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REPRODUCTION-DEVELOPMENT |
Dipartimento di Medicina Sperimentale, Sez. F. Bottazzi, II Università di Napoli, 80138 Naples, Italy
Address all correspondence and requests for reprints to: Dr. Riccardo Pierantoni, Dipartimento di Medicina Sperimentale, Sez. F. Bottazzi, II Università di Napoli, Via Costantinopoli 16, 80138 Naples, Italy. E-mail: riccardo.pierantoni{at}unina2.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ER knockout (ERKO) mice show impairment of sperm
production, and this has been ascribed to a defect of luminal fluid
reabsorption in the head of epididymis (2, 3). This seems
to be proved by transplantation of spermatogonia (SPG) from ERKO
mice in normal animals deprived of germ cells (4). Results
indicate that spermatogenesis arising from ERKO spermatogonia is not
disrupted; therefore, spermatogenic cells do not require ER for
development or function. However, a direct effect of estrogens on
tubular compartment is also considered. Indeed, E2 restores
spermatogenesis in hypogonadic mice (5), and ßER have
been found in germ cells (6). The use of nonmammalian vertebrate animal models has given insight into the mechanisms underlying spermatogenesis (1, 7, 8, 9). Several advantages can be ascribed to the use of these models. In particular, the presence of numerous seasonal breeder species allows the study of different stages of germ cells appearing progressively during the annual sexual cycle. Moreover, spermatogenesis occurs in cysts consisting of Sertoli cells enveloping germ cells at the synchronous stage (10); this allows an easy recognition of germ cell associations. Recently, we have used the frog, Rana esculenta, to localize protooncogene products appearing in the testis during the annual sexual cycle. R. esculenta is characterized, during the year, by a potentially continuous spermatogenesis (i.e. SPG never become refractory) (11) with a period of SPG proliferation (mid-winter, early spring) and a period in which spermatogenesis shuts down (late autumn, early winter) (12, 13). Interestingly, Fos protein immunoreactivity is localized in the cytoplasmic compartment of SPG during the resting period, whereas a nuclear localization is evidenced when spermatogenesis resumes (14); this process seems to be regulated by estrogens (15). Cytoplasmic localization of immediate-early gene products has also been described in other models, as, for example, during spermatogenesis in Vulpes vulpes (16), in cancer cells (17, 18), in ovine trophoblasts (19), in neuronal cells (20), and in Xenopus oocytes (21). Therefore, a change in the localization of Fos protein from the cytoplasm into the nucleus may switch on spermatogenesis. In this respect, possible differences in Fos forms between cytoplasm and nucleus can be hypothesized.
The scope of this paper was to determine whether these differences exist and to look at possible relationships with the progression of spermatogenesis. We describe the Fos protein profile in the testes of the frog, R. esculenta, during the annual sexual cycle. We show a 52-kDa Fos immunoreactive protein in cytosolic extracts, whereas two different Fos signals of 43 and 68 kDa are evidenced in the nuclear compartment. The 68-kDa signal increases in nuclear extracts concomitantly with the SPG proliferation period and also in animals in which SPG proliferation has been stimulated by thermal manipulation at 24 C. Experiments carried out using alkaline phosphatase (AP) and AP inhibitors indicate that the 68-kDa protein is a phosphorylated form. Furthermore, the thermal treatment suggests that this form derives from the 52-kDa Fos protein. Finally, we indicate that the presence of the 43-kDa form in nuclear extract is induced by E2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytoplasmatic and nuclear protein extraction
Cytoplasmic and nuclear extracts were prepared by modifications
of methods previously described (22). Briefly, 100 mg
testis tissue were gently homogenized using a type B pestle in 0.7 vol
buffer [10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 12% glycerol, 0.1
mM EGTA, 0.5 mM dithiothreitol (DTT), and 0.5
mM spermidine] in the presence of protease inhibitors [4
µg/ml leupeptin, aprotinin, pepstatin A, chymostatin,
phenylmethylsulfonylfluoride (PMSF), and 5 µg/ml
N--p-tosyl-L-lysine chloromethyl ketone
(TPCK)]. After centrifugation at 800 x g, the
supernatant was removed and centrifuged on glycerol at 100,000 x
g for 1 h at 4 C to obtain cytosolic extracts. The
nuclear pellet (presence and purity of the nuclei were checked under a
microscope), washed three times, was resuspended with 1.2 vol (1.2
µl/mg pellet) of buffer [10 mM HEPES (pH 7.9),
1.5 mM MgCl2, 420
mM NaCl, 15% glycerol, 0.1
mM EGTA, 0.5 mM DTT, and 2
mM spermidine] in the presence of protease
inhibitors and mix at 4 C for 30 min. Nuclei were pelleted by
centrifugation at 10,000 x g for 30 min at 4 C. The
supernatants, containing nuclear proteins, were collected. Protein
concentrations of cytoplasmic and nuclear extracts were determined
using the Lowry assay (23).
