Available online at www.sciencedirect.com
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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M. Godet et al. / Developmental Biology 315 (2008) 173–188
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).
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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
175
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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M. Godet et al. / Developmental Biology 315 (2008) 173–188
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.
M. Godet et al. / Developmental Biology 315 (2008) 173–188
177
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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M. Godet et al. / Developmental Biology 315 (2008) 173–188
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.
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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M. Godet et al. / Developmental Biology 315 (2008) 173–188
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.
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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
181
(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.
182
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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.
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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).
183
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).
184
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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
50 μM
12 μM
Number (×103) a % a
Number (×103) a % a
Number (×103) a % a
Number (×103) a % a
184 ± 4
100
167 ± 5
MPM-2 b 145 ± 7
79 ± 2
129 ± 7
PH3 c
5.8 ± 0.2 3.1 ± 0.2 5.7 ± 0.4
30 ± 2
100
27 ± 2
86 ± 8
23 ± 3
MPM-2 b 26 ± 2
PH3 c
5.2 ± 0.5 17.3 ± 0.5 4.4 ± 0.7
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
100
77 ± 4
2.6 ± 0.3
100
87 ± 4
13.7 ± 0.7⁎
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⁎
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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).
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.
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).
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)
Discussion
Cell
population
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
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
186
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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.
M. Godet et al. / Developmental Biology 315 (2008) 173–188
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