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BOR - Papers in Press, published online ahead of print January 10, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.055343
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BIOLOGY OF REPRODUCTION 76, 794–803 (2007)
DOI: 10.1095/biolreprod.106.055343
© 2007 by the Society for the Study of Reproduction, Inc.

Expression of the cAMP-Phosphodiesterase PDE4D Isoforms and Age-Related Changes in Follicle-Stimulating Hormone-Stimulated PDE4 Activities in Immature Rat Sertoli Cells1

Guénaëlle Levallet , Jérôme Levallet , Hélène Bouraïma-Lelong , and Pierre-Jacques Bonnamy 2

EA 2608, INRA USC 2006, Laboratoire Oestrogènes et Reproduction, Université de Caen, 14032 Caen, France

ABSTRACT

Major changes in the cAMP-dependent signal transduction pathway triggered by FSH take place during transition of rat Sertoli cells from proliferative to the quiescent/terminally differentiated state. Using Sertoli cell cultures isolated from 10-, 20-, and 30-day-old rats, we recorded a specific increase in PDE4 activity in both the soluble and particulate subcellular fractions of 20-day-old Sertoli cells, which also displayed the highest cAMP response to FSH and the highest FSH-induced increase in PDE4 activity in both subcellular compartments. RT-PCR and immunoblotting experiments showed that almost all the PDE4D isoforms, known as the main cAMP-regulated rolipram-sensitive PDE in Sertoli cells, were expressed throughout the early postpartum period, whereas only the short PDE4D isoforms (PDE4D1 and PDE4D2) were transcriptionally regulated by FSH. Unexpectedly, the immunoblot data also revealed that the soluble PDE4 activities were mainly related to the long PDE4D isoforms and that short PDE4D1 was predominantly particulate. The subcellular distribution and expression of PDE4D proteins were unaffected by the developmental status of the Sertoli cells. Only the expression of short PDE4D1 appeared to be upregulated by FSH and only in 20-day-old Sertoli cells, which suggests phenotype-dependent differential regulation of Pde4d1 mRNA translation. Resensitization of the cAMP response to FSH in 20-day-old Sertoli cells was also associated with the highest FSH-induced transient increase in both soluble and particulate PDE4 activities, which suggests developmental changes in the PKA-mediated upregulation of the catalytic activities of long PDE4D. Such alterations may be involved in the phenotype-dependent alterations in FSH receptor coupling with its associated G proteins in rat Sertoli cells.

cyclic adenosine monophosphate, follicle-stimulating hormone, phosphodiesterases, Sertoli cells, testis

INTRODUCTION

Most of the effects of FSH, which are required for proliferation and the final maturation of Sertoli cells [1], are either dramatically reduced or undetectable in the mature testes [2, 3]. However, the levels of circulating FSH and FSH receptor increase during Sertoli cell maturation [4] and sensitivity to this hormone is not markedly affected during this period [5]. Whereas FSH binding to its receptor is known to activate at least five different pathways [6], the cAMP pathway is thought to be the major one triggered by FSH in Sertoli cells. The regulation of intracellular cAMP levels is exerted through the modulation of both adenylate cyclase and cAMP-catabolizing phosphodiesterases (PDEs). A five-fold increase in PDE activity has been reported from 20 to 60 days postpartum in the whole testis [2, 3, 7], while in partially purified cultured Sertoli cells from 18- to 24-day-old rats, a decline in total PDE activity occurs concomitantly with a drastic decrease in the cAMP response to FSH [5, 8, 9]. Thus, the differences in the FSH responsiveness of immature and mature rats are still misunderstood due to the lack of information about the evolution of PDE activities in purified Sertoli cells during postnatal development.

PDEs constitute a family of more than 50 members of cyclic nucleotide-catabolizing enzymes, which has been subdivided in 11 subfamilies (from PDE1 to PDE11) according to substrate selectivity, inhibitor sensitivity, and molecular sequence [10–14]. Each member of the different PDE subfamilies is encoded by different genes, which can give rise to multiple variants by alternative splicing of mRNA or the use of alternate promoters [11–13]. In the mammalian testis, in situ hybridization experiments have identified PDE families 1, 2, 3, 4, 8, 9, 10, and 11 in Sertoli cells, pachytene spermatocytes, and round spermatids [15–20]. Whereas Sertoli cells have been shown to express multiple PDEs at both the mRNA and protein levels [21, 22], only Pde4 mRNAs exhibit cyclic variations throughout the seminiferous cycle [16], which suggests that PDE4 is the main, if not the sole, PDE subfamily that is subjected to regulation in the testis. PDE4 is effectively the target of cAMP feedback regulation mediated either by rapid phosphorylation or by changes in PDE expression [11, 13, 21], such that, in Sertoli cells, FSH may regulate PDE activity via changes in intracellular cAMP levels. The PDE4 isoenzymes are encoded by four independent genes (Pde4a to Pde4d), which generate more than 20 spliced variants that have been divided into three groups (the long, short, and super short isoforms) according to the presence or absence of two conserved regions at the N-terminus, termed upstream conserved region 1 (UCR1) and UCR2 [11, 13]. Each splice variant exhibits unique properties through the expression of these domains, which mediate protein-protein or protein-membrane interactions and thereby regulate the catalytic activities triggered by PKA and ERK mitogen-activated kinase (MAPK1, also known as ERK2) phosphorylation, phosphatidic acid binding, and dimerization [23–31]. Thus, members of the PDE4 subfamily appear to be critical for the formation of cellular dynamic microdomains, which generate localized cAMP signals and restrict cAMP diffusion within the cytoplasm [11, 13, 32].

For the whole testis, age-related changes in Pde4d expression have been reported [33]. Maximal expression of Pde4d1 and Pde4d2 mRNAs occurs at 15 days postpartum and declines thereafter, whereas the long Pde4d mRNA, which is barely detectable at 10–15 days postpartum, increases in older animals. These age-dependent PDE4D alterations in gene expression/activity, by modulating both the intensity and duration of the cAMP signal triggered by FSH in Sertoli cells, may be responsible for the extinction/activation of genes that direct the withdrawal of the cell cycle, followed by the expression of genes involved in the initiation of spermatogenesis.

