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1 Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA
2 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel
(Requests for offprints should be addressed to N Dekel; Email: nava.dekel{at}weizmann.ac.il)
(J D Hennebold is now at Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon 97006, USA)
* (A Hourvitz and E Gershon contributed equally to this paper) 
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Abstract |
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Introduction |
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-hydroxysteroid dehydrogenase (3HSD), a regulator of G protein signaling (RGS-2), tumor necrosis factor-induced gene-6 (TSO-6) and early growth regulator-1 (Egr-1). Even though the exact role of these genes in the ovulatory process is not clear yet, their diverse functions and spatial expression pattern in the ovary reaffirmed the complexity and global nature of the ovulatory process. Leo et al.(2001), in turn, have used DNA microarray technology. cDNAs prepared from ovarian RNA of rats, before and 6 h after the ovulatory trigger, were hybridized to DNA microarrays representing 600 known rat genes. Quantitative analysis identified a multitude of regulated genes. Several of these genes were involved in extracellular matrix degradation and in lipid/steroid metabolism. Three of these genes, those encoding C-FABP (cutaneous fatty acid-binding protein), the interleukin-4 receptor alpha chain, and preponociceptin, were validated by Northern blot hybridization analysis and further characterized.
Taken together, these and other studies demonstrate that there is a high diversity of yet uncovered genes involved in the complex process of ovulation. These genes, either restricted in their expression to the ovulatory phase or preferentially expressed during the ovulatory process, constitute critical molecular determinants of the cascade leading to follicular rupture. Therefore, the purpose of this work was to isolate systematically these genes that are expressed in an ovulation-selective/specific manner.
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Materials and Methods |
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Female C57BL/6 mice, 19 days of age upon arrival, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were initially quarantined for 3 days at the University of Utah Animal Resources Center. The latter adheres to the guidelines outlined by the Animal Welfare Act and by Institutional Animal Care and Use Committee (IACUC) protocols. At 25 days of age, one group of mice (n = 8) was killed by CO2 asphyxiation, thereby providing unstimulated ovarian material as well as nonovarian tissues. A second group of mice (n=38) was injected i.p. with 10 IU each of pregnant mare’s serum gonadotropin (PMSG; Sigma). At 48 h after PMSG injection, a group of mice were killed (n=8) to secure ovaries at the preovulatory phase of the reproductive cycle. The remaining mice (n=24) were injected i.p. with 10 IU each of human chorionic gonadotropin (hCG) (Sigma). Subgroups (n=6/subgroup) of the latter were killed at 2, 4, 6 and 8 h after hCG injection. Actual follicular rupture occurs approximately 10–14 h after the injection of hCG to PMSG-primed mice (Espey et al. 2000b, Robker et al. 2000a). Therefore, we defined preovulatory ovarian mRNA as one which is extracted from untreated mice and mice primed with PMSG for 48 h. Ovarian mRNA from untreated mice is included in the so-called preovulatory ovarian mRNA so as to minimize the isolation of genes, which are constitutively expressed throughout the reproductive life cycle. The ovulatory ovarian mRNA was represented by the pooled ovarian material collected 2, 4, 6 and 8 h after hCG. The ovulatory ovarian mRNA was selected, as such, so as to include a wide range of genes induced by hCG. We assumed that most ovulation-associated genes are turned on within 8 h of hCG administration. Other groups of mice were killed 12, 24 and 48 h after hCG treatment, the last two representing the ‘luteal’ phase of the ovarian cycle.
Indomethacin administration and ovulation rate assessment
We used the antiovulatory agent indomethacin, which blocks prostanoid synthesis, to verify that the new identified ovulatory gene was induced via the prostanoid pathway. A subgroup of mice (n=6) treated with PMSG and hCG was injected with indomethacin. Indomethacin (ICN-190217–25, Costa Mesa, CA, USA) was prepared as previously described (Espey et al. 2000b) and was injected s.c. 3 h after hCG in a dose of 0.7 mg per animal. The ovaries were extracted 8 h after hCG injection. Another subgroup of 16 animals similarly treated served for ovulation rate assessment. The ovulation rate in the experimental (n=5) animals (treated with PMSG/hCG and indomethacin) and control (PMSG/hCG-treated) animals (n=5) was determined by counting the oviductal ova at 24 h after hCG administration.
