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Centre de recherche en reproduction animale et Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, CP 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6
1 Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6
(Requests for offprints should be addressed to K Sayasith; Email: k.sayasith{at}umontreal.ca)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ovulation is a complex process that ultimately leads to the rupture of the follicle and release of the oocyte after the preovulatory LH surge (Armstrong 1981, Murdoch et al. 1993, Sirois et al. 2004). This process has been characterized as an acute inflammatory reaction (Espey 1980), in which the expression of several genes is stimulated (for reviews, see Richards et al. 2002, Sirois et al. 2004). The characterization of EGR-1 during the ovulatory process is of interest because of its obligatory role in reproduction, its induction in rat granulosa cells of ovulating follicles after the LH surge (Espey et al. 2000), and its ability to regulate the expression of many genes. However, these studies of EGR-1 have been limited to rodents, whereas there have been no studies describing the ovarian regulation of EGR-1 in large monoovulatory species, nor is there evidence that EGR-1-regulated genes are involved in the ovulatory process. Therefore, the objective of this study was to characterize the gonadotropin-dependent regulation of EGR-1 in bovine preovulatory follicles prior to ovulation and to investigate the effect of EGR-1 on the expression of genes known to be involved in ovulation.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To clone the bovine EGR-1 cDNA, RT-PCR, 3'- and 5'-RACE reactions were used. RT-PCR was performed using total RNA extracts (100 ng) isolated from preovulatory follicles (Sirois 1994), primers 1 (sense) and 2 (antisense), and the OneStep RT-PCR kit (Qiagen) as directed by the manufacturer (Fig. 1Aa
). These primers were designed from highly conserved coding regions of mouse (GenBank accession number: NM_007913
[GenBank]
) and human (GenBank accession number: NM_001964
[GenBank]
) EGR-1 homologs (Fig. 1B). RT-PCR products were subcloned into pGEM-T Easy vector and sequenced (Service de séquençage, Université Laval, Québec, Canada). A large bovine EGR-1 cDNA fragment of 1517-bp was obtained (Fig. 1Aa).
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). Primers 4 and 6 were designed from the 3'-end of a bovine EGR-1 cDNA fragment.
To obtain the 5'-missing end of bovine EGR-1 cDNA, 5'-RACE was performed with the 5'-RACE system version 2.0, in accordance with the manufacturer’s instructions (Invitrogen Life Technologies). Briefly, reverse transcription was carried out as directed using antisense primer 8 (Fig. 1Ac) and 5 µg of RNA extracted from a preovulatory follicle obtained 24 h post-hCG. The first PCR was performed with sense abridged anchor primer 9 (Invitrogen Life Technologies) and antisense primer 10, and the second PCR was done with sense abridged universal amplification primer 11 (Invitrogen Life Technologies) and antisense primer 12 (Fig. 1Ac). Primers 8, 10, and 12 were designed from the 5'-end of a bovine EGR-1 cDNA fragment. PCR consisted of 35 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, and PCR products were subcloned into pGEM-T Easy vector and sequenced. The complete bovine EGR-1 coding region was isolated by RT-PCR using 100 ng RNA from 24 h post-hCG preovulatory follicle, sense primer 13 and antisense primer 14 (Fig. 1Ad), and sequenced.
In vivo model of ovulation
Holstein heifers of 2–3 years of age, exhibiting normal estrous cycles were used as a model of hCG-induced ovulation to study the regulation of EGR-1 during the ovulatory process in vivo, as previously described (Sirois 1994). Briefly, bovine preovulatory follicles were obtained after induction of luteolysis on day 7 of the estrous cycle and i.v. administration of an ovulatory dose of hCG (3000 IU). The ovary bearing the preovulatory follicle was isolated by ovariectomy (via colpotomy) from individual heifers at 0–26 h after hCG (Sirois 1994). The interval of time from hCG administration to ovulation is 26–28 h in this animal model. The preovulatory follicles were dissected from the ovary with a scalpel and pieces of follicle wall (i.e. theca interna with attached granulosa cells) were prepared and further dissected into isolated preparations of granulosa and theca cells (Sirois 1994). All tissue samples were stored at –70 °C. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Université de Montréal and were consistent with the guidelines of the Canadian Council of Animal Care.
