Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells

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Developmental Biology 315 (2008) 173 – 188 www.elsevier.com/developmentalbiology

Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells Murielle Godet a,b,c,⁎, Odile Sabido d , Jérôme Gilleron e , Philippe Durand a,b,c a

d

Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Lyon F-69003, France b INRA, CNRS, Université Lyon 1, Ecole Normale Supérieure, Lyon F-69364, France c INSERM U418, Lyon, France Université Jean Monnet, Faculté de Médecine, Centre Commun de Cytométrie en flux, St. Etienne, France e INSERM 670, Université Paris V René Descartes, Paris, France Received for publication 30 May 2007; revised 6 December 2007; accepted 17 December 2007 Available online 29 January 2008

Abstract Progression of germ cells through meiosis is regulated by phosphorylation events. We previously showed the key role of cyclin dependent kinases in meiotic divisions of rat spermatocytes co-cultured with Sertoli cells (SC). In the present study, we used the same culture system to address the role of mitogen-activated protein kinases (MAPKs) in meiotic progression. Phosphorylated ERK1/2 were detected in vivo and in freshly isolated SC and in pachytene spermatocytes (PS) as early as 3 h after seeding on SC. The yield of the two meiotic divisions and the percentage of highly MPM-2-labeled pachytene and secondary spermatocytes (SII) were decreased in co-cultures treated with U0126, an inhibitor of the ERK-activating kinases, MEK1/2. Pre-incubation of PS with U0126 resulted in a reduced number of in vitro formed round spermatids without modifying the number of SII or the MPM-2 labeling of PS or SII. Conversely, pre-treatment of SC with U0126 led to a decrease in the percentage of highly MPM-2-labeled PS associated with a decreased number of SII and round spermatids. These results show that meiotic progression of spermatocytes is dependent on SC-activated MAPKs. In addition, high MPM-2 labeling was not acquired by PS cultured alone in Sertoli cell conditioned media, indicating a specific need for cell–cell contact between germ cells and SC. © 2007 Elsevier Inc. All rights reserved. Keywords: Testis; Spermatogenesis; Germ cells; Spermatocytes; Sertoli cells; Meiosis; MAPK; ERK1/2

Introduction Spermatogenesis is a dynamic process in which stem spermatogonia through a series of events involving mitosis, meiosis and cellular differentiation become mature spermatozoa. Sertoli cells are in close contact with germ cells in the seminiferous tubule; they play a critical role in these processes by providing the germ cells with physical support, nutrients, hormonal and paracrine signals, all of which are necessary for successful spermatogenesis (Griswold, 1998).

⁎ Corresponding author. INSERM U418, Hôpital Debrousse, 29 rue sœur Bouvier, 69322 Lyon cedex 05, France. Fax: +33 4 78 25 61 68. E-mail address: [email protected] (M. Godet). 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.12.019

The entry of eukaryotic cells into mitosis or meiosis is universally regulated by reversible phosphorylation events (Stukenberg et al., 1997; Cheung et al., 2000; Strahl and Allis, 2000). During meiosis, at the pachytene stage, chromosome condensation, synaptonemal complex assembly and disassembly (Lammers et al., 1995; Suja et al., 1999) and nuclear envelope breakdown (Tarsounas et al., 1999), which precede meiotic divisions and cytokinesis (Wang et al., 2006), are regulated by phosphorylation events. We have previously shown that the Ser/Thr kinase CDK1, associated with its regulatory partner cyclin B1 to form M-phase promoting factor (MPF), is a key regulator of the meiotic G2/M transition of rat spermatocytes. Pachytene spermatocytes (PS) or secondary spermatocytes (SII) pre-treated with roscovitine, a potent inhibitor of MPF (Meijer and Raymond, 2003), do not progress

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through the first or the second meiotic division under our culture conditions (Godet et al., 2004). However, it has been recently shown that other kinases, in particular extracellular signal-regulated protein kinases, ERK1 and ERK2, the two main isoforms of the mitogen-activated kinases (MAPKs) (Pearson et al., 2001; Katz et al., 2007), may also play a role in chromatin condensation during the first meiotic division of male germ cells in response to okadaic acid in vitro treatment (Wiltshire et al., 1995; Sette et al., 1999; Di Agostino et al., 2002). However, contradictory results were obtained by Inselman and Handel (2004) using similar experimental conditions. Two pathways can lead to MAPKs activation. One pathway depends upon tyrosine kinase receptors, activated by extracellular signal acting through the Ras GDP-exchange factor and activation of the MAPK kinases, MEK1/2 (Katz et al., 2007). The second pathway involves the MAPK kinase Mos (Fan and Sun, 2004), as shown in oocytes. However, the analysis of mutant mos−/− mice has shown that in this species, Mos is dispensable for spermatogenesis (Verlhac et al., 1996). Freshly isolated PS have almost undetectable levels of phospho-ERK1/2. However, MAPKs activation can be induced in PS by in vitro OA treatment and is correlated with progression of PS to meiotic metaphase I (Sette et al., 1999). It is worth observing that these cells do not progress beyond the diplotene/MI stages (Handel et al., 1995). In the seminiferous epithelium, there is a cross-talk between germ cells and Sertoli cells that involves paracrine regulations (Kierszenbaum, 1994; Griswold, 1998; Petersen and Soder, 2006), cell–cell adhesion (Siu et al., 2005) and cell–cell communication through gap junctions (Decrouy et al., 2004; Pointis and Segretain, 2005). Sertoli cells possess receptors for testosterone and FSH (Walker and Cheng, 2005) and consequently are the targets of hormonal signals that regulate spermatogenesis. It has been shown that ERK kinases are activated by FSH in cultured Sertoli cells from 5- and 11-dayold rats but not in more differentiated Sertoli cells from 19-dayold rats (Crepieux et al., 2001). Nevertheless, activated MAPK1/2 have been found in Sertoli cells of 19-day-old rats. Hence, it has been postulated that this FSH-regulated pathway may not play a large role in maintaining spermatogenesis in the adult rat. In contrast, TGFβ was shown to reversibly perturb blood–testis barrier integrity and Sertoli-germ cell adhesion via ERK signaling pathway (Xia and Cheng, 2005; Xia et al., 2006). We have established a system of co-culture of pachytene spermatocytes (PS) with Sertoli cells (SC), which allows these germ cells to complete the two meiotic divisions leading to in vitro formation of round spermatids (RS) (Weiss et al., 1997). The goal of our study was to investigate MAPKs activation in PS during the spontaneous (not pharmacologically induced) G2/ M transition in these cells. Our results show that MAPK activation in PS is deeply dependent of MAPKs activation in SC and requires close contacts between these germ cells and SC. Moreover, they demonstrate that progression of spermatocytes through the

