Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print August 30, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053769
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/6/868    most recent
biolreprod.106.053769v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.
Agricola
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.
BIOLOGY OF REPRODUCTION 75, 868–876 (2006)
DOI: 10.1095/biolreprod.106.053769
© 2006 by the Society for the Study of Reproduction, Inc.

research-article

Primate Granulosa Cell Response via Prostaglandin E2 Receptors Increases Late in the Periovulatory Interval1

Nune Markosyan 2, Brandy L. Dozier , Frank A. Lattanzio , and Diane M. Duffy 

Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507-1980

ABSTRACT

Successful ovulation requires elevated follicular prostaglandin E2 (PGE2) levels. To determine which PGE2 receptors are available to mediate periovulatory events in follicles, granulosa cells and whole ovaries were collected from monkeys before (0 h) and after administration of an ovulatory dose of hCG to span the 40-h periovulatory interval. All PGE2 receptor mRNAs were present in monkey granulosa cells. As assessed by immunofluorescence, PTGER1 (EP1) protein was low/nondetectable in granulosa cells 0, 12, and 24 h after hCG but was abundant 36 h after hCG administration. PTGER2 (EP2) and PTGER3 (EP3) proteins were detected by immunofluorescence in granulosa cells throughout the periovulatory interval, and Western blotting showed an increase in PTGER2 and PTGER3 levels between 0 h and 36 h after hCG. In contrast, PTGER4 (EP4) protein was not detected in monkey granulosa cells. Granulosa cell response to PGE2 receptor agonists was examined 24 h and 36 h after hCG administration, when elevated PGE2 levels present in periovulatory follicles initiate ovulatory events. PGE2 acts via PTGER1 to increase intracellular calcium. PGE2 increased intracellular calcium in granulosa cells obtained 36 h, but not 24 h, after hCG; this effect of PGE2 was blocked by a PTGER1 antagonist. A PTGER2-specific agonist and a PTGER3-specific agonist each elevated cAMP in granulosa cells obtained 36 h, but not 24 h, after hCG. Therefore, the granulosa cells of primate periovulatory follicles express multiple receptors for PGE2. Granulosa cells respond to agonist stimulation of each of these receptors 36 h, but not 24 h, after hCG, supporting the hypothesis that granulosa cells are most sensitive to PGE2 as follicular PGE2 levels peak, leading to maximal PGE2-mediated periovulatory effects just before ovulation.

granulosa cells, ovary, ovulation, signal transduction

INTRODUCTION

Prostaglandins (PGs) produced by the ovarian follicle are essential for ovulation to occur [1–3]. In primates as well as in other mammalian species, the ovulatory surge of LH acts in large periovulatory follicles to stimulate granulosa cell expression of PG synthesis enzymes [4–7]. Prostaglandin E2 (PGE2) has been identified as the key ovulatory PG in several mammalian species [8, 9]. Inhibition of prostaglandin synthesis by intrafollicular indomethacin injection prevented ovulation, whereas coadministration of indomethacin and PGE2 restored ovulation of monkey follicles, supporting a pivotal role for PGE2 in primate ovulation [9].

The actions of PGE2 are mediated by four different receptors: PTGER1, PTGER2, PTGER3, and PTGER4 (also known as EP1, EP2, EP3, and EP4, respectively). These G protein-coupled receptors differ in their signal transduction pathways. PTGER1 is coupled with intracellular calcium ([Ca2+]i) mobilization via G{alpha}q activation [10, 11]. PTGER2 and PTGER4 are coupled with cAMP production via activation of G{alpha}s and adenylyl cyclase [12]. Differential splicing of a transcript from a single gene yields multiple isoforms of the PTGER3 receptor, which either activate adenylyl cyclase via G{alpha}s, inhibit adenylyl cyclase via G{alpha}i, or increase [Ca2+]i via G{alpha}q [11, 13]. The presence and function of these PGE2 receptors have been studied in a variety of tissues and species [11, 14, 15]. However, very little is known about PGE2 receptors in the primate ovary and, more specifically, in periovulatory granulosa cells.

Successful ovulation requires functional PGE2 receptors to mediate the ovulatory effects of PGE2. PTGER2 receptors clearly play a critical role in ovulation and fertilization in mice. PTGER2 mRNA expression was induced in cumulus granulosa cells by the ovulatory gonadotropin surge [16]. Mice lacking PTGER2 expression had a severe deficiency in cumulus expansion, decreased ovulatory efficiency, and decreased fertilization rates [17–19]. Mice lacking expression of PTGER1 and PTGER3 had normal fertility, whereas PTGER4 knockout mice were not viable, leading to the conclusion that only PTGER2 receptors play a critical role in rodent ovulation [20]. The expression of functional PTGER1 and PTGER2 receptors has been reported in human luteinizing granulosa cells [21]. While the essential role of PGE2 in primate ovulation is well established, the expression and function of PGE2 receptors in primate periovulatory granulosa cells has received comparatively little attention.

The objectives of this study were to 1) determine which PGE2 receptors are expressed by primate granulosa cells during the periovulatory period and 2) identify the PGE2 receptors that are able to generate an intracellular response to PGE2 late in the periovulatory interval, when increased levels of PGE2 are crucial for successful ovulation.

MATERIALS AND METHODS

Animals

Granulosa cells and whole ovaries were obtained from adult female cynomolgus macaques at Eastern Virginia Medical School (Norfolk, VA). All animal protocols and experiments were approved by the Eastern Virginia Medical School Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Animal husbandry and sample collections were performed as described previously [22]. Briefly, adult females with regular menstrual cycles were checked daily for menstruation; the first day of menstruation was designated Day 1 of the menstrual cycle. Blood samples were obtained under ketamine chemical restraint (5–10 mg/kg body weight) by femoral venipuncture, and serum was stored at –20°C. Aseptic surgeries were performed in a dedicated surgical suite under isofluorane anesthesia and involved either midline laparotomy or laparoscopy.

