Environmental influence on testicular MAP kinase (ERK1) activity in the frog Rana esculenta

The amphibians are the first tetrapods and therefore occupy a phylogenetic position of great interest. As cold-blooded vertebrates, amphibians are greatly susceptible to environmental fluctuations. In fact, most of the anurans and urodeles exhibit a markedly seasonal testicular cycle (circannual)(Lofts, 1974; Rastogi, 1976; Rastogi et al., 1978, 1983). Among environmental factors involved in the amphibian breeding cycle the most relevant are the photoperiod and the temperature. Studies on the influence of light demonstrated that photoperiod may potentially modify testicular activity in the frog Rana esculenta (Rastogi et al., 1976); in particular, it has been suggested that the light has only a permissive role in facilitating the temperature response that directly influences the annual testicular cycle in R. esculenta (Di Matteo et al.,1981). Temperature, on the other hand, has been shown to play an important role in synchronizing the different phases of the seasonal testicular cycle in several amphibian species(Rastogi et al., 1978).

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, viatranslocation 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-β 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.

Adult male frogs Rana esculenta L. were captured in November and March (N=30/month) in the vicinity of Naples. Frogs under various treatments were maintained in plastic tanks and fed meal worms twice a week ad libitum. For each experiment animals were injected with 50 mg 100 ml^–1 of colchicine 24 h before evaluating mitotic index. Animals were killed by decapitation under anaesthesia with MS222 (0.05% in aqueous solution, Sigma Chemical Co., St Louis, MO, USA), and the testes were removed immediately and stored at –80°C (N=30 testes/month)until processed for western blot analysis, or quickly prepared for histological examinations (N=30 testes/month). For light microscopy,testes were fixed in Bouin's fluid and embedded in paraffin by standard procedures. 5 μm sections were stained with Hematoxylin and Eosin.

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.

Frozen frog testes (10 testes/month/experiment) were homogenised directly into lysis buffer containing 50 mmol l^–1 Hepes, 150 mmol l^–1 NaCl, 1 mmol l^–1 EDTA, 1 mmol l^–1 EGTA, 10% glycerol, 1% Triton-X-100 (1:2 w/v), 1 mmol l^–1 phenylmethylsulfonyl fluoride (PMSF) 1 mg aprotinin, 0.5 mmol l^–1 sodium orthovanadate, 20 mmol l^–1sodium 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).

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).

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 l^–1 Tris, pH 7.4; 200 mmol l^–1 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).

The primary spermatogonial mitotic index was expressed as the number of metaphases per total primary spermatogonia counted ×100 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.

In November (8°C; 8 h:16 h light:dark), the testis showed all stages of spermatogenesis, but the spermatogonial mitotic index was rather low and there were several cysts of degenerating primary and secondary spermatocytes (I and II SPC) and spermatids (SPT).

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 G₁–S 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).

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).

Male R. esculenta appear potentially to be continuous breeders,with annual testicular cycles depending upon environmental cues. It is well known that temperature is a primary factor in the regulation of the quiescent phase of the testicular cycle, whereas for stimulating and maintaining active spermatogenesis, both a relatively high temperature and long photoperiod are important (Rastogi et al.,1978); in particular, it has been demonstrated that the light only has a permissive role, facilitating the temperature response in the regulation of spermatogenesis of R. esculenta(Di Matteo et al., 1981).

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-β 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 hypotalamo–hypophyseal 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.

This work was supported by Grant of II Università di Napoli (ex 60%). We thank Fabrizio Fiorbianco(www.studiociotola.it)for skilful technical assistance with the artwork.