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Physiol. Genomics 27: 318-327, 2006. First published August 15, 2006; doi:10.1152/physiolgenomics.00309.2005
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Received 15 December 2005; accepted in final form 3 August 2006.
Physiological Genomics 27:318-327 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression

K. F. Rodriguez1, L. A. Blomberg2, K. A. Zuelke2, J. R. Miles1, J. E. Alexander1 and C. E. Farin1

1 Department of Animal Science, North Carolina State University, Raleigh, North Carolina
2 United States Department of Agriculture-Agricultural Research Service Biotechnology and Germplasm Laboratory, Beltsville, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In cultured cumulus oocyte complexes (COC), FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and to identify candidate mRNAs involved. Experiment I: murine COC were cultured 4 h in the presence of FSH with 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82 ± 7%). When DRB was added at 0, 5, or 10 min after culture initiation, oocyte maturation was blocked (17 ± 7, 14 ± 6, and 21 ± 6% GVBD, respectively). When DRB was added after 15, 20, or 30 min, progressively more COC underwent GVBD (37 ± 6, 39 ± 6, and 66 ± 6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P ≤ 0.01 and P ≤ 0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development, and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE MAMMALIAN OOCYTE BECOMES arrested in prophase I of meiosis during embryonic life or near birth. Resumption of meiosis must occur for the oocyte to support subsequent postfertilization development. This process can occur either in vivo in response to the gonadotropin surge (59) or in vitro in response to removal of the oocyte from the inhibitory follicular environment (43). In vitro, the oocyte will resume maturation spontaneously if cultured in the absence of gonadotropins. Maturation will also occur if cumulus oocyte complexes (COC) are cultured in the presence of gonadotropins, but the kinetics of maturation for these COC differ from kinetics observed for oocytes undergoing spontaneous maturation. When gonadotropins were included in the culture medium, germinal vesicle breakdown (GVBD) was initially inhibited in murine (8, 11, 51) and bovine (15) COC. This transient inhibition was followed by acceleration of the rate of GVBD compared with that seen during spontaneous maturation (8, 15). Gonadotropin-induced resumption of meiosis in vitro requires an initial transcriptional event in the mouse (47), pig (35), sheep (40), and cow (15, 26). When COC are cultured with specific inhibitors of RNA polymerase II, such as {alpha}-amanitin or 5,6-dicholoro-1-ß-D-ribofuranosylbenzimidazole (DRB), FSH-induced but not LH-induced oocyte maturation is blocked, and oocytes remain at the germinal vesicle (GV) stage. These inhibitors are effective only in intact COC, suggesting that the required gene transcription occurs in the cumulus cell compartment (15, 46, 47). The identities of genes whose expression is required for FSH-induced oocyte maturation are not known. Because in vitro oocyte maturation is utilized with increasing frequency in human assisted reproduction laboratories (5, 31) and with high frequency in embryo production systems for animals, a clearer understanding of the mechanisms that control the maturation of oocytes under in vitro conditions is needed.

Serial analysis of gene expression (SAGE) is a powerful technique that allows for the analysis of gene expression profiles at specific physiological stages without prior knowledge of the genes involved (61). SAGE relies on the generation of tags that correspond to specific mRNA transcripts. The major premise of SAGE is that every individual mRNA transcript in a sample has an equal chance of being counted as a tag in a SAGE library made from that sample. Thus the number of times a unique tag is repeated in a sample corresponds to the level of expression of that transcript (61). Databases, such as the National Center for Biotechnology Information (NCBI) SAGE Map, are then utilized for tag-to-gene identification. SAGE has been applied successfully in the quantitative and qualitative analysis of gene expression in a variety of different model systems (2, 3, 25, 34, 60). Once created, SAGE libraries can be added to public repositories for future reference (30).

