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Endocrinology, doi:10.1210/en.2007-1659
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Endocrinology Vol. 149, No. 6 3187-3195
Copyright © 2008 by The Endocrine Society

Role for Inhibitor of Differentiation/Deoxyribonucleic Acid-Binding (Id) Proteins in Granulosa Cell Differentiation

A. L. Johnson, Morgan J. Haugen and Dori C. Woods

Department of Biological Sciences and the Walther Cancer Research Center, The University of Notre Dame, Notre Dame, Indiana 46556

Address all correspondence and requests for reprints to: A. L. Johnson, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556. E-mail: johnson.128{at}nd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual inhibitor of differentiation (Id) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared with levels of Id1, Id3, and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3, and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, because factors that promote inhibitory MAPK signaling (TGF-{alpha} and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3, and Id4. Furthermore, overexpression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3, and Id4 mRNA but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knockdown studies using small interfering RNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side-chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BEFORE FOLLICLE SELECTION in the hen ovary, granulosa cells from prehierarchal (3–8 mm diameter) follicles are actively maintained in an undifferentiated state. The inability of granulosa cells to produce steroids (a primary determinant of differentiation) at this stage is attributed to the absence of steroidogenic acute regulatory (StAR) protein expression plus low levels of p450 cholesterol side-chain cleavage (P450scc) enzyme activity, both of which are actively inhibited by MAPK signaling (1, 2, 3). Signal transduction by MAPK in undifferentiated granulosa cells is, at least in part, initiated by one or more members of the epidermal growth factor (EGF) family of ligands (EGFL), which block FSH receptor (FSHR) and LH receptor (LHR) expression and function, both before and after activation of adenylate cyclase and cAMP formation (1, 2, 3, 4). By comparison, after follicle selection into the preovulatory hierarchy, active MAPK signaling within the granulosa layer declines, and cells begin the process of differentiation that includes the transition from FSHR to LHR dominance (2, 5, 6, 7). This progression is accompanied by increased expression of StAR protein and P450scc enzyme activity resulting from gonadotropin receptor-mediated cAMP accumulation (8, 9). Subsequently, granulosa cells from preovulatory follicles produce increasingly greater amounts of basal and gonadotropin-induced progesterone as they progress through the follicle hierarchy before ovulation. Although some of the earliest events after follicle selection and associated with the initiation of granulosa cell differentiation have been elucidated, little is known about cellular mechanisms that serve to regulate transcription of differentiation-related genes prior and immediately subsequent to follicle selection.

Members of the helix-loop-helix (HLH) superfamily of transcription factors broadly function in the coordinated regulation of transcriptional processes, including those involved in granulosa cell differentiation (10, 11, 12). The highly conserved HLH domain of such proteins enables them to homo- or heterodimerize, a process prerequisite for enabling HLH proteins to bind DNA and regulate transcriptional activity. Most HLH proteins belong to the basic HLH (bHLH) family and act as transcriptional enhancers or inhibitors through direct binding to the canonical E-box element (CANNTG) within the promoter region (13). The mammalian and avian inhibitor of differentiation/DNA-binding (Id) subfamily consists of four isoforms (Id1, Id2, Id3, and Id4) that lack a domain required for DNA binding. Consequently, Id/bHLH heterodimers are unable to bind DNA. Thus, Id proteins can act as negative antagonists of bHLH transcription factors and gene expression in both mammals (14) and birds (15). Id proteins are rapidly transcribed and translated (within 1–3 h) as well as rapidly degraded by the ubiquitin/proteasome pathway (t1/2 20–60 min), although they are stabilized by the formation of dimers with bHLH factors (16). These characteristics establish Id genes as immediate-early response genes and make them excellent candidates for non-epigenetic inhibitors of gene expression in a granulosa cell-specific and stage-of-development-dependent fashion.

