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Regular Article |
-Irradiated Rats During Testicular Development1
a Département de Radiobiologie et Radiopathologie, DSV/LRCG/CEA, 92265 Fontenay-aux-Roses Cedex, France
ABSTRACT
In fetal and newborn rat testes, gonocytes, which stop cycling for about 8 days, become highly radiosensitive. The presence of p53, p21, mdm2, and pRb, which are involved in cell cycle, apoptosis control, or both, were studied by immunohistochemistry to determine if their expression is related to this radiosensitivity. A strong cytoplasmic expression of p53 and p21 was detected. Cytoplasmic expression of p53 occurred only in arrested gonocytes, whereas that of p21 was observed before and after the block. P21 was found to colocalize with mitochondria. No expression of mdm2 was detected and pRb was present only when the gonocytes started cycling again. In animals exposed to 1.5 Gy of gamma-irradiation at Day 19 postcoitum, p53 expression was prolonged in time, whereas no change was observed in p21 amounts and localization, compared with controls. Using antibodies against 5-methyl cytosine, it was shown that gonocyte DNA passed from a hypomethylated to a methylated status 1 day after gonocytes stopped cycling. A prolonged survival of gonocytes after exposure to radiation was followed by their progressive apoptosis, which finally involved the entire gonocyte population between Days 6 and 12 postpartum. The elevated but delayed sensitivity of gonocytes to genotoxic stress may be related to the unusual expression of p53 and p21, which may itself be related to the large DNA methylation changes.
apoptosis, developmental biology, spermatogenesis, testes
INTRODUCTION
The establishment of spermatogenesis is a complex process involving cells from embryonic primordial germ cells to adult spermatozoa. In the seminiferous epithelium, germ cells are in close contact with Sertoli cells, which have an essential role as a nutritional source of growth factors and for structural support. Sertoli cells proliferate actively during fetal life with a maximum at Day 20 postcoitum (pc). They stop proliferating at Day 21 postpartum (pp) [1]. In rat fetuses, just after sexual differentiation, gonocytes start to proliferate in recently formed seminiferous cords from Day 14 pc. They stop proliferating from Day 18 pc until Day 4 pp, when they again begin to proliferate and progressively differentiate into type A spermatogonia. The testis is known to be sensitive to ionizing radiation, and in particular, during the period when gonocyte proliferation is arrested [2, 3]. Several proteins such as p53, mdm2, p21(WAF1/CIP1), and pRb are involved in the control of cell cycle progression, in response or not to DNA damage. The G1/S and G2/M checkpoints are under the control of p53 [4–6], which is highly expressed in the normal rat testis during the first round of spermatogenesis [7]. After irradiation, p53 expression increases in a time- and dose-dependent manner in adult rat germ cells [8, 9].
The amount of p53 is negatively regulated by the mdm2 oncoprotein, which is encoded by a p53-inducible gene [10, 11]. The nucleo-cytoplasmic shuttling of mdm2 is essential to promote p53 degradation. Recent data have shown that p19ARF blocks the shuttling of mdm2 and could stabilize p53 by inhibiting the nuclear export of mdm2 [12]. DNA damage can prevent the binding of mdm2 with p53 and induce p53 stabilization by inhibiting its degradation [13].
The p21 protein is a component of the quaternary cyclin-dependent kinase (cdk) complex, which includes proliferating cell nuclear antigen, cdk4 or cdk6, and D-cyclin [14]. p21 is a negative regulator of the cell cycle, inhibiting cdk2, cdk3, cdk4, and cdk6, which have a direct role in the control of the G1- to S-phase transition [15, 16]. Its expression, which is largely p53-dependent [17, 18], may be p53-independent as well [19–21]. In adult mouse testis, p21 is weakly expressed in spermatocytes at the pachytene stage and in spermatids. Ionizing radiation increases the amount of p21 in these cells, but not in spermatogonia, whereas that of p53 increases in both cell types [22].