A number of approaches are available for gene expression profiling. Of these, only microarray, oligonucleotide array, and SAGE methodologies offer rapid, cost-efficient analyses of large numbers of genes. cDNA microarrays offer the ability to examine 3,000–6,000 genes simultaneously (19, 36), whereas oligonucleotide arrays (GeneChip, Affymetrix) allow for the simultaneous analysis of >39,000 known genes using pairs of microchip-based arrays (19, 63, 64). These two approaches utilize nucleic acid hybridization strategies that, when used with appropriate controls, can be quantitative. Quantification of micro/oligonucleotide arrays is typically relative and thus can vary significantly between experiments both within and between laboratories (57). Outcomes of analyses using micro/oligonucleotide array techniques are limited to those genes or expressed sequence tags (ESTs) represented on the array. In comparison, the use of SAGE for analysis of global mRNA expression offers four distinct attributes that make these techniques particularly advantageous for gene discovery. These include 1) the capacity for high-throughput, cost-efficient analysis with data generation in digital format; 2) generation of quantitative comparisons independent of the use of protocols involving nucleic acid hybridization; 3) quantitative assessments independent of original mRNA sequence knowledge (open investigative system); and 4) creation of a permanent DNA sequence-based data set (49, 61, 63, 64). Because SAGE libraries provide absolute transcript numbers in a digital format, they can be used to provide statistical comparisons of data not only within and between laboratories but also across time (29, 49).

In general, direct comparisons of the quantitative accuracy of transcript profiling using the oligonucleotide array (GeneChip) hybridization and SAGE have correlated well (18, 23). However, discrepancies between the two approaches occurred when known (or rare) genes expressed at low levels were detected only using SAGE (23, 56) and when unknown genes were identified using SAGE and were not present on the oligoarray chip (18, 23). Thus SAGE would be considered the method of choice for experiments involving gene discovery, particularly in species that have full genomic sequence and gene annotations available.

The objectives of this study were 1) to determine the time of occurrence of the transcriptional event required for resumption of meiosis in cultured murine COC and 2) to identify potential candidate mRNAs associated with meiotic resumption during FSH-induced maturation in cultured murine COC. For this purpose, we have utilized SAGE to compare gene expression profiles of murine COC cultured in the presence or absence of the transcriptional inhibitor DRB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
COC Isolation and Culture
Isolation of mouse COC was described previously (47). Briefly, prepubertal Swiss Albino mice (20–23 days old; Taconic, Germantown, NY) were maintained on a 12:12-h light-dark cycle with access to food and water ad libitum. For each experimental replication, 15 female mice were given an intraperitoneal injection of 5 IU pregnant mare's serum gonadotropin (PMSG). Forty-eight hours later, ovaries were removed from the body cavity and placed into Waymouth medium supplemented with 5% fetal bovine serum, 0.23 mM pyruvate, 7 mM taurine, 50 mg/l streptomycin sulfate, 75 mg/l penicillin G, and 5 µg/ml FSH (maturation medium) where they were extracted from the bursa and cleaned of adhering fat. After preparation, ovaries were randomly distributed to dishes containing maturation medium containing the appropriate treatments. For experiments using DRB, a 20-mg/ml stock solution was prepared in DMSO and diluted in culture medium (1.92% vol/vol) to result in a final concentration of 120 µM. This dose of DRB has been shown previously to be effective in maintaining cultured murine COC in meiotic arrest (47). Antral follicles on each ovary were punctured with a sterile needle, and excellent quality COC displaying multiple layers of cumulus cells were collected. On average, 14 ± 0.6 excellent quality COC were collected from each mouse. Once collected, COC were washed once in fresh treatment medium and then cultured in multiwell dishes containing 0.5 ml of treatment medium. All cultures were conducted at 37°C in an atmosphere of 5% CO2 in air with 100% humidity (47). Unless otherwise noted, all reagents were purchased from Sigma Chemical, St. Louis, MO. All procedures involving animals were carried out in accordance with National Institutes of Health guidelines for the care and use of laboratory animals under the approval of the North Carolina State University Institutional Animal Care and Use Committee.

Experiment I: Time of Transcriptional Event Required for GVBD
This experiment was designed to determine the approximate time of occurrence of the transcriptional event required for GVBD. Murine COC were recovered, distributed to control (0-D) or DRB-containing maturation medium (0+D), and cultured as described above. In addition, separate groups of COC cultured in control maturation medium were assigned to treatments in which DRB was added at either 5, 10, 15, 20, 30, or 40 min after the initiation of culture (n = 18 ± 4 COC per treatment per replicate; 4 replicates). After 4 h of culture, all COC were denuded of their cumulus cells by manual pipetting and visually assessed for the resumption of meiosis using a stereomicroscope with a magnification of x60.