Therefore, we have evaluated the cAMP response to FSH and the FSH-stimulated PDE activities in proliferating (10 days postpartum), quiescent and early differentiated (20 days postpartum), and terminally differentiated (30 days postpartum) rat Sertoli cells. The kinetics of the cAMP-PDE activities, and the identities and subcellular distribution of the PDE activities were also evaluated in unstimulated and FSH-stimulated cultured Sertoli cells that originated from 10- to 30-day-old animals. Special attention was focused on the modulation of Pde4d expression through the study of its nine splice variants (PDE4D1 to PDE4D9) at the mRNA and protein levels under the aforementioned conditions.

MATERIALS AND METHODS

Materials

Ovine FSH (oFSH-20, 4453 IU/mg) was kindly provided by the National Institutes of Arthritis, Metabolic and Digestive Diseases (Pituitary Hormone Distribution Program, Bethesda, MD). Dulbecco modified Eagle medium (DMEM), Ham F12 medium, and trypsin (UPS grade) were from Gibco-BRL (Cergy-Pontoise, France). Collagenase and dispase were from Boehringer-Mannheim (Meylan, France). Ultroser SF (steroid-free serum substitute) was purchased from BioSepra (Cergy-Saint-Christophe, France). Adenosine 3',5'-cyclic monophosphate (cAMP), leupeptin, amphotericin, antipain, aprotinin, benzamidine, sodium chlorate, deoxyribonuclease I, hyaluronidase, kanamycin, penicillin, peptidase A, Dowex 1X2 resin (200–400 mesh), rolipram, streptomycin and the Crotallus atrox snake venom phosphatase were purchased from Sigma (Saint Quentin Fallavier, France). [2,8-3H]Adenosine 3',5'-cyclic phosphate (1.59 TBq/mmol) was from Amersham Biotech (Les Ulis, France). The cyclic AMP (3H) assay kit was from Amersham Biosciences (Buckinghamshire, UK). Moloney-Murine-Leukemia-Virus (M-MLV) reaction buffer 5x, M-MLV reverse transcriptase, random primers, dNTPs, RNasin, Thermus aquaticus (Taq) DNA polymerase reaction buffer 10x, and Taq DNA polymerase were from Promega (Charbonière-les-bains, France). The oligonucleotide primers were synthesized and purified by Invitrogen, Life Technologies (Cergy-Pontoise, France). The anti-PDE4D antibody, blocking peptide, and HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal mouse anti-actin (Ab-1) was from Calbiochem (Darmstadt, Germany). All others reagents were of analytical grade.

Cell Culture

All the procedures described herein were performed in accordance with French legal requirements for animal handling and welfare. Sprague-Dawley rats at 10, 20, and 30 days of age (Janvier, Le Genest Saint-Isle, France) were killed by cervical dislocation. Sertoli cells were isolated by sequential enzymatic digestion according to the procedures of Tung et al. [34], with the minor changes described previously [35]. Cells from 10-, 20-, and 30-day-old rats were seeded at densities of 125 000, 250 000 and 675 000 cells per cm2, respectively, in 75-cm2 plastic flasks and cultured at 32°C in 5% CO2 for 48 h in Ham F12- DMEM (1:1, v:v) supplemented with antibiotics (50 000 UI/L penicillin, 50 mg/L streptomycin, and 50 mg/L kanamycin), fungicide (0.25 mg/L amphotericin B), 2.2 g/L sodium bicarbonate, and 2% Ultroser SF. The cell densities were designed so as to obtain confluent cultures on day 5 after plating.

On Day 3 after plating, residual germ cells were lysed by hypotonic treatment with 20 mM Tris-HCl (pH 7.4) [36, 37]. Sertoli cells were then cultured for an additional 2 days in medium devoid of Ultroser before treatment. The Sertoli cell cultures were routinely assayed for peritubular cell contamination using histochemical detection of pericellular alkaline phosphatase [38, 39] and checking for the absence of fibronectin expression [40]. Contamination of Sertoli cell cultures by pericellular cells was less than 1%, regardless of the age of rats. Contamination by germ cells, as estimated by eye on Day 5 after plating, was undetectable in the 10-day-old Sertoli cell cultures, was less than 3% in 20-day-old Sertoli cell cultures, and did not exceed 5% in 30-day-old Sertoli cell cultures. Confluent Sertoli cells were used at Day 5 and were incubated for up to 24 h in the presence or absence of FSH (100 ng/ml).

Cyclic AMP Extraction and Measurement

Five days after plating, confluent Sertoli cells were incubated in the presence or absence of FSH (100 ng/ml) and with or without 100 µM 3-isobutyl-1-methylxanthine (MIX), which is a nonspecific PDE inhibitor [11, 14] for various periods of time up to 6 h. At the end of the incubation period, the Sertoli cells were scraped and washed with 2 ml of 100% ethanol. The suspension was centrifuged at 1000 x g for 5 min at room temperature, and the pellet was resuspended in 1 ml of 66% ethanol and then centrifuged at 1000 x g for 5 min at room temperature. The final supernatant was pooled with the first one before evaporation. Samples were resuspended in 110 µl of 5 mM Tris (pH 7.5) plus 4 mM EDTA and assayed for cAMP content using the cAMP assay kit (Amersham Biosciences).

PCR Primers

The oligonucleotides used for PCR amplification included a reverse primer located in the catalytic region common to all Pde4d genes (5'-CTGGTTGCCAGACCGACTCA-3'), and a sense primer specific for each of the following variants: Pde4d1 (GenBank accession no. U09455), 5'-GATGGCCCCCTTTGAACT-3'; Pde4d2 (U09456), 5'-CATCCGAGCATGGCGGGA-3'; Pde4d3 (NM017032), 5'-CCTTTAGAAGGCATTCCTGGAT-3'; Pde4d4 (AF031373), 5'-GCCAGGCCTGAAGAAATCTA-3'; Pde4d5 (AF012073), 5'-AGTCCAAGACAGCGAGGAAA-3'; Pde4d6 (AF536974), 5'- AATGGCTTTGTGGAAACTGG-3'; Pde4d7 (AF536979), 5'-GGAATGGAGCCCTATCTCGT-3'; Pde4d8 (AF536977), 5'-TGCCAGGACCATCTACAAGA-3'; and Pde4d9 (AY388961), 5'-CGAGATCCAGGTCCACAAGT-3' (for Pde4d9, a specific reverse primer located in the common PDE4D sequence was used: 5'-CGATACAGGAAGGACTCCCG-3'). All of the primers were of rat origin except for the Pde4d5 and Pde4d8 forward primers, which were of human origin. However, authentication of all PCR products, including Pde4d5 and Pde4d8, was carried out by sequencing and alignment with the published rat PDE4D sequences and/or the Rattus norvegicus chromosome 2 genomic contig (NW047620), which has been identified as a PDE4D locus. Moreover, the complete sequences of the rat Pde4d5 (EF102484) and rat Pde4d8 (EF121818) genes have been deposited in GenBank.

