Environmental influence on testicular MAP kinase (ERK1) activity in the frog Rana esculenta
Dipartimento di Medicina Sperimentale, II Università di Napoli, Naples, Italy
* Author for correspondence (e-mail: Paolo.Chieffi{at}unina2.it)
Accepted 24 March 2004
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Summary |
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Western blot analysis shows that under controlled experimental conditions an increase of temperature and photoperiod in November, characterized by a decrease in primary spermatogonial mitosis, induces ERK1 activity and spermatogonial proliferation, as confirmed using the proliferating cellular nuclear antigen (PCNA) as an early molecular marker. In contrast, a decrease in temperature and photoperiod in March, with an increase of primary spermatogonial mitosis, impairs ERK1 activity and spermatogonial proliferation.
In conclusion, our data clearly show for the first time in a non-mammalian vertebrate that the temperature and the photoperiod exert a role in the spermatogonial proliferation via ERK1 activity.
Key words: ERK1, spermatogenesis, photoperiod, proliferation, frog, Rana esculenta
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Introduction |
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Signal transduction mechanisms promoting mitosis of spermatogonia are poorly understood in vertebrate and invertebrate animals. Although little else is known of the early physiological consequences of this environmental cue on whole animals, downstream molecular pathways are demonstrably activated and are locally involved in initiating spermatogonial stem cell mitosis.
At least two signaling pathways are active; one is steroid based, the other
is mitogen-activated protein kinase (MAPK) based. Recent evidence also
suggests that these otherwise distinct pathways may interact in promoting
mitosis of spermatogonial stem cells (Chieffi et al.,
2000b,
2002
).
MAPKs play a crucial role in signal transduction, mainly by activating gene
transcription, including c-Myc, Ets1, Elk1 and c-Jun, via
translocation into the nucleus (Karin,
1995; Waskieewicz and Cooper,
1995
; Cobb, 1999
).
Among the members of this family the most extensively studied are p44 and p42
MAPK, also known as extracellular signal-regulated kinases (respectively ERK-1
and ERK-2), Jun amino-terminal kinase (JNK1/2) and p38. ERKs are expressed
ubiquitously and closely related to the yeast protein kinases involved in
pheromone induced mating (Nielsen et al., 1993). For example, in the frog
R. esculenta and in the lizard Podarcis s. sicula testis
different levels of ERK1/2 activity are present during the annual reproductive
cycle (Chieffi et al., 2001
,
2002
); in addition, it has been
shown that 17-ß estradiol induces ERKs phoshorylation in frog (R.
esculenta) (Chieffi et al.,
2000b
) and lizard (P. s. sicula) testis
(Chieffi et al., 2002
), and
JNK1 has different activity in frog (R. esculenta)
(Chieffi, 2003
) and lizard
(P. s. sicula) testis during the annual reproductive cycle
(Chieffi et al., 1999
).
The present work on R. esculenta (Amphibia, Anura) was undertaken to evaluate the influence of light and temperature, in different phases of the testicular cycle, on MAPK (ERK1) testicular activity. In this paper we present evidence that ERK1 testicular activity is also under temperature and photoperiodic control.
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Marerials and methods |
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Photo-thermal treatments
Frogs contained in plastic tanks were housed in photo-thermostatic chambers
where varying combinations of photoperiods and temperatures were controlled
with high precision (±5 min light; ±1°C)
(Rastogi et al., 1978).
Experiment A: in November 30 frogs were housed for 2 weeks under a 12 h:12 h (light:dark) photoperiod and a temperature of 22°C. Experiment B: in March 30 frogs were housed for 2 weeks under a 8 h:16 h (light:dark) photoperiod and a temperature of 4°C.
Protein extract preparations
Frozen frog testes (10 testes/month/experiment) were homogenised directly
into lysis buffer containing 50 mmol l1 Hepes, 150 mmol
l1 NaCl, 1 mmol l1 EDTA, 1 mmol
l1 EGTA, 10% glycerol, 1% Triton-X-100 (1:2 w/v), 1 mmol
l1 phenylmethylsulfonyl fluoride (PMSF) 1 mg aprotinin, 0.5
mmol l1 sodium orthovanadate, 20 mmol l1
sodium pyrophosphate, (Sigma), and clarified by centrifugation at 14 000
g 10 min. Protein concentrations were estimated using a
modified Bradford assay (Bio-Rad, Melville, NY, USA).
Antibody
The antibodies were purchased from the following sources: (1) polyclonal
anti-phospho-p44 MAP kinase (Thr202/Tyr204) antibody (#9101S, New England
Biolab, MA, USA), raised in rabbit, (2) polyclonal anti-ERK1 (#sc-94-G, Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA) raised in rabbit against epitope
corresponding to an amino acid sequence conserved in frog, chicken, murine and
human, (3) mouse monoclonal antibody against recombinant Proliferating
Cellular Nuclear Antigen (PCNA, Dako Corporation, Denmark).
Western blot analysis
25 or 40 mg of total protein extracts were boiled in Laemmli buffer for 5
min before electrophoresis. The samples were subjected to SDS-PAGE (10%
polyacrylamide) under reducing conditions. After electrophoresis, proteins
were transferred to nitrocellulose membrane (Immobilon Millipore Corporation,
Bedford, MA, USA); complete transfer was assessed using prestained protein
standards (Bio-Rad). The membranes were treated for 2 h with blocking solution
(5% no fat powdered milk in 25 mmol l1 Tris, pH 7.4; 200
mmol l1 NaCl; 0.5% Triton X-100, TBS/T), and then the
membranes were incubated for 1 h at room temperature with the primary
antibody, (1) against phospho-ERK1 (diluted 1:2000), (2) against ERK1 (diluted
1:2000), (3) against PCNA (diluted 1:1000). After washing with TBS/T and TBS,
membranes were incubated with the horseradish peroxidase-conjugated secondary
antibody (1:5000) for 45 min (at room temperature) and the reactions were
detected using the enhanced chemiluminescence (ECL) system (Amersham Life
Science, UK).