Experiment II: Identification of mRNAs Associated with GVBD Using SAGE
SAGE library construction.
COC were cultured for 25 min in medium containing FSH in the presence (plusDRB) or absence (minusDRB) of the transcriptional inhibitor DRB. Immediately after culture, COC from each replicate were snap-frozen in liquid nitrogen and stored at –80°C. A total of 20 replicates were performed (90 ± 13 COC per treatment per replicate). Within each replicate, additional groups of control COC were cultured in the presence or absence of DRB for 4 h and then assessed for resumption of meiosis (GVBD) as described above. Extraction of whole cell (wc) RNA was performed using Tri-reagent (Molecular Research Center, Cincinnati, OH). Fifty microliters of Tri-reagent were added to frozen COC from each treatment within each replicate and then pooled across all replicates within each treatment such that a final volume of 1 ml of Tri-reagent was used to extract a total of 1,771 ± 35 COC per treatment. Extractions were performed according to the manufacturer's instructions, and the resulting wcRNA was resuspended in diethylpyrocarbonate-treated water. The integrity of the pooled wcRNA from each treatment group was verified using a bioanalyzer (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA) at a concentration of 250 ng/ml. A total of 9 µg of COC wcRNA was recovered from the plusDRB treatment group and 10 µg from the minusDRB treatment group, representing an overall recovery rate of 5.57 ng wcRNA per COC extracted.

SAGE libraries were generated using all the pooled wcRNA extracted from the treatment group (9 µg and 10 µg of wcRNA from the plusDRB and minusDRB treatments, respectively). For SAGE library construction, an I-SAGE kit (Invitrogen, Carlsbad, CA) was used, and all of the manufacturer's instructions were followed. Briefly, mRNA was isolated from the samples using oligo-dT magnetic beads. cDNA was synthesized from the bead-bound RNA using Superscript II. Ditags were generated using NlaIII as an anchoring enzyme and BsmF1 as a tagging enzyme. Following PCR amplification, ditags (~26 bp in length) were realized by digestion with NlaIII, gel-purified using polyacrylamide gel electrophoresis, and ligated to form concatemers. The concatemers were size-fractionated (8% wt/vol PAGE) into three different groups: 300–500 bp, 500–800 bp, and >800 bp. These specifically sized concatemers were isolated and cloned into pZero-1 vector. Topo-10 electrocompetent cells were transformed with the vector constructs and cultured overnight at 37°C on low-salt LB agar plates containing 50 µg/ml Zeocin. Concatemers were sequenced by Agencourt Bioscience (Beverly, MA) either by submitting LB plates for automatic picking of the colonies or by submitting 96-well plates containing cells from individual clones that had been isolated, grown for 12 h at 37°C in low-salt LB agar containing 50 µg/ml Zeocin, and stocked in 10% glycerol at –80°C. Tag sequence information was analyzed using SAGE2000 4.5 Analysis software (Invitrogen). Tag-to-sequence mapping was performed using SAGE2000 software and the SageMap algorithm (http://www.ncbi.nlm.nih.gov/SAGE/), the Riken Mouse Encyclopedia Index (http://genome.rtc.riken.go.jp/) and the Mouse SAGE site (Institute of Molecular Genetics, Prague, Czech Republic, 2004; http://mouse.biomed.cas.cz/sage/taginfo.cgi). Functional analysis of differentially expressed genes was performed using Ingenuity Pathways Analysis software 3.0 (Mountain View, CA).

