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BOR - Papers in Press, published online ahead of print February 28, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.057927
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BIOLOGY OF REPRODUCTION 76, 1016–1024 (2007)
DOI: 10.1095/biolreprod.106.057927
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Molecular Mechanisms of Insulin-Like Growth Factor 1 Promoted Synthesis and Retention of Hyaluronic Acid in Porcine Oocyte-Cumulus Complexes1

Lucie Nemcová 3, Eva Nagyová 3, Michal Petlach 3, Milan Tománek 4, and Radek Procházka 2 3

Academy of Sciences of the Czech Republic,3 Institute of Animal Physiology and Genetics, Libechov 277 21, Czech Republic Research Institute of Animal Production,4 104 01 Prague 10-Uhríneves, Czech Republic

ABSTRACT

The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.

cumulus cells,, expansion, follicle-stimulating hormone, gamete biology, ovary, pig, signal transduction

INTRODUCTION

Mammalian oocytes are arrested at the dictyate stage of meiotic prophase I during growth. The resumption of meiosis occurs in preovulatory follicles as a result of interactions between the oocyte and surrounding cumulus and granulosa cells. The preovulatory surge of LH elicits signals in the somatic follicular cells that result in loss of their inhibitory activity and allow maturation of the oocyte to the ovulatory stage. LH may act directly on the cumulus cells [1] and/or indirectly via the production of specific mediators of the LH surge in granulosa cells [2, 3]. The morphology of cumulus cells changes rapidly after the LH surge. The number of gap junctions among the cumulus cells is reduced [4, 5], the cytoskeleton of cumulus cells undergoes complex rearrangement [6–9], and the cumulus cells start to synthesize hyaluronic acid (HA)-enriched extracellular matrix. The matrix is then deposited into extracellular spaces, leading to the process of expansion [10]. The production of HA is controlled by hyaluronan synthase 2 (HAS2), which is strongly expressed in cumulus cells shortly after the preovulatory surge of LH [11]. HA is anchored to the surface membranes of cumulus cells via the HA-binding receptor CD44 [12, 13]. The retention and organization of HA in the extracellular matrix is mediated by HA-binding proteins, which include versican [14], tumor necrosis factor alpha-induced protein 6 (TNFAIP6) [15], and serum-derived members of the inter-{alpha}-trypsin inhibitor (I{alpha}I) family [16, 17]. The expansion ensures detachment of the oocyte from the follicle wall, its ovulation into the oviduct, and successful fertilization [18, 19]. In addition, the binding of HA to CD44 activates a signaling pathway that is necessary for phosphorylation of connexin 43 and closure of the gap junctions in the cumulus compartment [20].

Under in vitro conditions, the expansion of cumulus cells can be induced by FSH in all mammalian species. FSH induces an increase in the cAMP concentration in cumulus cells [10, 21], followed by increased synthesis of key enzymes that are involved in the production of HA and in the binding of HA to the receptor, such as HAS2, prostaglandin-endoperoxidase synthase 2 (PTGS2), and TNFAIP6 [22, 23]. The expansion can also be induced in vitro by forskolin, which is a direct activator of adenylate cyclase [24, 25], or by 8-Br-cAMP, which is a membrane-permeable analogue of cAMP [26]. The increased level of cAMP results in activation of the cAMP-dependent protein kinase (PKA), which regulates transcription in cumulus cells via the transcription factor CREB [19]. However, an additional signal appears to be required for the expansion of cumulus cells. Mouse cumulus cells have a strict requirement for signaling from the oocyte, i.e., production of a protein that works in a paracrine manner to enable synthesis of HA by the cumulus cells [26]. The nature of this factor remains a matter of debate, although members of the transforming growth factor ß superfamily are among the most likely candidates [27]. In the pig, expansion of cumulus cells appears to be independent of oocyte-secreted factors [25, 28], while growth factors present in the follicular fluid may positively regulate this process. Epidermal growth factor (EGF) acts synergistically with FSH to promote synthesis of HA and the expansion of pig oocyte-cumulus complexes (OCCs) isolated from medium-size follicles [29]. In addition, EGF can efficiently stimulate the expansion of OCCs isolated from preovulatory follicles, as demonstrated for murine [30, 31] and porcine OCCs [9, 29]. Next, signaling in cumulus cells mediated by insulin-like growth factor 1 (IGF1) appears to be essential or at least beneficial for the FSH-stimulated expansion of porcine OCCs in vitro [32, 33]. It has been reported previously that IGF1 promotes FSH-stimulated synthesis and retention of HA in porcine OCCs cultured in serum-free medium [33].

The aims of the present study were to identify signaling pathways that are implicated in the promotion of FSH-induced expansion of porcine OCCs by IGF1 and to investigate whether the signals elicited by IGF1 increase the expression of HAS2 mRNA. For this purpose, the OCCs were cultured in vitro in serum-free medium and the effects of FSH and IGF1 on the expansion of cumulus cells and the synthesis of HA were assessed. In addition, the production of cAMP and the activation of PKA, mitogen-activated protein kinases 3 and 1 (MAPK3/1), and phosphoinositide-3-kinase (PIK3)/v-akt murine thymoma viral oncogene homolog (AKT; also known as protein kinase B) signaling pathways were examined in the stimulated OCCs. Finally, the effects of FSH and IGF1 on the expression of HAS2 mRNA were assessed in the present study.

MATERIALS AND METHODS

Isolation and Culturing of Oocyte-Cumulus Complexes

The ovaries of slaughtered gilts were collected at a local abattoir and transported to our laboratory in a thermos flask. OCCs were released from the medium-sized follicles (3–5 mm in diameter) by aspiration and washed three times in PBS that was supplemented with 3 mg/ml polyvinylpyrrolidone (PVP), as described previously [29]. OCCs with equal cumulus masses were randomly allocated to experimental groups and cultured in M-199 (Sigma, Prague, Czech Republic) that was supplemented with 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, 0.57 mM cysteine, antibiotics, and 3 mg/ml PVP. Ten OCCs were cultured in 100 µl of the medium in 96-well culture dishes (Corning) at 38.5°C in an atmosphere of 5% CO2 in air. To stimulate expansion of the cumulus cells, the culture medium was supplemented with 10 ng/ml of human recombinant FSH (Puregon; Organon, Oss, The Netherlands) alone or in combination with 50 ng/ml of IGF1 (Sigma). Cumulus cell expansion was assessed after 24 h of culture by measuring the maximum diameter of the expanded cumulus with an ocular micrometer.

In the kinase inhibition experiments, the culture medium was supplemented with 25 µM of the AKT inhibitor SH6 (Calbiochem, Merck Biosciences, Darmstadt, Germany), 10 µM of the MAPK3/1 inhibitor U0126 (Sigma) or 25 µM of the PIK3 inhibitor LY 294002 (Sigma). In these experiments, the control OCC groups were cultured in medium that was supplemented with 0.2% DMSO, which was used as the solvent for the inhibitors.

HA Synthesis

Groups of 10 porcine OCCs were cultured in 100 µl of the culture medium supplemented with 2.5 µCi of D-[6-3H] glucosamine hydrochloride (Amersham, Uppsala, Sweden) for 24 h. HA synthesis was measured using the procedure described by Eppig [34], with slight modifications [33]. Briefly, the cultures were terminated by adding 10 µl of a solution that contained 50 mg/ml pronase (Sigma) and 10 % Triton X-100 in 0.2 M Tris (pH 7.8). The samples were incubated for 2 h at 38.5°C and then transferred to Whatman 3MM filter paper circles. The circles were air-dried and then washed three times in a solution that contained 0.5% cetylpyridinium chloride and 10 mM of nonradioactive glucosamine hydrochloride (Sigma) for 45 min each. The circles were dried once again and the radioactivity was measured in a liquid scintillation counter. Synthesis of HA was measured either in medium with OCCs (total HA) or within the complexes alone (retained HA), which was achieved by simply transferring the complexes through three dishes of culture medium without labeled precursor before the addition of the pronase-Triton X-100 solution.

Production of cAMP and the PKA Assay

To assess the production of cAMP in OCCs after specific treatments, a two-factorial experiment with FSH (10 ng/ml) and IGF1 (50 ng/ml) was designed. Groups of 10 OCCs were cultured in 100 µl of the culture medium for 3 h, which according to our preliminary experiments is a sufficient period for the accumulation of cAMP and activation of downstream signaling pathways. The culture medium was aspirated at the end of the culture period and the concentration of cAMP was analyzed.

The levels of cAMP were quantified by radioimmunoassay using a commercially available cAMP RIA kit (Immunotech, Marseille, France). Briefly, aspirated culture media were diluted 1:11 with the assay buffer and 100 µl of each sample was pipetted into the antibody-coated tubes in duplicate. Thereafter, 500 µl of tracer (125I-cAMP) was added. The standard curve and positive and negative controls were prepared according to the kit instructions supplied. All tubes were incubated at 4°C for 18 h. After incubation, the contents of the tubes were removed by aspiration and the tubes were evaluated using the Berthold LB 2104 multi-crystal gamma counter (Berthold, Germany). The detection limit of the assay was 0.15–0.33 nM, and the intraassay and interassay coefficients of variation (%) were 6.1–7.7 and 9–11, respectively.

PKA activity was assessed using the PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase kit (Promega, Madison, WI) according to the manufacturers instructions. Briefly, 50 OCCs were cultured in control or FSH/IGF1-supplemented medium for 1 h, by which time the PKA activity had peaked, according to our preliminary experiments. The OCCs were then lysed in 10 µl of cell lysis buffer (Cell Signaling Technology, Danvers, MA) and mixed in a test tube with 5 µl of PKA reaction buffer (100 mM Tris [pH 7.4], 50 mM MgCl2, 5 mM ATP), 5 µl of PKA-specific fluorescent peptide substrate (Kemptide, 0.4 µg/µl stock) and 5 µl of deionized water. A negative control assay was run without the OCCs. In positive control assays, the sample was substituted with 25 ng of the catalytic subunit of PKA (diluted in 5 µl of 350 mM K3PO4 with 0.1 mM dithiothreitol) and 5 µl of PKA activator solution (5 µM cAMP in water). The reaction mixture was incubated at room temperature for 30 min and then stopped by placing the test tubes in boiling water for 10 min. The samples were loaded into the wells of a 0.8% agarose gel and electrophoresed at 100 V for 15 min. The phosphorylated substrate migrated towards the positive electrode, while the nonphosphorylated substrate migrated towards the negative electrode. The gel was photographed under UV light, and the images were assessed by densitometry for the proportions of phosphorylated and nonphosphorylated substrate.

Immunoblotting

Groups of OCCs were cultured in control or FSH/IGF1-supplemented medium for 1 h and then lysed in 15 µl of Laemmli sample buffer for SDS-PAGE, heated at 100°C for 3 min, and stored at –80°C. The proteins were separated on a 10% polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The membranes were blocked in 5% low-fat dry milk in Tris-buffered saline (TBS) with 0.5% of Tween-20 for 2 h at room temperature, and then incubated at 4°C overnight with primary antibody diluted 1:2000 in TBS-Tween plus 5% BSA. The following primary antibodies were used: anti-phospho-AKT (Ser 473) and anti-AKT (Cell Signaling Technology), and anti-p-ERK and anti-ERK (for the detection of MAPK3/1) (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibody (horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG; Amersham) was diluted 1:5000 in TBS-Tween plus 2% BSA. The membranes were incubated with the secondary antibody for 1 h at room temperature and washed intensively with TBS-Tween. The immune reaction was detected by enhanced chemiluminiscence (Pierce, Rockford, IL) according to the manufacturers instructions. Following detection, the antibodies were stripped by incubation of the membrane in 25 mM Tris with 2% ß-mercaptoethanol and 0.2% SDS at 60°C for 20 min, and reprobed with the next primary antibody. The intensities of the specific bands on the blots were analyzed by scanning densitometry using the Image J Version 1.29 free software (National Institute of Mental Health, Bethesda, MD).

Detection of HAS2 mRNA by RT-PCR

The total RNA from 30 OCCs was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. The concentration of total RNA in the samples was measured with a spectrophotometer NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). The RT-PCR was carried out with the One-Step RT-PCR Kit (Qiagen) using oligonucleotide primers directed against specific sequences of pig HAS2 (5'-GAATTACCCAGTCCTGGCCTT-3' and 5'-GGATAAACTGGTAGCCAACA-3') [13]. These primers were expected to generate a 581-bp cDNA fragment. For ß-actin (ACTB) as an internal control gene, RT-PCR was performed using the primers 5'- GACCCAGATCATGTTTGAGACC-3' and 5'-ATCTCCTTCTGCATCCTGTCAG-3', which generated a 593-bp fragment.

The total RNA of the samples was reverse-transcribed and amplified in a reaction mixture (total volume of 25 µl) that contained 5 µl of 5x reaction buffer, 1 µl dNTP mix (10 mM stock of each), 0.5 µl of both reverse and forward primers (0.02 mM stock), 0.15 µl RNasin (20 U/µl stock; Promega), 1 µl of enzyme mix, and RNA. For each sample, the amplification of both genes was run in separate tubes. The reaction conditions were as follows: cDNA synthesis at 50°C for 30 min, predenaturation at 95°C for 15 min, followed by various numbers of PCR cycles, each of which consisted of denaturation at 95°C for 30 sec, annealing at 57°C or 64°C for 30 sec for HAS2 and ACTB, respectively, extension at 72°C for 45 sec, and a final extension step at 72°C for 5 min. For semiquantitative RT-PCR, the number of cycles was optimized for each set of samples using a gradient method over the range of 20–40 cycles, to ensure that the amplification of cDNA for both primer sets was terminated in the exponential phase of the PCR. The products of the RT-PCR were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.

Quantification of HAS2 mRNA Expression by Real-Time RT-PCR

The relative abundance of HAS2 mRNA in porcine OCCs stimulated with FSH and IGF1 was assessed by a real-time RT-PCR using specific primers for the HAS2 sequence (GenBank accession no. XM 539153): 5'-GAAGTCATGGGCAGGGACATTC-3' and 5'-TGGCAGGCCCTTTCTATGTTA-3', which generated a 407-bp fragment.

The reaction mixture was the same as that described above. In addition, 0.5 µl of a 1000x stock solution of SYBR Green I (Molecular Probes, Eugene, OR) was added to each reaction. The amplification was performed on the RotorGene 2000 cycler (Corbett Research, Sydney, Australia) under the reaction conditions described above. Fluorescence data were acquired during an additional step at approximately 3°C below the product melting temperature (Tm), to distinguish potential primer-dimers. After the cycling, the melting curve was generated to verify the amplification of one specific target (one peak at a specific melting temperature demonstrates the specificity). No primer-dimers were generated during the 35 amplification cycles of real-time RT-PCR. In addition, the specificities of the RT-PCR products were assessed by gel electrophoresis and staining, as described above.

The relative concentrations of the templates in different samples were determined using comparative analysis software (Corbett Research). The results for individual target genes were normalized according to the relative concentration of the internal standard. The relative abundance of HAS2 mRNA is expressed as the HAS2:ACTB ratio.

Statistical Analysis

Each experiment was performed with at least three replicates. Differences in the percentages of expanding OCCs, differences between the amounts of HA and HAS2 mRNA in specific treatment groups, and the densitometrical quantifications of proteins and proportions of phosphorylated and nonphosphorylated substrate in the PKA assay were compared by ANOVA followed by the Tukey post-test. Error bars indicate the standard error of the mean (SEM).

RESULTS

IGF1 Promotes FSH-Stimulated Expansion of Cumulus Cells

Our previous study indicated that IGF1 increases FSH-stimulated synthesis of HA and its retention by pig cumulus cells [33]. In the present study, we have confirmed the results concerning HA synthesis and retention and expanded the data with an objective assessment of the degree of cumulus expansion by measuring the maximum diameter of the expanded cumulus. The mean diameter of the control OCCs matured for 24 h without FSH and IGF1 was 259 ± 5 µm (Fig. 1A), and the size and appearance of these OCCs were similar to those of intact OCCs isolated from the follicles (Fig. 2, A and B). OCCs cultured in medium with FSH underwent extensive expansion and their mean diameter was 371 ± 11 µm (P < 0.001) (Fig. 1A and Fig. 2C). The addition of IGF1 (50 ng/ml) together with FSH resulted in a further significant increase in the OCC diameter (442 ± 12 µm, P < 0.001; Fig. 1A and Fig. 2D). Correspondingly, the quantity of the synthesized and retained HA (Fig. 1B) was significantly higher for OCCs cultured with FSH than in the control group of nonstimulated OCCs. A further significant increase in the quantity of synthesized and the retained HA occurred in OCCs that were cultured with FSH and IGF1, as compared to OCCs cultured with FSH only (P < 0.05; Fig. 1B). These data show that IGF1 promotes FSH-stimulated synthesis and retention of HA in OCCs cultured in serum-free medium.


Figure 01
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  		FIG. 1. Effects of IGF1 and FSH on the expansion of porcine OCCs and on the synthesis and retention of HA. A) Diameters of OCCs cultured for 24 h in control (C) and FSH- and IGF1-supplemented media. Fifty OCCs were included in each group and the columns represent mean diameter ± SEM. B) Assessment of the total amount of HA and its retention in the cultured OCCs. The results of three independent experiments are summarized and expressed as mean ± SEM. Bars with no common letters indicate significant differences (P < 0.001 for A; P < 0.05 for B).


Figure 02
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  		FIG. 2. Expansion of OCCs cultured in vitro. A) Group of intact OCCs immediately after isolation from medium-sized follicles. B) Group of OCCs cultured in medium without FSH and IGF1 for 24 h. C) Group of OCCs cultured in medium supplemented with FSH for 24 h. D) Group of OCCs cultured in medium supplemented with FSH and IGF1 for 24 h. Original magnification x20.

IGF1 Does Not Increase Synthesis of cAMP and Activation of PKA in FSH-Stimulated OCCs

It has been shown that IGF1 has a synergistic effect with FSH on the production of cAMP in certain types of mammalian cells, especially with low doses of FSH [35, 36]. This finding led us to examine whether this mechanism is involved in IGF1-promoted synthesis of HA and expansion of OCCs cultured in FSH-supplemented medium. To answer this question, we carried out a two-factorial experiment to assess the effects of FSH and IGF1 on the production of cAMP (Fig. 3A). FSH alone stimulated the production of cAMP in the cultured OCCs. IGF1 alone neither stimulated the production of cAMP nor increased the FSH-stimulated production of cAMP. These data suggest that IGF1 does not promote FSH-stimulated synthesis and retention of HA by affecting cAMP production in OCCs. This conclusion was strengthened by the assay of PKA activity in the stimulated OCCs. FSH significantly increased the activity of PKA in the OCCs during 1 h of culture (Fig. 3, B and C), whereas IGF1 had no significant effect on PKA activity, irrespective of whether it was added to the culture medium alone or together with FSH.


Figure 03
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  		FIG. 3. Effects of IGF1 and FSH on PKA activity and cAMP production in cultured porcine OCCs. A) Assessment of cAMP produced by 10 OCCs during culture in vitro. The results of three independent experiments are summarized and expressed as mean ± SEM. B) Migration of the phosphorylated and non-phosphorylated PKA-specific substrate in an electric field following the PKA assay. Fifty OCCs were cultured in control (C) and IGF1- and FSH-supplemented media. Negative (N) and positive (P) control assays were run without the OCCs and with 25 ng of the catalytic subunit of PKA, respectively. C) Densitometric assessment of the three images shown in B. Different superscripts above the bars indicate significant differences (P < 0.001 for A; P < 0.05 for C).

IGF1Stimulates Phosphorylation of AKT in Cumulus Cells by a PIK3-Dependent Mechanism

Stimulation of OCCs with either FSH or IGF1 led to moderate phosphorylation of AKT (Fig. 4). Stimulation of the OCCs with FSH and IGF1 together resulted in a dramatic increase in AKT phosphorylation. This increase in AKT phosphorylation was dependent upon PIK3, since it was significantly (P < 0.05) reduced by LY 294002, which is a specific inhibitor of PIK3. Most of the phospho-AKT signal came from the cumulus cell compartment of the OCCs, since the signal elicited under the same experimental conditions by denuded oocytes was very faint (Fig. 4A).


Figure 04
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  		FIG. 4. Effects of IGF1 and FSH on AKT activation in cultured porcine OCCs. A) Immunoblotting of phosphorylated AKT (p-AKT, top panel) and total AKT (bottom panel) in samples of 25 OCCs cultured for 1 h in control (C) and FSH- and IGF1-supplemented media. An example of a p-AKT signal developed for 25 oocytes stripped of cumulus cells after the culture period is shown in the middle panel. The position of the 66-kDa molecular mass marker is on the right. Shown is a representative of three independent experiments. B) Quantification of activated AKT in OCCs by densitometry. The results are shown as proportions of the phosphorylated and total AKT and expressed in arbitrary units (AU) as fold-strength increases above the proportion found in OCCs cultured in the control medium. Different superscripts above the bars indicate significant differences (P < 0.05).

IGF1 Increases FSH-Stimulated MAPK3/1 Activity in Cumulus Cells

IGF1 promptly activates MAPK3/1 in a variety of cell types. We investigated whether IGF1 activates MAPK3/1 in cumulus cells and whether this kinase is involved in IGF1-promoted synthesis of HA. Treatment of the OCCs with FSH resulted in significant (P < 0.05) increase in the phosphorylation of MAPK3/1 within 1 h (Fig. 5). Stimulation of the OCCs with IGF1 alone did not increase MAPK3/1 phosphorylation above the control level. However, IGF1 enhanced significantly and in a synergistic manner the phosphorylation of MAPK3/1 caused by FSH (Fig. 5). The IGF1-induced increase in MAPK phosphorylation was not significantly (P > 0.05) reduced by LY 294002. Thus, the role of PIK3 in this process remains uncertain.


Figure 05
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  		FIG. 5. Effects of IGF1 and FSH on MAPK3/1 activation in cultured OCCs. A) Immunoblotting of phosphorylated MAPK3/1 (p-MAPK, top panel) and total MAPK3/1 (bottom panel) in samples of 25 OCCs cultured for 1 h in control (C) and FSH- and IGF1-supplemented media. Shown is a representative of three independent experiments. B) Quantification of activated MAPK3/1 in OCCs by densitometry. The results are shown as proportions of the phosphorylated and total MAPK3/1 and expressed in arbitrary units (AU) as fold-strength increases above the proportion found in OCCs cultured in the control medium. Bars with no common letter are significantly different (P < 0.05).

The AKT and MAPK3/1 Pathways Are Involved in IGF1-Promoted Production and Retention of HA

The previous experiments of the present study showed that IGF1 significantly increased the phosphorylation of AKT and MAPK3/1 induced by FSH. Consequently, the possible involvement of these kinases in the mechanism of IGF1-enhanced synthesis and retention of HA was investigated. We found that specific inhibitors of AKT (SH6; 25 µM) [37] and MAPK3/1 (U0126; 10 µM) significantly decreased both the synthesis (Fig. 6A) and retention (Fig. 6B) of HA that was stimulated by concomitant exposure of OCCs to FSH and IGF1. The production and retention of HA were reduced by SH6 and U0126 below the levels induced in OCCs by FSH alone. These data suggest that the AKT and MAPK3/1 pathways are involved not only in IGF1-induced promotion of HA, but also in the mechanism by which FSH itself stimulates the synthesis and retention of HA.


Figure 06
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  		FIG. 6. Effects of AKT and MAPK3/1 inhibitors on FSH- and IGF1-stimulated synthesis and retention of HA in cultured OCCs. The OCCs were stimulated with FSH and IGF1 and cultured in medium with AKT inhibitor SH6 (25 µM), MAPK3/1 inhibitor U0126 (10 µM) or both inhibitors, and the amount of total and retained HA was assessed after 24 h of culture. The results are shown in percentages of the amount of HA synthesized (A) or retained (B) in the OCCs stimulated with FSH (100%). Control OCCs (C) were cultured in medium with 0.2% DMSO. The results of four independent experiments are summarized and expressed as mean ± SEM. Different superscripts above the bars indicate significant differences (P < 0.05).

Effect of IGF1 on HAS2 Expression

To determine whether IGF1-promoted synthesis and retention of HA were caused by an increase in the expression of HAS2, the patterns of FSH-induced and FSH + IGF1-induced HAS2 mRNA expression were compared in porcine OCCs by semiquantitative RT-PCR. In both experimental groups, HAS2 mRNA was detected 2 h after stimulation and was increased at 4 h and 8 h of culture (Fig. 7, A and B). In OCCs stimulated with FSH only, HAS2 expression decreased rapidly after 20 h of culture and was undetectable at 24 h (Fig. 7A). In contrast, in OCCs stimulated simultaneously with FSH and IGF1, the expression of HAS2 was maintained after 20 h of culture and was still detectable at 24 h of culture (Fig. 7B). To assess the mechanism by which IGF1 maintains FSH-induced expression of HAS2, we investigated the relative abundance of HAS2 mRNA in stimulated OCCs using real-time RT-PCR. IGF1 itself neither stimulated the expression of HAS2 nor increased FSH-induced expression of HAS2 at 4 h of culture (Fig. 8A). At 24 h of culture, the total expression of HAS2 mRNA was maximal in OCCs that were stimulated simultaneously with FSH and IGF1 (Fig. 8B, top). However, the relative abundance of HAS2 mRNA, as calculated from the HAS2:ACTB ratio, was lower in this group of OCCs than in OCCs stimulated with FSH alone (Fig. 8B, bottom).


Figure 07
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  		FIG. 7. Detection of HAS2 mRNA expression in cultured OCCs A) Time course of FSH-stimulated expression of HAS2 mRNA assessed by RT-PCR. B) Time course of FSH- and IGF1-stimulated expression of HAS2 mRNA assessed by RT-PCR. Total RNA was isolated from OCCs cultured for the indicated periods of time, and an equivalent of two OCCs was used in each RT-PCR reaction (33 cycles for HAS2 and 24 cycles for ACTB). The positions of the DNA marker are indicated on the left. Shown are representatives of three independent experiments.


Figure 08
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  		FIG. 8. Assessment by real-time RT-PCR of the relative abundances of HAS2 mRNA in OCCs cultured for 4 h (A) or 24 h (B). The results are shown as the proportions of the mRNAs in stimulated OCCs, expressed in arbitrary units (AU) as fold-strength increases over the proportion found in control OCCs. The results of three independent experiments are summarized and expressed as mean ± SEM. Different superscripts above the bars indicate significant differences (P < 0.05).

DISCUSSION

IGF1 has been identified as the component of serum that enables cumulus cells to expand in response to FSH in vitro [32]. The effect of IGF1 was mediated through the receptor, since IGF1 receptor neutralizing antibody completely inhibited the FSH-stimulated expansion in the previous study. We have shown previously that FSH-stimulated porcine OCCs are capable of a certain degree of expansion in serum-free chemically defined medium, and we have demonstrated the ability of IGF1 to enhance expansion by stimulating both the synthesis of HA and its retention within the complex [33]. The data presented in the current study confirm the beneficial effects of FSH and IGF1 in promoting the expansion of porcine OCCs. Even though IGF1 alone did not stimulate the synthesis of HA, in combination with FSH it produced a significant increase in the diameter of the expanded cumulus and significantly enhanced the synthesis and retention of HA.

The signaling pathways associated with the IGF1-promoted expansion of OCCs have not been extensively studied to date. IGF1 stimulates the proliferation, survival, differentiation, and transformation of cells through interactions of the IGF1 receptor with several protein signaling cascades. The MAPK3/1 and PIK3/AKT pathways represent the principal signaling pathways that are activated after binding of IGF1 to its receptor in a wide variety of mammalian cell types [38]. In addition, in cultured granulosa cells, synergism of IGF1 with FSH in terms of the expression of steroidogenic enzymes has been found at the level of cAMP accumulation and/or distal to cAMP production and PKA activation [35, 36]. To clarify the pathways that are implicated in the IGF1-promoted synthesis of HA, we have studied the MAPK3/1 and PIK3/AKT signaling pathways, as well as cAMP production and PKA activity in the stimulated cumulus cells.

The results of the present study show that FSH rapidly stimulates the production of cAMP and the activation of PKA in the OCCs, whereas IGF1 neither stimulates an increase in cAMP level nor enhances the increase in cAMP level elicited by FSH treatment. These data indicate that the beneficial effects of FSH and IGF1 on synthesis and retention of HA are not due to increased production of cAMP and more potent stimulation of PKA in cumulus cells. Our results rather suggest the involvement of other IGF1-stimulated signaling pathways in the promotion of HA synthesis.

One of the signaling cascades activated upon binding of IGF1 to its receptor is the PIK3/AKT pathway. PIK3 generates the synthesis of phosphatydylinositol 3'-phosphate (PIP3) at the plasma membrane. Proteins with a PIP3-binding motif, such as 3-phospoinositide-dependent kinase 1 (PDK1) and AKT, are translocated to the plasma membrane, where PDK1 and PDK2 activate AKT by phosphorylation of the Thr 308 and Ser 473 residues [38]. There is an increasing body of evidence in the literature that PIK3/AKT signaling in cumulus cells plays multiple roles in the regulation of meiosis in mammals. A basal activity of AKT was reported to be associated with the preservation of meiotic arrest in pig oocytes, whereas high PIK3/AKT activities in cumulus cells are essential for the closure of gap junctions and the activation of MAPK3/1 following gonadotropin-stimulated resumption of meiosis [39, 40]. Our data suggest that the PIK3/AKT-dependent pathway is involved in the promotion of FSH-stimulated synthesis of HA in porcine OCCs.

Moderate activation of AKT in OCCs stimulated with FSH alone was observed in the present study, which is in accordance with the data published by Shimada et al. [40]. As expected, AKT phosphorylation was also observed in the present study following stimulation of the OCCs with IGF1 alone. However, a burst of AKT phosphorylation occurred in the OCCs only after simultaneous stimulation with IGF1 and FSH. This burst of phosphorylation was dependent upon the PIK3 signaling pathway and was essential for the promotion of synthesis and retention of HA, as documented by the ability of the PIK3/AKT signaling inhibitors LY 294002 and SH6 to abolish these HA-promoting processes.

The activation of MAPK3/1 in IGF1-stimulated cells may occur through Pyk2/Src/MEK signaling [41, 42] or alternatively from cross-talk between the ligand-activated IGF1 receptor and the EGF receptor [43, 44]. Activation of MAPK3/1 in cumulus cells seems to be important for the induction of both cumulus expansion and the resumption of meiosis in mammals. In the mouse, activation of MAPK3/1 in cumulus cells is required for gonadotropin-induced resumption of meiosis [45, 46]. Moreover, the induction of cumulus expansion by FSH, EGF, 8-bromo-cAMP, and growth differentiation factor 9 also requires the participation of MAPK3/1 [45]. In rat ovarian follicles, MAPK3/1 in cumulus cells mediates the LH-induced breakdown of cell-to-cell communication and maturation of oocytes [47]. The results of our present study indicate that the MAPK3/1 pathway is involved in the regulation of HA synthesis in porcine OCCs, since MAPK3/1 inhibition significantly decreased HA production. We found that MAPK3/1 in cumulus cells became rapidly phosphorylated after treatment with FSH but not with IGF1 alone. However, IGF1 enhanced the FSH-stimulated phosphorylation of MAPK3/1. The FSH-stimulated, cAMP- and PKA-dependent activation of MAPK3/1 has been described in several cell lines, including cultured pig granulosa cells [48]. In this case, the cross-talk between the PKA and MAPK3/1 pathways probably occurs at the level of v-raf murine sarcoma viral oncogene homolog B1 [49]. However, FSH-mediated activation of MAPK3/1 may also occur by relieving the inhibition imposed on MAPK3/1 by a phosphotyrosine phosphatase [50]. To date, it is unclear as to which of these mechanisms is involved in the FSH-induced activation of MAPK3/1 in cumulus cells and whether IGF1 signaling interferes with this mechanism.

The processes that regulate the synthesis and retention of HA by cumulus cells are not completely elucidated. In the mouse, maximal synthesis of HA in vitro requires the combined action of FSH (or EGF) and transforming growth factor ß1 (TGFB1) or a soluble oocyte factor [51]. FSH exerts its effect during the first 2 h of culture, whereas TGFB1 must be present continuously from 2 h onwards to achieve maximal production of HA [51]. Thus, it appears that the initial, FSH- or EGF-controlled phase is critical for the expression of a sufficient amount of HAS2 mRNA, and the delayed, TGFB1-controlled phase is critical for the production of factors that regulate further the synthesis and retention of HA. Our data show that IGF1 does not promote the synthesis of total HA by increasing the relative abundance of HAS2 mRNA. We assume that IGF1 instead increases the viability or the total number of cumulus cells that synthesize HA during the culture period. This assumption is supported by the finding that IGF1 efficiently reduces the apoptosis of cumulus cells cultured in vitro [52] and stimulates the proliferation of cumulus cells until they become terminally differentiated during the process of expansion [53]. As regards the IGF1-promoted retention of HA within the complex, we propose that IGF1 signaling increases the synthesis of proteins that are required for the stabilization of HA in the extracellular matrix [14–17]. This hypothesis is supported by the finding that FSH and IGF1 synergistically induce the upregulation of cartilage link protein in rat granulosa cells via the PIK3/AKT pathway [54]. The link protein belongs to the family of HA-binding proteins, and its addition to the culture medium dramatically increases the expansion of rat OCCs [55].

The data accumulated in the present study demonstrate a strong potential of IGF1 to affect FSH-stimulated expansion of porcine OCCs in vitro. We assume that IGF1 has similar physiological functions in the preovulatory follicles of the pig. Several lines of evidence support this hypothesis. First, IGF1 is present in the follicular fluid of porcine antral follicles in concentrations similar to that used in the present study [56]. Second, the concentration of insulin-like binding protein 2 (IGFBP2) decreases dramatically in porcine large antral follicles, and a further transient decrease occurs in the follicular fluid of preovulatory follicles after LH surge [56]. This pattern of IGFBP2 behavior enhances the binding of IGF1 to its receptor on cumulus cells, specifically during the period that is critical for remodeling cumulus cells towards expansion. Thus, IGF1 together with FSH, and presumably other growth factors produced by granulosa cells in response to LH surge [2, 3], may control the proliferation, survival, and eventual differentiation of cumulus cells.

In conclusion, the data of the present study support the hypothesis that multiple signaling pathways must be activated in cumulus cells to achieve full production of HA and its incorporation into the extracellular matrix. We show that FSH activates predominantly the cAMP/PKA signaling pathway in cumulus cells, and that it also induces moderate activation of AKT and MAPK3/1. IGF1 alone activates only the PIK3/AKT pathway (of the pathways followed in the present study), which proves to be insufficient stimulation of cumulus cells to initiate HAS2 expression and HA synthesis. However, IGF1 functions in vitro, and presumably also in vivo, in the intrafollicular environment as a potent promoter of signaling pathways activated by FSH. In our model, IGF1 acts together with FSH to activate the PIK3/AKT and MAPK3/1 pathways and consequently, to promote the synthesis and retention of HA.

FOOTNOTES

1Supported by grant no. 523/04/0574 from the Grant Agency of the Czech Republic and by research project AV0Z50450515 from IAPG. M. Tománek was supported by grant MZE 0002701401 from the Ministry of Agriculture of the Czech Republic. Back

Correspondence: 2FAX: 420 315 639 510; e-mail: prochazka{at}iapg.cas.cz

Received: 16 October 2006.

First decision: 22 November 2006.

Accepted: 27 February 2007.

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