Extraction of Total RNA and RT-PCR Analysis

Total RNA was isolated from Sertoli cells of different ages using an acid guanidinium thiocyanate-phenol-chloroform mixture according to the single step method of Chomczynski and Sacchi [41]. The integrity and quality of the purified RNA were controlled by 1% agarose gel electrophoresis, and measurements of the absorbance at 260 and 280 nm were performed to estimate the RNA concentration.

Total RNA (500 ng) was heat-denatured (60°C for 10 min) and then reverse-transcribed using oligo(dT) primers and 200 IU M-MLV reverse transcriptase at 37°C for 60 min, followed by 5 min of denaturation at 95°C. Similar amounts (2.5 µl) of the resulting cDNA were used as templates for 35 cycles of PCR performed with 10 pmol of the previously described primer sets and 0.25 IU Taq polymerase, under the following conditions : denaturation at 94°C for 1 min, annealing at 55°C to 60°C (according to the PCR primer set) for 40 sec, and extension at 72°C for 1 min. The expected sizes of the amplified fragments were 253 bp, 193 bp, 554 bp, 586 bp, 641 bp, 208 bp, 708 bp, 584 bp, and 172 bp for Pde4d1 to Pde4d9, respectively. Rat β-actin (GenBank accession no. V01217), used as a control to ensure equal amounts of total RNA under different conditions, was amplified with sense (5'-ACAGACTACCTCATGAAGAT-3') and antisense (5'-AGCCATGCCAAATGTCTCAT-3') primers, to yield a 665-bp fragment. The identities of the RT-PCR products were confirmed by Big Dye Terminator cycle sequencing in an ABI Prism 377 DNA sequencer (data not shown).

Positive standards and reaction mixtures that lacked the reverse transcriptase were used routinely as controls for each of RNA samples. No PCR product was detected in the absence of reverse transcriptase during the RT step, which indicates that the RNA preparations were free of intact genomic DNA. Rat RNA from the lung or heart was used as a positive control in RT-PCR when the Pde4d8 primers were used. All of the RT-PCR data were normalized to the rat cytoplasmic β-actin transcript level. The RT-PCR products were analyzed in 2.5% agarose gels that were stained with ethidium bromide. DNA bands were photographed and scanned before analysis with the Image J software (Framasoft, NIH).

Preparation of Cell Fractions

The fractionation of Sertoli cells was adapted from the method of Lobban and coworkers [42]. Briefly, at the end of the incubation, the Sertoli cell layer was washed with cold PBS and recovered by gentle scraping in homogenization buffer (20 mM Tris-HCl [pH 7.2], 1 mM EDTA, 250 mM sucrose). The cell suspension was briefly centrifuged (280 x g for 5 min) and resuspended in homogenization buffer that was supplemented with 0.1 mM phenylmethanesulfonyl fluoride, 2 mM benzamidine, and a mixture of protease inhibitors (antipain, aprotinin, leupeptin, pepstatin A) at a final concentration of 1 µg/ml. After several passages through a 27G needle, an aliquot of the total fraction was sampled and the remaining homogenate was centrifuged at 1000 x g for 10 min at 4°C. The supernatant was then collected and centrifuged at 100 000 x g for 1 h at 4°C. The supernatant (soluble fraction) and the pellet (particulate fraction) were resuspended in ice-cold complete homogenization buffer and then stored at –20°C. PDE assays were performed within two weeks of preparation and the protein content of each fraction was determined by the method of Bradford using BSA as a standard. The purity of each subcellular fraction was checked by assaying for both lactate dehydrogenase (soluble activity) and alkaline phosphatase (membrane-associated activities). In the 20-day-old Sertoli cells, only 11.7 ± 1.9% of the lactate dehydrogenase activity was measured in the particulate fraction, while contamination of the cytosolic fraction by membrane-associated alkaline phosphatase was estimated as 6.2 ± 1.1% (data not shown).

PDE Assay

PDE activities were assayed according to the two-step modified procedure of Thompson and Applemann [43]. A protein mixture (10 µg) was incubated at 34°C for 15 min in 200 µl of reaction buffer (40 mM Tris-HCl [pH 8], 1 mM MgCl2, 1.25 mM β-mercaptoethanol, 0.14 mg BSA) that contained 1 µM [3H]-cAMP as substrate (3.7 kBq of [2,8-3H]-cAMP per sample) [22, 44]. The reaction was then stopped by the addition of 200 µl of stop solution (40 mM Tris-HCl [pH 7.5], 10 mM EDTA) followed by heat denaturation at 100°C for 1 min. Crotallus atrox snake venom (50 µg) was added to each sample and incubated at 34°C for 20 min. The reaction products were separated by anion exchange chromatography using a freshly prepared solution of Dowex (Dowex:water:ethanol at 1:1:1, wt:v:v). The samples were gently mixed several times over 15 min and then centrifuged at 14 000 x g for 4 min at room temperature. An aliquot of the supernatant was counted by liquid scintillation. Samples that lacked proteins were used as blanks, and these values were subtracted from the values obtained for the Sertoli cell extracts.

For determination of the kinetic characteristics of the PDE activities, samples from cell culture homogenates were incubated with increasing concentrations (0.05 to 50 µM) of [2,8-3H]-cAMP and the mean Km and Vmax values were calculated using double reciprocal plots. To discriminate PDE4 activities from other PDE activities, identical amounts of protein from each sample were incubated either in absence (total PDE activities) or presence (rolipram-insensitive PDE activities) of 10 µM rolipram, which is a PDE4-specific inhibitor [13, 45]. Differences between the total and rolipram-insensitive PDE activities were considered to be the rolipram-sensitive PDE4 activities.

