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
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Abstract |
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Key words: granulosa cell/ovary/ovulation/prostaglandin/theca cell
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Introduction |
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The ovulatory surge of gonadotropin stimulates the production of PGs, including PGE2, by the periovulatory follicle (Wong and Richards, 1991; Sirois and Dore, 1997
; Duffy and Stouffer, 2001
). 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, 2001
). 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. 1997
). In contrast, a third form of PGES (cPGES) is described as a cytosolic enzyme, not associated with intracellular membranes (Tanioka et al., 2000
). 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., 2000
; Murakami et al., 2003
). 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., 2001) and rat (Guan et al., 2001
) 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.
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Methods |
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A controlled ovarian stimulation (COS) model developed for the collection of multiple oocytes for IVF was used to obtain monkey granulosa cells (n=45/time point) (Duffy et al., 2005). Briefly, monkeys received 68 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., 1996
). 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., 1973
), 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=34/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., 1973). 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., 1999). 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., 1999
). 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). 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., 1989
). 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, 2003
). 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 RTPCR
Levels of mRNA for PG synthesis enzymes as well as -actin were analysed by real time RTPCR 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, 1999
). Amplification of all mRNA was performed using the FastStart DNA Master SYBR Green I kit (Roche), with the exception of human
-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
-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
-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
-actin mRNA in each sample was determined in independent assays. All data were expressed as the ratio of PG synthesis enzyme mRNA to
-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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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, 2001). 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-hydroxylase (1:1000; Leavitt et al., 1999
) 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
-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 NewmanKeuls test. One data point (mPGES-1 mRNA 12 h HCG) was determined to be a statistical outlier (Dean and Dixon, 1951) 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 KruskalWallis 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.
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Results |
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To confirm PG synthesis enzyme expression and PGE2 production by monkey theca cells, theca cells were isolated from monkey follicles. Immunocytochemical detection of 17-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., 1993
). 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
-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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Discussion |
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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., 2001). 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, 2001
). 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, 1991
; Sirois, 1994
; Sirois and Dore, 1997
), although possible detection of COX-1 in rat theca cells has been reported (Wong and Richards, 1991
). The present study, and previous studies by other laboratories, have shown that theca cells from periovulatory follicles obtained from humans (Patwardhan and Lanthier, 1981
), rats (Zor et al., 1983
), pigs (Evans et al., 1983
) 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, 1997). 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, 1997
). 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, 2001
), 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.
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Acknowledgements |
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References |
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Submitted on November 4, 2004; resubmitted on January 12, 2005; accepted on January 17, 2005.