meiotic prophase and divisions requires activated MAPKs of both SC and the germ cells. Materials and methods Reagents and chemicals Roscovitine: 2-[R]-(1-ethyl-2-hydroxyethylamino-6-benzylamino-9-isopropylpurine) and U0126: 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene (Calbiochem, Euromedex, Mundolsheim, France) were dissolved (stock solution at 50 mM) in dimethylsulfoxide (DMSO) (Sigma-Aldrich, St. Quentin-Fallavier, France) and stored in aliquots at −20 °C until use. Ovine FSH (NIH-FSH-S1) was obtained from Dr. A.F. Parlow, Torrance, USA (lot no. AFP7028D). Polyacrylamide and other electrophoresis reagents were purchased from Bio-Rad Life sciences Group (Marnes La Coquette, France), and nitrocellulose membrane was purchased from Schleicher and Shull (Mantes La Ville, France). BCA kit, ECL Western Blotting Detection System (ECL-kit), HRP-conjugated sheep anti-mouse or donkey anti-rabbit immunoglobulins (IgG) and protease inhibitor cocktail were purchased from Amersham Biosciences (Orsay, France). Collagenase was obtained from Serva (Paris, France) and DMEM/F12 culture medium from Invitrogen (Cergy Pontoise, France). Kit for detection of apoptosis (in situ cell death detection kit) was purchased from Roche (Meylan, France); Vectastain ABC streptavidin horseradish peroxidase (HRP) kit, DAB (3, 3′Diaminobenzidine) substrat kit for peroxidase and Hematoxylin were from Vector Laboratories (Clinisciences, Montrouge, France). DakoCytomation (Trappes, France) provided anti-vimentin (clone V9), MPM-2 antibodies, phycoerythrin (PE)- or fluorescein (FITC)-conjugated rabbit anti-mouse IgG and normal mouse and rabbit serum. TRITC-conjugated anti-mouse IgG and FITCconjugated anti-rabbit antibodies were purchased from ImmunoResearch (Interchim, Montlucon, France). Anti-ERK1 (K-23) and anti-ERK2 (D-2) antibodies were obtained from Santa-Cruz Biotechnology (Tebu, Le Perray-enYveline, France). Anti-phosphorylated-ERK1/2 (E10) (anti-phospho-ERK1/2) was purchased from Cell Signaling (Ozyme, Montigny Le Bretonneux, France). Anti-phosphorylated serine 10 (ser10) of histone H3 (phospho-H3) and antiphosphorylated serine 139 of histone H2AX (γ-H2AX) were purchased from Upstate Biotechnology (Euromedex, Mundolsheim, France). Anti-Cx43 was obtained from Zymed Laboratories (Invitrogen, Cergy Pontoise, France). All other products for culture, Hoechst 33342 and phosphatase inhibitor cocktail were obtained from Sigma-Aldrich.

Animals Twenty-day-old male Sprague–Dawley rats were obtained from Charles River Laboratory (L'Arbresle, France); 90-day-old male Sprague–Dawley rats were obtained from a local animal facility and maintained under standard conditions. All procedure were approved by the Scientific Research Agency (Approval no. 69306) and conducted in accordance with the guidelines for Care and Use of Laboratory Animals.

Preparation of tissue sections and immunostaining Rat testes were fixed with PBS (pH 7.4) containing 4% paraformaldehyde for 24 h. After washes, tissues were cryoprotected in 18% sucrose in PBS, embedded in Tissue-Teck OCT compound (Bayer, Puteaux, France) then frozen at − 80 °C, cut into 8-μM-thick sections with a cryostat and mounted onto glass slides to be stored at −20 °C until used. Sections were treated for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) by irradiation in microwave (800 W) for 5 min; the sections were subsequently cooled for 30 min at RT, washed in PBS, treated with 0.5% Triton X-100 for 10 min then treated with blocking buffer (PBS/1% BSA/10% SVF) for 30 min. After washing, tissue sections were exposed to an anti-phospho-ERK1/2 antibody (1:150) in blocking buffer overnight at 4 °C. After washing, tissues sections were reacted with the reagents of the Vectastain ABC peroxidase kit and with the DAB substrate kit according to the manufacturer's instructions. For negative control, cells were incubated with non-immune mouse IgG. Stained testis sections were examined with an Axioskop microscope connected to a Color video Camera (Carl Zeiss, Oberkochen, Germany).

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Isolation and co-culture of rat Sertoli cells with pachytene spermatocytes (PS) Sertoli cells (SC) were isolated from the testes of 20-day-old rats as previously described (Weiss et al., 1997). Cells were plated onto the polyester membrane of the apical compartment of bicameral chambers (Greiner, Courtaboeuf, France) at a density of about 3.5 × 105 cells/cm2 in HEPES-buffered DMEM/F12 supplemented with insulin (10 μg/ml), transferrin (10 μ g/ml), vitamin C (10− 4 M), vitamin E (10 μg/ml), retinoic acid (3.3 × 10− 7 M), retinol (3.3 × 10− 7 M), pyruvate (1 mM), testosterone (10− 7 M) and FSH (1 ng/ml) (DMEM/F12-supplemented medium). Sertoli cells were cultured at 33 °C in a humidified atmosphere of 95% air and 5% CO2. On day 3 of culture, apical medium was saved as Sertoli cells conditioned medium. Pachytene spermatocytes (PS) were obtained from adult rats by centrifugal elutriation. The purity of the elutriated PS fraction was assessed by flow cytometry analysis: 95 ± 3% of cells were 4C cells, 3 ± 2% were 2C cells and 2.0 ± 0.5% were 1C cells (n = 5). Elutriated PS (4C cells) were composed of 13% of juvenile spermatocytes (stages XIV to IV of the rat seminiferous epithelium) (Clermont et al., 1959), 61% were middle PS (stages V to IX) and 26% were late PS (stages X to XIII) (Vigier et al., 2004). On day 3 of SC cultures (referred to as day 0 of co-culture), elutriated PS were seeded (3.5 × 10− 5 cells/cm2) on SC layers leading to the early reappearance of connexin 43, the most abundant gap junction protein identified in the testis (Batias et al., 2000), at the border between the germ cells and the SC (see Fig. 1). Protocols of cultures: (A) Elutriated PS were co-cultured with SC in the presence of DMSO (1:4000), 12 μM U0126 (U12), an inhibitor of the ERKactivating kinases MEK1/2 or 12 μM roscovitine (R12), an inhibitor of CDK1, CDK2 and CDK5, for 24 h. For western blot experiments, positive control of the induction of phospho-ERK1/2 was performed by incubating elutriated PS in Sertoli cell conditioned medium with 5 μM OA for 5 h. (B) Elutriated PS were cultured alone in Sertoli cell conditioned medium for 0 h, 3 h or 24 h in bicameral chambers. (C) Elutriated PS were pre-incubated in Sertoli cell conditioned medium with DMSO (1:2000) or 12 μM U0126 (U12), 25 μM (U25) and 50 μM (U50) for 3 h. After two washes with DMEM/F12supplemented medium to remove the inhibitors, PS were co-cultured with Sertoli cells for 3 h or 24 h. (D) SC were cultured in the presence of DMSO (1:4000) or U12 for 40 h. On day 0 of co-culture, apical and basal media containing inhibitors were discarded, the SC layers were washed twice with DMEM/F12-supplemented medium, then elutriated PS were seeded on SC. At the end of the culture period, cells were counted and viability was assessed by Trypan blue exclusion. At selected hours of co-culture, cells were prepared as follows: (i) for flow cytometry analyses, cells were trypsinized and fixed at 4 °C for 1 h in PBS containing 2% paraformaldehyde; (ii) for western blot analyses, germ cells were separated from adherent SC by vigorous horizontal shaking of the insert. Germ cells were centrifuged, washed with PBS and stored at −80 °C. SC layers on membrane inserts were stored at − 80 °C. The purity of the germ cell fractions

