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Endocrinology Vol. 145, No. 11 5384-5396
Copyright © 2004 by The Endocrine Society

Development and Application of a Rat Ovarian Gene Expression Database

Misung Jo, Mary C. Gieske, Charles E. Payne, Sarah E. Wheeler-Price, Joseph B. Gieske, Ignatius V. Ignatius, Thomas E. Curry, Jr. and CheMyong Ko

Departments of Obstetrics and Gynecology (M.J., S.E.W.-P., T.E.C.) and Clinical Sciences (M.C.G., C.E.P., J.B.G., I.V.I., C.K.), University of Kentucky, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Dr. CheMyong Ko, Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky 40536. E-mail: cko2{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pituitary gonadotropins play a key role in follicular development and ovulation through the induction of specific genes. To identify these genes, we have constructed a genome-wide rat ovarian gene expression database (rOGED). The database was constructed from total RNA isolated from intact ovaries, granulosa cells, or residual ovarian tissues collected from immature pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin-treated rats at 0 h (no PMSG), 12 h, and 48 h post PMSG, as well as 6 and 12 h post human chorionic gonadotropin. The total RNA was used for DNA microarray analysis using Affymetrix Rat Expression Arrays 230A and 230B (Affymetrix, Santa Clara, CA). The microarray data were compiled and used for display of individual gene expression profiles through specially developed software. The final rOGED provides immediate analysis of temporal gene expression profiles for over 28,000 genes in intact ovaries, granulosa cells, and residual ovarian tissue during follicular growth and the preovulatory period. The accuracy of the rOGED was validated against the gene profiles for over 20 known genes. The utility of the rOGED was demonstrated by identifying six genes that have not been described in the rat periovulatory ovary. The mRNA expression patterns and cellular localization for each of these six genes (estrogen sulfotransferase, synaptosomal-associated protein 25 kDa, runt-related transcription factor, calgranulin B, {alpha}1-macroglobulin, and MAPK phosphotase-3) were confirmed by Northern blot analyses and in situ hybridization, respectively. The current findings demonstrate that the rOGED can be used as an instant reference for ovarian gene expression profiles, as well as a reliable resource for identifying important yet, to date, unknown ovarian genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MAMMALIAN OVARY, the intricate yet regulated actions of peptide and steroid hormones at the hypothalamic, pituitary, and ovarian levels are crucial for the sequential and successful processes of follicular growth, maturation, and ovulation. FSH plays a key role in follicular recruitment and development of preovulatory follicles (1, 2, 3). The LH surge then acts on these preovulatory follicles to initiate the ovulatory process by triggering a cascade of multiple cellular and molecular pathways (2, 4, 5). These gonadotropins regulate the ovarian expression pattern of a myriad of downstream hormone-responsive genes, resulting in the synthesis of various proteins and steroid hormones by granulosa and theca cells of the follicle, as well as other ovarian cells.

A major focus of ovarian research has been the identification of downstream hormone-responsive genes induced in response to FSH or LH to fully understand the mechanism(s) by which these genes orchestrate the processes of follicular development and ovulation. In the last decade, rapid progress has been made in discovering key players in these processes by using molecular techniques such as differential mRNA display (6, 7), subtraction hybridization (8, 9), ovarian cDNA library screening (10), and DNA microarray (11, 12, 13). Results from these pioneering studies have reinforced the belief that follicular development and ovulation are highly complex, tightly regulated processes that require numerous hormone-induced factors. The actions of these factors are ovarian cell-type specific, temporally precise, and in many cases, interwoven. Furthermore, it is unknown as to how many of these downstream hormone-responsive genes have yet to be identified and/or characterized.

In an effort to rapidly and easily identify factors induced by FSH during follicular growth or by LH during the process of follicular rupture in a temporal and cell-specific manner, we have used an emerging molecular technology, DNA microarray, coupled with a specialized software program to develop a new rat ovarian gene expression database (rOGED). This novel database allows investigation of over 28,000 genes detected by the Affymetrix DNA microarray analysis (Affymetrix, Santa Clara, CA) in whole ovarian tissue, granulosa cells, and residual ovarian tissue across the period of gonadotropin-induced follicular growth and ovulation.

In the present study, we demonstrate the ease and the power of gene identification with rOGED, confirm its utility and specificity by comparing genes with known expression profiles to genes identified in rOGED, and then demonstrate the potential of this ovarian gene database in the identification of several genes not yet characterized in the periovulatory rat ovary. By optimizing the process of data mining, rOGED will enhance the efficiency of research, thereby facilitating the identification and characterization of genes involved in follicular development and the ovulatory process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Molecular biological enzymes, pCRII-TOPO Vector and Trizol, were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA).

Animals and tissue collection
All animal procedures were approved by the University of Kentucky Animal Care and Use Committee. Immature female Sprague Dawley rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and were provided with water and rat chow ad libitum and maintained on a 14-h light, 10-h dark cycle. At 21 d of age, rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG) sc to stimulate and synchronize follicular growth. Forty-eight hours later, the rats were injected with 10 IU human chorionic gonadotropin (hCG) ip to induce ovulation.