The retinoblastoma tumor suppressor gene (RB1) is a cell cycle regulator that inhibits the progression from G1 to S phase [23–25]. Its product, pRb, a nuclear phosphoprotein, is expressed in most tissues as hypophosphorylated or hyperphosphorylated forms. The hypophosphorylated, active form of pRb binds the transcription factors DP-1 and E2F, repressing their transactivation function. Phosphorylation inactivates pRb, which will release transcription factors that carry out the activation of cell cycle progression [26]. pRb is expressed in mouse [27], human [28], and rat testes [29, 30]. In adult rat seminiferous epithelium, pRb has been detected in Sertoli cells, spermatogonia at all stages of differentiation, and in the elongated spermatids, suggesting its involvement in the regulation of spermatogonia proliferation and in maintenance of the differentiation status of Sertoli cells and spermatids [30].
Methylation of deoxycytidine residues of CpG in mammalian DNA is involved in the regulation of gene expression during development [31]. DNA methylation is known to induce or maintain the inactivation of some genes [32]. In mouse testis, Coffigny and colleagues [33] have reported that between Days 16 and 17 pc, mouse gonocytes passed from a demethylated state to a strongly methylated state, which is maintained during the remaining period of mitotic arrest. Such a modification of the DNA methylation status may alter gene expression and may be involved in the unusual radiosensitivity of rat gonocytes.
Our purpose was to investigate the causes of the particular radiosensitivity of fetal and neonatal rat germ cells. By immunohistochemistry and Western blotting, we have studied the expression of p53, p21, mdm2, and pRb during the testicular development in control and irradiated rats, and the overall DNA methylation status using anti-5-methyl cytosine (5-mC) antibodies.
MATERIALS AND METHODS
Animals and Irradiation
Female rats from the Sprague-Dawley strain (IFFA-CREDO, France) were caged with males between 0800 h and 1100 h. The day of mating was counted as Day 0 pc. Birth occurred generally at the end of the 22nd day of pregnancy, corresponding to Day 0 pp, regardless of the effective date of parturition. Pregnant rats from gestational Day 19 were exposed to 
-irradiation using a 60>Co source with a total dose of 1.5 Gy, and a dose rate of 0.25 Gy/min. Immunohistochemical studies were performed on 16- to 21-day-old fetuses and 1- to 13-day-old newborns.
For p53 positive and negative immunohistochemical controls, wild-type and p53 knockout mice were kindly provided by Dr. E. May (CEA, Fontenay-aux-Roses, France).
Treatment with Actinomycin D and Cycloheximide
Rats at Days 2 and 6 pp were injected twice, at a 4-h interval, with actinomycin D or cycloheximide dissolved in saline, at a dose of 3 µg/g by body weight. Animals were killed 4 h after the second injection, and their testes were immediately removed and fixed.
Fixation
Immunohistochemistry Testes were fixed overnight at 4°C with 4% (w/v) paraformaldehyde-PBS or a diluted Bouins fluid (2% [w/v] picric acid, 25% [v/v] formaldehyde, and 5% [v/v] acetic acid), dehydrated, and embedded in paraffin. Sections (5 µm) were performed and mounted on poly-L-lysinated slides.
Electron microscopy Testes were fixed (1 h) in either buffered 1% (v/v) glutaraldehyde or buffered 2% (w/v) paraformaldehyde-0.5% (v/v) glutaraldehyde, and postfixed (1 h) in 1% (v/v) osmium tetroxide. Thin sections were stained with uranyl acetate and lead citrate, and observed with a Philips EM 300 electron microscope under 80 kV.
Measurement of Mitotic Index and Testicular Atrophy
In control and irradiated animals, gonocyte mitotic index was measured at Days 16, 17, 18, 19, 20, and 21 pc, and 2, 3, 4, 5, and 6 pp. Five pups from two or three distinct litters were killed for each developmental age. Mitoses were accounted on sections among 500–1500 germ cells after histological staining according to the Feulgen and Rossenbeck method [34].
