Induction of Early Growth Response Protein-1 (Egr-1) Gene Expression in the Rat Ovary at the Time of Ovulation¹

Lawrence L. Espey^2,3, Takeshi Ujioka³, Darryl L. Russell⁴, Molly Skelsey³, Bogdan Vladu³, Rebecca L. Robker⁴, Hitoshi Okamura⁵, JoAnne S. Richards⁴

Department of Biology³, Trinity University, San Antonio, Texas 78212; Department of Cell Biology⁴, Baylor College of Medicine, Houston, Texas 77030; Department of Obstetrics and Gynecology⁵, Kumamoto University School of Medicine, Kumamoto, Japan

Short Title: Ovarian Egr-1 expression during ovulation

¹Grant support: Supported by NSF Grant #9870793 (L.L.E.), by a Grant to support T. Ujioka as a Research Fellow of The Lalor Foundation, Providence, Rhode Island (L.L.E.), and by NIH Grant HD-16229 (J.S.R.)

²Correspondence: Lawrence Espey, Ph.D.

Department of Biology

Trinity University

San Antonio, TX 78212

tel: (210) 999-7237

fax: (210) 999-7229

e-mail: lespey@trinity.edu

ABSTRACT

Granulosa cells in a mature ovarian follicle have an abundance of LH/hCG receptors that respond rapidly to an ovulatory surge in gonadotropins. Within minutes, membrane signal transduction sets in motion metabolic changes that lead to follicular rupture. This study provides evidence that the initial ovarian response to such an ovulatory stimulus includes induction of the immediate-early transcription factor gene for early growth response protein-1 (Egr-1). Immature Wistar rats were primed with 10 IU eCG sc, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG, sc. Ovarian RNA was extracted at 0, 0.5, 1, 2, 4, 8, 12, and 24 h after injecting the primed animals with hCG. The RNA extracts were used for RT-PCR differential display for random detection of gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified cDNAs confirmed that it was part of a gene that was significantly up-regulated within 1 h after the ovaries had been stimulated by hCG. Maximum transcription was at 4 h after hCG, and expression declined to 0-h control levels by 24 h after hCG. Sub-cloning and sequence analysis revealed that the cDNA matched the gene for Egr-1. In situ hybridization indicated that the Egr-1 mRNA was in the granulosa layer of mature follicles. Western blotting confirmed the temporal pattern of Egr-1 expression detected by differential display, Northern analysis, and in situ hybridization. The Egr-1 protein was approximately 84 kDa. In conclusion, the data show that expression of the zinc-finger transcription factor Egr-1 is an early event in the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone.

Introduction

A mature ovarian follicle contains granulosa cells that express a substantial number of LH/hCG receptors (1, 2). The signal transduction processes that are initiated by these receptors at the time of the ovulatory surge in LH (and FSH) induce several dynamic changes in follicular cell function. Along with the resumption of meiotic activity in the oocyte, there is induction of granulosa cell differentiation into progesterone-secreting lutein cells. Also, the fibroblasts in the thecal layers around the periphery of a follicle undergo transformation from quiescence to motility as they proliferate through the membrana propria toward the interior of the follicle where they lay down a connective tissue framework to support the developing luteal tissue (1). Thus, acute hormonal stimulation of a mature ovarian follicle leads to substantial cellular changes that convert a cavernous ovulatory follicle into a solid mass of luteal cells within only 24-48 h in most mammals. This transformation of an ovarian follicle into a corpus luteum involves distinct ovarian cell types, diverse signaling pathways, and temporally controlled expression of specific genes (3).

When extracellular ligands such as trophic hormones stimulate cytokinetic phenomena such as the ovulation/luteinization transformation, the target tissue response involves alterations in gene expression in the activated cells. The genomic response usually includes the induction of immediate-early transcription factor genes such as early growth response protein-1 (Egr-1, a.k.a., Krox-24, NGFI-A, zif/268, cef5, or TIS8) gene, and/or the c-fos and c-jun genes (4, 5). While rapid (albeit transient) Egr-1 gene induction is a common component of the response to mitogenic hormones that stimulate cells to undergo G₀-G₁ transition (5, 6), this transcription factor is also known to increase in cells that are only differentiating without dividing, such as LH-secreting cells in the pituitary (7). After it is translated, the Egr-1 protein translocates into the nucleus and functions as a zinc finger transcription factor to regulate the expression of an estimated 80-100 other genes—increasing their transcription rates as much as 100-fold (4, 5).

The present report characterizes, for the first time, ovarian expression of the Egr-1 gene during the early stages of the ovulatory process in the gonadotropin-primed immature rat. This predictable gene expression was discovered by chance during differential display RT-PCR analysis of mRNAs that are expressed in the ovary at the time of ovulation. The report describes the temporal and spatial pattern of expression of the gene during a peri-ovulatory period ranging from 12 h before to 12 h after follicular rupture. It also assesses the effects of progesterone and prostaglandin synthesis on expression of the Egr-1 gene.

Materials and Methods

Animal tissue and animal injections

Immature Wistar rats were induced to super-ovulate by eCG and hCG treatment as described previously (8). Ovarian RNA was extracted primarily at the peri-ovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. However, in a subsequent experiment, RNA was also extracted at 0.5 and 1 h after hCG in order to identify more precisely the onset of Egr-1 mRNA expression. The nucleic acid extracts were used for differential display and for Northern blotting. Epostane and indomethacin were injected sc, also as described previously (8). These anti-ovulatory agents were administered at 3 h after hCG in doses of 5.0 mg and 1.0 mg, respectively. The ovulation rate in the various experimental animals was determined by a procedure that also has been described previously (8). For the determination of ovulation rate and the extraction of ovarian RNA, rats were killed by exposure to CO₂. The animals were acquired, retained, and used in compliance with the NIH Guide and with the approval of the institutional animal care review committee.

Differential display protocols for detection of Egr-1

The steps of the differential display were carried out as described previously (8). In brief, RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure. RT- PCR was performed using an RNAimage Kit (G502, GenHunter Corporation, Nashville, TN). The specific primer set that yielded differentially expressed cDNA for Egr-1 was 5'-HTTTTTTTTTA-3' and 5'-HTAGAGCG-3', where "H" represents a HindIII restriction site attached to the primers. After extraction and re-amplification of the differentially expressed cDNA, a standard Northern analysis was performed to confirm the ovulation-specific expression of the parent mRNA for Egr-1. The unique cDNA fragment was subcloned using a pCR-TRAP Cloning System (P404, GenHunter), and a cloning colony containing the Egr-1 cDNA was identified by secondary Northern analysis. Manual sequencing of the cDNA was performed using a Sequenase Version 2.0 DNA Sequencing Kit (US70770, Amersham Pharmacia Biotech, Inc., Piscataway, NJ). In situ hybridization was performed as described previously (8).

Western blot analysis of Egr-1

Ovaries were extirpated from rats at the indicated peri-ovulatory intervals, and granulosa cells were isolated from the residual ovarian compartment by puncture with a 26-guage needle. Whole cell extracts were prepared from cells and tissue by homogenizing in 10 mM Tris-buffer (pH 7.5) containing 0.4 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, phosphatase and protease inhibitors. Protein extracts (50 mg) were resolved by reducing SDS-PAGE on a 10% acrylamide gel and transferred to PVDF membranes (Immobilon-P, Millipore Corp, Bradford MA). Membranes were blocked by shaking for 1 h in 3% non-fat milk, by incubating for 1 h in 3% milk with 0.5 mg/mL Egr-1 antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and by subseqently washing in TBST (10 mM Tris at pH 7.5, 150 mM NaCl, and 0.05% Tween-20). Blots were then incubated for 1 h with 1:10,000 horse radish peroxidase-linked anti-rabbit IgG (Amersham). After incubation, the membranes were washed again in TBST, and Egr-1 detection was performed according to Amersham instructions.

Statistical analysis

Densitometric analysis of the intensity of the signals from the Northern blots were analyzed by the NIH-image program as described previously (8). Numerical data are presented as means ± SEM. The significance of the differences among the six principal time points of 0, 2, 4, 8, 12, and 24 h after hCG were determined by Duncan's multiple range tests after a completely randomized one-way analysis of variance of the means of the groups. The probability value used as the cutoff between "significant" and "not significant" was P = 0.05.

Results

Differential display of Egr-1 cDNA during the ovulatory process

Following RT-PCR, the sub-populations of radioactively labeled cDNAs that were generated from RNA extracts at each of the six stages of the peri-ovulatory period were separated from one another by electrophoresis on a polyacrylamide gel. The autoradiograph of these PAGE results revealed differentially expressed cDNA bands that were present at 2, 4, 8, and 12 h after hCG, but were not conspicuous at 0, or 24 h into the ovulatory process (Figure 1). Therefore, the most intense cDNA band (i.e., the band in the 4-h lane) was excised from the acrylamide gel and re-amplified for use as a probe in Northern analysis.

Northern analysis of Egr-1 mRNA expression during ovulation

The Northerns revealed a pattern of mRNA expression during ovulation that was comparable to the differential display autoradiograph (Figure 2). In view of the fact that the initial Northern analysis revealed that the onset of Egr-1 mRNA expression began at some point between 0 and 2 h after the hCG stimulus, a subsequent set of RNA extractions included time-points of 0.5 and 1 h. Since the most intense Northern signal was at 4 h, the value for this lane was arbitrarily set at 100%, and the densities at the other times during the peri-ovulatory period were expressed as fractions of that maximum. Accordingly, the NIH-image program was used to digitize all of the bands on the Northerns, and the ratio of the density of each experimental band to its corresponding b-actin control band was calculated for each lane. Means (± SEM) of the signal densities at 0, 2, 4, 8, 12, and 24 h after hCG were 2.6% ± 1.1, 86.1% ± 4.0%, 100%, 41.6 ± 17.0%, 37.8% ± 15.4%, and 1.7% ± 0.8%, respectively. (The averages of the two determinations at 0.5 and 1 h after hCG were 7.2% and 58.0% , respectively.) Thus, Egr-1 gene expression increased 39-fold within 4 h after initiation of the ovulatory process by injecting hCG into the animals. Subsequently, at 24 h after hCG (i.e., during early luteal development), Egr-1 gene expression declined to a level that was not significantly different from the 0-h control value.

Sequence of the cDNA fragment for Egr-1

After the hCG-induced expression of the Egr-1 gene was confirmed by Northern analysis, the cDNA fragment of this gene was sub-cloned and sequenced. The NCBI accession number for this fragment is #AF023087. The cDNA fragment is essentially identical to a segment of a gene that has been cloned from rat pheochromocytoma cells (NCBI accession #M18416). Also, the fragment is highly homologous with Egr-1 genes cloned from the mouse (NCBI accession #M20157) and the human (NCBI accession #NM_001964).

Effects of epostane and indomethacin on Egr-1 gene expression

For these tests, Northern blots were prepared from RNA extracted from control ovaries at 0 and 8 h into the ovulatory process, or extracted from experimental ovaries taken at 8 h after hCG from rats that had been treated 5 h earlier with ovulation-inhibiting doses of epostane or indomethacin. (These experimental intervals were selected in order to compare the present data with related studies using epostane and indomethacin (8)). The signal density (normalized against the b-actin control) of the 8-h control lane was arbitrarily set at 100% (Figure 3). There was minimal expression of Egr-1 mRNA at 0 h, but substantial expression at 8 h. In animals treated with the anti-ovulatory agent epostane, which blocks progesterone synthesis, the signal density of 139.3% ± 32.8% was not significantly different from the 8-h control value. Animals treated with the anti-ovulatory agent indomethacin, which blocks prostanoid synthesis, had a signal density that was 60.4% ± 11.2% of the 8-h control value. Although this was significantly lower than in the ovaries of animals treated with epostane, it was not significantly different from the 8-h control value. The ovulation rates in parallel groups of animals treated with indomethacin and epostane were significantly inhibited (Figure 3).

Localization of Egr-1 mRNA expression by in situ hybridization

In situ hybridization confirmed the temporal pattern of Egr-1 mRNA expression that was observed in the differential display autoradiograph and the Northern analysis. There was minimal signal from the 0-h control ovaries, a strong signal at 2-4 h after hCG treatment, a declining signal at 8-12 h, and negligible signal at 24 h (Figure 4). Hybridization was localized in the granulosa layer of the larger follicles (Figure 5). A number of smaller follicles (located mainly in the center of the ovaries) had no detectable Egr-1 mRNA. Most of the other smaller-looking follicles that did exhibit hybridization had thicker granulosa layers--indicating that these probably were large mature follicles that happened to be sectioned on a plane that was above or below their maximum diameter.

Western analysis of Egr-1 translation during ovulation

Western blotting revealed that translation of the Egr-1 gene product coincided with the temporal pattern of Egr-1 mRNA expression that was observed by Northern analysis and in situ hybridization. There was negligible translation at 0 h after hCG treatment, and the strongest signal was at 4 h (Figure 6). As predicted by the in situ data, the isolated granulosa cells evinced a substantial amount of Egr-1. The modest expression of Egr-1 in the residual ovarian tissue most likely arises from granulosa cells that invariably remain in the residual portion of the ovary. The size of the translated protein was 84 kDa.

Discussion

A wide variety of extracellular stimuli are known to activate phosphorylation-dependent membrane-signaling pathways that program cellular responses to provocation. Such diverse stimuli include mitogens, morphogens, antigens, neurotransmitters, serum, radiation, and hormones. In instances when a stimulus provokes reactions such as mitosis, differentiation, immune responses, action potentials, or inflammatory reactions, the signal transduction processes in the affected cells rapidly converge on the genome to induce the so-called immediate-early gene response. In essence, this early response consists of the expeditious induction of one or more genes that serve as transcription factors which function to activate a cell-specific repertoire of other genes to carry out the ultimate cellular response to the original stimulus. The best known early-response genes are c-fos, c-jun, and Egr-1 (4, 5, 9-11). Therefore, further characterization of the Egr-1 gene and its protein product is in order in view of the present evidence of ovarian expression of this gene in response to an ovulatory dose of gonadotropic hormone, and the subsequent inflammation and differentiation associated with ovulation and luteinization (1).

Transcription of the Egr-1 gene appears to be regulated by multiple signal transduction processes, including the protein kinase C (PKC) pathway (4). However, the protein kinase A pathway is the most likely path activated by the LH surge in granulosa cells (2, 3). The promoter region of the Egr-1 gene contains an Sp1 binding site that can confer transactivation by Sp1 as well as by COUP-TF (12). Sp1 is expressed at high levels in granulosa cells and Sp1 binding sites in the promoters of several ovarian-expressed genes have been shown to confer FSH and LH inducibility (2, 13). In some cells, the activation process occurs within minutes. For example, in quiescent fibroblasts that have been stimulated by fetal calf serum, there is detectable Egr-1 expression as early as 10 min. In this 3T3-cell model, expression reaches a peak within 30 min and then declines back to basal levels by 3-4 h (4). In contrast, ovarian expression of Egr-1 mRNA and its protein product appears to peak approximately 4 h after initial stimulation of the ovulatory process by hCG, and expression of this immediate-early gene does not return to basal level until sometime between 12 and 24 h after hCG. The difference in the relative time-course of Egr-1 expression in the ovary compared to that in 3T3 cells in culture may depend on the time required in vivo to increase intracellular levels of hormone, on the stage of cell differentiation, or on other contextual controls in ovarian cell function. Importantly, granulosa cells of preovulatory follicles respond to LH (or, to hCG) by rapid exit from the cell cycle and rapid entry into a program of terminal differentiation to luteal cells (14, 15), whereas the 3T3-cell model is poised for cell proliferation (4). It has been suggested that the post-stimulus return to basal level is the effect of some auto-regulatory mechanism (4).

The Egr-1 gene product is a member of the zinc-finger family of transcriptional regulators that bind specific sequence motifs in gene promoters. Egr-1 is unique in that it can regulate transcription of some genes positively, while effecting other genes negatively (4,10). Also, it should be noted that Egr-1 has a serine- and threonine-rich repressor domain that may dominate the transcriptional phenotype of the Egr-1 molecule in the absence of phosphorylation activity that is usually generated by signal transduction processes. This possible bi-functional nature of Egr-1 has been described in more detail elsewhere (4). The importance of the present discussion to future studies on ovarian genes regulated by Egr-1 is that target genes may be either activated or repressed, depending on the pattern of phosphorylation of the Egr-1 transcription factor—as well as depending on the promoter composition of the target genes.

It is interesting to note that the immediate early gene response that generates Egr-1 includes a slightly deferred induction of genes for NGFI-A-binding proteins (NAB), a family of corepressors that bind directly to Egr-1 and repress Egr-1-mediated transcription (16-18). Thus, the cascade of transcriptional activity that is induced by Egr-1 is transient, not just because Egr-1 gene expression is down-regulated, but also because the Egr-1 protein interaction with general transcriptional effectors becomes altered. Such transient transcriptional activity is relevant to the present experimental model because the ovulatory process has been likened to an early transitional phase of the luteinization process that is induced by gonadotropic hormones (1, 2). Therefore, it is possible that the downstream repertoire of Egr-1-induced growth signals could include a subset of genes that are responsible for temporary degradation of the ovarian follicle as it metamorphosis into a functional corpus luteum.

The ubiquitous nature of Egr-1 expression as an early response to growth signals suggests this zinc-finger transcription factor has a pivotal role in a cascade of gene expression in cells that have been induced to undergo proliferation, differentiation, or responses to inflammatory-like signals. There is a growing list of physiologically relevant genes that are now recognized as targets for Egr-1 (4, 5, 7, 19, 20). Some targets that are related to proliferative responses include the thimidine kinase gene that peaks during late G₁, various growth-factor genes like PDGF and FGF, the interleukin genes, and the TNF genes. Others, such as the LH-b gene, the family of cell-surface-adhesion protein genes for CD44, and genes for several matrix metalloproteinases (MMPs) are related more to differentiated functions of these cells. The reported relationship between Egr-1, CD44, and a number of MMPs (20-23) is especially intriguing since this family of proteases has been implicated in the degradative events of ovulation for quite some time (24). MT1-MMP (i.e., MMP14) is the only one to date that has been shown to have a functional Egr-1 site in its promoter (20). MMP14, which has been demonstrated in several different types of cells, initially decreases in granulosa cells within 4 h after stimulation by hCG, and then it increases at 12 h after hCG (25). Therefore, MMP14 might be negatively, or positively, regulated by Egr-1 in luteinizing granulosa cells. In contrast, ADAMTS-1 (24), MMP19 (25), and tissue inhibitor of metalloproteinase-1 (TIMP-1) (25) are all induced by hCG, making them possible targets of Egr-1 in granulosa cells. In any event, these and other genes that have been linked to Egr-1 are all candidates for investigation as potential mediators of Egr-1 action in granulosa cells during the ovulatory process.

In conclusion, Egr-1 expression is an immediate early gene response to gonadotropic hormone action on ovulatory follicles. This gene expression is not dependent on the well known increases in ovarian progesterone or prostaglandin synthesis during ovulation. The in situ hybridization data indicates that Egr-1 gene expression is localized in the granulosa layer of the larger antral follicles, suggesting the transcription factor that is translated from this gene may have a central role in regulating the rapid reprogramming of granulosa cells to luteal cells (2, 14). There is also evidence linking Egr-1 activity to sites of inflammation (9, 19, 26-28). Therefore, since the ovulatory process is comparable to an acute inflammatory reaction (29), Egr-1 might serve as a mediator of the transient events that cause degradation and rupture of a follicle. It will be of particular interest to assess the potential of Egr-1 protein as a transcription factor for MMP gene expression in follicular tissue at the time of ovulation. Finally, it has been reported that Egr-1 is important for female fertility because it regulates LH expression in the pituitary gland and LH receptor expression in the ovary (30, 31). The present results indicate that Egr-1 may also affect fertility by initiating a cascade of ovulation-specific gene expression in ovulatory follicles that have been stimulated by gonadotropic hormone.

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Legends

Figure 1. Autoradiograph of differentially displayed Egr-1 cDNA (arrows). Note that the cDNA is hardly visible in the 0 h RT-PCR product, and the greatest amplification was at 4 h.

Figure 2. Intensity of Northern blot signals at eight intervals of the peri-ovulatory period following hCG administration. Solid dots represent the mean value of six Northern blots prepared from three separate RNA extractions, while open dots represent the medium of two Northern blots from one RNA extraction. The signal density at 4 h was arbitrarily set at 100%. An actual Northern analysis of the Egr-1 cDNA, along with its b-actin control, is shown below the graph. Note that the greatest intensity is at 4 h after hCG.

Figure 3. Comparison of the % of signal from Northern blots containing RNA extracted at 8 h after hCG from animals that were also treated with either 5 mg of epostane (Epo), or 1 mg of indomethacin (Indo) administered at 3 h after hCG. Bar graphs are based on NIH-image analyses of four different Northerns. The signal from the 8-h control lane was arbitrarily set at 100% OD. In parallel groups of rats, the ovulation rate was determined at 24 h after hCG.

Figure 4. Change in intensity of the in situ hybridization signal during the six principal peri-ovulatory intervals following hCG administration. Light-field micrographs on the left show the histology of ovarian sections stained with hematoxylin and eosin (H & E), while the dark-field micrographs of the same sections show the localization of Egr-1 mRNA as detected by hybridization of a ³⁵S-labeled anti-sense probe derived from the Egr-1 cDNA.

Figure 5. Closer view of the distribution of the Egr-1 probe in the ovary. Black arrows pointing to the right in the 4-h H & E micrograph mark the granulosa layer where, as indicated by corresponding white arrows in the dark-field micrograph, the Egr-1 probe hybridized to the granulosa cells. The vertical arrows, pointing up and down, show that Egr-1 mRNA expression also occurred in the cumulus cells surrounding the oocyte. The array of black arrows from a central hub points to a number of small follicles that did not express Egr-1 mRNA.

Figure 6. Western blot of Egr-1 expression by granulosa cells and residual ovarian tissue at the designated intervals during the ovulatory process.