Mol. Hum. Reprod. Advance Access originally published online on June 12, 2007
Molecular Human Reproduction 2007 13(8):541-548; doi:10.1093/molehr/gam044
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Dynamic expression of epoxyeicosatrienoic acid synthesizing and metabolizing enzymes in the primate corpus luteum
1 Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, West Campus, Beaverton, OR 97006, USA 2 Department of Obstetrics and Gynecology, Oregon Health and Science University, Portland, OR 97239, USA 3 Present Address: Instituto de Biologia y Medicina Experimental (IBYME)-CONICET, Buenos Aires, Argentina
4 Correspondence address. Tel: +1 503 614 3720; Fax: +1 503 690 5563; henneboj{at}ohsu.edu
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Abstract |
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Epoxyeicosatrienoic acids (EpETrEs), produced from arachidonic acid via cytochrome P450 (CYP) epoxygenases, regulate inflammation, angiogenesis, cellular proliferation, ion transport and steroidogenesis. EpETrE actions are regulated through their metabolism to diols (dihydroxyeicosatrienoic acids; DiHETrE) via the enzyme soluble epoxide hydrolase (EPHX2). We set out to determine, therefore, whether EpETrE generating (epoxygenases CYP2C8, 2C9, 2C19, 2J2, 1A2 and 3A4) and metabolizing (EPHX2) enzymes are expressed in the primate corpus luteum (CL). CL were isolated from rhesus macaques during the early (day 3–5 post-LH surge), mid (day 6–8), mid-late (day 10–12), late (day 14–16) and very-late (day 17–19: menses) luteal phase of natural menstrual cycles. EPHX2 mRNA levels peaked in mid-late CL (5-fold when compared with early CL, P < 0.05) and remained elevated in the late CL. Ablation of pituitary LH secretion and luteal steroid synthesis significantly reduced (P < 0.05) EPHX2 mRNA levels in the mid-late CL, with progestin replacement being insufficient to restore its level of expression to control values. EPHX2 protein was localized to large and small luteal cells, as well as vascular endothelial cells. The EpETrE-generating CYP epoxygenase 2J2, 2C9 and 3A4 genes were also expressed in the macaque CL. While CYP2J2 mRNA levels did not significantly change through the luteal phase, CYP2C9 and CYP3A4 levels were significantly (P < 0.05) higher in the mid-late phase when compared with the early phase. CYP2C9, 2J2 and 3A4 proteins were each localized to the large luteal cells, with 2C9 and 2J2 also being present in the small luteal, stromal and endothelial cells. These studies demonstrate for the first time that an EpETrE generating and metabolizing system exists in the primate CL, with the latter being regulated by LH and steroid hormone(s).
Key words: epoxygenase/epoxyeicosatrienoic acid/soluble epoxide hydrolase/ovary/corpus luteum
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Introduction |
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It is well known that arachidonic acid can be converted to prostaglandins (PG) and leukotrienes via PG endoperoxide synthases (PGHS) and lipoxygenases, respectively. Additionally, certain cytochrome P450 (CYP) family members, collectively termed epoxygenases, convert arachidonic acid to four different regioisomers of epoxyeicosatrienoic acid (5,6-EpETrE; 8,9-EpETrE; 11,12-EpETrE and 14,15-EpETrE). The individual CYP epoxygenases, primarily belonging to the 2C and 2J family, preferentially form different EpETrE regio- and stereoisomers. EpETrEs, in turn, can be further metabolized to their corresponding vicinal diol (dihydroxyeicosatrienoic acids or DiHETrEs) via the enzyme soluble epoxide hydrolase (EPHX2).
EpETrEs affect numerous biological processes in a variety of different organ systems (Newman et al., 2005; Spector and Norris, 2006). For example, EpETrEs were reported to induce vasodilation and regulate vascular tone by activating large conductance K+ channels (Hecker et al., 1994; Larsen et al., 2006). EpETrEs also limit endothelial inflammatory responses by inhibiting NF-
B activation, enhance fibrinolysis by increasing tissue plasminogen activator expression and regulate renal ion transport by controlling Na+/K+ transporter activity (Node et al., 1999,2001; Zhao and Imig, 2003). At the molecular and cellular level, EpETrEs were demonstrated to serve as critical intermediates in epidermal growth factor signaling, inhibitors of apoptosis and pro-mitogenic factors (Kroetz and Zeldin, 2002; Spector and Norris, 2006). In addition, depending on the cell type analysed, EpETrEs either elevated or inhibited PGHS-2 activity or expression (Peri et al., 1997,1998; Fang et al., 1998). The ability of the EpETrEs to affect the above-described processes is also dependent upon the level of EPHX2 activity as their conversion to DiHETrEs results in decreased biological potency (Fang et al., 1998; Spector and Norris, 2006).
Despite the wealth of information regarding EpETrE synthesis, metabolism and action in various tissues, little information exists regarding these activities in the ovary. Human granulosa cells obtained from IVF procedures were reported to produce and metabolize EpETrEs in vitro (Zosmer et al., 1990; Van Voorhis et al., 1993; Zosmer et al., 2002). Additionally, CYP epoxygenase expression, EpETrE production and EPHX2 expression were demonstrated in the porcine ovulatory follicle, where it was noted that the level of EpETrEs and their metabolites, particularly 11,12- and 14,15- isomers, corresponds with the level of follicular fluid estradiol (E2) (Newman et al., 2004). Moreover, Van Voorhis and co-workers (1993) demonstrated that low doses (5–10 nM) of 14,15-EpETrE enhance E2 levels, whereas higher doses (1–50 µM) reduce E2 concentrations in human granulosa cell cultures.
We recently demonstrated that soluble EPHX2 mRNA is expressed at high levels in the mouse preovulatory follicle and developing corpus luteum (CL) (Hennebold et al., 2005). A parallel increase in EPHX2 enzyme activity was noted throughout a stimulated estrous cycle with the highest levels of activity being observed in the post-ovulatory interval. We set out, therefore, to determine whether a similar EpETrE metabolizing system exists in CL obtained from rhesus monkeys, a species that possesses a long, well-defined luteal phase (Stouffer, 2004). Studies were also conducted to characterize the presence and regulation of the genes encoding EpETrE synthesizing enzymes in the macaque CL through the luteal phase of natural menstrual cycles.
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Materials and Methods |
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Animals
The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC) was described previously (Wolf et al., 1990). All protocols were approved by the ONPRC Animal Care and Use Committee, and conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Menstrual cycles of adult female rhesus monkeys were monitored daily. Six days after menses, daily blood samples were collected by saphenous venipuncture. Serum was collected, and stored at – 20°C, until assayed for E2 and progesterone levels by specific electrochemiluminescent assays (Roche Elecsys 2010) through the Endocrine Services Laboratory, ONPRC (Young et al., 2002). The first day of low serum E2 following the mid-cycle peak has been demonstrated to correspond with the day after the LH surge, and was therefore termed Day 1 of the luteal phase (Duffy et al., 1999).
Tissues
CL (n = 3–4 stage) were isolated from anesthetized monkeys during an aseptic ventral midline laparotomy as previously described (Duffy et al., 2000). CL were collected during the early (days 3–5 post-LH surge), mid (days 6–8), mid-late (days 10–12), late (days 14–16) and very-late (days 17–19; mense) luteal phase. Each CL was divided into thirds, with 2 out of the 3 portions being frozen in liquid N2. The frozen samples were then stored at – 80°C until they were used for RNA and protein isolation. The remaining portion of each CL was fixed in 10% neutral buffered formalin for 5–7 days (Richard-Allen Scientific, Kalamazoo, MI), dehydrated in a series of ethanol solutions (50, 70 and 100%) and stored after being embedded in paraffin.
Hormone ablation and replacement protocol
Hormone ablation and replacement was performed in adult rhesus monkeys beginning on day 9 of the luteal phase (mid-late) as previously reported (Young et al., 2004). In this treatment protocol, females were randomly assigned to one of five groups (n = 4–5/group): control (no treatment); antide (ANT) (GnRH antagonist, 3 mg/kg body weight, Ares Serono; LH ablation); ANT + recombinant human (rh)LH (40 IU tid; Ares Serono; LH replacement); ANT + rhLH + Trilostane (TRL; 600 mg, a 3ß-hydroxysteroid dehydrogenase, or 3ß-HSD, inhibitor from Sanofi Research; steroid ablation); ANT + rhLH + TRL + R5020 (2.5 mg, a nonmetabolizable progestin; Promegestone; DuPont/NEN; progesterone replacement). Serum E2 and progesterone levels were analysed daily (Young and Stouffer, 2004). On day 12, CL were removed via laparotomy and flash frozen in liquid N2. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. RNA integrity was confirmed for each sample using an Agilent Technologies 2100 Bioanalyzer.
RT–PCR
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's directions. After RNA isolation, reverse transcription (RT) was performed on 1 µg DNase-treated RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) for 1 h at 37°C. PCR was performed in a 25 µl volume using Clontech reagents and the following cycling parameters (MJ Research, Watertown, MA): initial denaturation, 94°C for 1 min; denaturation, 94°C for 30 s; annealing (see Table 1 for primer sequences and optimal annealing temperatures) for 45 s and primer extension at 72°C for 1 min. The number of cycles necessary to ensure a linear phase of amplification was determined empirically. PCR products (5 µl) were electrophoresed through a 1.7% agarose gel stained with 0.1 µg/ml ethidium bromide. Gels were visualized using a gel documentation system (Gel Doc 2000, BioRad, Hercules, CA) and subsequently analysed densitometrically with Quantity One software (BioRad). All values were normalized to the internal standard peptidylprolyl isomerase A (PPIA; also known as cyclophilin A).
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Real-time PCR
PCR was performed using pooled luteal cDNA and primers designed against the human EPHX2 gene (Table 1). The resulting rhesus macaque EPHX2 PCR product was sequenced and used to design rhesus monkey-specific primers (Invitrogen) and a TaqMan© probe (Primer Express Software, Applied Biosystems, Foster City, CA) for the real-time PCR assay (for primer sequences see Table 1). The rhesus monkey EPHX2 TaqMan© probe sequence was 5'-6FAM-ACTGGCCACCGCCCTCACTCTCT-TAMRA-3'. A matrix of varying primer concentrations was employed to determine optimal primer concentrations.
Real-time PCR was performed using the TaqMan© PCR Core Reagent Kit with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as previously described (Young et al., 2002). 18S rRNA levels were monitored simultaneously in each sample to normalize the target signal. Amplifications were conducted in a 10 µl final volume containing 250 nmol/l TaqMan© EPHX2 probe (labeled with the reporter dye FAM), 300 nmol/l EPHX2 forward and reverse primers, 250 nmol/l TaqMan© 18S probe (labeled with the 5' reporter dye VIC) and 50 nmol/l forward and reverse 18S primers. The PCR reactions were conducted in sealed 96-well optical plates with thermal cycler conditions of: 2 min at 50°C, 10 min at 95°C and 40 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (primer annealing/extension). An internal standard curve, generated from five 10-fold dilutions of cDNA pooled from all CL samples, was used for relative mRNA quantification. The RNA equivalent values were divided by complimentary 18S RNA equivalent values derived from the same internal standard curve.
Western blot
Proteins were extracted from individual CL samples by homogenization in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10% glycerin and 1% Triton X-100) containing a protease inhibitor cocktail (Sigma, St. Louis, MO). Samples were centrifuged at 10 000g for 10 min at 4°C. The supernatant was collected and protein concentration was determined by Lowry assay (BioRad). Proteins (40 µg) were separated using 4–15% Tris–HCl sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Rabbit polyclonal anti-human EPHX2 (kindly provided by Dr. Bruce Hammock, University of California, Davis, CA) and anti-human ß-Actin (ACTB; Abcam, Cambridge, MA) were used at a 1:1000 dilution. ACTB antibody (1:5000 dilution) was used to verify equivalent protein loading. The blots were incubated for 1 h with peroxidase-conjugated anti-rabbit IgG antibody (1:4000; Santa Cruz Biologicals, Santa Cruz, CA) and the bands visualized using a chemiluminescent detection system (Santa Cruz Biologicals).
Immunohistochemistry
CL sections (6 µm) were deparaffinized with xylene and hydrated through a graded series of ethanol. Sections were incubated in phosphate-buffered saline (PBS) prior to pressure cooker antigen retrieval in citrate buffer (Citra; BioGenex Laboratories, Inc., San Ramon, CA). Endogenous peroxidase activity was then quenched with a 10 min incubation in 3% H2O2. Sections were placed in a blocking buffer (1.5% normal goat serum, NGS, in PBS) for 1 h at room temperature. Antibodies that recognize human CYP2C9 (Abcam, Cambridge, MA), CYP2J2 (kindly provided by Dr Zeldin DC, NIEHS), CYP3A4 (Abcam, Cambridge, MA) and EPXH2 were diluted to 1:100 in NGS/PBS and incubated with tissue sections at room temperature for 1 h or at 4°C overnight. Primary antibodies were detected using a biotinylated anti-rabbit IgG secondary antibody (7.5 µg/ml; Vector Laboratories) and a peroxidase substrate kit (ABC Elite Kit, Vector Laboratories). A non-specific, species and isotype-matched antibody was used as a negative control at the same concentration as the test antibody. The images were captured using an Olympus BX40 microscope equipped with a digital Olympus DP12 camera (40 x and 100 x magnification).
Statistical analysis
Statistical evaluation of mean differences among experimental groups was performed by ANOVA using the Sigma Stat software package (SPSS Inc., Chicago, IL). To isolate significant differences (P < 0.05) between groups, the Student–Newman–Keuls method was used for the pair-wise multiple comparisons.
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EPHX2 mRNA expression and protein immunolocalization in the rhesus macaque CL
We set out to determine whether EPHX2 is highly expressed in the rhesus macaque CL as was observed in the developing mouse CL (Hennebold et al., 2005). Using primers generated from the human EPHX2 gene sequence; EPHX2 mRNA was also detected in the primate CL (data not shown). Moreover, real-time PCR analysis of EPHX2 gene expression in the rhesus macaque CL revealed a significant increase in mRNA levels after the early luteal phase, with peak levels being observed in CL isolated from the mid-late luteal phase (5-fold relative to early CL, P < 0.05; Fig. 1A).
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To determine whether there was a corresponding change in EPHX2 protein levels, western blot analysis was performed. In parallel with EPHX2 gene expression, EPHX2 protein expression was minimal in homogenates isolated from early CL (Fig. 1B), but then increased to peak levels in mid-late and late CL.
The cellular distribution of EPHX2 was subsequently examined in the rhesus macaque CL by immunohistochemistry (IHC). Immunoreactivity was widely observed in the cells that comprise the CL, including the large luteal cells, and small luteal cells (Fig. 1C). Furthermore, EPHX2 expression was prominent in the endothelial cells lining individual blood vessels. The negative control (non-immune IgG) antibody exhibited no immunoreactivity (Fig. 1C, inset).
Regulation of EPHX2 gene expression in the rhesus macaque CL
Using a gonadotropin and steroid hormone ablation protocol, the regulation of EPHX2 gene expression in the macaque CL at the mid-late stage (days 9–12 post-LH surge) of the luteal phase was investigated. CL isolated from LH depleted animals (e.g. ANT-treated), showed a significant reduction (2.9-fold, P < 0.05) in EPHX2 gene expression when compared with untreated controls (Fig. 2). These levels were restored to control values after LH replacement, demonstrating a positive regulation of luteal EPHX2 gene expression by LH. The administration of Trilostane (steroid depletion, a 3ß-HSD inhibitor) to LH-replete animals (ANT + LH), however, inhibited EPHX2 mRNA expression to levels observed in the ANT-only treatment group (Fig. 2). Reduced EPHX2 gene expression in this group was not fully restored to control or ANT + LH levels following administration of a nonmetabolizable progestin (R5020). The analysis of circulating progesterone levels in the animals from which the individual CL were taken verified that the gonadotropin and steroid ablation and replacement treatments were effective for each group (data not shown, previously reported in Young and Stouffer, 2004).
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Epoxygenase gene expression and protein localization in the rhesus macaque CL through the luteal phase
As the CYP epoxygenases are responsible for converting arachidonate to EpETrEs, an endogenous substrate for EPHX2, studies were conducted to determine if the epoxygenase genes are also expressed in the primate CL. PCR primers were generated that correspond to known primate epoxygenase genes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, CYP2C19 and CYP2J2) based on the available human sequences. Of these, one CYP2J (CYP2J2) and one CYP2C (CYP2C9) subfamily member was expressed in the primate CL (Table 2). A member of the CYP3A subfamily (CYP3A4) was also expressed in the rhesus macaque CL. Rhesus macaque liver was used as a positive control (Ikeya et al., 1989; Wrighton et al., 1993; Schuetz et al., 1994; Enayetallah et al., 2004), with all of the primer sets for each epoxygenase gene amplifying a product of the appropriate size. To ensure that each primer set successfully amplified the correct cDNA, each PCR product was sequenced and compared with the corresponding human epoxygenase gene. All rhesus macaque CYP epoxygenase gene products were 96–99% homologous to the corresponding human sequence (data not shown).
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To determine if the CYP2J2, 2C9 and 3A4 epoxygenase genes are differentially expressed in the rhesus macaque CL through the luteal phase, semi-quantitative RT–PCR was performed. Semi-quantitative RT–PCR was utilized in place of real-time PCR due to the fact that the high degree of homology shared between the CYP epoxygenase genes (95–99%) is problematic for designing specific real-time Taqman© probes and primers. Also, through the use of this methodology, amplification of the correct product was established by amplicon size as well as the subsequent sequencing of the PCR product. As such, CYP2C9 expression was significantly higher (P < 0.05) during the mid-late luteal phase, when compared with the other stages of the luteal phase (Fig. 3). CYP2J2 mRNA levels, however, did not differ between CL obtained from any of the different luteal stages analysed (Fig. 3). Epoxygenase CYP3A4 expression in the rhesus monkey CL was significantly (P < 0.05) elevated in mid-luteal phase and remained high during mid-late and late stages when compared with early and very-late luteal phases (Fig. 3).
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To determine the potential cellular site of EpETrE synthesis within the primate CL, IHC was performed using antibodies that recognize human 2J2, 2C9 and 3A4 epoxygenases. IHC was performed using sections obtained from CL at the mid-late luteal phase, the stage at which a high level of expression for each epoxygenase gene was noted. Mid-late stage CL displayed positive CYP2C9 and 2J2 immunoreactivity within large luteal cells, small luteal and stromal cells, as well as endothelial cells lining blood vessels (Fig. 4A, B). CYP2C9 and 2J2 staining, while apparent in small luteal and stromal cells, was more intense in large luteal cells. CYP3A4 protein also localized predominantly in the large luteal cells, with limited expression present in the small luteal, stromal and endothelial cell populations (Fig. 4C).
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Discussion |
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The EPXH2 gene encodes soluble EPHX2, an enzyme, i.e. highly conserved between organisms (Newman et al., 2005) and is involved in the conversion of lipid epoxides (e.g. EpETrEs) to their corresponding vicinal diols (e.g. DiHETrEs) (Chacos et al., 1983; Beetham et al., 1995; Borhan et al., 1995; Yu et al., 2000). The EPHX2 enzyme serves to alter the EpETrE/DiHETrE ratio and, thus, the potential of these lipid epoxides to modulate various biological processes. In this regard, Fang et al. (1998) demonstrated that 14,15-EpETrE reduced PGE2 formation in smooth muscle cells, whereas 14,15-DiHETrE was incapable of eliciting this effect. EpETrEs also serve as regulators of vascular tone through their capacity to hyperpolarize the underlying smooth muscle cells by activating large conductance K+ channels (Hecker et al., 1994; Larsen et al., 2006). This vasodilatory effect has also been shown to be dependent on the level of endothelial cell EPHX2 activity and EpETrE metabolism (Imig et al., 2005).
Despite the increasing lines of evidence regarding the role of EpETrE formation and degradation in other tissues, little is known about the existence of an EpETrE synthesizing and metabolizing system in the ovary. Recently, we reported that the expression of the mouse Ephx2 gene increased significantly just prior to and after ovulation in animals undergoing a stimulated estrous cycle (Hennebold et al., 2005). Studies were conducted, therefore, to extend these findings to primate CL isolated throughout the luteal lifespan in the menstrual cycle. The expression of both EPHX2 mRNA and protein peaked in the rhesus macaque CL during the mid-late and late luteal stages of the macaque menstrual cycle. The mid-late luteal phase is a point of transition between when the CL is at peak function (i.e. maximal progesterone synthesis) to the onset of its physiological regression (i.e. during the late luteal phase). Therefore, the induction of EPHX2 expression during the mid-late and late luteal phases suggests that this enzyme plays a role as an initiator and/or effector of luteal regression. In the mouse ovary, increased Ephx2 gene expression after ovulation was observed primarily in luteinizing granulosa cells of the developing CL as determined by in situ hybridization (Hennebold et al., 2005). By immunohistochemical analysis, EPHX2 was localized to all cell types (e.g. large luteal, small luteal, stromal and endothelial cells) within the rhesus macaque CL.
Vertebrate EPHX2 is an inducible gene product and a number of studies have demonstrated hormonal regulation (Denlinger and Vesell, 1989; Inoue et al., 1993; Inoue et al., 1995; Pinot et al., 1995). For example, hepatic EPHX2 enzyme activity in mice is under the control of the hypothalamic-pituitary-gonadal axis (Inoue et al., 1995). In the present study, we demonstrate for the first time that rhesus macaque EPHX2 gene expression is regulated by the luteotropic hormone LH. Following the administration of the GnRH antagonist antide (ANT) to rhesus macaques for 3 days beginning on day 9 of the luteal phase, EPHX2 mRNA expression was significantly inhibited. With the replacement of LH in ANT-treated animals, however, EPHX2 mRNA levels were similar to those within CL taken from control animals. Moreover, ablation of steroid hormone synthesis through the administration of a 3ß-HSD inhibitor (Trilostane) significantly reduced EPHX2 gene expression to levels observed in CL obtained from gonadotropin-depleted (i.e. ANT-treated) rhesus monkeys. Supplemental progestin (R5020) only partially, but not significantly, restored EPHX2 mRNA levels to control values. EPHX2 gene expression, therefore, is likely indirectly regulated through steroid hormones other than progesterone, possibly including estrogens and androgens. A similar steroid dependent, but progesterone independent, regulation of gene expression in the rhesus monkey CL was reported for the metalloproteinase ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin repeats-1; Young and Stouffer, 2004). Additional in vivo steroid ablation and replacement studies, along with in vitro luteal cell experiments, will be required to define which steroids are primarily involved in regulating luteal EPHX2 gene expression. Furthermore, the partial restoration and variability of EPHX2 gene expression taken from animals receiving R5020 may be due to the limited number of CL available for analysis (n = 4) and, as such, may require additional samples from animals undergoing steroid ablation and replacement protocols to resolve this issue.
Since the EpETrEs are the primary endogenous substrates for EPHX2, we subsequently determined whether the EpETrE generating enzymes are also expressed in the rhesus macaque CL. Primate epoxygenase encoding genes include CYP2C8, 2C9, 2C19, 2J2, 1A2 and 3A4 (Rifkind et al., 1995; Wu et al., 1996; Zeldin et al., 1996; Bylund et al., 1998; Ayajiki et al., 2003). Previous studies demonstrated that ovarian samples taken from women of undetermined age and menstrual cycle stage express epoxygenase genes belonging to the CYP2C family (Klose et al., 1999). Additionally, cultured human granulosa cells possess an epoxygenase activity, that is up-regulated by LH (Zosmer et al., 1990). In the present study, the expression of the CYP2J2, 2C9 and 3A4 genes were detected in the rhesus macaque CL using a semi-quantitative RT–PCR approach. It was noted that CYP2C9 and 3A4 mRNA expression increases in the mid-late and mid-luteal phase, respectively, relative to CL isolated from the early luteal phase. In contrast to CYP3A4 gene expression, CYP2C9 drops at the late luteal phase. The CYP2J2 gene is constitutively expressed throughout the luteal phase. Moreover, there was no significant change in epoxygenase gene expression in CL isolated from animals undergoing the gonadotropin and steroid hormone ablation and replacement protocol (data not shown). When the cellular localization of the CYP2J2, 2C9 and 3A4 proteins was determined by IHC at the mid-late stage of the rhesus macaque luteal phase (i.e. at a point of high levels of gene expression), it was noted that these three epoxygenases are mainly expressed in large luteal cells, with variable staining in small luteal cells and endothelial cells. More importantly, however, these results demonstrate an overlap in CYP2C9, 2J2 and 3A4 expression with EPHX2 expression in the large luteal cells of the rhesus macaque CL. Additionally, co-localization of EPHX2 with CYP2C9 and 2J2 in the endothelial cells of blood vessels indicates that EpETrE actions may play a role in luteal vascular function, with any activity being regulated by their metabolism to DiHETrEs. Overlapping cellular expression between EPHX2 and CYP2C9 was reported in a number of human tissues, including the uterus (Enayetallah et al., 2004), thereby providing a system by which the synthesis, metabolism and consequently EpETrE-dependent activities are tightly controlled at a cellular level.
To our knowledge, this is the first time that the enzymes responsible for EpETrE synthesis and metabolism have been characterized in the primate CL. As such, these studies suggest a role for EpETrE synthesis and metabolism in primate luteal physiology. The epoxygenase genes CYP2C9 and 3A4 exhibit variable patterns of expression in the rhesus macaque CL, with CYP2J2 being constitutively expressed throughout the luteal phase of natural menstrual cycles. EPHX2 expression, on the other hand, begins to increase in the mid-late luteal phase and plateaus when luteal regression starts to take place (i.e. late luteal phase). The changes in EpETrE synthesizing and metabolizing enzyme expression during the CL lifespan likely alter the EpETrE/DiHETrE balance and, thus, the degree to which luteal cell processes are regulated by the arachidonate-derived epoxides.
Processes regulated by EpETrEs in other tissues that may be critical for various aspects of luteal development, function and/or regression include angiogenesis, inflammation, endothelial cell function and apoptosis. Specifically, inhibition of apoptosis by EpETrEs occurs through the activation of the phosphatidylinositol 3-kinase Akt signaling pathway (Chen et al., 1999). The angiogenic effects of the EpETrEs were also shown to utilize this pathway (Pozzi et al., 2005; Wang et al., 2005; Zhang et al., 2006). EpETrEs serve as endothelium-derived hyperpolarizing factors that cause relaxation of the underlying smooth muscle cells of the vessels thereby increasing blood flow (Fisslthaler et al., 1999; Fisslthaler et al., 2000; Campbell et al., 2001; Pratt et al., 2001). Furthermore, in non-ovarian cells, EpETrEs regulate production of PG through the regulation of PGHS-2 expression or activity (Peri et al., 1997; Fang et al., 1998; Peri et al., 1998). Lastly, EpETrEs produced in the rhesus macaque CL may regulate steroidogenesis as each of the EpETrE isomers (5,6-, 8,9-, 11,12- and 14,15-EpETrE) have been shown to induce steroid synthesis in human granulosa cells and mouse Leydig MA-10 cells (Van Voorhis et al., 1993; Zosmer et al., 2002; Wang et al., 2006). The latter was attributed to an EpETrE-dependent induction of steroidogenic acute regulatory protein expression (Wang et al., 2006). The above-described EpETrE activities taken together with the data presented herein supports a role for these arachidonate-derived compounds in luteal physiology. As such, future studies are warranted to further define the processes that are affected by EpETrEs in primate luteal cells.
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We are grateful for the contributions of the ONPRC Endocrine Services (Dr. Dave Hess), Imaging & Morphology (Barbra Mason) and Molecular & Cell Biology Core Laboratories (Yibing Jia). A special thanks to Dr. Richard Stouffer and coworkers for providing CL RNA from hormone ablation and replacement protocols. We also thank Dr. Stouffer for his comments and critique of the manuscript. The CYP2J2 and EPHX2 antibodies were kindly donated by Dr. D.C. Zeldin, NIEHS and by Dr B.D. Hammock, University of California-Davis, respectively. This work was supported by NICHD HD42 000, NICHD U54-18 185 and NCRR RR00 136. GI was supported through a Fogarty fellowship (NIH Fogarty International Center grant TW/HD-00 668 to P. Michael Conn).
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Submitted on April 16, 2007; resubmitted on May 8, 2007; accepted on May 14, 2007.
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