A controlled ovarian stimulation model developed for the collection of multiple oocytes for in vitro fertilization was used to obtain monkey granulosa cells [23, 24]. Beginning within 3 days of initiation of menstruation, recombinant human FSH (90 IU daily; Serono Reproductive Biology Institute, Rockland, MA, and Organon Pharmaceuticals USA, Inc., Roseland, NJ) was administered for 6–8 days, followed by daily administration of 90 IU of recombinant human FSH plus 60 IU r-hLH (Serono) for 2–3 days to stimulate the growth of multiple follicles. The GnRH antagonist Antide (0.5 mg/kg body weight; Serono) was also administered daily to prevent an endogenous ovulatory LH surge. Adequate follicular development was monitored by serum estradiol levels and ultrasonography [25, 26]. Follicular aspiration was performed before (0 h) or 12, 24, and 36 h after administration of 1000 IU r-hCG (Serono). In spontaneous menstrual cycles, follicle rupture in monkeys occurs approximately 40 h after the ovulatory gonadotropin surge [27], 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 larger than 4 mm in diameter were pooled. Whole ovaries also were obtained from monkeys undergoing ovarian stimulation. Additional whole ovaries were collected from monkeys experiencing spontaneous menstrual cycles around the expected time of the endogenous LH surge [27]. These ovaries were obtained before (n = 2), during (n = 3), 1 day after (n = 2), and 2 days after (n = 3) the LH surge as defined by serum estradiol, LH, and progesterone concentrations before and at the time of organ removal [28]. Monkey kidney tissue was obtained at necropsy.

Tissue Preparation

Monkey granulosa cells and oocytes were pelleted from follicular aspirates by centrifugation. Oocytes were mechanically removed, and a granulosa cell-enriched population of the remaining cells was obtained by Percoll gradient centrifugation [28]. The viability of granulosa cell-enriched preparations averaged 76% as assessed by trypan blue exclusion. Cells either were used immediately for cell culture or frozen in liquid nitrogen and stored at –80°C for preparation of total RNA or cell lysates. Whole stimulated ovaries were bisected, maintaining at least two periovulatory follicles greater than 4 mm in diameter on each piece. The pieces were covered with O.C.T. compound (Sakura, Tokyo, Japan), frozen in liquid propane, and stored at –80°C. Natural cycle ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Monkey kidney tissue either was frozen in liquid propane and stored at –80°C or fixed in 4% paraformaldehyde and embedded in paraffin.

Real-Time RT-PCR

PGE2 receptor mRNA levels were analyzed by real-time RT-PCR using a Roche LightCycler (Roche Diagnostics, Indianapolis, IN). Total RNA was obtained from granulosa cells using Trizol reagent (Invitrogen, Rockville, MD) treated with DNase and reverse transcribed as previously described [6]. PCR was performed using the FastStart DNA Master SYBR Green I kit (Roche) following the manufacturer's instructions and using 0.5 µM primers and 4 mM MgCl2. Primers were designed using LightCycler Probe Design software (Roche) based on the human or monkey sequences (Table 1) and span an intron whenever possible to prevent undetected amplification of genomic DNA. PCR products were sequenced (Microchemical Core Facility, San Diego State University, CA) to confirm amplicon identity. At least 5 log dilutions of the sequenced PCR product were included in each assay and used to generate a standard curve. The lower limit of detection of the assay (15 copies per 40 ng cDNA) was used for statistical analysis whenever amplification was not detected. For each sample, the content of each PGE2 receptor mRNA and beta actin mRNA (ACTB) was determined in independent assays. No amplification was observed when monkey cDNA was omitted. All data were expressed as the ratio of PGE2 receptor mRNA to ACTB for each sample. Intraassay and interassay coefficients of variation were less than 10%.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1. Primer information for real-time RT-PCR.

Immunofluorescent Detection of PGE2 Receptors

Frozen ovarian and kidney tissues were sectioned at 10 µm and fixed with buffered 10% formalin. After antigen retrieval using Antigen Retrieval Citra treatment (BioGenex Labs, San Ramon, CA), the sections were blocked with 5% nonimmune goat serum (Vector Laboratories, Burlingame, CA) in PBS containing 0.1% Triton (PBS-Triton). Sections then were incubated overnight with either rabbit anti-human polyclonal primary antibody against one of the PGE2 receptors (PTGER1 [1 µg/ml], PTGER2 [5 µg/ml], PTGER3 [2 µg/ml], PTGER4 [3 µg/ml]) or primary antibody plus blocking peptide (Cayman Chemicals, Ann Arbor, MI), followed by incubation with a Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:500; Molecular Probes, Eugene, OR). After incubation with 1% Sudan Black in 70% methanol, the slides were coverslipped using Vectashield medium (Vector Laboratories). All images were obtained using an Olympus BX41 fluorescent microscope fitted with a DP70 digital camera and associated software (Olympus, Melville, NY).

Paraffin-embedded ovarian and kidney tissue sections (5 µm) were deparaffinized, exposed to antigen retrieval as described above, and incubated with Image-iT signal enhancer (Molecular Probes). After exposure to 5% nonimmune serum block in PBS-Triton, sections were incubated with streptavidin and biotin (Vector Laboratories). After the overnight incubation with either primary antibodies against each PGE2 receptor (PTGER1 [3 µg/ml], PTGER2 [1 µg/ml], PTGER3 [3 µg/ml], PTGER4 [3 µg/ml]) or with primary antibody plus blocking peptide followed by biotinylated anti-rabbit secondary antibodies (1:200; Vector Laboratories), the sections were treated with Strepavidin-Alexa Fluor 488 (Molecular Probes) and then with 1% Sudan Black, coverslipped, and photographed as described above.

Western Blotting

Western blotting was performed as previously described [22]. Briefly, denatured granulosa cell lysates were loaded onto 4%–20% gradient polyacrylamide Tris-HCl gels (Bio-Rad, Hercules, CA). Proteins were transferred to polyvinylidene fluoride membranes (Imobilon; Millipore, Billerica, MA), and Western blotting was carried out with antibodies against PTGER1 (6 µg/ml), PTGER2 (5 µg/ml), and PTGER3 (4 µg/ml) and an anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase-conjugated secondary antibody (1:10 000; Amersham, Piscataway, NJ). Bands were detected by enhanced chemiluminescence (ECL; Amersham). Blots then were 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 antitubulin primary antibody (1:1000; Sigma) and anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody (1:20 000; Amersham). The molecular sizes of bands representing each PGE2 receptor and tubulin were determined by comparison to prestained standards (Bio-Rad). Films were scanned and analyzed densitometrically using SigmaGel software (Jandel Scientific, San Rafael, CA). The PGE2 receptor and tubulin protein contents for each sample were calculated based on standard curves generated from four lysate dilutions. The PGE2 receptor content of each granulosa cell sample is expressed as a receptor:tubulin ratio.

