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BOR - Papers in Press, published online ahead of print February 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037523
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BIOLOGY OF REPRODUCTION 72, 1305–1314 (2005)
DOI: 10.1095/biolreprod.104.037523
© 2005 by the Society for the Study of Reproduction, Inc.

Adipose Differentiation-Related Protein: A Gonadotropin- and Prostaglandin-Regulated Protein in Primate Periovulatory Follicles1

Carrie L. Seachord 3, Catherine A. VandeVoort 4, and Diane M. Duffy 2 3

Department of Physiological Sciences,3 Eastern Virginia Medical School, Norfolk, Virginia 23507; California National Primate Research Center Department of Obstetrics and Gynecology,4 University of California, Davis, California 95616


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The midcycle LH surge stimulates a rise in follicular fluid prostaglandin E2 (PGE2), which is necessary for normal ovulation. To examine PGE2-regulated processes in primate follicles, monkey granulosa cells were cultured with hCG alone or with hCG and PGE2, and the resulting total RNA was subjected to microarray analysis. Twenty PGE2-regulated mRNAs were identified, and we selected a lipid droplet protein, adipose differentiation-related protein (ADRP), for further study. To determine whether hCG and PGE2 regulate ADRP expression in vivo, monkeys received gonadotropins to stimulate multiple follicular development. Human chorionic gonadotropin was then administered alone or with the PG synthesis inhibitor celecoxib, and follicular aspirates or whole ovaries were obtained at times that span the 40-h periovulatory interval. Administration of hCG increased granulosa cell ADRP mRNA and protein, with peak levels measured just before the expected time of ovulation. Treatment with hCG and celecoxib decreased granulosa cell ADRP mRNA levels compared with those of animals treated with hCG only. ADRP was detected by immunocytochemistry in many monkey tissues that synthesize prostaglandins but was not consistently expressed by steroidogenic tissues. Granulosa cells of periovulatory follicles immunostained for ADRP after, but not before, hCG administration; ADRP colocalized with large lipid droplets within the granulosa cell cytoplasm. These studies identify ADRP as a novel gonadotropin- and PGE2-regulated protein in the granulosa cells of primate periovulatory follicles. Because ADRP facilitates arachidonic acid uptake in nonovarian cells, ADRP-associated lipid droplets may enhance arachidonic acid uptake by granulosa cells to provide a precursor for periovulatory prostaglandin production.

adipose differentiation-related protein, follicle, granulosa cells, monkey, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The key role of LH to initiate events leading to ovulation is well established, and LH-stimulated prostaglandins (PGs) produced within the periovulatory follicle are also essential for follicle rupture and oocyte release. PG synthesis inhibitor administration prevents normal ovulatory events in many mammalian species [1, 2], including monkeys [3, 4] and humans [5, 6]. A critical intraovarian role for PGE2 in ovulatory events was confirmed when PGE2, coadministered with a PG synthesis inhibitor, restored ovulation in rats [7] and monkeys [4]. Clearly, PGE2 plays a key role in mediating or modulating the effects of the ovulatory gonadotropin surge in primates and other mammals.

Little is known about the specific role of PGE2 in the regulation of ovulatory events. PGs may mediate gonadotropin-stimulated increases in ovarian blood flow and vascular permeability [8, 9]. Studies in mice lacking expression of the PGE2 receptor EP2 (also known as PTGER2) indicate that these vascular changes as well as the process of cumulus expansion can be attributed specifically to PGE2 action [10]. Previously, this laboratory [11] and others [12–14] have shown that PGE2 can stimulate progesterone synthesis by granulosa cells of large periovulatory follicles, though this effect is modest compared to the ability of gonadotropin to regulate granulosa cell progesterone production. Prostaglandins have been implicated in the regulation of ovarian matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) [11, 15, 16], which are involved in the breakdown of extracellular matrix, and which is necessary for tissue remodeling associated with follicle rupture and luteinization of the follicle wall. With the exception of specific MMPs and TIMPs, modulation of granulosa cell mRNA expression by ovulatory PGs has received little attention.

To identify specific granulosa gene products regulated by gonadotropin and PGE2 during the periovulatory interval, granulosa cells from large periovulatory follicles were cultured in the presence of an ovulatory dose of gonadotropin without and with PGE2. Granulosa cell mRNA was used to screen a microarray, and 20 hCG and PGE2-regulated mRNAs were identified. Because of our interest in PG synthesis by the periovulatory follicle, the lipid droplet protein adipose differentiation-related protein (ADRP, also known as ADFP or adipophilin) was selected for further study. The ovulatory surge of gonadotropin induces the expression of enzymes responsible for PGE2 synthesis by the periovulatory follicle [17–20], but PGE2 production also requires arachidonic acid as a precursor. ADRP is a lipid droplet protein involved in cellular uptake and storage of long-chain fatty acids, including arachidonic acid [21], the precursor for PGE2 synthesis. These studies were conducted to determine whether granulosa cell ADRP expression is regulated by gonadotropin and PGE2 in a manner consistent with a role for ADRP in periovulatory processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All animal protocols and experiments were approved by the appropriate institutional animal care and use committees and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Granulosa cells from large periovulatory follicles obtained in the absence of administration of an ovulatory dose of gonadotropin used for the microarray analysis were obtained from adult female rhesus macaques at the California National Primate Research Center as previously described [22]. After ultrasound-guided follicular aspiration, oocytes were mechanically removed from the aspirates, and granulosa cell suspensions were shipped at ambient temperature. Granulosa cells were received the day following follicle aspiration, enriched by Percoll gradient centrifugation, and placed in culture as described below. Viability of granulosa cells at initiation of culture averaged 80%.

