help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0980
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/10/5858    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duffy, D. M.
Right arrow Articles by Dozier, B. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duffy, D. M.
Right arrow Articles by Dozier, B. L.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CHORIONIC GONADOTROPIN
Related Collections
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5858-5865
Copyright © 2005 by The Endocrine Society

An Ovulatory Gonadotropin Stimulus Increases Cytosolic Phospholipase A2 Expression and Activity in Granulosa Cells of Primate Periovulatory Follicles

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

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

Address all correspondence and requests for reprints to: Diane M. Duffy, Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Road, Lewis Hall, Norfolk, Virginia 23507. E-mail: duffydm{at}evms.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Prostaglandins (PGs) produced within ovarian follicles in response to the ovulatory gonadotropin surge are essential for follicle rupture and oocyte release. Arachidonic acid, the common precursor for PG synthesis, is cleaved from membrane phospholipids via the activity of phospholipase A2 (PLA2).

Objective: The purpose of this study was to determine which PLA2 form(s) is involved in PG production by primate periovulatory follicles.

Design and Interventions: Gonadotropins were administered to cynomolgus monkeys to stimulate multiple follicular development; human chorionic gonadotropin (hCG) initiated periovulatory events. Granulosa cells and whole ovaries were obtained before (0 h), and 12, 24, and 36 h after hCG administration.

Patients: Granulosa-lutein cells were also obtained from women undergoing infertility treatment.

Outcome Measures and Results: mRNA for cytosolic (c)PLA2 and secretory (s)PLA2V, but not sPLA2IIA, was expressed by granulosa cells. cPLA2 mRNA levels were low at 0 h, elevated by 12 h, and remained high 24–36 h after hCG administration. sPLA2V mRNA levels were low at 0 h and did not change in response to hCG. cPLA2 and sPLA2V were detected by immunocytochemistry in granulosa cells of periovulatory follicles before and at all times after hCG administration. PLA2 activity was low in lysates of granulosa cells obtained 0–24 h after hCG and was elevated in granulosa cells obtained 36 h after hCG administration. A cPLA2-selective inhibitor decreased both PLA2 activity in monkey granulosa cell lysates and PGE2 accumulation in cultures of human granulosa-lutein cells.

Conclusions: cPLA2 is primarily or exclusively responsible for the gonadotropin-stimulated mobilization of arachidonic acid necessary for PG production by primate periovulatory follicles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) PRODUCED by the ovarian follicle are essential for ovulation to occur. In primates as well as in other mammalian species, the ovulatory surge of LH acts on large periovulatory follicles to stimulate granulosa cell expression of PG synthesis enzymes and increase follicular fluid PG concentrations (1, 2, 3), and the resulting elevated follicular concentrations of PGs are necessary for ovulation to occur (4). Both PGE2 and PGF2{alpha} have been implicated in ovulatory events in several mammalian species, including monkeys and women (5, 6, 7, 8).

PGs are produced by a series of enzymatic reactions, beginning with the cleavage of arachidonic acid from the second position of membrane phospholipids by phospholipase A2 (PLA2). Many forms of PLA2 have been identified to date (9). Only three forms of PLA2 are capable of cleaving arachidonic acid from membrane phospholipids: secretory (s)PLA2 groups IIA and V (PLA2G2A, also known as sPLA2IIA; PLA2G5, also known as sPLA2V) and the cytosolic, calcium-dependent PLA2 {alpha} [PLA2G4A, also known as cytosolic PLA2 (cPLA2)] (10). cPLA2 is specific for cleavage of arachidonic acid from the second position of membrane phospholipids, whereas sPLA2IIA and V will hydrolyze any fatty acid at the second position. Arachidonic acid is then converted to PGH2 by the activity of PG-endoperoxide synthase (also known as cyclooxygenase, COX) (11). PGH2 is the common precursor for the production of PGE2 and PGF2{alpha}, and specific PG synthases convert PGH2 into these bioactive PGs (12, 13).

Previous studies by this laboratory (3, 14) and others (1, 2, 15) have demonstrated that the ovulatory gonadotropin surge increases granulosa cell expression of the PG synthesis enzymes PTGS2 (also known as COX2) and membrane-associated PGE synthase (mPGES), so these enzymes are likely involved in PG production by the periovulatory follicle. Both cPLA2 and sPLA2IIA are expressed by granulosa cells of rat periovulatory follicles; gonadotropin exposure increased ovarian PLA2 expression in a manner consistent with a role for these enzymes in periovulatory PG production (16, 17). However, the form of PLA2 involved in periovulatory PG synthesis by granulosa cells of primates, which includes monkeys and women, has not been addressed. Therefore, the studies presented here were conducted to identify the form(s) of PLA2 involved in PG synthesis and to determine whether PLA2 expression and activity are regulated by the ovulatory gonadotropin surge in primate periovulatory follicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Granulosa cells and whole ovaries were obtained from adult female cynomolgus macaques (18). All animal protocols and experiments were approved by the Eastern Virginia Medical School (EVMS) Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health (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 d 1 of the menstrual cycle. Serum levels of estradiol and progesterone were determined using an automated chemiluminescent immunoassay system (Immulite; Diagnostic Products Corporation, Los Angeles, CA), with intra- and interassay coefficients of variation of less than 10% (19).