For DNA microarray analyses, rats were killed at 0 h (pre-PMSG injection), 12 h, and 48 h after PMSG administration or 6 and 12 h after hCG injection. As previously described (8), ovaries were harvested in cold 4F culture media without serum (n = 5 animals/time point). One ovary from each of four animals was collected and stored at –80 C for later extraction of total RNA (four ovaries per time point; performed in duplicate). To isolate granulosa cells, ovaries (six ovaries per time point) were pooled and processed as described previously (8) with a slight modification. Briefly, granulosa cells were isolated by the method of follicular puncture. The cells were pooled and filtered using a cell strainer (40-µm pore size) to remove oocytes. The granulosa cells were collected from the flow-through by centrifugation at 500 x g for 5 min, snap frozen, and stored at –80 C for later extraction of total RNA. To collect residual ovarian cells, the ovaries remaining after granulosa cell collection were dissected with a razor blade, sharp forceps, and needles. The residual cells were further dispersed by repeatedly flushing through a Pasteur pipette and then filtered using a 70-µm cell strainer. The majority of granulosa cells, oocytes, and small follicles passed through the strainer. The residual nongranulosa/oocyte cells (residual ovarian tissue) were retrieved from the surface of the cell strainer and resuspended in cold 4F culture media without serum. The cells were collected by centrifugation at 500 x g for 5 min, snap frozen, and stored at –80 C for later extraction of total RNA. For each time point, the whole isolation process was completed in less than 1 h. Due to heterogeneity and complexity of the ovary, it is nearly impossible to isolate completely pure populations of specific ovarian cell types. Thus, there is likely to be minor contamination of other cell types in our samples of granulosa cells and residual ovarian tissue.

For Northern blot analyses and in situ hybridization, animals were killed at 0 h (pre-PMSG injection), 12 h, and 48 h after PMSG administration or 6, 12, and 24 h after hCG injection (n = 3–6 animals/time point). One ovary from each animal was snap frozen and stored at –80 C until extraction of total RNA for Northern blot analysis, and the other ovary was placed in OCT (VWR Scientific, Atlanta, GA) and stored at –80 C until sectioned and processed for in situ hybridization.

DNA microarray
Total RNA was extracted from immature untreated or gonadotropin-treated rat ovaries or ovarian cells using Trizol reagent and further purified using a RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. The integrity of total RNA was verified by visualizing the intact and distinct 28S rRNA and 18S rRNA bands stained with ethidium bromide in a 1.5% agarose-formaldehyde gel.

Five micrograms of total RNA were used as a template for cDNA synthesis, and biotinylated antisense cRNA probe was prepared as described by the manufacturers of the SuperScript System kit (Invitrogen) and the ENZO BioArray HighYield RNA labeling kit (Enzo Diagnostics, Farmingdale, NY). Unincorporated nucleotides were removed from the riboprobe preparation using the RNeasy Mini kit (Qiagen). The integrity of the riboprobe was checked by gel electrophoresis. The Affymetrix Rat 230A and 230B oligonucleotide array sets were hybridized, washed, and scanned using Affymetrix equipment and protocols (Affymetrix; DNA Microarray Core Facility, University of Kentucky, Lexington, KY). A total of 15,924 and 15,333 probe sets were included in the Affymetrix Rat 230A and 230B genechip array, respectively. These genechips contained probes for 28,757 well-substantiated rat genes. The DNA microarray assays were done in duplicate with total RNA from intact ovaries obtained at each time point, whereas the microarray assays were done once with total RNA from pooled granulosa cells and pooled residual ovarian cells.

Development of rOGED
After the DNA microarray analysis was complete, the total data were compiled as an Access Database file in Excel (Microsoft, Redmond, WA). To create a program that would display the mRNA expression profile for any gene of interest with simple input processes, we have developed a specialized computer software program, referred to as the Gene Expression Display Tool (GEDT). Visual Basic 6.0 (Microsoft) was used as the base programming tool to develop the GEDT. The GEDT retrieves data from the Access Database file as an interface program, allowing the user to input a request for data display. The GEDT then connects to the DNA microarray Access Database, dynamically finds requested data, and calculates and displays the data on a computer screen in planned formats to represent the quantitative and temporal changes in ovarian gene expression in the different ovarian compartments (e.g. intact ovaries, granulosa cells, and residual ovarian tissue). The organized structure of the GEDT connected to the Access Database is the rOGED.

cDNA cloning
Partial cDNAs corresponding to each of the reference sequences listed in Table 1Go were generated by RT-PCR. Briefly, total RNA (1 µg) isolated from rat preovulatory ovaries obtained at 6 h after hCG injection was reverse transcribed at 42 C for 1 h using SuperScript II (Invitrogen) and Oligo dT primers (Invitrogen). First-strand cDNA samples were amplified using oligonucleotide primer pairs designed using published sequence data (the primer sequence of each gene is listed in Table 1Go). Amplification consisted of a preincubation at 94 C for 5 min before adding Taq polymerase and then 35 cycles at 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. A PCR product of the predicted size was cloned into the pCRII-TOPO Vector (Invitrogen). DNA sequences of cloned rat partial cDNAs for the selected genes were determined commercially (MWG Biotech, Inc., High Point, NC).


