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BOR - Papers in Press, published online ahead of print January 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.035246
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BIOLOGY OF REPRODUCTION 72, 1194–1204 (2005)
DOI: 10.1095/biolreprod.104.035246
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of CCAAT/Enhancer Binding Proteins Alpha and Beta in the Porcine Ovary and Regulation in Primary Cultures of Granulosa Cells1

Carolina Gillio-Meina , Yvonne Y. Hui , and Holly A. LaVoie 2 

Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CCAAT/enhancer binding proteins alpha and beta (CEBPA/ CEBPB) were evaluated in the porcine ovary during the estrous cycle. CEBPB mRNA was present in antral follicles and was significantly increased in healthy corpora lutea (CL), whereas CEBPA mRNA was constitutively expressed in these structures. Both isoforms of CEBPA (42 and 30 kDa) exhibited greater expression in preovulatory follicles, and the 42-kDa isoform increased in CL, whereas the 30-kDa isoform decreased. All major isoforms of CEBPB (38, 34, and 20 kDa) were expressed, with the 34- and 20-kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cAMP analogue on the distribution of CEBP isoforms were evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution, but cAMP treatment for 24 h tended to decrease the 30-kDa form of CEBPA and the 34-kDa form of CEBPB. The 34-kDa form of CEBPB was decreased by the protein kinase A inhibitor H89 at 4 h (with FSH treatment), and by both protein kinase A and phosphatidylinositol 3-kinase inhibitors at 24 h of treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a CEBP consensus sequence-reporter construct that was blocked by H89. These data implicate protein kinase A as the major regulator of CEBPB isoform distribution and CEBP-mediated transactivation in granulosa cells. The differential expression of specific CEBPA/B isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.

CCAAT/enhancer binding proteins, corpus luteum, cyclic adenosine monophosphate, follicle-stimulating hormone, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CCAAT/enhancer binding proteins (CEBPs) are a family of basic-leucine zipper transcription factors that interact with CCAAT box motifs present in numerous gene promoters. At least six members have been reported and characterized: {alpha}, ß, {delta}, {epsilon}, {gamma}, and {zeta} [1, 2]. These factors are highly conserved in their C-terminal DNA binding domain but differ in their N-terminal transactivation region. Although six genes exist, the number of CEBP protein isoforms is greater due to the use of alternative promoters, differential splicing, alternative translation initiation codon usage, and regulated proteolysis [1]. Several CEBP isoforms have been implicated in cell growth and differentiation [2].

At least two family members, CEBP alpha and CEBP beta (CEBPA and CEBPB) are expressed in the ovary [3, 4]. Previous studies using hormone-primed rats have shown that CEBPA mRNA and proteins are elevated in granulosa cells before hCG injection (an experimental LH surge), whereas shortly after hCG exposure, CEBPB increases dramatically and CEBPA is down-regulated [4]. In contrast, freshly isolated granulosa cells from bovine preovulatory follicles express high levels of CEBPB proteins before hCG exposure and gonadotropin injection resulted in a transient decrease in CEBPB, suggesting species differences [5]. CEBPs have been implicated in the regulation of prostaglandin endoperoxide synthase 2 gene (PTGS2), in which a CEBP site participates with an E-box to facilitate stimulation by forskolin and protein kinase C agonist in bovine granulosa cells and ovine luteal cells, respectively [5, 6]. The role of CEBP in the regulation of the rodent PTGS2 gene promoter is unclear [4, 7], but the PTGS2 mRNA fails to down-regulate in CEBPB knockout mice, suggesting an essential role for CEBPB in PTGS2 regulation [8]. CEBPA, CEBPB, or both have also been shown to participate in the gonadotropin/cAMP activation of the steroidogenic acute regulatory protein gene (StAR) in differentiating granulosa cells of several species [9–11]. Importantly, the ablation of the CEBPB gene causes infertility in female mice due in part to the inability of follicles to ovulate and the failure of granulosa cells to luteinize and up-regulate steroidogenesis [8]. These data suggest that CEBPs play a critical role in ovulation and the function of differentiated granulosa cells.

FSH and LH regulate the growth and differentiation of granulosa cells, with FSH action predominating before the midcycle gonadotropin surge [12]. Recent studies have shown that FSH acts via protein kinase A (PRKACA, also known as PKA)-independent as well as PKA-dependent pathways [13]. The classical PKA signaling pathway entails FSH-binding to its receptor, activation of adenylate cyclase, and the increased production of cAMP [14]. Cyclic AMP in turn activates PKA, leading to the phosphorylation of cellular proteins, including transcription factors [15]. FSH and cAMP can also activate members of the mitogen-activated protein kinase family [16–18]. Recent data have shown that FSH activates protein kinase B homologues 1/2 (AKT1/2, referred to hereafter as AKT) by a cAMP- and phosphatidylinositol 3-(PI3)-kinase-dependent mechanism that likely involves the activation of cAMP-regulated guanine nucleotide exchange factors [13, 19]. AKT is important in granulosa cell survival [20–22] and is required for FSH-induced differentiation in granulosa cells [23]. Active AKT can also inactivate glycogen synthase kinase-3ß (GSK3B; also known as GSK3ß) via phosphorylation [24]. When active, GSK3ß acts to negatively or positively regulate the activity of multiple transcription factors via altering their phosphorylation status [24]. In some cells types, GSK3ß can be inactivated by PKA as well [25].

In nonovarian cells, CEBPA and CEBPB expression, activity, or both are regulated primarily by two signaling pathways, the PI3-kinase/AKT pathway and the PKA pathway. Both the PI3-kinase/AKT and the PKA pathways are proposed to regulate CEBPA and CEBPB activity by modifying the phosphorylation status of each molecule [1]. Cyclic AMP/PKA signals have been shown to influence CEBP transcription or isoform expression (or both) [26, 27]. In addition, GSK3 has been shown to phosphorylate CEBPA, enhancing its activity [28], whereas GSK3 reduces CEBPB activity [24].

