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BOR - Papers in Press, published online ahead of print February 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026666
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BIOLOGY OF REPRODUCTION 70, 1782–1789 (2004)
DOI: 10.1095/biolreprod.103.026666
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Testicular Activity of Mos in the Frog, Rana esculenta: A New Role in Spermatogonial Proliferation1

Diana Ferrara3, Carmela Palmiero3, Margherita Branno4, Riccardo Pierantoni2,3, and Sergio Minucci3

Dipartimento di Medicina Sperimentale-sezione "F. Bottazzi,"3 Seconda Università degli Studi di Napoli, 80138 Napoli, Italy Laboratori di Biochimica e Biologia Molecolare,4 Stazione Zoologica "A. Dohrn"-Villa Comunale 121, 80132 Napoli, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.

spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proto-oncogenes are highly conserved cellular genes that play important roles during development and differentiation. They can be activated by quantitative or qualitative changes to oncogenes, which cause or contribute to the transformation of cells into a tumorigenic state [1].

The c-mos proto-oncogene is the cellular homologue of the Moloney murine sarcoma virus oncogene [2, 3]. It contains a long open reading frame lacking introns [4], and it is mainly expressed in ovaries, testes, and near-term embryos [5, 6]. The c-mos transcripts were also found in undifferentiated teratocarcinoma cell lines [7] and, at very low levels, in somatic tissues such as brain, kidneys, placenta, and mammary glands [5].

The product of the c-mos proto-oncogene is a serine- threonine kinase (Mos), the role of which has been mainly characterized in female gametogenesis. In Xenopus laevis immature oocytes, progesterone stimulation is followed by complex changes in the 3'-untranslated region of maternal c-mos RNA, leading to its polyadenylation and translation [8]. Mos, functioning as a MAPK kinase kinase, activates the MEK/MAPK/p90Rsk pathway that enhances maturation- promoting factor (MPF) activation, with subsequent entry into meiosis I inducing germinal vesicle breakdown [9]. In other vertebrate species, as well as in invertebrate ones, activation of the Mos/MAPK pathway does not have a critical role in initiating oocyte maturation [10–12]. Furthermore, it has been recently shown that the absence of c-mos in mice [13] and the inhibition of Mos synthesis by morpholino antisense oligonucleotides in Xenopus do not prevent germinal vesicle breakdown [14]. Therefore, it has been suggested that the unique conserved role of Mos is as a component of cytostatic factor, an activity that causes meiotic metaphase II arrest and that prevents parthenogenic activation of the female gamete until fertilization. The increase of intracellular Ca2+ that follows fertilization causes the drastic decrease of MPF activity; this event presumably results in the dephosphorylation of Mos Ser-3 residue [15] and in the ubiquitin-mediated degradation of the protein. Concomitantly, deadenylation of 3'-untranslated region occurs, and translation from maternal mos mRNA is inhibited [16]. As a consequence, the oocyte progresses through metaphase II arrest.

In opposition to the bulk of information concerning the role of Mos in female gametogenesis, to our knowledge the function of this protein during spermatogenesis remains to be clarified. In mouse, the mainly germinal cells expressing c-mos transcript are the haploid, postmeiotic round SPT [6], but both the transcript and the protein are also present in mouse as well as in rat pachytene spermatocytes (SPC). On the other hand, in contrast to the hypothesis that Mos may play a significant role during male meiosis, c-mos–/– mice lack of a detectable phenotype [17] and are not affected in sperm production and fertilizing ability [18]. Therefore, whether Mos is required during male germ cell progression is not known [19].

In this respect, nonmammalian vertebrates may represent a useful model to give insight regarding the testicular physiology because of the presence of many seasonal breeders showing a slow progression of germ cell stages throughout the year. Among elasmobranchs, the presence of a 1.7-kilobase c-mos mRNA as well as 106- and 32-kDa related products have been described in the testis of the dogfish Scyliorhinus canicula [20, 21]. Surprisingly, the proteins were not expressed in germ cells but were specifically localized in myeloid cells of the testicular interstitial compartment, probably coming from the epigonal organ [21]. Concomitantly, Mos has also been detected for the first time in another nonmammalian vertebrate, the anuran amphibian Rana esculenta, primarily in the cytoplasm of premeiotic germ cells [22]. All these data seem to suggest a possible nonmeiotic role of Mos during spermatogenesis, putting in evidence the need for further investigations.