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  		TABLE 1. cDNAs used for Northern blot analysis and in situ hybridization probes

 
Northern blot analyses
Northern blot analyses were carried out as described previously (14). Briefly, total RNA was extracted from ovaries using Trizol reagent according to the manufacturer’s protocol (Invitrogen) and quantified by spectrophotometry. Seven to ten micrograms of total RNA were separated by electrophoresis, capillary transferred to a nylon membrane (0.2-µm pore size, Nytran N; Schleicher & Schuell Inc., Keene, NH), and cross-linked to the membrane by baking in a vacuum oven at 80 C for 2 h. Plasmids containing rat cDNAs for selected genes and mouse cDNA for ribosomal protein L32 were linearized with appropriate restriction enzymes (i.e. BamHI, EcoRV, or EcoRI). Antisense riboprobes were transcribed using [{alpha}-32P]uridine triphosphate (10 mCi/ml; MP Biomedicals, Inc., Irvine, CA) and SP6 or T7 RNA polymerases (Ambion Inc., Austin, TX). The Northern membranes were hybridized with 1 x 106 cpm 32P-labeled antisense riboprobes for selected genes and L32 mRNA per milliliter of Ultrahyb hybridization buffer (Ambion Inc.) at 68 C for at least 16 h. Excess probe was removed by washing with a stringent buffer (0.1x standard saline solution, 0.1% sodium dodecyl sulfate) twice at 68 C for 1 h. The membrane was exposed to a phosphorimaging plate and quantified with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

In situ hybridization
Ovaries collected from immature gonadotropin-treated rats obtained at the selected time points described earlier were sectioned at 10 µm and mounted on Probe On Plus slides (Fisher Scientific, Pittsburgh, PA). In situ hybridization was carried out as described previously (15). Briefly, plasmids containing cDNA for selected genes were linearized with appropriate restriction enzymes to generate sense and antisense riboprobes. Linearized plasmids were labeled with [{alpha}-35S]uridine triphosphate (10 mCi/ml) and appropriate RNA polymerases. The sections were hybridized overnight with 1 x 106 cpm 35S-labeled riboprobes/slide in a humidified chamber at 55 C. The next day, the slides were washed and treated with RNase A (0.1 mg/ml; Amresco, Solon, OH) for 30 min at 45 C. Tissue sections were washed again at high stringency, dried, dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), and exposed at 4 C for 2–4 wk. To visualize the hybridized riboprobes, slides were developed with Kodak D19 and counterstained with hematoxylin solution. Tissues were examined with an Eclipse E800 Nikon microscope (Nikon Corp., Melville, NY) under bright- and dark-field optics. One ovary from each of three animals was used for in situ hybridization. At least 12 sections/ovary were analyzed for each antisense probe, making a total of at least 36 tissue sections analyzed for each time point. A sense riboprobe, used as a control for nonspecific binding, was included for each ovary and each time point.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Web version rOGED
After successfully constructing the database, we modified the original rOGED to develop a web-based version. In the web version of rOGED (http://web5.mccs.uky.edu/kolab/rogedendo.aspx), a user can search the database by entering a search phrase (e.g. gene names, GenBank identification numbers, or any word related to the genes of interest) into a textbox (Fig. 1AGo) and clicking the Search button. A separate dropdown box is provided so that one can select the Affymetrix probe identification number (Fig. 1BGo) instead of typing search phrases. Then, the rOGED searches gene descriptions for all possible matches, calculates the range of expression, and displays gene expression profiles in two formats, a Detection Call table (Fig. 1DGo) and a Gene Expression graph (Fig. 1FGo). The rOGED, by default, displays the data of the first match to the screen but also displays all of the other matches on the screen (Fig. 1CGo). From the list, the user can select any gene of interest, and then the ovarian expression profile of that gene is displayed. The Detection Call table provides a statistical evaluation of whether a transcript is reliably detected (Present), not detected (Absent), or detected but not reliably (Marginal), which is determined by a mathematical algorithm inherent to the Affymerix DNA microarray system. The Gene Expression graph depicts the temporal and quantitative expression pattern of a gene using raw data obtained from DNA microarray analysis. The data are displayed as the range of signal intensities (y-axis) detected against the various time points after hormonal treatment (x-axis). Three different-colored lines represent the three different sources of RNA (intact ovary, red; granulosa cells, blue; and residual ovarian tissue, green). The averages and SD of mRNA expression levels for each gene from intact ovarian samples (n = 2/time point) are provided as a table (E). Links to other online databases, such as National Center for Biotechnology Information-PubMed, National Center for Biotechnology Information-Nucleotide, Gene Expression Atlas, Ovarian Kaleiodoscope, NCBI-Serial Analysis of Gene Expression (SAGE), Affymetrix, and GeneCards, are made available to assist viewers who wish to explore known and/or potential functions for these genes in the ovary.



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  		FIG. 1. Web-accessible version of rOGED. The rOGED can be accessed at the following web site, http://web5.mccs.uky.edu/kolab/rogedendo.aspx. A, A textbox for the input of search phrases. B, A dropdown box for Affymetrix probe identification (ID) number selection. C, Display of alternative matches. D, A Detection Call table. E, A table showing averages and SDs for mRNA expression levels detected in intact ovaries. F, A graph of quantitative Gene Expression. A description of the selected gene is displayed in the Gene Description section of the rOGED screen, which provides GenBank ID numbers, gene names, and alternative names of the genes.

 
Comparison of rOGED profiles with the expression profile of known genes in the ovarian tissue
To evaluate the reliability of our database, we compared the expression profiles of over 20 known genes with those generated by our database. These genes that have been reported to have distinct expression patterns during follicular growth and/or the preovulatory period in gonadotropin-primed immature rat ovaries are listed in Table 2Go. The spatial and/or temporal expression pattern of mRNA for each of these genes is similar to the gene expression profile observed in our database, although fold changes in the levels of mRNA for each gene after gonadotropin treatment seen in our database may not be equivalent to values previously reported. This discordance is likely due to the fact that these values were obtained using different methodologies, such as DNA microarray analysis vs. Northern blot analysis or RT-PCR, or obtained from slightly different animal models (e.g. gonadotropin dose or time of hormone treatment). The representative mRNA profiles for four genes known to be selectively expressed in granulosa and/or theca cells after gonadotropin treatments are presented in Table 3Go and compared with those in rOGED (Fig. 2Go). For example, it is well known that LH receptor mRNA is dramatically increased in both granulosa and theca cells during follicular development but decreased sharply after hCG injection (16). Progesterone receptor mRNA has been reported to be induced exclusively in granulosa cells of preovulatory follicles after hCG injection (17). In contrast, previous studies documented that 17{alpha}-hydroxylase mRNA expression was restricted to theca cells and increased after PMSG injection to stimulate follicular development but then decreased after hCG injection (18). The expression profile for steroidogenic acute regulatory protein mRNA is unique in that there is an initial increase in mRNA levels in the theca layer during follicular growth, which is followed by induction of mRNA in granulosa cells by hCG administration (18, 19). The known profiles of these genes (TableGo 3) correspond well with the depicted graph of each gene in rOGED (Fig. 2Go).


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  		TABLE 2. Gene expression profiles identified by rOGED used for comparison with previously reported genes

 

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  		TABLE 3. Semiquantitative and temporal mRNA expression for each of four known genes after PMSG and/or hCG injection based on previously published data

 


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  		FIG. 2. Comparison of known gene profiles with rOGED profiles. Expression profiles of mRNA for the same four known genes in Table 3Go (LHR, PR, CYP17, and StAR) as generated by rOGED. The x-axis represents the time points of PMSG and hCG administrations as indicated. Each line in the graph represents ovary (red), granulosa cell (blue), or residual ovarian tissues (green). LHR, LH receptor; PR, progesterone receptor; CYP17, 17{alpha}-hydroxylase; StAR, steroidogenic acute regulatory protein.

 
Identification of new genes using rOGED
rOGED revealed numerous genes that display a dynamic pattern of expression but that have not yet been characterized in gonadotropin-treated immature rat ovaries. To test whether rOGED can be used as a tool to identify important genes in the ovulatory process, we selected several genes that displayed a transiently increased expression during the preovulatory period in the rOGED (Fig. 3Go). We have selected these genes based on their relatively high detection levels, transient expression pattern, and tissue-specific distribution in rOGED. The random selection of these genes was to validate the reliability of rOGED. The expression profile displayed by rOGED (Fig. 3Go) was compared with the temporal and spatial pattern of expression for these genes by Northern blot analysis (Fig. 4Go) and in situ hybridization (Fig. 5Go), respectively, and is described below.



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  		FIG. 3. mRNA expression profiles generated by rOGED. rOGED was used to generate mRNA expression profiles for STE (A), {alpha}1-macroglobulin (B), Runx 1 (C), Snap 25 (D), amphiregulin (E), calgranulin B (F), MKP-3 (G), and ribosomal protein L31. The y-axis represents the range of signal intensity detected by DNA microarray. The x-axis represents the time points of PMSG and hCG administrations as indicated. Each line in the graph represents ovary (red), granulosa cell (blue), or residual ovarian tissues (green).

 


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  		FIG. 4. mRNA expression profiles detected by Northern blot analysis. Northern blots show the expression profiles of transcripts for STE (A), {alpha}1-macroglobulin (B), Runx 1 (C), Snap 25 (D), amphiregulin (E), calgranulin B (F), MKP-3 (G), and ribosomal protein L32 in gonadotropin-primed immature ovaries collected from untreated rats (at the time of PMSG injection; 0 h), 12 or 48 h after PMSG injection, or 6, 12, or 24 h after hCG administration as indicated.

 


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  		FIG. 5. Cellular localization pattern of mRNA detected by in situ hybridization. In situ hybridization slides demonstrate the localization pattern of mRNA for STE (A), {alpha}1-macroglobulin (B), Runx 1 (C), Snap 25 (D), amphiregulin (E), calgranulin B (F), and MKP-3 (G) in serial tissue sections of PMSG-primed immature rat ovaries collected before or 6, 12, or 24 h after hCG administration as indicated. Representative panels for bright-field and dark-field (A–G) photomicrographs are depicted. Original magnification of all slides, x30. PF, Preovulatory follicles; CL, newly forming corpora lutea.

 
Estrogen sulfotransferase (STE)
STE is an enzyme that can sulfonate estrogen to biologically inactive estrogen sulfates, thus playing an important role in regulating the in situ estrogen activity (20, 21). STE mRNA expression in rOGED (Fig. 3AGo) is transiently up-regulated at 6 h after hCG administration. Northern blot analysis (Fig. 4AGo) confirmed the temporal expression pattern of STE mRNA during the preovulatory period. In situ hybridization analysis further verified this expression pattern by showing the high levels of STE mRNA exclusively in granulosa cells of preovulatory follicles (Fig. 5AGo).