Although CEBPA and CEBPB have been extensively studied in other tissues, only a few studies have examined these factors in the gonads. Most of these studies have used the hormone-primed immature rodent to evaluate CEBP induction in the ovary, including granulosa cells, with one study evaluating CEBPB in bovine granulosa cells [5]. Given that these previous studies implicate an essential role for CEBPs (especially CEBPB) in ovarian function, we sought to determine the developmental expression of CEBPA and CEBPB factors in the cyclic porcine ovary to determine whether their expression is associated with follicular maturation and luteinization. We also investigated whether the abundance of isoforms of both CEBPA and CEBPB were altered in primary cultures of porcine granulosa cells by the actions of FSH or cAMP analogue as a previous rat granulosa cell study showed FSH increased the abundance of CEBPB isoforms in short-term culture [9]. In addition, we determined whether PI3-kinase/AKT, PKA, or GSK3 signals (or a combination of these) alter the expression of isoforms. To test the ability of FSH and cAMP analogue to stimulate CEBP-mediated transactivation, we employed primary granulosa cell cultures transfected with a CEBP consensus sequence-reporter construct. Furthermore, we investigated the pathway or pathways responsible for CEBP transactivation ability in these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Unless otherwise indicated, general chemicals were purchased from Sigma (St. Louis, MO). Cell culture reagents, Trizol, Lipofectamine, and the pCRII plasmid were purchased from Invitrogen (Carlsbad, CA). Cell culture dishes were obtained from Falcon (Franklin Lakes, NJ). Ponceau S was purchased from ICN Biomedicals Inc. (Aurora, OH). The pCEBPLuc plasmid was purchased from Stratagene (La Jolla, CA). Luciferase assay reagents, restriction enzymes, the pRLtkLuc plasmid, and the anti-phospho(S473)-AKT antibodies were obtained from Promega Corp. (Madison, WI). Radioisotopes were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). NIDDK o-FSH-20 was obtained from the National Hormone and Pituitary Program (National Institutes of Health, Bethesda, MD). GSK3 inhibitor (SB216763) was purchased from Tocris Cookson Inc. (Ellisville, MO). Anti-caspase-3 was obtained from Active Motif (Carlsbad, CA). Other primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) unless noted. Lambda-phosphatase was obtained from New England BioLabs, Inc. (Beverly, MA). Porcine ovaries from cyclic animals were obtained from Caughman's Meat Packing Co. (Lexington, SC), whereas porcine ovaries from immature animals were purchased from Greenwood Packing Plant Inc. (Greenwood, SC).

Immunohistochemistry

Immediately following evisceration, porcine ovaries were placed in 4% paraformaldehyde fixative as previously described [29]. Ovarian tissue was paraffin-embedded by standard methodology. Approximately 8-µm serial sections were placed on poly-L-lysine-coated microscope slides. Sections were deparaffinized by standard methodology. Antigen retrieval followed by blockage of endogenous peroxidase activity were performed as previously described [29]. Slides were blocked for 1 h in PBS supplemented with 10% normal goat serum. CEBP family proteins were identified by incubation with either rabbit polyclonal CEBPA (2 µg/ml, sc-61X) or rabbit polyclonal CEBPB (0.5 µg/ml, sc-150X) antibodies at 4°C, overnight. To identify steroidogenic cells, serial sections were also incubated with cytochrome P450 cholesterol side-chain cleavage (CYP11A1) antibody (1: 1500, RDI-P450sccabr; Research Diagnostics, Inc.). Negative controls included substitution of primary antibodies with the same concentration of normal rabbit immunoglobulin G (IgG) (sc-2027) or rabbit serum. After washing in PBS, sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:500, 65–6120; Zymed, South San Francisco, CA). Diaminobenzidine staining and hematoxylin counterstaining were performed as previously described [29]. Images were captured using a Zeiss microscope and SPOT camera (Diagnostic Systems, Sterling Heights, MI). Final images were generated using Photoshop 5.5 (Adobe Systems, San Jose, CA). Staining for different follicle types and corpora lutea were repeated three times and with at least three different pigs for CEBPA and CEBPB. At least 15 animals were evaluated during this study. For several tissue blocks, two or more sections were probed using identical conditions to confirm the reproducibility of staining. Nuclei were scored as positive if a brown color was present. The percentage of positive cell nuclei was estimated by counting at least 100 cells per structure type from at least three different animals.

Partial cDNAs for CEBPA and CEBPB

RNA used for reverse transcription-polymerase chain reaction (RT-PCR) was isolated from porcine corpora lutea. RT-PCR was performed with a GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA). A partial, 157-base pair (bp) cDNA for porcine CEBPA was isolated by RT-PCR using primers corresponding to nt –1074 to –1056 and nt –1230 to –1211 of the bovine CEBPA cDNA (GenBank accession number D82984). A partial, 162-bp cDNA for CEBPB was also isolated by RT-PCR using primers corresponding to nt –945 to –961 and nt –1090 to –1116 of the bovine CEBPB cDNA (GenBank accession number D82984). The cDNAs were ligated into the pCRII plasmid and the amplicons were confirmed by sequencing (University of Maine DNA Sequencing Facility, Orono, ME). The CEBPA cDNA varies by two nucleotides (within the primer region) from a partial nucleotide porcine CEBPA cDNA subsequently submitted to GenBank (accession number AF103944). The isolated CEBPB cDNA (repeated with three animals) has been submitted to GenBank (accession number AY207000).

Ribonuclease Protection Assays

Ribonuclease protection assays (RPAs) were performed to detect CEBPA and CEBPB mRNA. RNA was isolated using Trizol reagent from granulosa cells (from 1- to 2-, 3- to 4-, and 8- to 10-mm antral follicles), small follicles (1–2 mm), medium follicles (3–4 mm), healthy preovulatory follicles (8–10 mm), corpora hemorrhagica (CH), midluteal phase corpora lutea (CL), and regressive corpora lutea (CA) dissected from ovaries of cyclic pigs. CH, CL, and CA were classified based on gross morphological appearance, including size, extent of vascularity, presence or absence of central clot, color, and overall appearance of the ovary [30]. CH used in this study were estimated to be at Days 3–4, midluteal phase CL were at Days 9–12, and regressive CL were at Days 18–21 of the estrous cycle.

The pCRII vectors containing the 157-bp cDNA of CEBPA or the 162-bp cDNA of CEBPB were linearized and transcribed using T7 or SP6 polymerase to yield 259- or 257-nucleotide riboprobes, respectively. Ribosomal 18S RNA template was obtained from Ambion (Austin, TX). Riboprobes were synthesized using the Maxiscript (CEBPs) or Megascript kit (low specific activity 18S; Ambion) and {alpha}-32P-UTP. Total RNA (5 µg) was cohybridized with gel-purified saturating amounts of CEBPA or CEBPB riboprobes and 18S riboprobe. RPAs were performed using the RPA II kit (Ambion). Hybridized products were electrophoretically separated on 6% acrylamide gels containing 8% urea. Dried gels were exposed to BioMAX MR film (Kodak, Rochester, NY) with intensifying screens.