In the present study, we investigate the presence of c- mos, both at the transcriptional and the protein level, in the testis of R. esculenta. Because the frog is a seasonal breeder, we studied the expression of this proto-oncogene during the reproductive annual cycle. In addition, the primary protein localization in the cytoplasm of spermatogonia (SPG) led us to hypothesize that Mos may also play a role in mitosis. For this reason, we have utilized ethane-dimethane sulphonate (EDS), an alkylating agent that has a cytotoxic effect on the interstitial Leydig cells (LC) of rats and R. esculenta [23, 24]. Interestingly, in R. esculenta, spermatogonial mitosis is subsequently arrested, resuming immediately after the differentiation of new LC [24, 25]. Therefore, we have used the above-quoted properties to investigate a putative role for Mos in the progression of spermatogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult males frogs (R. esculenta, n = 10) were collected monthly in the surroundings of Naples by a local dealer. Frogs were maintained in plastic tanks (23 x 16 x 11 cm) with food (mealworms) and water available ad libitum. The animals were killed by decapitation under anesthesia (MS-222; Sigma, St. Louis, MO), and the testes were dissected.

Left testes were used for RNA extraction; right testes were cut into halves, one of which was used for protein extraction and one of which was used for immunocytochemical analysis. Testes collected for molecular extractions were quickly frozen by immersion in liquid nitrogen and stored at –80°C. Testes collected for immunocytochemical analysis were fixed in Bouin fluid and processed; paraffin sections (thickness, 5 µm) were stained with hematoxylin-eosin for monthly check of the testicular activity.

This project was approved by the Italian Ministry of Education and Research (MIUR).

EDS Treatment

The animals (n = 30) were divided in two experimental groups as follows: 15 animals that received a single injection of dimethyl sulfoxide (DMSO-dH2O, 1:3 [v/v], 100 µl/animal) at Day 0 and 15 animals that received a single injection of EDS (100 mg/kg in DMSO-dH2O, 1:3 [v/ v], 100 µl/animal) at Day 0. Five additional animals were used as an initial control at the beginning of the treatment.

Animals were killed in groups of five at 4, 8, and 28 days after the EDS and DMSO injections, and the testes were dissected and stored as described above.

Preparation of Total RNA, Reverse Transcription- Polymerase Chain Reaction, Cloning, and Sequencing

Total RNA from R. esculenta testis was prepared with a modification of the procedure described by Chomczynski and Sacchi [26].

First-strand cDNA was synthesized using 5 µg of total RNA extracted from R. esculenta testes, 40 ng/µl of random exameric primer (Promega Corp., Madison, WI), and 200 U of Superscript II reverse transcription (RT) enzyme (Invitrogen, Paisley, U.K.) in a total volume of 20 µl according to the manufacturer's instructions (Invitrogen). Three microliters of this cDNA template were than used for the polymerase chain reaction (PCR; volume, 25 µl) with 1.5 mM MgCl2, 1x PCR buffer (10 mM Tris- HCl [pH 9.0], 50 mM KCl, and 1.5% Triton X-100), 0.2 mM dNTP, 7% DMSO, 0.3 U of Taq DNA polymerase (Promega). Five-picomole oligonucleotide primers, designed on the homologous c-mos sequence of R. japonica (forward primer, 5'-agcagtcctctggagctgag-3'; reverse primer, 5'- caaggtgacagcgaaggaat-3'), were used for the amplification. An appropriate region of frog ribosomal protein fP1 cDNA European Molecular Biology Laboratory [EMBL] data bank accession no AJ298875), amplified with specific oligonucleotide primers (forward primer, 5'-tacgagcgtccatcacacac- 3'; reverse primer, 5'-agaccaaagcccatgtcatc-3'), was used as control. The expected RT-PCR product sizes were 708 base pairs (bp) for R. japonica c-mos and 356 bp for fP1. Amplifications, carried out for 43 cycles, were as follows: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Amplification products were electrophoresed on 1.5% agarose gel in 1x Tris- acetate-EDTA (TAE) buffer.

The putative c-mos amplification product was purified by Qiagen gel extraction kit (Qiagen, Hilden, Germany) and cloned into the TOPO-TA vector according to the manufacturer's instructions (Invitrogen). The insert was sequenced on both strands using [35S]ATP (1000 Ci/mmol; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) by the dideoxy chain termination method [27]. The deduced protein sequence was compared with the EMBL gene bank database, and protein sequence alignments were generated using the CLUSTAL W Multiple Sequence Alignment Program (http://workbench.sdsc.edu/).

Oligonucleotide and cDNA Probes

A specific R. esculenta c-mos inner oligonucleotide (reverse primer, 5'-cagaagctctgccgggacgcc-3') was terminally labeled with [{gamma}-32P]ATP (3000 Ci/mmol; Amersham).

Ten micrograms of plasmid fP1 cDNA were digested with cloning restriction enzyme (EcoRI and XhoI), and the digestion product containing an insert of 517 bp was labeled with [{alpha}-32P]dATP and [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham) using the random priming labeling kit (Amersham).

Southern Blot Analysis

The R. esculenta c-mos and fP1 amplification products, obtained by RT-PCR as described above from total RNA extracted monthly during the annual cycle, were electrophoresed on 1.5% agarose gel in 1x TAE buffer and transferred to Hybond-N+ filters (Amersham) by overnight capillary blotting. The filter containing R. esculenta c-mos amplification products was incubated in prehybridization solution (5x SSC [1x SSC: 0.15 M sodium chloride and 0.015 sodium citrate], 5x Denhardt, 100 µg/ml of total RNA, 1% SDS, and 0.05% sodium pyrophosphate) for 1 h at 42°C and hybridized with radiolabeled R. esculenta c-mos reverse primer (2 x 106 cpm/ml) at 42°C overnight. The filter containing fP1 amplification products was incubated in prehybridization solution (5x SSC, 5x Denhardt, 100 µg/ml of sonicated salmon sperm DNA, 5 mM EDTA [pH 8.0], 0.5% SDS, and 50 mM sodium phosphate [pH 7.0]) for 1 h at 65°C and hybridized with radiolabeled fP1 cDNA (2 x 106 cpm/ml) at 65°C overnight. The filters were washed twice for 30 min at 42°C and 65°C, respectively, in 0.2x SSC and 0.1% SDS and then finally exposed to x-ray film (HR-H; Fuji Photo Film Co., Ltd., Tokyo, Japan).

Preparation of Total Protein Extracts

Total extracts were prepared by a modification of the method described by Xavier et al. [28]. Briefly, five half-testes were gently homogenized in 1:2 (w/v) lysis buffer (10 mM Hepes [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.42 mM NaCl, 12% glycerol, 0.1 mM EGTA [pH 8.1], 0.5 mM dithiothreitol, and 2 mM spermidine) in the presence of protease inhibitors (4 µg/ml each of leupeptin, aprotinin, pepstatin A, chymostatin, and PMSF and 5 µg/ml of N-p-Tosyl-L-phenylalanine chloromethyl ketone [TPCK]). The homogenate was forced through 22- and 26-gauge needles and mixed for 30 min at 4°C; after centrifugation for 30 min at 10 000 x g, and the supernatant was frozen at –80°C. Protein concentrations of total extracts were estimated using the method of Lowry et al. [29].

Western Blot Analysis

Proteins (25 µg/lane) were separated by 10% SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose (Amersham) for 2.5 h at 280 mA at 4°C. The filter was treated for 4 h with blocking solution (5% nonfat dried milk, 0.25% Tween 20 in Tris-buffered saline [TBS; pH 7.6]) to prevent nonspecific absorption and then incubated overnight at 4°C with anti-rabbit anti-Mos antibody (0.2 mg/ml; StressGen Biotechnologies Corp., Victoria, BC, Canada) diluted 1:400 in 4% nonfat dried milk in PBS (pH 7.5). The filter was washed three times in TBS containing 0.1% Tween 20 for 10 min and once in TBS for 10 min. It was then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig) G (DAKO, Copenhagen, Denmark) diluted 1:1000 in TBS containing 1% normal swine serum. The filter was washed as described above, and the immunocomplexes were detected using the enhanced chemiluminescence-Western blotting detection system (Amersham). The filter was stripped at 60°C for 30 min in a stripping solution (100 mM 2-ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl [pH 7.6]) and, as a control of protein loading, reprobed with anti-rabbit antiactin (0.2 mg/ml; Sigma) diluted 1:500. To check the specificity of the immunoreaction, the controls were treated with the primary antibody preabsorbed with an excess amount of the cognate peptide (10–6 M) or by omitting the primary antibody in the incubation reaction.

Immunocytochemical Analysis

Sections (thickness, 5 µm; n = 3 per animal) were dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubation in 3% H2O2 in methanol for 20 min. After a wash in water, slides were transferred into PBS (0.01 M, pH 7.2) and blocked for 30 min in normal swine serum (DAKO) diluted 1:30 in PBS. Incubations were performed at 4°C in a moist chamber for 16 h with the primary antisera. The primary antibody (anti-rabbit anti-c-mos polyclonal; StressGen) was applied at a dilution of 1:50 in PBS containing 1% v/v of normal swine serum. Sections were washed and incubated for 1 h at room temperature with a secondary antibody (anti-Ig raised in rabbit; DAKO) diluted 1:50 in the same buffer. Finally, the sections were incubated for 1 h with rabbit peroxidase anti-peroxidase (PAP) complex (DAKO). Immunoreaction products were visualized using 3,3'-diamonobenzidine tetrahydrochloride (Sigma) and 30% H2O2 in TBS (0.05 M, pH 7.6). To check the specificity of the immunoreaction, the controls were treated with the primary antibody preabsorbed with an excess amount of the cognate peptide (10–6 M) or by omitting the primary antibody in the incubation reaction.

Presentation of Data and Statistics

Data are expressed as the mean ± SD. The relative amount of c-mos/ fP1 cpm was obtained using an Instant Imager (Packard Instrument Company, Meriden, CT). The relative amount of Mos/actin densitometric analysis was obtained using a Gel Doc 1000 Instrument (Biorad Laboratories, Milan, Italy). One-way ANOVA followed by the Duncan test for multigroup comparison assessed the significance of differences at P < 0.05 and P < 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of c-mos cDNA from the Testis of R. esculenta

Identification of c-mos transcript in the testis of R. esculenta was investigated using two specific primers raised against the homologous c-mos sequence of Rana japonica. Subsequently, RT-PCR analysis on total RNA extracted from R. esculenta testes was carried out.

The amplification product was a single band of approximately 700 bp, which was subsequently cloned and sequenced. The insert was an uncompleted cDNA fragment of 696 bp (EMBL data bank accession no. AJ566900) lacking both start and stop codons (data not shown). The 232- amino acid sequence encoded from the open reading frame was compared with all nonredundant GenBank entries and showed a high similarity to Mos from different species. In particular, the sequence homology ranged from 90% as compared with R. japonica to 82% as compared with and X. laevis to 55% as compared with Rattus norvegicus (Fig. 1).



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  		FIG. 1. Multiple alignment of the Mos deduced amino acid uncompleted sequence of Rana esculenta with Homo sapiens, Sus scrofa, Rattus norvegicus, Gallus gallus, R. japonica, and Xenopos laevis homologues. The single, fully conserved residues are indicated by a star, conservation of strong groups by a colon, and conservation of weak groups by a full stop. Numbers indicate the percentage of identity

Temporal Expression of c-mos mRNA in the Testis of Rana esculenta

The RT-PCR and Southern blot analyses were performed on total RNA from testes collected monthly during the annual cycle.

Using the previously described primers, we amplified the expected 696-bp band in all the samples; the hybridization of the correspondent blot with a radiolabeled inner oligonucleotide, specific for R. esculenta c-mos cDNA, confirmed the presence of a single amplification product (Fig. 2A). We also performed control PCR using specific primers for the frog ribosomal protein fP1, and we obtained the expected 356-bp band, also evidenced by the hybridization of the correspondent blot with the labeled fP1 cDNA (Fig. 2B). Figure 2C shows the relative amounts (expressed as cpm) of R. esculenta c-mos cDNA versus fP1 cDNA: R. esculenta c-mos was always present at a low level in the testis during the annual sexual cycle and showed a single significant peak of expression in March (P < 0.01).



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  		FIG. 2. c-Mos mRNA expression in the testis of Rana esculenta during the annual sexual cycle from October 2000 to July 2001. A) Southern blot analysis of RT-PCR products using c-mos-specific primers and a labeled inner oligonucleotide as probe. B) Southern blot analysis of amplification products using fP1 specific primers and the labeled total fP1 cDNA as probe. The results in A and B are representative of one of three assays. C) Pattern of the relative amount of R. esculenta c-mos/fP1 cpm in the testis during the annual sexual cycle. a vs. b, P < 0.05; a vs. c, P < 0.01; b vs. c, not significant

Temporal and Spatial Expression of Mos in the Testis of R. esculenta

The temporal expression of Mos in the frog testis was studied by Western blot analysis of total protein extracted monthly during the annual sexual cycle.

Using a polyclonal anti-Mos antibody, we identified R. esculenta Mos as a 60-kDa protein (Fig. 3A); as a control, the same blot was also analyzed for the presence of actin, which was detected as a 42-kDa band (Fig. 3B). Figure 3C shows the relative densitometric analysis of R. esculenta Mos versus actin: R. esculenta Mos was always present at a low level in the testis during the annual sexual cycle and showed a single peak of expression in May (P < 0.01).



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  		FIG. 3. Mos protein expression in the testis of R. esculenta during the annual sexual cycle from October 2000 to July 2001. A) Western blot analysis of total protein extracts incubated with an anti-Mos antibody. B) The filter was reincubated with an antiactin antibody. The results in A and B are representative of one of three assays C) Densitometric analysis of Rana esculenta Mos:actin ratio in the frog testis during the annual sexual cycle. a vs. b, P < 0.01. D and E) Mos immunocytochemistry on the testis of a December frog. F and G) Mos immunocytochemistry on the testis of a May frog. H) Control section obtained by the omission of the primary antibody in the incubation reaction of the testis of a May frog. Positive staining is indicated by arrows. I, Interstitium; I SPG, Primary spermatogonia; I and II SPC, primary and secondary spermatocytes, respectively. Bar equals; 5 µm (D and F) and 22 µm (E, G, and H)

The spatial expression of Mos was also immunocytochemically studied during the annual sexual cycle. Mos immunoreactivity was localized throughout the year in SPC. Surprisingly, a very clear immunoreactivity was also detected in the cytoplasm of primary and secondary SPG. In particular, we report Mos localization in the testis of a December frog (Fig. 3, D [arrows] and E), when Mos is expressed at a low level, and in the testis of a May frog (Fig. 3, F [arrows] and G), when Mos expression reaches the maximal levels. A control of the immunoreaction, obtained by omitting the primary antibody in the incubation reaction of testes collected in May, is reported in Figure 3H.

Expression of c-mos in EDS-Treated Frogs

The expression of R. esculenta c-mos proto-oncogene was studied in frog testis in which spermatogenesis was temporary arrested by a single EDS injection [24, 25]. In particular, we analyzed its variation in testes of DMSO control and EDS-treated animals after 4, 8, and 28 days from the single injection.

In the testis of DMSO control animals, Mos was detected at a low level in the cytoplasm of primary and secondary SPG and SPC (Fig. 4A). On Day 4 from the EDS injection, when the majority of LC disappeared from the interstitial tissue, germ cells in the adjacent tubular compartments showed signs of degeneration, and primary SPG were sometimes the only cell type still distinguishable. In these testicular sections, a strong Mos signal was mainly detected in the cytoplasm of primary and secondary SPG (Fig. 4B). From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. No differences were detected between DMSO control and EDS-treated animals on Day 28 (data not shown).



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  		FIG. 4. c-Mos mRNA and Mos protein expression in the testis of Rana esculenta after EDS treatment. A) Mos immunocytochemical localization in the testis on Day 4 after a single DMSO injection. B) Mos immunocytochemical localization in the testis on Day 4 after a single EDS injection. C and D) Western blot analysis of total protein extracts from the testis of DMSO- and EDS-injected animals, respectively, incubated with an anti-Mos antibody. E and F) Agarose gel electrophoresis of RT-PCR products from the testis of DMSO-injected animals using R. esculenta c-mos- and fP1-specific primers, respectively. G and H) Agarose gel electrophoresis of RT-PCR products from the testis of EDS-injected animals using R. esculenta c-mos- and fP1-specific primers, respectively. C, Control PCR; I SPG, primary spermatogonia; II SPC, secondary spermatocytes; M, molecular weight marker VI (Roche Diagnostics, Mannheim, Germany). Bar = 20 µm (A) and 50 µm (B)

The immunocytochemical observations were confirmed by Western blot analysis. Indeed, the 60-kDa Mos signal was constantly detectable at low levels in the testis of DMSO control frogs (Fig. 4C). On the contrary, in EDS- injected animals, it strongly increased on Day 4, then decreased to control levels between Days 8 and 28 from the injection (Fig. 4D). As a control, the blots were analyzed for the presence of actin (data not shown).

We also analyzed c-mos mRNA levels by RT-PCR. A weak amplification product was always evidenced in DMSO control testis (Fig. 4E), whereas after EDS injection, it became more intense on Day 4 and progressively returned to the lower expression from Days 8 to 28 (Fig. 4G). Figure 4, F and H, shows control amplifications of fP1 cDNA in DMSO control and EDS-injected frogs, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The c-mos proto-oncogene is unusual in its highly restricted pattern of tissue-specific expression; in fact, it is mainly expressed in gonads and at very low levels in a few other tissues. Mos is a well-known protein, the function of which in female gametogenesis has been widely discussed [30], but very little is known about its role in spermatogenesis [19].

In spite of c-mos expression and Mos localization in meiotic and postmeiotic cells (SPC and SPT) of mammalian testis, investigations in other vertebrates give evidence of its presence also in nonmeiotic cells. Interestingly, in the testis of the dogfish (Scyliorhinus canicula), Mos is specifically localized in the myeloid cells of the testicular interstitial compartment, probably coming from the epigonal organ, whereas germ cells are immunonegative [20, 21]. In the testis of the frog (R. esculenta), Mos has been localized not only in the cytoplasm of SPC, but also in that of SPG [22], indicating a new role that Mos might play in nonmeiotic phases of spermatogenesis.

In the present paper, we report, to our knowledge for the first time, isolation of a c-mos cDNA fragment of 696 bp from R. esculenta testis. Its deduced amino acid sequence shows high similarity to Mos homologues of other vertebrates, with homology values ranging from 55% to 90% (see Fig. 1).

Rana esculenta is a seasonal breeder with a spermatogenic cycle, lasting 1 yr, that is regulated by endocrine and environmental factors [31, 32]. A proliferative phase is characterized by mitotic divisions of primary and secondary SPG in early spring, whereas a maturative phase shows the highest activity of meiotic divisions in late summer and early autumn, resulting in an increased number of secondary SPC and SPT [32]. For this reason, our investigations have to be performed throughout the frog annual sexual cycle.

Generally, c-mos mRNA expression is very low in the frog testis. This observation comes from the negative results obtained by Northern blot analysis (unpublished data) as well as from the high number of PCR cycles (n = 43) needed to detect a satisfactory signal. A more sensitive approach has already been used to evidence low levels of c- mos mRNA in other tissues, such as mouse brain, kidney, placenta, and mammary gland [5]. In addition, Yoshida et al. [33] isolated a c-mos cDNA from the frog R. japonica oocytes but failed to detect the protein; those authors concluded that if Mos exists in R. japonica oocytes, its content is very low. On the other hand, extremely low levels of v- mos RNA [34] and v-mos protein [35] are sufficient to transform NIH/3T3 cells. Therefore, it is easy to suppose that a lightly detectable expression of this proto-oncogene might be able to carry out its function in R. esculenta testis as well. This is clearly indicated by the RT-PCR and Southern blot analysis carried out in the present study. Indeed, we demonstrated the presence of c-mos mRNA at a low level throughout the year. Finally, the significant peak detected in March strongly suggests a physiological role.

Therefore, our next step was to focus on the temporal and spatial expression of Mos protein during the frog annual sexual cycle. By Western blot analysis, we have characterized, again to our knowledge for the first time, the presence of Mos in the R. esculenta testis as a 60-kDa protein throughout the year, with a significant peak in May. With respect to the size of Mos, in other species different molecular weights have been described according to the cellular source or to the characteristic of the antibodies. In particular, the most-mentioned Mos sizes are 39 kDa in Xenopus and mouse oocytes [36, 37], 43 kDa in mouse and rat testes [38, 39], 43 and 75 kDa in rat muscle [40], 40 and 37 kDa in human cells [41]. It is worth noting that the protein peak follows the transcript peak. Such a gap is well described in female gametogenesis, when maternal mRNAs are stored for translation only when necessary for embryo development [42], and it is not unusual in our context, if we consider the slow progression of spermatogenesis in R. esculenta. For example, in frog testis, estradiol starts to increase during April through May, but the subsequent c- fos mRNA expression is detectable in June [43].

The highest Mos expression in May has been confirmed by immunocytochemical observations performed on frog testicular sections collected throughout the year: Mos immunostaining appears stronger in the testis of May frogs. At the same time, these data also confirmed Mos localization in SPG and SPC cytoplasm [22]. It should be remembered that in May, when we observe the maximal Mos expression, spermatogonial proliferation slows down, and meiotic divisions increase. This seems to suggest that the function of Mos might be associated with these specific events of R. esculenta spermatogenesis. Alternatively, we cannot exclude that the highest level of Mos expression in May might result from a cytoplasmic accumulation associated to a lack of function.

In mouse pachytene SPC, ERK1, a downstream component of the Mos pathway, is specifically activated during the G2/M transition, contributes to MPF activation, and is essential for chromosome condensation associated with progression to meiotic metaphases [19]. Keeping this in mind, Mos localization in R. esculenta SPC might indicate a role of this proto-oncogene in meiotic maturation. On the other hand, Mos in SPC might be also considered as a residual protein in a cytoplasm directly inherited from SPG, where it has been synthesized. Thus, its function has already been completed. Interestingly, this hypothesis could be supported by the observation that Mos immunoreactivity is stronger in SPG and progressively decreases as cytoplasmic divisions go on.

Considering the above speculations, Mos localization in the cytoplasm of SPG represents the focal point of our investigation. In this respect, it is useful to compare the regulation of oogenesis with that occurring in the spermatogenesis of a seasonal breeder. In female gametogenesis, the progression of mature oocytes needs to be arrested until fertilization to avoid parthenogenic activation. In this context, the assessed function of Mos as a component of cytostatic factor is to induce, through the MEK/MAPK/p90Rsk pathway, the block of oocytes in metaphase II. On the other hand, a mechanism similar to that observed in the female also occurs in the spermatogenesis of a seasonal breeder, the frog R. esculenta, in which SPG proliferative activity progressively decreases until arrest, concomitantly with the increase of the maturative phase. For this reason, the presence of Mos in the cytoplasm of SPG seems to suggest that this proto-oncogene might block the mitotic activity as soon as meiosis occurs.

Therefore, to obtain functional data, we planned experiments with EDS. As a consequence, our hypothesis has been strongly supported by results obtained with the use of this substance. An alkylating agent, EDS has a selective cytotoxic effect on interstitial LC in several vertebrate species [44, 45]. In particular, in R. esculenta testis 4 days after a single EDS injection, the majority of tubules adjacent to the damaged areas are disorganized, and in some of them, nonproliferating primary SPG are the only germ cells still distinguishable. Eight days after the EDS injection, a new population of LC reappears in the interstitium, and spermatogenesis resumes. Twenty-eight days after the injection, spermatogenesis is completely restored [24, 45]. Our results concerning c-mos proto-oncogene expression in EDS-treated animals demonstrate an increased expression of both transcript and protein on day 4 after the EDS injection and the progressive resumption of the normal low levels of expression from days 8 to 28. Because an increased signal might be caused by an increased number of expressing cells, the immunocytochemical data are of particular interest. Indeed, on day 4, Mos immunoreactivity detected in the cytoplasm of SPG of EDS-injected frogs is stronger compared with that of the DMSO control animals. Therefore, the above-described results obtained by RT-PCR, Western blot analysis, and immunocytochemistry clearly show that c-mos is highly expressed concomitant with the temporary arrest of SPG mitosis and is detectable at low levels when the proliferative activity of these cells is restored.

In conclusion, in the present study, we isolated the cDNA and identified the protein homologue of the c-mos proto-oncogene in the testis of the frog R. esculenta. Our results showed that both are expressed throughout the frog annual sexual cycle. In particular, in March, we observe the maximal expression of the transcript, whereas in May, we detect the maximal expression of the protein. Mos is specifically localized in the cytoplasm of SPG as well as in that of SPC. Besides oogenesis, this suggests a possible role for this proto-oncogene in spermatogenesis. Finally, and to our knowledge for the first time in a vertebrate, we show an increased expression of c-mos in SPG during a temporary arrest of mitosis, whereas at the resumption of SPG proliferative activity, c-mos expression concomitantly decreases. This indicates that c-mos expression is involved in the regulation of spermatogonial proliferation.


   ACKNOWLEDGMENTS
 
We greatly thank Dr. Paolo Sassone Corsi for his critical support.


   FOOTNOTES
 
1 Supported by grants from MURST "ex 40% PRIN" and "Ricerca di Ateneo ex 60%." Back

2 Correspondence. FAX: 39 081 5667536; riccardo.pierantoni{at}unina2.it Back

Received: 17 December 2003.

First decision: 12 January 2004.

Accepted: 2 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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