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Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate "F.Bottazzi", Facoltà di Medicina e Chirurgia, II Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italia; Istituto Internazionale di Genetica e Biofisica (CNR), Via Marconi, 80100 Napoli, Italia
Address all correspondence and requests for reprints to: Riccardo Pierantoni, Seconda Universita degli Studi di Napoli, Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate, Universita di Napoli, Via Constantinopoli 16, Napoli 80138, Italy. E-mail: pieranto{at}unina.it
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Abstract |
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In conclusion, our results demonstrate that E2 induces ISPG multiplication in the frog, R. esculenta, and, for the first time in a vertebrate species, that it triggers c-fos activity in the testis. Moreover, E2 may be involved in mechanisms related to FOS transport in the nucleus of ISPG to induce the mitotic activity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proto-oncogene activity has been studied in vertebrate testis (5, 6, 7, 8) being oncoproteins generally involved in cellular growth, differentiation, and morphogenesis, all processes occurring during spermatogenesis. It is known that gonadotropins regulate proto-oncogene expression in cultured pig Leydig cells (9) and rat Sertoli cells (10), but the role played by steroids is not understood. However, it has been shown that estradiol and progesterone activate c-myc expression during sea star spermatogenesis (11), androgens and c-fos expression are interrelated in rat ventral prostate (12), progesterone rapidly decreases c-jun transcripts in the avian oviduct (13), and 17ß-estradiol induces c-myc, c-fos, and c-jun expression in rat uterus (14).
In the light of estrogen activity in the testis (15), it may be of interest to study whether or not c-fos expression is activated by estradiol. In this respect, the testis of the frog, Rana esculenta, may represent an useful model of investigation. Indeed, previous research has established that, similarly to mammals, estrogen receptors are present (16) and estradiol inhibits androgen production (17, 18) but enhances spermatogonial proliferation (19). Activity of c-fos has been detected during the annual sexual cycle in the frog testis (20) characterized by a slow progression of germ cell stages that occurs from February–March (spermatogonial proliferation) until September–October (spermatid and spermatozoa appearance) (21, 22). Interestingly, estradiol peaks concomitantly with spermatogonial proliferation (19, 23) and c-fos messenger RNA (mRNA) increase (20). Furthermore, MYC, FOS, and JUN proteins appear in germ cell nuclei during the occurrence of spermatogenesis (6).
Therefore, using the frog, R. esculenta, we confirm here that estradiol induces spermatogonial multiplication and, for the first time in a vertebrate species, that it also induces c-fos activity in the testis and the appearance of FOS protein in spermatogonial nuclei.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein extract preparations
Frozen tissue was placed in cold TEDG buffer (50 mM
Tris-HCl pH 7.5; 1 mM EDTA; 10% glycerol; 0.1
mM dithiothreitol; 5 mM MgCl2; 1
mM phenylmethylsulfonyl fluoride) (wt:vol, 1:2) containing
protease inhibitors (Leupeptin 5 mg/ml, pepstatin A 5 mg/ml,
chymostatin 5 mg/ml, Sigma Chemical Co.) and gently
homogenized. Large particulate material, included nuclei, was removed
by centrifugation at 800 x g for 30 min and retained
for preparation of nuclear protein extracts. The supernatant fraction
was carefully removed and centrifuged at 100,0000 x g
for 1 h at 4 C to obtain cytosolic extracts. The crude nuclear
pellet was subjected to a washing step to remove residual soluble
material and then resuspended in TKM buffer (5 mM Tris-HCl,
pH 7.4; 5 mM MgCl2; 2.5 mM KCl; 250
mM sucrose) in the presence of protease inhibitors,
filtered throughout a sterile gauze and sucrose concentration was
increased to 1.6 M. Samples were stratified on 2.2
M sucrose and centrifuged at 60,000 x g
for 1 h at 4 C. The residual pellets were resuspended in a small
volume of TKM buffer and sonicated. Total proteins from MCF7 cell line
were also extracted to be used as positive control in Western
analysis.
Protein concentrations of cytoplasmic and nuclear extracts were determined by Lowry method (24).
Antibody
The antibody used for Western blot was an anti c-FOS
(OA-11–822, CRB, UK) raised in sheep against a synthetic peptide
sequence derived from a conserved region of both mouse and human
c-FOS. For immunocytochemistry, OA-11–822 (CRB, UK) and Serva
(Heidelberg, Germany) anti c-FOS antibodies were used. Specificity
was tested by extinguishing the reaction with an excess amount
(10-6 M) of the cognate peptide (OP-11–3210;
CRB, UK) or by omitting one step of the reaction.
Western blots
Fifty micgrograms/lane of proteins were separated using 0.1%
SDS/12% polyacrylamide gel (7). After electrophoresis, the proteins
were blotted to nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 2.5 h at 250 mA at 4 C in
a mini trans-blot cell apparatus (Bio-Rad Laboratories, Inc., Hercules, CA).
After transfer and staining by Ponceau S (Sigma Chemical Co.) as control of protein loading, the membranes were treated for 3 h with blocking solution (5% nonfat powdered milk) to prevent nonspecific adsorption, washed several times in 25 mM Tris, pH 7.5; 200 mM NaCl; 0.5% Tween 20 (TBS I) and 25 mM Tris pH 7.5; 370 mM NaCl; 0.5% Tween 20 (TBS II) and then incubated for 2 h at room temperature with the primary antibody (diluted 1:7000) or with the primary antibody preabsorbed with 10-6 M of the antigen overnight at 4 C on an orbital shaker. After washing, the membranes were incubated with a horseradish peroxidase-conjugated antigoat IgG (1:1000). The immune complexes were detected using the electrochemiluminescence-Western blotting detection system (Amersham Pharmacia Biotech) following the manufacturer’s instructions.
Immunocytochemistry
Frog testes, rapidly removed and fixed in Bouin’s fluid, were
dehydrated in ethanol, cleared in xylene, and embedded in paraffin.
Tissue sections (5 µm) were processed by the
peroxidase-antiperoxidase technique (PAP). Four
sections/animal/treatment have been examined. The sections were treated
for 20 min with H2O2 to block endogenous
peroxidase. Incubations were performed at 4 C in a moist chamber for
14 h with the primary antiserum. The antiserum was diluted 1:50 to
1:200 in PBS 0.1 M at pH 7.4 containing 1% normal swine
serum. After washing in PBS, the sections were incubated for 1 h
at room temperature with a secondary antiserum (anti-Ig raised in
sheep; DAKO Corp., Denmark) diluted 1:80 in PBS.
After washing in PBS, the sections were incubated for 1 h with
goat PAP complex (DAKO Corp.). The antigen was visualized
using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.) and 0.3% H2O2 in PBS
solution.
RNA preparation and Northern blot
Total RNA was prepared from frog testes by the guanidium
isothiocyanate method (25). A 1.1 kb v-fos probe was
obtained by double digestion with pstI/salI from pBR322 (26). Northern
blot hybridization analysis employed a (32P)-dCTP (3000
Ci/mmol) by random priming (Amersham Pharmacia Biotech)
following the manufacturer’s instruction. Blots were prehybridized and
probed in 50% formamide, 5xSSPE, 2x Denhardt’s, 0.1% SDS, 40
µg/ml transfer RNA (Escherichia coli), and 100 µg/ml
denatured salmon sperm, overnight at 42 C and then blots were washed
(7, 20). A poly A- from frog testes was used as negative
control. Membranes were exposed for appropriate intervals using Kodak
x-ray autoradiographic films (Eastman Kodak Co.,
Rochester, NY).
Normalization employed a 32P-labeled 28S ribosomal (r)DNA probe from Drosophila melanogaster; the relative amounts of c-fos-like mRNA were determined by densitometric analysis of the autoradiographs of the Northern blots (20).
Autoradiographs were quantified by scanning densitometric and integration of peak areas with a transmitting scanning apparatus (Ultroscan XL, LKB, Sweden).
Seasonal cycle and in vivo experiments
To detect FOS immunoreactivity, cytosolic and nuclear extracts
were prepared from frog (n = 10/month) testes collected each month
for 1 yr and used for Western blot analysis.
To determine estradiol-17ß (E2) effect on germ cell proliferation, 40 animals were captured during February and divided in experimental groups as follows: a control group (n = 10) injected with 100 µl of amphibian Krebs Ringer bicarbonate buffer (KRB) pH 7.4, a group (n = 10) injected with 100 µl KRB containing 270 ng E2, a group (n = 10) injected with 100 µl KRB containing 270 ng E2 + 100 µl KRB containing 6 µg ICI 182–780 (Zeneca, UK) and a group (n = 10) injected with 100 µl KRB containing 270 ng E2 + 100 µl KRB containing 3.7 µg Tamoxifen (Sigma Chemical Co.). Animals received a single injection in the dorsal sac and were killed after 48 or 72 h (5 animals/time/group) after the injection. Twenty-four hours before they were killed, each frog was injected, sc, with 100 µg of colchicine for the evaluation of the mitotic index of primary spermatogonia (ISPG). Testes were fixed in Bouin’s fluid and embedded in paraffin; 5–6 µm serial sections were stained with hematoxylin-eosin. At least three sections, 0.05 mm apart, from each testis from each frog (n = 10 animals/experimental group) were used for quantitative analysis. The mitotic index of ISPG was expressed as the number of metaphase/total ISPG counted/section multiplied by 100.
To assess E2 effects on FOS production and distribution, 40 animals have been captured in February and divided in experimental groups as follows: a control group (n = 10) injected with 100 µl of amphibian Krebs Ringer bicarbonate buffer (KRB), pH 7.4, a group (n = 10) injected with 100 µl KRB containing 270 ng E2, a group (n = 10) injected with 100 µl KRB containing 270 ng E2 + 100 µl KRB containing 6 µg ICI 182–780 (Zeneca) and a group (n = 10) injected with 100 µl KRB containing 270 ng E2 + 100 µl KRB containing 3.7 µg Tamoxifen (Sigma Chemical Co.) for 2 weeks on alternate days. After 2.5 h from the last injection testes were removed and used for Western blot analysis or immunocytochemistry for evaluation of immunopositive ISPG. Immunopositive ISPG have been counted in three randomly chosen sections for each testis from each frog (n = 10 animals/time/experimental group). The number of cytoplasmic, perinuclear and nuclear immunopositive ISPG was expressed as number of immunopositive cells/total ISPG counted/section multiplied by 100.
In vitro experiments
Thirty animals have been captured in two different periods
(February and May) and testes removed and placed in cold KRB. After
washing in KRB at 20-22 C, 20 testes (time 0) have been homogenized in
guanidium isothyocyanate while the remaining 40 testes have been
incubated at 20–22 C in KRB containing E2
(10-6 M) or E2 (10-6
M) + ICI 182–780 (10-5 M) for 1
and 2 h. Twenty testes/time/treatment have been processed for
Northern blot analysis.
Statistics
Statistical analysis for the significance of differences was
performed using ANOVA followed by Duncan’s test for multigroup
comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and
B; P < 0.01; a vs. b and a' vs.
d) as compared with controls. The increase is more pronounced at
48 h (Fig. 1, A and B; P < 0.01; b vs.
d). This effect is counteracted by ICI 182–780 (Fig. 1, A and B;
P < 0.01; b vs. c and d vs. a').
A similar trend was obtained using Tamoxifen (not shown).
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In cytoplasmic preparations, FOS-related proteins are present during
the period lasting from October until March; three immunoreactive
specific bands of about 60, 80, and 100 kDa, respectively, have been
found (Fig. 2). In nuclear preparations,
FOS-related proteins are present from June until September, showing a
single specific band of about 60 kDa (Fig. 3). In MCF7 cell line, used as positive
control, a specific band of 80 kDa is present as expected (not
shown).
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).
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, lane 2
and 3), whereas testes incubated with E2 + ICI 182–780
show a weak signal of the same size either after 1 h (Fig. 5, lane
4) or 2 h incubation (not shown). No effects were found in May
testes (not shown).
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, A and B)
in ISPG, either in the cytoplasm (control group) or in the nuclear
compartment (E2-treated group). The antisera used fulfill
the criteria for investigating the specific localization. In fact,
immunoabsorption tests revealed that the labeling was totally blocked
by preincubation with excess amount of the peptide (Fig. 6C). In
control animals, the number of ISPG (Fig. 6D) showing cytoplasmic
localization of FOS is significantly higher as compared with the number
of the cells with both perinuclear and nuclear immunostaining
(P < 0.01 a vs. b and a vs.
c). Estradiol is able to increase the number of ISPG with both
perinuclear and nuclear FOS immunopositivity (P <
0.001 d vs. e and d vs. f). A scanty but
significant decrease of ISPG cytoplasmic localization is observed in
E2-treated animals (P < 0.01 a
vs. d). The combined treatment of E2 + Tamoxifen
shows an increase of the number of ISPG with cytoplasmic localization
of FOS and a decrease of the number of ISPG with both perinuclear and
nuclear localization (P < 0.01 g vs. d
and g vs. a). Similar results were obtained using ICI
182–780 (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With respect to E2 and its signal transduction mechanism in R. esculenta testis, Northern blot analysis reveals that in February c-fos mRNA increases after 1 h incubation, being this effect counteracted by ICI 182–780. Absence of E2 effects during May is in agreement with the slow down of SPG mitosis in this species (21). The c-fos transcript has identical size of that previously described during the sexual cycle of R. esculenta (20). Moreover, a high molecular weight (about 200 Kda) c-fos-related protein was also detected in cytosolic, but not nuclear, extract after estradiol treatment in vivo for two weeks. The detection of a c-fos-related protein with a molecular weight of about 200 kDa is difficult to explain. We remember that oncoproteins of molecular weight significantly higher than that estimated from the transcript size have already been found (7, 38, 39). Many hypotheses have been proposed to explain such discrepancies as for example physical association of the oncoproteins or the presence of distinct cell proteins physically associated to the oncoprotein in the immunocomplex (38). Moreover, denaturing conditions are often not sufficient in separating completely a protein complex (40).
The presence of immunoreactive FOS proteins in the cytoplasm of spermatogonia in the frog testis also occurs during the annual sexual cycle as observed by immunocytochemistry (6). Although contribution by somatic cells cannot be excluded in the Western blot data, it is important to remember that cytoplasmic localization has never been found in somatic cells in a previous study (6). Present results indicate that at least three immunoreactive specific bands (as indicated by the preabsorbed antiserum) of about 60, 80 and 100 kDa, respectively, have been evidenced in the cytoplasmic preparations of R. esculenta testis. Interestingly, the 60 and 80 Kda bands are within the range of the expected size previously described for FOS (see for example reference 41 and manufacturer’s instructions for the use of anti c-FOS OA-11–822). The protein of about 60 kDa is clearly available in the nucleus during June-September period when FOS immunoreactivity is observed in SPG nuclei by immunocytochemistry (6).
The possibility that "immediate early nuclear proto-oncogene" products are stored in cytoplasm and translocated in nuclei, although heretic, has recently been raised (6, 42, 43, 44). With respect to our results, the cytoplasmic localization of FOS occurs when spermatogenesis shuts down (autumn-winter), therefore it is conceivable to hypothesize that FOS may translocate in SPG nuclei inducing mitotic activity during the period characterized by active spermatogenesis (spring-summer). In this respect, we remember that in R. esculenta plasma and testicular estradiol peak in concomitance with the new annual spermatogenic wave (15, 16, 23). Moreover, we show here that estradiol treatment induces a significant increase of nuclear and perinuclear immunostaining in SPG; this effect is counteracted by antiestrogens. We have no explanation for the enormous increase of number of ISPG with cytosolic FOS immunostaining after E2 + antiestrogen treatment. We speculate that FOS synthesis may also be induced by other signals being the transport into the nucleus blocked by Tamoxifen or ICI 182–780. Thus, the spermatogonial proliferation may be triggered by an estradiol mediated mechanism involving FOS transport into the nucleus. Due to the in vivo experimental protocol we cannot exclude that estradiol may act via the pituitary gland. Indeed, gonadotropins have been claimed to induce SPG proliferation in R. esculenta (45). However, it has also been shown that estradiol strongly inhibits gonadotropin discharge in frogs (46). A positive feed back exerted by estradiol on frog pituitary has never been described so far.
In conclusion, using the frog model, which is characterized by a slow progression of spermatogenesis, we have shown for the first time in a vertebrate species that estradiol induces c-fos activity in the testis and that it may be involved in mechanisms related to the FOS transport into the nucleus of spermatogonia.
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Acknowledgments |
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Footnotes |
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Received September 2, 1998.
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References |
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-dihydrotestosterone,
testosterone, melatonin and serotonin. Gen Comp Endocrinol 64:405–410[CrossRef][Medline]
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