Mouse-specific germ cell J protein [MSJ (24)], also available in R. esculenta (25), and proliferating cell nuclear antigen (PCNA) were assayed to check for the absence of contamination (not shown). Indeed, the former is specifically available in the cytoplasm (26) and the latter in the nuclei (27) of testicular cells.
Western blot analysis
Proteins (25 µg/well) were separated by 8%, 10%, or 12%
(depending on the case) SDS-PAGE and then transferred to nitrocellulose
membrane (Amersham Pharmacia Biotech, Little Chalfont, UK)
for 2.5 h at 280 mA and 4 C. The filters, stained by Ponceau S
(Sigma, St. Quentinen- Yvelines, France) as the
control of protein loading, were treated for 3 h to prevent
nonspecific adsorption with blocking solution (5% nonfat powdered milk
and 0.25% Tween 20 in Tris-buffered saline, pH 7.6) and finally
incubated overnight at 4 C on orbital shaker with the primary antibody
in a blocking solution lacking Tween 20. Anti-Fos and anti-PCNA were
diluted 1:1000. The filters, washed three times in Tris-buffered
saline, pH 7.6, containing 0.25% Tween 20, were incubated for 1 h
with appropriate horseradish peroxidase-conjugated anti-IgG diluted
1:1000 (DAKO Corp., Copenhagen, Denmark) and then washed
again. The immune complexes were detected using the ECL Western
blotting detection system (Amersham Pharmacia Biotech)
following the manufacturer’s instructions. The membranes, stripped at
60 C for 30 min in stripping buffer (100 mM
2-mercaptoetanol, 2% SDS, and 62.5 mM Tris-HCl, pH 7.6),
were reprobed one time to check the specificity of immunoreaction using
preadsorbed antibody with 10-6 M
antigen.
Total protein extraction and immunoprecipitation
Frozen testes were homogenized in ice-cold lysis buffer [3 ml/g
tissue 10 mM HEPES, (pH 7.9), 1.5 mM
MgCl2, 420 mM NaCl, 15% glycerol,
0.1 mM EGTA, 0.5 mM DTT, and 2 mM
spermidine] in the presence of protease inhibitors (4 µg/ml
leupeptin, aprotinin, pepstatin A, chymostatin, PMSF, and 5 µg/ml
TPCK) and then centrifuged several times at 10,000 x g
for 10 min at 4 C to obtain a clarified lysate. Then, 600 µg total
protein were used for immunoprecipitation as follows: it was diluted by
adding 10 vol RIPA buffer [PBS, pH 7.6 (9.1 mM
Na2HPO4, 1.7
mM
NaH2PO4, and 150
mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS] and incubated with 10 µl primary
anti-Fos antibody (0.2 µg/µl; sc-253-G, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C. The
immunoprecipitation was carried out at 4 C on a rocker platform
overnight, adding 20 µl of the appropriate immunoprecipitation
reagent (0.25 µg/µl; Protein G-Plus-Agarose, sc-2002, Santa Cruz Biotechnology, Inc.). Immunoprecipitates were pulled down
by centrifugation at 1000 x g for 5 min at 4 C and
washed four times with PBS, pH 7.6, each time repeating the
centrifugation step. Finally, the pellet was dissolved in 20 µl
electrophoresis sample buffer [80 mM Tris-HCl
(pH 6.8), 10% glycerol, 4% SDS, 100 mM DTT, and 300
µg/ml bromophenol blue] and boiled for 5 min. Immunoprecipitates
were separated through 8% SDS-PAGE and analyzed by Western blot using
anti-Fos antibody (0.2 µg/µl; sc-253-G, Santa Cruz Biotechnology). The negative control was obtained as described
above using RIPA buffer instead of the primary antibody or instead of
the total protein extract.