Granulosa Cell Culture

Monkey granulosa cells were plated on 48-well or 96-well tissue culture plates or Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY) coated with fibronectin (Sigma, St. Louis, MO) at a concentration of 10 000 cells per 100 µl medium. The cells were cultured in serum-free Dulbecco modified Eagle medium (DMEM)-Ham F12 medium containing insulin (2 µg/ml), transferrin (5 µg/ml), selenium (0.25 nM), aprotinin (25 mg/ml), and human low-density lipoprotein (25 µg/ml) [29].

cAMP Measurements

Cells were cultured with the PTGER2 agonist butaprost, the PTGER3 agonist sulprostone, PGE2, PGE2 plus the PTGER1/PTGER2 antagonist AH6809 (Cayman Chemicals), or no treatment (control). Media were stored at –20°C until acetylated prior to assay for cAMP by enzyme immunoassay (Cayman Chemicals), which was performed and analyzed following kit instructions. The lower limit of detection (0.02 pmol cAMP/ml) was used for statistical analysis whenever cAMP levels were below detection. All cAMP levels were normalized to the protein content of cultured cells as determined by the bicinchoninic acid method (Sigma). Preliminary studies confirmed that media cAMP levels paralleled granulosa cell cAMP levels and that media cAMP accumulation was linear between 4 h and 24 h after agonist/antagonist addition (data not shown). For studies presented here, media for cAMP assay were collected 16 h after agonist/antagonist addition. Preliminary studies also demonstrated that vehicle (dimethyl sulfoxide [DMSO] ≤ 0.001%) did not affect cAMP accumulation. For each animal, data are presented as a ratio of agonist-stimulated cAMP level to the cAMP level in the untreated (control) cultures.

[Ca2+]i Measurements

For [Ca2+]i measurements, granulosa cells were incubated for 30 min in Hanks balanced salt solution containing 1.8 mM CaCl2 and the fluorescent dye Fluo 3AM (Molecular Probes) at 1:1000 dilution. The basal fluorescent emission of cells was determined before treatment (0 sec). Granulosa cells then were stimulated with PGE2 and imaged every 20 sec for 2 min. Finally, cells were exposed to the calcium ionophore calcimycin (Calbiochem, San Diego, CA); only cells with increased fluorescence after calcimycin treatment were included in quantitative analyses. A second culture of cells received 60-sec pretreatment with AH6809, after which PGE2 was added and the cells imaged as described above. Images of cells were obtained using a confocal microscope (Zeiss LSM–510; Carl Zeiss, Inc., Thornwood, NY) at absorption and emission wavelengths of 506 nm and 526 nm, respectively. For each treatment, 10 calcimycin-responsive cells were analyzed for fluorescence using MetaMorph software (Molecular Devices, Sunnyvale, CA), and [Ca2+]i was calculated using the following formula: [Ca2+]i = Kd (Ca2+) x (F – Fmin) / Fmax – F, where Kd (Ca2+) = 325 nM, Fmin is the fluorescence measured in the absence of Fluo 3AM in the medium, F is the fluorescence of the cells in the medium containing Fluo 3AM, and Fmax is the fluorescence after addition of calcimycin [30, 31]. For each animal, [Ca2+]i of cells before treatment was set equal to 1.0, and [Ca2+]i in the same cells after treatment was expressed relative to pretreatment levels.

Data Analysis

All data were assessed for heterogeneity of variance and were log-transformed when Bartlett test yielded a significance of less than 0.05. Data presented in Figure 1, C and D; Figure 4; and Figure 5 were log-transformed before further analysis. In all figures, untransformed data are presented. PCR and Western blot data were assessed by ANOVA. Data for cAMP and [Ca2+]i were assessed by ANOVA with one repeated measure. In all cases, posthoc analysis was performed using Duncan multiple range test. In addition, PTGER2 levels at 0 h and 36 h were compared by two-tail t-test. Statistical analyses were performed using StatPak v4.12 software (Northwest Analytical, Portland, OR). Data are presented as mean ± SEM, and significance was assumed at P < 0.05.


Figure 75060801
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1. PGE2 receptor mRNA expression in monkey granulosa cells. Granulosa cells were obtained from monkeys experiencing controlled ovarian stimulation before (0 h) and 12, 24, and 36 h after hCG administration. Levels of PTGER1 (A), PTGER2 (B), PTGER3 (C), and PTGER4 (D) mRNA were determined by real time RT-PCR; all mRNA levels are expressed relative to ACTB. Data are presented as mean ± SEM (n = 4 to 5 animals per timepoint). In C, groups with different superscripts are different by ANOVA and Duncan multiple range test, P < 0.05.


Figure 75060804
View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4. Production of cAMP by monkey granulosa cells. Granulosa cells obtained from monkeys experiencing controlled ovarian stimulation 24 h (A) and 36 h (B) after hCG administration were incubated for 16 h with butaprost (10 µM), sulprostone (1 µM), PGE2 (1 µM), PGE2 (1 µM) plus AH6809 (10 µM), or no treatment (control). Media cAMP was measured by enzyme immunoassay. For each animal the cAMP level in control cells was set equal to 1.0, and the cAMP level in treated cells was expressed relative to the control level. Data are presented as mean ± SEM (n = 5 to 6 animals per timepoint). Within each panel, groups with different superscripts are different by ANOVA and Duncan multiple range test, P < 0.05.


Figure 75060805
View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5. PGE2 regulation of [Ca2+]i in monkey granulosa cells. Granulosa cells were obtained from monkeys experiencing controlled ovarian stimulation either 24 h (A, B, C) or 36 h (D, E, F) after hCG administration. Cells were imaged before treatment (0 sec; A, D), 20 sec after addition of 1 µM PGE2 (B, E), and 60 sec after calcimycin addition (10 µM; C, F). In A, B, and C, arrows indicate two representative cells before and after treatment. Similarly, arrows indicate two representative cells in D, E, and F. Images are representative of 4 to 5 animals per timepoint. [Ca2+]i levels in 1 µM PGE2-treated cells (filled circles) and cells treated with 1 µM PGE2 plus 10 µM AH6809 (open circles) relative to basal [Ca2+]i level at 0 sec (represented by the horizontal line at intracellular calcium = 1.0) are plotted for the granulosa cells obtained 24 h (G) and 36 h (H) after hCG administration. Data are presented as mean ± SEM (n = 4 to 5 per timepoint). In H, groups with different superscripts are different by ANOVA and Duncan multiple range test, P < 0.05.