Although there have been several recent reports of one or more Id proteins regulating various functions within rat Sertoli cells (including FSHR expression and the initiation of differentiation) (13, 17, 18, 19, 20), to our knowledge, there are no published reports regarding the function or even regulated expression of Id proteins in the normal (noncancerous) vertebrate ovary. Accordingly, the present studies were conducted to initially characterize the expression of each Id isoform in granulosa cells during follicle development, then to establish the relationship between one of these isoforms (Id2) and the state of granulosa cell differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents
Single-comb white Leghorn hens (Creighton Bros., Warsaw, IN) 25–35 wk of age, and laying regular sequences of six or more eggs were used in all studies described. Hens were individually housed in laying batteries with free access to feed (Purina Layena Mash; Purina Mills, St. Louis, MO) and water, under a controlled photoperiod of 15 h light, 9 h dark (lights on at midnight). Approximate time of oviposition was monitored daily. Hens were euthanized by cervical dislocation 16–18 h before a mid-sequence ovulation, at which time ovarian follicles were removed and placed immediately in sterile 1% saline solution. 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 FSH and ovine LH were provided by the National Hormone and Pituitary Program (Torrance, CA). Recombinant human TGF{alpha}, betacellulin (BTC), and TGFβ1 were from PeproTech (Rocky Hill, NJ). U0126 [a selective MAPK ERK kinase (MEK) inhibitor] (21) was purchased from BioMol (Plymouth Meeting, NJ), whereas the selective ErbB1/ErbB4 tyrosine kinase receptor inhibitor, AG1478 (3), was from Calbiochem (San Diego, CA). The cAMP agonist 8-bromoadenosine cAMP (8-br-cAMP) and actinomycin D were from SigmaAldrich (St. Louis, MO).

Granulosa cells and cultures
Prehierarchal follicles (3–5 and 6–8 mm diameter), the most recently selected follicle (9–12 mm diameter), and the second largest preovulatory (F2) follicle were removed from the ovary for collection of granulosa layers. The germinal disc region (GDR) from the F2 follicle consisted of a 2- to 3-mm diameter sheet of granulosa cells immediately overlying the germinal disc (containing the genetic material and most organelles) of the germ cell (22, 23). The distal region represented a comparably sized layer directly opposite the GDR. For culture, layers of undifferentiated granulosa layers from 6- to 8-mm follicles were combined and cells dispersed as previously described (1, 24). Where appropriate, an aliquot of dispersed cells was immediately frozen at –70 C (0 h control). Cells were incubated for up to 4 h in 12- x 75-mm polypropylene culture tubes (Fisher Scientific, Pittsburgh, PA) in a shaking water bath or cultured for up to 24 h in six- or 12-well polystyrene culture plates (Becton Dickinson Labware, Franklin Lakes, NJ). Incubations and cultures were conducted at 40 C in an atmosphere of 5% CO2/95% air at a density of approximately 2.0–5.0 x 105 per well in DMEM containing 2.5% fetal bovine serum, 0.1 mM nonessential amino acids, and 1% antibiotic-antimycotic reagent (Invitrogen, Carlsbad, CA). Concentrations of TGF{alpha}, BTC, TGFβ1, U0126, AG1478, and 8-br-cAMP used were previously established (1, 21, 25).

Quantitative (real-time) PCR
Forward and reverse primers encoding the four Id isoforms (Id1–Id4), FSHR, LHR, p450scc, and StAR mRNAs, and 18S rRNA (used for the standardization of all target genes) were generated using MacVector software (Table 1Go) and were subsequently validated for use with real-time PCR by determining the optimal amplification efficiency and primer conditions as described by the system manufacturer (Applied Biosystems, Foster City, CA). Random-primed, reverse-transcribed cDNA synthesis reactions were performed using the Promega Reverse Transcription System (Promega, Madison, WI), according to conditions described by the manufacturer. For real-time PCR, primers were added to 25 µl total reaction volume using reagents provided in the ABgene Absolute QPCR Sybr Green Mix (ABgene, Rochester, NY). Reactions were completed on the ABI 7700 Thermocycler (Applied Biosystems). Amplification conditions included an initial denaturing for 15 min at 95 C followed by 15-sec denaturing at 95 C, 30 sec annealing at 60 C, and 30 sec extension at 72 C for 40 cycles. The Ct (defined as the cycle number at which the fluorescence exceeds a threshold level) was determined for each reaction (run in triplicate) using the Sequence Detection software (version 1.6.3), whereas quantification was accomplished using the {Delta}{Delta}Ct method (26). Results are expressed as fold difference compared with an appropriate control tissue (e.g. stroma) or treatment [freshly collected (0 h) or cultured control cells or mock-transfected cells].