Testicular atrophy was determined by weighing the left testis of 70-day-old rats, which were exposed to a 1.5-Gy dose of irradiation during the gestation or the postnatal period. Testes of 15 rats from three distinct litters were studied for each time point. The data were reported in percentage and compared with the testicular weight of 50 control rats, which represented 100%.
Evolution of Gonocyte Population in Irradiated Rats
BrdU incorporation 5-Bromodeoxyuridine (BrdU, Sigma, St. Quentin Fallavier, France) was injected i.p. at Days 3, 4, 6, 7, 9, 10, and 11 pp at a dose of 50 mg/kg of body weight. For each age, four rats from two distinct litters were killed 3 h later and the testes were fixed in Carnoy fluid. Incorporated BrdU was revealed by immunohistochemistry with an anti-BrdU antibody (Boehringer, Ingelheim, France) and a secondary antibody labeled with Texas red (Southern Biotechnology Associates, Birmingham, AL). Labeled gonocytes were accounted with an Olympus AX microscope.
Germ cell population after irradiation The number of gonocytes and type A spermatogonia were accounted at Days 6, 9, 10, 11, 12, and 13 pp in four control and four irradiated rats from two different litters each. A total of 300 sections of seminiferous tubules were observed for each age. The gonocytes were identified by their localization in the central region of the tubules and by their large and spherical nucleus containing two or more globular nucleoli. Type A spermatogonia have a spherical or ovoid nucleus with one or two nucleoli, and are located along the basement membrane.
DNA Content in Gonocyte and Sertoli Cell Nuclei from Fetuses at Day 21 Postcoitum
Sections (6 and 10 µm) of testes of three fetuses issued from three distinct litters were stained according to the Feulgen and Rossenbeck method [34]. Transmission of a monochromatic light was measured at 548 nm against 450 nm with a microhistophotometer (MPV1, Leitz). The percentage of transmitted light was measured through the nuclei, which were entirely included in the thickness of the section (light transmission was set to 100% on the cytoplasmic part of the section). DNA amounts were measured in the nuclei of 397 gonocytes and 770 Sertoli cells.
Immunohistochemistry
Tissue sections were rehydrated and boiled twice for 5 min in 10 mM sodium-citrate solution for antigen retrieval [35]. Endogenous peroxidase activity was blocked with 0.3% (v/v) H2O2 in methanol for 30 min, and nonspecific binding sites were blocked by incubating the sections with 5% normal goat serum (NGS) in PBS. Subsequently, the slides were incubated with primary antibodies overnight at 4° C. After washing in PBS, slides were incubated at 37°C for 60 min with a secondary biotinylated antibody (ABC-peroxidase staining kit; Vector Laboratories Inc., Burlingame, CA) diluted 1:200. The avidin-biotin complex reaction was performed according to the manufacturer's protocol. To visualize bound antibodies, sections were washed in PBS and covered with 3,3'-diaminobenzidine (DAB; substrate kit for peroxidase, Vector Laboratories Inc.). Negative control sections were treated as described above, except that primary antibody was omitted during the procedure and replaced by 5% NGS in PBS.
Antibodies Used for Immunohistochemistry
Anti-p53 antibodies Three anti-p53 antibodies were used: two mouse monoclonal antibodies, 240 nondiluted (kindly provided by E. May, CEA Fontenay-aux-Roses, France), and NCL-p53-1801 (Novocastra Laboratories Ltd, Newcastle, UK) diluted 1:20; and one sheep polyclonal antibody, anti-p53 protein, pan (BMG-1B1, Boehringer) diluted 1:5. Specificity of the three anti-p53 antibodies was tested on sections of wild-type and p53 knockout mouse testes.
Monoclonal Bax antibody SC-7480 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:100.
Polyclonal p21 antibody SC-397 (Santa Cruz Biotechnology) diluted 1:200. An additional control was performed by neutralizing the anti-p21 antibody with a fivefold excess of blocking peptide (SC-397P; Santa Cruz Biotechnology).
Monoclonal anti-mdm2 antibody SC-965 (Santa Cruz Biotechnology) diluted 1:50. This antibody was tested on sections of adult rat testis, which exhibited a positive mdm2 staining.