RNA extraction and semiquantitative RT-PCR/Southern blot
All tissues were obtained from a slaughterhouse, and total RNA was isolated from bovine preovulatory follicles, granulosa cell cultures, and tissues using the TRIzol reagent, and a Kinematica PT 1200C Polytron Homogenizer (Fisher Scientific, Montréal, Canada), according to the manufacturer’s instructions. RT-PCR/Southern blot was performed using extracts (100 ng), the OneStep RT-PCR kit as directed by the supplier, sense (5'-CCG ACT ATC TGT TTC CAC AAC-3') and antisense (5'-TTA GCA AAT TTC AAT TGT CCT GG-3') primers specific for bovine EGR-1 gene, producing a 871-bp DNA fragment, and sense (5'-GTT TCC AGT AGA TTC CAC CC-3') and antisense (5'-TCC ACC ACC CTG TTG CTG TA-3') primers specific for bovine GAPDH, generating a 850-bp DNA fragment (Tsai et al. 1996). The reaction conditions were one cycle of 50 °C for 30 min and 95 °C for 15 min, followed by 19 cycles for EGR-1 or 13 cycles for GAPDH of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 2 min. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification. PCR products were resolved on 2% Tris–acetate/EDTA–agarose gels, transferred to nylon membrane, and hybridized with corresponding 32P-labeled EGR-1 and GAPDH cDNA fragments using QuikHyb hybridization solution (Stratagene, LaJolla, CA). Membranes were exposed to X-OMAT Kodak films with the intensifier screen and signals were quantified by densitometer using the ImageQuant software.
Immunohistochemical localization of EGR-1 protein in bovine follicles
Immunohistochemistry was performed as previously described (Brûlé et al. 2003). Briefly, formalin-fixed tissues were paraffin-embedded and 3 µm-thick sections were prepared. The paraffin was removed by passage through graded xylene and alcohol solutions and sections were rehydrated, rinsed with TBS (150 mM NaCl, 0.1 M Tris, pH 7.5), and incubated for 10 min in TBS containing 0.1 M glycine. After the incubation, sections were heated in a pressure cooker for 14 min, incubated in blocking buffer containing TBS, 1% BSA, and 1% fat-free skim milk, and then incubated overnight at 4 °C with a rabbit polyclonal antibody against human EGR-1 (1:10 dilution; Santa Cruz Bio-technologies). The specificity of the antibody against bovine protein was verified by immunoblotting using extracts prepared from granulosa cells overexpressing bovine EGR-1 (data not shown). After three 5 min rinses with TBS, sections were incubated for 2 h at room temperature with a monoclonal anti-rabbit IgG conjugated to alkaline phosphatase (1:200 dilution; Sigma). After three washes in TBS, the reaction was revealed using the NBT/BCIP alkaline phosphatase substrate (Roche), and sections were mounted.
EGR-1 expression construct, granulosa cell cultures, and DNA transfection
To generate the EGR-1 expression construct, the full-length bovine EGR-1 was isolated by RT-PCR using RNA extracts (100 ng) of a preovulatory follicle, sense primer 13, and antisense primer 14. RT-PCR consisted of one cycle of 50 °C for 30 min and 95 °C for 15 min followed by 35 cycles of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 2 min. Reaction products were subcloned into the expression vector pcDNA 3.1 (+) (Invitrogen Life Technologies) and sequenced.
Granulosa cell cultures were prepared as previously described (Liu et al. 1999, Sayasith et al. 2004). Briefly, pairs of bovine ovaries bearing a newly formed corpora lutea and a follicle of 8–12 mm in diameter (dominant follicle of the first wave of the estrous cycle) were obtained from a slaughterhouse. Granulosa cells were isolated from the largest follicle as previously described (Liu et al. 1997), seeded at a density of 2 x 106 in the 100 mm Petri dish containing 10 ml of minimal essential medium (MEM; Invitrogen Life Technologies) supplemented with L-glutamine, non-essential amino acids, 2% fetal bovine serum, insulin (1 µg/ml), transferrin (5 µg/ml), and penicillin (100 units/ml)-streptomycin (100 µg/ml), and incubated at 37 °C in a humidified atmosphere of 5% CO2. To study the regulation of EGR-1 in vitro, cells were serum-starved overnight in fresh MEM and incubated with forskolin (10 µM; Calbiochem) for various times. After incubation, the cells were harvested and RNA and protein extracts were isolated. To examine the effect of EGR-1 on the transcript expression of other genes, serum-starved granulosa cells were transfected with the EGR-1 expression vector (4 µg/Petri dish) in serum-free MEM using LipofectAMINE PLUS, in accordance with the manufacturer’s protocol (Invitrogen Life Technologies). Three hours after transfection, cells were rinsed and incubated in serum-free MEM in the absence or presence of forskolin (10 µM) for 24 h, and the RNA extraction and RT-PCR analyses were performed.