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  		TABLE 1. Sequences of forward and reverse primers used for real-time PCR, together with the NCBI nucleotide accession number

 
Cell transfection for Id2 overexpression and small interfering RNA (siRNA)
The pCS2+ vector plus Gallus Id2-pCS2+ constructs were kindly provided by Dr. Marianne Bronner-Fraser (California Institute of Technology, Pasadena, CA) and have been described previously (15). siRNA specific for Gallus Id2 was designed using SciTools online software (Integrated DNA Technologies, Coralville, IA). Sequences for Id2 siRNA are as follows: sense, 5'-CCAUCGUCAGCCUCCACCACCAGCG-3'; antisense, 5'-CGCUGGUGGUGGAGGCUGACGAUGGAC-3'. All transfections were performed using Lipofectamine 2000 transfection reagent, as described by the manufacturer (Invitrogen). Briefly, 2 x 105 cells were seeded into 12-well plates with 200 µl serum- and antibiotic-free (transfection) medium. For each transfection, 2 µl Lipofectamine and 2 µg plasmid DNA or siRNA were incubated in 50 µl transfection medium for 30 min. After this incubation, 150 µl serum- and antibiotic-free cell culture medium was added to each Lipofectamine complex and the entire 200-µl solution added to the cells. Cells were cultured for 4 h, followed by the addition of 400 µl cell culture medium containing 10% fetal bovine serum and then cultured for an additional 20 h culture. For all transfection experiments, control cells were mock transfected with Lipofectamine alone or transfected with pCS2+ vector or noncoding RNA (Santa Cruz Biotechnology, Santa Cruz, CA) as experimental controls. Knockdown specificity and efficiency were evaluated by real-time PCR.

Western blot analyses
Western blots were performed as previously described using total cellular extracts (25). An affinity-purified polyclonal antibody for rabbit Id2 (Santa Cruz Biotechnology) was used at a dilution of 1:100, whereas a goat antirabbit secondary antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL) was diluted 1:10,000. Blots were subsequently incubated with ECL Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ) for 1 min, wrapped in plastic wrap, and exposed to x-ray film for 5–15 min. Blots were evaluated for equivalent protein loading using Ponceau stain.

Progesterone analysis
Progesterone in medium samples was measured by RIA as previously described (1) and expressed as nanograms per milliliter (mean ± SEM) for the combined replicate experiments.

Data analysis
All experiments were independently replicated a minimum of three times, unless otherwise stated. Standardized values were expressed as a fold difference (mean ± SEM) compared with an appropriate control. Data were analyzed either by the Student’s t test or using a one-way ANOVA without including data from the control group (arbitrarily set to 1.0) combined with the Fisher’s protected least significant difference test for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential expression of mRNAs encoding Id isoforms in granulosa cells during follicle development
Levels of Id2 mRNA levels progressively increase in granulosa cells during follicle development, with the highest levels found in fully differentiated granulosa cells (e.g. F2 follicle). By comparison, Id1, Id3, and Id4 mRNAs are highest in undifferentiated granulosa cells collected from prehierarchal (3–5 and 6–8 mm) follicles compared with actively differentiating granulosa cells from 9- to 12-mm follicles and differentiated granulosa cells from preovulatory follicles (Fig. 1AGo). The relationship between levels of mRNAs encoding the four Id isoforms and the extent of granulosa cell differentiation is maintained in the GDR compared with distal granulosa layer from the F2 preovulatory follicle. Specifically, Id2 mRNA is higher (P < 0.05) within differentiated granulosa cells from the distal region, whereas Id1, Id3, and Id4 mRNA levels are significantly lower (P < 0.05) (Fig. 1BGo).


Figure 1
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  		FIG. 1. A, Expression of Id isoform mRNAs in freshly collected granulosa layers from follicles during development standardized to ovarian stroma tissue (not shown): 3–5 and 6–8, prehierarchal (mm) follicles before follicle selection; 9–12, single follicle (mm) most recently selected; F2, second largest preovulatory follicle. Data represent the mean ± SEM from three replicate experiments. Different letters denote statistical significance (P < 0.05). B, Comparison of Id2 vs. Id1, Id3, and Id4 mRNA levels (mean ± SEM, n = 3 independent experiments) in the GDR compared with the region distal to the GDR (DR) from freshly collected F2 follicles. *, P < 0.05 compared with GDR, by t test using nontransformed data.