RESULTS

PGE2 Receptor mRNA Expression by Monkey Granulosa Cells

Monkey granulosa cell expression of PGE2 receptor mRNA was examined before and after administration of an ovulatory dose of hCG at times spanning the 40-h periovulatory interval in primates. PTGER1, PTGER2, PTGER3, and PTGER4 mRNA was detected in granulosa cells before (0 h) hCG administration (Fig. 1). Levels of PTGER1, PTGER2, and PTGER4 mRNA did not change in response to hCG exposure. In contrast, PTGER3 mRNA levels were low before (0 h), elevated at 12 h, and remained elevated 24–36 h after hCG administration.

PGE2 Receptor Proteins in Monkey Granulosa Cells

PTGER1 protein was not detected in granulosa cells of monkey periovulatory follicles by immunofluorescence at 0 h and at 12 h after hCG (Fig. 2A and data not shown). There was sporadic PTGER1 fluorescence in granulosa cells of ovaries obtained 24 h after hCG, but PTGER1 was detected consistently in granulosa cells of ovaries obtained 36 h after hCG (Fig. 2, B and C). PTGER1 detection was restricted to the extranuclear regions of granulosa cells. Ovarian stroma did not contain detectable PTGER1. PTGER1 immunofluorescence also was present in the monkey kidney (Fig. 2D), confirming previous reports [15, 32] and serving as a positive control. PTGER1 was not detected when the primary antibody was omitted (data not shown) or preincubated with the blocking peptide (Fig. 2, C and D, insets). PTGER1 was detected in monkey kidney lysates by Western blotting as a single band of 64 kDa, but PTGER1 was not detected in lysates of granulosa cells (data not shown).


Figure 75060802
View larger version (82K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2. Immunofluorescent detection of PGE2 receptors in monkey granulosa cells. Ovaries were obtained from monkeys experiencing controlled ovarian stimulation at 0 h (A, E, I, M), 24 h (B, F, J, N), and 36 h (C, G, K, O) after hCG administration. In each periovulatory follicle shown, the antrum (a) is in the upper right, granulosa cells (gc) are central, and the ovarian stroma (s) is in the lower left portion of the image. Immunodetection (bright green) of PTGER1 (A, B, C), PTGER2 (E, F, G), PTGER3 (I, J, K), and PTGER4 (M, N, O) in periovulatory follicles is shown. Immunodetection of PTGER1 (D), PTGER2 (H), PTGER3 (L), and PTGER4 (P) in monkey kidney served as a positive control. No fluorescence was observed when the primary antibodies were preabsorbed with the blocking peptide (C, D, G, H, K, L, O, P, insets). Ovarian images shown are representative of 3 to 4 animals per timepoint. Bar = 50 µm.

The PTGER2 protein was detectable by immunofluorescence in the extranuclear region of granulosa cells of periovulatory follicles obtained 0, 12, 24, and 36 h after hCG (Fig. 2, E, F, and G and data not shown). PTGER2 immunofluorescence was not observed in ovarian stroma. Monkey kidney tissue exhibited low levels of PTGER2 protein detection (Fig. 2H), as has been reported by others [15, 33]. As with PTGER1, PTGER2 immunofluorescence was absent when the primary antibody was omitted or preincubated with the blocking peptide (Fig. 2, G and H, insets, and data not shown). Western blotting using this PTGER2 antibody detected a single 52-kDa band in the lysates of granulosa cells obtained 0, 24, and 36 h after hCG (Fig. 3A); this band was eliminated when the primary antibody was preincubated with the blocking peptide (data not shown). While ANOVA showed a tendency for PTGER2 levels to increase with hCG exposure (P = 0.07), PTGER2 levels increased between 0 h and 36 h as determined by t-test (P = 0.02; Fig. 3C).


Figure 75060803
View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3. PTGER2 and PTGER3 proteins in monkey granulosa cells. Granulosa cells obtained before (0 h) and 24 h and 36 h after hCG administration were subjected to Western blotting for PTGER2 (A), PTGER3 (B), and tubulin, which was detected as a single band of 50 kDa in each sample. Protein levels for PTGER2 (C) and PTGER3 (D) are expressed relative to the tubulin content of each sample. Data are expressed as mean ± SEM (n = 3 to 4 animals per timepoint). In C, PTGER2 protein is different between 0 h and 36 h groups by two-tailed t-test (P = 0.02). In D, groups with different superscripts are different by ANOVA and Duncan multiple range test, P < 0.05.

PTGER3 was detected by immunofluorescence in granulosa cells of periovulatory follicles obtained 0, 12, 24, and 36 h after hCG; PTGER3 also was observed throughout the ovarian stroma (Fig. 2, I–K and data not shown). PTGER3 was localized to the extranuclear regions of granulosa and stromal cells. In the kidney, PTGER3 was detected in tubules (Fig. 2L), as previously reported for other species [34, 35]. PTGER3 immunofluorescence was not present in granulosa cells when the primary antibody was omitted or preabsorbed with the blocking peptide (Fig. 2, K and L, insets, and data not shown). PTGER3 protein was detected in granulosa cells by Western blotting as multiple bands, with the strongest band at 65 kDa (Fig. 3B). All bands were absent when blots were probed with primary antibody preincubated with the blocking peptide (data not shown). PTGER3 levels increased in response to hCG, with peak levels measured in granulosa cells obtained 36 h after hCG administration (Fig. 3D).

PTGER4 protein was not detected by immunofluorescence in granulosa cells of periovulatory follicles obtained 0, 12, 24, and 36 h after hCG administration; PTGER4 was, however, consistently detected throughout the ovarian stroma (Fig. 2, M–O and data not shown). As previously reported [15, 32, 35], PTGER4 protein showed high levels of expression in kidney glomeruli (Fig. 2P), which served as a positive control. PTGER4 immunofluorescence was not present when the primary antibody was preabsorbed or omitted (Fig. 2, O and P, insets, and data not shown). PTGER4 was not detected by immunofluorescence in monkey granulosa cells, so Western blotting for this PGE2 receptor was not performed.

Immunofluorescent detection of PGE2 receptors in sections of monkey ovaries obtained during natural menstrual cycles showed a pattern similar to that obtained when using stimulated ovaries as described above (data not shown). PTGER1 was not detected in the granulosa cells of follicles obtained before or immediately after the ovulatory LH surge, but PTGER1 was present in granulosa cells of periovulatory follicles obtained on Day 1 and Day 2 after the LH surge. PTGER2 and PTGER3 were detectable in granulosa cells of periovulatory follicles obtained before and after LH surge. PTGER4 was low/nondetectable in granulosa cells at all times examined. These data demonstrate that the expression and localization of PGE2 receptor proteins described above for stimulated monkey ovaries are consistent with PGE2 receptor localization and gonadotropin-regulated expression as seen in ovaries obtained during natural menstrual cycles.