SDS-PAGE Western Blot Analysis

Proteins from each subcellular fraction that originated from the 10-, 20-, and 30-day-old rat Sertoli cells were boiled for 5 min and separated by 8% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane (1 h at 100 V and 4°C). Western blotting was then performed using an affinity-purified goat polyclonal antibody raised against a peptide that maps near the C-terminus of the human PDE4D (Santa Cruz Biotechnology). Immunoblotting with antibody that was preincubated with an excess of the peptide used for immunization (Santa Cruz Biotechnology) was performed as a negative control, following the instructions of the supplier. Immunoreactive bands were detected using a donkey anti-goat IgG-horseradish peroxidase (HRP) complex and the enhanced chemiluminescence (ECL) Advance Western Blotting Detection Kit (Amersham Biosciences). For β-actin detection, the blots were stripped in a stripping buffer that contained 62.5 mM Tris-HCl (pH 6.7) 2% SDS, and 100 mM β-mercaptoethanol at 58°C for 30 min, and reprobed for actin with monoclonal mouse anti-actin antibody and goat anti-mouse IgG-HRP (Calbiochem). The immunoblots were scanned on the ProXPRESS Proteomic Imaging System (Perkin Elmer Life Science, Boston, MA) and analyzed with the TotalLab Image Analysis software (Nonlinear Dynamics Ltd., Newcastle, UK).

Statistical Analysis

The experimental data are presented as the means ± SEM of at least three independent cultures. Within each experiment, the values shown are the means of triplicate determinations for each experimental condition. Statistical differences were determined by one-way analysis of variance (ANOVA) followed by the Fisher t-test using the Sigma Stat software (SPSS Inc., Chicago, IL) and were considered significant at P < 0.05.

RESULTS

Age-Related Changes in Basal and FSH-Stimulated cAMP Production by Cultured Sertoli Cells

Changes were noted in the intracellular cAMP levels of Sertoli cells cultured from 10- to 30-day-old rats (Fig. 1A), with higher values recorded at 20 days of age (74.6 ± 7.8 fmol of cAMP per µg DNA).


Figure 01
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  		FIG. 1. Age-related changes in the endogenous levels of cAMP (A) and increases in these levels induced by FSH (B) in cultured rat Sertoli cells from 10- to 30-day-old rats. Sertoli cells from 10-, 20-, and 30-day-old rats were cultured in serum-free medium and stimulated with FSH (100 ng/ml) for periods of time that ranged from 0 to 360 min. At the end of the incubation, total cyclic AMP was measured after ethanol extraction as described in Materials and Methods. The values shown in B, which were first determined as fmol/µg DNA, are expressed relative to their respective endogenous levels in A. The values shown are means ± SEM from four independent experiments. The different superscripts in A indicate significant differences (P < 0.05). The asterisk in B indicates a significant difference (P < 0.05) between the FSH-induced cAMP accumulations and the levels in unstimulated Sertoli cells for each age studied.

The kinetics of FSH-stimulated cAMP accumulation in cultured Sertoli cells exhibited a pronounced age-related pattern (Fig. 1B). In the Sertoli cells from a 20-day-old rat, FSH induced a rapid increase in cAMP accumulation, which reached 80% of the maximal value as early as 1 h of incubation. The cAMP content (4.5-fold accumulation) was maintained for up to 6 h. The magnitude of the FSH-induced cAMP accumulation was significantly reduced in 10-day-old rat Sertoli cells and was almost totally abolished in 30-day-old rat Sertoli cells.

The addition to the incubation medium of MIX, which is a nonspecific inhibitor of PDE activities, potentiated the stimulatory effects of FSH on the cyclic AMP levels of Sertoli cells from 10- and 20-day-old rats and induced a low but distinct upregulation of the cAMP response to FSH in 30-day-old rat Sertoli cells (Fig. 2). For all the ages studied, the cAMP response to MIX of FSH-stimulated Sertoli cells peaked between 3 h and 6 h. As evidenced by the magnitude of the MIX-induced enhancement of cAMP levels in FSH-stimulated Sertoli cells, cAMP-hydrolyzing activities appeared to be maximal on Day 20. All these data suggest that significant changes in PDE activities occur in Sertoli cells during early postnatal development.


Figure 02
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  		FIG. 2. Age-related changes in the enhancement of FSH-stimulated cAMP synthesis by MIX in cultured Sertoli cells from 10- to 30-day-old rats. Sertoli cells were cultured for 5 days as described in Materials and Methods and further incubated for up to 6 h in serum-free medium with FSH (100 ng/ml) in the presence or absence of MIX (100 µM). Cyclic AMP was determined by radioimmunoassay as described in Materials and Methods. Gray areas indicate the MIX-induced increase in cAMP levels. The values shown are the means of three different cultures for all ages studied. The SEM values have been omitted for clarity.

Decreases in Maximal Velocities of Basal and FSH-Stimulated cAMP-PDE Hydrolyzing Activities in Sertoli Cells Between Day 10 and Day 30

To delineate the changes in PDE activities, we measured the basal and FSH-stimulated total cAMP-hydrolyzing activities of crude homogenates from 10- to 30-day-old cultured rat Sertoli cells. Analysis of double-reciprocal Lineweaver-Burke plots showed the presence of two different slopes over the range of substrate concentrations used in the present study (from 0.05 µM to 50 µM), which is in agreement with both the high and low Km PDE activities previously described for Sertoli cell homogenates [7]. The low-affinity cAMP-PDE activities (observable between 10 µM and 50 µM cAMP) displayed an estimated Km of 17.7 µM, whereas the high-affinity cAMP-PDE activities (observable between 0.05 µM and 5 µM) displayed a Km of approximately 0.4 µM. Since most of the cAMP-specific PDEs exhibited Km values in the submicromolar range [14], we focused our study on the high-affinity, low-Km PDE activities observable over the cAMP concentration range of 0.05 µM to 5 µM.

Analysis of the Lineweaver-Burke plot data showed that the Km value for total hydrolyzing activity towards cAMP in Sertoli cell homogenates was not affected by either the presence of FSH or the age of the rats (Table 1). In contrast, aging of rats was accompanied by a progressive decrease in the Vmax of the low-Km cAMP-PDE in unstimulated Sertoli cell homogenates between Days 10 and 30 (from 108.9 ± 14.6 on Day 10 to 54.2 ± 23.1 pmol min–1 mg–1 protein on day 30). Changes in the specific activities of cAMP-PDE according to rat age did not result from changes in the protein contents of the cell homogenates (45.5 ± 2.8, 54.9 ± 5.9, and 55.5 ± 6.8 µg per 9.6 cm2, respectively, for 10-, 20-, and 30-day-old rats). Irrespective of the age of the rats, the addition of FSH for 24 h induced a 2-fold increase in the maximal cAMP hydrolyzing activity in the cell homogenate (Table 1).