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and SC fractions recovered after trypsinization were assessed by counting vimentin-labeled negative or positive cells by microscopic examination; they were (72 ± 5%; n = 3) and (89 ± 3%; n = 3), respectively.

Immunolabeling of cells in co-culture On day 2, cells were fixed directly in bicameral chambers with methyl alcohol for 6 min at −20 °C. After washing with PBS, cells were permeabilized with PBS/0.5% saponin for 30 min at RT (room temperature) and exposed to an anti-vimentin antibody used at a dilution of 1:100 in blocking buffer (PBS/3% BSA) overnight at 4 °C. After washing, the cells were incubated with an antiCx43 antibody used at a dilution of 1:100 in blocking buffer for 3 h at 4 °C. After 3 washes with PBS, the cells were exposed to TRITC-conjugated rabbit antimouse IgG (1:100) and to PE-conjugated goat anti-rabbit IgG (1:100) in blocking buffer for 1 h at 4 °C. For negative control, cells were incubated with non-immune mouse and rabbit IgG. Cell nuclei were stained with DAPI in mounting medium and examined under a high resolution wide field deconvolution microscope (Nikon TE2000E) equipped with a Coolsnap HQ2 Camera (Ropper scientifique). Deconvolution was performed on AutoQuant software (Meyer instrument).

Immunolabeling of cells for flow cytometry Immunolabeling of co-cultured cells for flow cytometry analysis was performed essentially as previously described (Godet et al., 2000, 2004; Fouchecourt et al., 2006). After washing with PBS, fixed cells were permeabilized with PBS/1% BSA/0.25% Triton X-100 for 20 min on ice. The cells were exposed to an anti-vimentin antibody used at a dilution of 1:300 in blocking buffer (PBS/1% BSA/10% SVF) for 3 h at 4 °C. After 3 washes in PBS/1% BSA, the cells were exposed to FITC-conjugated rabbit anti-mouse IgG (1:60) in blocking buffer for 1 h at 4 °C. After washing, the cells were incubated with an anti-ERK1, anti-ERK2, anti-phospho-ERK1/2, anti-phospho-H3 or MPM-2 antibody, all used at a dilution of 1:200, in blocking buffer overnight at 4 °C. After washing, ERK1-, MPM-2- or phospho-H3-labeled cells were incubated with PE-conjugated rabbit antimouse IgG (used at a dilution of 1/60) and ERK2-, phospho-ERK1/2 or phospho-H3-labeled cells with PE-conjugated goat anti-rabbit IgG (1:100) in blocking buffer, for 1 h at 4 °C. Before analysis, cell nuclei were stained with Hoechst 33342 at a final concentration of 20 μg/ml for 20 min. For negative control, cells were incubated with non-immune mouse or rabbit IgG.

Flow cytometric analysis and cell sorting After immunolabeling, cells were analyzed using a FACSVantage SE cell sorter (BD Biosciences, le Pont de Claix, France) equipped with an Enterprise II

Fig. 1. Immunodetection of Cx43 in co-cultures of PS and SC on day 2. (A) Cx43 immunolabeling (green) was detected as punctuate staining between PS and SC (arrow). Nuclei were DAPI-labeled and appeared in blue. SC were identified by their vimentin labeling (red). The insert is a selected region at upper magnification. (B) Cells were incubated with mouse and rabbit IgG as a negative control. Scale bars: 10 μM.

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argon ion laser emitting simultaneously a 50-mW line tuned to UV (351 nm) and a second line tuned to 448 nm to respectively excite Hoechst and FITC or PE. Emission of PE and FITC fluorescence were acquired after logarithmic amplification through band-pass filters respectively BP 585/40 nm and BP530/ 30 nm. Emission of Hoechst fluorescence was acquired after linear amplification through a 424/44-nm BP filter. Acquisition and analysis were performed using CellQuest Pro™ 4.0.2 software. Cell sorting on slides was performed using CloneCyt™ Plus 3.1 software. Analyses were performed as previously described in details (Godet et al., 2000, 2004; Fouchecourt et al., 2006). Five data parameters were acquired and stored in list mode files: linear forward light scatter (FSC) and linear side angle light scatter (SSC), which roughly represent cell size and cellular granularity, respectively; logarithmic (log) PE (ERK1, ERK2, phospho-ERK1/2, MPM-2 or phospho-H3), log FITC (vimentin) to

detect the immunolabeling and linear Hoechst to measure the DNA content of the different population of cells. Contaminating events such as debris and clumped cells were eliminated from the analysis. Each acquisition was performed on 50,000 vimentin-negative cells. Flow cytometry using three different parameters “FSC/SSC and ploidy” allowed the identification of six germ cell populations in total tubular cell preparations (before elutriation) from adult rats (Godet et al., 2000) (Fig. 2). Juvenile spermatocytes (JPS), medium to late PS (mlPS) and round spermatids (RS) were in rather pure fractions, whereas secondary spermatocytes (SII) were contaminated by doublets of RS (Fig. 2J). Sorted cells of each fraction were identified according to their nuclear shape and cellular size, utilizing the description of the XIV steps of rat spermatogenesis by Russell et al. (1990) as in Vigier et al. (2004).

