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Dipartimento di Medicina Sperimentale, Sezione F. Bottazzi, II Università di Napoli, 80138 Naples, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AP-1, blood vessel, efferent duct, Fos family proteins, Fra1, male reproductive tract, male sexual function, peritubular myoid cell, proto-oncogenes, reproduction, spermiation, sperm motility and transport, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fra1 is involved in the cell cycle regulation of normal [9] and tumoral [10, 11] cells. A significant amount of Fra1 and Fra2 are synthesized during the cell cycle when neither c-Fos nor FosB are present [9, 12], suggesting that both Fra1 and Fra2 play a unique role in cell growth. Targeted disruption of the fra1 gene leads to early embryonic lethality due to impaired vascularization of the placenta [13]. Therefore, other AP-1 complexes, although present ([13] and references therein), do not compensate for the lack of Fra1.
The regulation of AP-1 activity occurs through increased expression of the specific AP-1 members as well as through phosphorylation of preexisting and newly synthesized AP-1 subunits [1, 4–6]. In Rana esculenta testis, the phosphorylation of c-Fos is involved in spermatogonial proliferation through the control of its cytoplasmic storage [14], nuclear translocation [15], and functional activation [14, 16]. Similarly, Fra1 undergoes posttranslational modifications that cause a significant shift in its gel mobility from 36 to 46 kDa or more [12]. In serum-stimulated fibroblasts, Fra1 undergoes extensive phosphorylation [7, 12, 17] and shows both nuclear and cytoplasmic localization [17]. Cytoplasmic and nuclear localizations of Fos family proteins, described in vitro [17, 18], have also been reported recently in in vivo models of amphibians [14–16, 19–21], reptiles [22], and mammals [23–25]. In particular, Fra1 has been localized in cytoplasmic and nuclear compartments of ovarian and testicular cells of pigs [25] and foxes [23], respectively. In mice and rats, it appears to be associated with spermatogonia [26], whereas in red fox, Vulpes vulpes, Fra1 immunoreactivity localizes to the cytoplasmic or perinuclear level in spermatogonia, and to the nuclear level in spermatocytes and in round and elongating spermatids [23].
In R. esculenta testis, using an anti-Fos family member antibody, we have recently described the presence of a nuclear, 43 kDa protein, hypothesized to be Fra1 [14, 15]. This animal model appears particularly suitable for studying the role of proto-oncogene activity in testicular physiology [27]. Indeed, R. esculenta is a seasonal breeder, characterized by a period of resumption of spermatogonial proliferation (late winter to early spring), a well-defined period of mating (March–April), and a postreproductive period [28]. With the assistance of a specific anti-Fra1 antibody, we have here extended the study of this protein in frog testis in order to 1) confirm the identity of the 43 kDa Fos-related antigen, 2) characterize its expression and localization, and 3) examine its possible role in male reproduction.
We provide evidence of differential expression of Fra1 in R. esculenta testis during its annual reproductive cycle, accounting for a novel role for Fra1 in the control of sperm transport from the tubular compartment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male frogs (R. esculenta) (n = 15) were captured near Naples, Italy, early in the afternoon on a monthly basis for two consecutive years. Frogs were killed under anesthesia with MS222 (Sigma-Aldrich, St. Louis, MO). Testes were removed and immediately processed for immunocytochemistry (n = 6) or nuclear protein preparation (n = 20). During the second-year sampling, in vivo and in vitro experiments were carried out using animals collected in March. For in vivo treatments, animals were maintained in plastic tanks (50 x 25 x 17 cm) with food (mealworms) and water ad libitum, and exposed to natural temperature (15°C) and photoperiod (9L:15D). The study protocol was approved by the Italian Ministry of Health.
Rat testes (n = 3) were also collected and used as a positive control tissue for the Fra1 signal.
Nuclear Protein Preparation
Nuclear protein extracts were prepared with modifications to methods used previously by Dignam et al. [29]. Briefly, six testes per experiment were gently homogenized using a type B pestle in 7 volumes (w/v) of hypotonic Hepes buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 12% glycerol, 0.1 mM EGTA, 0.5 mM dithiothreitol, and 0.5 mM spermidine) in the presence of protease inhibitors (4 g/ml of leupeptin, aprotinin, pepstatin A, chymostatin, and PMSF; and 5 µg/ml of N-tosyl-L-phenylalanine chloromethyl ketone). After centrifugation at 800 x g, the supernatant was removed. The nuclear pellet was washed three times, resuspended in 1.2 volumes (1.2 ml/mg pellet) of hypertonic Hepes buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 15% glycerol, 0.1 mM EGTA, 0.5 mM dithiothreitol, and 2 mM spermidine) in the presence of protease inhibitors (see above), and mixed at 4°C for 30 min. Nuclei were pulled down by centrifugation at 10 000 x g for 30 min at 4°C. The supernatants containing nuclear proteins were then collected. Protein concentrations were determined using the Lowry assay [30]. A similar protocol was used to prepare nuclear protein extracts from rat testis.