Granulosa cells and whole ovaries used for all other experiments were obtained from adult female cynomologus macaques at Eastern Virginia Medical School (EVMS) as previously described [23]. Adult females with regular menstrual cycles were checked daily for menses; the first day of menstruation was designated as Day 1 of the menstrual cycle. Blood samples were obtained under ketamine chemical restraint by femoral or saphenous venipuncture, and serum was stored at –20°C. Aseptic surgeries were performed in a dedicated surgical suite under isofluorane anesthesia.

A controlled ovarian stimulation model developed for the collection of multiple oocytes for in vitro fertilization [24] was used to obtain granulosa cells and follicular fluid (n = 4–5/time point). Beginning within 3 days of initiation of menstruation, monkeys received 60 IU of recombinant human (r-h) FSH (also known as FSHB; Serono Reproductive Biology Institute, Rockland, MA; Days 1–6, i.m.), followed by 60 IU of r-hFSH plus 30 IU r-hLH (also known as LHB; Serono; Days 7–8, i.m.) to stimulate the growth of multiple follicles. Animals also received the GnRH antagonist Antide (Serono; 0.5 mg/kg body weight, s.c.) daily to prevent an endogenous ovulatory LH surge. Follicular development was monitored by serum estradiol levels and by ultrasonography [25]. Follicular aspiration was performed before (0 h) and 12, 24, or 36 h after administration of 1000 IU r-hCG (also known as CGB5; Serono; Day 9, i.m.).

In spontaneous menstrual cycles, follicle rupture in monkeys occurs approximately 40 h after the ovulatory gonadotropin surge [26], so these times span the periovulatory interval. Previous studies in rhesus monkeys verified ovulation sites on ovaries and oocytes in the oviducts following this protocol [24, 27], and preliminary experiments in cynomologus monkeys confirmed ovulation sites in response to this protocol. To obtain undiluted follicular fluid as well as 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. To inhibit follicular PG production during the periovulatory interval, additional animals were treated with gonadotropins and Antide as described above. These animals also received a cyclooxygenase-2 (COX-2, also known as PTGS2) selective inhibitor (celecoxib [Celebrex]; Pfizer, New York, NY; 32 mg orally every 12 h) beginning with hCG administration until follicles were aspirated 36 h later (n = 4). Whole ovaries were also obtained from monkeys undergoing controlled ovarian stimulation at the times described above (n = 3–4/time point) and after treatment with hCG and celecoxib for 36 h (n = 3).

To obtain monkey ovaries with corpora lutea, blood samples were taken daily from monkeys beginning on Day 8 of natural menstrual cycles. Day 1 of the luteal phase was defined as the first day of low serum estradiol after the midcycle estradiol surge [26]. Ovaries were removed on Luteal Days 4 (n = 2), 6 (n = 1), 10 (n = 1), and 15 (n = 1) to span the luteal phase of the primate menstrual cycle. Additional monkey tissues (adrenal, kidney, testis, epididymis, heart, lung, liver, spleen, seminal vesicle, and uterine endometrium) were obtained from male and female cynomologus monkeys at necropsy. Baboon placental tissue sections obtained at midgestation (n = 1) and term (n = 1) were a gift from Dr. Gerald Pepe, EVMS.

Due to the limited availability of monkeys for research purposes, two different macaque species were used for these studies. Data generated with these two macaque species have been combined in previous publications [28, 29], demonstrating the similarity of ovarian function in these two species.

Tissue Preparation

Granulosa cells and follicular fluid were obtained from follicular aspirates as described previously [30]. Briefly, the aspirates were centrifuged to pellet oocytes and granulosa cells; the resulting supernatant (i.e., follicular fluid) was removed and stored at –80°C. Oocytes were mechanically removed, and a granulosa cell-enriched population of the remaining cells was obtained by Percoll gradient centrifugation [30]. Total RNA was obtained from granulosa cells using Trizol reagent (Invitrogen, Rockville, MD) and was stored at –20°C. Whole ovaries were bisected, maintaining at least two periovulatory follicles greater than 4 mm in diameter on each piece. One piece was fixed in 4% paraformaldehyde for 48 h, transferred to 5% sucrose in PBS for 1–3 days, and embedded in paraffin. The other piece was coated in embedding compound (Tissue-Tek O.C.T.; Sakura, Tokyo, Japan) and frozen in liquid propane before storage at –80°C. Natural cycle ovaries and nonovarian monkey tissues were also fixed and embedded as described above.

Preparation of Granulosa Cell RNA for Microarray Analysis

Granulosa cells obtained from large periovulatory follicles (0 h hCG; n = 4) were plated on tissue culture plates coated with fibronectin (Sigma, St. Louis, MO) and maintained in serum-free conditions as previously described [31] in the presence of an ovulatory dose of hCG (100 ng/ml, Serono) for 40 h, the length of the periovulatory interval in primates. Previous studies demonstrated that this dose of hCG stimulates luteal progesterone production and other periovulatory functions of granulosa cells but does not stimulate significant PGE2 production in vitro [11]. Therefore, PGE2 at the concentration measured in monkey follicles just before the expected time of follicle rupture (1 µg/ml; [18]) was added for the final 10 h to some cultures to simulate periovulatory exposure of granulosa cells to PGE2. At the end of the culture period, granulosa cells were lysed in situ with Trizol reagent (Invitrogen). Total RNA was prepared according to the manufacturer's instructions and then further enriched using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). Equal amounts (1.75 µg) of total RNA from each animal were pooled within treatment group for the preparation of biotinylated cRNA. A T7-(dT)24 oligomer (Genset, La Jolla, CA) primer and Superscript II double-stranded cDNA kit (Invitrogen) was used for first-strand cDNA synthesis from mRNA. Second-strand synthesis was completed in the presence of DNA ligase (67 U/ml), DNA polymerase I (267 U/ml), and RNase H (13 U/ml), followed by phenol:chloroform extraction and ethanol precipitation. Biotin-labeled antisense cRNA was prepared using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA) followed by passage through RNeasy spin columns and ethanol precipitation. ResGen (Invitrogen) performed RNA fragmentation and screening of the GeneChip Human Gene FL array (Affymetrix). This array was selected because the oligonucleotides included on the array were obtained from the coding regions of human genes, providing the best opportunity for a cross-species match with monkey RNA, as has been previously reported [32, 33].

Additional Granulosa Cell Cultures

Granulosa cells from large periovulatory follicles (0 h hCG) were maintained in vitro as described above in the presence of the general COX inhibitor indomethacin (10–7 M) to prevent endogenous PGE2 production. Cells were maintained in culture with no additional treatment (control), hCG (100 ng/ml), PGE2 (1 µg/ml), or hCG + PGE2 for 24 or 48 h. Cells were then lysed in situ with Trizol for preparation of total RNA.

Real-Time Reverse Transcription-Polymerase Chain Reaction

Granulosa cell levels of the mRNAs for ADRP, ß-actin (also known as ACTB), vascular cell adhesion molecule 1 (VCAM1), and cold-inducible RNA binding protein (CIRBP) were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) using a LightCycler (Roche, Indianapolis, IN). Total RNA was incubated with DNase, and RT was performed as described previously [34]. PCR was performed using the FastStart DNA Master SYBR Green I kit (Roche) following the manufacturer's instructions using 0.5 µM of each primer and 3–4 mM MgCl2. Granulosa cell content of each mRNA was determined in a separate assay. PCR primers for ADRP (up, 5'-GCCAGGAAGAATGTGTATAG; down, 5'-CAGATCGCTGGGTCTC), ß-actin (up, 5'-ATCCGCAAAGACCTGT; down, 5'-GTCCGCTAGAAGCAT), VCAM1 (up, 5'-GTTCCTAGCGTGTACCC; down, 5'-GCTGACCAAGACGGTT), and CIRBP (up, 5'-AATGAGCAGTCGCTGG; down, 5'-CCTGGTCTACTCGGAT) were designed based on the human sequences (GenBank accession numbers X97324, NM 00101, M30257, and D78134, respectively) using LightCycler Probe Design software (Roche). All primer pairs span an intron, and product length confirmed amplification of mRNA but not genomic DNA. PCR products were sequenced by the Microchemical Core Facility at San Diego State University (San Diego, CA; accession numbers for monkey ADRP [AY770628], ß-actin [AY765990], VCAM1 [AY856076], and CIRBP [AY856078]). Nucleic acid identity between the monkey and human sequences were 97.6% for ADRP, 97.4% for ß-actin, 95.6% for VCAM1, and 95.3% for CIRBP. At least five log dilutions of the PCR product used for sequencing were included in each assay, and the crossing points for these samples were used to generate a standard curve. All data were expressed as the ratio of the mRNA of interest to ß-actin mRNA for each sample. Intraassay and interassay coefficients of variation were less than 10%.

Western Blotting for ADRP

Granulosa cells were lysed and thoroughly homogenized in PBS + 0.5% SDS + 0.1% Triton X-100, mixed with denaturing sample buffer, heated to 95°C for 10 min, and loaded onto 4%–20% gradient polyacrylamide Tris-HCl gels (Bio-Rad, Hercules, CA). Proteins were transferred to polyvinylidene membranes (Immobilon; Millipore, Billerica, MA), and Western blotting proceeded as previously described [23]. The anti-ADRP primary antibody was a mouse monoclonal antibody generated against a synthetic peptide based on the human ADRP sequence (Research Diagnostics, Flanders, NJ). The anti-ADRP antibody was used at a concentration of 1.75 µg/ml; an anti-mouse immunoglobulin G (IgG)-horseradish peroxidase conjugate (Amersham, Piscataway, NJ) was used as a secondary antibody at a 1:20 000 dilution.

Primary and secondary antibody incubations were performed for 2 h at room temperature. Bands were detected by chemiluminescence (ECL kit; Amersham). Blots were then stripped of primary and secondary antibodies following instructions provided by the membrane manufacturer, and Western blotting was performed on the stripped membranes using a mouse anti-tubulin primary antibody (1:1000 dilution, Sigma) and secondary antibody as described above; tubulin levels in granulosa cell preparations were not different between different treatment groups in this study. Identity and molecular size of bands representing ADRP and tubulin proteins were determined by comparison to prestained standards (Bio-Rad). Films were scanned and analyzed densitometrically using SigmaGel software (Jandel Scientific, San Rafael, CA). In each experiment, different amounts of granulosa cell protein from a single sample were loaded onto different lanes and analyzed densitometrically to generate a standard curve for ADRP and tubulin quantification as previously described [23]. Tubulin levels were not different between treatment groups. For each granulosa cell sample, the amounts of ADRP and tubulin were determined by comparison to the standard curves. All data are expressed as a ratio of ADRP: tubulin content for each granulosa cell sample.