Fig. 2. Representative flow cytometry analysis of populations of testis germ cells obtained from 50-day-old rats. (A) Distribution of vimentin-positive diploid (R1) Sertoli cells or vimentin-negative diploid (R3), tetraploid (R4) and haploid (R2) populations of germ cells. (B–D) The different germ cell populations were subsequently characterized according to their forward light scatter (FSC) and side scatter (SSC). (E–L) Gated populations were sorted on slides and (E, F) immunolabeled for γ-H2AX or (G–L) stained with May–Grunwald–Giemsa (MGG). Sorted cells were identified as Juvenile spermatocytes (E, G: R5), middle to late PS (F, H: R6), spermatogonia and preleptotene spermatocytes (I: R7), secondary spermatocytes and doublet of round spermatids (J: R8), elongated spermatids (K: R9) and round spermatids (L: R10). (E–F) γ-H2AX-labeled cells were identified as leptotene (lepto.), zygotene (zygo.) or pachytene (pachy.) spermatocytes. Highly γH2AX-labeled sex vesicles are indicated by arrows. Scale bars: 10 μM.

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Fig. 3. Distribution of endogenous phospho-ERK1/2 proteins in the rat testis and effect of U0126, an MEK inhibitor, and roscovitine, an MPF inhibitor, on the two meiotic divisions of pachytene spermatocytes (PS) co-cultured with Sertoli cells (SC). (Aa–b) Indirect immunofluorescence staining of phospho-ERK1/2 in crosstubule sections of adult rats. Note that the periphery of the tubule section (SC and spermatocytes) appears more labeled than the adluminal part of the tubule section (SC and spermatids). (d–f) Hoechst labeling of cross-sections. (c) Tubule sections were incubated with mouse IgG as a negative control. Scale bar: 10 μM. (B) Western blot analysis of ERK1, ERK2 and phospho-ERK1/2 in SC and germ cells separated after 3 h or 24 h of co-culture in the absence (DMSO) or presence of 12 μM U0126 (U12). Freshly isolated SC (purified SC) were obtained from 20-day-old rats and cultured for 3 days alone (0 h of co-culture). Freshly elutriated PS (elut. PS) were obtained from 90-day-old rats and cultured with DMSO (ctl) or 5 μM okadaic acid (5 μM OA) for 5 h as a positive control. 100 μg of total protein were loaded per lane. (C, D) Flow cytometry analysis of the relative levels of ERK1, ERK2 (C) and phospho-ERK1/2 (D) in elutriated PS; juvenile spermatocytes (JPS), medium to late PS (mlPS), secondary spermatocytes (SII) or round spermatids (RS) after a 24-h co-culture period with SC. (E–G) Flow cytometry analysis of the numbers of mlPS (E), SII (F) or RS (G) per well in co-culture of PS with SC in the presence of DMSO (ctl), U12 or 12 μM roscovitine (R12). (H, I) Proportions (%) of TUNEL-labeled mlPS (H) or SII (I) were determined after TUNEL reaction on slides of cells sorted by flow cytometry. Results are the mean ± SEM of 4 different experiments. Different letters indicate significant differences (p b 0.05). (J) Western blot analysis of MPM-2 epitopes in freshly elutriated PS (elut. PS) and in germ cell fractions after 24 h of co-culture with SC in the absence (DMSO) or presence of 12 μM roscovitine (R12) or 12 μM U0126 (U12).

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Fig. 3 (continued). In order to differentiate between JPS and mlPS, γ-H2AX detection was performed. Spots of one thousand JPS and mlPS were sorted on glass slides and incubated with an anti-γ-H2AX antibody used at a dilution of 1:200 in blocking buffer for 1 h at RT. After washing, cells were reacted with the reagents of the Vectastain ABC peroxidase kit and the DAB substrate kit according to the manufacturer's instructions. Stained cells were examined with a microscope connected to a color video camera (Figs. 2E and F). H2AX is phosphorylated throughout the chromatin in leptotene spermatocytes. As the DNA doublestrand breaks are processed by the recombination machinery and synapsis is established during zygonema, γ-H2AX gradually decreases and, by pachynema, it is thin on autosomal chromatin. During pachynema and diplonema, the sex body is strongly labeled (Mahadevaiah et al., 2001; Chicheportiche et al., 2007). In culture experiments, the percentage of each category of germ cells was multiplied by the total number of cells per well in order to obtain the absolute number of mlPS, SII and RS.

reducing conditions (Laemmli, 1970). Proteins were electroblotted onto nitrocellulose membranes. After transfer, the membranes were saturated for 1 h in TBS-T (TBS/0.1% Triton X-100) with 5% fat milk and probed overnight at 4 °C with anti-ERK1 and anti-ERK2, anti-phospho-ERK1/2 or MPM-2 antibodies used at a dilution of 1:500 in TBS-T with 5% fat milk. After washing in TBS-T, membranes were incubated with an anti-mouse or an anti-rabbit HRP antibody (1:1000). An enhanced chemiluminescence kit (ECL-kit) was used to detect immune complexes and blots were exposed to Kodak autoradiographic films.

Statistical analysis Analysis of variance followed by Fisher's test was used to assess statistical differences between controls and treated cells. Differences were considered significant at p b 0.05.

Detection of apoptosis on sorted cells and counting

Results In order to perform apoptosis detection, two spots of one thousand mlPS, SII or SC were sorted on glass slides and incubated with 20 μl of TUNEL reaction mixture according to the manufacturer's instructions. TUNEL-positive and -negative cells were counted by microscopic examination.

Electrophoresis and western blotting Proteins of germ cells or SC were solubilized on ice in lysis buffer: 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 0.5% NP40, protease inhibitors and 1% phosphatase inhibitors. After cell lysis and centrifugation, protein concentration in the supernatant was determined using BCA kit from Pierce (Perbio, Brebières, France). One hundred micrograms of proteins was resolved by 10% or 12% SDS–PAGE under

ERK1, ERK2 and their phosphorylated forms are present in Sertoli cells and in meiotic germ cells In a first series of experiments, the presence of ERK1 and ERK2 together with their activated phosphorylated (phospho) forms was investigated in cross sections of seminiferous tubules, in isolated SC and meiotic germ cells (Fig. 3). Phospho-ERK1/2 were observed both in SC and in germ cells on seminiferous tubule sections of rat testes (Fig. 3A). ERK1, ERK2, phospho-ERK1 and phospho-ERK2 were present in

Fig. 4. Relative levels and cellular localization of MPM-2 in primary and secondary spermatocytes co-cultured with Sertoli cells. (A–D) Flow cytometry analysis of the relative levels of MPM-2 in JPS (A), mlPS (B), SII (C) or RS (D) after a 0-h (blue line) or a 24-h (red line) co-culture period. Co-cultured cells incubated with mouse IgG as a negative control (black line). (E–M) mlPS and (N–P) SII were sorted on glass slides and examined for Hoechst (E, H, K, N); MPM-2 (F, I, L, O) or merged (G, J, M, P) by fluorescent confocal microscopy. Co-cultured cells were incubated with anti-IgG-conjugated with PE as a negative control (F–G). MPM-2 epitopes were present both in the cytoplasm and the nucleus of elutriated PS (H–J), whereas after 24 h of co-culture with SC, MPM-2 co-localized with chromatin in both mlPS (K–M) and SII (N–P). Scale bars: 10 μM.

