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BOR - Papers in Press, published online ahead of print December 15, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.035899
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BIOLOGY OF REPRODUCTION 72, 968–975 (2005)
DOI: 10.1095/biolreprod.104.035899
© 2005 by the Society for the Study of Reproduction, Inc.

Identification and Characterization of an Ovary-Selective Isoform of Epoxide Hydrolase1

Jon D. Hennebold2,3,4,, Kuni Mah3, Wilma Perez3, Jessica E. Vance3, Richard L. Stouffer3, Christophe Morisseau5, Bruce D. Hammock5, and Eli Y. Adashi6

Division of Reproductive Sciences,3 Oregon National Primate Research Center, Beaverton, Oregon 97006 Department of Obstetrics and Gynecology,4 Oregon Health & Science University, Portland, Oregon 97239 Department of Entomology,5 University of California-Davis, Davis, California 95616 Department of Obstetrics and Gynecology,6 University of Utah Health Sciences Center, Salt Lake City, Utah 84132


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel ovary-selective gene was identified by suppression subtractive hybridization (SSH) that is expressed only during the mouse periovulatory phase of a stimulated estrous cycle. Analysis of the protein encoded by the full-length cDNA revealed that the majority of it, with the exception of the first 44 amino acids, matched soluble epoxide hydrolase (Ephx2, referred to as Ephx2A). By comparing the cDNA sequence of this newly identified variant of soluble epoxide hydrolase (referred to as Ephx2B) with the mouse genome database, an exon was identified that corresponds to its unique 5' cDNA sequence. Through the use of an Ephx2A-specific probe, Northern blot analysis revealed that this mRNA was also expressed in the ovary, with the highest level of expression occurring during the luteal phase of a stimulated estrous cycle. In situ hybridization revealed that Ephx2B mRNA expression was restricted to granulosa cells of preovulatory follicles. Ephx2A mRNA expression, however, was detectable in follicles at different stages of development, as well as in the corpus luteum. Total ovarian epoxide hydrolase activity increased following the induction of follicular development, and remained elevated through the periovulatory and postovulatory stages of a stimulated estrous cycle. The change in enzyme activity paralleled the combined mRNA expression profiles for both Ephx2A and Ephx2B, thus supporting a role for epoxide metabolism in ovarian function.

corpus luteum, granulosa cells, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To date, ovary-selective genes (i.e., genes expressed preferentially or possibly exclusively in the ovary) have been identified on a case-by-case basis. Special mention is made of Moloney sarcoma virus oncogene (Mos), a regulator of meiosis [1]; {alpha}-inhibin [2], growth differentiation factor-9 (Gdf9) [3], and bone morphogenetic protein 15 (Bmp15) [4], members of the transforming growth factor-ß (TGFß) superfamily; factor in the germline-{alpha} (Fig{alpha}) [5]; the genes encoding the zona pellucida proteins 1–3 [6]; and the FSH receptor [7]. The critical importance of some of these ovary-selective genes to murine ovarian function was unequivocally established through the generation of null mutants displaying a markedly aberrant ovarian phenotype and consequent female sterility [5, 6, 8–12]. It is highly likely that additional, potentially novel ovary-selective genes exist that may represent critically important regulators of ovarian function.

Therefore, systematic identification of ovary-selective genes was achieved through the use of suppression subtractive hybridization (SSH). Genes common to brain, heart, lung, liver, spleen, kidney, muscle, and ovaries in the unstimulated, follicular, ovulatory, and luteal phases of a stimulated estrous cycle were effectively removed by SSH [13]. Of the 340 nonredundant clones isolated by SSH, 83 nonredundant cDNAs were determined by Basic Local Alignment Search Tool nucleotide (BLASTn) analysis to represent putative novel genes [13]. The 83 cDNAs were designated as novel given the lack of homology to sequence entries deposited in publicly accessible, nonredundant nucleotide databases. Complementary DNAs were designated as novel even if they matched a full-length or partial sequence whose function has not been determined (i.e., enhanced sequence tag sequences).

One such novel cDNA (clone P2D2) was chosen for further analysis due to its ovary-selective and hormonally dependent expression profile. Identification of the full-length cDNA and the putative open reading frame revealed that the novel gene potentially encodes a previously uncharacterized isoform of soluble epoxide hydrolase (Ephx2). The protein encoded by Ephx2 is a member of a family of enzymes that convert endogenously produced epoxide substrates to their corresponding vicinal diols [14]. One such class of substrates for EPHX2 includes epoxyeicosatrienoic acids (EETs), molecules generated by cytochrome P450 (Cyp) epoxidation of arachidonic acid. EETs regulate numerous biological processes [15] including several that are important for proper ovarian function (e.g., the regulation of prostaglandin and estradiol synthesis) [16, 17]. EPHX2, therefore, serves as a key regulator of EET actions by metabolizing these compounds to diols that have limited biological activity. In this study, we detail the expression and cellular localization of two Ephx2 genes, one of which is a novel ovary-selective isoform (Ephx2B), through the course of a stimulated estrous cycle in the mouse ovary.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Protocols

Female C57BL/6 mice, 21 days of age at arrival, were purchased from Jackson Laboratories (Bar Harbor, ME). All protocols were approved by the Oregon National Primate Research Center's Institutional Animal Care and Use Committee. At 25 days of age, one group of mice (n = 8) was killed by CO2-induced asphyxiation to provide unstimulated ovarian material as well as nonovarian tissues. A second group of mice was injected with 5 IU of eCG. Forty-eight hours after the administration of eCG, a group of mice was killed (n = 7) to provide ovaries at the preovulatory phase of a reproductive cycle. The remaining mice were each injected with 5 IU of hCG. Subgroups (n = 5/subgroup) of the latter were killed at various intervals between 3 and 48 h post-hCG injection. Ovaries removed between 3 and 12 h post-hCG treatment encompass the interval preceding follicular rupture, whereas the 18- to 48-h post-hCG treatment groups encompass the postovulatory (i.e., luteal) phase of the stimulated ovarian cycle. Hormones were purchased from the National Institute of Diabetes and Digestive and Kidney Disease's National Hormone and Pituitary Program.

RNA Isolation

The isolation of total RNA was performed using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. For use in the rapid amplification of cDNA ends (RACE) protocol, PolyA+ RNA was isolated using an oligo(dT) magnetic sphere-based separation system (RNAatract; Promega, Madison, WI) according to the manufacturer's directions.

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 as previously described [18]. Prior to transfer, RNA quality and concentration were assessed by ethidium bromide staining and visualization under UV light. Nonovarian tissue blots (20 µg of total RNA) were purchased from Origene Technologies (Rockville, MD). Nylon membranes were prehybridized for 2–6 h at 42°C in 5x SSPE, 50% formamide, 5x Denhardt solution, and 0.25% SDS. Probes were generated by radiolabeling individual polymerase chain reaction (PCR)-amplified cDNA inserts with 32P-dCTP using the random-priming method (Amersham Pharmacia, Piscataway, NJ). The P2D2 probe was generated from the previously described ovary-selective cDNA library [13]. 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 solution used for prehybridization. The membranes were washed in 2x SSC and 0.1% SDS at room temperature three times for 5 min each, followed by two washes in 0.125x SSC and 0.25% SDS for 15 min at 60°C. The blots were rinsed in 4x SSC and imaged through the use of a phosphorImager (Bio-Rad, Hercules, CA). The signal intensity was quantified using Quantity One software (Bio-Rad). Equivalent RNA loading was verified by probing the same (stripped) blots with radiolabeled, sequence-verified PCR products of the housekeeping genes ß-actin (Actb) or glyceraldehyde-3-phosphate dehydrogenase (Gapd).

Multiple Tissue Array

A dot blot containing ~2 µg of mRNA isolated from 18 different tissues and 4 whole embryos at different stages of development was purchased from BD Biosciences (San Jose, CA). The 32P-dCTP labeled P2D2 probe was generated as described above. All blot hybridization reactions and washing procedures were carried out according to the manufacturer's directions (BD Biosciences). Hybridization signal was assessed using a phosphorImager (Bio-Rad).

Rapid Amplification of cDNA Ends

RACE was performed using the BD Biosciences SMART RACE cDNA Amplification Kit for identifying both the 5' and 3' ends of the P2D2 cDNA. Touchdown PCR parameters were those indicated by the manufacturer's instructions. Two separate cDNA templates were created possessing different adapters for use in either the 5' or 3' RACE reaction from polyA+ RNA isolated from the ovaries of eCG-treated (48 h) mice injected with hCG for 6 h. For 5'-RACE, Universal Primer Mix (UPM; BD Biosciences) and the P2D2-Rev primer (5'-TGGTGTCTGTGTCCTCCTGAGAAG-3') were used to amplify the 5' product. For 3'-RACE, the UPM (BD Biosciences) was used in combination with the P2D2-forward primer (5'-AAATCCTCATGCGGTTTGCAG-3'). Touchdown PCR was performed using the following parameters: denaturation at 94°C for 30 sec and annealing/extension at 72°C for 3 min (five cycles); followed by five cycles of denaturation at 94°C for 30 sec, annealing at 70°C for 30 sec, and extension at 72°C for 3 min; and finally, 27 cycles of denaturation at 94°C for 30 sec, annealing at 68°C for 30 sec, and extension at 72°C for 3 min. Products were analyzed by electrophoresis using a 1% agarose gel and visualized by ethidium bromide staining. PCR products were excised from the gel, purified with a QIAquick gel extraction kit, and subcloned into pCR-SCRIPT vector (Stratagene, La Jolla, CA). Multiple clones were isolated and the inserts were sequenced.

In Situ Hybridization

PCR products corresponding to the unique regions of the Ephx2A or Ephx2B genes were used to produce 35S-UTP-labeled antisense and sense probes with the Lign'Scribe no-cloning promoter addition kit (Ambion, Austin, TX). Ovaries from eCG/hCG-stimulated mice were isolated at selected intervals, embedded in OCT compound (VWR, West Chester, PA), and frozen using liquid propane. Tissue sections (10 µm) were placed onto microscope slides (SuperFrost Plus, Fisher Scientific, Pittsburgh, PA) and fixed in 4% paraformaldehyde for 15 min at 4°C. Tissue sections were rinsed in 0.5x SSC, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0 for 16 min, and then air-dried. The slides were prehybridized at 55°C overnight in hybridization solution (10 mM dithiothreitol, 0.3 M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA, 1x Denhardt solution, 10% dextran sulfate, and 50% formamide) containing the appropriate concentration of probe (1–5 x 106 cpm/ml). Representative slides were also incubated with a sense probe as a negative control. After hybridization, all the slides were treated with RNase A at 37°C for 30 min to inactivate nonhybridized probe, rinsed in a descending series of SSC concentrations (2x SSC, 1x SSC, and 0.5x SSC), and then washed in 0.1x SSC at 65°C (high stringency) for 30 min. Following dehydration and drying, a coat of photographic NTB-2 emulsion (VWR) was added to each slide and exposed for 2 wk at 4°C. Slides were developed with D-19 developer and fixer (Sigma, St. Louis, MO) and counterstained with hematoxylin. Both brightfield and darkfield illumination were used to visualize tissue histology and probe hybridization, respectively.

Epoxide Hydrolase Enzyme Assay

Ovaries were removed from mice undergoing stimulated estrous cycles, and ovarian homogenates were prepared in sodium phosphate buffer (100 µl, 100 mM, pH 7.4). Protein concentration was determined using the Bio-Rad protein assay reagent with BSA serving as a standard. Soluble epoxide hydrolase activity was performed as previously described [19]. Homogenates were diluted with sodium phosphate buffer (100 mM, pH 7.4) containing 0.1 mg/ml BSA and placed in glass tubes on ice until the assay was initiated. The enzyme assay was initiated by the addition of 1 µl of a 5 mM solution ([S] final: 50 µM) of substrate ([2-3H]-trans-1,3-diphenylpropene oxide; 3H-tDPPO) in N,N'-dimethyl-formamide. The tubes were incubated at 30°C for 5 min. The reaction was then quenched by adding 60 µl of methanol and 200 µl of iso-octane. To control for any glutathione-transferase activity, the iso-octane was substituted with hexanol as previously described [19]. The samples were vortexed vigorously and centrifuged for 5 min at 2800 x g. An aliquot (40 µl) of the remaining aqueous phase was removed and analyzed for the presence of the diol product by liquid scintillation counting. Reactions were performed in triplicate. Results are means ± SEM of three separate experiments.

Statistical Analysis

Multiple data points per experiment were analyzed by one-way analysis of variance with post hoc analysis using the Fisher least significant difference test. Data consisting of two separate groups were analyzed by a Student t-test. All statistical analysis was performed using SigmaStat software (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We previously isolated a number of novel genes that are selectively or specifically expressed in the mouse ovary via the differential screening technique SSH [13]. The expression of one such novel cDNA, originally designated P2D2, was analyzed through the course of a stimulated estrous cycle. As determined by Northern blot analysis, the expression of P2D2 was observed only 3 to 12 h post-hCG injection in eCG-primed mice (Fig. 1).



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  		FIG. 1. Phase-specific expression pattern of the novel clone P2D2. RNA (20 µg) isolated from various phases of a stimulated estrous cycle in C57BL/6 mice was assessed by Northern blot analysis for expression of the novel gene P2D2 as described in Materials and Methods. A ß-actin (Actb) probe was used to verify equivalent loading of RNA. Error bars represent the SEM of three separate experiments

During the initial characterization of clone P2D2, the expression of this gene could not be detected by Northern blot analysis in any of the 12 nonovarian tissues tested [13]. To expand the number of nonovarian tissues analyzed for P2D2 expression, the use of an mRNA dot-blot was employed. The mRNA samples used in the commercially prepared blot were isolated from 18 tissues, including the ovary, as well as from embryos at 4 different stages of development. Expression was detected in the ovarian mRNA (Fig. 2; spot 15), with limited expression also being detected in the epididymis (spot 17).



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  		FIG. 2. Tissue-selective expression profile of the novel clone P2D2. Using a commercially prepared tissue array (Multiple Tissue Array; BD Biosciences), mRNA isolated from 18 different adult and 4 different embryonic stages of development was analyzed for P2D2 expression

Because SSH yields only partial cDNA fragments (209 base pairs [bp] for clone P2D2), another methodology must be employed to isolate the full-length sequence. For P2D2, RACE was used to accomplish the identification of the complete 5' and 3' sequence. Using a 3' gene-specific primer and a 5' RACE primer, a product of ~250 bp corresponding to the 5' end of the P2D2 sequence was isolated. These results indicate that the 209-bp P2D2 fragment is at the 5' end of the cDNA sequence. A PCR product of ~1800 bp was isolated from the RACE reaction that used a 5' gene-specific primer and the 3' RACE primer. The 5' and 3' RACE products were isolated, cloned, and several randomly isolated clones were sequenced. The full-length cDNA sequence was subjected to BLASTn analysis and found to possess significant homology to the gene soluble epoxide hydrolase (Ephx2). In fact, only the first 211 nucleotides differ from the reported Ephx2 sequence (Fig. 3A). The SSH procedure utilizes the restriction enzyme RsaI to reduce the average size of the cDNAs and increase the hybridization efficiency before the amplification of differentially expressed genes. In this particular case, there is an RsaI site close to the junction (nucleotide 217) at which the original Ephx2 differs from the ovary-selective Ephx2 (nucleotide 211) sequence and thus explains why this fragment was obtained from our SSH-derived ovary-selective cDNA library [13]. The existence of the ovary-selective form of Ephx2 was verified by using primers in reverse transcription-PCR experiments that amplifies a product from ovarian cDNA spanning the junction between the two Ephx2 forms (data not shown). Based on the significant level of overlap between the two sequences, the ovary-selective cDNA sequence is subsequently referred to as Ephx2B (GenBank accession number AY098585), with the original sequence being termed Ephx2A (GenBank accession number NM_007940). From the full-length Ephx2B cDNA sequence, a single open reading frame was identified that would encode a protein of 536 amino acids (Fig. 3B). Accordingly, EPHX2A and EPHX2B overlap through most of the carboxyl end of the protein and differ only within the N-terminus. The EPHX2B isoform is 18 amino acids shorter than the previously characterized EPHX2A protein. It is also important to note that the first 44 amino acids of EPHX2B do not match the EPHX2A isoform.



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  		FIG. 3. The full-length cDNA sequence of clone P2D2, as determined by RACE, corresponds to a novel isoform of soluble epoxide hydrolase. A) Nucleotide and deduced amino acid sequence of Ephx2B. Underlined nucleotide sequences represent primer sequences used for RACE. The shaded region denotes the RsaI restriction site and boxed nucleotides represent the consensus polyadenylation signal sequence. B) Amino acid alignment of EPHXA and EPHX2B proteins. Symbols (*) indicate the key catalytic Asp residue required for lipid phosphatase activity, () divalent cation or phosphate-binding amino acids, and ({diamondsuit}) catalytic triad necessary for epoxide hydrolase activity. Arrow indicates approximate junction between phosphatase and epoxide hydrolase domains. C) Schematic representation of the exon/intron organization for Ephx2A and Ephx2B

Because the two Ephx2 variants overlap through the majority of their nucleotide sequences, it is highly likely that alternative splicing is involved in the generation of Ephx2B. The unique region of the Ephx2B sequence was therefore compared against the complete mouse genome sequence. From this analysis, it was observed that an exon corresponding to the unique region of Ephx2B was 1910 bp upstream of exon 3 (Fig. 3C).

It was reported that the expression of Ephx2A can be up-regulated via activation of the steroid receptor superfamily member PPAR{alpha} during agonist binding [20, 21]. To determine whether the Ephx2B splice variant was regulated in a similar fashion, mice were fed either a control diet or the PPAR{alpha} agonist clofibrate, as previously reported, for 5 days before and during the induction of a stimulated estrous cycle [20]. Ovaries were removed from control and clofibrate-treated mice at different phases of a cycle and analyzed for expression of both Ephx2A and Ephx2B mRNAs by Northern blot analysis. Using a radiolabeled probe specific for either Ephx2A or Ephx2B, it was apparent that clofibrate treatment did not increase the expression of either gene in the ovary (Fig. 4). Expression of Ephx2B mRNA was not observed in the livers of untreated or clofibrate-treated mice (data not shown). While some level of Ephx2A mRNA expression could be detected in all the samples analyzed (Fig. 4B), Ephx2B exhibited the same periovulatory-restricted expression profile (Fig. 4C). There was a consistent increase in Ephx2A mRNA expression following the administration of eCG that subsequently decreased following the administration of hCG. Ephx2A levels remained low up until just before ovulation (12 h post-hCG), at which point its expression increased and reached a maximum when corpora lutea are the dominant structure in the ovary. Ephx2A expression significantly increased in the liver of clofibrate-treated animals, thereby demonstrating that the clofibrate treatment protocol was effective (Fig. 4B).



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  		FIG. 4. Ephx2A and Ephx2B expression in the mouse ovary is regulated by gonadotropins and not by the PPAR{alpha} agonist clofibrate. A) Ovarian Ephx2A or Ephx2B expression was determined in control or clofibrate-treated C57BL/6 mice undergoing a stimulated estrous cycle by Northern blot analysis. Each lane was loaded with 20 µg of total RNA and hybridized with radiolabeled probes specific for each isoform. Equivalent loading was determined by reprobing the blots with a radiolabeled ß-actin (Actb) probe. B) Results represent the combined densitometric data for Ephx2A mRNA expression obtained from three independent experiments (± SEM). a) P < 0.05 versus CTRL samples from untreated animals whereas (b) signifies P < 0.05 versus CTRL samples from clofibrate-treated animals. *P < 0.05, liver Ephx2A expression in untreated and clofibrate-treated mice. C) Results represent the combined densitometric data for Ephx2B mRNA expression obtained from three independent experiments (±SEM)

Using a riboprobe specific for each isoform, in situ hybridization was used to determine which cell type or types within the ovary express Ephx2A and Ephx2B mRNA. Ephx2A mRNA expression was primarily localized to the granulosa cells of antral follicles before and after ovulation induction (Fig. 5, A–D). A high degree of Ephx2A mRNA expression was also observed in the cells that comprise the corpus luteum (Fig. 5E). Ephx2B mRNA expression was restricted to the granulosa cells of preovulatory follicles (Fig. 5, I and J). Early antral/antral-stage follicles from unstimulated (data not shown) or eCG-primed mice (Fig. 5H), as well as corpora lutea of postovulatory mice (Fig. 5K) exhibited little or no Ephx2B mRNA expression. The sense Ephx2A and Ephx2B riboprobe controls (Fig. 5, F and G, respectively) lacked detectable signal.



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  		FIG. 5. Granulosa cells express Ephx2A and Ephx2B mRNA. Frozen sections of C57BL/6 ovaries obtained from control (A); eCG-treated for 48 h (B and H); and eCG-primed mice treated with hCG for 4 h (C and I), 8 h (D and J), or 48 h (E and K) were probed with 35S-labeled antisense riboprobes specific for Ephx2A (A–E) or Ephx2B (H–K). Background sense probe hybridization was determined for Ephx2A (F) and Ephx2B (G) using sections from eCG-primed mice treated with hCG for 48 h and 8 h, respectively. CL, corpus luteum. Magnification for all panels x20

The level of soluble epoxide hydrolase enzyme activity was determined in whole ovarian homogenates. There was a significant increase (2.2-fold, P < 0.05) in soluble epoxide hydrolase activity 48 h after eCG administration and before hCG injection (hCG 0 h) (Fig. 6). Through the periovulatory phase of a stimulated estrous cycle (3–12 h post-hCG administration), soluble epoxide hydrolase activity was ~2-fold higher than in unstimulated mice (P < 0.05). Enzyme activity remained elevated through the postovulatory phase of the stimulated cycle (24 h and 48 h post-hCG administration). Soluble epoxide hydrolase activity increased 2.4-fold and 3-fold at 24 h and 48 h post-hCG, respectively, relative to controls (P < 0.05). Thus, total ovarian enzyme activity paralleled the combined gene expression profiles of both Ephx2A and Ephx2B as observed in Northern blot and in situ hybridization studies.



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  		FIG. 6. Epoxide hydrolase enzyme activity in whole ovarian homogenates correlates with the combined Ephx2A and Ephx2B expression profiles. Ovarian homogenates were generated from different stages of a stimulated estrous cycle using C57BL/6 mice and tested for soluble epoxide hydrolase enzymatic activity as previously described [19]. Results are from three experiments (± SEM). *P < 0.05 compared with control homogenates


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we identified a novel soluble epoxide hydrolase transcript (Ephx2B) that is expressed selectively in the ovary. A lower level of Ephx2B mRNA expression was also observed in the epididymis, a tissue recently demonstrated to possess soluble epoxide hydrolase enzyme activity [22]. It is not known at this time whether Ephx2B contributes to this activity, nor the biological role that EPHX2 enzymatic activity plays in the male reproductive tract. The first 43 amino acids of the putative EPHX2B protein lack significant homology to the N-terminal region of the previously characterized EPHX2A isoform. The EPHX2B isoform is also shorter than the EPHX2A isoform by 18 amino acids. The remaining 492 amino acids of the EPHX2B isoform, however, share complete identity with the EPHX2A protein. The part of the protein that is conserved between the EPHX2A and EPHX2B isoforms contains the catalytic domain responsible for epoxide hydrolase activity [23–25]. The N-terminal region not conserved between the two isoforms was previously reported to be involved in homodimerization of the enzyme and to possess lipid phosphatase activity [26–28]. The best substrates so far reported for the lipid phosphatase activity were dihydroxy lipid phosphates, with monohydroxy lipid phosphates being hydrolyzed at a lower rate. Through site-directed mutagenesis studies, it was subsequently demonstrated that an Ala-to-Asp substitution at position 9 of the human EPHX2A protein abolished phosphatase activity [27]. The different N-terminal amino acid sequence of the EPHX2B isoform may therefore affect protein-protein interactions and lipid phosphatase activity. Epoxide hydrolase activity should remain intact, because the key amino acid residues involved in this activity are identical in both EPHX2A and EPHX2B. We are currently in the process of producing a recombinant form of EPHX2B to compare directly with recombinant EPHX2A.

The newly identified EPHX2B isoform described in the present study appears regulated by the ovulatory surge of gonadotropins, particularly LH/hCG, because its expression was restricted to the periovulatory phase of a stimulated estrous cycle. No expression was detected by Northern blot analysis during the follicular or luteal phases of a stimulated estrous cycle. These results were confirmed by in situ hybridization studies that revealed primarily the granulosa cells of periovulatory follicles express Ephx2B mRNA. A similar granulosa cell-restricted pattern of expression was also noted for Ephx2A, with a high level of mRNA expression also being observed in the cells that comprise the corpus luteum. It has been reported that Ephx2A mRNA expression in the liver is induced following the activation of PPAR{alpha} by the selective agonist clofibrate [20, 21]. Increased hepatic Ephx2A expression was also observed in the present study following in vivo administration of clofibrate. Ovarian Ephx2A or Ephx2B expression, however, did not change in response to clofibrate at any of the stimulated estrous cycle stages tested. The inability of clofibrate to induce Ephx2A, and perhaps Ephx2B, may be the result of PPAR{alpha} expression being primarily restricted to the stroma/theca cell compartment [29, 30], while both Ephx2Aand Ephx2B are expressed selectively in granulosa cells.

It is therefore likely that granulosa cell EPHX2 enzymes would serve to hydrolyze endogenously synthesized EETs. It is well established that EPHX2 efficiently utilizes endogenously produced EETs as substrates converting them to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) [25, 31]. Four different EET regio-isomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) are generated from arachidonic acid via several different cytochrome P450 (CYP) mono-oxygenase family members [15]. EETs are involved in the regulation of numerous biological processes, including vascular tone, renal ion transport, angiogenesis, and inflammation [32–38]. The ability of each EET isomer to regulate various physiological events, however, is also dependent on EPHX2 activity as conversion to their corresponding diol (DHET) results in an altered potency [16]. Therefore, the combined CYP and EPHX2 activity within a given tissue, including ovarian follicles, would be the critical factor in determining the potential for EET-induced activities.

Limited information exists regarding the role the epoxide metabolites of arachidonic acid (i.e., EETs) play in ovarian physiology. It was reported that human granulosa cell cultures recovered from in vitro fertilization procedures produce significant quantities of EETs, particularly 5,6- and 14,15-EET, along with prostaglandins (PGs), following activation of the LH receptor [17, 39–41]. EET metabolism by EPHX2 occurs within granulosa cells, as significant amounts of DHETs were also identified within the culture supernatants [17]. The addition of an EPHX2 inhibitor to the granulosa cell cultures led to an increase in EET levels with a parallel decrease in the amount of DHETs [17]. It was also noted in this study that 14,15-EET regulates basal and LH-induced human granulosa cell estrogen production [17]. At lower concentrations (0.001 to 0.05 µM) of 14,15-EET, granulosa cell estradiol production increased ~2-fold to 3-fold. At higher concentrations (100 to 500 µM), however, 14,15-EET inhibited estradiol production by 40% to 50%. Recently, Newman and coworkers demonstrated that EET-generating CYP epoxygenases and EPHX2 are expressed in the porcine ovary [42]. CYP epoxygenase activity was detected in both theca and granulosa cells, whereas EPHX2 activity was higher in granulosa cells relative to theca cells. It was also determined in this study that the ratio of 14,15-EET to 11,12-DHET in follicular fluid correlated with estradiol levels. Taken together, these studies suggest that EETs may be involved in follicular development.

EETs were also shown to modulate prostaglandin E2 (PGE2) production [16, 43–45]. In a study by Kozak et al., 11,12-EET inhibited the production of PGE2 by cultured rat monocytes stimulated with lipopolysaccharide [46]. The effect of 11,12-EET on monocyte PGE2 production was believed to be mediated via a direct mechanism, because no change in PTGS2 protein expression was observed [46]. It was also reported that 8,9-EET and 14,15-EET inhibited platelet cyclooxygenase activity in a dose-dependent manner, with the corresponding diols (8,9-DHET and 14,15-DHET) being inactive [43]. In another study, however, PG production was enhanced by the addition of EETs to primary rat intestinal epithelial cell cultures [44, 45]. It was subsequently determined that this effect was due to an up-regulation of Ptgs2 mRNA expression. In testing the different EET regio-isomers, 14,15-EET was found to be the only one with the capacity to induce the expression of Ptgs2. The addition of an epoxide hydrolase inhibitor along with 14,15-EET to the cell cultures potentiated the efficacy of 14,15-EET in inducing Ptgs2 expression [45]. The potential regulation of PTGS2 expression or activity in the ovary by EETs would be significant because PGs have been demonstrated to play a critical role in ovulation in a number of mammalian species [47–49].

Epoxide hydrolase activity was analyzed in mouse ovaries through the course of a stimulated estrous cycle and found to parallel the combined mRNA levels for Ephx2A and Ephx2B. Enzyme activity increased 2.2-fold following eCG administration, a period when Ephx2A is predominantly expressed. During the time points at which the Ephx2B gene is predominantly expressed (3–12 h post-hCG administration), there was a significant (~2-fold) increase in activity relative to unstimulated control mice. The highest level of activity, however, was observed during the postovulatory phase of the stimulated cycle. After ovulation (hCG 48 h), there was a 3-fold increase in enzymatic activity compared with untreated control mice. This increase in enzyme activity correlates with the increased expression of Ephx2A gene at this time point. It is clear from this study that there is an increase in epoxide hydrolase enzymatic activity through the course of a stimulated estrous cycle, likely the result of the two different EPHX2 isoforms. The question arises, therefore, as to why two different isoforms of soluble epoxide hydrolase are expressed in the ovary at nonoverlapping phases of an estrous cycle. One possible explanation may be that there are distinct substrates for each of the two isoforms. Events associated with ovulation may require only epoxide hydrolase activity as the EPHX2B isoform presumably lacks lipid phosphatase activity, whereas some aspect of luteal function may require both epoxide hydrolase and lipid phosphatase activity.

Recently, null mutant Ephx2A mice were generated by targeting its first exon [50]. Null mutant male mice exhibit lower blood pressure, similar to the levels found in wild-type females. No notable phenotype was reported in the null mutant females, including fertility. A lack of an ovarian phenotype may be due to the deletion of the first exon, which in theory, would leave the exon that is used in the generation of the Ephx2B transcript untouched. Thus, the EPHX2B gene product may play the critical role in ovarian physiology or it may compensate for the loss of the EPHX2A enzyme. Studies are currently underway using Ephx2A-null mutants to test this hypothesis directly and to test for alterations in ovarian function.


   ACKNOWLEDGMENTS
 
We thank Julianne White for help in preparing the manuscript for submission and Dr. Robert Brenner for helpful comments and suggestions.


   FOOTNOTES
 
1 Supported in part by National Institutes of Health grants HD42000 (to J.D.H.), NCRR00163 (to J.D.H. and R.L.S.), HD30288 and 37845(to E.Y.A.), HD20869 (to R.L.S.), HD19182 (to K.M.), and by grants ES02710 and ES04699 from the National Institute of Environmental Health Sciences (to C.M. and B.D.H.). Back

2 Correspondence: Jon D. Hennebold, Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690-5563; henneboj{at}ohsu.edu Back

Received: 3 September 2004.

First decision: 5 October 2004.

Accepted: 16 November 2004.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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