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BOR - Papers in Press, published online ahead of print July 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030155
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BIOLOGY OF REPRODUCTION 71, 1646–1651 (2004)
DOI: 10.1095/biolreprod.104.030155
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Peroxiredoxin 6 Is Upregulated in Bovine Oocytes and Cumulus Cells During In Vitro Maturation: Role of Intercellular Communication1

Gregory Leyens3, Benjamin Verhaeghe3, Marie Landtmeters4, Joëlle Marchandise3, Bernard Knoops4, and Isabelle Donnay2,3

Unité des Sciences vétérinaires,3 Laboratoire de Biologie cellulaire,4 Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins are peroxidases involved in antioxidant defense and intracellular signaling. Expression of transcripts coding for peroxiredoxin 6 (PRDX6) has been previously described to be upregulated in oocytes after in vitro maturation, a period during which general transcription decreases dramatically in oocytes. The aim of the present work was to evaluate PRDX6 regulation in bovine cumulus-oocyte complexes in relation to maturation and intercellular communication. PRDX6 expression was analyzed by reverse transcription-PCR and Western blotting in oocytes and cumulus cells before and after in vitro maturation. PRDX6 was found to be upregulated at the mRNA and protein levels in both cell types after maturation. The effect of paracrine and gap junctional communication on PRDX6 expression was then assessed by culturing cumulus clusters in the presence or absence of denuded oocytes. While PRDX6 upregulation in oocytes required intact cumulus-oocyte junctions, the presence of denuded oocytes was necessary but sufficient for the upregulation to occur in cumulus cells. Finally, the influence of recombinant mouse growth differentiation factor-9 (GDF-9) on PRDX6 expression in cumulus cells was studied. GDF-9 induced cumulus expansion and PRDX6 upregulation in bovine cumulus clusters. Altogether, our data suggest that PRDX6 upregulation in cumulus-oocyte complexes during in vitro maturation is mutually regulated by both cell types: PRDX6 upregulation in oocytes would require gap junctions with cumulus cells, while upregulation in cumulus would depend on secretion of oocyte paracrine factor(s) with GDF-9 being a likely candidate.

cumulus cells, gene regulation, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins (PRDXs) are peroxidases involved in antioxidant defenses and intracellular signaling through alkyl and hydrogen peroxide reductase activities. The family is composed of six members in mammals: PRDX1–PRDX6 [1, 2]. They have two catalytically active cysteines, except PRDX6, which exhibits only one. After peroxide reduction, oxidized PRDXs are reduced through electron transfer from thiol-containing donor molecules such as thioredoxin for PRDX1–PRDX5 and glutathione for PRDX6 [2]. Recently, cyclophilin A has also been described as a potential electron donor for cytosolic PRDXs [3].

Besides being the only 1-cys PRDX in mammals and being a glutathione peroxidase, PRDX6 has been reported to exhibit peroxynitrite [4] and phospholipid hydroperoxide reductase activities [5]. Furthermore, it is the only PRDX to possess an acidic calcium-independent phospholipase A2 activity. This particular activity has been mainly studied in lungs, where it has been associated to surfactant phospholipid turnover [6]. In a previous work, we showed that the expression of PRDX6 transcripts was upregulated in bovine oocytes during in vitro maturation [7]. This was unexpected because general transcription in oocytes declines dramatically during maturation [8]. Because PRDX6 transcripts are upregulated in oocytes during in vitro maturation and cumulus-oocyte complexes (COC) form a functional entity, PRDX6 could also be modulated in cumulus cells. Furthermore, cumulus cells might regulate PRDX6 expression in oocytes and vice versa.

Such modulation of oocyte meiotic regulation (arrest and resumption) and cytoplasmic maturation by the cumulus is well known [9]. Recently, there has been a growing interest in the communication between oocyte and cumulus cells, which includes oocyte control of cumulus proliferation and differentiation [10]. The communication from the cumulus to the oocyte appears to occur mostly through gap junctions [11]. These junctions are located on cumulus cytoplasmic processes that traverse the zona pellucida and make contact with the oolemma [12]. On the other hand, the communication from the oocyte to the cumulus cells appears to occur mainly via paracrine pathways. GDF-9 is one of those oocyte-secreted factors. This member of the transforming growth factor-beta superfamily is implicated in folliculogenesis [13]. In the mouse, GDF-9 is already required for development beyond the primary follicle stage and is responsible for the proliferation and differentiation of cumulus cells surrounding the fully grown oocyte. In the bovine, GDF-9 has been detected in oocytes at the primordial follicle stage up to eight-cell embryos [14, 15].

The main objective of this work was to study PRDX6 regulation in COCs. We first analyzed PRDX6 abundance at the mRNA and protein levels both in oocytes and cumulus cells, before and after in vitro maturation, using reverse transcription (RT)-PCR and Western blotting. The necessity for junctional or paracrine communication was then assessed by maturing cumulus clusters (CC) either alone or in the presence of denuded oocytes (DO) or as COC. Finally, the possible involvement of GDF-9 in PRDX6 expression in cumulus cells was investigated on CC cultured with recombinant mouse GDF-9.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte Collection and Cumulus Clusters

COCs were collected from abattoir ovaries by aspiration of 3- to 6-mm follicles and washed in HEPES buffered TCM-199 (Sigma-Aldrich, Steinheim, Germany). Some complexes were kept intact, while others were either stripped of their cumulus cells by repeated pipetting through a 200-µl tip to obtain completely denuded oocytes, either aspirated through a 75-µm glass pipette to prepare CC.

Maturation Conditions

The oocytes and/or the cumulus cells were matured for 24 h in a humidified atmosphere of 5% CO2 in air at 39°C in bicarbonate buffered TCM-199 (Sigma-Aldrich) supplemented with 104 IU/ml penicillin, 10 mg/ml streptomycin, and 10 ng/ml epidermal growth factor (EGF), which allows proper nuclear maturation and good embryonic development potential [16]. In some experiments, the maturation medium consisted of TCM-199 supplemented with various concentrations of either GDF-9 conditioned medium or control medium (CTRL), both kindly provided by Dr. M.M. Matzuk (Baylor College of Medicine, TX). The GDF-9-conditioned medium was obtained by incubating Chinese hamster ovary (CHO) cells transfected with murine GDF-9 in DME:F12 (1:1) supplemented with 0.1% BSA [17]. The final concentration of mouse GDF-9 in the batch of medium was 0.75 ng/µl. CTRL-conditioned medium was obtained by incubating mock-transfected CHO cells in the culture medium. No bovine GDF-9 was available but the amino acid sequence of the mature mouse GDF-9 is 87% identical to the bovine protein [18].

Western Blot Analysis

Pools of 20 denuded oocytes collected before and after in vitro maturation were obtained by repeated pipetting of the COCs in PBS supplemented with 1 mg/ml polyvinyl pyrrolidone (PVP). These oocytes were further washed in PBS-PVP, while the corresponding cumulus cells were transferred to 500-µl tubes and centrifuged for 2 min at 425 x g prior to removing the excess PBS-PVP. Until assayed, oocytes and cumulus cells were conserved at –80°C in a minimal volume of washing solution. The samples were then treated for tissue lysis as described by Nuttinck and collaborators [19]. Proteins were then separated on 15% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were incubated overnight with 1:1000 mouse anti-bovine PRDX6 monoclonal antibody kindly provided by Dr. A.B. Fisher (University of Pennsylvania Medical Center, Philadelphia, PA) followed by incubation for 1 h with 1:2000 peroxidase-conjugated goat anti-mouse IgG. Detection by chemiluminescence reaction was carried out using ECL Western blotting detection reagent (Amersham Biosciences, Roosendaal, Netherlands) followed by exposure to Hyperfilm (Amersham). Quantification was made by densitometric analysis of band intensity using Kodak 1D v. 3.5.3 software (Kodak Scientific Imaging Systems). PRDX6 quantification in oocytes was normalized to the number of oocytes per sample. For cumulus cells, band intensities were normalized to total proteins charged on the gel evaluated by staining proteins on the nitrocellulose membrane with a solution of 0.5% Ponceau S and 1% glacial acetic acid in water.

RNA Extraction and Reverse Transcription

Denuded oocytes and cumulus cells were obtained as for Western blot analysis and frozen at –80°C by groups of 10 oocytes or corresponding cumulus cells in a minimal volume of washing solution. Prior to RNA extraction, 20 µg of glycogen were added as a carrier to the thawed samples. Total RNA was extracted with 100 µl TriPure Isolation Reagent (Roche Molecular Biochemicals, Mannheim, Germany) as already described [7]. The samples of RNA were then diluted in 6 µl of diethyl pyrocarbonate (DEPC)-treated water with 250 ng Hexanucleotide Mix (Boehringer Ingelheim GmbH, Germany) prior to annealing for 10 min at 65°C. The RNA was reverse transcribed for 1 h at 42°C after addition of Expand Reverse Transcriptase Tris-HCl buffer (Roche) and DEPC-treated water supplemented with 10 mM dithiothreitol, 1 mM of each dNTP (final concentrations), 25 U Expand Reverse Transcriptase (Roche) and 10.6 U RNA Guard (Amersham Pharmacia Biotech, Piscataway, NJ). The total volume after reverse transcription was 15 µl of cDNA template, which was then diluted five times by addition of 60 µl DEPC-treated water.

Semiquantitative Polymerase Chain Reaction

To amplify bovine PRDX6 (GenBank accession number: NM_ 174643), the forward 5'-ATT GCT CTT TCC ATA GAC AG-3' and reverse 5'-GAA CAT TTT GGT CAA CAC AG-3' primers were designed using Amplify 1.2 [20] as previously described [1]. The amplicon is 830 base pairs (bp) long and includes 4 exons. To amplify bovine histone H2A (H2A), which was used as positive internal control for the RT-PCR, the forward 5'-GTC GTG GCA AGC AA G GAG-3' and the reverse 5'-GAT CTC GGC CGT TAG GTA CTC-3' were chosen according to Robert and colleagues [21]. The amplicon is 192 bp long and includes 1 exon. Each pair of primers was diluted to 0.2 µM in the final PCR solution. Ex Taq polymerase (0.8 U; Takara, Madison, WY) was used according to the manufacturer' instructions with 5 µl of diluted cDNA template in a hot-start PCR: denaturing for 2 min at 94°C, addition of the polymerase mix at 80°C for 2 min, then 26–34 cycles of denaturing for 1 min at 94°C, annealing for 1 min at 54°C, and extension for 1 min at 72°C; cycles were followed by a final extension step of 5 min at 72°C. Twenty-six or 34 cycles were used to amplify PRDX6, and 30 or 32 cycles were used to amplify H2A in cumulus or oocytes, respectively. These numbers of cycles were chosen because they corresponded to the linear range of amplification of the various samples. The PCR products were separated for 2 h in a 2% agarose gel and stained for 30 min in 0.05% ethidium bromide in Tris-borate EDTA.

The gels were scanned with Kodak Image Station 440CF Chemiluminescent Imaging System (Kodak Scientific Imaging Systems). The density of the PCR products were quantified using Kodak 1D v. 3.5.3 software. For each sample, the signal intensity of the PRDX6 band was normalized to the basal level of the H2A band.

Real-Time Polymerase Chain Reaction

Transcripts coding for PRDX6 and H2A were also quantified using real-time PCR as previously described [22]. Briefly, forward 5'-GGC AAG AAA TAC CTC CGC TAC-3' and reverse 5'-GGC AGC TCC AGA ACC ATC TC-3' primers were used for PRDX6, and forward 5'-AGA AGA CGC GCA TCA TCC C-3' and reverse 5'-ACT TTG CCC AGC AGC TTG TT-3' for H2A. The 5'-AGC CAT AGG CTC GCC AT-3' and 5'-CAT CCG CAA CGA CGA GGA GCT CA-3' hybridization probes with TAMRA quencher at the 5' end and FAM fluorescent dye at the 3' end were used, respectively, for PRDX6 and H2A. PCRs were performed on an ABI Prism 7700 (Applied Biosystems, Foster City, CA). The amplification reaction used 5 µl cDNA and Platinum quantitative PCR Super mix-UDG (2X) (Invitrogen Life Technologies, Carlsbad, CA). Four hundred nanomoles of each primer (forward and reverse) and 200 nM of the hybridization probe were added to the reaction. The PCR protocol included a first step at 50°C (2 min) for the activity of uracil-N-glycosylase (UNG), preventing carryover contamination from previous PCR products, then 10 min at 95°C for the activation of the Taq polymerase, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. For both transcripts, a standard curve of amplification was established using five serial dilutions (in triplicate) of a reference cDNA, revealing slopes of –3.31 for PRDX6 and –3.17 for H2A.

Experimental Design

Experiment 1: PRDX6 Expression in Cumulus-Oocyte Complexes During In Vitro Maturation COCs were matured for 24 h in four-well culture plates. Relative quantification by semiquantitative PCR of PRDX6 transcripts in oocytes and cumulus cells was done, respectively, on 20 and 22 samples before maturation and on 16 and 25 samples after in vitro maturation. The samples originated from a minimum of five batches of COC collected at different days. At the end of our study, validation of our semiquantitative technique was performed using real-time PCR on oocyte and cumulus samples both before (seven and eight samples, respectively) and after (nine and seven samples, respectively) in vitro maturation. These samples originated from a minimum of two different batches. PRDX6 expression was also studied at the protein level by Western blotting of four samples originating from a minimum of two distinct batches of oocytes or cumulus cells before and after in vitro maturation.

Experiment 2: Involvement of Gap Junctional and Paracrine Communication between Oocyte and Cumulus Cells in the Regulation of PRDX6 Expression During In Vitro Maturation Eight to 14 pools of oocytes and cumulus cells from a minimum of four distinct batches were matured by groups of five in 30-µl droplets of maturation medium under oil. The samples were matured either as COC or as DO in the presence of CC (Fig. 1). The effect of these different maturation conditions on PRDX6 expression was evaluated both in oocytes and cumulus cells by RT-PCR and relative quantification.



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  		FIG. 1. Schematic representation of the different maturation conditions used in the various experiments. Cumulus clusters were matured alone (CC), in the presence of denuded oocytes (CC + DO), or as cumulus-oocyte complexes (COC)

In another set of experiments, PRDX6 expression was evaluated in eight pools of CC from two distinct batches cultured in the presence or absence of DO in 30 µl maturation medium with or without EGF under oil (2 x 2 factorial arrangement). Analysis was made by RT-PCR and relative quantification.

Experiment 3: Effect of GDF-9 on PRDX6 Expression in Cumulus Cells Eight pools of CC from two distinct batches were cultured in four-well culture plates for 24 h in TCM-199 supplemented with increasing amounts of recombinant mouse GDF-9-conditioned medium or increasing amounts of CTRL-conditioned medium: 6.7% (v/v), 13.3%, and 26.7%, corresponding respectively to 50, 100, and 200 ng/ml GDF-9 in maturation medium. PRDX6 expression in CC was evaluated by RT-PCR and relative quantification. Cumulus expansion was estimated under a stereomicroscope.

Statistical Analysis

For each experiment, results are presented as mean ± SEM of a minimum of eight replicates obtained from at least two distinct batches of COCs. Data were analyzed either by unpaired, two-tailed t-test or by one-way ANOVA followed by Newman-Keuls post hoc test using Prism 2.0 (GraphPad Software Inc., San Diego, CA). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PRDX6 Expression in Cumulus-Oocyte Complexes During In Vitro Maturation

PRDX6 transcripts showed a significant upregulation both in oocytes (P < 0.05) and in cumulus cells (P < 0.001) after maturation (Fig. 2). This pattern observed using semiquantitative PCR was confirmed using real-time PCR (Fig. 2, B and C; curve). In both cases, PRDX6 upregulation was higher in cumulus cells (around 10-fold) than in oocytes (around 1.6-fold). Western blotting also revealed an upregulation of PRDX6 at the protein level in oocytes (3.55- ± 1.22-fold) and cumulus cells (1.62- ± 0.14-fold) after maturation (mean ± SEM; n = 4) (Fig. 3).



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  		FIG. 2. PRDX6 expression in oocytes and cumulus cells before (0 h) and after (24 h) in vitro maturation of cumulus-oocyte complexes. A) Detection of PRDX6 and H2A on agarose gel after RT-PCR. B) Relative quantification results in oocytes by semiquantitative PCR (white bars; n = 20 pools before and 16 pools after maturation) and confirmed by real-time PCR (grey bars; n = 7 pools before and 9 pools after maturation). C) Relative quantification results in cumulus cells by semiquantitative PCR (white bars; n = 22 pools before and 25 pools after maturation) and confirmed by real-time PCR (grey bars; n = 8 pools before and 7 pools after maturation). Data are presented as mean ± SEM. a, b: For each PCR technique, columns with different superscripts are significantly different (P < 0.05)



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  		FIG. 3. Detection of PRDX6 by Western blotting on oocytes (A) and cumulus (B) cells before (0 h) and after (24 h) in vitro maturation of cumulus-oocyte complexes. PRDX6 was normalized in cumulus cells by staining of total proteins on the nitrocellulose membrane with Ponceau S

Involvement of Gap Junctional and Paracrine Communication Between Oocyte and Cumulus Cells in the Regulation of PRDX6 Expression During In Vitro Maturation

PRDX6 transcripts were upregulated in oocytes only when matured as COC (P < 0.05), but not when cumulus-oocyte junctions were disrupted before maturation (DO in the presence of CC) (P > 0.05) (Fig. 4). In cumulus cells, no difference was observed after maturation as COC or as CC in the presence of DO (P > 0.05) (Fig. 4): in both cases, a significant upregulation was observed (P < 0.001). However, after maturation of CC alone, PRDX6 transcripts were not upregulated (P > 0.05) (Fig. 5). The absence of EGF in the maturation medium had no impact on PRDX6 expression in CC.



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  		FIG. 4. Influence of cell junctions and paracrine communication between oocytes and cumulus cells on PRDX6 expression in both cell types. Relative quantification of PRDX6 transcripts normalized to the basal level of H2A before maturation (0 h), after maturation for 24 h as cumulus-oocyte complexes (24 h as COC), in denuded oocytes matured for 24 h in the presence of cumulus clusters (24 h + CC), and in cumulus clusters matured for 24 h in the presence of denuded oocytes (24 h + DO). Data are presented as mean ± SEM. a, b: Within cell types, columns with different superscripts are significantly different (P < 0.05). Numbers in the columns indicate the number of analyzed samples



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  		FIG. 5. Effect of oocytes or EGF on PRDX6 expression in cumulus clusters. Relative quantification of PRDX6 transcripts normalized to the basal level of H2A in cumulus cells before maturation (0 h), in cumulus clusters matured alone (–DO) or in the presence of denuded oocytes (+DO) for 24 h (24 h) in the absence (–EGF) or presence of EGF (+EGF). Data are presented as mean ± SEM. a, b: Columns with different superscripts are significantly different (P < 0.001). n, Number of analyzed samples

Effect of GDF-9 on PRDX6 Expression in Cumulus Cells

When CC were matured alone in TCM-199 supplemented with GDF-9, PRDX6 transcripts were upregulated in cumulus cells in a dose-dependent way, with maximum effect from 100 ng/ml (Fig. 6). The upregulation induced by the increasing concentrations of GDF-9-conditioned medium was significantly higher than the upregulation induced by the corresponding concentrations of CTRL-conditioned medium (P < 0.05 for each concentration). Furthermore, CC cultured in the presence of GDF-9-conditioned medium presented an important cumulus expansion, which was not observed in CC cultured in the presence of CTRL-conditioned medium (Fig. 7).



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  		FIG. 6. Effect of GDF-9 on PRDX6 expression in cumulus cells. Relative quantification of PRDX6 transcripts normalized to the basal level of H2A in cumulus clusters after 24 h maturation in TCM-199 (without EGF) supplemented with increasing amounts of medium conditioned either by GDF-9 transfected cells (GDF-9) or by mock transfected cells (CTRL). n = 8 pools of cumulus clusters at each concentration of both conditions. Data are presented as mean ± SEM. Results significantly different from the control at the same dilution: *, P < 0.05; **, P < 0.005



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  		FIG. 7. Effect of GDF-9 on cumulus expansion. Cumulus clusters were matured for 24 h in TCM-199 (without EGF) supplemented with medium conditioned by mock transfected cells (A) or supplemented with medium conditioned by GDF-9 transfected cells and corresponding to 100 ng/ml recombinant mouse GDF-9 (B). Bar = 300 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present work shows that PRDX6 upregulation occurs after in vitro maturation both in oocytes and cumulus cells. Furthermore, the upregulation at the mRNA level was correlated with an upregulation at the protein level in both cell types. PRDX6 expression in COC therefore appears to be regulated at the transcriptional level, as already described in lens and lung epithelial cells [23, 24].

The oocyte is a peculiar cell. It accumulates transcripts for its own use, but also to be used by the early embryo, the genome of which is mostly inactive up to the fourth cell cycle in the bovine [25]. Deadenylation is involved in the storage of those dormant mRNAs, while their translation can be activated by cytoplasmic polyadenylation [26]. This polyadenylation may be responsible for enhanced detection of the PCR products when oligo(dT) are used for reverse transcription [22]. It is not the case in our study because the transcripts were reverse transcribed using random hexamers, and PRDX6 mRNA has recently been shown to be deadenylated in oocytes during maturation [22]. Furthermore, the reliability of our semiquantifications has been validated by real-time PCR analysis of oocytes and cumulus cell samples before and after maturation.

The fact that PRDX6 transcripts are accumulated in the deadenylated form suggests a storage for later use in the early embryo. Indeed, PRDX6 transcripts have been previously observed in embryos up to the five- to eight-cell stage [7]. No transcripts were detected from the 9- to 16-cell to the morula stages, but PRDX6 transcripts were again detected in blastocysts. This pattern of replacement of maternal transcripts pool by mRNAs of embryonic origin after the major activation of the embryonic genome has been observed for several PRDXs [7]. PRDX6 upregulation in oocytes at the protein level and the accumulation of deadenylated transcripts suggest that this enzyme plays a role in oocyte maturation but also in embryo development.

This increased expression after in vitro maturation appeared to be differentially regulated in oocytes and in cumulus cells. Indeed, upregulation only occurred in oocytes when the integrity of the junctions with the cumulus cells was conserved. Our results suggest, therefore, that the cumulus cells control PRDX6 expression in oocyte via gap junctions. Nevertheless, it must be pointed out that cumulus cells play major roles in oocyte maturation [9] and that isolating the oocyte from its surrounding cumulus may affect its response to paracrine factors secreted by the cumulus. Even so, 50% of the denuded oocytes progressed normally to metaphase II, versus 77% of the oocytes matured as COC (data not shown). The efficacy of medium 199 supplemented with EGF for the maturation of denuded oocytes had already been described [16]. Regarding regulation by the oocyte of PRDX6 expression in cumulus cells, as long as CC were cultured in the presence of oocytes, there was no need for cell-to-cell contact. The oocyte thus likely secretes paracrine factor(s) responsible for PRDX6 upregulation in cumulus cells during in vitro maturation. Removing EGF from the maturation medium did not affect this upregulation in CC.

Among the various factors secreted by oocytes, GDF-9 has been shown in vitro to regulate gene expression in granulosa cells [17]. Besides its role in follicular development, GDF-9 would be responsible for the cumulus cells phenotype, differentiating them from mural granulosa cells [27]. Recombinant mouse GDF-9 was tested in this work for the first time on bovine CC, and it induced a dose-dependent effect (culminating from 100 ng/ml) on the upregulation of PRDX6 transcripts in cumulus cells. Therefore, GDF-9 might be one oocyte paracrine factor responsible for the upregulation of PRDX6 in CC cultured in the presence of DO. The slight upregulatory effect observed with the control maturation medium could be due to the secretion of growth factors active on PRDX6 expression by CHO cells.

We also evaluated the effect of GDF-9 on the expansion of CC in the absence of added EGF known to induce cumulus expansion in the bovine [28]. Cumulus clusters matured in TCM-199 supplemented with CTRL-conditioned medium showed no expansion, while cumulus expansion was observed when CC were matured in the presence of conditioned medium containing GDF-9. A similar effect was observed in the mouse [29], even if the mechanisms leading to cumulus expansion in both species seem different [30].

Genes of which expression is modulated by GDF-9 in granulosa cells are known to play a role in COC maturation: Cox2 and Ptgerep2 (also called EP2) are involved in prostaglandin signaling, while Has2 is involved in cumulus expansion like Plau (also called uPA), the downregulation of which prevents degradation of the cumulus matrix [18]. The fact that PRDX6 can also be upregulated by GDF-9 reinforces the possibility of an involvement of PRDX6 in COC maturation. For example, the phospholipase A2 activity of PRDX6 in cumulus could be implicated in producing arachidonic acid for prostaglandin production, which is involved in the maturation process [31, 32]. PRDX6 might also be accumulated in oocytes to be used during fertilization or early embryo development. Regarding those potential roles, in addition to antioxidant properties, PRDX6 peroxide reductase activity might be involved in fine-tuning hydrogen peroxides levels in COC and early embryos. Shifts in the intracellular redox state may indeed contribute to fertilization and genome activation [33]. PRDX6 could use the glutathione accumulated both in the oocyte and cumulus cells during maturation [34] to reduce its oxidized form during the peroxidation process. These putative functions will be addressed in a future work aimed at evaluating PRDX6 role(s) during cytoplasmic and nuclear maturation of the oocyte, cumulus expansion, and early embryo development.

In conclusion, our results suggest that, during in vitro maturation, cumulus cells transmit a signal through gap junctions to the oocyte, where it induces PRDX6 upregulation. The upregulation in cumulus cells seems to be due to oocyte paracrine factor(s), with GDF-9 being a likely candidate. This upregulation of PRDX6 transcripts is related to an increase in PRDX6 protein in both oocytes and cumulus cells after in vitro maturation.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Stephanie A. Pangas and Dr. Martin M. Matzuk (Baylor College of Medicine, Houston, TX) for providing us with recombinant mouse GDF-9 and to Dr. Yefim Manevich and Dr. Aaron B. Fisher (University of Pennsylvania Medical Center, Philadelphia, PA) for providing us with the mouse anti-bovine PRDX6 monoclonal antibody. Thank you to Cecile Marchand for her technical expertise.


   FOOTNOTES
 
1 Supported by grants from the Fonds National de la Recherche Scientifique (Belgium), from the European Commission (contract QLK3-CT1999-00104), and from the Communauté française de Belgique-Action de Recherches Concertées (contract 02/07-275). G.L. is a Research Fellow of the Fonds National de la Recherche Scientifique (Belgium). Back

2 Correspondence: Isabelle Donnay, Veterinary Unit, Place Croix du Sud 5, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium. FAX: 32 10 47 37 17; donnay{at}vete.ucl.ac.be Back

Received: 29 March 2004.

First decision: 13 April 2004.

Accepted: 29 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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S. Mazerbourg and A. J.W. Hsueh
Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands
Hum. Reprod. Update, July 1, 2006; 12(4): 373 - 383.
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