{alpha}1-Macroglobulin
{alpha}1-Macroglobulin belongs to a family of high-molecular weight plasma globulins and has been known to inhibit a broad range of proteinases (22). In rOGED, {alpha}1-macroglobulin mRNA expression (Fig. 3BGo) is transiently up-regulated at 6 h post hCG and, unlike the majority of selected genes, its expression is confined to residual ovarian cells. Northern blot analysis confirmed the transient increase in levels of {alpha}1-macroglobulin mRNA at 6 h after hCG administration (Fig. 4BGo). Also, in situ hybridization showed the expression of {alpha}1-macroglobulin mRNA exclusively in the theca-interstitial layer of preovulatory ovaries (Fig. 5BGo).

Runt-related transcription factor 1 (Runx 1; or acute myeloid leukemia 1)
The Runx 1 gene encodes the {alpha}-subunit of a heterodimeric transcription factor, core binding factor (CBF) or polyomavirus enhancer binding protein 2 (23), which has been shown to regulate cellular proliferation and differentiation (24). rOGED indicated that Runx 1 mRNA expression is up-regulated at 6 h after hCG injection (Fig. 3CGo). Northern blot analysis confirmed the increases in levels of ovarian Runx 1 mRNA at 6 h after hCG administration, and the levels rapidly declined between 12 and 24 h post hCG (Fig. 4CGo). Moreover, the strong hybridization signal for Runx 1 mRNA was localized to granulosa and theca cell layers of preovulatory follicles at 6 h after hCG administration, and by 12 h after hCG, the expression was predominantly confined to the granulosa cells of periovulatory follicles (Fig. 5CGo).

Synaptosome-associated protein 25 kDa (Snap 25)
Snap 25 is a key component of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are required for intracellular membrane fusion events during exocytosis of vesicles (25). rOGED indicated that Snap 25 mRNA expression increases in granulosa cells at 6 h post hCG but rapidly declines by 12 h after hCG administration (Fig. 3DGo). Unexpectedly, Northern blot analysis revealed a marked increase in levels of Snap 25 mRNA expression at 12 h after hCG administration (Fig. 4DGo). Furthermore, high expression of Snap 25 mRNA was localized to both granulosa cells of periovulatory follicles and the theca-interstitial layer at 12 h after hCG (Fig. 5DGo). This discrepancy in the spatiotemporal expression of Snap 25 is due to two isoforms of this gene and is discussed in detail in the Discussion.

Amphiregulin
Amphiregulin is a member of the epidermal growth factor (EGF) family that has been shown to be involved in normal tissue development (26), as well as tumor growth (27). At the time of the selection of this gene for further characterization, the expression pattern of amphiregulin in the ovary had not been characterized. However, a recent study by Park et al. (28) has demonstrated rapid and transient induction of three members of the EGF family, amphiregulin, epiregulin, and ß-cellulin, in mural granulosa cells of preovulatory follicles in PMSG-primed immature mouse ovaries after hCG injection. Similar to the expression pattern seen in the mouse ovary, rOGED showed the profile of transient expression of amphiregulin mRNA in PMSG-primed immature rat ovaries after hCG treatment (Fig. 3EGo). Northern blot analysis validated this transient expression profile of amphiregulin mRNA (Fig. 4EGo). Furthermore, the present data from in situ hybridization (Fig. 5EGo) revealed the highest level of amphiregulin mRNA accumulation in granulosa cells of preovulatory follicles at 6 h and, to a lesser extent, in cells scattered in the stroma layer at 6 and 12 h after hCG injection in the rat ovary.

Calgranulin B (S100A9)
Calgranulin B forms a heterodimer with calgranulin A (S100A8) in the presence of calcium (29). This heterodimeric complex, also known as calprotectin or calgranulin, has shown to be involved in inflammatory processes (30). The expression of calgranulin B mRNA was described by rOGED to be up-regulated in both granulosa cells and residual ovarian cells at 6 h after hCG injection and then to sharply decline by 12 h post hCG (Fig. 3FGo). The temporal changes in levels of ovarian calgranulin B mRNA as determined by Northern blot analysis (Fig. 4FGo) closely resembled the profile of rOGED. However, in situ hybridization revealed that calgranulin B mRNA was localized to cells scattered in the interstitial layer and stroma of preovulatory ovaries obtained at 6 and 12 h after hCG administration but not to follicular cells (Fig. 5FGo). The possible explanation for this discrepancy between the profile observed in rOGED and the in situ hybridization data is outlined in the Discussion.