RNA isolation
Total RNA was isolated from the following nonovarian tissues of immature (25-day) female C57BL/6 mice: brain, heart, kidney, liver, spleen, stomach, small intestine, large intestine, adrenal, uterus, muscle, uterus and lung. Total RNA was also isolated from the ovaries of 25-day-old female C57BL/6 mice undergoing the above-mentioned superovulation protocol. The isolation of total RNA was performed with the RNAeasy Kit (Qiagen) according to the manufacturer’s directions. PolyA+ RNA was subsequently isolated with an oligo-dT magnetic sphere-based separation system (RNAatract; Promega).
Suppression subtractive hybridization (SSH)
SSH was performed with the PCR-Select Kit (Clontech) according to the manufacturer’s directions. Briefly, an equal amount of PolyA+ RNA isolated from each of the preovulatory ovaries was combined to generate a total of 1 µg PolyA+ RNA. This mRNA was used to generate the driver cDNA with the SMART cDNA synthesis kit (Clontech) according to the manufacturer’s instructions. Ovulatory PolyA+ RNA (1 µg) isolated from mice undergoing the above-described superovulation protocol was used to construct the tester cDNA (2, 4, 6 and 8 h after hCG). Twenty-five primary and 12 secondary PCR cycles were used to amplify the target (subtracted) ovulatory-selective cDNAs.
Cloning and sequencing of cDNAs
The PCR products generated by SSH were digested with RsaI to generate blunt ends and to remove the adapters previously ligated to both ends of the target cDNAs. These cDNAs were subsequently purified by the Qiagen PCR system, ligated into the vector pGEM-T Easy (Promega) and transformed into the Epicurian coli strain XL2- Blue MRF’ Ultracompetent Cells (Stratagene, San Diego, CA, USA). The individual cDNA inserts were isolated by PCR amplification with flanking T7 and SP6 primer sites. The plasmid template used in the PCR reaction was obtained by direct use of the bacterial cultures lysed in ddH2O at a dilution of 1:50. Purified/PCR-amplified cDNAs were sequenced with T7 primers at the DNA-sequencing core facility of the Huntsman Cancer Institute at the University of Utah Health Sciences Center with Perkin Elmer ABI 377 automated sequencers (Boston, MA, USA). After the adapter and vector sequences were trimmed, the obtained sequence data was analyzed for homology with previously characterized mRNA deposited in the National Center for Biotechnology Informatics (NCBI) database, which includes entries from Genbank, European Molecular Biology Laboratory (EMBL), and DNA Database of Japan (DDBJ) databases using the BLASTn program. Clones not matching entries within the nonredundant database were matched to the NCBI EST database.
Analysis of subtraction efficiency
An equal amount of cDNA from the (presubtraction) tester pool and final SSH-subtracted product were used as a template to amplify the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The forward (5'-TGAAGGTCGGTGTGAACGGATTT GGC-3') and reverse G3PDH primers (5'-CATGTAG GCCATGAGGTCCACCAC-3') were used to amplify a 983 bp product within the following PCR parameters: denaturation – 94 °C for 45 s; annealing – 56 °C for 45 s; and extension – 72 °C for 1 min and 30 s. Samples were removed after the completion of 16, 20, 24 and 28 cycles. The resultant amplicon was resolved on a 2% agarose gel stained with ethidium bromide.
Northern blot analysis
Total RNA (20 µg) isolated from ovaries at different stages of the superovulation protocol was separated on denaturing 1% agarose-formaldehyde gels and transferred to nylon membranes (Magna Graph; MSI, Westboro, MA, USA) by the protocol of Sambrook et al.(1989). Before transfer, RNA quality and concentration were assessed by ethidium bromide staining and visualization under UV light. Nylon membranes were prehybridized for 2–6 h at 42 °C in 5 SSPE (sodium chloride–sodium phosphate–EDTA), 50% formamide, 5 Denhardt’s solution (0.2% BSA, 0.2% polyvinylpyrrolidone and 0.2% Ficoll), 0.25% SDS and 100 µg/ml denatured salmon sperm DNA. Probes were generated by radiolabeling individual PCR-amplified cDNA inserts with 5 µCi [32P]dCTP by the random-hexanucleotide-primed, second-strand synthesis method (Rediprime II; Amersham Pharmacia Biotech). The probes were denatured in a boiling water bath for 5 min before quenching with ice. Membranes were hybridized with the relevant probe overnight at 42 °C in the same (above-mentioned) solution used for prehybridization. Thereafter, membranes were sequentially washed three times for 5 min at room temperature with 5 SSC (standard saline citrate) and 0.5% SDS, followed by two washes for 15 min at 60 °C with 1 SSC and 0.75% SDS. The blots were ultimately rinsed with 4 SSC. To quantify the extent of hybridization, the membranes under study were exposed to a phosphor screen (Molecular Imager System; Bio-Rad), and the resultant digitized data were analyzed with Molecular Analyst software (Bio-Rad). The membranes were then stripped by heating to 95 °C in 0.2 SSC/0.5% SDS and reprobed with a 32P-labeled PCR product corresponding to the mouse ß-actin cDNA to correct for possible variation in RNA loading and/or transfer. Each experiment was carried out at least three times with three different sets of animals in an effort to minimize possible errors introduced by a given individual experiment.
Semiquantitative RT–PCR
First-strand cDNA was synthesized from total ovarian RNA. Briefly, 1 µg total RNA and 0.5 µg oligo (dT)12–18 (Amersham Pharmacia Biotech) were mixed in diethyl ester pyrocarbonic acid (DEPC)-treated water to a final volume of 30 µl and heated to 70 °C for 2 min, and the reaction was finally quenched on ice for 2 min. Reverse-transcription reactions (total volume of 50 µl) were carried out with final concentrations of 50 mM Tris–HCl (pH 8.3), 15 mM MgCl2, 75 mM KCl, 1 mM deoxynucleotide triphosphates, 37 units of RNAguard Ribonuclease Inhibitor from human placenta (Amersham Pharmacia Biotech), 10 mM DTT, 0.1 mM each deoxynucleotide triphosphates (d-NTP), 0.1 mM oligo(dT)12–18, and 400 units Moloney murine leukemia virus reverse transcriptase (M-MLV reverse transcriptase; Gibco BRL). This mixture was incubated at 37 °C for 1 h and inactivated at 70 °C (10 min). A 1:20 dilution of the resultant cDNA was stored at –20 °C until used.
cDNAs corresponding to the different experimental time points or different tissues were used for PCR amplification. Included were a primer set for ß-actin (0.5 µM each; forward primer, 5'-CCCCATTGAACAT GGCATTGTTAC-3'; reverse primer, 5'-TTGATGTCA CGCACGATTTCC-3') or fatty acid elongase 1 (FAE-1) homolog (0.5 µM each; forward primer, 5'-CGATAG GTGCTGAATTGTGG-3'; reverse primer, 5'-AGTGG TGGGAAGTCGAATGG-3') in a 25 µl reaction volume with 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100 (Promega), 2.5 mM MgCl2, 400 µM each d-NTP and 0.625 units of Taq DNA Polymerase (Promega). PCR was performed for 27 cycles (initial denaturation at 94 °C for 3 min, and then 27 cycles at 94 °C for 1 min, 59 °C for 1 min, 72 °C for 1 min and a final incubation at 72 °C for 7 min). The number of cycles used was determined to be in the log phase of the amplification reaction. The reaction mix (23 µl) was run on a 1.5% agarose gel stained with ethidium bromide, and quantified by UV imaging (Gel Doc 1000; Bio-Rad) and Molecular Analyst software (Bio-Rad). Signals corresponding to FAE-1 expression were normalized relative to ß-actin for each sample. Experimental replication of each time point was performed in triplicate for all three sets.