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  		TABLE 1. Age-related changes in kinetic characteristics of low Km cAMP hydrolyzing activities in crude homogenates from control and FSH-stimulated Sertoli cells.*

Increases in Particulate and Cytosolic Rolipram-Sensitive PDE4 Activities in Rats

Aging of the rats resulted not only in a decrease in total cAMP-PDE activity, but also in changes in the subcellular distributions of the rolipram-sensitive (PDE4) and rolipram-insensitive PDE activities. In cultured Sertoli cells from 10- to 30-day-old rats (Table 2), cAMP-PDE activities were mainly present (80% of total PDE) in the particulate fractions. Rolipram-sensitive PDE4 accounted for less than 25% of the total PDE activities of the particulate and soluble fractions. In contrast, in Sertoli cells that originated from 20-day-old rats, the cAMP-PDE activities shifted from the particulate to the cytosolic fraction, which accounted for 40% of the total PDE. Moreover, the relative percentage of rolipram-sensitive PDE4 was significantly increased in the particulate fraction and especially in the soluble fraction (25% and 15% of the total cAMP-PDE in the homogenate, respectively). Such changes resulted from both a marked decrease in particulate rolipram-insensitive PDE and a drastic increase in soluble rolipram-sensitive PDE (Table 3). The particular distribution of PDE activities recorded in 20-day-old Sertoli cells appeared transitory during the early postnatal period, since the subcellular distributions of rolipram-sensitive and rolipram-insensitive PDE activities observable in 10-day-old Sertoli cells were restored in the 30-day-old Sertoli cells.


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  		TABLE 2. Age-dependant alterations in subcellular distribution of total low Km PDE activities in rat Sertoli cells.*


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  		TABLE 3. Age-related changes in subcellular distribution of rolipram-sensitive (PDE4) and rolipram-insensitive (other PDE) PDE activities in cultured rat Sertoli cells.*

Particulate and Soluble PDE4 Are the Sole FSH-Stimulated PDE Activities in Sertoli Cells

The kinetics of the rolipram-sensitive and rolipram-insensitive PDE activities in the subcellular fractions of FSH-stimulated Sertoli cells revealed gonadotropin-induced increases in the activities of the particulate and soluble fractions of Sertoli cells from 10- to 30-day-old rats (Fig. 3, A and B). The increases in total PDE activities induced by FSH in Sertoli cells are entirely attributable to the upregulation of rolipram-sensitive PDE4 activities. Indeed, for all ages studied, FSH did not affect significantly the level of rolipram-insensitive PDE in either the soluble or particulate fraction (Fig. 3, C and D). Regardless of the age and subcellular compartment, the PDE4 activities peaked 6 h after FSH stimulation, and subsequently declined up to 24 h, albeit at levels above the initial values, especially in the 20-day-old rat Sertoli cells. In the particulate fraction, there was no significant age-related difference in the kinetic pattern of FSH-stimulated PDE4 (Fig. 3A), whereas a significant increase in the magnitude of stimulation was apparent at Day 20 (6.4 ± 0.6-fold on Day 20 vs. 2.7 ± 0.5-fold and 3.3 ± 0.6-fold on Day 10 and Day 30, respectively). The magnitude of stimulation of soluble PDE4 after 6 h in the presence of FSH was also increased in the 20-day-old Sertoli cells (approximately 4-fold vs. 3-fold on Days 10 and 30). Moreover, in contrast to the 10- and 30-day-old rat Sertoli cells, the temporal pattern of FSH-stimulated soluble PDE4 activities was similar to that of the FSH-stimulated particulate PDE4 activities, i.e., showing a rapid decrease in the 6-h peak values.


Figure 03
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  		FIG. 3. Time course of rolipram-sensitive and -insensitive PDE4 activities in FSH-stimulated Sertoli cells from 10- to 30-day-old rats. Sertoli cells were cultured for 5 days, and then incubated for up to 24 h in the presence of FSH (100 ng/ml). At the indicated time-point, cells were homogenized and fractionated into particulate and soluble fractions as described in Materials and Methods. Cyclic AMP PDE activities were measured as described in Materials and Methods by incubating 10 µg of proteins of each subcellular fraction with or without 10 µM rolipram, which is a specific inhibitor of PDE4. The PDE activity determined in the presence of rolipram (rolipram-insensitive) was subtracted from the total activity to quantify the rolipram-sensitive PDE4 activity. Rolipram-sensitive (A, B) and rolipram-insensitive (C, D) PDE activities in the particulate (A, C) and soluble (B, D) fractions are expressed as pmoles of cAMP hydrolyzed per min and per mg protein. The values shown are means ± SEM of three independent experiments for each age studied.

Pde4d Gene Expression in Control and FSH-Stimulated Sertoli Cells

Since the product of the Pde4d gene has been previously described as the major rolipram-sensitive PDE4 involved in cAMP regulation in Sertoli cells [22], we determined the expression pattern of Pde4d splice variants in cultured Sertoli cells. The mRNAs of eight of the nine known variants of Pde4d were identified by RT-PCR (Fig. 4). The short variants of Pde4d (Pde4d1, Pde4d2, and Pde4d6) were significantly expressed in the Sertoli cells from 10- to 30-day-old rats. Some of the long isoforms, such as Pde4d5, Pde4d7, and Pde4d9, were also expressed in the Sertoli cells. The expression of other long isoforms was either undetectable (Pde4d8) or barely detectable (Pde4d3 and Pde4d4). No significant alteration in the expression pattern of Pde4d splice variants in Sertoli cells was recorded during the early postnatal period.


Figure 04
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  		FIG. 4. Identification of Pde4d variants expressed in immature (A), premature (B) and mature (C) Sertoli cells by RT-PCR. For each age, 500 ng of total RNA from 5 day-cultured Sertoli cells was subjected to RT-PCR with the Pde4d variant-specific forward primer (see Materials and Methods). The agarose gel illustrates a representative result of at least three different experiments with 10-, 20- and 30-day-old rat Sertoli cells (M: 100-bp DNA ladder). All the RT-PCR data were normalized to the β-actin levels measured in an RT-PCR performed in parallel with the housekeeping gene primers. No signal was detected in the RT-PCR performed without the M-MLV reverse transcriptase or in the negative PCR control reactions (data not shown).