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freshly isolated SC and their levels were maintained when SC were cultured for 24 h in control medium either alone or together with PS (Fig. 3B). Addition of U0126 (Favata et al., 1998; Davies et al., 2000), at a concentration of 12 μM at the time of addition of PS on SC, abolished the presence of phospho-ERK1 and phospho-ERK2 in SC from 3 h onwards, without modifying the levels of ERK1 or ERK2. Both ERK1 and ERK2 were present in freshly elutriated PS but their phosphorylated forms were close to the threshold of detection (compare Fig. 3B with 9A). By contrast, both phospho-ERK1/2 were observed in cultured middle to late PS (mlPS) as early as 3 h after seeding on SC and U0126 completely

prevented this activation (Fig. 3B). As expected (Sette et al., 1999), okadaic acid (5 μM OA) increased the levels of phosphoERK1/2 in freshly purified PS. The fraction of in vitro formed SII was sorted on glass slides and the purity was assessed by counting the number of mono-nucleated cells (SII) and bi-nucleated cells (doublets of spermatids) under microscopic examination: (95 ± 5%; n = 6) of the cells were SII. It was observed that ERK1/2 were also present in SII and in RS formed in culture, though at lower levels than in mlPS (Fig. 3C). Phospho-ERK1/2 were present in SII at similar levels than in mlPS but not detected in RS (Fig. 3D).

Fig. 5. MPM-2 labeling of elutriated PS incubated in Sertoli cells conditioned medium or co-cultured with SC. Indirect immunofluorescence staining of MPM-2 of (B) freshly elutriated PS (D) elutriated PS incubated in Sertoli cells conditioned medium for 3 h or (F) 24 h or (H) cultured for 24 h with SC. (A, C, E, G) Nuclei were stained with Hoechst. Scale bars: 10 μM.

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U0126 and roscovitine act on different steps of the meiotic progression of PS co-cultured with SC Pachytene spermatocytes were co-cultured with SC either in the absence or presence of U0126 or roscovitine, and the number of SII and RS formed in vitro was determined 24 h after treatment. None of the treatments affected the number of mlPS or the percentage of apoptotic mlPS (Figs. 3E and H). However, U0126 or roscovitine both lowered the numbers of in vitro formed SII and RS (Figs. 3F and G) without changing the percentage of apoptotic SII (Fig. 3I). In an attempt to determine whether these pharmacological compounds act at similar or different steps of the meiotic progression, we studied their effect on the maintenance and/or appearance of the (pSer or pThr)-Pro epitope (Westendorf et al., 1994) recognized by the MPM-2 antibody (Davis et al., 1983) and of phosphorylated histone H3 (phospho-H3). The MPM-2 monoclonal antibody was risen against mitotic HeLa cells (Davis et al., 1983) and specifically recognizes a subset of proteins that are phosphorylated from early prophase onwards

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(Vandre et al., 1984; Kuang et al., 1994; Conicella et al., 2003; Escargueil and Larsen, 2007). Conversely, histone H3 becomes phosphorylated at the end of the meiotic prophase at diplotene (Hendzel et al., 1997; Cobb et al., 1999b). As expected (Cobb et al., 1999a), the MPM-2 antibody recognized many protein bands in freshly elutriated PS fractions (Fig. 3J). When PS were cultured for 24 h in the presence of DMSO, roscovitine or U0126, many bands were also recognized in the recovered germ cell fraction but no clear-cut changes were observed irrespective the treatment. The germ cell fraction recovered after culture was composed not only of primary spermatocytes, but also of secondary spermatocytes together with round spermatids (see above Figs. 3E, F and G) and roscovitine or U0126 treatment changed the proportion of these meiotic germ cells in the fraction. Therefore, these populations were analyzed by flow cytometry in order to quantify the MPM-2 labeling in each germ cell population. On day 0 of culture, JPS were less labeled by the MPM-2 antibody than mlPS (compare Figs. 4A with 4B). After 1 day of

Fig. 6. Populations of mlPS or SII in co-culture with SC exhibit lower MPM-2 labeling in the presence of 12 μM U0126 (U12). (A, B) Flow cytometry analysis of the relative levels of MPM-2 in mlPS (A) and SII (B) co-cultured in the absence (green line) or presence (red line) of U12. Co-cultured cells incubated with mouse IgG as a negative control (black line). (C–E) G1-gated mlPS and (F–H) SII were sorted on glass slides and examined for (C, F) Hoechst, (D, G) MPM-2 or (E, H) merged by fluorescent confocal microscopy. MPM-2 was mainly present in the cytoplasm of these mlPS and SII. Scale bars: 10 μM.

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Table 1 Effect of U0126 and roscovitine on the numbers and percentages of MPM-2 or phospho-H3-labeled middle to late PS (mlPS) and secondary spermatocytes (SII) cocultured with Sertoli cells Cell population

mlPS mlPS mlPS SII SII SII

Labeled cells

MPM-2 b PH3 c MPM-2 b PH3 c

Treatment 12 μM U0126

DMSO

12 μM roscovitine

Number (×103) a

%a

Number (×103) a

%a

Number (×103) a

%a

325 ± 31 296 ± 5 7.5 ± 0.8 37 ± 4 33 ± 2 5.0 ± 0.7

100 90.8 ± 0.8 2.3 ± 0.2 100 89.1 ± 1.4 13.5 ± 0.2

304 ± 42 230 ± 4⁎ 4.2 ± 1.0⁎ 32 ± 5⁎ 23 ± 2⁎ 1.5 ± 0.8⁎

100 75.6 ± 0.3⁎ 1.3 ± 0.3⁎ 100 71.8 ± 1.2⁎ 4.6 ± 1.2⁎

317 ± 50 283 ± 8 3.5 ± 0.6⁎ 17 ± 2⁎ 15 ± 3⁎ 0.6 ± 0.1⁎

100 89.3 ± 0.8 1.1 ± 0.3⁎ 100 88.2 ± 0.2 3.5 ± 1.0⁎

a

Values are the mean ± SEM; n = 4. Highly MPM-2-labeled mlPS or SII populations (Gate G2; see Figs. 4A and B). c Phospho-H3-labeled mlPS or SII (see Fig. 7). ⁎ p b 0.05 vs. control DMSO. b