Monoclonal anti-pRb antibody NCL-RB (Novocastra Laboratories Ltd.) diluted 1:20.
Detection of 5-Methyl Cytosine (5-mC)
Sections were either labeled with 4',6'-diamidino-2-phenylidole (DAPI) or treated for immunohistochemistry according to the following protocol: sections were rehydrated, digested with pepsin (0.2 mg/ml), and treated with 2 N HCl. Anti-5mC antibody [36] was revealed by a second antibody labeled with rhodol green (Molecular Probes, Eugene, OR).
Double Labeling of Mitochondria and p21
Testes at Day 16 pc were incubated at 37°C with 500 ng of Red Mito Tracker (M-7512; Molecular Probes), permitting a fluorescent staining of mitochondria. After 3 h, testes were fixed overnight with Carnoy fluid, dehydrated, and embedded in paraffin. P21 was detected by immunohistochemistry and revealed after incubation with streptavidin-fluorescein (RPN 1232; Amersham).
In Situ End-Labeling Method
Detection of apoptotic cells was performed with the Apoptag kit (Appligene Oncor, Illkirch, France). Tissue sections, fixed in a diluted Bouins fluid, were treated with proteinase K (20 µg/ml Tris HCl 10 mM pH 7.4). Endogenous peroxidase activity was blocked with 3% (v/v) H2O2 in PBS for 5 min. After equilibration, sections were incubated for 1 h at 37°C in a buffer containing TdT enzyme and digoxigenin-dNTP. Slides were washed in PBS and incubated with an anti-digoxigenin peroxidase conjugate for 30 min at room temperature. The slides were stained with diaminobenzidine and counterstained with Mayer hemalun.
Western Blot Analysis
Seminiferous tubules were isolated by incubating rat testes for 10 min at 37°C in collagenase (type IA-S [Sigma], 1 mg/ml). After a wash in PBS and centrifugation, the pellet was recovered. Proteins were extracted with the TriInstaPure reagent (Eurogentec s.a., Belgium) and stored in 0.5% (w/v) SDS after quantification according to the Bradford method [37].
Proteins (30 µg/lane) were denatured, separated on a 10% SDS-polyacrylamide gel, and blotted onto a nitrocellulose membrane. Nonspecific sites were blocked with 10% (w/v) nonfat milk in TBS-T (20 mM Tris-HCl, 137 mM NaCl pH 7.6, and 0.1% [v/v] Tween-20). Blots were probed with rabbit p53 polyclonal antibody (NCL-p53-CM1, Novocastra Laboratories) or antiactin as a loading control. After extensive washings, proteins were tagged using a goat anti-rabbit secondary antibody, conjugated to horseradish peroxidase (Pierce, Rockford, IL), and then visualized by enhanced chemiluminescence (ECL; Pierce). Blots were exposed to hyperfilms (Amersham), and intensity of p53 protein signals was measured using the "NIH Image" program. The Western blot was replicated three times.
Statistics
All values are means ± SD. The significance of the differences between mean values was evaluated by the Student t-test.
RESULTS
Effect of 1.5-Gy -Irradiation at Day 19 Postcoitum on Rat Testis
After irradiation at 1.5 Gy of rays at different ages of development, an important testicular atrophy, characterized by sterile tubules, was observed when irradiation occurred between Days 18 pc and 3 pp (Fig. 1A). In control animals, the mitotic index of gonocytes strongly decreased between Days 16 and 18 pc. No mitosis was observed between Days 18 pc and 3 pp (Fig. 1B). At Day 3 pp, a small proportion of gonocytes started entering S phase, and at Day 4 pp, gonocytes abruptly resumed mitosis. After a small decrease at Day 5 pp, the mitotic index increased again at Day 6 pp in a biphasic manner. Thus, the period of hyperradiosensitivity precisely corresponded to the cell cycle arrest of gonocytes in the controls.