Verification by semiquantitative PCR.
A second independent set of murine COC were isolated and cultured for 25 min in the presence or absence of 120 µM DRB as described above. COC, collected over 11 replicates, were pooled within treatment. wcRNA was extracted as previously described from the plusDRB (n = 1,260 COC) and minusDRB (n = 1,168 COC) treatment groups. Samples were treated with 15 U of DNase I (Promega, Madison, WI) for 20 min at 37°C. DNA-free wcRNA was then reversed transcribed using Superscript II RNase H– (Invitrogen). The final 50-µl reaction included 2.5 µg of sample wcRNA, 50 ng of random primers (Promega), 0.8 mM deoxyribonucleotide triphosphate (dNTP) mix, 1x first-strand buffer (50 mM Tris·HCl, 75 mM KCl, 3 mM MgCl2), 0.01 mM dithiothreitol, and 400 U of Superscript II RT. Reactions were carried out for 1 h at 42°C. cDNA samples were cleaned to remove reverse transcription reagents using a Qiaquick PCR purification kit (Qiagen, Valencia, CA) and then resuspended in diethylpyrocarbonate-treated water at a concentration of 20 ng/µl. Primers for semiquantitative PCR (Table 1) were designed using Gene Amplify 1.2 (Madison, WI) and Oligo 4.0.2 Primer Analysis software (Plymouth, MA). For each PCR assay, the individual 20-µl reactions contained 60 ng of sample, 10 µM each appropriate primer, 10 mM dNTPs, 10x BV buffer (166 mM ammonium sulfate, 670 mM Tris·HCl, pH 8.8, 67 mM MgCl, 100 mM ß-mercaptoethanol), and 1 U of Platinum Taq DNA polymerase (Invitrogen). Within each assay, samples were run in duplicate. All reactions were assembled on ice, overlaid with oil, and briefly spun at 4°C before placement into a PTC-100 thermal cycler and denaturation for 2 min at 94°C. The standard PCR amplification was as follows: denaturation at 94°C for 30 s, annealing at the appropriate temperature for each primer set for 30 s (Table 1), and extension at 72°C for 30 s. The final cycle included a final extension at 72°C for 5 min. The number of cycles that resulted in linear amplification for each primer set is listed in Table 1. The band intensity of each PCR product was determined using computer-assisted video densitometry (Optimus 6.1; Media Cybernetics, Bothell, WA). The ratio of the signal intensities for the specific amplicon products was expressed relative to the signal intensity of connexin43.


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  		Table 1. Primer sequences and PCR conditions used for semiquantitative RT-PCR of murine COC wcRNA

 
Statistical Analysis
Percentage data from Experiment I were analyzed using general linear model procedures after arcsin transformation (50). The data are expressed as means ± SE, and all means were considered statistically different at P < 0.05. Differential expression of tag frequencies between SAGE libraries was analyzed using statistical tools included in the SAGE2000 software package and were based on chi-square analysis combined with Monte Carlo simulations (Invitrogen).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiment I: Time of Transcriptional Event Required for GVBD
At 4 h of culture, the majority of COC in the control group (0-D) had resumed meiosis (84 ± 6% GVBD; Fig. 1). When COC were cultured in the presence of DRB from the onset of culture (0+D) or when DRB was added after 5 or 10 min after the initiation of culture, the majority of COC remained at the GV stage (Fig. 1). However, when DRB was added to culture after 15, 20, 30, and 40 min, progressively greater proportions of COC resumed meiosis (32 ± 6, 44 ± 6, 66 ± 6, and 72 ± 6% GV, respectively; Fig. 1). The critical period for transcription of genes required for GVBD was determined to be between 15 and 30 min after the initiation of culture.


Figure 1
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  		Fig. 1. Effect of time of addition of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) to cultured murine cumulus oocyte complexes (COC) on the incidence of germinal vesicle breakdown (GVBD). 0-D, COC cultured in the absence of DRB; 0+D, COC cultured in the presence of DRB. a,b,c,dP < 0.05

 
Experiment II: Identification of mRNAs Associated with GVBD Using SAGE
On the basis of the results of Experiment I, COC were cultured for 25 min to allow for the transcription of genes whose expression was required for GVBD. A total of 20 experimental replications were performed to collect sufficient numbers of COC for SAGE library construction. In each replicate, additional groups of control COC were cultured for 4 h in the presence (n = 13 ± 2 COC per replicate) or absence (n = 13 ± 2 COC per replicate) of DRB to verify treatment effectiveness. Positive control COC cultured in the absence of DRB resumed meiosis (95 ± 6% GVBD), whereas negative control COC cultured with DRB were prevented from undergoing maturation (6 ± 6% GVBD). COC used for library construction were pooled from each replicate only if DRB was effective in blocking ≥90% of the negative control COC (≤10% GVBD) from that replicate. A total of 1,795 COC were pooled to represent the minusDRB treatment, and 1,746 COC were pooled to represent the plusDRB treatment.