co-culture with SC, both populations exhibited a higher labeling but the shift was much more marked in the mlPS population (Fig. 4B) than in the JPS population (Fig. 4A). By contrast, when PS were cultured in Sertoli cell conditioned medium but in the absence of SC, the MPM-2 labeling was dramatically decreased as early as 3 h after seeding (compare Fig. 5B with 5D) and lost at 24 h (Fig. 5F). The MPM-2 labeling appeared to decrease after each meiotic division, as shown by the levels of MPM-2 labeling in the population of in vitro formed SII and RS on day 1 of co-culture (Figs. 4C and D). In freshly elutriated PS, MPM-2 labeling appeared equally distributed between the cytoplasm and the nucleus (Figs. 4H–J). After 1 day of co-culture with SC, the labeling was

mainly nuclear (Figs. 4K–M). Similarly, MPM-2 labeling of in vitro formed SII was observed mainly in the nucleus (Figs. 4N–P). In the 24-h co-cultures of PS and SC, U0126 12 μM partially prevented the acquisition of a high level of MPM-2 labeling by mlPS (Fig. 6A), whereas roscovitine did not (Table 1). In the gated population (G1), the MPM-2 labeling localized mostly in the cytoplasm of mlPS (Figs. 6C–E). Conversely, MPM-2 labeling of mlPS in the G2 population was mostly nuclear, as expected (see above and Figs. 4K–M). For SII, both U0126 (Fig. 6B) and roscovitine decreased the number of gated G2 MPM-2-labeled cells (Table 1); however, the percentage of highly MPM-2-labeled SII was decreased

Fig. 7. Histone H3 phosphorylation at Ser10 in populations of mlPS and SII after 24 h of co-culture with SC. (A, C) Flow cytometry analysis of mlPS (A) and SII (C) immunolabeled with an anti-phospho-H3 antibody (—) or with rabbit IgG as a control (⋯). (B, D) Gated phospho-H3-positive mlPS (B) and SII (D) were sorted on glass slides and examined by fluorescent microscopy. Identical results were obtained in three separate experiments. Scale bars: 10 μM.

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only by U0126 (Fig. 6B and Table 1), and this was associated with delocalization of MPM-2 toward the cytoplasm of these cells (Figs. 6F–H).

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Very few cells, if any, were labeled with the anti-phospho-H3 antibody in the population of PS on day 0 of culture (1.0 ± 0.1%; n = 4). However, a distinct population of labeled cells was

Fig. 8. Effects of PS incubation with U0126 or roscovitine on the two meiotic divisions. PS were pre-incubated with DMSO (ctl); 12 μM (U12), 25 μM (U25), 50 μM (U50) U0126 for 3 h, washed and seeded on SC. (A) Western blot analysis of ERK1, ERK2 and phospho-ERK1/2 in germ cells after 3 h or 24 h of co-culture. One hundred micrograms of total protein was loaded per lane. (B–D) Flow cytometry analyses of the numbers of mlPS (B), SII (C) or RS (D) per well after 24 h of coculture. (E, F) Proportions (%) of TUNEL-labeled mlPS (E) and SII (F) were determined after TUNEL reaction, on slides, of cells sorted by flow cytometry. (G–I) Flow cytometry analysis of the numbers of SII (G), phospho-H3-positive SII (H) and number of RS (I) per well after 48 h of co-culture with the SC showing the reversibility of the U25 treatment on the second meiotic division. Results are the mean ± SEM of 6 different experiments. Different letters indicate statistically significant differences (p b 0.05).

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observed on day 1 of co-culture with SC (Figs. 7A and B). Similarly, a population of anti-phospho-H3-labeled cells was identified in the population of in vitro formed SII on day 1 of co-culture (Figs. 7C and D). U0126 and roscovitine decreased both the number and the percentage of anti-phospho-H3-labeled mlPS (Table 1). Similarly, both compounds decreased significantly the number and the percentage of phospho-H3positive SII (Table 1). Given that both the SC and the germ cells co-cultured with SC contain phosphorylated ERK1/2, in the following experiments either germ cells or SC were pre-treated with U0126 before being cultured together. Incubation in the presence of roscovitine served as a positive control of the inhibition of the meiotic divisions.

labeled SII, without any change in the level of MPM-2 labeling (Table 2). Roscovitine also decreased significantly both the number and percentage of anti-phospho-H3-labeled SII (Table 2). In order to determine whether the inhibitory effect of 25 μM U0126 on the second meiotic division was reversible, cultures were prolonged up to day 2 (Figs. 8G–I). At that time, both the numbers of phospho-H3-labeled SII and RS were no longer different from those of control cells, indicating that the inhibition of the second meiotic division observed on day 1 was not due to a toxic effect of U0126 on PS.

Pre-incubation of PS in the presence of U0126 decreases the yield of the second meiotic divisions in a reversible manner

Since phospho-ERK1/2 were observed not only in germ cells but also in SC, in the next experiments, cultured SC were treated with 12 μM U0126 for 40 h, washed and then cocultured for 3 h or 24 h with PS (Fig. 9). Phospho-ERK1/2 was undetectable in SC treated for 40 h with U0126 (time 0 of coculture) (Fig. 9A). The levels of phospho-ERK1/2, however, re-appeared as soon as 3 h after washing, reaching control levels after 24 h of co-culture with PS. PS seeded on SC pretreated with U0126 did not exhibit phospho-ERK1/2 activation at 3 h as opposed to PS seeded on control SC (Fig. 9A). However, after 24 h of co-culture, phospho-ERK1/2 levels were similar in PS cultured on either control or pre-treated SC. It is important to note that in PS cultured for 24 h in the absence of SC, but in Sertoli cells conditioned media (viability of 85 ± 5%; n = 3), a hyper phosphorylation of ERK1/2 was observed (Fig. 9A). This was not correlated to high MPM-2 labeling (see Fig. 5F). A 40-h treatment of SC with 12 μM U0126 did not enhance SC death as assessed by Trypan blue exclusion and TUNEL assay (data not shown). However, the delay in phospho-ERK1/2 activation in PS cultured on treated SC (see Fig. 9A) led to a decrease in both numbers and percentages of highly MPM-2-