Intracellular Signal Generation in Response to PGE2 Receptor Agonists

In order to determine which PGE2 receptors respond to PGE2 in a manner consistent with a role in ovulation, we examined the ability of each PGE2 receptor to respond to a receptor-specific agonist with the generation of an appropriate intracellular signal. Follicular PGE2 concentrations are elevated in response to gonadotropin 24–36 h after hCG administration [22], and PGE2 is hypothesized to mediate the essential ovulatory effect of prostaglandins during this time interval. For this reason, the ability of granulosa cells to respond to PGE2 receptor agonists was examined specifically in granulosa cells obtained 24 h and 36 h after hCG administration. Because PTGER4 was not consistently detected in granulosa cells of ovaries obtained after controlled ovarian stimulation or during natural menstrual cycles, the ability of granulosa cells to respond via PGE2 receptors was examined only for PTGER1, PTGER2, and PTGER3.

cAMP Generation

To determine if stimulation of either PTGER2 or PTGER3 regulates cAMP levels via interactions with G{alpha}s or G{alpha}i, granulosa cells were cultured with PTGER2 and PTGER3 agonists. In granulosa cells obtained 24 h after hCG, the PTGER2 agonist butaprost did not increase cAMP to levels above those in controls (Fig. 4A). Similarly, the PTGER3 agonist sulprostone did not alter cAMP levels in granulosa cells obtained 24 h after hCG administration. While stimulation of either PTGER2 or PTGER3 did not increase cAMP above control levels, addition of PGE2 to cultures of granulosa cells obtained 24 h after hCG increased cAMP levels 5-fold above levels measured in control cells. The ability of PGE2 to elevate media cAMP was abolished by co-incubation with the PTGER1/PTGER2 antagonist AH6809. In the same cells, treatment with butaprost plus sulprostone increased cAMP to levels that were not different from cAMP levels measured after stimulation with PGE2 (3.38 ± 1.23 and 7.02 ± 2.59 fold above control, respectively, n = 3). In summary, these studies indicate that stimulation of either PTGER2 or PTGER3 did not increase intracellular cAMP in granulosa cells obtained 24 h after hCG. However, activation of both PTGER2 and PTGER3 with either PGE2 or the combination of butaprost and sulprostone was able to increase intracellular cAMP above control levels.

The ability of PGE2 receptor agonists to alter cAMP accumulation in granulosa cells obtained 36 h after hCG also was examined. Butaprost stimulated a 4-fold increase in cAMP, whereas sulprostone doubled cAMP levels compared with controls (Fig. 4B). PGE2 treatment induced an 8-fold increase in media cAMP, which was eliminated by coincubation with AH6809. In the same cells, treatment with butaprost plus sulprostone yielded cAMP levels that were not different from PGE2-stimulated cAMP levels (5.64 ± 1.98 and 6.12 ± 1.98 fold above control, respectively, n = 3). Therefore, stimulation of both PTGER2 and PTGER3 with either PGE2 or the combination of butaprost plus sulprostone also increased cAMP above the levels measured in control cultures. Most importantly, stimulation of either PTGER2 or PTGER3 resulted in significantly increased cAMP levels.

Intracellular Calcium

To determine whether PTGER1 stimulation leads to changes in granulosa cell [Ca2+]i, response to treatment with PGE2 or PGE2 plus the PTGER1/PTGER2 antagonist AH6809 was assessed. Granulosa cells obtained 24 h after hCG administration failed to increase [Ca2+]i in response to PGE2, but increased fluorescence was measured in response to calcimycin treatment (Fig. 5, A–C). Treatment with PGE2 or PGE2 plus AH6809 did not alter [Ca2+]i when compared to basal levels (Fig. 5G).

In granulosa cells obtained 36 h after hCG, PGE2 treatment doubled [Ca2+]i after 20 sec (Fig. 5, D and E). As anticipated, this increase in [Ca2+]i was transient and declined after the initial peak. However, by 120 sec after treatment [Ca2+]i levels remained elevated above the levels in the same cells before the treatment. The PTGER1/PTGER2 antagonist AH6809 abolished the PGE2-stimulated increase in [Ca2+]i (Fig. 5H).

DISCUSSION

The present study is the first to examine the expression and function of all PGE2 receptors in primate periovulatory granulosa cells. Much attention has focused on the identification of PGE2 as the essential ovulatory PG as well as the ability of the ovulatory gonadotropin surge to increase granulosa cell expression of PG synthesis enzymes, resulting in peak follicular PGE2 levels just before follicle rupture [5–7, 36]. The data presented in this report suggest that the ovulatory gonadotropin surge also regulates the ability of granulosa cells to respond to PGE2 during the periovulatory period. Exposure to an ovulatory dose of hCG increased granulosa cell expression of PTGER2, PTGER3, and, possibly, PTGER1 receptors. In addition, granulosa cells obtained 24 h after hCG administration did not respond to stimulation of PTGER1, PTGER2, or PTGER3 with altered intracellular signals; granulosa cells obtained 36 h after hCG responded to agonists specific for these three PGE2 receptors. Taken together, these data support the hypothesis that the ovulatory gonadotropin surge increases PGE2 receptor expression by granulosa cells to achieve peak granulosa cell response to PGE2 in the hours just before ovulation.

In the present study, mRNA for PTGER1, PTGER2, PTGER3, and PTGER4 was detected in the granulosa cells of monkey periovulatory follicles before and after hCG administration. Studies by others have shown that mRNA for PTGER1, PTGER2, and PTGER4 are expressed in either freshly isolated or long-term cultured human luteinizing granulosa cells [21, 37]; PTGER3 expression in human granulosa cells has not been reported. In bovine cumulus granulosa cells, expression of PTGER2, PTGER3, and PTGER4 mRNA has been detected [38, 39]. Reports of subfertility in PTGER2 knockout mice [17] focused attention on PTGER2 expression and function in rodent granulosa cells [40]. However, recent detection of PTGER2 and PTGER4, but not PTGER1 and PTGER3, mRNA in mouse granulosa cells supports the hypothesis that multiple PGE2 receptors mediate the ovulatory effects of PGE2 [16]. Clearly, granulosa cell PGE2 receptor expression differs between species. These data, combined with our detection of mRNA for all PGE2 receptors in primate granulosa cells, suggest that the ovulatory effects of PGE2 may require multiple PGE2 receptors.