A total of 93,798 SAGE tags were sequenced, with 48,431 sequenced from the plusDRB library and 45,367 sequenced from the minusDRB library. The tag sequences and frequencies from these two SAGE libraries have been deposited for public access via the NCBI Gene Expression Omnibus (minusDRB, GSM 128917; plusDRB, GSM128919). For comparison purposes, total tag counts for each SAGE library were normalized to 45,000 tags. To correct for sequencing errors, only tags with a total of at least five sequence counts between both libraries were included in the analysis (Table 2). By use of this criterion, the combination of both libraries represented a total of 1,876 unique transcripts, with 1,732 (92.3%) transcripts shared between both libraries, 75 transcripts (4%) expressed only in the absence of DRB (minusDRB library), and 69 transcripts (3.7%) expressed only in the plusDRB library. If more conservative minimum tag counts were used to account for potential sequencing errors, the number of differentially expressed tags between the plusDRB and minusDRB libraries progressively decreased (Table 2), suggesting that many of the differentially expressed tags may represent relatively rare mRNAs.


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  		Table 2. Frequencies of unique tags found in the plusDRB or minusDRB libraries

 
Of the total unique tags identified (n = 1,876 based on 5-tag minimum), only 44 (2.3% of total tags) could not be annotated. Thus >97% of tags were successfully assigned a Unigene ID using the available databases. Of the 40 most-abundant transcripts found in both libraries, more than one-half of these (n = 27) encoded various ribosomal proteins or their subunits. Tags corresponding to mRNA transcripts for nonribosomal proteins that were expressed in highest abundance in both libraries are listed in Table 3. Criteria used to identify transcripts of particular interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. By use of these criteria, 39 and 27 transcripts were identified as differentially expressed at the P ≤ 0.01 and P ≤ 0.001 levels, respectively (Table 4). Tags corresponding to oocyte-specific mRNAs that were identified in the plusDRB and minusDRB libraries are listed in Table 5.


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  		Table 3. Highest-abundance (nonribosomal) transcripts identified in both plusDRB and minusDRB libraries

 

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  		Table 4. Candidate mRNAs whose expression was associated with the resumption of meiosis (GVBD) in murine COC

 

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  		Table 5. Oocyte-specific genes found in SAGE libraries from COC cultured for 25 min in the presence or absence of DRB

 
All transcripts expressed across both libraries were grouped into functional classifications using Ingenuity Pathway Analysis (Fig. 2). In addition, mRNAs whose expression was specifically blocked (P ≤ 0.05) by culture with DRB were also grouped by functional classification (Fig. 3).


Figure 2
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  		Fig. 2. Functional analysis of all genes mapped from tags sequence in serial analysis of gene expression (SAGE) libraries generated from murine COC cultured for 25 min in the presence or absence of the transcriptional inhibitor DRB. Graph includes the 10 most populous functional groups in order of gene frequency.

 

Figure 3
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  		Fig. 3. Functional analysis of genes mapped from tags whose expression was significantly decreased (P < 0.05) in SAGE libraries generated from murine COC cultured for 25 min in the presence of the transcriptional inhibitor DRB. Graph includes most populous functional groups in order of gene frequency.

 
A total of six genes were selected for confirmation of expression by semiquantitative RT-PCR (sqRT-PCR) using a second, independent set of wcRNA pools made from COC cultured in the presence or absence of DRB. The selected genes and the magnitude of their differential expression between the plusDRB and minusDRB SAGE libraries were as follows: early growth response-1 (Egr1; 5.2-fold difference, P < 0.001), nuclear receptor subfamily 4 group A member 1 (Nr4a1; 40-fold difference, P < 0.001), histone H3 (H3; 3.3-fold difference, P < 0.001), glyceraldehyde-3-phosphate dehydrogenase (Gapd; 1.32-fold difference, P = 0.003), FOS osteosarcoma (Fos; 7.4-fold difference, P < 0.001), and connexin43 (Mm. 370184; 1.01-fold difference, P = 0.284). For the sqRT-PCR assays, expression of connexin43 mRNA was utilized as an internal control (housekeeper) because it was one of the most abundant tags found in both libraries, and its expression did not differ significantly between treatments. This observation was subsequently confirmed by sqRT-PCR using the independent set of COC wcRNA pools (data not shown). When the expression of the selected mRNAs was compared between the plusDRB and minusDRB treatments using semiquantitative (sqRT-PCR) assays, there was good agreement between the semiquantitative (sq)RT-PCR and SAGE analyses with regard to the effect of treatment on the directional change in gene expression (Fig. 4).