Elutriated PS incubated for 3 h in the presence of 12, 25 or 50 μM U0126, then washed and cultured with SC for 3 h, exhibited lower levels of phospho-ERK1/2 than control PS (Fig. 8A). By contrast, after 24 h of culture, similar levels of phospho-ERK1/2 were observed in control and treated cells. When PS pre-incubated for 3 h in the presence of 12 or 25 μM U0126 were co-cultured for 24 h with SC, the number of SII formed was similar to that of control cultures (Fig. 8C). However, the number of RS was lower in the presence of 25 μM U0126 (Fig. 8D). Preincubation in the presence of a higher concentration of this compound (50 μM) did not change the number of apoptotic mlPS but resulted in a dramatic increase in the number of apoptotic SII, precluding a clear-cut interpretation of these results. Neither the number nor the percentage of highly MPM-2-labeled nor that of anti-phospho-H3-labeled mlPS were significantly modified by U0126 (Table 2). By contrast, pre-incubation of PS with 25 μM U0126 decreased both the number and the percentage of anti-phospho-H3-

Meiotic progression of PS requires activated MAPKs of Sertoli cells and close contacts between germ cells and Sertoli cells

Table 2 Effect of pre-treatment of PS with U0126 or roscovitine on the numbers and percentages of MPM-2 or phospho-H3-labeled middle to late PS (mlPS) and secondary spermatocytes (SII) co-cultured with Sertoli cells Cells

Labeled cells

Treatment DMSO Number (×103) a

mlPS mlPS mlPS SII SII SII a

U0126 %a

Roscovitine

12 μM

25 μM 3 a

Number (×10 )

184 ± 4 100 167 ± 5 79 ± 2 129 ± 7 MPM-2 b 145 ± 7 5.8 ± 0.2 3.1 ± 0.2 5.7 ± 0.4 PH3 c 30 ± 2 100 27 ± 2 86 ± 8 23 ± 3 MPM-2 b 26 ± 2 5.2 ± 0.5 17.3 ± 0.5 4.4 ± 0.7 PH3 c

%

a

3 a

Number (×10 )

100 169 ± 8 77 ± 4 130 ± 13 3.4 ± 0.2 4.5 ± 0.7 100 24 ± 2 85 ± 4 21 ± 2 16.2 ± 0.7 3.3 ± 0.5⁎

Values are the mean ± SEM; n = 6. Highly MPM-2-labeled mlPS or SII populations (Gate G2; see Figs. 4A and B). c Phospho-H3-labeled mlPS or SII (see Fig. 7). d Not determined: “TUNEL-positive cells”. ⁎ p b 0.05 vs. control DMSO. b

50 μM %

a

100 77 ± 4 2.6 ± 0.3 100 87 ± 4 13.7 ± 0.7⁎

12 μM 3 a

a

Number (×103) a % a

Number (×10 )

%

168 ± 10 130 ± 10 4.8 ± 0.4 nd d nd nd

100 168 ± 14 77 ± 5 131 ± 12 2.8 ± 0.2 3.2 ± 0.2⁎ nd 19 ± 1 nd 17 ± 1⁎ nd 2.3 ± 0.2⁎

100 77 ± 3 1.9 ± 0.3⁎ 35 ± 5 89 ± 4 12.1 ± 0.3⁎

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Fig. 9. Effects of pre-treatment of SC with 12 μM U0126 (U12) on the two meiotic divisions. SC were cultured alone for 48 h (0 h of co-culture) with DMSO or U12, washed and co-cultured with PS. (A) Western blot analysis of ERK1, ERK2 and phospho-ERK1/2 in SC and germ cells separated after 3 h or 24 h of co-culture. The last lane (PS alone 24 h) is elutriated PS cultured alone in conditioned medium of SC for 24 h. One hundred micrograms of total protein was loaded per lane. (B–D) Flow cytometry analyses of the numbers of mlPS (B), SII (C) or RS (D) per well after 24 h of co-culture. (E–F) Proportion (%) of TUNEL-labeled mlPS (E) and SII (F) was determined after TUNEL reaction, on slides, of cells sorted by flow cytometry. Results are the mean ± SEM of 3 different experiments. Different letters indicate statistically significant differences (p b 0.05).

labeled (G2) and also phospho-H3-labeled mlPS (Table 3) together with a slight increase in the percentage of apoptotic mlPS (10 ± 2%; n = 4) vs. control (3 ± 1%; n = 4) (Fig. 9E). These results were associated with a decrease in the number of SII and RS (Figs. 9C, D) and a threefold increase in the percentage of apoptotic SII (45 ± 9%; n = 4) vs. control (13 ± 3%; n = 4) (Fig. 9F). Similar results were obtained on day 2 (data not shown). Discussion The present results indicate that meiotic progression requires close contact between spermatocytes and SC and involves MAPK activity. Both ERK1 and ERK2 were detected in meiotic cells from JPS to RS present in co-culture of PS with SC, as in freshly isolated germ cell populations (present results and Inselman and Handel, 2004. Moreover, their activated (phosphorylated) forms increased during meiotic progression, suggesting a role of MAPKs in this process. The presence of U0126 an inhibitor of

185

MEK (a MAPK kinase) decreased the yield of the meiotic division of PS co-cultured with SC. Although phospho-ERK1/2 were observed in germ cells on seminiferous tubule sections, very few amounts of phosphoERK1/2 were detected in freshly prepared PS. But PS cultured on SC exhibited high levels of phospho-ERK1/2 as early as 3 h after seeding. Conversely, high amounts of phospho-ERK1/2 were detected in freshly isolated SC, as observed on tubule sections, but phospho-ERK1/2 of cultured SC exposed to U0126 was no longer detected as soon as 3 h after treatment, indicating a short half-life of phospho-ERK1/2 (present results and Crepieux et al., 2001; Salazar and Hofer, 2007). Taken together, these results should indicate that phospho-ERK1/2 is indeed present in vivo in PS but is lost during germ cell preparation due to loss of their contact with SC. In support of this hypothesis, the nuclear localization of MPM-2 labeling observed in freshly elutriated PS was enhanced in PS cocultured with SC but decreased when phosphorylation of ERK1/2 was prevented. In addition, it must be underlined that whereas the isolation of SC requires about 2 h in our hands, PS isolation and purification requires almost 4 h. It should also be noted that in seminiferous tubule sections phospho-ERK1/2 labeling appeared higher at the periphery of the tubules (which contains SC and PS) than in the inner part of the tubule (which contains SC and spermatids). This fits rather well with the higher levels of phospho-ERK1/2 observed by cytometry in cultured PS than in vitro formed RS. Accordingly, the results described above suggest that the data obtained under our culture conditions are relevant to the physiological process. MAPKs and MPF have complementary actions in meiotic progression Co-culture of PS and SC in the presence of either U0126 or roscovitine, a potent inhibitor of CDK1 whose association with cyclin B1 forms the MPF, decreased the yield of the first and the second meiotic divisions. This result was correlated to a decrease in the numbers and percentages of phospho-H3labeled primary and secondary spermatocytes. Unlike roscovitine, U0126 also decreased the numbers and percentages of highly MPM-2-labeled primary and secondary spermatocytes. Table 3 Effect of the pre-treatment of Sertoli cells with U0126 on the numbers and percentages of MPM-2 or phospho-H3-labeled middle to late PS (mlPS) Cell population