The present study also demonstrates that primate granulosa cells express PTGER1, PTGER2, and PTGER3 proteins. Granulosa cell levels of PTGER1 were too low for detection by Western blotting, which is consistent with previous reports that PTGER1 levels are very low except in epidermis, kidney, and cell lines transfected to express abundant PTGER1 [41, 42]. However, consistent immunofluorescent detection of PTGER1 in granulosa cells obtained 36 h after hCG but low/nondetectable PTGER1 in granulosa cells obtained 0–24 h after hCG suggests that the levels of PTGER1 in granulosa cells may increase in response to the ovulatory gonadotropin surge. PTGER2 and PTGER3 both were detected in granulosa cells obtained before and at all times after hCG administration. Granulosa cell levels of PTGER2 increased in response to hCG despite unchanged levels of PTGER2 mRNA, whereas levels of both PTGER3 mRNA and protein increased in response to hCG administration. Levels of PTGER2 and PTGER3 receptor proteins peaked 36 h after hCG administration, just before ovulation. Interestingly, PTGER4 protein was not detected in monkey granulosa cells, despite the presence of PTGER4 mRNA at every timepoint examined. To our knowledge, this study represents the first examination of PGE2 receptor proteins in primate granulosa cells. Our data indicate that the ovulatory gonadotropin surge stimulates granulosa cell expression of at least two, and possibly three, PGE2 receptors to achieve maximal receptor concentrations just before ovulation, when follicular PGE2 levels reach their peak.

The PGE2 receptors that mediate the ovulatory PGE2 signal in primates have not been identified. Mice lacking expression of PTGER2 have decreased ovulation and fertilization efficiency [17, 19]. In contrast, deficiencies of PTGER1 and PTGER3 expression did not negatively impact reproductive function [17–19, 43]. In the primate periovulatory follicle, PGE2 begins to accumulate in follicular fluid 24 h after hCG administration and reaches peak levels 36 h after hCG, or just before ovulation [5–7, 22]. Therefore, the PGE2 receptors that mediate the ovulatory PGE2 signal should be both present on granulosa cells and capable of intracellular signal generation 24–36 h after hCG administration. The present study demonstrates that primate granulosa cells have PTGER1, PTGER2, and PTGER3 receptors available to mediate the ovulatory effects of PGE2. Therefore, additional studies were performed to determine which PGE2 receptors were capable of transducing the PGE2 signal during this period of elevated PGE2 that precedes primate ovulation.

PTGER1 couples with G{alpha}q to activate phospholipase C and produce inositol trisphosphate, which increases [Ca2+]i by releasing calcium from intracellular stores [11, 14]. PGE2 treatment increased [Ca2+]i when granulosa cells were obtained 36 h after hCG administration in vivo. However, granulosa cells obtained 24 h after hCG did not respond to PGE2 with increased [Ca2+]i. These data are supported by the consistent immunofluorescent detection of PTGER1 protein in granulosa cells at 36 h, but not 24 h, after hCG administration. The ability of PGE2 to increase [Ca2+]i was abolished by the PTGER1/PTGER2 receptor antagonist AH6809. It is unlikely that PGE2 acts via PTGER2 receptors to increase [Ca2+]i, as PTGER2 receptors couple exclusively to G{alpha}s to increase cAMP [14, 44]. The ability of AH6809 to block the PGE2-stimulated increase in [Ca2+]i also suggests that PGE2 does not act via PTGER3 receptors coupled with G{alpha}q. Taken together, these data best support the hypothesis that PGE2 increases [Ca2+]i solely via PTGER1 receptors. The presence of functional PTGER1 receptors only 36 h after hCG, late in the periovulatory interval, suggests that PTGER1 may mediate some of the ovulatory effects of PGE2 in primate granulosa cells.

PTGER2 couples with G{alpha}s and activates adenylyl cyclase to stimulate cAMP production [14]. While granulosa cells obtained 24 h after hCG expressed PTGER2, the PTGER2-specific agonist butaprost did not increase intracellular cAMP. In contrast, stimulation of PTGER2 with butaprost increased cAMP levels above control levels in granulosa cells obtained at 36 h; these cells also had the highest levels of PTGER2 protein. The PTGER1/PTGER2 antagonist AH6809 reduced or abolished PGE2-stimulated cAMP production. Because PTGER1 receptors couple exclusively to G{alpha}q, these data support the conclusion that PGE2 stimulates adenylyl cyclase activity at least partially through the PTGER2 receptor. While PGE2 may be more efficacious than butaprost as a ligand for the monkey PTGER2 receptors, PGE2 also may act through other PGE2 receptors in addition to PTGER2 to achieve maximal cAMP production. Taken together, our data are consistent with studies in mouse and human granulosa cells [16, 21] and demonstrate that primate periovulatory granulosa cells express functional PTGER2 receptors. These data also support the hypothesis that primate granulosa cells respond to stimulation of PTGER2 receptors with increased cAMP at 36 h, but not 24 h, after hCG, or just before ovulation.

PTGER3 receptors in monkey granulosa cells couple primarily, if not exclusively, with G{alpha}s. Numerous isoforms of PTGER3 have been characterized to date [45]. The PCR primers used in this study were designed to detect all 10 known human PTGER3 mRNA splice variants, and Western blotting consistently identified multiple PTGER3 isoforms in monkey periovulatory granulosa cells. PTGER3 receptors, which couple to G{alpha}i and G{alpha}q, have been reported in human cells [46]. Granulosa cells obtained 24 h after hCG did not alter intracellular cAMP in response to sulprostone, the PTGER3-specific agonist. In granulosa cells obtained 36 h after hCG administration, treatment with sulprostone increased cAMP, suggesting that one or more PTGER3 isoforms couple with G{alpha}s in primate granulosa cells. Additionally, sulprostone did not decrease basal or butaprost-stimulated cAMP, so it is unlikely that PTGER3 couples with G{alpha}i in these cells. As discussed above, PTGER3 receptors coupled with G{alpha}q were not deteceted in monkey granulosa cells. Therefore, granulosa cells likely respond to the ovulatory gonadotropin surge with an increase in the expression of G{alpha}s-coupled PTGER3 receptors, with PGE2 acting in part via PTGER3 to increase cAMP production late in the periovulatory interval.