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After radiation exposure, there was no mitosis recovery in gonocytes at Day 4 pp (Fig. 1B). BrdU incorporation at Days 3, 4, 6, 7, 9, 10, and 11 pp indicated that some rare gonocytes entered into S phase from Days 4 to 11 pp (Fig. 1C). The histological examination indicated that the number of germ cells decreased from Day 6 pp and reached zero at Day 12 pp (Fig. 2A). The use of the in situ end labeling (ISEL) method demonstrated that this progressive gonocyte disappearance occurred by apoptosis (Fig. 2B). The presence of Bax, the proapoptotic protein, was also observed in gonocytes by immunohistochemistry, but no difference was detected between control and irradiated animals (data not shown). Thus, gonocytes failed to differentiate into spermatogonia, and continuously underwent apoptosis. Their small number (average 
0.8 per tubular cross-section) did not allow us to determine the rate of apoptosis.
The relative amount of DNA in testicular cells from 21-day-old fetuses from three distinct litters was evaluated by measuring the light transmission through cell nuclei. In gonocyte nuclei, the light transmission decreased from 70% to 56%. This corresponded to a single peak of cells (Fig. 3). Immature Sertoli cell nuclei transmitted light from 70% to 37%, which corresponded to cells in different phases of the cell cycle. The first peak was the most important. It overlapped the gonocytes peak and corresponded to the diploid content. The second peak corresponded to the largest DNA amount (i.e., the tetraploid content). The absence of a second peak in gonocytes indicates a lack of a tetraploid content, which is indicative of G2/M.
Expression of p53 and mdm2 in Normal and Irradiated Rat Testes
In control testes, a Western blot analysis indicated that p53 expression is high at Day 21 pc and decreases at Day 6 pp. In irradiated animals, the high expression, observed at Day 21 pc also decreased at Day 6 pp, but to a lesser extend than in controls (Fig. 4). These results were complemented by a p53 immunohistochemical study. In controls, at Day 16 pc, no specific staining of p53 was detected in proliferating germ or somatic cells (Fig. 5A). From Day 18 pc on, a strong p53 cytoplasmic staining was observed in arrested gonocytes (Fig. 5, B and C). At Day 6 pp, this strong staining was lost in a proportion of gonocytes, concomitantly with proliferation resuming (Fig. 5D). In irradiated animals, the strong p53 cytoplasmic staining was maintained from Days 21 pc to 6 pp in all gonocytes, which remained quiescent (Fig. 5E). The three different anti-p53 antibodies used, which recognize distinct epitopes of the protein, gave similar results. Moreover, no labeling was observed in p53 knockout mouse testes and a nuclear staining was detected in germ cells of wild-type adult mouse testes (Fig. 5F). No staining of Sertoli cells was observed at any time in both control and irradiated animals. A double labeling for p53 and BrdU incorporation was performed in BrdU-treated animals. This allowed us to show that the p53 labeling was lost in cycling gonocytes that had incorporated BrdU (Fig. 6, A–D).
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Despite repeated trials, it remained impossible to detect any mdm2 protein expression in control gonocytes at Days 16 and 21 pc and Day 6 pp by immunostaining. In irradiated animals, mdm2 was also undetectable at Days 21 pc and 6 pp (data not shown).
Expression of p21 in Control and Irradiated Rat Testes
In controls, the p21 protein was detected in the cytoplasm of gonocytes at Days 16 and 17 pc (i.e., before their complete cell cycle arrest; Fig. 7, A and B). It was also detected in arrested gonocytes at Day 21 pc as well as in proliferating gonocytes at Days 6 and 12 pp, when gonocytes were all differentiated into spermatogonia (Fig. 7, C–E). In irradiated animals, no change of p21 staining was observed. The protein was detected in the cytoplasm of all gonocytes, and at Day 12 pp, gonocytes were completely eliminated (Fig. 7F). The p21 labeling was not homogeneous in gonocyte cytoplasm. At Days 16 and 17 pc, it was concentrated at one pole of the cells (arrows, Fig. 7, A and B), whereas at Day 21 pc, it was located around the nucleus (Fig. 7C). Using electron microscopy, mitochondria were found to follow the same localization as p21, polar or perinuclear at Days 16 or 20 pc, respectively, suggesting a colocalization (Fig. 8, A and B). To validate this hypothesis, a double labeling was performed on rat testis at Day 16 pc. Mitochondria were identified by a red staining after incorporation of Mito Tracker (Fig. 9A), and p21 by a green staining (Fig. 9B). Mitochondria and p21 were effectively colocalized in gonocytes. In Sertoli cells, no p21 staining was observed at any time in both control and irradiated animals.