mlPS mlPS mlPS a

Labeled cells

MPM-2 b PH3 c

Treatment 12 μM U0126

DMSO a

Number (×103) a

%

289 ± 26 244 ± 18 4.9 ± 0.4

100 84 ± 4 1.7 ± 0.1

Number (×103) a

%a

281 ± 29 135 ± 10⁎ 2.8 ± 0.3⁎

100 48 ± 4⁎ 1.0 ± 0.1⁎

Values are the mean ± SEM; n = 4. Highly MPM-2-labeled mlPS or SII populations (Gate G2, see Figs. 4A and B). c Phospho-H3-labeled mlPS and SII (see Fig. 7). ⁎ p b 0.05 vs. control DMSO. b

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There is a tight correlation between H3 phosphorylation on Ser 10 with chromosome condensation and segregation during mitosis (Wei et al., 1999) and meiosis (Wei et al., 1998; Prigent and Dimitrov, 2003). Histone H3 becomes phosphorylated at the end of the meiotic prophase in the diplotene stage with peak level at metaphase and is dephosphorylated at telophase (Manzanero et al., 2000). By contrast, MPM-2 labeling is observed throughout the meiotic prophase, with the highest levels in mlPS and in SII. The MPM-2 antibody reacts with over 40 or 50 different proteins (Davis et al., 1983; Renzi et al., 1997). Early works of Tombes et al. (1991) have suggested that the MPM-2 antigens may be substrates of CDK1. However, neither histone H1 nor lamin and cyclin B1 are reactive to MPM-2 after phosphorylation, suggesting that the MPM-2 epitope kinases may be different from CDK1 (Davis et al., 1983; Ottaviano and Gerace, 1985; Kuang et al., 1991). Until now, only few MPM-2 recognized epitopes have been identified as substrates of CDK1 (Westendorf et al., 1994; Ye et al., 1995; Proteau et al., 2005; Bengoechea-Alonso and Ericsson, 2006). Taken together, these results suggest that MAPKs and CDK1 have complementary actions during the meiotic prophase. MAPKs present in Sertoli cells or in germ cells have different effects on meiosis In a previous study, we showed that the effect of roscovitine on the meiotic divisions of spermatocytes was exerted directly on the germ cells (Godet et al., 2004). MAPKs are present in both the SC and the meiotic germ cells; therefore, it could not be decided if the effect of U0126 observed in co-cultures of SC and PS resulted from an inhibition of MAPKs present in the SC, the germ cells or both. Pre-incubation of PS for only 3 h with 25 μM U0126, before seeding on SC, resulted in a lower number of in vitro formed RS without modifying significantly the number of SII. In this case, the percentage of phospho-H3-labeled SII was decreased but not that of highly MPM-2-labeled SII. It must be emphasized that even at 50 μM, U0126 affected neither MPM-2 nor phospho-H3 labeling of primary spermatocytes. These data indicate that, under these experimental conditions, the second meiotic division is deeply dependent on MAPK-induced biochemical reactions which occur in PS. Our results also suggest that the appearance of the epitopes recognized by the MPM-2 antibody is not directly dependent on the activation of MAPKs present in the germ cells but should depend on the activation of MAPKs present in the SC. Conversely, to the results obtained when pre-treating the germ cells (see above), pre-treatment of SC with U0126 resulted in a decrease in both the number and the percentage of highly MPM-2-labeled PS on day 1 of co-culture with SC. In co-cultures of PS with SC, low levels of phospho-ERK1/2 in JPS were correlated with relatively low MPM-2 labeling, whereas higher levels of phospho-ERK1/2 in mlPS were associated with a higher MPM-2 labeling. Similarly, PS treated with OA exhibited high levels of phospho-ERK1/2 (Sette et al., 1999; Inselman and Handel, 2004) and high MPM-2 labeling

(Cobb et al., 1999a). By contrast, PS cultured in Sertoli cells conditioned media for 24 h exhibited very high levels of phospho-ERK1/2 but very few MPM-2 labeling, if any, and were unable to complete meiotic divisions (unpublished results). These data indicates that it is not MAPK activation per se, which is important to allow the meiotic divisions but their ability to induce phosphorylation of the epitopes recognized by the MPM-2 antibody. Taken together, the present results suggest that a star role in the meiotic maturation of rat spermatocytes is devoted to activated MAPKs present in the SC. It must also be underlined that close contacts between the SC and the germ cells are required for this maturation. When PS were cultured in Sertoli cell conditioned media (but in the absence of SC), phosphorylation of the antigens recognized by the MPM-2 antibody decreased dramatically, whereas it increased when PS were cocultured with SC. Likewise, the nuclear localization of the MPM-2 labeling in the PS was induced by the contacts of the germ cells with SC. Many studies have correlated changes in kinase activity with changes in gap junctional communication and SC-germ cells anchoring junctions dynamics (Longin et al., 2001; Roscoe et al., 2001; Cheng and Mruk, 2002; Lee and Cheng, 2005). Now, functional ectoplasmic specialization structures and desmosomes like junction (Lee and Cheng, 2005; Siu et al., 2005) and connexin 43 (present study) are detected between spermatocytes and SC within 24–48 h of co-culture. Moreover, a very recent study (Winterhager et al., 2007) has shown that replacement of connexin 43 by connexin 26 in transgenic mice impairs spermatogenesis leading to the absence of germ cells beyond spermatocytes type I. Taken together, such data reinforce the view that the co-culture system used in the present work enables to highlight mechanisms pertinent to the physiological processes. Identification at the molecular level of the pathway leading to MAPK activation in male germ cells should help to decipher the mechanisms regulating the onset of the meiotic divisions. Acknowledgments We are grateful to Michèle Vigier for help with elutriation of germ cells; Denis Resnikov for confocal microscopy analysis performed at the Centre Commun de Quantimétrie, Université Claude-Bernard Lyon I, the Centre Commun de Microscopie, Université Renée Descartes Paris and Noëlline Coudouel for testis sections. We thank Séverine Mazaud for advices and critical reading of the manuscript and Dominic Fullman, Belinda Smale, Linda Durand and Paula Milne for English revision. This work was supported by the Institut National de la Recherche Agronomique, INRA and the Institut National de la Santé et de la Recherche Médicale, INSERM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2007.12.019.

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