These studies focused on the expression and activity of PGE2 receptors in granulosa cells. Follicular aspirates used for detection of mRNA and proteins by Western blotting likely contain primarily mural granulosa cells, with some cumulus cells present. However, immunofluorescent detection of PGE2 receptors in tissue sections was restricted to mural granulosa cells, as the large size of the macaque follicle results in very few tissue sections containing a portion of the cumulus oocyte complex. While PTGER3 and PTGER4 were detected in ovarian stroma, localization to theca cells was not performed. Monkey theca cells do not exist as a multicell layer but instead are present sporadically outside the granulosa cell basement membrane [47], so PGE2 receptor expression cannot be confirmed for theca cells using the data presented here. Future studies will be required to examine PGE2 receptor expression by these important follicular cell types.

In summary, this study is the first to demonstrate expression and function of three distinct PGE2 receptors in primate periovulatory granulosa cells. Exposure of granulosa cells to an ovulatory dose of hCG increased levels of PTGER2, PTGER3, and, possibly, PTGER1 receptor proteins; granulosa cells responded to stimulation of each of these PGE2 receptors with increased intracellular signal generation only just before ovulation. These PGE2 receptors may regulate different intracellular events, suggesting that PGE2 may use multiple pathways to stimulate periovulatory events in primate granulosa cells. The present data support the hypothesis that the ovulatory gonadotropin surge increases the expression and function of PGE2 receptors as well as PGE2 levels within the follicle. Both PGE2 levels and PGE2 receptor responses peak just before ovulation, ensuring the maximal ovulatory effect of PGE2.

ACKNOWLEDGMENTS

The authors would like to thank Kim Hester for her role in animal training and animal protocols, and Carrie Seachord and Marcia Burch for their help in protein detection studies. Recombinant human gonadotropins and Antide were generously provided by Serono Reproductive Biology Institute, Rockland, MA, and Organon Pharmaceuticals USA, Inc., Roseland, NJ.

FOOTNOTES

1Supported by National Institutes of Health grant HD39872 to D.M.D., and Eastern Virginia Medical School. Back

Correspondence: 2 Nune Markosyan, Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Rd., Norfolk VA 23507-1980. FAX: 757 624 2269; e-mail: markosn{at}evms.edu

Received: 10 May 2006.

First decision: 2 June 2006.

Accepted: 27 August 2006.