RT-PCR analysis
RNA extracts (100 ng) isolated from granulosa cells were analyzed by RT-PCR to study the expression of EGR-1, PGHS-2, PGES, EP2, P450scc, P450arom, and LH-R using the OneStep RT-PCR kit and various primers: 5'-CAC AGT GCA CTA CAT ACT TAC C-3' (sense) and 5'-GTC TGG AAC AAC TGC TCA TCG C-3' (antisense) primers, generating a 735-bp fragment of PGHS-2; 5'-TGA TGA ACG GCC AGG TGC TC-3' (sense) and 5'-ATG CCA CGG TGT GTA CCA TAC GG-3' (antisense) primers, generating a 330-bp fragment of PGES; 5'-CTG CTG GAT CAT TGG AAG TAT GC-3' (sense) and 5'-TCC ATC TCG CTG TTC CAC GTG-3' (antisense) primers, generating a 439-bp fragment of EP2; 5'-AGA GAA TGG ACT TTC GCC ACA TC-3' (sense) and 5'-AGG GAC ACT GGT GTG GAA CAT C-3' (antisense) primers, generating a 525-bp fragment of P450scc; 5'-CAC ATC CTC AAT ACC AGG TCC C-3' (sense) and 5'-AGA GGT GTC CAG CAT GAT GCG-3' (antisense) primers, generating a 469-bp fragment of P450arom; and 5'-CTT CTG CAT GGG GCT CTA-3' (sense) and 5'-TCA CGT TGC GAA TCA GGA TGG-3' (antisense) primers, generating a 395-bp fragment of LH-R. Each reaction was performed at one cycle of 50 °C for 30 min and 95 °C for 15 min followed by 35 cycles of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 2 min. PCR products were electrophoresed on 2% Tris–acetate/EDTA–agarose gels.
Cell extracts and immunoblot analysis
Cell extracts were prepared from granulosa cell cultures as described previously (Sayasith et al. 2004), and protein concentrations were determined by the method of Bradford (Bradford 1976) (Bio-Rad Protein Assay). Samples (100 µg/well) were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to PVDF membranes (Amersham Pharmacia Biotech). Membranes were incubated with a rabbit polyclonal antibody against human EGR-1 (1:200 dilution), and immunoreactive proteins were visualized by incubation with the horseradish peroxidase-linked donkey anti-rabbit secondary antibody (1:6000 dilution) and the enhanced chemiluminescence system (ECL plus) according to the manufacturer’s protocol (Amersham Pharmacia Biotech).
Statistical analyses
One-way ANOVA was used to test the effect of different times of hCG on levels of EGR-1 mRNA in samples. EGR-1 mRNA levels were normalized with the control gene GAPDH prior to analysis. Statistical analyses were performed using JMP software (SAS Institute, Inc., Carry, NC). When ANOVA indicated a significant F value (P < 0.05), Dunnett’s test was used for multiple comparisons of individual means.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The bovine EGR-1 cDNA was cloned by a combination of RT-PCR, 3'- and 5'-RACE using RNA extracts isolated from preovulatory follicles 24 h after hCG treatment (Sirois 1994) and primers designed from highly conserved coding regions of mouse and human EGR-1 homologs. Results showed that full-length bovine EGR-1 cDNA contains an ORF of 1623 bp (including stop codon), a 5'-untranslated region (UTR) of 5 bp, and a 3'-UTR of 529 bp. The nucleotide sequence of bovine EGR-1 was submitted to GenBank with accession number AY924307
[GenBank]
and its predicted complete coding region was isolated by RT-PCR, subcloned and confirmed by sequencing. This coding region encodes a 540-amino acid protein that is three amino acid residues shorter than human EGR-1, and 7, 32, 30, and 25 amino acid residues longer than mouse, rat (GenBank accession number: NM_012551
[GenBank]
), chicken (GenBank accession number: AY034140
[GenBank]
) and fish (GenBank accession number: XM_688016
[GenBank]
) homologs respectively (Fig. 2). Comparative analyses indicated that bovine EGR-1 amino acid sequence is relatively similar to human (93%), mouse (89%), rat (84%), chicken (73%), and fish (62%) homologs, and contains conserved structural domains putatively involved in its functions, including three zinc-finger motifs responsible for the binding with the promoter of genes, a transactivation domain, a glycine-rich region, two serine-rich regions, a nuclear localization signal, and an inhibitory domain (Fig. 2).