 
Short-term regulation of Id mRNA expression in undifferentiated granulosa cells
Short-term incubation of undifferentiated granulosa cells with TGF{alpha} (50 ng/ml) for 4 h significantly decreased levels of Id2 mRNA compared with control cells (Fig. 2AGo) but increased levels of Id1, Id3, and Id4 mRNA (Fig. 2BGo). Additionally, FSH (100 ng/ml) treatment induced a 3.5-fold increase in Id2 mRNA but significantly decreased levels of Id1, Id3, and Id4 (P < 0.05). Treatment with TGFβ (10 ng/ml) did not alter mRNA levels for any Id isoform.


Figure 2
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  		FIG. 2. Levels of Id2 (A) and Id1, Id3, and Id4 (B) mRNA in undifferentiated granulosa cells collected from 6- to 8-mm follicles after a 4-h treatment with TGF{alpha} (25 ng/ml), TGFβ (10 ng/ml), or FSH (100 ng/ml). Data represent the mean ± SEM for three to five replicate experiments. * and {dagger}, P < 0.05 vs. untreated control cells (Con) by t test using original data.

 
Treatment for 4 h with either the MEK inhibitor U0126 (10 µM) or the ErbB1/ErbB4 receptor tyrosine kinase inhibitor AG1478 (10 µM) increased levels of Id2 mRNA by 3.1- and 3.8-fold compared with control cultured cells, whereas U0126 and AG1478 decreased levels of Id1, Id3, and Id4 mRNA compared with the control (Fig. 3Go).


Figure 3
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  		FIG. 3. Levels of Id2 (A) and Id1, Id3, and Id4 (B) mRNA in undifferentiated granulosa cells after a 4-h treatment with the MEK inhibitor U0126 (10 µM) or the ErbB tyrosine kinase inhibitor AG1478 (10 µM). Data represent the mean ± SEM for four or five replicate experiments. *, P < 0.05 vs. the control (Con) by t test using nontransformed data.

 
Expression of mRNA encoding Id isoforms in undifferentiated granulosa cells after a 24-h culture
During a 24-h culture, basal levels of Id2 mRNA gradually increased by 3.2-fold compared with 0 h (Fig. 4AGo, – BTC). Notably, levels of LHR mRNA also increased after culture for 24 h in the absence of 10 ng/ml BTC (a 2.7-fold increase vs. 0 h), and the increase in both Id2 and LHR mRNA was blocked by coculture with BTC. By comparison, basal levels of Id1, Id3,and Id4 mRNA decreased after 6 h (P < 0.05), and then levels remained suppressed up to 24 h of culture (Fig. 4BGo). Treatment for 24 h with BTC (+ BTC) significantly enhanced the level of Id1, Id3, and Id4 compared with the comparable control (– BTC). Increased levels of Id1, Id3, and Id4 mRNA in response to cotreatment with BTC are the result of de novo transcription, as determined by the ability of actinomycin D (1 µg/ml) to block this stimulatory effect (Fig. 4CGo). Taken together, the results of these initial experiments demonstrate a negative relationship between the expression of Id1, Id3, and Id4 vs. Id2 mRNA expression during follicle development and provide an association between elevated levels of Id2 expression and a differentiated phenotype in granulosa cells.


Figure 4
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  		FIG. 4. A and B, Levels of Id2 and LHR (A) plus Id1, Id3, and Id4 (B) mRNA in undifferentiated granulosa cells before and after 6 and 24 h of culture in the absence or presence of BTC (10 ng/ml); C, de novo transcription of Id1, Id3, and Id4 mRNA in response to BTC is indicated by the ability of actinomycin D (1 µg/ml) to block each BTC-induced Id isoform after a 24-h culture. Data represent the mean ± SEM of three replicate experiments standardized to ovarian stroma tissue (not shown). Different letters denote statistical significance (P < 0.05).

 
Overexpression of Id2 in undifferentiated granulosa cells
To establish a causative relationship between Id2 expression and a differentiated phenotype, undifferentiated granulosa cells from prehierarchal follicles were transfected with pCS2+ vector or Id2-pCS2+. Basal levels of Id2 protein were not detectable by Western blot analysis in undifferentiated mock- and vector-transfected cells but were readily detected in Id2-pCS2+-transfected cells (Fig. 5AGo). Interestingly, transfection of cells with the Id2-pCS2+ construct significantly decreased levels of Id1, Id3, and Id4 mRNA compared with cells transfected with vector alone (Fig. 5BGo).