In situ hybridization
Mouse ovaries were obtained from immature gonadotropin-primed animals (at the indicated time points). Freshly dissected ovaries were immediately fixed in 4% paraformaldehyde in PBS, overnight, at 4 °C. Paraffin-embedded tissues were sectioned at 10 µm and mounted onto poly-L-lysine-coated slides. Sections were deparaffinized, rehydrated, rinsed with DEPC water, and digested with proteinase K. The SSH-generated cDNA was ligated into the vector of pGEM-T Easy Vector (Promega). The vector was used to generate digoxigenin (DIG)-labeled RNA antisense/sense probes of a mouse FAE-1 (300 bp) using the Riboprobe-combination system SP6/T7 (Promega) and the DIG RNA labeling mix (Roche). Tissues were hybridized for 16 h at 60 °C with 100 µl hybridization solution (50% formamide, 1 Denhardt’s solution, 5 SSC, 10% dextran sulfate, 0.25 mg/ml tRNA and 0.5 mg/ml salmon sperm DNA) and 1 µg/ml of the DIG-labeled FAE-1 mouse antisense or sense probe. At the conclusion of the hybridization phase, the sections were washed, treated with ribonuclease (20 µg/ml RNase A for 30 min, at 37 °C), and gradually desalted (2 SSC, 0.1 SSC and Tris). Staining of the sections was performed with anti-DIG antibody (1:500; Roche), conjugated to alkaline phosphatase overnight at 4 °C. Finally, the ovarian sections were washed and incubated with chromogen (Zymed, Eugene, OR, USA) until color appeared. The sections were visualized by an E-800 microscope (Nikon, Kanagawa, Japan).
Statistical analysis
Each experiment was carried out at least three times with 3–4 mice at each time point. Data points are presented as mean ± S.E. Statistical significance (Fisher’s protected least significance difference) was determined by the analysis of variance (ANOVA) to assess differences between multiple experimental groups. All analyses were performed using Statview for Macintosh (SAS Institute, Cary, NC, USA).
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Results |
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Ovulatory cDNAs were isolated by SSH. The efficiency of the SSH procedure was determined by PCR amplification of the housekeeping gene G3PDH. In the subtracted (target) ovarian cDNA population, the amount of G3PDH was significantly reduced relative to the unsubtracted ovarian cDNA (Fig. 1
). An additional six PCR cycles were required for the subtracted (target) cDNA to achieve the same level of G3PDH amplification as in the unsubtracted ovulatory cDNA. Since PCR amplification is an exponential process, this difference in the number of cycles translates into a 64-fold depletion of G3PDH cDNA in the subtracted ovulatory material.
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Sequence analysis of the ovulatory cDNAs
Each sequenced clone was analyzed after trimming the adapter and vector ends, using the BLASTn program. The corresponding accession number of the best match in the publicly accessible, nonredundant database of NCBI, its E probability value, and the degree of matching were recorded (Table 1). Of the 485 clones analyzed, 252 were determined to be nonredundant sequences. All 252 non-redundant clones sequenced shared homology with entries in the nonredundant database of NCBI, although 12 of these clones possessed significant homology to genomic clones only (i.e. BAC clones), and one clone (4-E5) shared the best homology with entries within the NCBI EST database. Except for two rat homologs, all cDNAs were of mouse origin (Table 1).
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To verify that inserts representing subtracted cDNA are expressed in an ovulatory manner, preovulatory ovarian mRNA (48 h after the administration of PMSG) and ovulatory ovarian mRNA (2, 4, 6 and 8 h after hCG) were subjected to Northern blot analysis. Confirmation of equivalent cDNA loading was accomplished by probing for the housekeeping gene ß-actin. To date, we have analyzed 98 genes. In this analysis, 25 clones (26%) failed to show any signal. Of the 73 hybridizations with a positive signal, 30 clones (41%) displayed an ovulation-selective expression, in that their expression proved higher after hCG than their limited expression 48 h after PMSG. Thirteen clones (18%) were determined to have an ovulation-specific expression pattern, in that their expression occurred after hCG administration only, without any signal 48 h after PMSG. Thirty clones were observed to hybridize equivalently to both preovulatory and ovulatory cDNA populations, thereby giving a false-positive rate of 41%. The full list of genes isolated from the SSH-derived ovulation ovulation-selective cDNA library and confirmed thus far to be expressed in an ovulation-dependent manner is described in Table 2.
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. An additional gene, the FAE-1 homolog, was selected for further evaluation. The expression of FAE-1 signal was very low in the preovulatory ovarian RNA samples, but was significantly (P<0.05) increased 4 h after hCG administration. Specifically, the FAE-1 transcript gradually increased up to a peak of approximately 2.4-fold at 12 h after hCG relative to ovaries preceding hCG treatment (Fig. 3). Equivalent RNA loading was verified by reprobing the same membrane for ß-actin transcripts.
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The cDNA fragment of the FAE-1 is 362 bp. This cDNA fragment is highly homologous (E-value=0) with a segment of the mouse FAE-1 gene, accession nos AK085696 [GenBank] , AK085663 [GenBank] , AK051580 [GenBank] , AK045274 [GenBank] , AK031743 [GenBank] , AK028761 [GenBank] and AK004319 [GenBank] , which was originally cloned from Mus musculus embryos, skin and mammary glands. Additionally, a fragment of the FAE-1 gene has homology with a gene named ELOVL family member 5 (Elovl5; accession nos. NM_134255 [GenBank] and BC022911 [GenBank] ).
The effect of indomethacin administration on ovulation rate and FAE-1 homolog expression
To confirm the anticipated effect of indomethacin, a prostaglandin synthesis inhibitor, on ovulation rate, parallel groups of animals were treated with or without an inhibitory dose of indomethacin 3 h after hCG administration. The mean ovulation rate (oocytes numbers) in the 24-h post-hCG control animals (without indomethacin) was 42.75 ± 5.30 as compared with 5.20 ± 1.13 in the 24-h indomethacin-treated animals (Fig. 4A). Moreover, in the control group, all the animals ovulated (8/8), while in the 24-h indomethacin-treated animals only 5/8 animals ovulated. Taken together, these data confirm the ovulation inhibitory effect of indomethacin injection.
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). Mouse tissue-specific FAE-1 gene expression
To assess the FAE-1 gene expression in diverse mouse tissues, RNA was extracted from 14 different tissues and subjected to semiquantitative RT–PCR analysis with specific primers of this gene. As shown in Fig. 5, FAE-1 gene expression could be detected in six of the 14 tissues tested (mouse brain, kidney, adrenal, liver, testis and ovary). The strongest signal was detected in the brain and ovary (8 h after hCG). No signal was detected in the heart, spleen, stomach, small intestine, large intestine, uterus, muscle and lung.
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The signal of the in situ hybridization reaction localized the FAE-1 to the granulosa cells of preovulatory follicles (Fig. 6). Time-course studies revealed ovarian FAE-1 mRNA expression to rise from undetectable levels at the time of hCG injection (48 h after PMSG) to maximal levels within 12 h after treatment with hCG, in accordance with the aforementioned Northern blot results.
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, great heterogeneity was noted in labeling intensity among granulosa cells of PMSG-primed/hCG-triggered antral follicles. The message encoding FAE-1 localized exclusively to the inner peri-antral granulosa (granulosa cells adjacent to the antrum) and to the cumulus cells of developing antral follicles. No detectable signal was noted for the mural granulosa cells. |
Discussion |
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We define preovulatory ovarian mRNA as one that was extracted from untreated mice and mice primed with PMSG for 48 h. Ovarian mRNA from untreated mice was included in the preovulatory ovarian mRNA so as to minimize the isolation of genes constitutively expressed throughout the reproductive life cycle. Actual follicular rupture occurs approximately 10–14 h after the injection of PMSG-primed mice with hCG (Espey 1980, Espey et al. 2000b). In preliminary studies, we found ovulation to occur as early as 8 h after hCG, peaking at 12–14 h (data not shown). Therefore, the ovulatory ovarian mRNA was represented by pooled ovarian material collected 2, 4, 6 and 8 h after hCG. The ovulatory ovarian mRNA was selected, as such, so as to include a wide range of genes induced by hCG. We assumed that most ovulation-associated genes would be expressed within 8 h after hCG administration.
Several techniques are currently available to identify new genes (Lisitsyn & Wigler 1993, Schena et al. 1995, Velculescu et al. 1995, Chee et al. 1996, Diatchenko et al. 1999, Espey et al. 2000b, Wang & Feuerstein 2000). We chose to use the SSH technique, since the relative advantages of SSH include the fact that it does not rely on an existing cDNA library and therefore is not limited by its quality. Other advantages are the normalization of the representation of high and low abundance transcripts, and the elimination of the physical subtraction step in the isolation of target cDNAs (Lee et al. 2000, Levesque et al. 2003, Fayad et al. 2004, Rebrikov et al. 2004). Moreover, the successful use of this PCR-based method has previously been reported in the context of constructing testis-specific library (Diatchenko et al. 1996) and by our laboratory in constructing an ovary-specific library (Tanaka et al. 2003). The discovery of new ovulatory genes in this study confirms the potential of this technique.
Although the utilization of SSH in the current study successfully yielded previously characterized, ovulation-specific genes (such as tumor necrosis factor-stimulated gene-6, steroidogenic acute regulatory protein (StAR), early growth response protein-1 and 3ß-HSDI), several expected genes were not present within the target cDNA library. For example, C/EBP-ß (Pall et al. 1997), Cox-2 (Lim et al. 1997, Davis et al. 1999) and the progesterone receptor (Lydon et al. 1995, 1996) were not found within the subtracted ovulation library. The absence of these genes from the library may be due to the fact that the screening of the subtracted ovulation cDNA library was not complete. It also may be due to an incomplete representation of the relevant mRNA in the tester cDNA pool that was used in the subtraction process. Both the tester and driver cDNA pools were generated by the SMART (Switching Mechanism At 5' end of RNA Transcript) cDNA synthesis kit (Clontech). This process relies on the addition of unique adapter oligonucleotides to the first-strand cDNA. The unique adapters can then be used to prime the PCR amplification and the generation of double-stranded cDNA. The advantage of this procedure is that it allows the generation of large amounts of cDNA from limited quantities of RNA. Due to the utilization of PCR, however, some of the cDNAs may not be amplified as efficiently as others and may thus be lost from the SSH starting material. A similar inability to identify all expected known genes after a differential screen was recently reported by others and ascribed to the incomplete representation of the total cDNA repertoire (den Hollander et al. 1999, Tanaka et al. 2003).
The ovulatory cDNAs isolated from the (subtracted/ SSH-generated) library included several cDNAs that have previously been reported to be involved in the murine ovulatory process (Espey & Richards 2002). Examples include StAR (Espey & Richards 2002), 3ß-HSDI, early growth response protein-1 (Espey et al. 2000a), epiregulin (Espey & Richards 2002), cathepsin-L (Robker et al. 2000a, 2000b) and tumor necrosis factor-stimulated gene-6 (Brannstrom et al. 1994, Yoshioka et al. 2000). During the validation process, 26% of the tested cDNA could not be detected by the Northern blot technique. This negative outcome may reflect the low level of sensitivity of the Northern blot methodology employed, as compared with the capability of the SSH technique, to identify low abundant genes. Verification of ovulation-selective or -specific expression of these 25 negative clones will require the use of a more sensitive methodology, such as real-time RT–PCR. Thirty clones were expressed at a same or higher level in the 48-h PMSG (preovulatory) ovarian mRNA relative to the post-hCG (ovulatory) mRNA, giving a false-positive rate of 41%. This rate is within the accepted range of the reported false-positive rate for the SSH technique, as it varies very much depending on experimental circumstances (Lee et al. 2000, Tanaka et al. 2003, Fayad et al. 2004).