The addition of FSH to Sertoli cell cultures induced a transient upregulation of the expression of the short isoforms (Pde4d1 and Pde4d2), which generally peaked at 6 h, with the exception of Pde4d1 (peaked at 3 h), in 20-day-old Sertoli cells (Fig. 5). In contrast, FSH did not exeret any significant effect on the expression of the long Pde4d splice variants, such as Pde4d5 (Fig. 5) or the super short Pde4d6 variant (data not shown).


Figure 05
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  		FIG. 5. Upregulation of short but not long Pde4d isoform mRNA expression in FSH-stimulated Sertoli cells from 10- to 30-day-old rats. Sertoli cells were cultured for 5 days and then incubated with FSH (100 ng/ml) for an additional period of up to 24 h. For all three age groups studied, 500 ng of total RNA was reverse-transcribed and amplified with the variant-specific forward primer in a separate tube from an aliquot of the same RT. The Pde4d1, Pde4d2, and Pde4d5 mRNA levels were adjusted to that of β-actin and the results are expressed as percentages of the corresponding mRNA expression level at the start of the incubation period. The values shown are means ± SEM of three different cultures for each age studied.

Expression Patterns of PDE4D Proteins in Sertoli Cells

The expression patterns of PDE4D isoforms in particulate and soluble fractions of Sertoli cells were analyzed by immunoblotting using purified goat polyclonal antibody raised against a peptide that maps near the common C-terminus of human PDE4D. To ensure that this antibody was suitable for the detection of rat PDE4D isoforms, we performed immunoblotting of protein homogenates from different rat tissues (heart, lung, and brain). The patterns of expression of immunoreactive proteins were similar to those observed for the same tissues by Richter and coworkers [29]. The specificities of the immunoreactive bands were further confirmed by their disappearance when the anti-PDE4D antibody was preincubated with an excess of the peptide used for immunization (data not shown).

As shown in Figure 6A, five immunoreactive bands were detected in the subcellular fractions of cultured Sertoli cells that originated from 10- to 30-day-old rats. The apparent molecular weights of these proteins (68 kDa, 75 kDa, 90 kDa, 100 kDa, and 110 kDa) fit well with those described for PDE4D2/D6, PDE4D1, PDE4D3/D8/D9, PDE4D5/D7, and PDE4D4, respectively [29]. All of these proteins appeared to be specific, as evidenced by the lack of immunoreactivity with antibody preincubated with an excess of the peptide used for immunization (data not shown). An additional and specific band with an apparent molecular weight of about 95 kDa was observed in the soluble fraction of the Sertoli cells. Its exact nature is unknown but this type of immunoreactive PDE4D protein has also been observed in COS7 cells transfected with a plasmid that encodes the Pde4d5 variant [29]. As postulated by the authors, this protein may be generated by proteolytic cleavage of PDE4D5 or by the use of an alternative translational start site.


Figure 06
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  		FIG. 6. Expression of PDE4D proteins in the particulate and soluble fractions of cultured rat Sertoli cells from 10- to 30-day-old rats. A) Representative immunoblot of particulate (50 µg) and soluble (20 µg) proteins fractions probed with goat polyclonal human anti-PDE4D antibody. Arrowheads indicate the molecular weights of the immunoreactive proteins. For quantification, the intensities of the immunoreactive bands in the particulate (B) and soluble (C) fractions were determined and first normalized against those of actin. The value obtained for the 110-kDa immunoreactive protein in each subcellular fraction of the 10-day-old Sertoli cells was set at 1, and the values for the other immunoreactive proteins in all the ages studied were then calculated relative to this value. The densitometry values are the mean ± SEM of three experiments.

The pattern of PDE4D protein expression differed according to the subcellular fraction. In the soluble fraction, the 90–100-kDa immunoreactive proteins predominated, which suggests that the long PDE4D isoforms (i.e., PDE4D3, PDE4D5, PDE4D7, PDE4D8, and PDE4D9) constitute the main soluble PDE4D activities. In contrast, the predominance of the 75-kDa immunoreactive protein suggests, on the basis of its electrophoretic mobility, that a large proportion of the particulate PDE4D activities is sustained by the PDE4D1 short isoform, and to a lesser extent, by the 90-kDa long PDE4D isoforms. As reflected by the densitometric analyses (Fig. 6, B and C), the patterns of the putative PDE4D isoforms did not display any significant age-related changes in either subcellular compartment.

Similar experiments were performed on the subcellular fractions from cultured Sertoli cells that were stimulated or not stimulated with FSH (100 ng/ml) for 6 h. This incubation period was chosen because it coincided with the peak FSH-stimulated PDE4 activities in both subcellular compartments (Fig. 3). The addition of FSH induced a moderate but reproducible increase in the 75-kDa immunoreactive protein in the particulate and soluble fractions from 20-day-old Sertoli cells (Fig. 7, A and B). Interestingly, the magnitude of the FSH-induced increase in putative PDE4D1 expression varied according to both the age of the rats and the subcellular compartment. Gonadotropin upregulated soluble PDE4D1 expression in 10- and 20-day-old, but not in 30 day-old, Sertoli cells, whereas particulate PDE4D1 expression was only increased in 20-day-old Sertoli cells (Fig. 7C). In contrast to the short PDE4D1 isoform, expression of the long PDE4D immunoreactive proteins, as illustrated by the 90-kDa immunoreactive protein (Fig. 7D), did not exhibit any significant alteration during FSH stimulation, regardless of the age of the rats.


Figure 07
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  		FIG. 7. Effects of FSH on the expression of the particulate and soluble PDE4D isoforms in cultured rat Sertoli cells from 10- to 30-day-old rats. Sertoli cells were cultured for 5 days and then incubated without (control) or with 100 ng/ml FSH for 6 h. Particulate proteins (50 µg) (A) and soluble proteins (20 µg) (B) were immunoblotted with purified goat polyclonal anti-PDE4D antibody. The age-related changes in the relative intensities of the 75-kDa (C) and 90-kDa (D) immunoreactive proteins (normalized against actin) were determined for both subcellular fractions. The values shown are mean ± SEM of two (soluble fraction) or three (particulate fraction) experiments.

DISCUSSION

The present results clearly demonstrate that significant changes in the regulation of cAMP levels occur after FSH binding in rat Sertoli cells during the early postnatal period, and that these changes are accompanied by alterations in the subcellular distributions of PDE4 activities and the expression of PDE4D proteins. Although the number of FSH receptors actually increased between Days 10 and 30 [4], the cAMP response to the gonadotropin displayed an age-related, bell-shaped pattern that reached a nadir at Day 20. These data confirm the well-known decline in cAMP responses to FSH at 18–20 days postpartum [5, 9] and provide supplementary information on Sertoli cell responses to FSH during their proliferative period.