Figure 5
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  		FIG. 5. A, Western blot of Id2 protein in mock-transfected, pCS2+-transfected and Id2-pCS2+-transfected (for 18 h) granulosa cells from 6- to 8-mm follicles. This blot was independently replicated with similar results. B, Expression of Id1, Id3, and Id4 mRNA 18 h after transfection with Id2-pCS2+ compared with pCS2+ in undifferentiated granulosa cells from 6- to 8-mm follicles. Data are expressed relative to mock-transfected cells (not shown) and represent the mean ± SEM of three replicate experiments. *, P < 0.05 compared with pCS2+-transfected cells.

 
Overexpression of Id2 for a total of 24 h resulted in a slight, but significant, increase in FSHR and LHR mRNA levels compared with vector alone, whereas FSH treatment during the last 20 h of culture enhanced this response (Fig. 6Go). Moreover, although Id2 overexpression by itself failed to induce progesterone production, FSH treatment in the presence of elevated Id2 enhanced progesterone production compared with FSH treatment alone (Fig. 7AGo) (P < 0.01). To assess whether the Id2 plus FSH-induced increase in LHR mRNA (Fig. 6BGo) reflected functional LHR protein, Id2-pCS2+-transfected cells were challenged with LH (100 ng/ml) during the last 4 h of culture. This challenge induced an approximate 3-fold increase in progesterone production (15.6 ± 4.7 ng/ml) compared with the absence of elevated Id2 (5.1 ± 1.2 ng/ml; Fig. 7BGo). Taken together, these overexpression studies provide evidence that elevated levels of Id2 are sufficient to enhance FSH-induced expression of key markers (e.g. LHR expression and progesterone production) that characterize a differentiated granulosa cell phenotype.


Figure 6
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  		FIG. 6. Expression of FSHR (A) and LHR (B) mRNA after transfection with pCS2+ or Id2-pCS2+ (Id2). Cells were cultured without or with FSH for the last 20 h. Data were standardized to mock-transfected cells (not shown) and represent the mean ± SEM from four (FSHR) or five (LHR) replicate experiments. Different letters denote statistical significance (P < 0.05).

 

Figure 7
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  		FIG. 7. A, Progesterone production after pCS2+ or pCS2+-Id2 transfection, followed by a 20-h treatment with FSH; B, transfected cells challenged with LH (100 ng/ml) for the last 4 h of culture. Data represent the mean ± SEM from six replicate experiments. Different letters denote statistical significance (P < 0.01).

 
siRNA knockdown of Id2 mRNA in undifferentiated granulosa cells
Finally, to evaluate whether some minimal level of Id2 expression is required for granulosa cell differentiation, undifferentiated granulosa cells were transfected with siId2 duplexes over a total of 24 h. siId2 transfection significantly decreased basal and gonadotropin-induced Id2 mRNA expression compared with the noncoding duplex controls (Fig. 8AGo), whereas there was no significant effect of siId2 on levels of Id1, Id3, or Id4 mRNA (Fig. 8BGo). Transfection with siId2 also decreased basal levels of FSHR mRNA as well as FSH-induced levels of LHR mRNA expression (Fig. 9AGo), plus attenuated FSH-induced StAR mRNA and p450scc mRNA expression (Fig. 9BGo). Significantly, siId2 did not block 8-br-cAMP-induced StAR or p450scc mRNA. Finally, siId2 transfection for 24 h blocked FSH-induced (treated for final 20 h) progesterone production and greatly attenuated progesterone production induced by FSH (treated for the final 20 h) plus LH (challenged for the last 4 h) (Fig. 9CGo).


Figure 8
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  		FIG. 8. A, Levels of Id2 mRNA after transfection for 24 h with noncoding (Nc) siRNA duplexes or with duplexes specific for siId2. Cells were cultured without or with FSH during the last 20 h of culture, with or without LH challenge during the last 4 h. Data represent the mean ± SEM for three replicate values standardized to the mock control (not shown). Different letters denote statistical significance (P < 0.05). B, Transfection with siId2 did not alter levels of mRNA encoding Id1, Id3, or Id4 compared with cells transfected with Nc sequences. Data represent the mean ± SEM for three replicate values standardized to the mock control (not shown). ns, P > 0.05; n = 3 replicate experiments by t test.