REFERENCES

  1. Sirois J, Sayasith K, Brown KA, Stock AE, Bouchard N, Dore M. Cyclooxygenase-2 and its role in ovulation: a 2004 account. Hum Reprod Update 2004; 10:373–385[Abstract/Free Full Text]
  2. Murdoch WJ, Hansen TR, McPherson LA. A review–role of eicosanoids in vertebrate ovulation. Prostaglandins 1993; 46:85–115[CrossRef][Medline]
  3. Pall M, Friden BE, Brannstrom M. Induction of delayed follicular rupture in the human by the selective COX-2 inhibitor rofecoxib: a randomized double-blind study. Hum Reprod 2001; 16:1323–1328[Abstract/Free Full Text]
  4. Sirois J. Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo. Endocrinology 1994; 135:841–848[Abstract]
  5. Duffy DM and Stouffer RL. The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and prostaglandin production by the monkey follicle. Mol Hum Reprod 2001; 7:731–739[Abstract/Free Full Text]
  6. Duffy DM, Seachord CL, Dozier BL. An ovulatory gonadotropin stimulus increases cytosolic phospholipase A2 expression and activity in granulosa cells of primate periovulatory follicles. J Clin Endocrinol Metab 2005; 90:5858–5865[Abstract/Free Full Text]
  7. Duffy DM, Seachord CL, Dozier BL. Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle. Hum Reprod 2005; 20:1485–1492[Abstract/Free Full Text]
  8. Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, Langenbach R. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta. Endocrinology 1999; 140:2685–2695[Abstract/Free Full Text]
  9. Duffy DM and Stouffer RL. Follicular administration of a cyclooxygenase inhibitor can prevent oocyte release without alteration of normal luteal function in rhesus monkeys. Hum Reprod 2002; 17:2825–2831[Abstract/Free Full Text]
  10. Funk CD, Furci L, FitzGerald GA, Grygorczyk R, Rochette C, Bayne MA, Abramovitz M, Adam M, Metters KM. Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype. J Biol Chem 1993; 268:26767–26772[Abstract/Free Full Text]
  11. Asboth G, Phaneuf S, Europe-Finner GN, Toth M, Bernal AL. Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: involvement of EP1 and EP3 receptor subtypes. Endocrinology 1996; 137:2572–2579[Abstract]
  12. Coleman RA, Eglen RM, Jones RL, Narumiya S, Shimizu T, Smith WL, Dahlen SE, Drazen JM, Gardiner PJ, Jackson WT, et al. Prostanoid and leukotriene receptors: a progress report from the IUPHAR working parties on classification and nomenclature. Adv Prostaglandin Thromboxane Leukot Res 1995; 23:283–285[Medline]
  13. Yang J, Xia M, Goetzl EJ, An S. Cloning and expression of the EP3-subtype of human receptors for prostaglandin E2. Biochem Biophys Res Commun 1994; 198:999–1006[CrossRef][Medline]
  14. Breyer RM, Bagdassarian CK, Myers SA, Breyer MD. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 2001; 41:661–690[CrossRef][Medline]
  15. Breyer MD and Breyer RM. Prostaglandin E receptors and the kidney. Am J Physiol Renal Physiol 2000; 279:F12–F23[Abstract/Free Full Text]
  16. Segi E, Haraguchi K, Sugimoto Y, Tsuji M, Tsunekawa H, Tamba S, Tsuboi K, Tanaka S, Ichikawa A. Expression of messenger RNA for prostaglandin E receptor subtypes EP4/EP2 and cyclooxygenase isozymes in mouse periovulatory follicles and oviducts during superovulation. Biol Reprod 2003; 68:804–811[Abstract/Free Full Text]
  17. Hizaki H, Segi E, Sugimoto Y, Hirose M, Saji T, Ushikubi F, Matsuoka T, Noda Y, Tanaka T, Yoshida N, Narumiya S, Ichikawa A. Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2). Proc Natl Acad Sci U S A 1999; 96:10501–10506[Abstract/Free Full Text]
  18. Kennedy CR, Zhang Y, Brandon S, Guan Y, Coffee K, Funk CD, Magnuson MA, Oates JA, Breyer MD, Breyer RM. Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor. Nat Med 1999; 5:217–220[CrossRef][Medline]
  19. Tilley SL, Audoly LP, Hicks EH, Kim HS, Flannery PJ, Coffman TM, Koller BH. Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor. J Clin Invest 1999; 103:1539–1545[Medline]
  20. Prostaglandins Other Lipid Mediat Kobayashi T and Narumiya S. Function of prostanoid receptors: studies on knockout mice. 2002;557–573
  21. Harris TE, Squires PE, Michael AE, Bernal AL, Abayasekara DR. Human granulosa-lutein cells express functional EP1 and EP2 prostaglandin receptors. Biochem Biophys Res Commun 2001; 285:1089–1094[CrossRef][Medline]
  22. Duffy DM, Dozier BL, Seachord CL. Prostaglandin dehydrogenase and prostaglandin levels in periovulatory follicles: implications for control of primate ovulation by prostaglandin E2. J Clin Endocrinol Metab 2005; 90:1021–1027[Abstract/Free Full Text]
  23. Vandevoort CA, Baughman WL, Stouffer RL. Comparison of different regimens of human gonadotropins for superovulation of rhesus monkeys: ovulatory response and subsequent luteal function. J In Vitro Fert Embryo Transf 1989; 6:85–91[Medline]
  24. Hibbert ML, Stouffer RL, Wolf DP, Zelinski-Wooten MB. Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates. Proc Natl Acad Sci U S A 1996; 93:1897–1901[Abstract/Free Full Text]
  25. Wolf DP, Alexander M, Zelinski-Wooten M, Stouffer RL. Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles. Mol Reprod Dev 1996; 43:76–81[CrossRef][Medline]
  26. VandeVoort CA and Tarantal AF. The macaque model for in vitro fertilization: superovulation techniques and ultrasound-guided follicular aspiration. J Med Primatol 1991; 20:110–116[Medline]
  27. Weick RF, Dierschke DJ, Karsch FJ, Butler WR, Hotchkiss J, Knobil E. Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology 1973; 93:1140–1147[Medline]
  28. Chaffin CL, Hess DL, Stouffer RL. Dynamics of periovulatory steroidogenesis in the rhesus monkey follicle after ovarian stimulation. Hum Reprod 1999; 14:642–649[Abstract/Free Full Text]
  29. Schipper I, Fauser BC, van Gaver EB, Zarutskie PW, Dahl KD. Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness. Hum Reprod 1993; 8:1380–1386[Abstract/Free Full Text]
  30. Lipp P and Niggli E. Modulation of Ca2+ release in cultured neonatal rat cardiac myocytes. Insight from subcellular release patterns revealed by confocal microscopy. Circ Res 1994; 74:979–990[Abstract/Free Full Text]
  31. Bootman M, Niggli E, Berridge M, Lipp P. Imaging the hierarchical Ca2+ signalling system in HeLa cells. J Physiol 1997; 499(Pt 2):307–314[Medline]
  32. Sugimoto Y, Namba T, Shigemoto R, Negishi M, Ichikawa A, Narumiya S. Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney. Am J Physiol 1994; 266:F823–F828[Medline]
  33. Nemoto K, Pilbeam CC, Bilak SR, Raisz LG. Molecular cloning and expression of a rat prostaglandin E2 receptor of the EP2 subtype. Prostaglandins 1997; 54:713–725[CrossRef][Medline]
  34. Breyer MD, Jacobson HR, Davis LS, Breyer RM. In situ hybridization and localization of mRNA for the rabbit prostaglandin EP3 receptor. Kidney Int 1993; 44:1372–1378[Medline]
  35. Breyer MD, Davis L, Jacobson HR, Breyer RM. Differential localization of prostaglandin E receptor subtypes in human kidney. Am J Physiol 1996; 270:F912–F918[Medline]
  36. Sirois J and Dore M. The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 1997; 138:4427–4434[Abstract/Free Full Text]
  37. Narko K, Saukkonen K, Ketola I, Butzow R, Heikinheimo M, Ristimaki A. Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells. J Clin Endocrinol Metab 2001; 86:1765–1768[Abstract/Free Full Text]
  38. Calder MD, Caveney AN, Sirard MA, Watson AJ. Effect of serum and cumulus cell expansion on marker gene transcripts in bovine cumulus-oocyte complexes during maturation in vitro. Fertil Steril 2005; 83(suppl 1):1077–1085[CrossRef][Medline]
  39. Calder MD, Caveney AN, Westhusin ME, Watson AJ. Cyclooxygenase-2 and prostaglandin E(2)(PGE(2)) receptor messenger RNAs are affected by bovine oocyte maturation time and cumulus-oocyte complex quality, and PGE(2) induces moderate expansion of the bovine cumulus in vitro. Biol Reprod 2001; 65:135–140[Abstract/Free Full Text]
  40. Ochsner SA, Russell DL, Day AJ, Breyer RM, Richards JS. Decreased expression of tumor necrosis factor-alpha-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 2003; 144:1008–1019[Abstract/Free Full Text]
  41. Konger RL, Billings SD, Thompson AB, Morimiya A, Ladenson JH, Landt Y, Pentland AP, Badve S. Immunolocalization of low-affinity prostaglandin E receptors, EP1 and EP2, in adult human epidermis. J Invest Dermatol 2005; 124:965–970[CrossRef][Medline]
  42. Fang KM, Shu WH, Chang HC, Wang JJ, Mak OT. Study of prostaglandin receptors in mitochondria on apoptosis of human lung carcinoma cell line A549. Biochem Soc Trans 2004; 32:1078–1080[CrossRef][Medline]
  43. Ushikubi F, Sugimoto Y, Ichikawa A, Narumiya S. Roles of prostanoids revealed from studies using mice lacking specific prostanoid receptors. Jpn J Pharmacol 2000; 83:279–285[CrossRef][Medline]
  44. Ushikubi F, Hirata M, Narumiya S. Molecular biology of prostanoid receptors; an overview. J Lipid Mediat Cell Signal 1995; 12:343–359[CrossRef][Medline]
  45. Irie A, Sugimoto Y, Namba T, Harazono A, Honda A, Watabe A, Negishi M, Narumiya S, Ichikawa A. Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase. Eur J Biochem 1993; 217:313–318[Medline]
  46. Schmid A, Thierauch KH, Schleuning WD, Dinter H. Splice variants of the human EP3 receptor for prostaglandin E2. Eur J Biochem 1995; 228:23–30[Medline]
  47. Sanders SL and Stouffer RL. Localization of steroidogenic enzymes in macaque luteal tissue during the menstrual cycle and simulated early pregnancy: immunohistochemical evidence supporting the two-cell model for estrogen production in the primate corpus luteum. Biol Reprod 1997; 56:1077–1087[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/6/868    most recent
biolreprod.106.053769v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.
Agricola
Right arrow Articles by Markosyan, N.
Right arrow Articles by Duffy, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS