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. 2008 Feb 21;149(6):2782–2789. doi: 10.1210/en.2007-1662

Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells

Ping Zhao 1, Ananya De 1, Zeng Hu 1, Jing Li 1, Sabine M Mulders 1, Maarten D Sollewijn Gelpke 1, En-Kui Duan 1, Aaron J W Hsueh 1
PMCID: PMC2408816  PMID: 18292196

Abstract

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3β-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

ALTHOUGH OVARIAN FOLLICLE development is mainly regulated by gonadotropins, recent studies have demonstrated the importance of multiple intraovarian ligand-receptor signaling systems in mediating or modulating gonadotropin actions (1,2,3). These factors are regulated by gonadotropins and play paracrine or autocrine roles in diverse ovarian functions including oocyte maturation, ovulation, and luteinization. For example, IGF-I, IL-1, and estrogen augment the actions of FSH and LH (4,5), whereas epidermal growth factor family ligands (6) and insulin-like factor 3 (7) promote oocyte maturation. Based on DNA microarray analyses of ovarian gene expression during the preovulatory period, we demonstrated the induction of the brain-derived neurotrophic factor (BDNF) ligand and the TNF-related weak inducer of apoptosis (TWEAK) receptor after the preovulatory LH/human chorionic gonadotropin (hCG) stimulation. BDNF acts as paracrine factor to promote first polar body extrusion and cytoplasmic maturation of the oocyte for optimal development into blastocysts (8), whereas the TWEAK ligand protects preovulatory follicles from excessive luteinization (9). Identification of these key ovarian paracrine /autocrine factors improves our understanding of the hormonal mechanisms underlying gonadotropin actions.

Using a genome-wide search for paracrine factors based on DNA microarray analyses (10), we found major increases in ovarian transcripts for a chemokine fractalkine during the preovulatory period and investigated its potential role as an ovarian paracrine factor. Chemokines are small secreted proteins capable of stimulating the directional migration of leukocytes during the inflammation reaction. Fractalkine, also known as CX3CL1 or neurotactin, was originally identified based on its sequence homology to other chemokine family members (11,12). Unlike other chemokines, fractalkine has a unique cysteine pattern, Cys-X-X-X-Cys, and is a transmembrane protein with its chemokine domain linked to a mucin-like stalk. The extracellular domain of fractalkine could be cleaved by proteases to produce a soluble form. Besides immune cells, fractalkine is highly expressed in neurons of the central nervous system (13). Similar to other chemokines, recombinant fractalkine was found to activate a seven transmembrane receptor CX3CR1 (14,15). This receptor is expressed in microglia cells that mediate inflammatory reactions in the central nervous system and fractalkine treatment protects microglial cells from apoptosis (16,17). Here we demonstrated the gonadotropin induction of ovarian fractalkine expression during the preovulatory period and identified the ovarian cell types expressing fractalkine and its receptor CX3CR1. Treatment with fractalkine was found to augment progesterone biosynthesis, stimulate key steroidogenic enzymes, and phosphorylate the P38 MAPK in cultured granulosa cells.

Materials and Methods

Animals

Immature Sprague Dawley rats at 21 d of age were obtained from Vital River Laboratories (Beijing, China). Animals were housed in an environment with constant photoperiod (14-h light, 10-h dark cycle), humidity, and temperature based on the Guideline of the Animal Care and Use Committee of the Institution of Zoology, Chinese Academy of Sciences. After housing for 4 d, rats were given a sc injection with 15 IU PMSG (Sigma-Aldrich, St. Louis, MO) at 25 d of age to induce follicular maturation. Forty-eight hours later, some rats received a sc administration of 10 IU hCG (Sigma-Aldrich) to induce ovulation.

DNA microarray analysis

The procedure for DNA microarray has been described previously (8). Briefly, 108 mice of the B6D2F1 strain were treated with 7.5 U Humegon (containing equivalent doses of human FSH and LH; Organon, Oss, The Netherlands) at 21 d of age to stimulate follicular growth. Forty-eight hours later, some animals were injected ip with 5 U Pregnyl (comprising of hCG; Organon) to induce ovulation. Ovaries of three mice were dissected every 2 h after Humegon treatment, and one mouse was dissected hourly after Pregnyl treatment. Under this protocol, each animal ovulated 20 oocytes after gonadotropin inducement. The RNA of ovary samples was extracted with TRIZOL reagents (Invitrogen, Carlsbad, CA) and hybridized to the Affymetrix mouse MGU74v2 arrays (Affymetrix, Santa Clara, CA) according to standard Affymetrix protocols.

Real-time RT-PCR analysis

SybrGreen-based quantitative RT-PCR was carried out to measure transcript levels of fractalkine (GenBank accession no. NM 134455) and CX3CR1 (GenBank accession no. NM 133534) using the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Immature rat ovaries were collected at different intervals after gonadotropin treatments. Some ovaries were punctured to obtain cumulus-oocyte complexes and granulosa cells. Follicle shells were also obtained from mechanically isolated preovulatory follicles (>400 μm in diameter) after puncturing to minimize granulosa cells. To study fractalkine regulation of steroidogenic enzymes, granulosa cells isolated from PMSG-primed immature rats were treated with 20 ng/ml hCG in the presence or absence of 150 ng/ml fractalkine for 48 h. Cells were washed and collected for subsequent extraction of total RNA.

Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, and DNA was removed using the Turbo DNA-free kit (Ambion, Austin, TX). Based on absorbance at 260 nm, RNA samples were adjusted to 1 μg/μl before performing reverse transcription using Superscript reverse transcriptase (Invitrogen). Real-time PCR was preformed in 20 μl reaction volume contained 10 μl 2× Brilliant SYBR Green quantitative PCR master mix (Stratagene, La Jolla, CA), 2 μl template cDNA, 0.5 μm primers, and 300 nm reference dye. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization whereas the reserve transcription reaction was omitted for negative controls. Plasmids of fractalkine, CX3CR1, and GAPDH were amplified to generate standard curves after serial dilutions. Real-time PCR results were analyzed using the ABI Prism 7000 SDS software (Applied Biosystems). For all transcripts, melting curve analysis and agarose gel electrophoresis were used to verify the specificity of amplification. The following primers were used: fractalkine, sense, 5′-GTG GCA AGT TTG AGA GCG-3′, antisense, 5′-CCT GGG AAA TAG CAG TCG GT-3′; CX3CR1, sense, 5′-CAA CAC GGC GTC ACC ATC A-3′, antisense, 5′-AGG ACG AAG CCC AGG ATG T-3′; cytochrome P45011A (CYP11A), sense, 5′-CCA AGT TCA ACC TCA TCC TGA-3′, antisense, 5′-CGT GTG ACT GCA GCC TGC AA-3′; steroidogenic acute regulatory protein (StAR), sense, 5′-AGA TGA AGT GCT AAG TAA GGT GGT G-3′, antisense, 5′-CCA GTT CTT CAT AGA GTC TGT CCA T-3′; 3β-hydroxysteroid dehydrogenase (3β-HSD), sense, 5′-AGA CCA TCC TAG ATG TCA ATC TGA A-3′, antisense, 5′-CAG GAT GAT CTT CTT GTA GGA GT-3′; GAPDH (18), sense, 5′-CTC ATG ACC ACA GT C CAT GC-3′, antisense, 5′-TTC AGC TCT GGG ATG ACC TT-3′.

Tandem RT-PCR for different ovarian compartments

Oocytes, cumulus cells, mural granulosa cells, and follicle shells were isolated from ovaries of immature PMSG-primed rats before and at 3 h after hCG treatments. Due to the low number of oocytes and cumulus cells, tandem RT-PCR was preformed to detect transcripts for fractalkine and CX3CR1 with 28 cycles using the first set of primers and another 28 cycles using the second primer set. The first PCR primer sets were: fractalkine (19), sense 5′-CTC GCC AAT CCC AGT GAC CTT GCT C-3′, antisense, 5′-GAT TGG TAG ACA GCA GAA CTC CAA ATG-3′; CX3CR1, sense, 5′-TGG GAC CAT CTT CCT ATC T-3′, antisense, 5′-CCT CTT CAT GCC ACA ACT A-3′; GAPDH, sense, 5′-CGT GGA GTC TAC TGG CGT CTT-3′, antisense 5′-TCA TCA TAC TTG GCA GGT TTC T-3′. The second PCR primers were same as the primers used for real-time RT-PCR described above.

Immunohistochemistry

To identify cell types expressing fractalkine and CX3CR1, ovaries from immature rats at 48 h after PMSG administration or 5 h after hCG injection were fixed overnight in 4% (wt/vol) paraformaldehyde before dehydration and embedding in paraffin. After deparaffinization and rehydration, 5-μm sections were pretreated in EDTA buffer (pH 8.0) for 15 min at 95 C to retrieve antigens. Tissue sections were then immersed in 1.5% peroxide/methanol for 15 min to remove endogenous peroxidase activity followed by blocking in 5% BSA for 60 min. Slides were incubated with goat antirat fractalkine antibodies (R&D Systems, Minneapolis, MN) at a 1:250 dilution or rabbit antirat CX3CR1 (Torrey Pines Biolabs, San Diego, CA) at a 1:500 dilution for 18 h at 4 C. After three washes in PBS, slides were incubated with biotin-conjugated antigoat or antirabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37 C. Tissue sections were then washed three times and incubated with horseradish peroxidase-conjugated streptavidin (Jackson Laboratories) for 30 min at 37 C. Signals were developed using the diaminobenzidine kit (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and cell nuclei were stained using the hematoxylin solution (Sigma-Aldrich). For negative controls, the primary antibodies were replaced by preimmune goat or rabbit IgG.

Granulosa cell cultures and measurement of steroid and cAMP production

Granulosa cells were collected from ovaries of immature rats 48 h after PMSG injection by punching ovaries in L-15 Leibovitz media (Life Technologies, Inc.-Invitrogen, Carlsbad, CA) and removing cell debris and small follicles. After centrifugation at 500 × g for 10 min, granulosa cells were dispersed by repeated washing before resuspension into McCoy’s 5a media (Modified; Invitrogen) supplemented with 10−7 m androstenedione, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in 48-well plates (Nunclon, Roskilde, Denmark) and treated with increasing doses of hCG (Fitzgerald, Chelmsford, MA) with or without recombinant rat fractalkine (R&D Systems). To test the dose-dependent effects of fractalkine, cells were treated with 20 ng/ml hCG and varying concentrations of recombinant fractalkine. Two days after culture, media were collected and stored at −80 C until progesterone and estradiol determination using a 125I-labeled RIA kits (Beijing North Institute of Biological Technology, Beijing, China). After repeated freeze-thawing of cells and media in the lysate buffer, total cAMP content was determined using a cAMP ELISA kit (R&D Systems).

Culture of preovulatory follicles

Preovulatory follicles (>400 μm in diameter) were dissected from ovaries of immature rats at 48 h after PMSG injection. Follicles (three per group) were incubated in 400 μl α-MEM (Invitrogen) supplemented with penicillin, streptomycin, l-glutamine and 0.1% BSA (Sigma-Aldrich) and treated with graded doses of hCG (National Hormone and Pituitary Program, Torrance, CA) in the absence or presence of 500 ng/ml recombinant fractalkine (R&D Systems). Follicles were placed in a chamber gassed at the beginning of the culture and at 12 h after culture initiation with 95% O2-5% CO2. Culture media were collected at 24 h after incubation and the progesterone content was determined using RIA.

Protein extraction and immunoblotting analyses

To measure the protein content of fractalkine, ovaries dissected from PMSG-primed immature rats before and at 5 h after hCG treatment were homogenized in PRO-PREP protein extraction solution (SBS Genentech, Beijing, China) containing protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, pepstatin A, leupeptin, aprotinin). For studies on MAPK signaling, granulosa cells from PMSG-injected rats were cultured in six-well plates coated with 5 μg/ml fibronectin (Sigma-Aldrich). After the cells were adhered to the plates, some cells were treated with increasing dose of recombinant rat fractalkine with or without purified hCG for 30 min. After the treatment, cells were washed twice and incubated with the PRO-PREP lysis buffer by adding phosphatase inhibitor cocktail (Sigma-Aldrich). Cell lysates were then centrifuged and supernatants were separated for immunoblotting analysis. Different samples were loaded on 12% SDS-polyacrylamide gels and transferred onto polyvinyl difluoride membranes. After blocking with 5% fat-free milk in Tris-buffered saline/Tween 20 buffer (pH 7.4) for 1 h at room temperature, membranes were incubated with different antibodies diluted in Tris-buffered saline buffer containing 0.1% Tween 20 and 5% BSA at 4 C overnight. The antibodies include antifractalkine antibody (1:1000 dilution; R&D Systems), anti-β-actin antibody (1:500 dilution; Neomarker, Fremont, CA), anti-p38 MAPK, anti-p42/44 MAPK, antiphospho-p38 MAPK (Thr180/Tyr182), and antiphospho-p42/44 MAPK (Thr202/Tyr204) antibodies (1:1000 dilution; Cell Signaling Technology, Beverly, MA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (antigoat, antirabbit, or antimouse antibodies; Jackson Laboratories) before signal development using an enhanced chemiluminescence kit (Santa Cruz Biotechnology).

Statistical analysis

All data are present as mean ± se of at least three independent experiments. Results were analyzed by one-way ANOVA and P < 0.05 was considered to be statistically significant.

Results

Expression of fractalkine and CX3CR1 transcripts in rodent ovaries after gonadotropin treatment

To provide a genome-wide survey of ovarian gene expression, immature mice were treated with Humegon to stimulate follicle maturation, followed by a single injection of Pregnyl to induce ovulation. DNA microarray analyses indicated transient increases in the expression of ovarian fractalkine transcripts shortly after Humegon (Fig. 1A, line graph). Furthermore, treatment with Pregnyl also dramatically increased fractalkine mRNA levels. In contrast, transcript levels for the CX3CR1 receptor displayed minimal changes during gonadotropin treatment (Fig. 1B, line graph). To confirm the DNA array data during the preovulatory period, we carried out real-time RT-PCR using ovarian samples from rats. Immature rats were administrated with PMSG (15 IU) followed by 10 IU hCG 48 h later. As shown in Fig. 1A (bar graph), fractalkine transcript levels were increased after hCG treatment with a peak at 3 h followed by a gradual decrease. Again, CX3CR1 transcripts levels showed minimal changes (Fig. 1B, bar graph).

Figure 1.

Figure 1

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Gonadotropin regulation of fractalkine and its receptor CX3CR1 in rodent ovaries. Immature mice or rats were treated with a single does of PMSG or Humegon to induce follicle maturation, followed at 48 h with a single injection of hCG or Pregnyl to induce ovulation. Line graphs represent DNA microarray data depicting the expression intensity of each transcript in gonadotropin-treated mouse ovaries (y-axis on the left), whereas bar graphs depict quantitative real-time RT-PCR results of fractalkine and CX3CR1 transcript levels in ovaries of gonadotropin-stimulated rats expressed as the ratio of target gene and GAPDH levels (y-axis on the right). Values for expression intensity were obtained after integration of hybridization signals from multiple probe sets. A, Fractalkine. B, CX3CR1. Data are represented as the mean ± se of three independent experiments. *, P < 0.05 as compared with ovarian transcripts at 48 h after PMSG treatment.

Localization of fractalkine and CX3CR1 transcripts in different ovarian compartments

To identify the cell types expressing fractalkine and its receptor, we performed tandem RT-PCR by isolating different ovarian compartments 3 h after hCG treatment from PMSG-primed rats. As shown in Fig. 2A, fractalkine transcripts were found in oocytes, cumulus cells, and granulosa cells as well as follicle shells consisting of mainly theca cells. In addition, CX3CR1 transcripts were detected in cumulus cells, mural granulosa cells, and follicle shells but not the oocyte.

Figure 2.

Figure 2

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Localization of fractalkine and CX3CR1 transcripts in immature rat ovaries. A, Expression of fractalkine and CX3CR1 mRNAs in isolated ovarian compartments were determined by tandem RT-PCR from PMSG-primed rats before and at 3 h after hCG injection. Levels of GAPDH served as loading controls, total ovarian cDNA served as positive controls, and samples without the reverse transcription reaction served as negative controls. OV, Ovary; C, negative controls; Oo, oocyte; CC, cumulus cells; GC, mural granulosa cells; FS, follicle shell. B, Quantitative determination of fractalkine transcript levels in whole ovaries (OV), cumulus-oocyte complexes (COC), granulosa cells (GC), and follicle shell (FS). Samples were obtained from rats before and at 3 h after hCG treatment and transcript levels were determined using real-time RT-PCR. All data were normalized based on GAPDH levels. Mean ± se of four independent experiments. *, P < 0.05; **, P < 0.001 as compared with the 0 h group.

To accurately identify ovarian cell types stimulated by hCG, we also performed real-time RT-PCR to estimate increases of fractalkine transcripts after hCG stimulation in different ovarian compartments. As shown in Fig. 2B, hCG treatment stimulated fractalkine expression in all ovarian compartments (P < 0.05) with granulosa cells displaying the highest increase (P < 0.01).

Expression of fractalkine and CX3CR1 antigens in rat ovaries

To extend findings on message levels, we also analyzed fractalkine and CX3CR1 protein expression in ovaries using immunohistochemistry. Fractalkine signals were found in oocyte, cumulus cells, mural granulosa cells, and theca cells before hCG injection (Fig. 3A). At 5 h after hCG treatment, stronger signals were detected in all these compartments. For CX3CR1 staining, comparable levels of signals were found in cumulus cells, mural granulosa cells, and theca cells before and after hCG injection (Fig. 3B). Unlike fractalkine, no CX3CR1 signal was detected in the oocyte.

Figure 3.

Figure 3

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Expression of fractalkine and CX3CR1 antigens in rat ovaries. A, Localization of fractalkine in ovaries of PMSG-primed rats before and at 5 h after hCG treatment. Fractalkine was localized in oocytes, cumulus cells (arrowheads), mural granulosa cells (thin arrow), and theca cells (thick arrow) and displayed increased staining after hCG treatment. The present graphs were derived using antirat fractalkine antibodies from R&D Systems. Similar staining was found using another antifractalkine antibody from Torrey Pines Biolabs (data not shown). B, Immunohistochemical analysis of CX3CR1 antigens. Signals were detected in cumulus cells (arrowhead), mural granulosa cells (thin arrow), and theca cells (thick arrow). No signal was detected on an adjacent control sections stained with rabbit or goat IgG. C, Immunoblotting analysis of fractalkine in rat ovaries before and at 5 h after hCG treatment. Levels of β-actin serve as sample loading controls. D, Densitometric analyses of immunoblotting results (mean ± se of three independent experiments). *, P < 0.05 as compared with ovaries before hCG treatment.

We further performed immunoblotting analyses to validate fractalkine protein expression in the ovary. As shown in Fig. 3C, a major band of approximately 50 kDa molecular mass is evident together with several minor bands with smaller sizes. Although the nature of these minor bands is unclear, they could represent degradation products. Densitometric analyses indicated that treatment with hCG increased the major fractalkine band by 135% (n = 3, P < 0.05).

Stimulatory effects of treatment with fractalkine on progesterone production in cultured granulosa cells and preovulatory follicles

To study possible effects of fractalkine on steroidogenesis, we isolated granulosa cells from immature rat ovaries 48 h after PMSG treatments and treated them with graded dose of hCG in the presence or absence of recombinant fractalkine (150 ng/ml). After culturing for 48 h, media progesterone and estradiol levels were detected using RIA. As shown in Fig. 4A, progesterone secretion was stimulated dose dependently by hCG. When cells were cotreated with fractalkine and hCG, progesterone production was further increased by approximately 2-fold (P < 0.05). Although fractalkine augmented hCG-stimulated progesterone production, no changes in either media estradiol or total cAMP production were found in the same cultures (Fig. 4, C and D).

Figure 4.

Figure 4

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Enhancing effects of fractalkine on hCG-stimulated progesterone biosynthesis by cultured granulosa cells and preovulatory follicles. Granulosa cells obtained from PMSG-primed rats were treated with increasing doses of hCG with or without recombinant fractalkine (150 ng/ml) for 48 h. Media progesterone content (A), media estradiol content (C), and total cAMP levels (D) are shown. Progesterone and estradiol contents were measured using RIAs, whereas cAMP levels were determined using ELISA. B, Dose-dependent effect of fractalkine on progesterone production. Granulosa cells were treated with 20 ng/ml hCG with or without varying doses of fractalkine for 48 h. E, Media progesterone concentrations from preovulatory follicles. Follicles were collected from PMSG-primed immature rats and cultured with graded doses of hCG. Some follicles were cotreated with recombinant fractalkine. After 24 h of culture, media progesterone content was measured by RIA. Data are represented as the mean ± se of three independent experiments, and n represents the number of follicles analyzed per group. *, P < 0.05; **, P < 0.001 as compared with corresponding cells or follicles treated with hCG alone.

The augmenting effect of fractalkine was dose dependent. As shown in Fig. 4B, cells were treated with 20 ng/ml hCG with or without cotreatment with increasing concentrations of fractalkine for 48 h. RIA analyses indicated that fractalkine enhanced the hCG-induced progesterone biosynthesis with a minimal effective dose at 150 ng/ml (Fig. 4B) (P < 0.05).

We performed preovulatory follicle cultures to further extend findings of enhanced progesterone production by fractalkine in granulosa cells. As shown in Fig. 4E, treatment with graded doses of hCG stimulated progesterone and the stimulatory effects of hCG were further augmented by fractalkine with a 4.1-fold increase at 30 ng/ml hCG.

Regulation of hCG-induced steroidogenic enzymes by fractalkine in granulosa cells

To investigate the mechanisms underlying fractalkine augmentation of hCG-stimulated progesterone production, we preformed real-time RT-PCR of key steroidogenic enzymes which are important for cholesterol transport (StAR), conversion of cholesterol to pregnenolone (CYP11A), and the synthesis of progesterone from pregnenolone (3β-HSD). Based on the dose dependency of progesterone stimulation induced by fractalkine, 150 ng/ml of fractalkine were chosen. Granulosa cells from PMSG-primed rats were incubated with 20 ng/ml hCG and/or 150 ng/ml fractalkine for 48 h. quantitative RT-PCR analyses (Fig. 5) indicated that fractalkine cotreatment significantly augmented the hCG stimulation of transcript levels for StAR, cytochrome P45011A (CYP11A), and 3β-HSD by 59, 60, and 71%, respectively (P < 0.05). In contrast, treatment with fractalkine alone did not affect the levels of these enzymes (P > 0.05).

Figure 5.

Figure 5

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Enhancing effect of fractalkine on the hCG stimulation of transcript levels for different steroidogenic enzymes in cultured granulosa cells. Isolated granulosa cells were cultured with 20 ng/ml hCG and/or 150 ng/ml fractalkine for 48 h. Transcript levels of StAR (A), CYP11A (B) and 3β-HSD (C) were quantitated using real-time RT-PCR and normalized using GAPDH levels. Data are represented as the mean ± se of three independent experiments. *, P < 0.05 as compared with cells treated with hCG alone.

Fractalkine treatment enhanced p38 MAPK activation in cultured granulosa cells

We further tested possible signaling pathways activated by the ovarian fractalkine/CX3CR1 system. In diverse tissues, fractalkine was found to activate different MAPK components (20,21). We tested three well-characterized subfamilies of the MAPK system including P42/44 MAPK (ERK1/2), c-Jun N-terminal kinase (JNK) MAPK, and P38 MAPK using granulosa cells. Cells were treated with increasing doses of fractalkine with or without 20 ng/ml hCG for 30 min before immunoblotting analyses. As shown in Fig. 6A, treatment with hCG stimulated p38 MAPK activation. In addition, both basal and hCG-stimulated p38 MAPK phosphorylation was enhanced by fractalkine cotreatment. As shown in Fig. 6B, densitometric analyses indicated approximately 2-fold increases in the phosphorylation of p38 MAPK after fractalkine treatments compared with basal and hCG-treated group (n = 3). In contrast, fractalkine treatment did not affect ERK1/2 and JNK phosphorylation under the same treatment conditions (data not shown). These findings suggest that, in preovulatory granulosa cells, p38 MAPK pathway can be activated shortly after fractalkine treatment.

Figure 6.

Figure 6

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Fractalkine treatment enhances the phosphorylation of p38 MAPK in cultured granulosa cells. A, Isolated granulosa cells were cultured for 2 h to allow attachment to the plates before treating cells with graded doses of fractalkine with or without 20 ng/ml hCG for 30 min. Representative immunoblotting results of phospho-P38 MAPK (Thr180/Tyr182) (upper lane), total P38 MAPK protein (middle lane), and β-actin (lower lane) are shown. B, Densitometric analyses of phospho-P38 MAPK levels from three independent experiments. Data are represented as mean ± se. *, P < 0.05, compared with controls or groups treated with hCG alone.

Discussion

Our data demonstrate major increases in fractalkine expression after the preovulatory LH/hCG stimulation. RT-PCR using isolated ovarian cell types and immunohistochemical analyses further localized major increases of fractalkine expression to granulosa cells. In vitro treatment of preovulatory granulosa cells and isolated follicles with fractalkine augmented hCG-stimulated progesterone biosynthesis. This augmentative effect is not associated with changes in either estradiol or cAMP secretion and is likely mediated by a stimulation of key steroidogenic enzymes including CYP11A, StAR, and 3β-HSD. The observed increases in fractalkine expression and its augmentation of progesterone biosynthesis by cultured granulosa cells indicated that this intraovarian hormone could be produced in concert with the preovulatory LH surge to facilitate optimal progesterone biosynthesis and the eventual luteinization process.

In the ovary, resident leukocytes were postulated to be involved in inflammation-like processes related to ovulation and luteal functions (22,23,24,25). During ovulation, several chemokines (IL-8, monocyte chemotactic protein-1, thymus-expressed chemokine, and others) were found in the ovary and could recruit leukocytes to the ovary (26,27,28). Fractalkine is a chemokine with the unique C-X3-C motif and expressed in two forms either membrane-bound or soluble. The soluble form of fractalkine acts as a chemoattractant for monocytes and lymphocytes in the immune system (16,21,29), whereas the membrane-bound form promotes adhesion of leukocytes to endothelial cells (12,14,30). Our immunoblotting analyses suggested the majority of fractalkine (∼50 kDa) in the preovulatory ovary is likely a soluble form based on its molecular size. In many tissues, soluble fractalkine can be released via proteolysis by the TNF-α converting enzyme (31) or ADAM 10 (32) Of interest, both of these enzymes have been reported in ovary (33), and future studies on their roles in fractalkine processing are important. In the preovulatory rat ovary, Wong et al. (34) identified and quantified more than a dozen chemokines. Among them, fractalkine was found to be expressed in the PMSG-primed ovaries at 6 h after hCG treatment. Although the authors suggested that intraovarian chemokines may be responsible for the cyclic intraovarian residence of representatives of the white blood cell series, no functional analyses were performed.

Although previous studies focused on chemokines secreted from ovarian resident leukocytes, few studies dealt with chemokines secreted from ovarian somatic cells. Our data demonstrated a preovulatory increase in the expression of fractalkine at both mRNA and protein levels. Although we cannot rule out its expression in ovarian lymphocytes, fractalkine is expressed mainly in ovarian somatic cells including cumulus cells, mural granulosa cells, and theca cells. We further demonstrated major increases of fractalkine expression in granulosa cells after treatment with an ovulatory dose of hCG. In addition, CX3CR1, the receptor for fractalkine, is also expressed in cumulus cells, granulosa cells, and theca cells. However, the levels of this receptor did not vary during the preovulatory period.

In the ovary, the preovulatory gonadotropin surge induces profound changes in the preovulatory follicles, including breakdown of the extracellular matrix at follicle apex for the release of the cumulus-oocyte complex, differentiation of follicular cells to luteal cells, and oocyte maturation in preparation for fertilization and early embryo development. These events are mediated or modulated though diverse paracrine factors. The preovulatory LH/hCG stimulates the expression of BDNF to promote first polar body extrusion and cytoplasmic maturation of oocytes (8). Similarly, preovulatory increases in epithelial growth factor-like growth factors by granulosa/cumulus cells facilitate cumulus expansion and oocyte maturation (6). Additionally, increases in preovulatory LH stimulate theca cells to produce insulin-like factor 3 important for germinal vesicle breakdown of the oocyte (7).

Our data indicate that in both cultured granulosa cells and preovulatory follicles, treatment with fractalkine augments the hCG stimulation of progesterone biosynthesis. This is accompanied by increases in the levels of three key steroidogenic enzymes important for progesterone synthesis, including StAR, CYP11A, and 3β-HSD (35,36,37). Earlier studies indicated that the preovulatory LH surge initiates a program in follicular cells to undergo structural and genomic changes needed for their differentiation into nondividing progesterone-producing luteal cells (38). We found the preovulatory increases of LH/hCG rapidly induced transcript and protein levels for fractalkine at 3 and 5 h after hormonal treatment, respectively. Due to the essential role of luteal progesterone for pregnancy, a complex series of luteinization stimulators and inhibitors are involved in maintaining optimal progesterone biosynthesis. Ovarian growth differentiation factor-9, bone morphogenetic protein-6, endothelin-1, stanniocalcin-1, and TWEAK have been postulated as luteinization inhibitors for their progesterone-suppressing actions (39,40,41,42,43). In contrast, IL-1β, prolactin, and pituitary adenylate cyclase activating polypeptide were believed to be luteinization stimulators due to their ability to augment progesterone biosynthesis from preovulatory follicles (44,45,46). Among the luteinization stimulators, fractalkine is similar to pituitary adenylate cyclase activating polypeptide (46,47,48); both of these intraovarian factors were found to be increased by the preovulatory LH/hCG surge and could act in concert with LH to promote progesterone biosynthesis and luteinization.

To further elucidate molecular mechanisms underlying fractalkine actions in preovulatory granulosa cells, we analyzed signaling mediated by MAPKs represented by the well-characterized ERK1/2, SAPK-1/JNK, and SAPK-2/p38MAPK pathways (49). Although treatment with fractalkine did not affect ERK1/2 and JNK phosphorylation, it stimulated basal and hCG-stimulated p38 MAPK activation in cultured granulosa cells. The observed effects of hCG on p38 MAPK phosphorylation is consistent with earlier reports showing p38 MAPK activation by hCG in a protein kinase A- and C-independent manner (50). Although hCG treatment also activated ERK1/2 phosphorylation in cultured granulosa cells (51,52), treatment with fractalkine was ineffective. In microglial cells, treatment with soluble fractalkine also stimulates the p38 MAPK pathway (53), whereas this hormone activates both ERK1/2 and p38 MAPK pathways in a monocytic cell line (29). Although p38 MAPK signaling is generally considered to be activated in response to environmental stresses, recent studies have shown that activation of p38 MAPK can also lead to other biological responses (54,55). Because the fractalkine augmentation of p38 phosphorylation showed an all or none relationship, the exact relationship between p38 MAPK activation and progesterone biosynthesis from preovulatory granulosa cells is presently unclear. A role of this signaling pathway in luteinization has been postulated because intraluteal administration of a p38 MAPK inhibitor (SB203580), but not a ERK1/2 inhibitor (PD98059), suppressed luteal functions in monkeys (56).

The present findings suggest that the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles. After the preovulatory increases in LH, fractalkine transcripts and proteins are induced mainly in preovulatory granulosa cells to augment progesterone production by granulosa cells and to promote the luteinization process. It is becoming clear that a diverse group of hormonal factors are induced by the preovulatory surge of LH to exert intraovarian actions for optimal luteinization and pregnancy.

Acknowledgments

We thank Professor Lin Zhi Zhuang for her kind help in setting up the RIA and providing the antiestrogen antiserum.

Footnotes

This work is supported by the National Basic Research Program of China (2007CB947401 and 2006CB0F1006) and the National Institute of Child Health and Human Development, National Institutes of Health, Cooperative Agreement U54 HD31398 as part of the Specialized Cooperative Centers Program in Reproduction Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 21, 2008

Abbreviations: BDNF, Brain-derived neurotrophic factor; CYP11A, cytochrome P45011A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; 3β-HSD, 3β-hydroxysteroid dehydrogenase; JNK, c-Jun N-terminal kinase; StAR, steroidogenic acute regulatory protein; TWEAK, TNF-related weak inducer of apoptosis.

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