Figure 4
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  		Fig. 4. Comparison of SAGE and semiquantitative (sq) PCR analyses for directional changes in expression of selected genes in murine COC after culture with DRB. Data represent fold changes in expression of transcripts following 25-min culture of COC in the presence of DRB with respect to COC cultured for 25 min in the absence of DRB, based on SAGE or sqRT-PCR analyses. A negative fold change indicates that expression of the mRNA decreased with DRB treatment. Egr1, early growth response-1; Nr4a1, nuclear receptor 4a1; H3, histone H3; Gapd, glyceraldehyde-3-phosphate dehydrogenase; Fos, osteosarcoma-related oncogene.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
SAGE allows for the comparison of mRNA expression profiles between samples in a qualitative as well as quantitative manner. In this study, two SAGE libraries were constructed to compare the transcriptional profiles of mouse COC cultured in FSH-supplemented medium in the presence or absence of a transcriptional inhibitor, DRB, to identify mRNAs whose expression is associated with FSH-induced maturation.

In vitro, it is FSH rather than LH that is the gonadotropin responsible for driving oocyte maturation. Although the initiating gonadotropin may differ between in vivo and in vitro oocyte maturation, the in vitro model using FSH-induced maturation has been recognized as useful for assessing potential mechanisms of oocyte maturation (53, 54). Furthermore, while mechanisms regulating oocyte maturation in vivo may differ from those used for oocyte maturation in vitro, an understanding of the mechanisms regulating oocyte maturation in vitro has important application to the area of clinical assisted reproduction in both humans and animals. Therefore, in this study, we focused on the identification of transcripts associated with gonadotropin-induced resumption of meiosis of in vitro-matured oocytes.

Cumulus cells are required for transcriptional inhibitors to block GVBD in a variety of species including sheep, cattle, and mice (15, 40, 47). Thus an interaction between the oocyte and the cumulus cells is necessary for the FSH-induced transcriptional event to initiate oocyte GVBD in vitro. Because the oocyte and its surrounding cumulus cells form a single physiological unit that must be maintained as an intact structure for gonadotropin-induced oocyte maturation to occur, we chose to use COC, rather than denuded oocytes, to generate the SAGE libraries for this experiment. mRNAs regulating the initiation of GVBD would be expected to be products of the cumulus cells, rather than products of the oocyte (46).

In bovine, porcine, and ovine COC, the transcriptional event required for gonadotropin-induced GVBD occurs within 1–2 h of the initiation of culture (26, 35, 40). In the mouse, addition of DRB at the start of culture (47) or 5 to 10 min after initiation of culture in FSH-supplemented medium (present study) prevented GVBD. In contrast, addition of the transcriptional inhibitor 30 min after the initiation of culture in FSH-supplemented medium was ineffective in preventing GVBD. Thus, in mice, the key transcriptional events required for FSH-induced maturation began after 15 min of culture and continued through at least 30 min of culture.

In this in vitro model system, FSH triggers a transcriptional event that is required for resumption of meiosis (47). Although the precise mechanisms underlying the reinitiation of oocyte meiotic maturation in vivo and in vitro have not been clearly defined, COC recovered from both murine and bovine follicles matured in vivo contain receptors for FSH but not LH (12, 13, 38). Similarly, LH receptors have not been identified on bovine COC matured in vitro (38). Consistent with this observation, we did not identify any tags mapping to the LH receptor (LHcgr; Mm. 1644) in either of the SAGE libraries produced in the present study. To reconcile the long-standing hypothesis that LH is the gonadotropin responsible for reinitiating oocyte maturation in vivo with the observed lack of LH receptors on COC, it has been suggested that LH binding to mural granulosa cells within the preovulatory follicle leads to the production of EGF-like growth factors, including amphiregulin, epiregulin, and betacellulin (42). It has been proposed that, through a paracrine mechanism, these factors act to stimulate oocyte maturation (1, 6, 42). It remains to be determined whether exposure of COC to FSH in vitro will also upregulate expression of these paracrine factors. We performed a search of the SAGE libraries generated in this experiment for tag sequences corresponding to murine mRNAs for amphiregulin, epiregulin, and betacellulin and found that no tags for these mRNAs were present in either library. This is consistent with the observed lack of expression for these mRNAs in COC of preovulatory follicles from human chorionic gonadotropin-treated animals (42). It is possible that harvest of COC after only 25 min in culture with FSH may have been too soon to see an increase in expression of these factors. In whole murine follicles, mRNAs for amphiregulin and epiregulin were apparent at 1 h but did not peak along with the mRNA for betacellulin until 3 h after exposure to LH in vivo (42). The absence of these mRNAs in COC after 25 min of culture, a time interval during which key transcriptional events regulating the initiation of FSH-induced GVBD occur, suggests that expression of these specific mRNAs does not play a critical role in regulating at least the initiation of GVBD in vitro.