Western Blot Analysis
Thirty micrograms of proteins/lane were separated on 10% SDS-polyacrylamide gel. After electrophoresis, the gel was cut around the 100 kDa size; the upper part was immediately stained with Coomassie brilliant blue (Sigma, St. Louis, MO) to quantify the content of loaded proteins, and the remaining part was transferred to a nitrocellulose filter (Amersham Pharmacia Biotech, U.K.) at 280 mA for 2.5 h at 4°C to evaluate the Fra1 signal. To prevent nonspecific adsorption, the filter was treated for 3 h with blocking solution (5% nonfat powdered milk, 0.25% Tween-20 in Tris-buffered saline [TBS] pH 7.6) and then incubated with the primary antibody (anti-Fra1 R-20 sc-605 diluted 1:1000; anti-c-Jun N sc-45 and pan-Fos antibody sc-243-G, both diluted 1:500; all three purchased from Santa Cruz Biotechnology, Heidelberg, Germany) diluted in a PBS-3% nonfat powdered milk solution overnight at 4°C on an orbital shaker. The filter, washed in TBS-0.25% Tween-20, was then incubated with 1:1000 horseradish peroxidase-conjugated immunoglobulin G (IgG; DAKO, Denmark) (we used goat anti-rabbit IgG to recognize Fra1 antibody and rabbit anti-goat IgG to recognize c-Jun and pan-Fos antibodies) in TBS-1% normal swine serum (NSS; DAKO) and then washed three times in TBS-0.25% Tween-20. The immune complexes were detected using the enhanced chemiluminescence-Western blotting detection system (Amersham Pharmacia Biotech) following the manufacturer's instructions. Specificity of the reactions was tested through competition studies using antibody previously preabsorbed for 18 h at 4°C on an orbital shaker with a large excess (10–6 M) of the corresponding peptide (Fra1 peptide, sc-605P; c-June peptide, sc-45P; Fos-peptide, sc-253P; Santa Cruz, Biotechnology).
Immunocytochemistry
Testes that had been rapidly removed and fixed in Bouin fluid were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Tissue sections (5 µm) were processed by a modification to the method already described [15, 20]. Briefly, the sections were treated for 20 min with H2O2 to block endogenous peroxidases. Incubations were performed at 4°C in a moist chamber for 14 h with anti-Fra1 antibody (R20 sc-605; Santa Cruz Biotechnology) diluted 1:50 in PBS buffer (0.01 M PBS pH 7.1 containing 1% NSS [DAKO] and 2% BSA). Sections were washed in PBS-0.1% Triton X-100 and processed with a Vectastatin ABC System Universal Quick kit (Vector Laboratories Inc., Burlingame, CA) following the manufacturer's instructions. The immune complexes were detected using 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 3% H2O2 in 50 mM Tris-HCl (pH 7.6). To check the specificity of the immunoreactions, controls were treated with anti-Fra1 antibody, diluted 1:50 in PBS buffer (see above) that had been previously preabsorbed with a large excess (10–6 M) of the corresponding peptide (sc-605P; Santa Cruz Biotechnology). The antibody was preabsorbed for 18 h at 4°C on an orbital shaker.