Protein Extraction and Immunoblots

Nuclear, cytoplasmic, and whole cell extracts (WCEs) were prepared and immunoblotting was performed as previously described [31]. Cytoplasmic extracts were obtained during nuclear protein isolation from the buffer A supernatant fraction after pelleting nuclei [31]. Protein concentrations were determined using Bio-Rad dye reagent (Bio-Rad Laboratories, Hercules, CA). Twenty-five micrograms of WCE and 15–20 µg of nuclear and cytoplasmic proteins were separated by 10% or 12% SDS-PAGE minigels and electrotransferred to PVDF membranes (Amersham). Nuclear proteins were separated in an 8 x 9.5 cm gel size Mini-Vertical Gel Electrophoresis Unit (Amersham). After blocking in 5% nonfat milk-Tris-buffered saline containing 0.05% Tween-20, membranes were incubated with rabbit anti-CEBPA (4 µg/ml) or rabbit anti-CEBPB (2 µg/ml) overnight at 4°C. Goat anti-CEBPA primary antibody (2 µg/ml, sc-9314) was used to confirm CEBPA immunoreactive bands. Secondary antibodies included HRP-goat anti-rabbit IgG (1:2500, Zymed) or HRP-donkey anti-goat IgG (1:3000, sc-2304). Immunoreactive bands were detected by enhanced chemiluminescence (Amersham). Controls included substitution of CEBP primary antibody with normal IgG (2–4 µg/ml) or incubation of primary antibodies with fivefold excess of their respective blocking peptides. To assess protein loading, membranes were stained after transfer with Ponceau S.

To determine whether CEBPA or CEBPB exist as phosphorylated isoforms, 20–25 µg of nuclear extracts from granulosa cells and antral follicles, CH, and CL were isolated as described above. Nuclear extracts were incubated with or without 2500 units/ml of lambda-phosphatase at 30°C for 1 h followed by electrophoresis on 8 x 9.5 cm minigels as previously described [29].

Primary Culture of Porcine Granulosa Cells and Signaling Pathway Studies

Porcine granulosa cells were isolated from 1- to 5-mm ovarian follicles of prepubertal gilts by needle aspiration and cultured in minimal essential medium (MEM) supplemented with antibiotics and 3% fetal calf serum as previously described [32]. Cells were plated at a concentration of 8 x 106 or 2 x 107 viable cells in 6-well plates for WCE isolations, and in 60-mm culture dishes for nuclear protein isolations, respectively. Medium was changed after the initial 24 h. After 39–43 h of culture, medium was replaced by serum-free medium plus antibiotics, and cells were maintained in these conditions for 5.5 h before treatment [33].

To determine whether the PI3-kinase/AKT, PKA, or GSK3ß signaling pathways (or a combination of these) are involved in the basal, FSH, or 8-Br-cAMP-mediated distribution of CEBPA or CEBPB protein isoforms, granulosa cells were treated in the absence (vehicle alone) or presence of PI3-kinase inhibitor wortmannin (0.1 µM), LY 294002 (10 µM), PKA inhibitor H89 (10 µM), or GSK3 inhibitor SB216763 (5 µM) for 1 h in serum-free medium plus antibiotics followed by treatment with 8-Br-cAMP (1 mM) or FSH (15 ng/ml) for 4 and 24 h. Concentrations were chosen according to our preliminary studies and from previous studies in rat or porcine granulosa cells [13, 22]. Preliminary time-course experiments indicated that 4- and 24-h treatments were required to observe changes in CEBP isoforms. Nuclear proteins were subjected to immunoblotting for CEBPA and CEBPB as above using 18 µg of protein.

To verify that both AKT and GSK3ß pathways are activated by FSH or 8-Br-cAMP under our culture conditions, granulosa cells were treated (after the 5.5-h incubation in serum-free MEM) in the presence or absence of FSH or 8-Br-cAMP for 30 min, and for 1, 2, 4, and 24 h in serum-free medium. Twenty micrograms of WCEs were subjected to immunoblotting with anti-phospho(S473)-AKT (1:2500, G744A; Promega), anti-phospho(Ser9)-GSK3ß (2 µg/ml, sc-11757), anti-AKT1/2 (1 µg/ml, sc-8312), and anti-GSK3ß (0.2 µg/ml, sc-9166). Membranes were stripped between each probing.

To evaluate inhibitor toxicity (apoptosis) during the culture period, granulosa cells were treated in the presence or absence of all inhibitors and hormones for 4 or 24 h in serum-free medium as described above, and WCEs were collected at the end of each experiment. Proteins (15 µg) were separated on 15% SDS-PAGE minigels, followed by immunoblotting with mouse IgG1 anti-caspase-3 (40924; 2 µg/ml) and HRP-rabbit anti-mouse IgG1 (1:5000, 61-0120; Zymed) secondary antibody.

Transfections

Granulosa cells were plated at a density of 5 x 106 cells/well in 12-well dishes and cultured as described above. Immediately before transfection, cells were rinsed with free-serum MEM and then transfected using 12 µl Lipofectamine, 1.99 µg of pCEBPLuc plasmid (containing 3 CEBP consensus binding sites), and 0.01 µg of ptkRL/luc plasmid, as a transfection control, in 1 ml per well in MEM for 5.5 h. Following transfection, medium was replaced with MEM supplemented with antibiotics and cells were allowed to recover for 1 h. Cells (duplicate wells) were then pretreated with vehicle, PI3-kinase, PKA, or GSK3 inhibitors for 1 h before the addition of vehicle, FSH, or 8-Br-cAMP at the same concentrations as above. Cells were incubated for 6 h (as determined in preliminary experiments). Cells were then lysed in 1x Passive Lysis buffer and frozen at –70°C. Lysate supernatants were assayed using the Dual Luciferase Assay kit. Firefly luciferase activity was normalized to its corresponding Renilla luciferase activity.