 

Figure 9
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  		FIG. 9. A, Transfection with siId2 over a 24-h culture period inhibits basal and FSH-induced FSHR mRNA and LHR mRNA expression in undifferentiated granulosa cells. Nc, Noncoding control duplexes. Data are expressed as the mean (±SEM) fold difference compared with mock-transfected (not shown) cells. n = 3. B, siId2 attenuates FSH-induced but not 8-br-cAMP (8br)-induced StAR mRNA and p450scc mRNA expression in undifferentiated granulosa cells. Data are expressed as the mean (±SEM) fold difference compared with mock-transfected (not shown) cells. Note the split ordinate for 8-br-cAMP-treated cells. n = 3 replicate experiments. Different letters denote statistical significance (P < 0.05). C, Additionally, si-Id2 inhibits FSH-induced progesterone production after a 24-h culture and greatly attenuates FSH-primed (for last 20 h) plus LH-challenged (for final 4 h of culture) progesterone production. Data are expressed as the mean (±SEM) compared with mock-transfected cells. n = 3. Different letters denote statistical significance (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this report represents the first to evaluate a functional relationship between the expression of all four Id isoforms and the status of granulosa cell differentiation in the ovary of a vertebrate species. These studies demonstrate that although elevated levels of Id1, Id3, and Id4 isoforms are associated with an undifferentiated granulosa phenotype, enhanced expression of Id2 is related to the initiation and progression of a differentiated phenotype. Moreover, results establish that Id2 expression is both sufficient and necessary to promote gonadotropin receptor expression. Consequently, Id2 facilitates the transition to a differentiated granulosa cell phenotype at or immediately subsequent to the time when a hen prehierarchal follicle is selected into the preovulatory hierarchy. A proposed model for the role of Id isoforms in undifferentiated granulosa cells from prehierarchal follicles (before selection) and in differentiated granulosa cells from selected (preovulatory) follicles is presented in Fig. 10Go.


Figure 10
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  		FIG. 10. Working models for the role of Id proteins in undifferentiated granulosa cells from follicles before selection and in differentiating granulosa cells from selected (>9-mm) follicles. These models are based primarily upon results described herein and do not preclude a role for additional mechanisms of action or alternative interpretations. Top panel, Before follicle selection, granulosa cells are maintained in an undifferentiated state, in part, by elevated expression of Id1, Id3, and Id4 (Fig. 1Go). These Id isoforms are proposed to inactivate transcription factors (TFs) required for mediating transcription of FSH-induced (but not TGFβ-induced) genes (24 ) that are required for differentiation (e.g. FSHR and LHR). EGFL induce Id1, Id3, and Id4 mRNA expression (Figs. 2Go and 4Go) and suppress differentiation via MAPK signaling (1 2 ). Bottom panel, Increasing Id2 expression at the stage of follicle selection (Fig. 1Go) is proposed to indirectly or directly attenuate signaling by paracrine and/or autocrine signals responsible for maintaining granulosa cells in an undifferentiated state. Such factors may include one or more EGFL. Factor(s) that promote Id2 expression at follicle selection include FSH and potentially additional factors signaling via cAMP.

 
Initial studies established an inverse pattern of expression for Id2 mRNA compared with Id1, Id3, and Id4 mRNA (Fig. 1Go). Specifically, elevated levels of Id1, Id3, and Id4 mRNA are associated with an undifferentiated stage of granulosa cell development, whereas increasing levels of Id2 mRNA expression are found in differentiating (9- to 12-mm follicle) granulosa cells, and the highest levels are found in fully differentiated (e.g. F2 follicle) granulosa cells. These expression patterns exist in granulosa cells not only relative to stage of follicle development but also regionally within a preovulatory follicle (e.g. germinal disc vs. distal granulosa regions). It has previously been established that granulosa cells from the GDR of the most mature (F1, F2, and F3) follicles are phenotypically undifferentiated (e.g. are highly responsive to EGFL, steroidogenically inactive, and proliferative), whereas cells distal to the germinal disc are LHR dominant and steroidogenically active (23, 27).