Functional analysis of all mRNAs expressed across both libraries indicated that the majority of de novo transcripts synthesized within 25 min of the initiation of COC maturation are involved in basic cellular functions such as cellular growth and proliferation, apoptosis (cell death), regulation of gene transcription, and cell development. Although seemingly contradictory, the concurrent activities of cell growth and apoptosis do occur within the COC in response to the gonadotropin stimulus. FSH induces cumulus expansion, which requires transcription of genes regulating hyaluronic acid synthesis and secretion by cumulus granulosa cells (4). Following expansion, the outer layers of cumulus cells surrounding the oocyte undergo apoptosis after FSH exposure. Thus it may not be surprising that mRNAs involved in cell growth, development, and apoptosis are all induced within the COC in response to the maturation stimulus. Of the transcripts that were specifically blocked in COC by exposure to DRB, ~70% were classified into functional groups that included cell growth and proliferation, cell development, regulation of gene expression, cell morphology, and cell cycle regulation. These groupings reflect the scope of physiological activities that COC undertake at the time of ovulation and resumption of meiosis when gonadotropin exposure induces differentiation of granulosa cells into luteal cells, affecting cell hypertrophy, cell morphology, cell division, and the reinitiation of meiosis within the oocyte.

The murine oocyte is estimated to contain 0.47 ng of RNA (39). This represents ~8.5% of the average amount of wcRNA obtained from individual COC in this study (5.57 ng per COC). Therefore, of the ~10 µg wcRNA used for construction of each SAGE library, oocyte wcRNA represented ~850 ng. Thus the SAGE technique was sufficiently sensitive to identify transcripts specific to the oocyte, although only a limited amount of oocyte wcRNA was present in the pool from which the library was constructed, and no procedures were used to selectively enrich the wcRNA pool for rare mRNAs. A number of oocyte-specific transcripts were detected in both libraries, including the following: oocyte specific histone H1 (H1foo; Mm. 21899, Ref. 58), oocyte-specific homeobox 5 (Obox5; Mm. 222738, Ref. 44), oocyte-specific homeobox 1 (Obox1; 358932, Ref. 44), oocyte-secreted protein 1 (Oosp1; Mm. 24754, Ref. 62), growth differentiation factor 9 (Gdf9; Mm. 9714, Ref. 32), and bone morphogenic protein-15 (BMP-15; Mm. 42160, Ref. 10). Interestingly, expression of both Gdf9 and BMP-15 was significantly upregulated (P ≤ 0.01 for both) when COC were cultured with the transcriptional inhibitor. This observation suggests that the expression of these two mRNAs is actively suppressed in COC cultured with FSH.

Data from the SAGE analysis of the minusDRB library represent the transcriptional profile of murine COC during the first 25 min of culture following isolation from the follicle. As expected, transcripts associated with cumulus cells such as the FSH receptor (Mm. 57155, P = 0.4), FSH primary response 1 (Fshpr1; Mm. 34903, P = 0.5), and follistatin (Mm. 4913, P = 0.3) were detected. Furthermore, expression of inhibin B mRNA (Mm. 3092, P < 0.01), which is induced in granulosa cells by FSH treatment (9, 17), was shown to be blocked by exposure of the COC to the transcriptional inhibitor. Many of the early response genes activated during the first 25 min of maturation in vitro were associated with regulation of gene expression. Two key transcriptional regulators whose expression was blocked by culture with DRB were activator of transcription 4 (Atf4; Mm. 641, P < 0.01) and early growth response-1 (Egr1; Mm. 181959, P < 0.001). Egr1 is rapidly and transiently induced by FSH in rat granulosa cells (14, 48). On the basis of the data from this SAGE analysis, DRB treatment not only reduced expression of Egr1 mRNA but also blocked the expression of genes known to be regulated by Egr1 such as cathepsin L (Ctsl; P = 0.01, Mm. 130; Ref. 22), prostaglandin E2 synthase (Ptges2; P = 0.05, Mm28048; Ref. 37), and ADAMTS (P = 0.02, Mm. 1421; Ref. 28). These genes each have been previously recognized as involved in the response of granulosa cells to gonadotropins (16, 45, 48).