The similarity of the age-related patterns of Sertoli cell response to FSH in the presence or absence of MIX, which is a nonspecific inhibitor of PDE activities, suggests that: (i) alterations in the Sertoli cell response to FSH during the early postnatal period results from lesions in the signaling pathway located upstream of cAMP catabolism, as previously hypothesized [22, 46]; and (ii) PDE activities are closely regulated by cAMP levels. These data are in apparent contradiction with the results obtained for cell homogenates in which FSH, after a 24-h incubation period, retained its ability to induce a 2-fold increase in the maximal cAMP hydrolyzing capacity, although this latter parameter decreased progressively and significantly (–50%) between Days 10 and 30, in agreement with the decline in total PDE, as well as the high-affinity, low-Km activities previously described during rat testis maturation [5, 9]. Differences in the substrate concentrations, i.e., intracellular cAMP concentrations under culture conditions versus added cAMP concentrations in homogenate experiments, may explain these discrepancies and strengthen the hypothesis that changes in FSH responses during sexual maturation result from alterations in cAMP synthesis rather than changes in cAMP catabolism.

The time-point of 20 days postpartum appears to be a pivotal juncture between early proliferating (10 days) and late quiescent and differentiated (30 days) Sertoli cells. Both high cAMP responsiveness to FSH and high PDE activities confer a unique response profile towards FSH on the 20-day-old rat Sertoli cells. These cells also displayed particular subcellular distributions of rolipram-sensitive and rolipram-insensitive PDEs. While Sertoli cells at Day 10 and Day 30 shared similar distributions of PDEs between the soluble and particulate fractions, 20-day-old rat Sertoli cells exhibited a sharp increase in soluble PDE4 activities concomitant with a drastic decrease in particulate rolipram-insensitive PDEs. A slight but nonsignificant increase in particulate rolipram-sensitive PDE4 was also recorded. The retargeting of PDE activities in 20-day-old Sertoli cells was accompanied by changes in the magnitude of FSH-stimulated PDE4 activities in both the particulate and soluble compartments.

While on Days 10 and 30, FSH induced a broad but transient increase in soluble PDE4 activities, which returned to the basal levels within 24 h, the gonadotropin elicited a sharp increase in these activities by Day 20. Moreover, rolipram-sensitive soluble PDEs were maintained at well above basal levels even after 24 h. The lack of significant changes in rolipram-insensitive PDE elicited by FSH in both the soluble and particulate compartments during the postnatal period studied indicates that the PDE4 activities are the only ones that are upregulated by the gonadotropin in rat Sertoli cells. Among the PDE4 subfamilies, it is well established that PDE4D isoforms are the main cAMP-hydrolyzing activities regulated by cAMP in Sertoli cells [22, 47].

As evidenced by the RT-PCR experiments, almost all of the previously described PDE4D isoforms were expressed in cultured rat Sertoli cells. The sole exception was the long Pde4d8 isoform, which was not expressed throughout the early postnatal period but was highly expressed in rat heart and lung tissues (data not shown), as previously shown [29]. The pattern of Pde4d mRNAs expressed by cultured Sertoli cells coincided with that described by Richter and coworkers [29] for the whole adult testis, with one noteworthy exception, which is likely due to differences in the biological material used (purified cells vs. whole tissues) and/or the age of the animals (prepubertal vs. adult). Indeed, we demonstrated the expression of pde4d6, which was initially described as brain-specific [48] but was later identified in lung and kidney tissues [29]. The expression of the long Pde4d isoforms (Pde4d3, Pde4d4, Pde4d5, Pde4d7, Pde4d9) was sustained throughout the postnatal period and no significant age-related changes in gene expression were recorded in the present experiments. This expression pattern differed from that reported for the whole testis, in which the expression of long Pde4d isoforms increased in growing animals while that of the short Pde4d isoforms decreased [33].

As reflected in the Western blot analysis, most of the Pde4d variants mRNAs were translated. Although the PDE4D isoforms were not easily distinguishable on the basis of their electrophoretic mobilities [29], at least five immunoreactive species were easily detectable with apparent molecular weights of 68 kDa, 75 kDa, 90 kDa, 100 kDa, and 110 kDa, which fit well with those described for D2/D6, D1, D3/D8/D9, D5/D7, and D4, respectively [29]. As evidenced by the immunoblotting analyses, the short isoforms, and especially PDE4D1, accounted for a large proportion of the particulate PDE4D proteins, while the long isoforms, and especially the 90-kDa PDE4Ds, were the major PDE4D proteins in the soluble fraction. This unexpected result differed from those obtained in transfected COS cells [49] and FRTL-5 thyroid cells [50], demonstrating the soluble localization of the PDE4D1/D2 isoforms. The predominant particulate localization of the short PDE4D isoforms may result from the presence of binding partners in the Sertoli cell membrane, which are not expressed in other cell types.

According to their size, the multiple variants of PDE4D are known to be regulated either at the transcriptional level or by phosphorylation [24–29]. Among the isoforms expressed by Sertoli cells, only short PDE4D1 and PDE4D2 appear to be transcriptionally regulated by FSH, in agreement with the cAMP-induced upregulation of these PDE4D isoforms described previously [33, 46, 47]. Since the mRNA that encodes PDE4D2 is generated by splicing of the PDE4D1 primary transcript, the similitude of the expression patterns of PDE4D1 and PDE4D2 under FSH conditions suggests that FSH does not regulate the splicing of PDE4D1 transcripts. Interestingly, FSH retained its ability to upregulate Pde4d1 mRNA levels throughout the early postnatal period but increases in the corresponding protein levels occurred only at Day 20, as reflected in the significant increase in the amount of the 75-kDa immunoreactive protein. Since this period coincided with the strongest activation of the cAMP pathway in Sertoli cells, these data are consistent with the idea that differential regulation of short PDE4D protein expression in Sertoli cells throughout the early postpartum period resides at the level of cAMP-mediated activation of PKA. This phenotype-dependent differential regulation of short PDE4D variants in response to cAMP-elevating agents has been previously reported for vascular smooth muscle cells [51] but appeared to be due, in contrast to our data, to differences in gene expression consecutive to changes in the levels of histone acetylation at the intronic promoter. In contrast, the long PDE4D isoforms corresponding to the 90-kDa, 100-kDa, and 110-kDa immunoreactive bands did not display significant changes during the early postnatal period either in absence or presence of FSH, in agreement with the lack of gonadotropin-induced upregulation of long Pde4d expression.