Data Analyses

Autoradiographic and chemiluminescent bands for RPA and immunoblot analyses were quantified using UN-SCAN-IT gel version 5.1 software (Silk Scientific, Orem, UT). Optical density units of CEBPA and CEBPB mRNA bands were normalized to their corresponding 18S rRNA. Optical density units of the 42- and 30-kDa isoforms of CEBPA; and of the 38-, 34-, and 20-kDa isoforms of CEBPB chemiluminescent bands were expressed as a percentage of total CEBPA and CEBPB protein, respectively. Optical density units of P-AKT and P-GSK3ß bands were normalized with the corresponding total AKT and GSK3ß protein bands. Data from RPA studies (CEBP mRNA/18S rRNA), transfections, and AKT/ GSK3 immunoblot analyses were subjected to analysis of variance (ANOVA) followed by a Tukey HSD difference test. Transfection data, AKT, and GSK3ß data were normalized for vehicle control and were transformed to natural logarithms before ANOVA. Comparisons were made with GraphPad Prism version 3.00 (GraphPad software, San Diego, CA). P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical analyses were conducted to localize CEBPA and CEBPB immunoreactivity in sections of cyclic porcine ovaries (Fig. 1). Granulosa cells (80%– 100%) of healthy unilaminar and multilaminar preantral and antral follicles of different sizes exhibited mostly nuclear staining for both CEBPA and CEBPB proteins. Strong immunoreactivity of both factors was observed in both cumulus and mural granulosa cells of antral follicles (not shown). Most of the theca cells exhibited primarily nuclear staining for CEBPA and CEBPB. After ovulation, in CH and healthy CL, most luteal cells displayed nuclear immunoreactivity for both proteins. Strong cytoplasmic staining for CEBPB was also observed in preovulatory follicles, CH, and CL, whereas relatively weaker cytoplasmic staining was observed for CEBPA. Both CEBP proteins were also detected in some stromal cells. Approximately 75%– 95% of cells in CA also stained for both factors. CYP11A1 protein, a steroidogenic cell marker, was used to identify steroidogenic cell populations in CH and CL (Fig. 1). In addition, CYP11A1 was localized in the cytoplasm of some granulosa and theca cells (theca > granulosa) from preovulatory follicles. Although the number of immunopositive cells was high in CA for all three proteins, most of these cells lacked the appearance of luteal cells (Fig. 1, M, N, and O). These cells in CA may represent lymphocytes that cross-react with the CYP11A1 antibody or macrophages that have ingested luteal cell remnants [34]. Control staining of sections using normal IgG in place of the CEBP antibody produced only weak cytoplasmic staining of granulosa cells, theca cells, stromal cells, and oocytes (not shown). Preabsorption of the CEBP antibody with its respective blocking peptide or substitution of normal rabbit serum for CYP11A1 antibody abolished all staining. Atretic follicles exhibited the same pattern of expression for CEBPA and CEBPB (data not shown). Within atretic follicles, granulosa cells retained expression of both factors until their nuclei appeared pyknotic. Theca cells (which remained healthy) retained expression of both CEBPA and CEBPB even at advanced stages of follicular atresia.



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  		FIG. 1. Immunohistochemical localization of CEBPA, CEBPB, and CYP11A1 proteins in porcine ovarian structures. Positive staining is shown in brown. Ovaries were stained with CEBPA (left column), CEBPB (center column), or CYP11A1 (right column) primary antibodies, and goat anti-rabbit secondary antibody. A–C) Section of ovary including a preantral follicle (on the right) and a 1- to 2-mm antral follicle (on the left). D–F) Section of the ovary including an 8-mm antral follicle (preovulatory follicle). G–I) Section of the ovary including corpus hemorrhagicum. J–L) Section of the ovary including a midluteal phase corpus luteum. M–O) Section of the ovary including a regressing corpus luteum. P–R) Section of the ovary including a midluteal phase corpus luteum using blocking peptides or preimmune serum in place of the CEBP or CYP11A1 primary antibodies, respectively (control slide). GC, granulosa cells; Tc, theca cells; St, stromal cells; LC, luteal cells; Oc, oocytes; Bv, blood vessels; An, antrum. In all panels, bar = 20 µm

The abundance of porcine CEBPA and CEBPB mRNA in granulosa cells (isolated from antral follicles), whole antral follicles, CH, CL, and CA was assessed by RNase protection assay (Fig. 2). No significant differences in CEBPA mRNA expression were found among the structures analyzed. CEBPB mRNA levels were significantly higher than follicles (P < 0.05) after ovulation in CH and CL, and CA mRNA levels were decreased again compared with midluteal phase CL. CEBPB mRNA expression was similar among granulosa cells from different sized follicles. One predominant protected fragment of the correct size was observed with each probe in tissues and no protected fragments were observed with yeast RNA.



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  		FIG. 2. Detection of CEBPA and CEBPB mRNAs in porcine ovarian cells and structures. The graphs represent the expression of CEBPA (A) and CEBPB (B) transcripts in granulosa cells (left) and tissues (right) of the porcine ovary as assessed by ribonuclease protection assay. Data are expressed as the mean ratios of CEBPA mRNA:18S rRNA (A) or CEBPB mRNA:18S rRNA (B) optical densities and SEM for three independent determinations. On the left (A and B), numbers represent granulosa cells isolated from 1- to 2-, 3- to 4-, and 8- to 10-mm antral follicles. On the right (A and B), numbers represent the diameter of the antral follicles (mm) isolated. Also shown are corpus hemorrhagicum (CH), midluteal phase corpus luteum (CL), and regressing corpus luteum (CA). The letter a indicates the expression is significantly higher than all follicle sizes and the letter b indicates expression was decreased compared with CL; P < 0.05

Various CEBP protein isoforms can be produced from single CEBPA and CEBPB mRNAs by differential initiation of translation. CEBPA mRNA can give rise to two polypeptides, a full-length isoform of 42 kDa and an N-terminal truncated form of 30 kDa; whereas CEBPB mRNAs can produce at least three isoforms, a full-length of approximately 38 kDa (also known as LAP I, or liver stimulatory protein), 34 kDa (LAP II), and an N-terminal truncated form 20 kDa (LIP, or liver inhibitor protein). Immunoblot analyses were conducted to examine the cyclic expression of CEBPA and CEBPB protein isoforms using whole cell (data not shown), nuclear, and cytoplasmic extracts from granulosa cells from antral follicles (1–2, 3–4, and 8–10 mm), intact follicles, and CL (Fig. 3). Two predominant proteins with molecular masses of 42 and 30 kDa for CEBPA and three proteins with molecular masses of 38, 34, and 20 kDa for CEBPB were detected in all structures examined. Whole cell extracts (not shown) exhibited the same developmental changes in CEBPs as nuclear extracts. Comparison of nuclear and cytoplasmic fractions showed that the 42-kDa and the 30-kDa isoforms of CEBPA were predominantly in the nuclear fraction, with the exception of CL, in which the 42-kDa isoform was strongly present in both fractions (Fig. 3A). This 42-kDa isoform of CL produced a strong mobility shift on the immunoblots when compared with antral and preovulatory follicles that exhibited only a small amount of this higher mobility 42-kDa form. The 20- and 34-kDa forms of CEBPB were predominantly found in the nuclear fraction of antral follicles and CL, and increased in preovulatory structures (8–10 mm) and CL (Fig. 3B). Although the 38-kDa form was present in both nuclear and cytoplasmic fraction, it was predominant in the cytoplasmic fraction of antral follicles.



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  		FIG. 3. Nuclear and cytoplasmic distribution of CEBPA and CEBPB isoforms in ovarian structures and their nuclear phosphorylation status. Nuclear and cytoplasmic fractions from granulosa cells (GC) isolated from antral follicles or follicles of the indicated diameter (mm), and midluteal phase corpus luteum (CL) were separated by SDS-PAGE followed by immunoblotting for CEBPA (A) or CEBPB (B). On the left (A and B), numbers represent granulosa cells isolated from 1- to 2-, 3- to 4-, and 8- to 10-mm antral follicles. On the right in (A, B, and C), numbers represent the diameter of the antral follicles (mm) isolated. The approximate molecular masses (MW) of the proteins shown in this figure were approximately 42 and 30 kDa for CEBPA; and 38, 34 (doublet), and 20 kDa for CEBPB. Nonspecific bands (ns) are also shown. C) Representative immunoblots of nuclear extracts of granulosa cells from 1- to 2-mm or 8- to 10-mm follicles or CL incubated with phosphatase and separated on long minigels. The symbol (–) indicates the nuclear extract was incubated without phosphatase enzyme and (+) indicates a similar amount of extract incubated with phosphatase enzyme. The band indicated with ns for CEBPB was not visible on the GC 1–2 exposure. Dephosphorylation (of CEBPB) was evident as a faster migration of isoforms on blots. For (A–C) immunoblot analyses for each structure were repeated three times with similar results

Treatment of tissue nuclear extracts with phosphatase produced a mobility shift on immunoblots in the 38-, 34-, and 20-kDa isoforms of CEBPB, which is characteristic of dephosphorylation, but no mobility shifts in CEBPA isoforms (Fig. 3C). These experiments indicated that the isoforms of CEBPB but not CEBPA were phosphorylated in the ovarian structures.

GSK3ß activity has not been previously examined in the porcine ovary. To determine whether AKT and GSK3ß were regulated under our culture conditions, granulosa cells were cultured in serum-free medium in the presence or absence of FSH and 8-Br-cAMP at different time points. Full activation of AKT protein by PI3-kinase requires phosphorylation at two different sites, Thr308 and Ser473 [35], whereas inactivation of GSK3ß by AKT requires the phosphorylation at Ser9 [24]. Phospho-AKT (P-AKT, Ser473, active form) and phospho-GSK3ß (P-GSK3ß, Ser9, inactive form) were detectable in granulosa cells with vehicle treatment (Fig. 4A) and at time zero (data not shown). FSH produced a significant increase in AKT phosphorylation versus vehicle control at 2 h of treatment, but activation was not significant at other times measured (Fig. 4A). Treatment with 8-Br-cAMP resulted in a numerical increase in P-AKT at 30 min, and at 1, 2, 4, and 24 h compared with vehicle control levels, however, these changes were not statistically significant. With both treatments, this lack of significance was likely due to the great variability in the P-AKT response at a particular time point, because the maximal response time varied between experiments. GSK3ß inactivation as identified by the P-GSK3ß antibody was significantly increased by FSH at 30 min compared with control levels (Fig. 4B). Both FSH and 8-Br-cAMP produced a significant increase in the levels of P-GSK3ß after 1 and 2 h of treatment. Although the levels of P-GSK3ß remained high after 4 and 24 h of treatment, they were not significantly higher than the control due to the greater variability in response at these later time points. GSK3 phosphorylation was more consistent than AKT phosphorylation at the earlier time points.



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  		FIG. 4. FSH and 8-Br-cAMP effects on phosphorylation of AKT and GSK3ß in porcine granulosa cells. Porcine granulosa cells were treated in serum-free medium in the absence (vehicle only) or presence of FSH or 8-Br-cAMP and WCEs were collected as described in Materials and Methods. A) Representative immunoblot (upper) and graph (lower) summarizing the results of the immunoblots probed for P(Ser473)-AKT protein. Relative AKT activation represents the optical density of P-AKT corrected for its corresponding total AKT then normalized for vehicle control at the same time point. B) Representative immunoblot (upper) and graph (lower) summarizing the results of the immunoblots probed for P-GSK3ß protein. Relative GSK3ß inactivation represents the optical density of P-GSK3ß corrected for its corresponding total GSK3ß then normalized for vehicle control at the same time point. Data represent mean and SEM for four independent immunoblots. Asterisks indicate values significantly different from vehicle treatment at the same time point

The ratio of different isoforms of CEBPA and CEBPB has been observed to change during a variety of developmental and differentiation processes [1]. Therefore, we tested whether FSH or cAMP analogue mediates changes in the isoform distribution of CEBPA or CEBPB proteins (or both) in granulosa cells and whether isoform distribution involved GSK3, PKA, or PI3-kinase (or a combination of these). Porcine granulosa cells were cultured in the presence or absence of LY-294002 and wortmannin (PI3-kinase inhibitors), H89 (PKA inhibitor), or SB216763 (GSK3 inhibitor) for 1 h followed by FSH or 8-Br-cAMP treatment for 4 and 24 h. Isoform distribution was considered to change if the means varied by more than 50% from their respective vehicle control or the same treatment without inhibitor. Figure 5 shows the distribution of CEBPA isoforms in cells treated with FSH or 8-Br-cAMP, inhibitors, or both. At 24 h, 8-Br-cAMP tended to decrease the 30-kDa isoform of CEBPA compared with vehicle control after 24 h of treatment (Fig. 5B). The GSK3 inhibitor blocked the decrease in the 30-kDa isoform produced by 8-Br-cAMP. Wortmannin also appeared to block the decrease by 8-Br-cAMP in the 30-kDa isoform, but there was a large variability in this response (Fig. 5C). Total CEBPA protein tended to be lower at 24 h in the presence of FSH and 8-Br-cAMP (not shown).