Statistical analysis
The primary spermatogonial mitotic index was expressed as the number of
metaphases per total primary spermatogonia counted x100 in three
randomly chosen sections/animal. Values are expressed as means ± s.d.
Significance of differences was evaluated using one-way analysis of variance
(ANOVA) followed by Duncan's test for multigroup comparisons.
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Results |
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Frogs were housed for 2 weeks under a 12 h (12 h:12 h light:dark)
photoperiod at 22°C. In order to assess spermatogonial proliferation we
analyzed activation of the ERK1 isoform; in fact, a recent paper reports that
ERK1 protein is present in the SPG of the frog R. esculenta and its
activation is necessary for spermatogonial proliferation
(Chieffi et al., 2000b). These
conditions induced an increase of ERK1 phosphorylation status (Thr202/Tyr204)
after 1 and 2 weeks with respect to the control
(Fig. 1). The spermatogonial
mitotic index (SPG-MI) was consistent with the increased ERK1 activity. An
increase of SPG-MI after 1 and 2 weeks with respect to the control was
observed (Fig. 2A). In
addition, the increase of primary SPG proliferation was confirmed using PCNA
as an early molecular marker (Chieffi et
al., 2000a
); in fact, this nuclear protein is expressed in
G1S phases of SPG. Western blot analysis showed an increase
in the amount of PCNA after 1 and 2 weeks with respect to the control
(Fig. 2B,C).
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Experiment B
The initial controls captured in March at a temperature of ca. 18°C (12
h:12 h light:dark photoperiod) showed very active spermatogenesis. Treatment
at 4°C (8 h:16 h light:dark) induced a decrease of ERK1 phosphorylation
status (Thr202/Tyr204) after 1 and 2 weeks with respect to the control
(Fig. 3). The SPG-MI was
consistent with the decreased P-ERK1 activity, and decreased after 1 and 2
weeks with respect to the control (Fig.
4A). I SPG proliferation was monitored by western blot analysis,
which showed a decrease in the amount of PCNA after 1 and 2 weeks with respect
to the control (Fig. 4B,C).
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Discussion |
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Using the male green frog R. esculenta spermatogenic system, we experimentally manipulated spermatogenesis using environmental cues. The objective of this study was to analyse MAPK (ERK1) spermatogonial activity in the regulation of proliferation induced by temperature and photoperiod. In experiment A, our data clearly show that an increase of photoperiod and temperature induces a ISPG proliferation through ERK1 activation. In addition, experiment B shows that a decrease of temperature and photoperiod in spring caused an intense spermatogonial proliferation, with a downturn in ERK1 activation and, consequently, mitotic spermatogonial division.
In testicular germ cells ERKs are predominantly confined to the nuclei,
where they might also regulate gene transcription and substrate
phosphorylation which, in turn, regulate cell proliferation (Chieffi et al.,
2000, 2002). Since changes in
the state of activation of ERKs also correlate with spermatogenetic activity,
it is likely that these enzymes play a key role in the regulation of the
testicular epithelium proliferation
(Chieffi et al., 2000b
).
Recently, it has also been reported that the mitogenic c-Src/p21ras/MAP kinase
signal transducting pathway, which is known to be stimulated by different
growth factors in different systems, is activated by 17-ß estradiol
(Migliaccio et al., 1996
;
Watters et al., 1997
); for
example, it has been demonstrated that estrogens induce spermatogonial
proliferation by activation of the MAP kinase cascade which, in turn, promotes
transcription of the immediate early genes (Chieffi et al., 2000,
2002
).
In R. esculenta, not only the pineal complex but also the eyes
intervene in the normal testicular activity
(Rastogi, 1976). In fact, the
light influences the hypotalamohypophyseal system, with the mediation
of pineal gland/eyes (Rastogi,
1976
; d'Istria et al.,
2003
), in the secretion of gonadotropins that regulate the
synthesis of mitogen factors (i.e. growth factors, estrogens) by Sertoli and
Leydig cells that induce and regulate spermatogonial proliferation
(Rastogi, 1976
). In this
scenario MAPK (ERK1) seems to be a primary factor in the phosphorylation of
different nuclear transcriptional factors necessary for the spermatogonial
proliferation (Chieffi et al., 2000,
2002
). It is important to note
that our data clearly demonstrate that ERK1 activation of spermatogonial
proliferation in the frog R. esculenta is also under temperature and
photoperiodic control. It is well documented that temperature plays an
important role in the control of spermatogenesis
(Rastogi, 1976
); in fact,
under natural conditions temperatures of up to 25°C and photoperiod up to
ca. 16 h of light daily are favorable for active winter spermatogenesis
(Di Matteo et al., 1981
).
Experiments performed in autumn and winter revealed the importance of high
temperature and normal photoperiod in the stimulation and maintenance of
normal spermatogenesis (Rastogi et al.,
1978
).
In conclusion, our data demonstrate for the first time in a non-mammmalian vertebrate that exogenous factors such as temperature and photoperiod exert a role in the induction of spermatogonial proliferation through increased ERK1 activity.
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Acknowledgments |
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References |
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