We have previously reported that undifferentiated granulosa cells collected from prehierarchal follicles begin to differentiate during a 24-h culture period (21). This process is reflected, in part, by increasing expression of functional LHR plus progesterone accumulation following an LH challenge. This initiation of differentiation, in vitro, is proposed to occur as a result of the alleviation from inhibitory MAPK signaling. The presence and magnitude of increasing LHR mRNA expression in cells cultured for 24 h (compared with freshly collected cells) is confirmed in the present studies (Fig. 4Go). Moreover, this increase in LHR mRNA expression is associated with elevated levels of Id2 mRNA and decreased levels of Id1, Id3, and Id4 mRNA. The ability of BTC treatment to prevent these effects after a 24-h culture provides support for the ability of EGFL/MAPK signaling to modulate Id protein expression and, consequently, gonadotropin receptor mRNA transcription related to granulosa cell differentiation. The rapid decline in levels of Id1, Id3, and Id4 mRNA levels after 6 h in culture suggests that tonic expression of these isoforms is maintained, in situ, by one or more EGFL and possibly additional factors and is consistent with the previously described short (20- to 60-min) half-life of Id isoforms (16, 28).

Inhibition of MAPK signaling, using either a selective MEK inhibitor (U0126) or an ErbB receptor tyrosine kinase inhibitor (AG1478) (3), rapidly facilitates Id2 mRNA expression (within 4 h of culture) but results in decreased expression of Id1, Id3, and Id4 (Fig. 3Go). This enhancing effect on Id2 expression by these inhibitors in undifferentiated granulosa cells, in vitro, temporally precedes the previously described increase in LHR mRNA expression after a 20-h culture (21). Also related to the regulation of FSHR mRNA expression is the finding that culture with TGF{alpha} suppresses, whereas FSH induces, Id2 mRNA expression (Fig. 2Go). The ability of FSH to induce Id2 transcription has previously been reported for rat Sertoli cells, and this occurs primarily via cAMP signaling (20). It has also been determined that TGFβ1 promotes granulosa cell differentiation by inducing FSHR mRNA and FSH-induced LHR mRNA expression (24, 25). However, treatment with TGFβ1 has no effects on either Id2 or Id1/Id3/Id4 mRNA expression, which indicates that the regulation of Id protein expression in granulosa cells occurs independent of the Smad 2/3 signaling pathway.

Conversely, it is speculated that the ability of EGFL (e.g. TGF{alpha} and BTC) to enhance Id1, Id3, and Id4 mRNA expression (Figs. 2Go and 4Go) can, at least in part, explain the previous findings that activated MAPK signaling prevents FSHR and LHR mRNA expression (1, 24). Specifically, Id1, Id3, and/or Id4 may serve as negative regulators of both FSHR and LHR transcription (Fig. 10Go). This proposal is supported by the recent finding that overexpression of Id1, Id3, or Id4 blocks LH-induced LHR mRNA expression and greatly attenuates LH-stimulated progesterone production in differentiated granulosa from preovulatory follicles (unpublished data).

Nevertheless, these initial data provide only an associative, not a cause-effect, relationship between elevated levels of Id2 expression and the progression of granulosa cell differentiation during follicle development. Subsequent experiments determined that transfection with a Gallus Id2-pCS2+ construct not only elevated levels of Id2 protein but also significantly decreased levels of Id1, Id3, and Id4 mRNA compared with the vector-transfected control (Fig. 5Go). Although reports describing a negative correlation between expression patterns of one Id isoform vs. another in a variety of cell types are common (e.g. elevated Id1 occurring in the absence of detectable Id2 in growing mammary epithelial cells, compared with elevated expression of Id2 occurring in the absence of Id1 after differentiation) (29), to our knowledge, the ability of one Id protein to directly or indirectly inhibit transcription of another has not previously been reported. The present findings are suggestive of elevated Id2 protein acting indirectly (perhaps via inhibition of EFGL transcription) as a negative regulator of the remaining Id isoforms, whereas suboptimal levels of Id2 are permissive of Id1, Id3, and Id4 expression (Fig. 8BGo). Although these proposed transcriptional actions for Id2 remain to be experimentally demonstrated in granulosa cells, this could in part explain the negative relationship between the decreasing levels of Id1, Id3, and Id4 mRNA in the face of increasing Id2 mRNA expression during follicle development (Fig. 1Go).