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  		FIG. 5. Evaluation of FSH and cAMP-analogue treatment on the expression of CEBPA isoforms in porcine granulosa cells and effects of GSK3, PI3-kinase, and PKA inhibitors. Porcine granulosa cells were cultured in serum-free medium in the presence of control solvents or GSK3 (SB216763), PI3-kinase (wortmannin, LY-294002), or PKA (H89) inhibitors followed by treatment with vehicle (C), FSH (15 ng/ml), or 8-Br-cAMP (8-Br, 1 mM) for 4 and 24 h. Nuclear extracts were collected and immunoblot analyses were conducted as described in Materials and Methods. A) A representative immunoblot showing the expression of the CEBPA isoforms (42 and 30 kDa) in cultured porcine granulosa cells treated for 4 h in the absence (OH) or presence of GSK3 inhibitor (GSKi). Numbers on the right indicate the molecular mass in kilodaltons (MW) of the CEBPA isoforms. Nonspecific bands (ns) are indicated. Relative protein loading is indicated with membrane stained with Ponceau S (ponc). B) Summary of the effects of the inhibitor vehicle control, ethanol (OH), and the GSK3 inhibitor (GSKi) treatment on CEBPA isoform distribution. C) Summary of the effects of the inhibitor vehicle control (DMSO), wortmannin (Wort), LY-294002 (LY), and H89 treatments on CEBPA isoform distribution. Data represent mean and half of the range for two independent immunoblots. Relative expression of the 42- and 30- kDa CEBPA isoforms were calculated by the ratio of the optical densities between the specific isoform and total CEBPA protein. The letter a indicates mean values were >50% different from vehicle treatment, and b indicates values varied by >50% from the same treatment with inhibitor vehicle

Figure 6 shows the results of similar treatments on the distribution of CEBPB isoforms. Both FSH and 8-Br-cAMP tended to increase the 38-kDa isoform of CEBPB compared with vehicle control at 4 h of treatment and the GSK3 inhibitor was able to increase basal levels of the 38-kDa isoform (Fig. 6B). 8-Br-cAMP tended to decrease the 34-kDa isoform of CEBPB after 24 h of treatment compared with vehicle control. Figure 6C indicates that the PKA inhibitor, H89, produced a decrease in the levels of the 34-kDa form in the presence of FSH at 4 h. No changes were detected in the 20- or 38-kDa isoforms at this time point. After 24 h of treatment and in the presence of 8-Br-cAMP, both LY-294002 and wortmannin decreased the levels of the 38-kDa (Fig. 6C). At this time point, not only H89 but also LY-294002 and wortmannin produced a decrease in the levels of the 34-kDa isoform when the cells were treated in the presence of FSH or 8-Br-cAMP. Basal levels of the 34-kDa isoform were also decreased more than 50% by wortmannin and H89. Total CEBPB protein tended to be lower than vehicle at 24 h in the presence of FSH and 8-Br-cAMP (not shown).



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  		FIG. 6. Evaluation of FSH and cAMP-analogue treatment on the expression of CEBPB isoforms in porcine granulosa cells and effects of GSK3, PI3-kinase, and PKA inhibitors. Porcine granulosa cells were treated as described in Figure 5. A) A representative immunoblot showing the expression of the CEBPB isoforms (38, 34, and 20 kDa) in cultured porcine granulosa cells treated for 4 h in the presence of wortmannin, LY-294002, and H89 inhibitors. Numbers on the right indicate the molecular mass in kilodaltons (MW) of CEBPB isoforms. Nonspecific bands (ns) are indicated. Relative protein loading is indicated with membrane stained with Ponceau S (ponc). B) Summary of the effects of the inhibitor vehicle control, ethanol (OH), and the GSK3 inhibitor (GSKi) treatment on CEBPB isoform distribution. C) Summary of the effects of the inhibitor vehicle DMSO (D), wortmannin (Wort/or W), LY-294002 (LY), and H89 treatments on CEBPB isoform distribution. Data represent mean and half of the range for two independent immunoblots. Relative expressions of the 38-, 34-, and 20-kDa CEBPB isoforms were calculated by the ratio of the optical densities between the specific isoform and total CEBPB protein. The letter a indicates mean values were >50% different from vehicle treatment and b indicates values varied by >50% from the same treatment with inhibitor vehicle

To determine whether LY-294002, wortmannin, H89, or SB216763 (or a combination of these) exhibited toxicity, as indicated by alterations in the active forms of caspase-3, an apoptotic marker, treated granulosa cells extracts were analyzed for caspase-3 isoforms by immunoblot. Figure 7 shows changes in the expression of caspase-3 (the 35- and 17-kDa isoforms) in the presence of FSH, 8-Br-cAMP, inhibitors, or a combination of these. There was a slight increase in active caspase-3 (17 kDa) when cells were treated in the presence of SB216763, LY-294002, wortmannin, and H89 inhibitors compared with vehicle control at 4 h. After 24 h of treatment, H89 produced a notable increase in the active caspase-3, and LY-294002 yielded minimal active caspase-3 (Fig. 7B). FSH and 8-Br-cAMP were both able to slightly reduce the intensity of the active caspase-3 band in the presence of H89 compared with vehicle control with the inhibitor (Fig. 7B). Differences were observed in the amount of active caspase-3 depending on whether ethanol or dimethyl sulfoxide (DMSO) was used as the solvent. Ethanol (0.5%, Fig. 7A) tended to give higher basal caspase-3 than DMSO (0.05%, Fig. 7B).



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  		FIG. 7. Effects of GSK3, PI3-kinase, and PKA inhibitors on caspase-3 activation in porcine granulosa cells. Granulosa cells were cultured in serum-free medium in the presence of inhibitor vehicle or GSK3, PI3-kinase, or PKA inhibitors followed by treatment with vehicle, FSH, or 8-Br-cAMP for 4 and 24 h. Immunoblots of whole cell extracts were probed with anti-caspase-3 to monitor apoptosis. On the left, pro-caspase (35 kDa) and active caspase-3 (17 kDa) are indicated as pro and active, respectively. A) An immunoblot showing the expression of active caspase-3 in porcine granulosa cells in the presence or absence of GSK3 inhibitor. Labels indicate the presence (+) or absence (–) of ethanol vehicle (OH) or GSK3 inhibitor (GSK3i) and treatment with control vehicle (C), FSH, or 8-Br-cAMP (8-Br). B) An immunoblot showing the expression of active caspase-3 in porcine granulosa cells in the presence or absence of PI3-kinase and PKA inhibitors. Labels indicate the presence (+) or absence (–) of DMSO vehicle or inhibitors including wortmannin (Wort) and LY-294002 (LY), or H89 and treatment with control vehicle (C), FSH (F), or 8-Br-cAMP (8-Br). Time 0 is indicated as 0 h. Each experiment was repeated twice with similar results

To our knowledge there are no known ovarian gene promoters exclusively regulated by CEBPs. To assess whether FSH and 8-Br-cAMP can stimulate CEBP transactivation in granulosa cells and to identify the major pathway or pathways involved in CEBP reporter activity, primary cultures were transfected with an artificial promoter construct containing consensus sequences for CEBP linked to a luciferase reporter gene. After transfection, cells were treated in the presence or absence of PI3-kinase, PKA, and GSK3 inhibitors followed by treatment with FSH and 8-Br-cAMP. The results shown in Figure 8 indicated that in the absence of inhibitors, FSH and 8-Br-cAMP significantly stimulated CEBP reporter gene activity when compared with control. The GSK3 or the PI3-kinase inhibitors did not significantly affect basal, FSH-, or 8-Br-cAMP-mediated activation of the CEBP reporter construct with the exception that the combination of FSH and wortmannin did not result in statistically significant stimulation compared with wortmannin and vehicle control. The presence of H89 in the culture media produced a significant decrease (P < 0.05) in basal, FSH-, and 8-Br-cAMP-mediated stimulation of CEBP reporter activity when compared with the same treatment without inhibitor (Fig. 8B). These data implicate the PKA pathway as the major regulator of basal, FSH-, and 8-Br-cAMP-mediated CEBP transactivation in porcine granulosa cells.



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  		FIG. 8. FSH and cAMP analogue-stimulated CEBP reporter gene activity is regulated primarily by the PKA signaling pathway. Primary cultures of porcine granulosa cells were transfected with the pCEBPLuc reporter construct (containing three CEBP consensus binding sites) and ptkRL/luc control. Cells were pretreated for 1 h with control solvent or inhibitor followed by treatment with control vehicle (C), FSH (15 ng/ml), or 8-Br-cAMP (1 mM) for 6 h. A) Summary of transfections experiments with GSK3 inhibitor (GSKi) or ethanol vehicle (OH). B) Summary of transfection experiments with PI3-kinase (Wort, LY), PKA (H89), or DMSO vehicle. Relative luciferase activity indicates light units normalized to the vehicle-treated control without inhibitor. Data represent the mean fold activation and SEM for three to four independent experiments. Asterisks indicate the (FSH or 8-Br-cAMP) treatment was significantly different from control with the same inhibitor or DMSO; P < 0.05. Crosses indicate that the treatment was significantly different than the same treatment with DMSO vehicle; P < 0.05


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our tissue data indicated that both CEBPA and CEBPB are present at all stages of follicular and luteal development in the cyclic pig. CEBPA protein increased during the preovulatory period (8- to 10-mm structures) and the 42-kDa form was further increased in functional corpora lutea; however, the corresponding CEBPA mRNA was constitutively expressed. These studies differ from those with the hormone-primed immature rat in which CEBPA mRNA increased during follicular development, decreased after an experimental gonadotropin (hCG) surge, and increased again in CL [3, 4]. Also in contrast to the rat studies, the 42-kDa form of porcine CEBPA increased in CL compared with that of preovulatory follicles [3, 4]. However, the 30-kDa form of CEBPA was reduced in CL compared with follicles, consistent with its expression in rat follicles after hCG administration [4]. Immunoblotting of porcine CL showed that the mobility of the 42-kDa isoform shifted slightly to a lower molecular weight form. This mobility shift was most likely not due to dephosphorylation because no mobility shifts were observed with any CEBPA molecule subjected to phosphatase treatment. One possibility is that the slightly smaller CEBPA isoform observed with the 42-kDa form is generated from a second in-frame start codon noted in the cDNA of some species [36]. The relevance of the shift in 42-kDa isoform mobility at this time is unclear, but could also represent a change in some other post-translational modification or possibly enhanced cleavage associated with the CL. Increased CEBPA 42-kDa isoform abundance has been associated with cessation of cell proliferation and enhanced differentiation, suggesting a possible function in porcine CL [36].

CEBPB mRNA significantly increased after ovulation in CH and CL. These porcine data are consistent with the rodent ovary in which CEBPB transcripts and proteins increase dramatically in granulosa cells after an experimental LH surge with hCG [4, 37]. However, in the pig, increases in CEBPB protein were observed in preovulatory follicles (8–10 mm) before detectable changes in mRNA. High levels of CEBPB protein were also observed in bovine granulosa cells of preovulatory follicles before and 20 h after hCG injection [5]. The authors of the bovine study suggested that species differences between the rodent and cow might be due to the longer ovulatory process in the cow. Like the cow, the pig has a longer ovulatory process that might account for its differences from the rodent species. In addition, female CEBPB knockout mice fail to ovulate [8], indicating that CEBPB is needed before actual rupture of the follicle in the periovulatory period. Thus in the pig, increased CEBPB translation may occur in the periovulatory period before detectable changes in the CEBPB transcript to accommodate such needs.

Our studies indicate that CEBPA and CEBPB expression are also post-transcriptionally modified in porcine ovarian cells. Phosphatase treatment of fresh tissue extracts showed all CEBPB isoforms were phosphorylated, suggesting that phosphorylation could potentially regulate these isoforms in porcine ovarian tissue. We recently reported that in cultured porcine granulosa cells that all CEBPB isoforms are present as phosphoproteins [11]. The identities of phosphorylation sites in ovarian cells are unknown. Endocrine factors have been shown to regulate both phosphorylation and dephosphorylation of CEBPB in preadipocytes [38]. In addition, in adipocytes dephosphorylation of CEBPA protein occurs via inhibition of GSK3 [39, 40]. Due to the limited availability of phosphospecific CEBP antisera that recognize the porcine sequences, we are unable at this time to thoroughly explore whether one or more specific phosphorylations of these factors are regulated by FSH, or cAMP analogue (or both). However, one phospho-specific antiserum used for immunoblotting, anti-P(Thr235)-CEBPB (Cell Signaling Technology, Beverly, MA) did cross-react with porcine CEBPB. This phosphorylation site was unregulated by FSH and 8-Br-cAMP treatment at the 4- and 24-h time points (data not shown).

Production of different CEBPA and CEBPB protein isoforms by use of alternative initiation codons represents a major form of translational control [1]; therefore, we evaluated this endpoint in cultured granulosa cells. Studies in other cell types have indicated that the ratios between different specific CEBP isoforms can change and regulate the net activity of each factor [36, 41]. In fresh tissue, the 34- and 20-kDa forms of CEBPB increased with follicular differentiation, while the 34-kDa form of CEBPB was labile in cultured granulosa cells in the presence of PI3-kinase and PKA inhibitors. Reduction in the 34-kDa isoform compared with total CEBPB could be by selective inhibition of the 34-kDa isoform translation, or by increased degradation (or both). The mechanism remains to be determined. The 38- and 34-kDa forms (LAP I and LAP II, respectively) of CEBPB are the transcriptionally active forms, whereas the 20 kDa (LIP) contains the DNA binding domain, but lacks the transactivation domain. Alteration in the ratio of active to inactive isoforms can determine the efficacy of CEBPB, because LIP can dimerize with LAP isoforms on DNA, resulting in reduced transactivation [1].