PGE2 Concentrations in Follicular Fluid

Follicular fluid was acidified and extracted before assay as previously described [18]. To allow determination of PG recovery, [3H] PGE2 was added to each sample; total mass of PGE2 added was less than 0.1% of the PGE2 content of follicular fluid samples. Samples were resuspended in assay buffer (see below), and an aliquot was subjected to scintillation counting for calculation of PG recovery, which averaged 84%. Concentrations of PGE2 in monkey follicular fluid extracts were determined by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI), and the PGE2 content of each sample was corrected based on PGE2 recovery calculated for that sample. Intraassay and interassay coefficients of variation were 14.6% and 13.4%, respectively.

Immunocytochemistry

Immunocytochemical detection of ADRP in ovarian tissues was performed with 5-µm sections of 4% paraformaldehyde-fixed, paraffin-embedded tissues as previously described [18] but omitting antigen retrieval and using the anti-ADRP antibody described above for Western blotting. Briefly, endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol, and sections were blocked with 3% horse serum, followed by incubation with the primary antibody (1 µg/ml) at room temperature for 1 h and overnight incubation at 4°C. After rinsing with PBS, sections were incubated with biotinylated horse anti-mouse IgG secondary antibody and then with the avidin-biotinylated peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). All nonimmune serum and antibody incubations were performed in PBS with 0.1% Triton X-100. Peroxidase activity was visualized using Nova Red chromagen (Vector). Images were obtained using an Olympus BX41 microscope fitted with a DP70 digital camera and associated software (Melville, NY).

Confocal Microscopy

For colocalization of ADRP and lipids, 10-µm sections of frozen ovaries were fixed in 10% formalin in phosphate buffer (pH 7.0) for 30 min. After incubation for 1 h with nonimmune serum in PBS + 0.1% Triton as described above, sections were incubated with the primary antibody against ADRP (1 µg/ml in PBS + 0.1% Triton with 5% nonimmune serum) for 1 h at room temperature, and then overnight at 4°C. After washing in PBS, sections were incubated with Alexa Fluor 488-conjugated secondary antibody directed against mouse IgG (1:500 dilution in PBS + 0.1% Triton + nonimmune serum; Molecular Probes, Eugene, OR) for 1 h at room temperature. After washing in PBS, sections were exposed to Nile red (10 ng/ml in PBS + 0.1% Triton + 0.1% acetone; Sigma) for 1 min. Nile red was selected to visualize lipid droplets because other lipid dyes have been reported to promote fusion of adjacent lipid droplets in unfixed and fixed tissues [35]. Sections were briefly rinsed in PBS, mounted with Vectashield aqueous mounting medium (Vector), and examined using a Zeiss 510 laser scanning confocal microscope with LSM5 software for image acquisition (Carl Zeiss Inc., Thornwood, NY). Fluorescence imaging was performed simultaneously using 488 nm excitation with a 505/ 550 band-pass filter (green channel) and 543 nm excitation with a 560 nm long-pass filter (red channel).

Data Analysis

All data were assessed for heterogeneity of variance using a Bartlett test and log-transformed when the Bartlett test yielded a significance of <0.05. Data presented in Figure 1 and Figure 3 as well as follicular fluid PGE2 levels were log-transformed before further analysis. Data obtained from real-time RT-PCR used to confirm the results of the microarray analysis were assessed by paired t-test. ADRP mRNA levels in cultured granulosa cells shown in Figure 1 were compared by one-way analysis of variance (ANOVA) with one repeated measure (blocked for individual animal), followed by a Newman-Keuls test. ADRP mRNA and protein levels in granulosa cells obtained before and after hCG administration in vivo were compared using one-way ANOVA, followed by a Newman-Keuls test. Comparison of PGE2 content in follicular fluid from animals treated with hCG and celecoxib as well as granulosa cell ADRP mRNA levels in these animals was performed by unpaired t-test. Tubulin levels as determined by Western blotting were assessed by the Kruskal-Wallis test. All statistical tests described above were performed using StatPak v4.12 (Northwest Analytical, Portland, OR). One sample identified as a statistical outlier using the Q-test [36] was excluded from the data shown in Figure 2. Data are presented as mean ± SEM, and significance was assumed at P < 0.05. Fold change and P values for microarray samples as reported in Table 1 were determined by GeneChip Operating Software (Affymetrix).



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  		FIG. 1. ADRP mRNA levels in monkey granulosa cells in vitro. Granulosa cells were obtained from large periovulatory follicles before administration of an ovulatory dose of hCG (0 h) and maintained in culture in the absence of treatment (control) or with hCG (100 ng/ml), PGE2 (1 µg/ ml), or hCG + PGE2 for 24 or 48 h; all cultures received indomethacin (10–7 M) to prevent endogenous PGE2 synthesis. Total granulosa cell RNA was assessed for ADRP mRNA levels by real-time RT-PCR; data are expressed relative to the ß-actin mRNA content of each sample. For each animal, ADRP mRNA content of control cells was set equal to 1.0; ADRP mRNA content of treated cells was expressed relative to control levels. For the five animals examined, four had higher ADRP mRNA after hCG treatment, three had higher ADRP mRNA after PGE2 treatment, and five had higher ADRP mRNA after hCG + PGE2 treatment for 24 h in vitro. Groups with different superscripts are different by ANOVA and Newman-Keuls test, P < 0.05. No differences between treatment groups were identified after 48 h of culture. Data are expressed as mean ± SEM; n = 5/ group



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  		FIG. 3. ADRP protein content of monkey granulosa cells obtained throughout the periovulatory interval. A) Granulosa cells obtained from large periovulatory follicles before (0 h) and 12, 24, and 36 h after administration of an ovulatory dose of hCG were subjected to Western blotting for ADRP and tubulin. ADRP levels are expressed relative to the tubulin content of each sample and are shown in (B). ADRP protein was nondetectable in three of four samples obtained at 0 h hCG (*), so this group was excluded from the parametric statistical analysis. Groups with different superscripts are different by ANOVA and Newman-Keuls test, P < 0.05. Data are expressed as mean ± SEM; n = 4/group



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  		FIG. 2. ADRP mRNA levels in monkey granulosa cells obtained throughout the periovulatory interval. Granulosa cells were obtained from large periovulatory follicles before (0 h) and 12, 24, and 36 h after administration of an ovulatory dose of hCG. ADRP mRNA levels were assayed by real-time RT-PCR; data are expressed relative to the ß-actin mRNA content of each sample. Groups with different superscripts are different by ANOVA and Newman-Keuls test, P < 0.05. Data are expressed as mean ± SEM; n = 3–4/group


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  		TABLE 1. Granulosa cell mRNAs identified by microarray.*


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of hCG- and PGE2-Regulated Granulosa Cell mRNAs

To identify granulosa cell mRNAs regulated by the ovulatory gonadotropin surge and resulting elevated intrafollicular PGE2 levels, monkey granulosa cells obtained from large periovulatory follicles (0 h hCG) were maintained in vitro and treated with an ovulatory dose of hCG only or hCG with an ovulatory concentration of PGE2 as described in Materials and Methods. At the end of the 40-h culture period, total RNA was prepared from granulosa cells treated with hCG only and with hCG + PGE2, and cRNA probes were prepared for microarray analysis. Twenty hCG + PGE2-regulated granulosa cell gene products were identified (Table 1).

Three gene products identified by the microarray analysis were selected for further study. RT-PCR for specific gene products was performed using the four granulosa cell preparations cultured for preparation of mRNA and microarray analysis. ADRP mRNA levels were higher in granulosa cells treated with hCG + PGE2 compared with that of hCG-treated cells (4.19 ± 1.93 versus 3.88 ± 1.89, P < 0.05), resulting in a mean 1.2-fold increase in response to hCG + PGE2 treatment and supporting the results of the microarray (+2.4). CIRBP mRNA was lower in hCG + PGE2 treated granulosa cells compared with that of hCG-treated cells (0.46 ± 0.12 versus 0.70 ± 0.16, P < 0.05); the 1.6-fold decrease in response to hCG + PGE2 treatment supported the results of the microarray (–2.2). VCAM1 mRNA levels tended to be lower in hCG + PGE2 treated granulosa cells compared with levels in cells treated with hCG alone (2008 ± 813 versus 7629 ± 4261, P = 0.09). Three of four granulosa cell samples examined decreased VCAM1 expression in response to PGE2 treatment (mean decrease 6.5-fold), supporting the results of the microarray (–2.3). These data support a role for PGE2 in the regulation of ADRP, CIRBP, and VCAM1 expression by monkey granulosa cells during the periovulatory interval.

Due to our interest in PG synthesis by the periovulatory follicle, ADRP, a lipid droplet protein involved in cellular uptake of the PG synthesis precursor arachidonic acid [21], was selected for further study. To support findings from the microarray analysis and to determine whether hCG or PGE2 alone could increase granulosa cell ADRP mRNA levels in vitro, monkey granulosa cells were obtained as described above and maintained in culture with hCG, PGE2, hCG + PGE2, or no treatment (control) for 24 or 48 h in the presence of indomethacin to prevent endogenous PGE2 synthesis. Treatment with hCG + PGE2, but not hCG or PGE2 alone, increased ADRP mRNA levels above control in granulosa cells cultured for 24 h (Fig. 1). No effect of any treatment was evident after 48 h of culture.

Regulation of ADRP by Gonadotropin and PGE2 In Vivo

To determine whether granulosa cell ADRP expression was enhanced in response to an ovulatory dose of gonadotropin in vivo, granulosa cells were obtained from monkeys experiencing controlled ovarian stimulation at 0, 12, 24, and 36 h after hCG administration. ADRP mRNA levels were low at 0–12 h hCG, elevated 24 h after hCG administration, and remained high 36 h after hCG (Fig. 2).

Granulosa cell ADRP protein levels showed a similar pattern. Monkey granulosa cell lysates subjected to Western blotting for ADRP yielded a single band at approximately 50 kDa, consistent with the expected size for ADRP [42]; tubulin was also detected as a single band of approximately 50 kDa (Fig. 3A). ADRP protein levels were low or nondetectable before (0 h) hCG administration; low levels were present 12 h after hCG administration, intermediate levels were present 24 h after hCG, and the highest levels were observed 36 h after administration of hCG (Fig. 3B).

Previous studies demonstrated that the ovulatory gonadotropin surge stimulates PGE2 production by the monkey periovulatory follicle [18]. To determine whether hCG-induced expression of ADRP by granulosa cells is mediated or modulated by PGE2 in vivo, additional animals experiencing controlled ovarian stimulation received only hCG or hCG in addition to the COX-2 selective inhibitor celecoxib for 36 h before follicle aspiration to obtain granulosa cells and follicular fluid. PGE2 levels in follicular fluid were 7-fold lower in hCG + celecoxib treated monkeys than in monkeys receiving hCG only for 36 h (55 ± 45 ng/ml versus 414 ± 168 ng/ml, n = 3–5/group, P < 0.05) and similar to PGE2 levels measured in monkeys receiving hCG for 24 h (54 ± 22 ng/ml, n = 6). Granulosa cell expression of ADRP mRNA was lower in hCG + celecoxib treated animals compared with levels in animals receiving hCG only (0.76 ± 0.16 versus 1.5 ± 0.4, n = 4/group, P < 0.05). PGE2 levels in follicular fluid obtained from monkeys receiving only hCG have been previously reported [23].

Immunocytochemistry was used to localize ADRP within monkey tissues (Fig. 4). ADRP immunostaining was cytoplasmic and punctate in appearance. Strong ADRP immunostaining was observed in monkey testis (Fig. 4I). ADRP-positive testicular cells were located inside the seminiferous tubule along the basement membrane, consistent with identification as either Sertoli cells or spermatogonia. Luminal cells of the seminal vesicle expressed ADRP near the basolateral membrane (Fig. 4J). Modest ADRP immunostaining was observed in the zona fasciculata, but not the zona glomerulosa or zona reticularis, of the monkey adrenal (Fig. 4G); adipose tissue adhered to ovaries also showed modest ADRP expression (not shown). Liver, lung, and the syncytiotrophoblast of midgestation and term placenta showed occasional ADRP immunostaining, while the majority of cells in these tissues were ADRP negative (Fig. 4K; and not shown). Many tissues, including kidney, heart, uterine endometrium, epididymis, and spleen showed no ADRP immunostaining (Fig. 4L; and not shown). Immunostaining was not present in any tissue when the primary antibody was omitted (Fig. 4H; and not shown).



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  		FIG. 4. Immunodetection of ADRP in monkey tissues. All immunocytochemical staining is cytoplasmic and appears red (A–L); tissues are not counterstained. In all periovulatory follicles (A–D and H), ovarian stroma (st) is on the left, granulosa cell layer (gc) is central, and the follicle antrum (an) is on the right of the image. Ovaries obtained before hCG administration (0 h) showed little or no ADRP immunostaining in granulosa cells or stromal cells of large periovulatory follicles (A). Immunostaining was present in the mural granulosa cells (arrow) of follicles from ovaries obtained after 12 h of exposure to hCG (B); mural and antral granulosa cells immunostained for ADRP after 36 h of hCG exposure (C). Follicles from monkeys treated with hCG + celecoxib for 36 h showed ADRP immunostaining in granulosa cells (D), with strongest immunostaining in mural granulosa cells (arrow). Occasional stromal cells consistent in location with theca cells immunostained for ADRP at all times examined (B and not shown, arrowhead). No immunostaining was observed in follicles obtained 36 h after hCG (H) or any other tissue (not shown) when the anti-ADRP primary antibody was omitted. Immunostaining for ADRP was also observed in the cells of the corpus luteum on Days 4 (E) and 15 (F) after the midcycle LH surge as well as the zona fasciculata (f) but not the zona glomerulosa (g) or zona reticularis (not shown) of the monkey adrenal (G). ADRP immunostaining was present in cells near the basement membrane of the seminiferous tubules of the testis (tu) but not in the interstitium (*), where Leydig cells are located (I). Luminal epithelial cells (lu) of the seminal vesicle also showed ADRP immunostaining (J), as did syncytiotrophoblast cells of the midgestation baboon placenta (K). Kidney showed no ADRP immunoreactivity (L). Confocal laser microscopy images of Nile red lipid stain (M, P, and S) and ADRP immunofluorescence (green, N, Q, and T) in granulosa cells of follicles obtained 0 h (M–O), 12 h (P–R), and 36 h (S–U) after hCG administration. Overlay of red and green images (O, R, and U) shows ADRP (green) at the periphery of many large (arrowheads) and small (double arrowheads) lipid droplets after hCG exposure; some smaller lipid droplets did not colocalize with ADRP (arrows). All confocal images represent a single image of 0.5 µm thickness. For A–H and L, bar in L = 50 µm. For I–K, bar in L = 100 µm. For M–U, bar in U = 5 µm

ADRP protein was localized to the cells of the monkey ovary by immunocytochemistry (Fig. 4). No ADRP immunostaining was observed in granulosa cells of primordial, primary, and secondary follicles, but granulosa cells of small (1–2 mm) antral follicles occasionally immunostained for ADRP (not shown); stromal cells consistent in location with theca cells also occasionally showed ADRP immunostaining (Fig. 4B, arrowhead). ADRP immunostaining was primarily observed in granulosa cells of large periovulatory follicles. In the granulosa cells of large periovulatory follicles obtained after controlled ovarian stimulation, little or no ADRP immunostaining was observed after exposure to 0 h of hCG (Fig. 4A). ADRP immunostaining was observed in granulosa cells of ovaries obtained after 12 and 24 h of hCG treatment (Fig. 4B; and not shown). At these times, ADRP immunostaining was most often observed in mural granulosa cells near the basement membrane (arrow) and was less prominent near the follicle antrum. After 36 h of exposure to hCG, granulosa cells throughout the granulosa cell layer showed ADRP immunostaining, including those near the follicle antrum (Fig. 4C). ADRP was also detected in the cells of corpora lutea obtained throughout the luteal phase of the menstrual cycle (Fig. 4, E and F; and not shown).

ADRP immunostaining was also observed in granulosa cells of ovaries from animals treated with hCG + celecoxib (Fig. 4D, arrow). In many follicles from these ovaries, ADRP was localized to mural, but not antral, granulosa cells as was observed in ovaries exposed to hCG for 12– 24 h in the absence of celecoxib.

Granulosa cells of periovulatory follicles contained cytoplasmic lipid droplets associated with ADRP. Confocal microscopy using sections of ovarian tissue sections flash-frozen to preserve intracellular lipid deposits showed ADRP and lipid colocalized to round cytoplasmic structures, with ADRP located on the periphery of the larger lipid droplets (Fig. 4, M–U). In granulosa cells of large periovulatory follicles obtained before hCG administration (0 h hCG), few lipid droplets and little ADRP protein were detected (Fig. 4, M–O). After 12–24 h of hCG exposure, granulosa cells contained many lipid droplets; some of the smaller and most of the larger droplets colocalized with ADRP (Fig. 4, P–R; and not shown). By 36 h after hCG, granulosa cells contained small and large droplets; ADRP was located on the surface of the majority of these lipid droplets.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report is the first to identify ADRP as a gonadotropin- and PGE2-regulated gene product in the granulosa cells of periovulatory follicles. Granulosa cell ADRP mRNA and protein levels were low before the ovulatory gonadotropin surge, increased during the periovulatory interval, and peaked 36 h after the ovulatory gonadotropin surge, just before the expected time of ovulation. ADRP was located within the cytoplasm of granulosa cells at the periphery of lipid droplets. In studies of nonovarian cells, ADRP is also found on the surface of intracellular lipid droplets and is involved in intracellular transport of long-chain fatty acids, including arachidonic acid [21], the common precursor for all PG synthesis. In addition to the well-established ability of gonadotropin to increase granulosa cell expression of PG synthesis enzymes such as COX-2 [18], the data presented here suggest that the ovulatory gonadotropin surge, mediated in part by increased follicular PGE2, may enhance periovulatory PG synthesis through additional pathways, including increased arachidonic acid availability within primate granulosa cells through an ADRP-dependent mechanism.

ADRP-associated lipid droplets have been proposed to play a role in cellular accumulation of the steroid hormone precursor cholesterol [43] as well as long-chain fatty acids, which are precursors for PG synthesis [21]. In monkey tissues, ADRP was expressed in lipid-accumulating tissues, including adipose, but not in tissues that do not accumulate lipids, such as kidney, heart, spleen, epididymis, and uterine endometrium. In the present study, several classical steroidogenic cell types that use cholesterol as a precursor for steroid synthesis (e.g., the zona glomerulosa and zona reticularis of the adrenal, Leydig cells of the testis, and theca cells of the periovulatory follicle [44]) showed limited or no ADRP immunostaining. Granulosa cells of follicles obtained before the ovulatory gonadotropin surge, which synthesize steroid hormones from precursors hormones and not from cholesterol [45], also showed little or no ADRP immunostaining. However, certain steroidogenic cells that produce steroid hormones from cholesterol (e.g., the corpus luteum, the adrenal zona fasciculata, and the syncytiotrophoblast of the placenta [44, 46]) did express ADRP. Therefore, there was no correlation between cholesterol utilization for steroid hormone synthesis and ADRP expression in the monkey tissues examined. However, ADRP was localized to many cell types that express PG synthesis enzymes and produce PGs, including the luminal epithelium of the seminal vesicle, placenta, granulosa cells of the periovulatory follicle after the ovulatory gonadotropin surge, and the corpus luteum [18, 47–49]. Of interest, strong ADRP immunostaining was observed in the seminiferous tubule of the testis in a location consistent with either Sertoli cells or spermatogonia; PG synthesis enzymes have also been localized to spermatogonia [48, 50]. These findings are consistent with and extend previous reports of ADRP expression by lipid-accumulating rat [51] and human [52] tissues, and are most consistent with a role for ADRP-associated lipid droplets in PG synthesis rather than steroidogenesis.

ADRP is a structural protein of lipid droplets [53]. In the present study, ADRP immunodetection was cytoplasmic in location and punctate in appearance; confocal microscopy localized ADRP to the surface of intracellular lipid droplets. While ADRP-associated droplets may contain cholesterol, other laboratories have demonstrated that ADRP preferentially binds to and enhances cellular uptake of long-chain fatty acids, including arachidonic acid, the common precursor for PG synthesis [54]. ADRP may shuttle fatty acids to and from lipid droplets [55] and facilitate carrier-mediated active transport of fatty acids across the plasma membrane [56], suggesting an important functional role for ADRP in the regulation of lipid uptake, intracellular transport, and utilization. ADRP-containing lipid droplets can move rapidly within a cell and are often found in close association with the plasma membrane [56] as well as the endoplasmic reticulum [57], where enzymes involved in PG synthesis are located [58–61]. Lipid droplets containing ADRP also contain RAB proteins involved in shuttling lipid droplets to the endoplasmic reticulum, Golgi, and other intracellular membranes [62], so ADRP-containing lipid droplets may shuttle lipids such as arachidonic acid from the plasma membrane to intracellular membranes containing PG synthesis enzymes, providing an ample supply of precursor for efficient PG synthesis. However, further studies will be required to determine whether arachidonic acid supplied via ADRP-associated lipid droplets is involved in granulosa cell PG synthesis.

The ability of gonadotropin to stimulate ADRP expression by primate periovulatory granulosa cells may be mediated in part by PGE2. Administration of an ovulatory dose of gonadotropin stimulated monkey granulosa cell ADRP mRNA and protein expression in vivo, but hCG treatment also elevates follicular concentrations of PGE2 and other eicosanoids [63]. To examine the individual roles of hCG and PGE2 in the regulation of granulosa cell ADRP expression, both in vitro and in vivo approaches were used in the present study. Granulosa cells used for microarray analysis had higher ADRP mRNA levels after exposure to hCG + PGE2 for the final 10 h of culture compared with granulosa cells exposed to hCG only. In a separate study, granulosa cells maintained in vitro with either hCG or PGE2 had ADRP mRNA levels similar to that of controls, while treatment with hCG + PGE2 elevated ADRP expression above control levels. Additionally, in vivo administration of the PG synthesis inhibitor celecoxib with hCG decreased follicular fluid PGE2 concentrations and decreased ADRP mRNA levels in granulosa cells. While celecoxib likely reduces follicular levels of all eicosanoids produced via the COX-2 pathway, these findings, in combination with our in vitro studies, suggest that PGE2 mediates at least part of the ability of hCG to stimulate granulosa cell ADRP expression. Gonadotropin initiates ADRP expression before PGs accumulate in follicular fluid in periovulatory follicles (12–24 h after hCG administration) and is likely a primary stimulus for granulosa cell ADRP expression early in the periovulatory interval. However, PGE2 may enhance ADRP expression late in the periovulatory interval to maximally elevate ADRP levels just before ovulation.

Gonadotropin and PGE2 may stimulate ADRP expression in different subpopulations of granulosa cells. ADRP immunostaining was most prominent in granulosa cells near the basement membrane in monkeys receiving 12–24 h of hCG administration as well as those treated for 36 h with hCG + celecoxib. Because follicular fluid PGE2 concentrations did not rise until late in the periovulatory interval and did so only in the absence of celecoxib, the primary stimulus for ADRP expression in mural granulosa cells is likely gonadotropin. Granulosa cells throughout the granulosa cell layer in periovulatory follicles express ADRP 36 h after hCG administration, so elevated PGE2 present in the follicles of these animals may be responsible for ADRP expression in granulosa cells near the follicle antrum. LH receptors have been localized primarily to mural granulosa cells of the periovulatory follicle [64], so ADRP expression by mural granulosa cells is likely gonadotropin- and LH receptor-mediated. Human granulosa cells also express several subtypes of PGE2 receptors [65, 66]. While the location of PGE2 receptors on subtypes of primate granulosa cells is unknown, the PGE2 receptor EP2 is located primarily on cumulus and possibly antral granulosa cells of rodent follicles [67]. The absence of ADRP expression in antral granulosa cells until late in the periovulatory interval may be due to the lack of LH receptors on antral granulosa cells and suggests that ADRP expression by antral granulosa cells may be PGE2- and, possibly, EP2-dependent.

A key role for gonadotropin in the induction of periovulatory PG synthesis enzyme expression is well established. However, the data presented here suggest an additional role for gonadotropin to regulate periovulatory PG synthesis by increasing ADRP expression. While the ovulatory gonadotropin surge likely initiates ADRP expression in granulosa cells, gonadotropin-stimulated ADRP expression is further enhanced by PGE2 late in the periovulatory interval. ADRP may increase the ability of granulosa cells to acquire and use the PG precursor arachidonic acid and, therefore, facilitate follicular PG synthesis. Elevated granulosa cell arachidonic acid availability late in the periovulatory interval may provide the precursor needed for the rapid increase in follicular PGE2 synthesis, which is necessary for successful ovulation.


   ACKNOWLEDGMENTS
 
We thank Ms. Kim Hester for her role in animal training and animal protocols. Recombinant human gonadotropins and Antide were generously provided by the Serono Reproductive Biology Institute, Rockland, MA.


   FOOTNOTES
 
1 Supported by National Institutes of Health grants HD39872 and RR13439 to C.A.V., by grant RR00169 from the California National Primate Research Center, and by a grant from The Andrew W. Mellon Foundation to D.M.D. Back

2 Correspondence: Diane M. Duffy, Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Road, Lewis Hall, Norfolk VA 23507. FAX: 757 624 2269; duffydm{at}evms.edu Back

Received: 1 November 2004.

First decision: 18 November 2004.

Accepted: 18 January 2005.


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