The intracellular redistribution of PDE4 activities at Day 20 without simultaneous changes in protein expression may result from the loss of rolipram-insensitive PDE activities (see Table 3) and/or the redistribution of PDE4 activities into different discrete locations within the same subcellular compartment, thus affecting accessibility of the substrate. Moreover, an increase in the FSH-induced stimulation of PDE4 activities occurred in both the soluble and particulate fractions at this age. A gonadotropin-induced increase in the de novo synthesis of short PDE4 isoforms probably accounts for part of this effect, although both the timing and amplitude of the stimulation suggest that enhancement of PDE4 activities in response to FSH at Day 20 also resulted from an increase in PKA-mediated phosphorylation of long PDE4D isoforms. This could occur by developmental changes in the expression of scaffolding proteins, such as A-kinase anchoring proteins (AKAPs). These docking proteins, by binding RII subunits of PKA holoenzymes with high affinity [52, 53], can target a kinase close to the particulate PDE4D isoforms at the time of the surge in cAMP concentration, i.e., at 20 days of age. The expression of some AKAPs has been shown to be regulated in a cAMP-dependent manner by TSH in thyroid cells [54] and by FSH in granulosa cells [55]. In addition, the expression of PKA subunit RIIb mRNA was increased several fold by cAMP in Sertoli cells [56, 57] and the resulting de novo synthesis induced a redistribution of the kinase, tethering it to specific subcellular locations through interactions with specific AKAPs. Finally, the post-translational regulation of PDE4 activities by PKA phosphorylation is strengthened by the transience of a large part of the FSH-induced upregulation of PDE4 activities, especially in 20-day-old rat Sertoli cells, which suggests that phosphorylated long PDE4s are rapidly deactivated by a still-unknown phosphatase.

The period of resensitization of the cAMP pathway towards FSH, i.e., 20 days postpartum, corresponds to the timing of ablation of gonadotropin-induced MAPK3/1 activation. Indeed, the transition between the proliferative (10 days) and quiescent and early differentiated (20 days) states is associated with switching of signaling of the FSH receptor from MAPK3/1 to adenylate cyclase activation, as evidenced by the fact that FSH activated MAPK3/1 in Sertoli cells from 5-day-old and 11-day-old, but not 19-day-old rats [58]. Thus, it seems likely that resensitization of the cAMP pathway triggered by FSH at Day 20 postpartum resulted from the switching of FSH receptor coupling from MAPK3/1 to adenylate cyclase. A central role has already been attributed to β arrestin-recruited specific PDE4D (PDE4D5) in decreasing the ability of membrane-bound PKA to phosphorylate β2 adrenergic receptor and to switch receptor coupling from Gs (cAMP pathway) to Gi (MAPK 3/13 pathway) in HEK 293 cells [59, 60].

In growing Sertoli cells, resensitization was temporally related to increases in PDE4 activities in the particulate fraction, which suggests that PDE4, and especially PDE4D, may be involved in this process. The hypothesis of PDE4D involvement in the regulation of FSH receptor/protein Gs coupling was further strengthened by data obtained in the female counterparts of Sertoli cells. PDE4D–/– granulosa cells, in contrast to PDE4D+/+ cells, display a decreased response to gonadotropins (both LH and FSH) due to a lesion located at the level of receptor/G protein coupling [61]. That unpaired coupling has been interpreted as an adaptative mechanism to the loss of PDE4D feedback required to generate a transient response to gonadotropin. Such uncoupling could be observed between Days 20 and 30 in rat Sertoli cells when FSH responsiveness, even in the presence of MIX, was decreased despite an increase in the number of FSH receptors at the cell surface [4]. Since desensitization in 30-day-old Sertoli cells was also associated with a decrease in FSH-induced PDE4D expression/activation in the particulate fraction, it is tempting to speculate that a mechanism similar to that observed in granulosa cells is involved. Decreases in PDE4D activities at the membrane level in Sertoli cells of growing rats could sustain high PKA activity close to the gonadotropin receptor. The resulting increase in receptor phosphorylation could trigger FSH receptor uncoupling with Gs, thus attenuating the cAMP pathway. By altering transcription in a specific manner and by preserving the integrity of the blood-testis barrier, which is known to be altered by high cAMP levels [62], attenuation of cAMP levels in response to circulating FSH would allow the creation within the seminiferous tubules of a microenvironment suited to the onset of spermatogenesis.

In conclusion, our results provide some evidence for age-related changes in: (i) the subcellular distributions of PDE4 activities; (ii) FSH-stimulated PDE4D1 expression; and (iii) FSH-induced activation of long PDE4D isoforms. Since these changes coincide with alterations in the cAMP response to FSH, it could be hypothesized that the role of PDE4D, and especially particulate PDE4D, is not confined to feedback regulation of the cAMP pathway but may be extended to the regulation of coupling efficiency between the FSH receptor and the Gs heterotrimeric protein. Since the cell membrane localization of long, and probably short PDE4D isoforms requires protein-protein interactions, the nature of the binding partner(s) in Sertoli cells is an interesting challenge for the future. The resensitization of the cAMP pathway observed in vitro in 20-day-old Sertoli cells coincides with the acquisition in vivo of the epithelial polarized phenotype by Sertoli cells [63]. Whether the interactions between cell surface receptors for extracellular matrix components (e.g., integrins) and the basal lamina can organize signalization scaffolds in the vicinity of FSH receptors and thus regulate FSH receptor/G proteins interactions remains to be demonstrated. The obvious dependence of FSH-induced Gs activation [64] and MAPK 3/1 inactivation [65] on cell adhesion for proliferative Sertoli cells is a strong argument in favor of this hypothesis, although further studies will be required to address this issue.

FOOTNOTES

1Supported by a fellowship from the Ministère de l'Education Nationale et de la Recherche (G.L). Back

Correspondence: 2Pierre-Jacques Bonnamy, Laboratoire OEstrogènes et Reproduction, UPRES-EA 2608, INRA USC 2006, Université de Caen, 14032 Caen cedex, France. FAX: 33 2 3156 5120; e-mail: pierre-jacques.bonnamy{at}unicaen.fr

Received: 8 July 2006.

First decision: 4 August 2006.

Accepted: 4 January 2007.

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