A controlled ovarian stimulation model, developed for the collection of multiple oocytes for in vitro fertilization, was used to obtain monkey granulosa cells (18). Briefly, monkeys received 6 d of recombinant human (r-h)FSH (120 IU; Serono Reproductive Biology Institute, Rockland, MA), followed by 2 d of r-hFSH and r-hLH (120 and 60 IU, respectively; Serono) to stimulate the growth of multiple follicles. Daily GnRH antagonist administration (Antide, 0.5 mg/kg body weight; Serono) prevented an endogenous ovulatory LH surge. Follicular development was monitored via serum estradiol levels and ultrasonography (20). Follicular aspiration was performed via laparotomy or laparoscopy before (0 h) or 12, 24, and 36 h after administration of 1000 IU r-h chorionic gonadotropin (hCG) (Serono). In spontaneous menstrual cycles, follicle rupture in monkeys occurs approximately 40 h after the ovulatory gonadotropin surge (21), 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 (n = 3–4 per time point) were also obtained from monkeys experiencing controlled ovarian stimulation. Monkey spleen, liver, and heart were obtained at necropsy.

Tissue preparation

Monkey granulosa cells were obtained from follicular aspirates via Percoll gradient centrifugation after mechanical oocyte removal (22). Total granulosa cell RNA was obtained using Trizol reagent (Invitrogen, Rockville, MD). Ovarian tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Additional monkey tissues were paraffin embedded or flash frozen in liquid nitrogen for preparation of total RNA.

Granulosa cell culture

Monkey granulosa cells obtained from large periovulatory follicles (0 h hCG) were plated at a density of 100,000 cells/well in 48-well plates coated with fibronectin (Sigma, St. Louis, MO) and maintained in serum-free conditions (23) in the absence or presence of an ovulatory dose of hCG (100 ng/ml; Serono). For preparation of total RNA, cells were lysed with Trizol reagent, and total RNA was prepared with 20 µg glycogen (Roche Diagnostics Corp., Indianapolis, IN) to improve RNA recovery.

Human granulosa-lutein cells were obtained from women undergoing infertility treatment at the Jones Institute for Reproductive Medicine at EVMS, with approval of the EVMS Institutional Review Board. Owing to the exempt nature of the protocol, no specific information is available regarding patients’ demographics, diagnoses, or treatments. Granulosa-lutein cells were obtained after oocyte removal by Percoll gradient centrifugation and maintained in culture as described above for monkey granulosa cells.

To assess PLA2 activity, human granulosa-lutein cells were cultured for 4 h with either vehicle (0.1% DMSO, control) or the cPLA2 inhibitor arachidonyl trifluoromethyl ketone [ATFK; 3–30 µM (24, 25)]. Media were stored at –20 C until assayed for PGE2 by enzyme immunoassay (Cayman Chemical Company, Ann Arbor, MI) (26).

Real-time PCR

mRNA levels were analyzed by real-time PCR using a LightCycler (Roche). Total RNA was incubated with DNase and reverse transcribed (27). Amplification was performed using the FastStart DNA Master SYBR Green I kit (Roche) with an annealing temperature of 55 C. Primers were designed based on human cDNA sequences (Table 1Go) using LightCycler Probe Design software (Roche) and span an intron within the coding region to prevent undetected amplification of genomic DNA. All PCR products were sequenced (Microchemical Core Facility, San Diego State University, CA). At least four log dilutions of sequenced PCR product were included in each assay to generate a standard curve; this technique allows determination of the number of copies of each mRNA (relative to ß-actin) present in each sample. All data were expressed as the ratio of enzyme to ß-actin mRNA for each sample. Intra- and interassay coefficients of variation were less than 10%. PCR products resulting from amplification of 10 ng reverse transcribed mRNA were separated by electrophoresis using 2% agarose gels containing 0.08 µg/ml ethidium bromide and photographed under UV illumination. COX2 mRNA was not detected in two of four and three of four control samples assayed after 24 and 48 h in vitro, respectively; the assay limit of detection was used for data analysis.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Reaction conditions for real-time PCR

 
Immunocytochemistry

Immunocytochemical detection of PLA2 and COX proteins in ovarian tissues was performed using 5-µm sections and citrate antigen retrieval (3). Primary antibodies were mouse monoclonal antibodies used at the following concentrations: cPLA2, 50 µg/ml; sPLA2IIA, 30 µg/ml; sPLA2V, 30 µg/ml; COX1, 10 µg/ml; and COX2, 10 µg/ml (cPLA2 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; others from Cayman). Immunocytochemical detection was achieved using a biotinylated bovine antimouse IgG secondary antibody, peroxide conjugated avidin solution, and Nova Red chromagen (Vector Laboratories, Inc., Burlingame, CA). For COX1 and COX2, preabsorption of the primary antibody with the appropriate peptide/protein yielded no staining as previously reported (3). Because peptides were not available for preabsorption of PLA2 antibodies, omission of the primary antibody served as a negative control.

PLA2 activity

PLA2 activity was determined using lysates of Percoll-enriched populations of monkey granulosa cells. Cells were sonicated, following manufacturer’s instructions, and the clear supernatant (lysate) obtained was assayed for PLA2 activity (cPLA2 Assay Kit, Cayman). Briefly, PLA2 hydrolyzes arachidonoyl thio-PC at the sn-2 position; the thiol released is detected by the addition of 5,5'-dithiobis(2-dinitrobenzoic acid), followed by reading at 405 nm in a plate reader. This method measures all PLA2 activity present in a sample. Background absorbance for each sample was determined after adding reagents but omitting incubation. Preliminary experiments using the calcium-independent PLA2 (PLA2G6, also known as iPLA2) inhibitor bromoenol lactone (5 µM) confirmed that iPLA2 activity was not detected in granulosa cells. To determine whether PLA2 activity detected was the result of cPLA2 activity, aliquots of each granulosa cell lysate were incubated without and with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP, 5 µM). The lower limit of detection in the assay was used for calculations when PLA2 activity was not detected in a sample. PLA2 activity for each sample was normalized to the total protein concentration of the lysate as determined by the bicinchoninic acid method (Sigma), so the resulting data are expressed as units of enzyme activity per milligram lysate protein. Intra- and interassay coefficients of variation for the PLA2 activity assay were 15.0 and 12.2%, respectively.

Data analysis

All data were assessed for heterogeneity of variance and log transformed when Bartlett’s test yielded a significance of P < 0.05; all data representing mRNA, PLA2 activity, and media PGE2 levels were log transformed before further analysis. In all figures, untransformed data are presented. Enzyme mRNA and PLA2 activity levels in monkey granulosa cells obtained before and after hCG administration were compared using one-way ANOVA, followed by Newman-Keuls’ test. PGE2 production by human granulosa-lutein cells in vitro was assessed by ANOVA with 1 repeated measure, followed by Duncan’s multiple-range test. Within each time point, mRNA levels in cultured granulosa cells as well as PLA2 activity the absence vs. the presence of MAFP were compared using a paired t test. Levels of cPLA2 in control cultures of granulosa cells after 24 and 48 h in vitro were compared by unpaired t test; similar analysis was performed for COX2 mRNA levels in these cultures. Statistical analyses were performed using StatPak v4.12 software (Northwest Analytical, Inc., Portland, OR). Data are presented as mean + SEM, and significance was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PLA2 and COX expression by monkey granulosa cells

Monkey granulosa cell expression of cPLA2, sPLA2IIA, and sPLA2V was examined before and after administration of an ovulatory dose of hCG at times that span the 40-h periovulatory interval in primates (21). cPLA2 mRNA levels were low before (0 h), elevated at 12 h, and remained elevated 24–36 h after hCG administration (Fig. 1AGo), whereas sPLA2V mRNA levels did not change in response to hCG exposure (Fig. 1BGo). In contrast, sPLA2IIA mRNA was low/nondetectable in all granulosa cell samples examined. However, sPLA2IIA mRNA was detected in other monkey tissues, including liver and heart (Fig. 1CGo). cPLA2 was the most highly expressed PLA2 mRNA in granulosa cells at all times examined; copies of cPLA2 mRNA were present in 50- to 200-fold greater number than were copies of sPLA2V mRNA.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. PLA2 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. Granulosa cell levels of cPLA2 mRNA (A) and sPLA2V mRNA (B) as determined by real-time PCR; all mRNA levels are expressed relative to ß-actin. Data are expressed as mean + SEM (three to five samples per group). Groups with different superscripts are different by ANOVA and Newman-Keuls test; P < 0.05. C, sPLA2IIA mRNA was detected by 35 cycles of real-time PCR in monkey liver and heart but was low/nondetectable in granulosa cells obtained before and at specific times after hCG (representative of n = 4 per time point). No amplification was observed when monkey cDNA was omitted (H2O); positions of size standards (base pairs) are indicated at left.

 
Granulosa cells of cynomolgus monkey follicles were also assessed for expression of COX1 and COX2 mRNA. COX2 mRNA levels were low at 0 h hCG, rose 50-fold by 12 h hCG, and remained high through 36 h after hCG administration (Fig. 2AGo; P < 0.05). COX1 mRNA was not detected in any granulosa cell sample assayed, but COX1 mRNA was detected in other monkey tissues, including spleen and liver (Fig. 2BGo). These findings are similar to our previous report of gonadotropin-regulated expression of COX2, but not COX1, by rhesus monkey granulosa cells during the periovulatory interval (3).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 2. COX 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. A, Granulosa cell levels of COX2 mRNA were determined by real-time PCR (relative to ß-actin). Data are expressed as mean + SEM (four to five samples per group). Groups with different superscripts are different by ANOVA and Newman-Keuls test; P < 0.05. B, COX1 mRNA was detected by 35 cycles of real-time PCR in monkey spleen and liver but was low/nondetectable in granulosa cells obtained before and at specific times after hCG (representative of n = 4 per time point). No amplification was observed when monkey cDNA was omitted (H2O); positions of size standards (base pairs) are indicated at left.

 
Expression of cPLA2, sPLA2V, and COX2 in vitro

To determine whether gonadotropin acts directly on granulosa cells to stimulate cPLA2 expression, granulosa cells obtained from large periovulatory follicles before hCG administration (0 h) were maintained in vitro in the absence (control) and presence of an ovulatory dose of hCG. After 24 h of culture, levels of cPLA2 mRNA were 4-fold greater in hCG-treated cells than in controls; COX2 mRNA increased 48-fold in response to hCG (Fig. 3Go; P < 0.05). In contrast, cPLA2 and COX2 mRNA levels were not different between control and hCG-treated cells after 48 h in vitro. cPLA2 mRNA levels did not differ between control cells maintained 24 and 48 h in vitro; COX2 mRNA levels also did not differ between control cells cultured for 24 and 48 h. sPLA2V mRNA content was below the level of detection in all granulosa cell cultures examined (n = 4 per group; data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3. cPLA2 and COX2 mRNA levels in monkey granulosa cells in vitro. Granulosa cells obtained from monkeys experiencing controlled ovarian stimulation before administration of an ovulatory dose of hCG (0 h) were maintained in culture in the absence of treatment (control) or with hCG (100 ng/ml) for 24 or 48 h. Total granulosa cell RNA was assessed for cPLA2 (A) and COX2 (B) mRNA levels by real-time PCR; data are expressed relative to the ß-actin content of each sample. For each animal at each time point in vitro, cPLA2 or COX2 mRNA content of control cells was set equal to 1.0; mRNA content of hCG-treated cells was expressed relative to control level. After 24 h in vitro, control was less than hCG-treated for each mRNA by paired t test; P < 0.05. No effect of hCG treatment was observed after 48 h in vitro. Data are expressed as mean + SEM (three to four samples per group).

 
Localization of PLA2 and COX proteins to the cells of the monkey ovary

Immunocytochemistry was used to localize PLA2 and COX proteins in monkey tissues (Fig. 4Go). cPLA2, sPLA2IIA, and sPLA2V were detected in luminal epithelial cells (but not stromal cells) of the seminal vesicle (Fig. 4Go, G–I), which served as a positive control; no immunostaining was present in the seminal vesicle when the primary antibodies were omitted (data not shown).



View larger version (191K):
[in this window]
[in a new window]
  		FIG. 4. Immunocytochemical detection of PLA2 in monkey tissues. Ovaries with periovulatory follicles were obtained from monkeys experiencing controlled ovarian stimulation before (0 h, A–C) and 36 h after (D–F) administration of an ovulatory dose of hCG. In each periovulatory follicle shown, the follicle antrum (an) is in the upper right, granulosa cells (gc) are central, and the ovarian stroma (st) is in the lower left portion of the image. Immunodetection of cPLA2 (A and D), sPLA2IIA (B and E), and sPLA2V (C and F) in periovulatory follicles is shown. Periovulatory follicles shown are representative of three to four ovaries per time point. No immunostaining was observed when the primary antibody was omitted (B, inset). Luminal epithelial cells (lu) of the monkey seminal vesicle immunostained for cPLA2 (G), sPLA2IIA (H), and sPLA2V (I), whereas the seminal vesicle stroma (st) was consistently devoid of immunostaining. Immunostaining appears red/brown; sections are not counterstained. Scale bar, 25 µm for all panels.

 
Immunodetection of both cPLA2 and sPLA2V were observed in granulosa cells of periovulatory follicles obtained at 0, 12, 24, and 36 h after hCG exposure (Fig. 4Go, A, C, D, and F for 0 h and 36 h; 12 h and 24 h not shown). However, immunostaining for sPLA2IIA was very low in granulosa cells (Fig. 4Go, B and E and not shown), consistent with the low/nondetectable levels of sPLA2IIA mRNA detected by real-time PCR. No form of PLA2 was observed in stromal cells underlying the basement membrane of the periovulatory follicles. Because theca cells are located in this portion of the ovarian stroma, theca cells may not express cPLA2, sPLA2IIA, or sPLA2V proteins.

COX2, but not COX1, was also detected in the granulosa cells of cynomolgus monkey follicles by immunocytochemistry as previously reported, by this laboratory, for rhesus monkeys (3). COX2 was inconsistently detected in granulosa cells of large periovulatory follicles at 0 and 12 h after hCG administration but was consistently detected in granulosa cells of follicles exposed to hCG for 24–36 h (n = 3–4 per group; not shown). In contrast, COX1 was not detected in granulosa cells of monkey follicles before (0 h) or at any time after hCG administration (n = 3–4 per group; not shown).

PLA2 activity in monkey and human granulosa cells

PLA2 enzyme activity was assessed in monkey granulosa cells obtained before (0 h) and at 24 and 36 h after hCG administration in vivo (Fig. 5AGo). PLA2 activity in granulosa cell lysates was low at 0 and 24 h after hCG; only one of four lysates obtained at 0 h hCG had total PLA2 activity above the limit of detection of the assay. In contrast, PLA2 activity was higher 36 h after hCG administration (P < 0.05) when compared with 0 and 24 h granulosa cell lysates. When MAFP (an inhibitor of cPLA2 but not sPLA2IIA or sPLA2V; 5 µM) was included in the assay, PLA2 activity in 36 h hCG-treated cells was reduced (P < 0.05), whereas activity in 0 h and 24 h hCG-treated cells was unchanged. PLA2 activity attributed to cPLA2 for each group is shown in Fig. 5BGo.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5. PLA2 activity in monkey granulosa cells. Granulosa cells were obtained from monkeys experiencing controlled ovarian stimulation before (0 h) and 24 and 36 h after hCG administration. A, Granulosa cell PLA2 activity (black bars) and PLA2 activity during treatment with the cPLA2 inhibitor MAFP (5 µM; white bars) were expressed relative to granulosa cell lysate protein concentrations. B, Data in A expressed as cPLA2 activity (PLA2 activity – PLA2 activity in the presence of MAFP). Data are expressed as mean + SEM; three to four samples per group. For panel A, groups with different superscripts are different as determined by ANOVA and Newman-Keuls test; P < 0.05. Within each time point, MAFP reduction of PLA2 activity was assessed by paired t test; MAFP significantly reduced PLA2 activity in 36-h hCG granulosa cells as indicated (*, P < 0.05).

 
To determine whether cPLA2 activity is required for PGE2 production by primate granulosa cells, human granulosa-lutein cells (estimated 34–36 h post hCG) were maintained in vitro in the absence (control) or presence of the cPLA2 inhibitor ATFK (Fig. 6Go). ATFK was selected for these studies because MAFP is a potent ligand for cannabinoid (CB1) receptors and inhibits fatty acid amine hydrolase in addition to its ability to inhibit cPLA2 activity (28). Media harvested after 4 h of culture showed a dose-dependent decrease in PGE2 concentration in response to increasing doses of ATFK. The concentration of ATFK reported to result in half-maximal inhibition of cPLA2 activity ranges from 1–10 µM (24, 25), similar to the finding in the present study that half-maximal inhibition of PGE2 production occurs between 3–30 µM.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 6. Inhibition of cPLA2 activity reduces PGE2 production by human granulosa-lutein cells. Human granulosa-lutein cells obtained from patients undergoing treatment for infertility were maintained in vitro in the absence (0 µM ATFK, control) or presence of the cPLA2 inhibitor ATFK (3–30 µM) for 4 h; media were collected and assessed for PGE2 content by enzyme immunoassay. PGE2 concentration in cultures treated with ATFK was expressed relative to the PGE2 concentration in control cultures. Data are expressed as mean + SEM; three samples per group. Groups with different superscripts are different by ANOVA and Duncan’s multiple-range test; P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report represents the first demonstration that cPLA2 is involved in gonadotropin-stimulated PGE2 production by the primate periovulatory follicle. Expression of all forms of PLA2 known to cleave arachidonic acid from membrane phospholipids was examined. Granulosa cells of monkey periovulatory follicles expressed both cPLA2 and sPLA2V. cPLA2 mRNA was present at 50- to 200-greater copy number than was sPLA2V, and both cPLA2 expression and activity were increased in response to hCG exposure. Both granulosa and theca cells obtained from primate follicles produce PGs in vitro (14, 26, 29), but our observation that PLA2 expression is likely restricted to granulosa cells indicates that granulosa cells are the major source of periovulatory PGs in mammalian follicles. Primate follicular fluid PG levels increase 100-fold in response to the ovulatory gonadotropin surge (3, 18), so arachidonic acid must be rapidly mobilized for periovulatory PG production. Increased granulosa cell cPLA2 expression and activity in response to an ovulatory dose of hCG in parallel with rising follicular PGE2 levels supports the hypothesis that cPLA2 is the primary PLA2 form responsible for mobilizing arachidonic acid in granulosa cells, leading to the rise in follicular PG levels observed just before ovulation.

In the present study, we demonstrated that an ovulatory dose of gonadotropin increased granulosa cell cPLA2 mRNA as well as cPLA2 activity, supporting a key role for cPLA2 in periovulatory PG production by the follicle. Studies in rodents support a pivotal role for cPLA2 in ovulatory processes; rat granulosa cell expression of both cPLA2 and sPLA2IIA, as well as granulosa cell PLA2 activity, increased following exposure to an ovulatory dose of gonadotropin (17, 30, 31). Mice without functional sPLA2IIA or sPLA2V had normal-size litters with healthy pups (32, 33), whereas fertility defects were noted in mice lacking expression of cPLA2 (34). Taken together, these data suggest that cPLA2 is the PLA2 form required for normal reproductive function. Indeed, cPLA2 knockout mice often reentered the estrous cycle 4–5 d after mating, and reduced litter size in successful pregnancies was also noted (35). A role for ovarian cPLA2 activity in normal rodent ovarian function was supported by studies in which the cPLA2 inhibitor ATFK administered directly into the ovarian bursa reduced both ovarian PGE2 content and ovulation rate (16). Our observation that inhibition of cPLA2 activity with ATFK significantly reduced human granulosa-lutein cell PGE2 production, by 85%, supports the hypothesis that cPLA2 activity is required for arachidonic acid mobilization and periovulatory PG production by primate granulosa cells.

The ovulatory gonadotropin surge is likely a key regulator of cPLA2 expression by granulosa cells in primate follicles. Gonadotropin stimulated expression of cPLA2 by primate granulosa cells both in vivo and in vitro (present study), consistent with a previous report of increased granulosa cell cPLA2 expression in rat preovulatory follicles in response to the ovulatory gonadotropin surge (30). Stimulation of the LH/hCG receptor activates both adenylyl cyclase and phospholipase C (36). The promoter region of the human cPLA2 gene did not possess a classical cAMP response element but did contain several response elements stimulated by phorbol esters (37, 38), suggesting that hCG may increase cPLA2 expression by activation of protein kinase C through the phospholipase C pathway (36).

Cytokines produced by granulosa cells may also regulate granulosa cell cPLA2 expression. The long-standing hypothesis that ovulation shares many characteristics with inflammatory reactions (39) includes the observation that cytokines regulate ovarian PG production. Reported stimulation of cPLA2 promoter activity in response to proinflammatory cytokines, including IL-1ß and TNF{alpha}, in nonovarian tissues likely results from activation of response elements sensitive to nuclear factor-Kß (38). Human granulosa-lutein cells produce both IL-1ß and TNF{alpha} (40, 41), and both IL-1ß and TNF{alpha} can regulate granulosa cell processes essential for ovulation, including increased COX2 expression and PGE2 production (42, 43, 44). Additionally, IL-1ß increased cPLA2 expression and PG production by the rat ovary (45). Further studies will be required to identify specific cytokines that mediate the ability of the ovulatory gonadotropin surge to stimulate cPLA2 expression and PG production by the primate periovulatory follicle.

The activity of enzymes involved in follicular PG production may be regulated by factors in addition to the demonstrated role of gonadotropin to stimulate gene expression. cPLA2 must be phosphorylated to cleave arachidonic acid from membrane phospholipids, and elevated intracellular calcium stimulates cPLA2 activity independent of phosphorylation status (46). In addition, cPLA2 interacts with vimentin filaments, which transport cPLA2 to the perinuclear region; disruption of the interaction between cPLA2 and vimentin decreases PG production (47). COX2 is also preferentially located in the perinuclear region, and the close physical proximity of cPLA2 and COX2 within the cell may contribute to maximal PG production (48, 49). Although cPLA2 and COX2 are cytoplasmic enzymes, sPLA2V is secreted and active extracellularly (10). However, sPLA2V can be concentrated on the plasma membrane, brought back into the cell in vesicles, and transported to the perinuclear region to participate in PG synthesis (50). Cooperation between cPLA2 and sPLA2V to enhance PGE2 production has been reported; transfection of PLA2 forms into mesangial cells demonstrated that, whereas sPLA2V did not efficiently mobilize arachidonic acid in the absence of cPLA2, the presence of both cPLA2 and sPLA2V increased free arachidonic acid above levels measured with cPLA2 alone (51). The possible contribution of physical proximity or cooperation among cPLA2, sPLA2V, and COX2 in granulosa cell PG synthesis has not been addressed to date.

In summary, we have demonstrated that primate granulosa cell expression of the PG synthesis enzymes COX2, mPGES-1 (PTGES), and now cPLA2 increases 12 h after the ovulatory gonadotropin surge [(3, 14, 18) and present study; Fig. 7Go], suggesting that gonadotropin initiates coordinated expression of the enzymes that constitute the primary pathway for synthesis of PGE2 by the primate periovulatory follicle. cPLA2 activity, however, does not increase above 0 h levels until 36 h after hCG administration, and the correlation between cPLA2 activity and follicular fluid PGE2 levels supports the hypothesis that posttranslational control of cPLA2 activity may be the primary regulator of granulosa cell PGE2 production and, therefore, ovulation in primates.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 7. Granulosa cell cPLA2 activity increases in parallel with rising PGE2 in primate follicles. The ovulatory dose of hCG increases granulosa cell levels of mRNAs for the PG synthesis enzymes cPLA2 (white), COX2 (gray), and mPGES-1 (black); levels for all enzyme mRNAs are significantly elevated 12 h after hCG and remain high without additional significant elevation until ovulation. Granulosa cell cPLA2 activity (white circles) is low 0–24 h after hCG and elevated 36 h after hCG, just before ovulation. Follicular fluid PGE2 levels (black circles) parallel changes in cPLA2 activity. [Granulosa cell mPGES-1 mRNA levels are reproduced from D. M. Duffy et al.: Hum Reprod 20:1485–1492, 2005 (14 ). ©European Society of Human Reproduction and Embryology. Permission was provided by Oxford University Press/Human Reproduction. Follicular fluid PGE2 levels are reproduced with permission from D. M. Duffy et al.: J Clin Endocrinol Metab 90:1021–1027, 2005 (18 ). ©The Endocrine Society.]

 


   Acknowledgments
 
The authors thank Ms. Kim Hester for her role in animal training and animal protocols and Dr. Reinhart Billiar for his critical reading of this manuscript. Recombinant human gonadotropins and Antide used for these studies were generously provided by Serono.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD39872 (to D.M.D.).

First Published Online June 21, 2005

Abbreviations: ATFK, Arachidonyl trifluoromethyl ketone; c, cytosolic; CG, chorionic gonadotropin; COX, cyclooxygenase; h, human; MAFP, methyl arachidonyl fluorophosphonate; mPGES, membrane-associated PGE synthase; PG, prostaglandin; PLA2, phospholipase A2; r-h, recombinant human; s, secretory.

Received May 4, 2005.

Accepted June 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wong WYL, Richards JS 1991 Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary. Mol Endocrinol 5:1269–1279[Abstract]
  2. Sirois J, Dore M 1997 The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 138:4427–4434[Abstract/Free Full Text]
  3. Duffy DM, Stouffer RL 2001 The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and prostaglandin production by the monkey follicle. Mol Hum Reprod 7:731–739[Abstract/Free Full Text]
  4. Janson PO, Brannstrom M, Holmes PV, Sogn J 1988 Studies on the mechanism of ovulation using the model of the isolated ovary. Ann NY Acad Sci 541:22–29
  5. Murdoch WJ, Hansen TR, McPherson LA 1993 A review-role of eicosanoids in vertebrate ovulation. Prostaglandins 46:85–115[CrossRef][Medline]
  6. Duffy DM, Stouffer RL 2002 Follicular administration of a cyclooxygenase inhibitor can prevent oocyte release without alteration of normal luteal function in rhesus monkeys. Hum Reprod 17:2825–2831[Abstract/Free Full Text]
  7. Priddy AR, Killick SR, Elstein M, Morris J, Sullivan M, Patel L, Elder M 1990 The effect of prostaglandin synthetase inhibitors on human preovulatory follicular fluid prostaglandin, thromboxane, and leukotriene concentrations. J Clin Endocrinol Metab 71:235–242[Abstract]
  8. Wallach EE, Bronson R, Hamada Y, Wright KH, Stevens VC 1975 Effectiveness of prostaglandin F2{alpha} in restoration of hMG-hCG induced ovulation in indomethacin-treated rhesus monkeys. Prostaglandins 10:129–138[Medline]
  9. Murakami M, Kudo I 2002 Phospholipase A2. J Biochem 131:285–292[Abstract/Free Full Text]
  10. Balsinde J, Winstead MV, Dennis EA 2002 Phospholipase A2 regulation of arachidonic acid mobilization. FEBS Lett 531:2–6[CrossRef][Medline]
  11. Vane JR, Bakhle YS, Botting RM 1998 Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 38:97–120[CrossRef][Medline]
  12. Murakami M, Nakatani Y, Tanioka T, Kudo I 2002 Prostaglandin E synthase. Prostaglandins Other Lipid Mediat. 68–69:383–399
  13. Watanabe K 2002 Prostaglandin F synthase. Prostaglandins Other Lipid Mediat. 68–69:401–407
  14. Duffy DM, Seachord CL, Dozier BL 2005 Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle. Hum Reprod 20:1485–1492[Abstract/Free Full Text]
  15. Filion F, Bouchard N, Goff AK, Lussier JG, Sirois J 2001 Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo. J Biol Chem 276:34323–34330[Abstract/Free Full Text]
  16. Kurusu S, Iwao M, Kawaminami M, Hashimoto I 1998 Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats. Prostaglandins Leukot Essent Fatty Acids 58:405–411[CrossRef][Medline]
  17. Ben-Shlomo I, Kol S, Ando M, Altman KR, Putowski LT, Rohan RM, Adashi EY 1997 Ovarian expression, cellular localization, and hormonal regulation of rat secretory phospholipase A2: increased expression by interleukin-1 and by gonadotropins. Biol Reprod 57:217–225[Abstract]
  18. Duffy DM, Dozier BL, Seachord CL 2005 Prostaglandin dehydrogenase (PGDH) and prostaglandin levels in periovulatory follicles: implications for control of primate ovulation by PGE2. J Clin Endocrinol Metab 90:1021–1027[Abstract/Free Full Text]
  19. Albrecht ED, Aberdeen GW, Pepe GJ 2000 The role of estrogen in the maintenance of primate pregnancy. Am J Obstet Gynecol 182:432–438[CrossRef][Medline]
  20. Wolf DP, Alexander M, Zelinski-Wooten MB, Stouffer RL 1996 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 43:76–81[CrossRef][Medline]
  21. Weick RF, Dierschke DJ, Karsch FJ, Butler WR, Hotchkiss J, Knobil E 1973 Periovulatory time course of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology 93:1140–1147[Medline]
  22. Chaffin CL, Hess DL, Stouffer RL 1999 Dynamics of periovulatory steroidogenesis in the rhesus monkey follicle after controlled ovarian stimulation. Hum Reprod 14:642–649[Abstract/Free Full Text]
  23. Duffy DM, Molskness TA, Stouffer RL 1996 Progesterone receptor messenger ribonucleic acid and protein in luteinized granulosa cells of rhesus monkeys are regulated in vitro by gonadotropins and steroids. Biol Reprod 54:888–895[Abstract]
  24. Riendeau D, Guay J, Weech PK, Laliberte F, Yergey J, Li C, Desmarais S, Perrier H, Liu S, Nicoll-Griffith D 1994 Arachidonyl trifluoromethyl ketone, a potent inhibitor of 85-kDa phospholipase A2, blocks production of arachidonate and 12-hydroxyeicosatetraenoic acid by calcium ionophore-challenged platelets. J Biol Chem 269:15619–15624[Abstract/Free Full Text]
  25. Bartoli F, Lin HK, Ghomashchi F, Gelb MH, Jain MK, Apitz-Castro R 1994 Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets. J Biol Chem 269:15625–15630[Abstract/Free Full Text]
  26. Duffy DM, Stouffer RL 2003 Luteinizing hormone acts directly at granulosa cells to stimulate periovulatory processes: modulation of luteinizing hormone effects by prostaglandins. Endocrine 22:249–256[CrossRef][Medline]
  27. Chaffin CL, Stouffer RL 1999 Expression of matrix metalloproteinases and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid regulation. Biol Reprod 61:14–21[Abstract/Free Full Text]
  28. Deutsch DG, Omeir R, Arreaza G, Salehani D, Prestwich GD, Huang Z, Howlett A 1997 Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase. Biochem Pharmacol 53:255–260[CrossRef][Medline]
  29. Patwardhan VV, Lanthier A 1981 Prostaglandins PGE and PGF in human ovarian follicles: endogenous contents and in vitro formation by theca and granulosa cells. Acta Endocrinol (Copenh) 97:543–550[Medline]
  30. Kurusu S, Motegi S, Kawaminami M, Hashimoto I 1998 Expression and cellular distribution of cytosolic phospholipase A2 in the rat ovary. Prostaglandins Leukot Essent Fatty Acids 58:399–404[CrossRef][Medline]
  31. Leigh AJ, Poyser NL, Bonney RC, Whitehead SA, Wilson CA 2000 Heterogeneity of the bioactivity of LH secreted by peripubertal rats. J Reprod Fertil 118:187–193
  32. Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, Cromlish WA 1995 A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains. J Biol Chem 270:22378–22385[Abstract/Free Full Text]
  33. Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka Y, Grusby MJ, Arm JP 2004 Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption. J Biol Chem 279:16488–16494[Abstract/Free Full Text]
  34. Bonventre JV, Huang Z, Teheri MR, O’Leary E, Li E, Moskowitz MA, Sapirstein A 1997 Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390:622–625[CrossRef][Medline]
  35. Sapirstein A, Bonventre JV 2000 Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts. Biochim Biophys Acta 1488:139–148[Medline]
  36. Davis JS 1994 Mechanisms of hormone action: luteinizing hormone receptors and second-messenger pathways. Curr Opin Obstet Gynecol 6:254–261[Medline]
  37. Dolan-O’Keefe M, Chow V, Monnier J, Visner GA, Nick HS 2000 Transcriptional regulation and structural organization of the human cytosolic phospholipase A2 gene. Am J Physiol. 278:L649–L657
  38. Morii H, Ozaki M, Watanabe Y 1994 5'-flanking region surrounding a human cytosolic phospholipase A2 gene. Biochem Biophys Res Commun 205:6–11[CrossRef][Medline]
  39. Espey LL 1994 Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod 50:233–238[Abstract]
  40. Roby KF, Weed J, Lyles R, Terranova PF 1990 Immunological evidence for a human ovarian tumor necrosis factor-{alpha}. J Clin Endocrinol Metab 71:1096–1102[Abstract]
  41. Hurwitz A, Loukides J, Ricciarelli E, Botero L, Katz E, McAllister JM, Garcia JE, Rohan R, Adashi EY, Hernandez ER 1992 Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation. J Clin Invest 89:1746–1754
  42. Ando M, Kol S, Kokia E, Ruutianen-Altman K, Sirois J, Rohan RM, Payne DW, Adashi EY 1998 Rat ovarian prostaglandin endoperoxide sythase-1 and -2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes. Endocrinology 139:2501–2508[Abstract/Free Full Text]
  43. Brannstrom M, Bonello N, Wang LJ, Norman RJ 1995 Effects of tumour necrosis factor {alpha} (TNF{alpha}) on ovulation in the rat ovary. Reprod Fertil Dev 7:67–73[CrossRef][Medline]
  44. Hurwitz A, Lavy Y, Finci-Yeheskel Z, Milwidsky A, Shimonovitz S, Yagel S, Adashi EY, Lauffer N, Mayer M 1995 Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures. J Clin Endocrinol Metab 80:3018–3030[Abstract/Free Full Text]
  45. Kol S, Ben-Shlomo I, Ando M, Payne DW, Adashi EY 1997 Interleukin-1ß stimulates ovarian phospholipase A2 (PLA2) expression and activity: up-regulation of both secretory and cytosolic PLA2. Endocrinology 138:314–321[Abstract/Free Full Text]
  46. Schievella AR, Regier MK, Smith WL, Lin LL 1995 Calcium-mediated translocation of cytosolic prospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270:30749–30754[Abstract/Free Full Text]
  47. Nakatani Y, Tanioka T, Sunaga S, Murakami M, Kudo I 2000 Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A2{alpha}. J Biol Chem 275:1161–1168[Abstract/Free Full Text]
  48. Morita I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, Smith WL 1995 Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 270:10902–10908[Abstract/Free Full Text]
  49. Murakami M, Kambe T, Shimbara S, Kudo I 1999 Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways. J Biol Chem 274:3103–3115[Abstract/Free Full Text]
  50. Kim JY, Kim KP, Rhee HJ, Das S, Rafter JD, Oh YS, Cho W 2002 Internalized group V secretory phospholipase A2 acts on the perinuclear membranes. J Biol Chem 277:9358–9365[Abstract/Free Full Text]
  51. Han WK, Sapirstein A, Hung CC, Alessandrini A, Bonventre JV 2003 Cross-talk between cytosolic phospholipase A2 {alpha} (cPLA2 {alpha}) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 {alpha} activity that is responsible for arachidonic acid release. J Biol Chem 278:24153–24163[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ReproductionHome page
B. L Dozier, K. Watanabe, and D. M Duffy
Two pathways for prostaglandin F2{alpha} synthesis by the primate periovulatory follicle
Reproduction, July 1, 2008; 136(1): 53 - 63.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
P. Feuerstein, V. Cadoret, R. Dalbies-Tran, F. Guerif, R. Bidault, and D. Royere
Gene expression in human cumulus cells: one approach to oocyte competence
Hum. Reprod., December 1, 2007; 22(12): 3069 - 3077.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
D. L. Russell and R. L. Robker
Molecular mechanisms of ovulation: co-ordination through the cumulus complex
Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
N. Markosyan, B. L. Dozier, F. A. Lattanzio, and D. M. Duffy
Primate Granulosa Cell Response via Prostaglandin E2 Receptors Increases Late in the Periovulatory Interval
Biol Reprod, December 1, 2006; 75(6): 868 - 876.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/10/5858    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duffy, D. M.
Right arrow Articles by Dozier, B. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duffy, D. M.
Right arrow Articles by Dozier, B. L.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CHORIONIC GONADOTROPIN
Related Collections
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals