Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle

Diane M. Duffy1, Carrie L. Seachord and Brandy L. Dozier

Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Road, Lewis Hall, Norfolk, VA 23507, USA

1 To whom correspondence should be addressed. Email: duffydm{at}evms.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Prostaglandin E2 (PGE2) has been identified as the key ovulatory PG in the primate follicle. Follicular PGE2 levels increase just before the expected time of ovulation, suggesting that the midcycle LH surge induces the expression of enzymes involved in PGE2 synthesis. METHODS: To identify the specific form(s) of prostaglandin E synthase (PGES) expressed by the primate periovulatory follicle, we examined granulosa and theca cell expression of the three microsomal (m) and cytosolic (c) forms of PGES (mPGES-1, mPGES-2 and cPGES) identified to date. Monkey granulosa cells and whole monkey ovaries were obtained from animals receiving exogenous gonadotropins to stimulate multiple follicular development; monkeys then received an ovulatory dose of HCG to initiate periovulatory events. RESULTS: Expression of mPGES-1 mRNA and protein by granulosa cells of periovulatory follicles increased in response to HCG administration, peaking just before the expected time of ovulation. Immunocytochemistry showed that mPGES-1 protein was present in both granulosa and theca cells of monkey periovulatory follicles. Monkey granulosa cells also expressed mPGES-2 and cPGES mRNA, but mRNA levels did not change in response to HCG administration. Isolated monkey theca cells expressed both mPGES-1 and cyclooxygenase-2 mRNA, and produced PGE2 in vitro. Human granulosa-lutein cells obtained from women undergoing treatment for infertility expressed mRNAs for mPGES-1, mPGES-2 and cPGES. CONCLUSIONS: These data indicate that mPGES-1 is a gonadotropin-regulated PG synthesis enzyme expressed by granulosa cells of primate periovulatory follicles and suggest that mPGES-1 may be the primary PGES responsible for the increased follicular PGE2 levels necessary for primate ovulation.

Key words: granulosa cell/ovary/ovulation/prostaglandin/theca cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Prostaglandins (PGs) produced within the periovulatory follicle are essential for ovulation to occur. Administration of PG synthesis inhibitors has been shown to decrease follicular fluid PG levels and block ovulatory events in monkeys and women (Wallach et al., 1975GobGo; Pall et al., 2001Go; Duffy and Stouffer, 2002Go). Systemic administration of PGF2{alpha} has been reported to restore ovulation in monkeys in one article (Wallach et al., 1975GobGo), but the specific site of PG action in this study cannot be determined. More recently, replacement of PGE2 directly into the periovulatory follicle restored follicle rupture and oocyte release in PG synthesis inhibitor-treated monkeys (Duffy and Stouffer, 2002Go), establishing a central role for elevated ovarian PGE2 concentrations in primate ovulation.

The ovulatory surge of gonadotropin stimulates the production of PGs, including PGE2, by the periovulatory follicle (Wong and Richards, 1991Go; Sirois and Dore, 1997Go; Duffy and Stouffer, 2001Go). Expression of cyclooxygenase-2 (COX-2), which catalyses a key step in PGE2 production, increases soon after the ovulatory gonadotropin surge in granulosa and theca cells of primate follicles (Duffy and Stouffer, 2001Go). COX-2 converts arachidonic acid into PGH2, the common precursor for synthesis of numerous PGs including PGE2. A PGE synthase (PGES) is then required to convert PGH2 into PGE2. To date, three forms of PGES have been reported in humans. Two forms of microsomal PGES (mPGES-1 and mPGES-2) are so named because of their association with intracellular membranes (Watanabe et al. 1997Go). In contrast, a third form of PGES (cPGES) is described as a cytosolic enzyme, not associated with intracellular membranes (Tanioka et al., 2000Go). Additional studies have shown that co-localization of PG synthesis enzymes within the cell contributes to efficient PG synthesis. In these studies, mPGES-1 and -2 often preferentially colocalize with COX-2, while cPGES is most often found associated with COX-1 (Tanioka et al., 2000Go; Murakami et al., 2003Go). Because COX-2 is the key cyclooxygenase in periovulatory PG production, we hypothesized that a form of mPGES is involved in the production of periovulatory PGE2.

An unidentified isoform of PGES has been identified in granulosa cells of bovine (Filion et al., 2001Go) and rat (Guan et al., 2001Go) follicles. However, little else is known regarding the enzyme(s) within follicular cells that converts PGH2 into PGE2. This study was designed to identify the form(s) of PGES present in cells of the primate periovulatory follicle and to determine whether PGES expression increases in response to the ovulatory gonadotropin surge in a manner consistent with a role in periovulatory PGE2 production.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Granulosa cells, follicular fluid and whole ovaries were obtained from adult female cynomologus macaques at the Eastern Virginia Medical School (EVMS) (Duffy et al., 2005Go). All animal protocols and experiments were approved by the EVMS Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adult females with regular menstrual cycles were checked daily for menses; the first day of menstruation was designated day 1 of the menstrual cycle. Blood samples were obtained under ketamine chemical restraint by femoral or saphenous venipuncture, and serum was stored at –20 °C. Serum levels of estradiol (E2) and progesterone were determined using an automated chemiluminescent immunoassay system (Immulite; Diagnostic Products Corp., Los Angeles, CA, USA), with intra- and interassay coefficients of variation of <10% (Albrecht et al., 2000Go). Serum LH levels were determined by Leydig cell bioassay (Oregon National Primate Research Center, Beaverton, OR, USA) (Resko et al., 1975Go) with intra- and interassay coefficients of variation of <19%.

A controlled ovarian stimulation (COS) model developed for the collection of multiple oocytes for IVF was used to obtain monkey granulosa cells (n=4–5/time point) (Duffy et al., 2005Go). Briefly, monkeys received 6–8 days of recombinant human (rh) FSH (60 IU twice daily; Serono Reproductive Biology Institute, Rockland, MA, USA) followed by 2 days of rhFSH and rhLH (60 and 30 IU, respectively, twice daily; Serono) to stimulate the growth of multiple follicles. Daily GnRH antagonist administration (Antide, 0.5 mg/kg body weight; Serono) beginning up to 3 days before FSH administration and continuing until the day of HCG administration prevented an endogenous ovulatory LH surge. Adequate follicular development was monitored by daily serum E2 levels and transabdominal ultrasonography beginning on day 6 of FSH administration (Wolf et al., 1996Go). Follicular aspiration was performed before (0 h) or 12, 24 and 36 h after administration of 1000 IU rHCG (Serono). In spontaneous menstrual cycles, follicle rupture in monkeys occurs ~40 h after the ovulatory gonadotropin surge (Weick et al., 1973Go), so these times span the periovulatory interval. To obtain granulosa cells, each follicle was pierced with a 22-gauge needle, and the aspirated contents of all follicles >4 mm in diameter were pooled.

Whole ovaries (n=3–4/time point) were also obtained from monkeys experiencing COS. Additional whole ovaries were collected from monkeys experiencing spontaneous menstrual cycles around the expected time of the endogenous LH surge (Weick et al., 1973Go). These ovaries were obtained before the LH surge (n=2), the day of the LH surge (n=3), 1 day after the LH surge (n=2) or 2 days after the LH surge (n=2). Cynomologus monkey seminal vesicle was obtained at necropsy.

Tissue preparation
Monkey granulosa cells were obtained from follicular aspirates as described previously (Chaffin et al., 1999Go). Briefly, oocytes were mechanically removed from follicular aspirates, and a granulosa cell-enriched population of the remaining cells was obtained by Percoll gradient centrifugation (Chaffin et al., 1999Go). Total RNA was obtained from granulosa cells using Trizol reagent (Invitrogen, Rockville, MD, USA) and stored at –80 °C. Ovarian tissue was fixed in 4% paraformaldehyde and embedded in paraffin.

Isolation and culture of monkey theca cells
Monkey theca cells were obtained essentially following the method of McAllister et al. (1989)Go. Briefly, a whole monkey ovary was obtained after COS as described above, except that 6 days of rhFSH administration was followed by HCG treatment (1000 IU); ovariectomy was performed 48 h after HCG administration. This protocol was designed to stimulate theca cell proliferation; follicle rupture was not observed at the time of ovariectomy. Follicles >2 mm in diameter were dissected free from the ovarian stroma and bisected. Using a dissecting scope, the follicle wall was manually separated from the theca externa. Remaining granulosa cells were removed from the follicle wall by scraping with a platinum loop. The follicle wall (i.e. theca interna) was minced and digested with collagenase I for one hour (5.0 mg/ml) at 37 °C. The resulting cell suspension was plated and maintained in cell culture in serum-containing medium (McAllister et al., 1989Go). Cells were plated on chamber slides (Nalge Nunc International, Naperville, IL, USA) for immunocytochemistry or 48-well plates (Corning, Corning, NY, USA) for analysis of mRNA and media PGE2, grown to ~70% confluence in serum-containing medium, and then switched to serum-free medium 24 h before use in experiments. Some cultures received treatment with HCG (100 ng/ml; Serono) for 24 h prior to harvest of cells and media. Total RNA was obtained as described for granulosa cells above. Media was stored at –20 °C until PGE2 levels were determined using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) as described previously (Duffy and Stouffer, 2003Go). Replicate experiments were performed on passages two, three and four of these cells.

Human granulosa-lutein cells
Granulosa-lutein cells were obtained from women (n=4) undergoing treatment for infertility at the Jones Institute for Reproductive Medicine at the EVMS. Approval for collection and use of human cells for this study was obtained from the Institutional Review Board at the EVMS. Owing to the exempt nature of this protocol, no information is available regarding patients' demographics, diagnoses or treatments. Follicular aspirates were obtained after removal of the oocytes. A granulosa-enriched population of cells was obtained by Percoll gradient centrifugation, and total RNA was prepared as described for monkey granulosa cells above.

Real time RT–PCR
Levels of mRNA for PG synthesis enzymes as well as {beta}-actin were analysed by real time RT–PCR using a Roche LightCycler (Roche, Indianapolis, IN, USA). Total RNA was incubated with DNase, and reverse transcription was performed as described previously (Chaffin and Stouffer, 1999Go). Amplification of all mRNA was performed using the FastStart DNA Master SYBR Green I kit (Roche), with the exception of human {beta}-actin, which was performed using the Quantitect SYBR Green kit (Qiagen, Valencia, CA, USA); all reactions used 0.5 µmol/l of each primer and an annealing temperature of 55 °C. Primers were designed based on human or monkey complementary DNA sequences (Table I) using LightCycler Probe Design software (Roche). All primer pairs are located within the coding region and span an intron to prevent undetected amplification of genomic DNA. PCR products for all primer pairs were sequenced (Microchemical Core Facility, San Diego State University, San Diego, CA, USA). For mPGES-1, mPGES-2, COX-2 and {beta}-actin, at least 4 log dilutions of the sequenced PCR product were included in each assay and used to generate a standard curve; this technique allows quantitative analysis and determination of the number of copies of each mRNA (relative to {beta}-actin) present in each sample assayed. Because the sequenced PCR product for cPGES would not reamplify efficiently, a relative standard curve of at least 4 log dilutions of reverse-transcribed monkey testis total RNA was utilized. Expression of mPGES-1, mPGES-2, cPGES, COX-2 and {beta}-actin mRNA in each sample was determined in independent assays. All data were expressed as the ratio of PG synthesis enzyme mRNA to {beta}-actin mRNA for each sample. Intra- and interassay coefficients of variation were <10%. PCR products were separated by electrophoresis using 2% agarose gels containing 0.08 µg/ml ethidium bromide and photographed under ultraviolet illumination with Polaroid 667 film (Eastman Kodak Company, Rochester, NY, USA).


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Table I. Reaction conditions for real time RT–PCR

 
Western blotting
Granulosa cells were thoroughly lysed on ice in phosphate-buffered saline (PBS) containing 0.5% sodium dodecyl sulphate and 0.1% Triton X-100, mixed with denaturing sample buffer, heated to 95 °C for 10 min and loaded onto 4–20% gradient polyacrylamide Tris–HCl gels (Bio-Rad, Hercules, CA, USA). Each experiment included at least four lanes of serial-diluted granulosa cell lysate; detection and densitometric analysis of the protein of interest in these samples was used to generate a standard curve for semi-quantitative analysis of granulosa cell lysates. Proteins were transferred to polyvinylidine difloride membranes (Immobilon, Millipore, Billerica, MA, USA), and western blotting was carried out as previously reported (Duffy et al., 2005Go). The anti-mPGES-1 primary antibody (1 µg/ml) was a rabbit polyclonal generated against a synthetic human mPGES-1 peptide (Cayman Chemical); an anti-rabbit immunoglobulin G (IgG)–horseradish peroxidase conjugate secondary antibody (Amersham, Piscataway, NJ, USA) was used at a dilution of 1:10 000. Bands were detected by chemiluminescence (ECL; Amersham). Blots were then stripped of primary and secondary antibodies following instructions provided by the membrane manufacturer, and western blotting was performed on the stripped membranes using a mouse anti-tubulin primary antibody (1:1000 dilution; Sigma) and an anti-mouse IgG–horseradish peroxidase conjugate secondary antibody (Amersham). Tubulin levels in granulosa cell lysates were not different between treatment groups examined in this study. Molecular size of bands representing mPGES-1 and tubulin proteins were determined by comparison to prestained standards (Bio-Rad). Films were scanned and analysed densitometrically using SigmaGel software (Jandel Scientific, San Rafael, CA, USA). Data are expressed as a ratio of mPGES-1/tubulin content for each granulosa cell sample.

Immunocytochemistry
Immunocytochemical detection of PG synthesis enzymes in ovarian tissues was performed with 5-µm sections of paraffin-embedded monkey tissues as described previously (Duffy and Stouffer, 2001Go). All primary antibodies were rabbit polyclonal antibodies generated against synthetic human peptides (Cayman Chemical) and were used at the following concentrations: mPGES-1, 2 µg/ml; mPGES-2, 0.5 µg/ml; and cPGES, 2 µg/ml. Immunocytochemical detection was achieved using a biotinylated bovine anti-rabbit IgG secondary antibody and peroxide conjugated avidin solution (Vector Laboratories, Burlingame, CA, USA); peroxidase activity was visualized with Nova Red (red stain; Vector) or nickel-3,3-diaminobenzidine chromagens (blue stain; Vector). In some experiments the primary antibody was preabsorbed with the peptide used to generate the antibody according to manufacturer's instructions before incubation with tissue sections. Slides were not counterstained.

For immunocytochemical detection of PG synthesis and steroidogenic enzymes in isolated monkey theca cells, cells were plated on chamber slides. After 24 h in serum-free media in vitro, cells were fixed in 19% formaldehyde in PBS containing 0.3% Triton X-100 for 30 min and processed for immunocytochemical detection using primary antibodies directed against mPGES-1 (Cayman Chemical; 2 µg/ml), COX-2 (Cayman Chemical; 2 µg/ml), 17{alpha}-hydroxylase (1:1000; Leavitt et al., 1999Go) and aromatase (SeroTec, Raleigh, NC, USA; 1:250), as described for ovarian tissue sections above. Vector ABC kits were used for detection of mouse (COX-2, aromatase) and rabbit (mPGES-1, 17{alpha}-hydroxylase) primary antibodies, followed by Nova Red chromagen (Vector). Slides were counterstained with haemotoxylin. All images were obtained using an Olympus BX41 microscope fitted with a DP70 digital camera and associated software (Olympus, Melville, NY, USA).

Data analysis
All data were assessed for heterogeneity of variance using Bartlett's test and log-transformed when Bartlett's test yielded a significance <0.05. Enzyme mRNA and protein levels in monkey granulosa cells obtained before and after HCG administration were compared using one-way analysis of variance, followed by the Newman–Keuls test. One data point (mPGES-1 mRNA 12 h HCG) was determined to be a statistical outlier (Dean and Dixon, 1951Go) and was eliminated from the dataset prior to analysis. Theca PG synthesis enzyme mRNA levels and media PGE2 concentrations in the absence and presence of HCG were compared by paired t-test. Tubulin levels as determined by western blotting were assessed by the Kruskal–Wallis test. Except where indicated, all statistical tests were performed using StatPak v4.12 (Northwest Analytical, Portland, OR, USA). Data are presented as mean ± SEM, and significance was assumed at P<0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
PG synthesis enzyme expression in monkey follicles
Monkey granulosa cell expression of mPGES-1, mPGES-2 and cPGES was examined before and after administration of an ovulatory dose of HCG at times that span the 40-h periovulatory interval in primates (Weick et al., 1973Go). mPGES-1 mRNA was detected in all granulosa cell samples examined. mPGES-1 mRNA levels were low before (0 h), elevated at 12 h, and remained elevated 24–36 h after, HCG administration (Figure 1A). mPGES-1 protein levels were also low at 0 h HCG and remained low until 36 h after HCG administration, when mPGES-1 protein levels were 25-fold above 0-h levels (Figure 1B and C). mPGES-2 and cPGES mRNA were also detected in all granulosa cell samples examined. However, granulosa cell levels of mPGES-2 and cPGES mRNA were not altered in response to HCG administration at any time point examined (Figure 2).



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Figure 1. mPGES-1 expression in monkey granulosa cells. Granulosa cells were obtained from monkeys experiencing COS before (0 h), and 12, 24 and 36 h after HCG administration. (A) Granulosa cell mPGES-1 mRNA levels (relative to {beta}-actin) as determined by RT–PCR. (B) Detection of mPGES-1 and tubulin proteins in monkey granulosa cell lysates by western blotting. Representative lanes demonstrate chemiluminescent detection of mPGES-1 and tubulin in an individual monkey granulosa cell lysate from each time point examined; position of molecular weight (MW) standards for mPGES-1 (expected MW 16 kDa) and tubulin (expected MW 50 kDa) are shown at right. (C) Granulosa cell mPGES-1 protein levels (relative to tubulin) as determined by western blotting. Data expressed as mean ± SEM, n=4/group. Within each panel, groups with different superscripts are different, P<0.05.

 


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Figure 2. mPGES-2 and cPGES expression in monkey granulosa cells. Granulosa cells were obtained from monkeys experiencing COS before (0 h), and 12, 24 and 36 h after HCG administration. Granulosa cell levels of mPGES-2 mRNA (A) and cPGES mRNA (B) were determined by RT–PCR. Data are expressed as mean ± SEM, n=3–5/group.

 
Immunocytochemistry was used to localize mPGES-1, mPGES-2 and cPGES protein to the cells of the monkey ovary. mPGES-1 was detected in granulosa cells of periovulatory follicles obtained 0, 12, 24 and 36 h after HCG administration; immunostaining was low at 0 h and clearly detectable 36 h after HCG administration (Figure 3A and D; and data not shown). Stromal cells surrounding periovulatory follicles demonstrated mPGES-1 immunostaining at all times examined; the location of these stromal cells is consistent with identification as theca cells. Granulosa cells of primary and secondary follicles occasionally showed mPGES-1 immunostaining, but stromal cells surrounding these smaller follicles were consistently mPGES-1-negative (Figure 3J). mPGES-2 immunostaining was observed in granulosa cells of monkey periovulatory follicles obtained 0, 12 and 24 h after HCG administration, but was low/non-detectable in granulosa cells of follicles exposed to HCG for 36 h (Figure 3B and E; and data not shown). Granulosa cells of primary and secondary follicles often displayed immunostaining for mPGES-2 (Figure 3K). Ovarian stromal cells surrounding follicles of all stages were consistently devoid of mPGES-2 immunostaining. Periovulatory follicles also expressed cPGES; immunostaining for cPGES was observed in granulosa, but not theca, cells of periovulatory follicles obtained 0, 12, 24 and 36 h after HCG administration (Figure 3C and F; and data not shown). cPGES was also occasionally detected in granulosa, but not theca, cells of primary and secondary follicles (Figure 3L). For mPGES-1, mPGES-2 and cPGES, immunostaining was not present when the primary antibody was preabsorbed with the peptide used to generate the antibody or when primary antibody was omitted (Figure 3GI; and data not shown). Luminal epithelial cells of monkey seminal vesicle immunostained for mPGES-1, mPGES-2 and cPGES, confirming previous reports in various mammalian species (Filion et al., 2001Go; Lazarus et al., 2002Go) and serving as a positive control tissue (Figure 3MO).



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Figure 3. Immunocytochemical detection of PGE synthesis enzymes in monkey tissues. Ovaries with periovulatory follicles were obtained from monkeys experiencing COS before (0 h) (AC) and 36 h (DI) after administration of an ovulatory dose of HCG. In each periovulatory follicle shown, the follicle antrum is in the upper right and the ovarian stroma is in the lower left portion of the image. The position of the granulosa cell layer is indicated by the arrow (A). Immunodetection of mPGES-1 (A, D), mPGES-2 (B, E) and cPGES (C, F) is shown; stromal cells immunostaining for mPGES-1 are indicated (A, D, PQ; arrowheads). Periovulatory follicles shown are representative of n=3–4 ovaries/time point. Tissue sections showing granulosa cell immunostaining for mPGES-1 (J), mPGES-2 (K) and cPGES (L), in primary (K) and secondary (J, L) follicles, were obtained 24, 0 and 12 h after HCG, respectively. Preabsorption of the primary antibody against mPGES-1 (G), mPGES-2 (H) and cPGES (I) eliminated granulosa cell immunostaining in ovarian tissues obtained 36 h after HCG. Staining of oocytes was not consistently eliminated with preabsorption, so oocyte staining is not considered specific. Luminal epithelial cells (arrowhead, M) of the monkey seminal vesicle immunostained for mPGES-1 (M), mPGES-2 (N) and cPGES (O), while the seminal vesicle stroma (st) was consistently devoid of immunostaining. Monkey periovulatory follicles obtained during spontaneous menstrual cycles on the day of (P) and 2 days after (Q) the endogenous LH surge also immunostained for mPGES-1. Granulosa cell staining in the luteinizing follicle (Q) is indicated with the arrow; the antrum is not shown. Stromal cells immunostaining for mPGES-1 are indicated with arrowheads (P, Q). Isolated theca cells (passage two) immunostained for mPGES-1 (S), COX-2 (T) and 17{alpha}-hydroxylase (U), but not aromatase (V); no staining was observed when the primary antibody was omitted (R). Immunostaining performed with Nova Red chromagen appears reddish-brown (AO, RV), while immunostaining with nickel-3,3'-diaminobenzidine chromagen appears blue (P, Q). AP, bar = 25 µm; for Q, bar = 50 µm; for RV, bar = 10 µm. Use bar in O for all images.

 
Additional ovaries obtained from monkeys experiencing natural menstrual cycles were immunostained for mPGES-1, mPGES-2 and cPGES. Serum levels of estrogen, progesterone and LH on the days leading up to and including the day of ovary removal confirmed normal menstrual cyclicity (data not shown). Immunostaining for mPGES-1, mPGES-2 and cPGES was detected in granulosa cells of large periovulatory follicles obtained the day before the LH surge, the day of the LH surge, the day after the LH surge and 2 days after the LH surge (Figure 3P and Q; and data not shown). Stromal cells immediately outside the granulosa cell basement membrane of large periovulatory follicles immunostained for mPGES-1 but not mPGES-2 or cPGES. Immunostaining for mPGES-1, mPGES-2 and cPGES was also occasionally observed in primary, secondary and small antral follicles in ovarian tissues obtained during spontaneous menstrual cycles (not shown).

To confirm PG synthesis enzyme expression and PGE2 production by monkey theca cells, theca cells were isolated from monkey follicles. Immunocytochemical detection of 17{alpha}-hydroxylase (expressed by theca, but not granulosa, cells) but not aromatase (expressed by granulosa, but not theca, cells) in >99% of cells supports the identification of the stromal cells isolated from this single ovary as theca cells (Figure 3U and V) (Suzuki et al., 1993Go). mRNA for mPGES-1 and COX-2 was detected in theca cells maintained in vitro. However, treatment in vitro for 24 h without and with an ovulatory concentration of HCG (100 ng/ml) did not alter mRNA content of mPGES-1 (0.047 ± 0.010 versus 0.052 ± 0.008) or COX-2 (0.041 ± 0.012 versus 0.033 ± 0.008) when expressed relative to {beta}-actin mRNA. Cultured theca cells immunostained for both mPGES-1 and COX-2; no immunostaining was observed when the primary antibody was omitted (Figure 3RT). PGE2 was detected in media obtained after 24 h of culture of monkey theca cells without and with an ovulatory concentration of HCG, but no effect of treatment was observed (5.4 ± 1.6 versus 4.3 ± 1.9 pg/ml). Similarly, preliminary experiments indicated that LH and FSH also did not alter PG synthesis enzyme expression and media PGE2 levels (data not shown).

PG synthesis enzyme expression by human granulosa-lutein cells
Granulosa-lutein cells from human periovulatory follicles expressed mPGES-1, mPGES-2 and cPGES mRNA, confirming the expression of all three PGES forms in the human follicle (Figure 4).



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Figure 4. Detection of mPGES-1, mPGES-2 and cPGES mRNA in human granulosa-lutein cells. RT–PCR amplification of each PGES and {beta}-actin mRNA from human granulosa-lutein cells (H) and monkey granulosa cells obtained 36 h after HCG administration (M) is shown; no amplification was observed with any primer pair in the absence of reverse-transcribed RNA (H2O). Expected sizes of PCR products (major band in each lane) are given in the Table I. Results shown are representative of n=4 human patients and monkeys. Sizes of bands in standard lanes are shown on left (base pairs). PCR products shown are the result of 45 amplification cycles and do not permit quantitative comparisons.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
These studies provide the first demonstration that all PGES forms identified to date are expressed by the periovulatory follicle. mPGES-1 is the only PGES form that demonstrated gonadotropin-stimulated expression in vivo in primate granulosa cells. Granulosa cell levels of mPGES-1 mRNA and protein increased in response to the ovulatory gonadotropin surge and were maximal just before the expected time of ovulation. These findings are consistent with previous reports of expression of an unidentified form of PGES in granulosa cells of mouse small antral follicles (Guan et al., 2001Go) and bovine periovulatory follicles (Filion et al., 2001Go). Follicular fluid concentrations of PGE2 in primate follicles are low early in the periovulatory interval but peak just before the expected time of ovulation (Duffy and Stouffer, 2001Go), and monkey and human granulosa cells (Patwardhan and Lanthier, 1981Go; Duffy and Stouffer, 2003Go), as well as granulosa cells from non-primate species (Evans et al., 1983Go; Zor et al., 1983Go), have been shown to produce PGE2 in vitro. Therefore, the expression of mPGES-1 by human granulosa-lutein cells and the pattern of mPGES-1 expression in monkey granulosa cells suggests that mPGES-1 is likely the PGES form responsible for conversion of PGH2 to PGE2 in primate granulosa cells during the periovulatory interval.

In addition to granulosa cells, theca cells of monkey periovulatory follicles express mPGES-1 and produce PGE2. Immunocytochemistry demonstrated that stromal cells surrounding periovulatory follicles expressed mPGES-1, while mPGES-2 and cPGES were not detected. Isolated monkey theca cells expressed mPGES-1 mRNA and protein and produced PGE2 in vitro. Interestingly, mPGES-1 was expressed by theca cells of monkey periovulatory follicles obtained from both stimulated and spontaneous menstrual cycles, contrasting with a previous report of an unidentified form of mPGES in theca cells of bovine follicles obtained after ovarian stimulation but not during spontaneous estrous cycles (Filion et al., 2001Go). Isolated monkey theca cells also expressed COX-2, supporting our previous immunodetection of COX-2 in theca cells surrounding monkey periovulatory follicles after, but not before, exposure to an ovulatory dose of HCG in vivo (Duffy and Stouffer, 2001Go). However, studies examining COX expression in periovulatory follicles of rats, horses and cows showed no evidence of COX-2 expression by theca cells (Wong and Richards, 1991Go; Sirois, 1994Go; Sirois and Dore, 1997Go), although possible detection of COX-1 in rat theca cells has been reported (Wong and Richards, 1991Go). The present study, and previous studies by other laboratories, have shown that theca cells from periovulatory follicles obtained from humans (Patwardhan and Lanthier, 1981Go), rats (Zor et al., 1983Go), pigs (Evans et al., 1983Go) and now monkeys produce PGE2 in vitro, suggesting that theca cells of most, if not all, mammalian species express all enzymes needed for PGE2 synthesis. While COX-2 and mPGES-1 are likely responsible for PGE2 production by primate theca cells within the periovulatory follicle, expression of these specific enzymes by theca cells may not occur in all mammalian species. The preliminary studies presented here indicate that the expression of PG synthesis enzymes, as well as PGE2 production in vitro, by theca cells from the large antral follicles of a single ovary obtained 48-h post-HCG appear to be insensitive to gonadotropin exposure in vitro, perhaps as a result of prior gonadotropin exposure in vivo. While gonadotropin-stimulated COX-2 expression may increase theca PGE2 production after the ovulatory gonadotropin surge in vivo, the contribution of PGE2 produced by theca cells to ovulatory processes remains equivocal.

While PGE2 is well established as the key ovulatory PG in mammals, most studies have focused on examination of gonadotropin regulation of COX-2 expression within granulosa cells of periovulatory follicles. COX-2 converts arachidonic acid to the common PG precursor PGH2; in many cells this conversion is the rate-limiting step in PG production. In rodents and domestic animals, the ovulatory gonadotropin surge stimulates COX-2 expression by the granulosa cells of periovulatory follicles, and COX-2 protein levels peak just a few hours before increased PGE2 levels are measured in the follicular fluid of these species (Sirois and Dore, 1997Go). While the time between the ovulatory gonadotropin surge and initiation of COX-2 expression varied between species, the time between initiation of COX-2 expression, elevated follicular fluid PGE2 and follicle rupture was the same for several non-primate species, leading to the hypothesis that initiation of COX-2 expression was the primary determinant of the time at which ovulation would take place (Sirois and Dore, 1997Go). However, there is no direct evidence that COX-2 catalyses the rate-limiting step in periovulatory PGE2 production. In monkey periovulatory follicles, granulosa cell COX-2 expression is initiated soon after the ovulatory gonadotropin surge, but follicular fluid PGE2 levels do not peak until just before ovulation (Duffy and Stouffer, 2001Go), suggesting that an enzyme capable of converting the COX-2 product PGH2 into PGE2 may control the rate of PGE2 production by primate periovulatory follicles. While granulosa cell expression of mPGES-2 and cPGES, as well as theca production of PGE2, may play a role in periovulatory processes, our identification of mPGES-1 as a gonadotropin-stimulated gene product expressed by granulosa cells late in the periovulatory interval supports the hypothesis that mPGES-1 is the primary PGES form responsible for PGE2 production by the primate periovulatory follicle.


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to thank Dr Jerome F.Strauss 3rd for his assistance in the isolation of monkey theca cells and Ms Kim Hester for her role in animal training and animal protocols. Recombinant human gonadotropins and Antide used for these studies were generously provided by Serono Reproductive Biology Institute, Rockland, MA, USA. This study was supported by NIH grant HD39872 (D.M.D.).


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 4, 2004; resubmitted on January 12, 2005; accepted on January 17, 2005.