Antibody
Polyclonal anti c-Fos antibody (sc-253-G, Santa Cruz Biotechnology, Inc.), used for Western blot and
immunoprecipitation, was raised in goats against an acid sequence
within a highly conserved domain in Fos family members (c-Fos, FosB,
Fra-1, and Fra-2) of human origin, identical to corresponding mouse,
rat, and chicken sequences (as reported in the manufacturer’s
instructions).
Anti-PCNA (PC10; sc-56, Santa Cruz Biotechnology, Inc.) was a mouse monoclonal antibody. It is reported in the manufacturer’s instruction that the antibody reacts with PCNA p36 protein of mouse, rat, human, insect, and yeast origin by Western blot.
The specificity of the Fos signals found by Western blot was tested by extinguishing the reaction with excess amount (10-6 M) of the cognate peptide (sc-253-P, Santa Cruz Biotechnology, Inc.) and through immunoprecipitation as described above.
Seasonal cycle
To detect Fos immunoreactivity, cytosolic and nuclear extracts
were prepared from frog (n = 10/month) testes collected monthly
for 1 yr and used for Western blot analysis. During the second year,
the cycle was checked to perform in vivo and in
vitro experiments.
Induction of spermatogonial proliferation by thermal
treatment
The experiment was carried out in October, when SPG
proliferation slows. Ten fresh animals were killed, whereas the
remaining 20 animals were kept for 3 months at low temperature (4 C)
and injected on alternate days with homologous hypophysis homogenate
(100 µl amphibian Krebs-Ringer bicarbonate buffer containing one
third hypophysis equivalent per injection) in the dorsal sac. It is
well known that this treatment empties the testis of meiotic cells, and
only slow proliferating SPG cells are detectable (12, 13).
After 3 months of treatment, 10 animals were killed, and 10 animals
were treated for an additional week at 24 C with hypophysis homogenate
to induce SPG proliferation. Testis fragments from control frogs and
from 4- and 24 C-treated animals were prepared for routine histological
observation (hematoxylin/eosin staining) to check germ cell stage
composition. Furthermore, nuclear extracts were analyzed by Western
blot for PCNA content to check the status of the cell cycle.
Dephosphorylation experiments
In the first experiment, nuclear proteins (140 µg), extracted
from January testes, were incubated at 37 C in the presence of protease
inhibitors (4 µg/ml leupeptin, aprotinin, pepstatin A, chymostatin,
PMSF, and 5 µg/ml TPCK: 80 µg) were incubated with phosphatase
inhibitors (0.3 nM okadaic acid, 10 mM
p-nitrophenylphosphate, 10 mM sodium
pyrophosphate, and 0.1 mM sodium orthovanadate),
and 60 µg were incubated without phosphatase inhibitors. Aliquots of
20 µg proteins were stored on ice for different times (0, 5, 15, and
30 min) and analyzed by Western blot.
In the second experiment, five aliquots (20 µg each) of a nuclear protein extract (still extracted from January testes) were dephosphorylated by increasing quantities (0, 4, 10, and 20 U) of AP from calf intestine (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at 30 C. In an additional tube, 20 µg nuclear protein extract were incubated with a cocktail of AP inhibitors, used at a 50-fold dilution (phosphatase inhibitor cocktail 1, Sigma-Aldrich Corp.) and with a maximal quantity of AP from calf intestine (20 U) for 15 min at 30 C.
17ß-E2 in vivo treatment
Animals collected during February were separated into three
experimental groups (n = 10): the control group was injected with
100 µl KRB (pH 7.4), the E2-treated group was injected with 100 µl
KRB containing 10-5 M 17ß-E2, the
E2+antagonist-treated group was injected first with 50 µl KRB
containing 10-3 M of the estrogen
antagonist ICI182–780 (Zeneca Pharmaceuticals,
Macclesfield, UK) and then, after 1 h, with 50 µl KRB
containing 10-5 M 17ß-E2.
Injections were given in the dorsal sac on alternate days for 2 wk.
Statistical analysis
Quantification of 52- and 68-kDa signals detected by Western
blot analysis during the annual sexual cycle was carried out using a
transmitting scanning densitometer (Ultrascan XL, LKB,
Bromma, Sweden), evaluating the integration of peak areas of
each month. The relative amounts of the signals are expressed as the
fold increase over the minimal value registered.
ANOVA followed by Duncan’s test for multigroup comparison were carried out to assess the significance of differences. Data were considered significantly different at P < 0.05 and are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
intensity of this band (Fig. 1B) was higher from September through
December (P < 0.01) than in the January through July
period, with June excluded (P < 0.01, June
vs. July).
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). In particular, the 43-kDa signal
was present in September through October only, whereas the 68-kDa
signal was present from January until December. The intensity of this
band (Fig. 1D) was higher (P < 0.01) during the
January through July period (June excluded, P < 0.05)
and decreased thereafter.
Fos-immunoreactive protein expression in testes with and without
proliferating SPG after thermal treatment
To study the role of Fos protein in spermatogenesis, Fos pattern
expression has been analyzed in total protein extract from testes of
October animals, from testes of animals showing only slow proliferating
SPG (animals at 4 C), and from testes resuming normal proliferating SPG
(animals at 24 C). Histological observations indicated that in control
testes all spermatogenic stages were present, whereas 4 C frog testes
were almost exclusively composed of SPG (Fig. 2). Signs of spermatogenesis progression
(appearance of very few spermatocytes) were evidenced in 24 C-treated
animals (not shown). Moreover, to check the resumption of the SPG cell
proliferative cycle at 24 C, testicular PCNA content has been analyzed
by Western blot. As expected, PCNA signal was stronger in testes of 24
C-treated animals, indicating that SPG proliferation resumes (Fig. 3A). The lower amount of PCNA in control
group is consistent with meiotic and postmeiotic germ cell populations
available in the testis of October animals compared with the 4 C group,
in which SPG predominate. PCNA detection by immunocytochemistry has
previously been reported in R. esculenta testis
(27). With respect to Fos analysis, Western blot indicated
that the 52- and 68-kDa immunoreactive proteins were present in all
testicular preparations (Fig. 3B). In particular, 52-kDa signal was
strong in control frogs and in those kept at 4 C. A decrease in signal
was found in animals treated at 24 C when SPG proliferation restarts.
Conversely, 68-kDa signal increased at 24 C (Fig. 3B). To further check
the presence of specific 52- and 68-kDa Fos related proteins in the
total protein extract of October animals, immunoprecipitation was
carried out with the same Fos antibody as that used for Western blot
analysis. The two proteins, showing reverse relations in the thermal
treatment, were found (Fig. 4).
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, lane 1). In the first
experiment the extract was incubated for 5, 15, and 30 min with or
without phosphatase inhibitors (Fig. 5, lanes 2–4 and 5–7,
respectively). The 68-kDa signal was present only in the nuclear
extracts incubated with phosphatase inhibitors, whereas it disappeared
when the inhibitors were absent. In the second experiment the extract
was dephosphorylated by increasing concentration of AP. The intensity
of the 68-kDa band decreased in the presence of increasing
concentrations of AP (Fig. 6, lanes
2–4), but it was still present when the nuclear extract was incubated
with AP in combination with specific inhibitors (Fig. 6, lane 5). These
results indicated that the 68-kDa Fos immunoreactive protein was a
phosphorylated form.
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). Fifty-two- and 68-kDa immunoreactive
proteins appeared in cytoplasmic (Fig. 7A) and nuclear (Fig. 7B)
preparations, respectively, and were not affected by E2 treatment.
Conversely, a 43-kDa Fos protein appeared in testicular nuclear
extracts of animals treated with E2. The specificity of the signals was
determined through the use of preabsorbed antibody (Fig. 7, lower panels).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fos family proteins are constituted by several members, such as c-Fos,
FosB, Fra-1, and Fra-2. These proteins dimerize with Jun family members
to constitute a transcription factor, named activator protein 1.
Different homodimers (Jun-Jun) and heterodimers (Fos-Jun) have been
proved to have different transcriptional activity (28).
c-Fos activity has been detected in mammalian testis, particularly in
premeiotic germ cells during spermatogenesis in the mouse
(29). In the frog, R. esculenta, we have shown
that the appearance of Fos first in the cytoplasmic and then in the
nuclear compartment may trigger SPG proliferation and resumption of the
spermatogenic wave during the annual sexual cycle (15). In
this paper, using an antiserum raised against all Fos family members,
we characterize the different Fos forms available in cytoplasmic and
nuclear compartments and show relationships with SPG proliferation. By
Western blot analysis, we confirm the presence of Fos immunoreactivity
in the cytoplasm and nuclei of testicular cells (Ref. 15
and present data). As previous results indicate that the cytoplasmic
localization is exclusively ascribed to spermatogonial cells
(14), the presence of Fos protein is attributable to these
germ cells. Immunoprecipitation (Fig. 4) and preabsorption (Fig. 7) of
Fos antibody have been carried out to test the antiserum used. However,
the Fos protein pattern detected here shows some differences compared
with previous observations (15). Some changes in the
methods used in the present work may explain these results.
Cytosolic Fos signal available during the annual sexual cycle is represented by 52-kDa protein, showing a weak intensity during the active SPG proliferation period (January–July) and a strong intensity during the resting phase (September–December). Interestingly, the 68-kDa signal in the nucleus mismatches the 52-kDa immunoreactivity available in the cytoplasm. In fact, a strong signal is detected between January and July. We have no explanation for the strong immunoreaction related to the 52-kDa protein in cytosolic extracts during June, but we confirm that concomitantly with a strong signal in one cellular compartment, a weak signal is detectable in the other (note the decrease in 68-kDa intensity during June in the nuclear compartment as the strong 52-kDa signal in the cytoplasm appears). Overall, these results suggest that the 68-kDa Fos-related protein is involved in the regulation of SPG proliferation.
To support this hypothesis, we have treated frogs under thermal conditions that slow (4 C) or trigger (24 C) SPG proliferation. Indeed, the lack of refractoriness of SPG to environmental stimuli in potentially continuous spermatogenesis (11) may be particularly useful in this animal model to study SPG proliferation. Western blot analysis of whole protein extract of October animals shows a strong 52-kDa signal. This is attributable to a cytoplasmic localization, as evidenced by the results obtained during the annual sexual cycle. Animals kept at 4 C for 3 months and treated with homologous hypophysis homogenate have testes primarily showing slow proliferating SPG, as checked by histological observation. It is well known that the above treatments slow SPG proliferation due to the low temperature while allowing postmitotic germ cells to mature until spermiation produced by gonadotropin treatment (12, 13). Therefore, results from testes of 4 C-treated frogs (deprived of postmeiotic and meiotic stages) further confirm that the 52- and 68-kDa signals found in controls are ascribed to SPG cells. Moreover, the analysis carried out using whole protein extract permits a better understanding of the relationship between the two molecular forms because they are observed in the same preparation. Interestingly, when 4 C-treated frogs, showing primarily SPG and deprived of other stages, are transferred to a condition (24 C) that accelerates the SPG proliferative cell cycle, as indicated by PCNA content, and triggering spermatogenesis (12, 13), we detect a weak 52-kDa immunoreactivity and a highly intense 68-kDa signal. As this signal has been shown to be typical of the nuclear compartment when spermatogenesis resumes during the annual sexual cycle, it is easy to conclude that the 68-kDa protein in 24 C-treated frogs should be present in SPG nuclei. Therefore, we indicate that the regulatory role of the 68-kDa protein in the nuclei during spermatogenesis may be concerned with the induction of SPG mitosis. In addition, a reverse relationship exists between cytoplasmic and nuclear Fos forms.
The inverse relationship between the 52- and 68-kDa Fos signals, available both during the annual sexual cycle and in thermal treated animals, may indicate that the higher molecular form of Fos-immunoreactive protein is due to a modification of the 52-kDa protein. Phosphorylation may account for this. If so, the addition of phosphatase inhibitors to the nuclear extract must protect the putative phosphorylated 68-kDa protein. Conversely, disappearance of the signal should be detected in the absence of phosphatase inhibitors. As this is the case, we strongly indicate that the 68-kDa signal is constituted by a phosphorylated protein. To further support this conclusion, nuclear testicular extracts were incubated with increasing concentrations of AP alone or in combination with specific inhibitors. Our results unequivocally show that the 68-kDa signal gradually decreases with concomitant increase in AP concentration, whereas the inhibitors counteract this effect. Thus, phosphorylation was definitely proved. Surprisingly, the 52-kDa form was not observed after dephosphorylation. We have no explanation for this phenomenon, but we speculate that after dephosphorylation the protein is rapidly digested in the nuclear extract. Dephosphorylated c-Fos forms have been reported to have a short half-life (30). Furthermore, c-Fos degradation can be observed despite the presence of protease inhibitors (31). In this respect, persistence of the 52-kDa form (see thermal experiment) when a total (cytoplasmic plus nuclear) protein extract is used may indicate protection exerted by cytosolic substances. It is worthy of note that the inverse relationship between 52- and 68-kDa forms is clearly evidenced with thermal treatment. In addition, both proteins can be immunoprecipitated from a total protein extract.
To gain further information on nuclear Fos proteins, we treated frogs with E2, which induces strong Fos localization in SPG nuclei. Western blot analysis confirmed the presence of 52- and 68-kDa proteins in cytosolic and nuclear testicular extracts, respectively. Interestingly, the 43-kDa protein is the form found in nuclear extracts of E2-treated animals. The antiestrogen ICI counteracts the E2 effect, indicating that the appearance of the 43-kDa protein is dependent on an ER-mediated mechanism. The presence of ER in amphibian, R. esculenta included, testis has been proved previously (32). Furthermore, this experiment, together with specific markers of cytosolic and nuclear extracts (MSJ and PCNA), respectively, can be used to asses the absence of contamination in nuclear and cytosolic extracts.
Although E2 treatment confirms the presence of the 43-kDa signal described during the annual sexual cycle, we have no explanation for the appearance of this signal during September through October, when endogenous estrogens are undetectable (33, 34). Probably very low estrogen levels, below the sensitivity of the RIA method used, may explain this result. Alternatively, regulation by other factors during September and October in frog testis may induce the presence of the 43-kDa Fos-related protein. For example, modulation of the 43-kDa protein has been obtained by treating frogs with a GnRH analog (Cobellis, G., R. Meccariello, S. Minucci, C. Palmiero, R. Pierantoni, and S. Fasano, unpublished).
A final speculation that can be made from the present results concerns the identities of the different Fos-related signals. Indeed, an antiserum that recognizes all Fos family members has been used in the present study. It is well known that c-Fos protein may have different molecular masses depending on phosphorylation status. Indeed, 52- to 68-kDa c-Fos proteins have been found in other systems (35, 36, 37, 38). As in our system, 52 and 68 kDa are well in the range of the size of c-Fos proteins reported in literature, we suppose that the 68-kDa signal is a hyperphosphorylated c-Fos form derived from the 52-kDa c-Fos protein. Therefore, the absence of 52-kDa signal in the nuclear extract of E2-treated frog testes may be explained by its rapid transformation into the 68-kDa form after translocation into the nuclear compartment (see also data from 4- and 24 C-treated frogs showing the inverse relationship between the two signals and absence of the 52-kDa form in nuclear extracts after dephosphorylation experiments). Which factors are responsible for the putative translocation of the 52-kDa form should be matter of further investigation.
The 43-kDa signal is well within the size range of Fra proteins, which are members of the Fos family (39, 40). Interestingly, Fra-1 and Fra-2 have been localized in spermatids during spermatogenesis in mammals (16). The appearance of spermatids in the frog, R. esculenta, testis has been described to occur during September through October (12, 13) when 43-kDa signal is available during the annual sexual cycle.
In conclusion, our results show for the first time in a vertebrate species that storage and phosphorylation of Fos proteins in the cytoplasm and nucleus of germ cells, respectively, regulate testicular activity. In particular, a phosphorylated 68-kDa Fos form may be involved in the mechanisms underlying SPG proliferation.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP, Alkaline phosphatase; DTT, dithiothreitol;
ERKO, ER knockout; PCNA, proliferating cell nuclear antigen; PMSF,
phenylmethylsulfonylfluoride; TPCK,
N--p-tosyl-L-lysine chloromethyl
ketone.
Received June 22, 2001.
Accepted for publication July 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for
development or function. Endocrinology 141:1273–1275
expression and early
implantation process. Mol Reprod Dev 46:127–137[CrossRef][Medline]
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