FSH and the cAMP analogue did not notably stimulate CEBPB isoform expression in porcine granulosa cell cultures (with the exception of 38-kDa form at 4 h). After 24 h, 8-Br-cAMP actually reduced (69%) the 34 kDa form expression in the presence of ethanol vehicle and tended to lower this isoform (30%) in the presence of DMSO vehicle. These results differ considerably from data with rat granulosa cells, which demonstrated an increased expression of all three CEBPB isoforms after 6 h of FSH treatment [9]. In addition, cultured rat Sertoli and Leydig cells also showed increased LAP I or LAP II expression (or both) in response to cAMP analogues [27, 42]. These studies indicate species-specific differences between rat and pig gonadal cell expression of CEBPB. An additional explanation for the minimal increase in CEBPB observed with cAMP agonists in the pig may be the duration of culture used. Studies with rat granulosa cells found LAP II (and LAP I to a lesser extent) increased between the time of plating and 9 h in culture without treatment [9]. Our porcine cells have been established in culture for 44–48 h before treatment. These culture conditions were found in previous studies to be required for FSH-stimulation of porcine StAR gene transactivation by CEBPB and GATA4 [11]. Because porcine granulosa cells placed in culture begin to differentiate spontaneously, our cells have most likely up-regulated CEBP during pretreatment culture. This interpretation is supported by the immunoblot data with freshly isolated granulosa cells from 1- to 2-mm and 3- to 4-mm follicles that exhibit lower overall CEBPB isoform abundance. Cells from similar sized follicles (1–5 mm) represent the population of granulosa cells that are used for cell culture and would reflect their lower CEBP status at the time of plating.

Other intracellular signaling pathways including PI3-kinase/AKT are required for FSH-induced granulosa cell differentiation in addition to PKA [23]. In rat granulosa cells, GSK3 inhibition (Ser9 phosphorylation) was increased by FSH via a PI3-kinase-dependent mechanism [13]. Our studies also show that both FSH and 8-Br-cAMP inactivate GSK3Bß in porcine granulosa cells. GSK3 activity has been shown to enhance or repress CEBPA or CEBPB activity, respectively [24]. However, treatment of granulosa cells with the GSK3 inhibitor had no effect on CEBPA or CEBPB isoform distribution (except 30 kDa CEBPA at 24 h in the presence of cAMP). PI3-kinase and PKA inhibitors reduced the levels of the 38- or 34-kDa isoforms (or both) of CEBPB after 24 h of treatment when compared with inhibitor vehicle, and the decrease was also observed in the presence of FSH and 8-Br-cAMP. Taken together, these data indicate that basal PKA (4 and 24 h) and PI3-kinase (24 h) activity influence the distribution of predominantly CEBPB isoforms, and that GSK3ß is not involved.

In our granulosa cells, the PI3-kinase and PKA inhibitors produced differential activation of caspase-3, a marker of apoptosis, suggesting that some of the changes in the distribution of CEBP isoforms could be due to inhibitor toxicity. However, the activation of caspase-3 was higher for PI3-kinase inhibitors at 4 h than at 24 h. At 4 h, no change in CEBPB isoform distribution was observed with PI3-kinase inhibitors, whereas at 24 h there was an effect on isoform distribution, suggesting that apoptosis was not responsible for isoform alterations. The PKA inhibitor H89 had some caspase-3 activation at 4 h that was greater at 24 h, suggesting that toxicity was a possible complicating factor. However, the concentration of H89 (10 µM) used in this study has been commonly used in numerous granulosa cell studies and has been shown to affect PKA-dependent pathways only [13, 23, 43]. However, we cannot exclude the possibility that H89 may interfere with other protein kinases in all our assays because the compound has been shown to inhibit MSK1, S6K1, and ROCK2 kinases with similar efficacy in in vitro kinase assays, and to partially reduce protein kinase C activity as well [44]. It should be noted that the differences in caspase-3 activation observed between 4- and 24-h time points, such as with PI3-kinase inhibitors, is because cells at advanced stages of apoptosis detach from culture plates, are removed with the medium at the end of the experiments, and therefore are not part of the protein analyses.

CEBP reporter gene activity was up-regulated by FSH and cAMP in transfected granulosa cells. The GSK3 inhibitor did not affect reporter gene activity, suggesting that this signal is not required for CEBP activation by FSH or cAMP; however, we cannot exclude the possibility that because GSK3 can positively regulate CEBPA and negatively regulate CEBPB activity that their contrasting actions are balanced by the addition of the inhibitor. LY294002 or wortmannin did not decrease or had minimal effect on the FSH- or 8-Br-cAMP-stimulated CEBP promoter activity. On the other hand, complete inhibition of CEBP reporter activity occurred in the presence of H89, suggesting that cAMP-dependent PKA pathway is the major signaling pathway involved in basal, FSH-, and 8-Br-cAMP-mediated regulation of CEBP transactivity in granulosa cells. Although our transfection data do not discriminate between CEBPA and CEBPB-mediated transactivation, the results with the PKA inhibitor are consistent with an alteration in CEBPB isoform distribution observed in immunoblot studies (4 h).

In summary, our data show that increased CEBPA and CEBPB expression in vivo is associated with the luteinization of granulosa cells. In vitro studies with granulosa cells support PKA as the major regulator of CEBPB isoform abundance (34-kDa form) and as the primary pathway mediating FSH and cAMP-stimulated CEBP activity as measured by reporter gene assay. Our data suggest that CEBPA and CEBPB play a role not only in follicular development, but also during the transition period from follicle to corpus luteum where they might facilitate the increase in gene expression associated with differentiation and steroidogenesis.


   FOOTNOTES
 
1 Supported in part by National Institutes of Health grants HD38945 and MD00233, and by the University of South Carolina School of Medicine Research Development Fund. Back

2 Correspondence. FAX: 803 733 3212; hlavoie{at}med.sc.edu Back

Received: 13 August 2004.

First decision: 5 October 2004.

Accepted: 29 December 2004.


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 RESULTS
 DISCUSSION
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