The expression of genes involved in cytoskeletal organization such as tubulin-{alpha}2 (Tuba2; Mm. 379036, P < 0.001), tubulin-ß2 (Tubb2; Mm. 246377, P = 0.04), ß-actin (Actb; Mm. 297, P = 0.02), {gamma}-actin (Actg1; Mm. 196173, P = 0.04), and profilin 1 (Pfn1; Mm. 2647, P = 0.01) was also blocked by treatment with DRB. These observations suggest that key cytoskeletal rearrangements may be involved in FSH-induced GVBD in COC. The expression of mRNAs for heat shock proteins-1 (Mm. 215667, P < 0.001), -5 (Mm. 330160, P < 0.001), and -8 (Mm. 336743, P < 0.001) was also blocked by treatment with DRB. The expression of these transcripts in the first 25 min of culture points to either a major response of COC to the in vitro culture conditions or a true role for these proteins in the process of FSH-induced maturation.

A 40-fold difference in expression of Nr4a1 (Mm. 119) occurred in the control (minusDRB) COC library compared with the transcriptionally inhibited library. This was the largest magnitude of differential expression found for any mRNA transcript. Nr4a1 is a member of the steroid-thyroid hormone-retinoid receptor superfamily. In sertoli cells, the rat homolog of Nr4a1 is known as Ngfi-b and is positively regulated by FSH (33). In the rat ovary, Ngfi-b is rapidly induced by LH, and its expression peaks after LH in granulosa cells of preovulatory follicles (41). Treatment of human granulosa cells with FSH or forskolin results in an induction of Ngfi-b, which is followed by an increase in 3-ß-hydroxysteroid dehydrogenase type 2, which may result in an increase in progesterone production (21). Furthermore, Nr4a1 has been shown to increase synthesis of progesterone in mouse Leydig cell tumor cell line, K28 (52). The magnitude of its differential expression, noted between the two SAGE libraries in the present study, and the known roles of Nr4a1 in regulating steriodogenenesis in ovarian and testicular cells support the suggestion that part of the mechanism of FSH in stimulating meiosis is to upregulate steriodogenic activity within the cultured COC. In this respect, at least part of the mechanism of action of FSH in vitro may be very similar to that used by LH in vivo, since LH also rapidly induces Nr4a1 expression in granulosa cells of preovulatory follicles (41). Interestingly, this induction of Nr4a1 by LH also requires nacent gene transcription, since it is blocked by treatment with {alpha}-amanitin (41). Thus Nr4a1 may be an important mRNA candidate, warranting further investigation of its potential role in gonadotropin-mediated oocyte maturation.

One of the most abundant differentially expressed transcripts was translationally controlled tumor protein 1 (Tpt1; Mm. 296922). Tpt1 has been described in rat and human testis as having a potential role in spermatogenesis (20). To our knowledge, it is described here for the first time in ovarian cells. Tpt1 is involved in proliferation events and may be involved in the regulation of Na,K-ATPase activity and intracellular calcium levels (24, 27). Therefore, this transcript may have an important role in the resumption of meiosis, since mobilization of intracellular calcium has been suggested to be required for FSH-dependent resumption of meiosis (7, 54, 55).

Because COC remain arrested at the GV stage when cultured with a transcriptional inhibitor, some or all of these transcripts identified may be required for the oocyte to resume meiosis. Future studies utilizing targeted disruption of specific mRNA expression will be required to distinguish the transcript or group of transcripts ultimately responsible for mediating FSH-induced GVBD in cultured murine COC. The identification of key transcription factors, nuclear receptors, and structural proteins whose mRNA expression was blocked by treatment with DRB make them strong candidates for further investigation of their role in regulating oocyte maturation.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The research presented in this paper was supported by National Institute of Child Health and Human Development Grant 1-R03-HD-043875 to C. E. Farin.


   FOOTNOTES
 
Address for reprint requests and other correspondence: C. E. Farin, North Carolina State Univ., Dept. of Animal Science, Box 7621, Raleigh, NC 27695 (e-mail: Char_Farin{at}ncsu.edu)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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