In this report, we chose to focus on FAE-1 as a representative of a new ovulation-selective gene. FAE-1 was found to increase significantly after an ovulatory dose of hCG, reaching a peak 8–12 h after hCG, when follicles first begin to rupture. FAE-1 (FAE1, SSC 1, ELOVL 1) is a ß-ketoacyl-CoA synthase that belongs to the ELO family. The ELO family consists of eukaryotic, evolutionarily related, integral membrane proteins involved in fatty acid elongation. As these genes were identified only recently, not much is known on their function. The family includes the mammalian proteins ELOVL1–4 (Tvrdik et al. 2000) and the yeast proteins ELO1–3 (Oh et al. 1997). They seem to be components of membrane-bound, fatty acid elongation systems that catalyze the initial step of very long-chain fatty acids and produce the 26-carbon precursors for ceramide and sphingolipid synthesis (Oh et al. 1997). According to the ExPASy protein analysis tools, they may catalyze one or both of the reduction reactions in fatty acid elongation, that is, conversion of beta-ketoacyl CoA to beta-hydroxyacyl CoA or reduction of trans-2-enoyl CoA to the saturated acyl CoA derivative. The proteins have 271–435 amino-acid residues. Specifically, FAE-1 consists of 299 amino acids. Structurally, they seem to be formed of three sections: an N-terminal region with two transmembrane domains, a central hydrophilic loop and a C-terminal region that contains from one to three transmembrane domains.
The PSORT (http://psort.nibb.ac.jp:8000) cellular localization prediction algorithm suggests that FAE-1 is an endoplasmic reticulum (ER)-associated protein (reliability: 94.1), containing a KKXX-like motif in its C-terminus that is an ER membrane retention signal. The related gene, yeast ELO3, affects plasma membrane H(+)-ATPase activity, and may act on a glucose-signaling pathway that controls the expression of several genes that are transcriptionally regulated by glucose, such as PMA1.
It has been previously shown that the metabolism of membrane sphingolipids (such as sphingomyelin or ceramide) may be an important regulatory pathway in the control of steroid metabolism and steroid hormone synthesis (Sender Baum & Ahren 1988, Hattori & Horiuchi 1992, Degnan et al. 1996, Budnik et al. 1999, Soboloff et al. 1999). It has also been shown that in cultured fibroblasts, exogenous sphingomyelinase decreases cholesterol synthesis (Degnan et al. 1996). Moreover, LH-receptor expression is modulated by ganglioside-specific ligands (Lee et al. 1977, Chatelain et al. 1979, Hattori et al. 1994). We therefore suggest that FAE-1 may be involved in the regulation of steroid hormone synthesis during the ovulation process through the action of sphingolipid synthesis. Another role for FAE-1 may be related to a protective effect from carbon fragments formed in the ovary during or after ovulation. It was reported (O’Meara et al. 1985) that elongation of essential fatty acids by the ovary is an important mechanism in disposing of carbon fragments generated by the incomplete oxidation of fatty acids during steroidogenesis. The ovarian level of FAE-1 returns to the nonsignificant control levels at 24 h after hCG, confirming FAE-1 as a representative of an early gene response to gonadotropic hormone action on the ovulatory follicle. The dose of indomethacin that inhibited ovulation did not block the transcription of mRNA for this enzyme. Moreover, the early expression of the gene, before the ovulatory peak in PG production, suggests that prostanoid synthesis is not required for the induction of FAE-1 ovarian expression. However, this does not exclude a role for this enzyme in the ovulatory process, since the gonadotropin-induced expression of FAE-1 can be either a direct effect preceding the prostanoid expression or one mediated through ovarian steroids. The signal localized chiefly in the inner periantral granulosa (that is, granulosa cells adjacent to the antrum) and cumulus granulosa cells of developing antral follicles may suggest a role in follicular development. Further studies are needed to elucidate the exact role of this gene in the ovulation process.
In summary, this work demonstrates that the SSH technique can be used to identify new hCG-induced genes suspected to be involved in the ovulatory process. These ovulation-selective/specific genes may contribute to a better understanding of the molecular mechanisms of ovulation, and to the development of new strategies for either the promotion of fertility or its control.
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Acknowledgements |
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Received 22 November 2005
Accepted 17 December 2005
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