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Expression of p53 and p21 after Treatments with Actinomycin D or Cycloheximide
Expression of p53 and p21 was studied after actinomycin D or cycloheximide treatment in testes from 2- and 6-day-old rats, in arrested and cycling gonocytes, respectively. Both treatments at both periods induced a decreased p21 immunostaining compared with controls. This decrease was very important after cycloheximide, and less marked after actinomycin D treatment (data not shown). No modification of p53 expression could be detected by either treatment (data not shown).
Expression of pRb in Control and Irradiated Testes
Control and irradiated animals were studied at Days 21 pc and 6 pp using an anti-pRb antibody that recognizes both hypophosphorylated and hyperphosphorylated forms of the protein. In controls, pRb was not or was barely detectable in arrested gonocytes at Day 21 pc (Fig. 10A). At Day 6 pp, a proportion of gonocyte nuclei became strongly labeled (Fig. 10C). In irradiated animals, pRb remained almost undetectable at Day 21 pc (Fig. 10B), but also at Day 6 pp (Fig. 10D), except in some very rare gonocytes (data not shown). Proliferating Sertoli cells always displayed a strong nuclear labeling in both irradiated and control animals.
DNA Methylation Status in Developing Rat Testis
DNA methylation was studied in testes from 16-, 19-, and 20-day-old rat fetuses. At Day 16 pc, when gonocytes that were observed in the seminiferous tubules after DAPI labeling (Fig. 11A) were cycling, their interphase nuclei appeared to be dull after anti-5mC antibody staining compared with those of Sertoli cell (Fig. 11B). At Day 19 pc, whereas gonocytes were blocked in G0-G1 phase, nuclei had the same staining pattern as they did at Day 16 pc (data not shown). At Day 20 pc, while gonocyte cell cycle was still arrested, their nuclei became labeled by anti-5mC antibody (Fig. 11C, arrows). This methylation, which occurred abruptly between Days 19 and 20 pc, took place in gonocytes about 5 days before the beginning of S phase. From Day 5 pp, the intensity of labeling of interphase nuclei of germ cells decreased. An immunohistochemical 5-mC labeling of gonocyte metaphase chromosomes demonstrated that this decrease was a consequence of the semiconservative loss of methylated chromatids (data not shown), as it was shown for the mouse during the corresponding period of testis development [33]. In gonocytes of rats irradiated at Day 19 pc, the DNA methylation occurred as in controls.
DISCUSSION
In both fetal and neonatal rat testes (i.e., from Days 18 pc to 3 pp), the two main cell populations behave differently. Sertoli cells are continuously cycling, whereas gonocytes remain blocked in a prereplicative stage, as shown by DNA content studies. Paradoxically, these noncycling cells are highly radiosensitive. According to the different results obtained in this work, the following chronology can be proposed for control rat gonocytes:
Both periods during which gonocytes progressively stop cycling or begin cycling again are about 3 days long. It is not known whether or not gonocytes, which are arrested first, also resume cycling first. If that is the case, all gonocytes would be arrested for about 8 days. In any case, the population of gonocytes is not synchronous.
