter region contains several transcription factor-binding sites that may represent the target of EGF actions [35]. In our ongoing studies of hen granulosa cells, we routinely utilize recombinant human TGF to promote EGF receptor activation because human TGF has been reported to bind the chicken EGF receptor with a considerably higher affinity compared with human EGF [36]. Accordingly, we speculate that the inhibitory effects of MAP kinase signaling on TGFß1- and activin A-induced Smad2-P may result from increased i-Smad7 expression (Fig. 9).
One approach to understanding the process of follicle selection and why but a single follicle from a cohort of 8– 12 prehierarchal follicles is recruited per day into the preovulatory hierarchy is to elucidate mechanisms that can prevent premature differentiation of the granulosa layer. This concept of release from inhibition is relevant because it has recently been established that all hen follicles within the cohort from which selection occurs (both whole follicles as well as the isolated granulosa layers from these follicles) express FSH-R mRNA (Woods and Johnson; unpublished data). Assuming similar levels of receptor translation and functional coupling to signaling pathways prior to the selection process, one would expect equal follicle exposure to circulating FSH [37], and thus the potential for multiple follicles to simultaneously begin FSH-promoted differentiation. Results presented previously and herein are consistent with MAP kinase/Erk activity serving as an effective inhibitor of premature granulosa cell differentiation. Specifically, it has been determined that TGF-induced MAP kinase/Erk signaling can block FSH-induced P450scc, LH-R, and StAR expression in cultured, undifferentiated granulosa cells [1, 4]. Moreover, inhibition of constitutive MAP kinase activity using the selective pharmacologic inhibitors, U0126 or PD98059, promotes LH-R expression in cultured cells while the combination treatment of either MAP kinase inhibitor plus FSH results in an additive increase in LH-R expression [1]. Novel results from the present report are consistent with this hypothesis in that TGFß- and activin A-mediated LH-R expression is also blocked by active MAP kinase/Erk signaling (Fig. 8).
Finally, it is important to consider potential interrelationships with additional factors proposed to regulate granulosa cell function during early follicle growth. For instance, Lovell et al. [16] have recently quantitated levels of inhibin A, inhibin B, follistatin, and activin A in granulosa and theca cells during hen follicle development. Because both inhibins and follistatin are proposed to antagonize the actions of activin A, they propose activin tone as an estimate of activin bioavailability with changing levels of the related antagonists. According to this concept, they predict that activin tone progressively decreases as prehierarchal follicles grow from 1–2 mm to approximately 9 mm, the stage of follicle selection. Clearly, it is difficult to predict the physiological consequences of such interrelationships, yet interestingly, they suggest that high activin tone in 1- to 2-mm follicles may aid in predisposing such follicles to the subsequent acquisition of LH responsiveness.
In summary, the related paracrine/autocrine factors, activin A and TGFß1, represent additional factors (together with circulating FSH and possibly paracrine-acting vasoactive intestinal peptide) [38] that are proposed to initiate hen granulosa cell differentiation and, by implication, follicle selection into the preovulatory hierarchy. Accordingly, a current focus is to investigate additional cellular sites of MAP kinase inhibition together with mechanisms that are ultimately responsible for releasing granulosa cells from such inhibition within those follicles selected for ovulation.
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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
3 Current address: Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, OR 97403
Received: 25 May 2004.
First decision: 18 June 2004.
Accepted: 21 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ßA-subunits in the domestic hen. Biol Reprod 1996 54:429-435[Abstract]-hydroxylase enzyme activity in hen granulosa cells. Biol Reprod 1994 51:327-333[Abstract]This article has been cited by other articles:
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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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 Y.-C. Chen, R. K Cochrum, M. T Tseng, D. T Ghooray, J. P Moore, S. J Winters, and B. J Clark Effects of CDB-4022 on Leydig Cell Function in Adult Male Rats Biol Reprod, December 1, 2007; 77(6): 1017 - 1026. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Li, C. Ying Wang, A. H. Yan Kwok, and F. C. Leung Epidermal Growth Factor (EGF) Receptor Ligands in the Chicken Ovary: I. Evidence for Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Potential Oocyte-Derived Signal to Control Granulosa Cell Proliferation and HB-EGF and Kit Ligand Expression Endocrinology, July 1, 2007; 148(7): 3426 - 3440. [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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 T. M Lovell, S. L Al-Musawi, R. T Gladwell, and P. G Knight Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles Reproduction, June 1, 2007; 133(6): 1159 - 1168. [Abstract] [Full Text] [PDF]
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D. C Woods and A L Johnson Protein kinase C activity mediates LH-induced ErbB/Erk signaling in differentiated hen granulosa cells Reproduction, April 1, 2007; 133(4): 733 - 741. [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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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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