Although FSH treatment does not by itself induce levels of its own receptor in undifferentiated hen granulosa cells, in vitro (1), the presence of elevated Id2 protein is sufficient to facilitate FSHR mRNA expression both in the absence and presence of FSH treatment (Fig. 6Go). This finding may provide a mechanism for the selective elevation of FSHR mRNA levels observed in the single follicle within the cohort of 6- to 8-mm prehierarchal follicles. It has been proposed that this follicle has recently been selected to enter the preovulatory hierarchy (1). By comparison, FSH is a well-recognized inducer of LHR mRNA expression (21, 24), and elevated expression of Id2 further enhances this response (Fig. 6Go). In turn, the ability of FSH to promote Id2 mRNA expression (Fig. 2Go) provides for an efficient feedback loop to ensure maximal levels of LHR expression, as is characteristic of preovulatory follicles (5).

It has also been established that undifferentiated hen granulosa cells cultured immediately after collection produce negligible amounts of steroid, yet coculture with FSH for 18 h initiates a low level of progesterone production (8, 22). In the present studies, overexpression of Id2 protein in undifferentiated granulosa enhances FSH-induced progesterone production compared with FSH or FSH plus vector (Fig. 7Go), at least in part by its ability to enhance FSHR expression. In addition, by promoting the expression of functional LHR, overexpression of Id2 in the presence of FSH for 16 h significantly enhances progesterone production after a 4-h challenge with LH. Collectively, these data provide evidence that the increasing levels of Id2 expression in granulosa cells during follicle development are sufficient to promote gonadotropin receptor (particularly LHR) expression, followed by the initiation of steroidogenesis.

Lastly, transfection with siId2 significantly reduced not only basal and gonadotropin-induced levels of Id2 mRNA but also FSHR and LHR mRNA expression (Figs. 8Go and 9Go). This finding is consistent with the requirement for some critical level of Id2 to promote gonadotropin receptor expression and, by implication, the initiation of differentiation. Reduced Id2 levels failed to alter the already minimal basal expression of StAR and p450scc mRNA but did attenuate FSH-induced expression of each. Similarly, gonadotropin-induced progesterone production was attenuated by reduced Id2 expression. This is likely a direct effect of reduced gonadotropin receptor and, indirectly, a result of minimal StAR and p450scc expression due to suboptimal adenylyl cyclase/cAMP signaling. By comparison, the inability of reduced Id2 to attenuate 8-br-cAMP-induced StAR mRNA expression indicates that the effect of Id2-regulated transcription is dependent upon enhanced LHR expression. This finding further supports the conclusion that Id2 serves as a direct modulator of gonadotropin receptor expression but not the expression of key genes related to the steroidogenic pathway.

Collectively, these experiments are consistent with the working model that tonic suppression of FSH and TGFβ-induced differentiation by MAPK signaling (Fig. 10Go) (21) is mediated via enhanced expression of Id1, Id3, and Id4. Although yet to be directly tested in undifferentiated granulosa cells from prehierarchal follicles, elevated Id1, Id3, and Id4 expression is proposed to directly prevent transcription of FSHR and possibly LHR mRNAs. Conversely, after the removal of inhibitory MAPK signaling at follicle selection (30), enhanced signaling via FSHR and the adenylyl cyclase/cAMP pathway can rapidly (within 4 h) induce expression of Id2 protein. Although studies are currently ongoing to identify additional target genes, the results presented herein provide evidence that Id1, Id2, and Id3 are among those indirectly or directly inhibited by Id2.


   Acknowledgments
 
We thank Dr. Marianne Bronner-Fraser, Division of Biology, California Institute of Technology, for the generous gift of Id-pCS2+ constructs.


   Footnotes
 
This work was supported by National Science Foundation Grant No. 0445949 to A.L.J. D.C.W. was supported by National Research Initiative Competitive Grant 2007-35203-18085 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service.

Disclosure Statement: The authors A.L.J., M.J.H., and D.C.W. have nothing to declare related to the material being published.

First Published Online March 6, 2008

Abbreviations: bHLH, Basic HLH; 8-br-cAMP, 8-bromoadenosine cAMP; BTC, betacellulin; EGF, epidermal growth factor; EGFL, EGF ligand; FSHR, FSH receptor; GDR, germinal disc region; HLH, helix-loop-helix; Id, inhibitor of differentiation/DNA-binding; LHR, LH receptor; MEK, MAPK ERK kinase; P450scc, p450 cholesterol side-chain cleavage; siRNA, small interfering RNA; StAR, steroidogenic acute regulatory.

Received November 30, 2007.

Accepted for publication February 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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