MAPK phosphotase-3 (MKP-3)
MKP-3 is a dual-specificity phosphatase that selectively inactivates one subfamily of MAPK, the ERKs (31, 32), and thus suppresses the activation state of MAPK. In rOGED, MPK-3 mRNA expression is up-regulated in both granulosa cells and residual ovarian cells at 6 h after hCG injection (Fig. 3GGo). This expression pattern was confirmed by Northern blot analysis (Fig. 4GGo) and in situ hybridization (Fig. 5GGo). Unlike the other selected genes, moderate levels of MKP-3 mRNA were detected in both immature untreated or PMSG-treated rat ovaries before hCG injection by Northern blot analysis (Fig. 4GGo), which could be due to the expression of MPK-3 mRNA in oocytes detected by in situ hybridization (Fig. 5GGo). In addition, MKP-3 mRNA was also localized to newly forming corpora lutea.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rOGED reveals in vivo ovarian gene expression profiles across the rat genome in a dynamic and quantitative manner at a cell and tissue level during follicular growth and ovulation using a well-established gonadotropin-treated rat model. In developing rOGED, it was critical to ensure that the RNA from the isolated primary cells accurately reflected the in vivo status of mRNA expression, in that specific cell types should be isolated without substantial changes in mRNA content. For example, a significant change in cellular RNA content could occur from either the degradation of existing RNA or the induction of gene expression during the granulosa and residual tissue isolation and collection period. In the present study, the whole cell/tissue isolation procedure at each time point was completed in less than 1 h, minimizing these potential problems. A second important consideration in the construction of rOGED is potential cross-contamination of different types of cells present in the ovary during the granulosa or residual nongranulosa/oocyte cell (residual ovarian tissue) isolation process. It is nearly impossible to isolate completely pure populations of specific ovarian cell types within a limited time period; therefore, the possibility exists for the cross-contamination of different cell types in our granulosa cells and residual ovarian tissue samples. However, to minimize this cross-contamination, we used multiple cell sorting techniques that allowed us to isolate relatively pure populations of granulosa cells and residual ovarian tissue. Furthermore, in the current study, we compared granulosa cell and residual tissue gene expression profiles seen in rOGED with in situ hybridization images of well-characterized ovarian genes that display cell-type specific mRNA expression patterns. For instance, expression of mRNA for aromatase and progesterone receptor is confined to granulosa cells of growing follicles (33) and granulosa cells of preovulatory follicles (17), respectively, which is in agreement with rOGED profiles of mRNA for these genes. Previously, a disintegrin and metalloproteinase with thrombospondin motifs was localized to granulosa cells of periovulatory follicles and the theca-interstitial layer of periovulatory ovaries (34). Our rOGED profile for a disintegrin and metalloproteinase with thrombospondin motifs-1 mRNA is virtually identical to the previously reported in situ hybridization expression pattern. Moreover, the cellular localization pattern of mRNA for STE, {alpha}1-macroglobulin, Runx 1, amphiregulin, and MKP-3 determined by the present in situ hybridization analysis correlated well with the gene expression profiles by rOGED, confirming the efficiency of our cell/tissue isolation procedure and thus the accuracy of the rOGED’s in vivo gene expression pattern.

Using rOGED, we found that the expression patterns of hundreds of ovarian genes are changed after PMSG and/or hCG injection. The induction and/or alteration of the expression patterns of these gonadotropin-induced genes may be crucial for successful follicular development, maturation, and ovulation in the rat ovary. Previously, similar approaches using DNA microarrays have identified many genes that are regulated after PMSG and/or hCG treatment, resulting in the elucidation of key players involved in follicular development (11) and the ovulatory process (12). However, the current study has built upon the technological foundation of DNA microarrays to construct a database that allows rapid and simple retrieval of any of the over 28,000 genes that may potentially be expressed in the rat ovary. The expression pattern of these genes can be displayed in a temporal and cell type-specific (granulosa cells vs. residual ovarian cells) manner during follicular growth and ovulation in immature, gonadotropin-treated rats.

In the present study, the utility of rOGED was demonstrated by selecting several genes that are dramatically and transiently increased after hCG injection in rOGED but that had not, to date, been reported in the rat periovulatory ovary. We hypothesized that the genes induced by hCG that were identified by rOGED and confirmed by Northern blot and in situ hybridization may have significant functions during follicular maturation and the ovulatory process in the rat ovary. Each of the potential roles for these genes is discussed below.

STE can regulate in situ estrogen activity by sulfonating estrogen to an inactive form (20, 21). In the ovary, developing preovulatory follicles synthesize copious amounts of estradiol, which is essential for inducing the LH surge from the anterior pituitary. After the LH/FSH surge, follicular estradiol decreases dramatically. So far, little is known about the mechanism of rapid clearance of follicular estradiol. The current findings show that STE mRNA is transiently up-regulated in granulosa cells of preovulatory follicles after hCG administration, suggesting that the up-regulation of STE expression may ensure the rapid decline in bioactive estrogen, thereby protecting ovulatory follicles from the undesirable effect of residual estrogen.

{alpha}-Macroglobulins inhibit a broad spectrum of proteinases by functioning as large molecule traps (22, 35). Upon binding to its specific membrane receptor, the {alpha}-macroglobulinproteinases complex undergoes endocytosis, which permits rapid clearance of proteinases from the circulation or extracellular space (35). The rat has three related macroglobulins, {alpha}1-macroglobulin, {alpha}2-macroglobulin, and {alpha}1-inhibitor 3. Both the {alpha}1- and {alpha}2-macroglobulins are structurally homologous to human {alpha}2-macroglobulin (22), but they have a few critical differences (36). For example, rat {alpha}2-macroglobulin has been known as an acute-phase protein because, under normal conditions, its levels in the plasma are low (<0.01 mg/ml in plasma) but increase during inflammation, whereas {alpha}1-macroglobulin remains relatively constant at high concentrations in the plasma (>2–4 mg/ml) (22, 37, 38). In the rat ovary, {alpha}2-macroglobulin has been shown to be selectively induced in luteinizing granulosa cells of periovulatory follicles and in corpora lutea by LH and prolactin (39). Interestingly, another study demonstrated that the concentrations of ovarian {alpha}-macroglobulins increase dramatically by 4 h after ovulatory hCG injection to PMSG-primed immature rats (40). Because the expression of {alpha}2-macroglobulin mRNA was only detectable in periovulatory follicles at 12 h post hCG (39), these authors suggested a vascular origin of {alpha}-macroglobulins in the rat ovarian tissue during the early period of the ovulatory process. The present data revealed, for the first time, the transient induction of {alpha}1-macroglobulin mRNA expression in the theca-interstitial layer of preovulatory ovaries. Therefore, this up-regulation of {alpha}1-macroglobulin mRNA by 6 h post hCG may, in part, account for the increase in ovarian {alpha}-macroglobulins during the early ovulatory period. Furthermore, the current findings suggest that {alpha}1-macroglobulin may be involved in tissue remodeling processes of the theca-interstitial layer by regulating the activity of proteinases that escalate during the ovulatory process to bring about rupture of the follicle wall.

The Runx 1 gene encodes the DNA binding subunit of heterodimeric transcription factor, polyomavirus enhancer binding protein 2/CBF (23). Runx 1, together with its partner CBFß, has been shown to play a critical role in hematopoietic cell differentiation and proliferation, and its functional dysregulation leads to leukemia (reviewed in Ref. 41). It is also reported that Runx 1 is involved in T-cell differentiation (42), nerve-cell innervation (43), and fibroblast transformation (44). The current study revealed the transient up-regulation of Runx 1 mRNA expression in granulosa and theca-interstitial cells of preovulatory follicles after hCG injection. Interestingly, a recent study has demonstrated that in hepatic stellate cells, Runx 1 can regulate transcription of tissue inhibitor of metalloproteinases 1 (TIMP-1) (42). In the ovary, TIMP-1 mRNA expression dramatically increased in ovulating follicles, which transform into corpora lutea (45). Considering the rapid changes in follicular cell characteristics from proliferating to differentiating cell types after the LH surge, Runx 1 may play an important role in the differentiation of follicular cells to luteal cells via regulating the transcription of the specific genes, such as TIMP-1.

Snap 25 has been identified as a key component of the SNARE complex (25). The assembly of the SNARE core complex, which comprises Snap 25, syntaxin, and vesicle-associated membrane proteins/synaptobrevin, is necessary for synaptic vesicle exocytosis in neurons and for intracellular membrane fusion in nonneuronal secretory cells (46). Recently, Grosse et al. (47) have reported the presence of Snap 25 in follicular cells and oocytes of adult rat ovaries. However, nothing is known about the expression of the Snap 25 gene during the ovulatory period. In the present study, the rOGED documented that Snap 25 mRNA was up-regulated at 6 h after hCG injection in granulosa cells of periovulatory follicles. Yet, the present Northern blot data indicated that the levels of Snap 25 mRNA were highest at 12 h after hCG. Based on the intensity of the in situ hybridization signal, high expression of Snap 25 mRNA was localized to both granulosa cells of periovulatory follicles and theca-interstitial layers. Through sequence analysis, we found that there are two isoforms of Snap 25, Snap 25a and Snap 25b. Also, results from the BLAST search indicated that the partial cDNA probe for Snap 25 used for the present study perfectly matched Snap 25a (GenBank accession no. U56261), whereas only 75% of the probe sequence matched Snap 25b (GenBank accession no. AB003992). Moreover, the oligonucleotide primers for Snap 25 used for the Affymetrix microarray assay were designed based on the sequence for Snap 25b mRNA (GenBank accession no. NM_030991). Therefore, the Snap 25 mRNA expression detected by Northern blot analysis and in situ hybridization is for Snap 25a, whereas rOGED showed the expression profile of Snap 25b. This apparent discrepancy in the time course appearance of Snap 25 gene expression between the rOGED and the Northern blot data, therefore, is easily accounted for by the two different isoforms present in the ovary. Nevertheless, the present findings suggest that both isoforms of Snap 25 are transiently up-regulated in distinct temporal patterns in response to hCG injection. Given the fact that ovulatory follicles secrete a wide variety of factors that are involved in the ovulatory process and/or corpora lutea formation and that this secretory process involves intracellular membrane fusion and vesicle exocytosis (25, 48), the up-regulation of Snap 25 expression may be important for facilitating the timely release of these factors.

Amphiregulin is a member of the EGF/TGF-{alpha} family that can activate the EGF receptor (49). A recent report by Park et al. (28) has documented the sequential and transient induction of mRNA for the EGF family members, amphiregulin, epiregulin, and ß-cellulin, in the mouse ovary. In the rat ovary, epiregulin mRNA has been reported to be transiently induced in granulosa cells of preovulatory follicles after hCG injection (6). In rOGED, levels of mRNA for both amphiregulin and epiregulin were transiently increased between the time of hCG injection and ovulation, but the rapid induction of ß-cellulin mRNA expression was not observed in response to hCG injection. Moreover, the present study showed that, in rat preovulatory ovaries, amphiregulin expression was high at 6 h after hCG injection, whereas in the mouse ovary, amphiregulin mRNA expression was highest at 3 h and then became undetectable by 6 h post hCG (28). Another difference between these two species revealed by the present in situ hybridization data is that, in the rat ovary, amphiregulin mRNA was localized to granulosa cells of preovulatory follicles as well as to the interstitial layer, whereas the expression was restricted to mural granulosa cells of preovulatory follicles in the mouse ovary (28). Taken together, these findings implicate conserved roles of EGF family members in the ovary but also show divergence between two close species, although whether these differences would have any functional consequence needs to be further determined. Nonetheless, in the mouse ovary, amphiregulin has been shown to promote resumption of oocyte meiosis and cumulus expansion partially via inducing Ptgs2, Tnfaip6, and Has2 mRNA (28). Amphiregulin was also found to be one of the progesterone-regulated genes in the mouse uterus (50) and increased urokinase-type plasminogen activator in transformed breast epithelial cells (51). Collectively, the present data are suggestive of a crucial role(s) for amphiregulin in the final stages of oocyte maturation and ovulation.

Calgranulin B is a low-molecular weight acidic protein that interacts with calgranulin A to form a heterodimer in the presence of calcium (29). This heterodimer complex is highly expressed in neutrophils and monocytes that are found in a variety of inflammatory conditions, thus suggesting that this protein is an important player in inflammatory processes (30). Furthermore, calgranulins have been shown to function as regulatory molecules in adhesion and migration of leukocytes to the sites of inflammation (52, 53). In rOGED, calgranulin B was observed as being transiently up-regulated in both granulosa and residual ovarian cells at 6 h after hCG injection. Unexpectedly, the current in situ hybridization data revealed that calgranulin B mRNA is expressed predominately in cells scattered in the interstitial and stroma layer of preovulatory ovaries. Considering the irregular cellular localization pattern of calgranulin B mRNA expression (e.g. a lack of distinct association with the theca), we speculate that these cells may be leukocytes. Furthermore, an increase in macrophages and neutrophilic granulocytes in the medullary region and in the thecal layer of the ovary around the time of ovulation has been reported (54). Thus, it is possible that these leukocytes are associated with the vasculature and, during the granulosa cell isolation procedure, may be distributed into the granulosa and residual ovarian cell populations, resulting in the expression of calgranulin B mRNA in granulosa cells observed with rOGED. However, the present finding of up-regulation of calgranulin B expression in preovulatory ovaries suggests a role for this protein in leukocyte trafficking during the inflammatory responses of ovulation.

MKPs are dual-specificity phosphatases that are capable of dephosphorylating both Tyr(P) and Thr(P) in the MAPKs (55). MKP-3 was found to selectively inactivate one subfamily of MAPK, the ERKs (31, 32), and thus plays a specific role in regulating the activation state of MAPKs. In the ovary, both LH and FSH have been shown to induce a rapid increase in ERK tyrosine phosphorylation (56, 57, 58). The gonadotropin-induced phosphorylation of ERK was detected as early as 1 min after stimulation, but this activation was only maintained for up to 30 min, and then it started to decline after 30–60 min in cultured granulosa cells (57, 58). Similar to the results from in vitro studies, Salvador et al. (57) have shown that the phosphorylation of ERK was clearly observed in whole ovarian extracts obtained at 1 h after hCG injection to PMSG-primed immature rats but was barely detectable by 8 h post hCG, although the phosphorylation of the kinase upstream of ERK, MAPK kinase, appeared to be increased. These findings imply the presence of the tight regulation of activation/deactivation of ERK signaling pathways in the ovary. In the present study, we found that MKP-3 mRNA is up-regulated in granulosa cells of preovulatory follicles and theca-interstitial layers after hCG injection, suggesting that up-regulation of MKP-3 may be important for regulating intracellular signaling pathways, specifically ERKs, that are activated by the LH surge.

The present results of identifying relatively unknown genes demonstrated the utility of rOGED in the identification of genes that are regulated by hormones and involved in the ovulatory process. Yet, it remains to be determined whether the changes observed in rOGED are equally reflected at the levels of protein or their activity. Furthermore, it needs to be verified whether such changes in gene expression pattern observed in the rat ovary using the PMSG/hCG-induced model also occur in the ovaries of naturally cycling animals.

In summary, the rOGED has multiple applications. It is an effective tool to identify novel and/or key factors involved in follicular development and ovulation. In addition, rOGED can be used as a web-based source or reference, which will provide valuable information when constructing new hypotheses, designing experiments, and evaluating experimental results. The development of rOGED is a novel approach to display the DNA microarray data in such a manner that the use of this web-based database will have broad applications to further our understanding of numerous aspects of ovarian physiology.


   Acknowledgments
 
The authors thank Drs. O. K. Park-Sarge for providing plasmids containing mouse cDNAs for L32 and A. C. McDonnel for critical reading of the manuscript.


   Footnotes
 
This work was supported by Grant NIH P20 RR15592 from the National Institutes of Health, Grant NIA AG17164 from the National Institute on Aging, and the University of Kentucky Microarray Facility Pilot Award, and start-up fund for CheMyong Ko.

Abbreviations: CBF, Core binding factor; EGF, epidermal growth factor; GEDT, Gene Expression Display Tool; hCG, human chorionic gonadotropin; MKP-3, MAPK phosphotase-3; PMSG, pregnant mare serum gonadotropin; rOGED, rat ovarian gene expression database; Runx 1, runt-related transcription factor 1; Snap 25, synaptosome-associated protein 25 kDa; STE, estrogen sulfotransferase; TIMP-1, tissue inhibitor of metalloproteinases 1.

Received March 30, 2004.

Accepted for publication July 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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