Specificity of Anti-Fra1 Antibody
In this study we used a rabbit polyclonal anti-Fra1 antibody (R-20 sc-605; Santa Cruz Biotechnology) raised against a peptide mapping at the N-terminus of Fra1 of rat origin. The antibody was specific for Fra1 of mouse, rat, and human (according to the data sheet accompanying the antibody). Therefore, we used rat tissue as a positive control for the Fra1 signal. The specificity of the signal has been tested by extinguishing the immunoreaction with an excess amount (10–6 M) of the cognate peptide (sc 605-P; Santa Cruz Biotechnology). Immunoprecipitation and coimmunoprecipitation studies were also carried out. Briefly, testicular nuclear extracts (0.5 mg) representing the annual reproductive cycle were diluted in ice-cold lysis buffer (50 mM Tris-HCl, 4 mM EDTA, and 1% Triton X-100 pH 7.6) in the presence of protease inhibitors (see above) at a protein concentration of about 0.5 mg/ml and processed for immunoprecipitation. Anti-Fra1 antibody (2 µg) was incubated with (positive sample) and without (negative sample) nuclear extract for 1 h at 4°C on an orbital shaker. Afterward, a 20-µl suspension of protein G PLUS-agarose (sc-2002; Santa Cruz Biotechnology) was added and further incubated at 4°C. After 
18 h, immunoprecipitates were pulled down by centrifugation at 1000 x g for 5 min at 4°C and washed four times with lysis buffer pH 7.6. Finally, the pellet was dissolved in 40 µl of electrophoresis buffer and boiled for 5 min. Immunoprecipitates were separated through 9% SDS-PAGE and analyzed by Western blot for Fra1 presence using anti-Fra1 (R-20 sc-605; Santa Cruz Biotechnology) and anti-pan-Fos (sc-253-G; Santa Cruz Biotechnology; the antibody was raised in goat against an acid sequence within a highly conserved domain in Fos family members of human origin). Proteins, pulled down by anti-Fra1 antibody, were finally analyzed by coimmunoprecipitation study for c-Jun presence using goat c-Jun antiserum (N-sc-45-G; Santa Cruz Biotechnology) raised against a peptide mapping within the N-terminus domain of c-Jun of mouse origin.
Immunoprecipitation was also carried out using pan-Fos antibody (sc-253-G; Santa Cruz Biotechnology). Fra1 presence was checked by Western blot using Fra1 antiserun (R-20 sc-605; Santa Cruz Biotechnology).
In Vivo and In Vitro Hypophysis Homogenate Treatments
Animals (n = 14) were injected with 100 µl of amphibian Krebs Ringer bicarbonate buffer (KRB; n = 7; controls) or with one-third of hypophysis (pars distalis, PD) gently homogenized in 100 µl of KRB (n = 7; PD-treated group). As already demonstrated, PD induces spermiation [31]. Thus, the quality of the hypophysis homogenate was verified by the presence of spermatozoa in the cloaca. Injections into the dorsal sac were carried out for 2 wk on alternate days. Two hours after the last injection, animals were killed under anesthesia with MS222 (Sigma-Aldrich); testes were removed and immediately processed for hematoxylin-eosin staining (n = 3), immunocytochemistry (n = 3), or nuclear protein preparation (n = 8).
In in vitro experiments, testes from 10 animals were removed, washed, and then incubated in 10 ml of KRB for 1 h with (PD group; n = 10) or without (control group; n = 10) hypophysis homogenate (1/6 hypophysis/ testis) at 22°C. After treatment, testes were processed for nuclear protein preparation and analyzed by Western blot.
Data Presentation and Statistics
The Fra1 signal and the content of loaded proteins were quantified by densitometry analysis carried out using the GELDOC1,00-UV system (Bio-Rad, Hercules, CA). Nuclear Fra1 levels were plotted as quantitative densitometry analysis of Fra1 signal corrected on the basis of protein content (Fra1 signals/protein content) evaluated by Coomassie brilliant blue staining (see Western Blot Analysis). Values were expressed as optical density (OD) units.
Testes of in vivo treated animals were analyzed for number of peritubular myoid cells (PMCs) expressing Fra1. Counting was carried out on three randomly chosen sections per testis from different animals. Values are expressed as Fra1 immunopositive PMCs per total tubules per section, multiplied by 100.
A Student t-test and analysis of variance followed by a Duncan test for multigroup comparison was carried out, where appropriate, to evaluate the significance of differences. Data were expressed as the mean ± SEM from at least three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polyclonal anti-Fra1 antibody was tested using Western blot analysis on nuclear extracts of rat (positive control) and R. esculenta testes (Fig. 1). Antiserum detected a 43 kDa signal in both nuclear extracts. Binding was removed by preincubation of the antibody with an excess amount of cognate peptide, suggesting a specificity of the immunoreaction.
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Immunoprecipitation experiments, analyzed by Western blot, showed that anti-Fra1 antibody pulls down a 43 kDa protein that is recognized by both anti-Fra1 (Fig. 2A) and pan-Fos (Fig. 2B) antibodies. The anti-Fra1 antibody also pulls down a small protein that is specifically recognized by anti-c-Jun antibody (Fig. 2C). Specificity of the Fra1 signal was finally demonstrated also changing the antibodies used for immunoprecipitation and Western blot. In fact, anti-Fra1 antibody also recognized the 43 kDa protein when it was pulled down by the pan-Fos antibody (Fig. 2D). Therefore, the 43 kDa signal was recognized by the different uses of the two different antisera. Furthermore, the coimmunoprecipitation of c-Jun together with the 43 kDa protein indicated a protein-protein interaction. Because Fra1 interacts with c-Jun in the AP-1 complex [3], this provided additional evidence that the 43 kDa protein was Fra1. However, although our data strongly indicate that we have detected Fra1 in the frog testis, the possibility of cross-reaction with some other Fos family members (e.g., FosB, Fra2) should be investigated.
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Expression of Fra1 During the Annual Reproductive Cycle in Frog Testis
Figure 3 shows Fra1 expression in R. esculenta testis during the annual reproductive cycle. In particular, Western blot analysis carried out on nuclear proteins (Fig. 3A) showed a 43 kDa signal always present from January until December. The intensity of signals (Fig. 3B) was higher during the March–October period (P < 0.05 at least) compared with that of other months.
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Immunocytochemistry revealed Fra1 immunostaining restricted to efferent ducts (ED) and blood vessels (BV; Fig. 4A). In ED, Fra1 was localized in smooth muscle cells and in epithelial cells (Fig. 4B) where immunostaining was often observed in the cytoplasm, but rarely in the nucleus. In the cytoplasm, it appeared to be organized in small spots localized around the nucleus. In blood vessels, endothelial and vascular smooth muscle cells were immunopositive. In endothelial cells (Fig. 4B), rarely did we find Fra1 immunoreactivity in the nucleus, because it was present mainly in the cytoplasm. Finally, a clear immunostaining was detected in PMCs of testis collected in the March–April period (Fig. 4D). Contrary to that of the epithelial cells of ducts and cells of blood vessels, the immunoreactivity of PMCs during the year was, curiously, restricted to the March–April period. The specificity of the signals was tested through the use of preabsorbed antibody (Fig. 4E).
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Rat testis (Fig. 5) was used as a positive control for the Fra1 immunoreaction. Immunocytochemistry localized Fra1 antigen in PMCs and blood vessels (Fig. 5A). In endothelial cells, the immunoreaction was present mainly in the cytoplasm, and rarely in the nucleus. In vascular smooth muscle cells (VSMCs), the signal was present at both cytoplasmic and nuclear levels. In particular, the immunoreaction was compartmentalized to a small spot, very similar to that showed in frog samples. The specificity of signals was tested through the use of preabsorbed antibody (Fig. 5B).
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Hypophysis Homogenate Treatments
To confirm testicular morphological changes induced by PD [31] (lumen of efferent ducts, spermiation, presence of spermatozoa in external ducts), testes of animals treated with and without PD homogenate were first analyzed by histology.
Testicular sections of untreated animals showed tubules full of mature spermatids (mSPT). Efferent ducts lacked spermatozoa and were characterized by a very reduced lumen (Fig. 6A). Conversely, after PD treatment, testes showed more tubules full of free spermatozoa (Fig. 6B), and lumen of efferent ducts appeared enlarged (Fig. 6C). Sperm cells exiting tubules were often observed in efferent ducts (Fig. 6D). Many tubules appeared empty (Fig. 6E), but then, spermatozoa were observed in external ducts (Fig. 6F), and finally in the cloaca. Thus, the presence of spermatozoa in the cloaca, observed 2 h after the last PD injection, was normally used to check spermiation.
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In testes of both control and PD-treated animals, nuclear extracts expressed the 43 kDa protein (Fig. 7A). The intensity of the signals was significantly stronger (P < 0.05) in PD-treated animals compared with that of control frogs (Fig. 7B).
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Immunocytochemistry localized Fra1 in efferent ducts and in PMCs of testes in both experimental groups. In the control group, Fra1 antigen was scantly visible in epithelial cells of efferent ducts and randomly in PMCs (Fig. 8A). In PD-treated animals, the immunoreaction was strongly present in epithelial cells and in PMCs (Fig. 8B). In epithelial cells, the signal was organized in spots, but was spreading in the cytoplasm around the nucleus (as shown in Fig. 4B), which rarely appeared immunopositive. Finally, the number of PMCs showing immunopositivity increased (P < 0.01) after PD treatment (Fig. 9).
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Hypophysis Homogenate Effect on Incubated Testis Fragments
Testis pieces washed with KRB were incubated for 1 h with (PD group) or without (control group) hypophysis homogenate. Western blot analysis detected the 43 kDa Fra1 antigen in nuclear preparations of both experimental groups (Fig. 10A). Densitometry analysis (Fig. 10B) showed that PD treatment significantly increased (P < 0.05) Fra1 signals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain information about the expression and localization of Fra1 in R. esculenta testis, we investigated its presence during the annual reproductive cycle. Western blot detected Fra1 in nuclear protein preparations from January until December. Quantitative densitometry analysis of the signals revealed a higher expression during the March–October period. Immunocytochemistry showed Fra1 immunostaining in efferent ducts and blood vessels all year around. Conversely, in PMCs, its appearance was restricted to the March–April period. Thus, we conclude that the higher expression of the 43 kDa signal detected by Western blot in this period is mainly related to PMCs.
In efferent ducts and blood vessel cells, Fra1 immunostaining was often observed in the cytoplasm. Of interest, the blood vessels in frogs and rats showed a very similar pattern of immunolocalization. Indeed, in both species, the signal appeared to be compartmentalized in small spots, spreading in the cytoplasm around the nucleus. Although the presence of Fra1 in the cytoplasm has already been reported in mammalian fibroblasts [3], gonads [23], and blood vessels [25], the unusual cytoplasmic localization requires further experiments to characterize this signal.
Regarding the immunolocalization of Fra1 in PMCs, it is noteworthy to remark on its cyclic appearance during the annual reproductive cycle in R. esculenta. In mammals, PMCs undergo rhythmic contractile activity immediately after spermiation. In fact, the main biological function of PMCs is the generation of impulses, starting after spermiation, that propel spermatozoa through the tubules toward the rete testis and efferent ducts [32]. This transport is believed to result from forces that are not intrinsic to the sperm cells [33, 34], but due to tubule contraction induced by PMCs. Given the cyclic appearance of Fra1 in PMCs during the March–April period when frog mating occurs [28], we hypothesized a relationship between Fra1 expression and the transport of spermatozoa through the tubules. Because this transport naturally occurs immediately after spermiation [31], we carried out in vivo and in vitro experiments with PD homogenate. Indeed, spermiation is primarily under gonadotropin control [31]. After PD treatment, spermatozoa travel from the tubular lumen through efferent ducts to the cloaca. Simultaneously, the number of PMCs expressing Fra1 increased, together with the nuclear, 43 kDa signal as was also shown by the in vitro experiment. Therefore, the above-described results strongly indicate for the first time in a vertebrate species, a close relationship between Fra1 expression and PMC activity. This of course may be a consequence of several paracrine/endocrine interactions occurring in the testis. In this respect, it should be emphasized that Pmods, a factor produced by the peritubular cells, modulates Sertoli cell function after androgen stimulation [16]. In the future, an in vitro experiment using phase contrast microscopy should be designed to establish a correlation between Fra1 expression and seminiferous tubule peristalsis.
The presence of Fra1 in blood vessels was consistent with observations previously reported in pig [25] and rat [35]. Comparative analysis, carried out here on an evolutionary distant species, showed a very similar Fra1 intracellular localization in VSMCs and endothelial cells. This also suggests that in blood vessels, in addition to PMCs, Fra1 function is highly conserved in evolution. Although the presence of Fra1 at cytoplasmic and nuclear levels in these cells has already been reported [25], its function is still unknown. Due to the contractility of VSMCs, we speculate that the biological function might be the regulation of vessel tone. The additional Fra1 localization in smooth muscle cells of efferent ducts, whose partial contraction is supposed to be required to maintain the tone of duct, may support this hypothesis.
Regarding the endothelial cells, the impaired placental vascularization in Fra1–/– mice suggests that Fra1 is also involved in vasculogenesis and angiogenesis. Analysis of the annual testicular activity of the frog testis indicates that the interstitial compartment develops cyclically. Therefore, whether or not Fra1 is involved to form new or more efficient blood vessels (or both) during the increase in the interstitial compartment in the postreproductive period [28] needs further investigation.
In conclusion, we have used the frog, R. esculenta, as a useful animal model for studying the activity of Fra1 in the testis. Besides the demonstration of Fra1 presence in a nonmammalian vertebrate gonad, we report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells. We confirm the effect of PD on sperm release and we demonstrate that PD treatment increases the number of PMCs expressing Fra1. This occurs when spermatozoa are propelled toward tubules and ducts. Therefore, our results strongly indicate a novel role for Fra1 in the control of sperm transport.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX 39 081 5667500; riccardo.pierantoni{at}unina2.it
Received: 27 September 2004.
First decision: 1 November 2004.
Accepted: 16 December 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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