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To examine the expression of bovine EGR-1 mRNA in various tissues, RT-PCR/Southern blot was performed using RNA extracts isolated from bovine tissues. Results showed that levels of EGR-1 mRNA were high to very high in corpora lutea, uterus, kidney, pituitary, spleen, and preovulatory follicle, and moderate to low in other tissues (Fig. 3A), whereas levels of GAPDH mRNA (control gene) remained relatively constant in tissues tested (Fig. 3B). To study the regulation of EGR-1 mRNA in bovine follicles prior to ovulation, semiquantitative RT-PCR/Southern blot was performed using RNA extracts obtained from the wall of preovulatory follicles isolated at 0, 6, 12, 18, and 24 h after hCG treatment. Representative results revealed that levels of EGR-1 mRNA were very low at 0 h, increased at 6 h, and decreased from 12 to 24 h post-hCG (Fig. 3C). Results from several follicles expressed as ratios of EGR-1 to GAPDH showed a significant increase of EGR-1 mRNA at 6 h post-hCG when compared to 0 h (P < 0.05; Fig. 3D).
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To investigate cellular localization of bovine EGR-1 mRNA expression in preovulatory follicles, RT-PCR/Southern blot was performed using RNA preparations of isolated granulosa and theca cells prepared from preovulatory follicles obtained at 0, 6, 12, 18, 20, 22, and 24 h post-hCG. In granulosa cells, representative results indicated that levels of EGR-1 transcript were very low at 0 h, increased at 6 h after hCG, and decreased thereafter (Fig. 4A). When results from several follicles were expressed as EGR-1 and GAPDH ratios, a high increase of EGR-1 was observed at 6 h post-hCG when compared to 0 h (P < 0.05; Fig. 4B). In theca cells, representative results showed that levels of EGR-1 mRNA were low at 0 h, increased at 6 h, and decreased between 12 and 24 h post-hCG (Fig. 4C). Results from several follicles expressed as ratios of EGR-1 to GAPDH revealed a significant increase of transcripts at 6 h post-hCG when compared to 0 h (P < 0.05; Fig. 4D). Interestingly, the induction of EGR-1 mRNA predominantly occurred in granulosa cells.
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), in keeping with very low levels of EGR-1 mRNA detected in preovulatory follicles obtained at this time, but markedly increased in granulosa cells of a follicle isolated at 24 h post-hCG (Fig. 5B and C). As control of immunohistochemical reaction, no immunostaining was observed when the primary antibody was replaced with PBS (Fig. 5D).
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To study the regulation of EGR-1 in vitro, granulosa cells were isolated from preovulatory follicles and cultured in the absence or presence of forskolin for various times. These primary cultures have been previously established as a valuable model to study the regulation of bovine PGHS-2 observed in vivo (Liu et al. 1999, Sayasith et al. 2004). RT-PCR/Southern blot analyses showed that levels of EGR-1 mRNA were low at 0 h, increased at 6 h, and declined to steady-state levels from 12 to 72 h post-forskolin (Fig. 6A). When results from three independent experiments were expressed as ratios of EGR-1 to GAPDH, a significant and transient increase of EGR-1 mRNA was observed at 6 h post-forskolin when compared with 0 h (P < 0.05; Fig. 6B). To investigate if the induction of EGR-1 mRNA was associated with its protein expression, immunoblotting was performed using extracts isolated from granulosa cells after forskolin treatment. Results revealed that the levels of EGR-1 protein were low at 0 h, and increased from 6 to 24 h post-forskolin (Fig. 6C).
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, lanes 2 vs 1). In EGR-1-transfected cells, overexpression of EGR-1 increased basal expression of PGHS-2, PGES, and EP2 mRNA, initially observed in untransfected cells (Fig. 6D, lanes 3 vs 1), whereas the treatment with forskolin enhanced the latter EGR-1-dependent increase (Fig. 6D, lanes 4 vs 3). Overexpression of EGR-1 in the absence or presence of forskolin had no effect on the expression of P450arom and P450scc mRNA when compared to untransfected cells (Fig. 6D, lanes 3 vs 1, and lanes 4 vs 2). Basal levels of LH-R mRNA were very low, and not affected by forskolin treatment in untransfected cells (Fig. 6D, lanes 2 vs 1), but highly increased by EGR-1 overexpression (Fig. 6D, lanes 3 vs 1 and 2). The latter increase was markedly stimulated by forskolin (Fig. 6D, lanes 4 vs 3). |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ovulation is a complex process initiated by LH stimulation, which is accompanied by an increase in the expression of several genes, including PGHS-2, PGES, an inhibitor of the cell cycle p21CIP1, membrane type 1-metalloproteinase (MT1-MMP), MMP19, tissue inhibitor of MMP1, and cathepsin L (for reviews, see Richards et al. 2002, Sirois et al. 2004). Some of these genes contain GC-rich regions in their promoters, which are recognized as targets for EGR-1. Many studies have shown that EGR-1 can bind directly to GC boxes or act in harmony with other proteins to mediate transactivation of several genes or its own expression in different cell types. Indeed, binding of EGR-1, Sp1, and CREB transcription factors to their corresponding sites plays a key role in the regulation of cathepsin L in rat granulosa cells (Sriraman & Richards 2004), whereas interactions between EGR-1, Sp1/Sp3, and NFAT (nuclear factor of activated T cells) c1 are required for the MT1-MMP expression in glomerula mesanglial cells (Alfonso-Jaume et al. 2004). In contrast, EGR-1 alone was needed for the induction of MT1-MMP and p21CIP1 in other cell types (Haas et al. 1999, Aicher et al. 2003). It has been reported that EGR-1 acts as a key regulator of CD44 expression, a major cell-surface receptor of hyaluronan (HA) in several cell types (Maltzman et al. 1996, Abdel-Latif et al. 1999, Fitzgerald & O’Neill 1999, Mishra et al. 2005). HA is the main component of cumulus expansion (for review, see Richards et al. 2002), and its synthesized amount is closely correlated with the degree of cumulus expansion (Chen et al. 1996). Recent studies have indicated that CD44 was expressed in the membrane of cumulus cells (Kimura et al. 2002, Yokoo et al. 2002), and identified a HA-binding protein required for expansion of the cumulus–oocyte complex (Yokoo et al. 2002). Thus, it is of interest to know whether EGR-1 is involved in CD44 expression in cumulus cells during the ovulatory process. It has also been suggested that the binding of EGR-1 to two CG-rich regions on the rat LH-R gene promoter plays a critical role in the expression of LH-R in a tumor cell line derived from mouse Leydig cells (Yoshino et al. 2002), while genetic disruption of EGR-1 resulted in a loss of LH-R expression in ovaries and infertility of female mice, suggesting the involvement of EGR-1 in ovarian functions (Lee et al. 1996, Topilko et al. 1998). Supporting this presumption, our findings report for the first time that EGR-1 overexpression induced the expression of LH-R transcripts in granulosa cells. However, it remains unknown whether this induction was followed by a mechanism similar to that observed for mouse LH-R expression in Leydig cells. The isolation of bovine LH-R gene promoter and its functional characterization should help unravel the molecular basis for the species-specific regulation of LH-R in granulosa cells. Further studies are needed to determine whether EGR-1 is directly involved in the regulation of genes implicated in the ovulatory process.
The present study is the first to report the influence of EGR-1 on the expression of genes involved in ovulation. Indeed, our preliminary findings revealed that over-expression of EGR-1 also increased the levels of mRNA for PGES, PGHS-2, and EP2, but not for P450arom and P450scc in granulosa cells. As the expression of PGHS-2 and EP2 is required for ovulation, at least in rodents, our data suggest the involvement of EGR-1 in the ovulatory process, playing an important role in prostaglandin biosynthesis pathway. Previous studies have reported that the binding of EGR-1 to two GC-rich boxes of the PGES promoter regulates the expression of rat PGES in non-ovarian cells (Naraba et al. 2002, Subbaramaiah et al. 2004). Since two GC-rich regions are also present within the bovine PGES promoter (our unpublished observation), this suggests that the increase of PGES mRNA by EGR-1 overexpression in granulosa cells may be mediated by a mechanism similar to that observed for rat PGES expression. Sequence analyses indicated that no EGR-1 sites are found within bovine PGHS-2, whereas the bovine EP2 promoter has not been cloned. Therefore, the increase in levels of PGHS-2 mRNA after EGR-1 overexpression in granulosa cells may be through mechanisms that implicate the interactions of EGR-1 with other cofactors. Interestingly, the follicular induction of PGHS-2 (Sirois 1994) and PGES (Filion et al. 2001) occurred (18 to 24 h post-hCG) much later than those of EGR-1 (6 h post-hCG), thereby supporting the concept of the EGR-1 involvement in the increase of PGHS-2 and PGES mRNA in bovine preovulatory follicles. The characterization of their promoter activities should help to clarify this contention. Previous studies have shown that bovine granulosa cells isolated from small follicles express mRNA for P450arom and P450scc in cultures, which were stimulated by FSH (Sahmi et al. 2004). Similarly, our data also indicated that treatment with forskolin increases P450arom and P450scc mRNA abundance in primary granulosa cells, similar to that observed in differentiating follicles in vivo (Xu et al. 1995; Bao et al. 1997), suggesting the maintenance of granulosa cells in a non-luteinizing state. This state appeared unaffected by EGR-1 over-expression, since levels of P450arom and P450scc mRNA remained unchanged when compared to untransfected cells. This suggested a direct effect of EGR-1 on the expression of genes in granulosa cells, but not by a change in differentiation status of the cells, thereby strengthening the role of EGR-1 in the ovulatory process.
Studies on the molecular control of EGR-1 induction have been limited to rodents. In this species, interactions of EGR-1, in association with Sp1, with the proximal GC box within its promoter, and together with CREB and serum response factor have been shown to play a critical role in the induction of EGR-1 and its promoter activity in granulosa cells (Russell et al. 2003). Treatment with PGE2 has been suggested to regulate EGR-1 synthesis in non-ovarian cells (Danesch et al. 1994, Fujino et al. 2003, Faour et al. 2005), whereas the administration of an ovulation-inhibiting dose of indomethacin (an inhibitor of PGHS, which significantly reduces ovarian prostanoid production) did not alter EGR-1 expression during the early stage of the ovulatory process (Espey et al. 2000). This suggests that the pronounced increase of PGE2 in follicular fluid observed prior to ovulation is not involved in EGR-1 synthesis in preovulatory follicles. Moreover, this increase occurred much later than that of EGR-1 (4 h post-hCG in rodents (Espey et al. 2000) and 6 h post-hCG in cattle observed from this study), which thereby does not support the role of PGE2 in the increase of EGR-1 expression.
In summary, this study is the first to report the bovine EGR-1 cDNA and its gonadotropin-dependent regulation in preovulatory follicles of large monoovulatory species prior to ovulation. Levels of EGR-1 were high in granulosa cells, and low in theca cells after hCG treatment. Using primary granulosa cell cultures, we were able to replicate the regulation of EGR-1 as observed in vivo. Interestingly, overexpression of EGR-1 stimulated transcript expression of PGHS-2, PGES, EP2, and LH-R in these cultures, suggesting a potential involvement of EGR-1 in the ovulatory process, including prostaglandin biosynthesis pathway. Further investigation will be needed to study the molecular mechanisms behind the gonadotropin induction of EGR-1 and its stimulating effect on the other genes expressed during the important physiological process.
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Acknowledgements |
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Funding |
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Canadian Institutes of Health Research (CIHR) Grant MT-13190 (to J S) supported this work. The nucleotide sequence reported in this paper has been submitted to GenBank/EBI Data Bank with accession number AY924307 [GenBank] .
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Received in final form 18 May 2006
Accepted 8 June 2006
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