In testes from animals irradiated at Day 19 pc (i.e., when all gonocytes are arrested), no characteristic changes are observed until Day 3 pp, compared with gonocytes from nonirradiated controls. In particular, the amount and localization of p53 are not modified. At Day 4 pp, a much smaller proportion of gonocytes enter S phase, but none or very few of them reach mitosis. Both p53 and p21 stainings remain cytoplasmic. No nuclear localization is observed, but a faint nuclear staining might have been masked by the strong cytoplasmic labeling. Apoptosis was detected from Day 6 pp on, and finally, the entire gonocyte population was eliminated at Day 12 pp. Thus, germ cells display a very unusual radiosensitivity during the prenatal and neonatal periods. They seem to be highly resistant during the first days following the exposure to radiations, but all undergo a delayed apoptosis over a period of 7 days. Thus, they are hypersensitive to radiation. This unusual behavior of gonocytes may be partially explained by their metabolic characteristics studied here.
The strong cytoplasmic expression of p21, shown to be colocalized with mitochondria, is followed by a cell cycle arrest. However, cell cycle inhibitory effect of p21, which is shown to be strictly dependent on its nuclear localization, should not be responsible. Suzuki and colleagues [38] reported that cytoplasmic p21, associated with procaspase 3 and mitochondria, exhibits antiapoptotic properties in HepG2 cells. This may account for the observed delayed death of gonocytes.
The strong cytoplasmic expression of p53 is roughly concomitant with the cell cycle arrest. The involvement of p53 protein in cell cycle arrest also requires a nuclear localization [39, 40], However, Marchenko and colleagues [41] have reported that cytoplasmic p53 can contribute, in vitro, to apoptosis via mitochondria. Thus, apoptosis observed in gonocytes after irradiation may be a consequence of the interaction between p53 and mitochondria. This apoptotic pathway would be Bax-independent because its expression is not increased in gonocytes after irradiation. This was confirmed by Beumer and colleagues [42], who have demonstrated that Bax was localized in adult mouse germ cells, but that its expression did not change after irradiation. Moreover, Li and colleagues [43] have shown that p53-induced apoptosis is not necessarily Bax-dependent.
Accumulation of p53 shortly precedes DNA methylation. DNA methylation is commonly assumed to inhibit transcriptional activity of most genes [44]. Thus, the cytoplasmic maintenance of large p53 amounts may be related to the lack of proteins involved in its degradation, translocation, or both. This may be the case for mdm2 that we could not detect in gonocytes. This hypothesis of a stabilization of p53 is strengthened by a lack of effect of actinomycin D and cycloheximide treatments on its expression, suggesting the absence of protein turnover. The strong decrease of p21 amounts induced by these treatments suggests that p21 is continuously synthesized, and that DNA methylation does not affect p21 gene transcription.
How gonocytes escape from cell cycle arrest is not known. Appearance of the pRb protein in control gonocytes, while none of the hypophosphorylated and hyperphosphorylated forms were detectable in these cells after irradiation, suggests a role of this protein in the cell cycle arrest. However, pRb may either represent the main actor of the cell cycle resuming, or it may be only a consequence of an upstream signal. Because DNA methylation is maintained during the next cycle [33], active DNA methylation changes are not the cause. The exact moment of the G0-G1 phase at which gonocytes are arrested is not easy to determine. At microscopic examination, gonocyte size progressively increases during the mitotic arrest. This, as well as the effect of cycloheximide and actinomycin D on p21 amounts, indicates that some metabolic activities are maintained, despite DNA methylation. Thus, the cycle of gonocytes is rather slowed down in G1 phase rather than blocked in G0 phase. Cellular radiation sensitivity is largely cycle-dependent, and noncycling cells are generally considered to be less sensitive than cycling cells [45, 46]. Among cycling cells, those in a transition period, and especially in G1- to S-phase transition, are particularly sensitive. The observed delayed death of gonocytes may be a consequence of the uncoupling of cellular functions, as a possible consequence of their slow progression into G1 phase and DNA methylation.
When irradiated at Day 19 pc, no death message is immediately emitted, although DNA lesions were formed. Several days later (i.e., at Day 4 pp), a proportion of gonocytes pass through the transition period before reaching S phase, while some DNA lesions may persist. They further undergo apoptosis. During the next days, other gonocytes progressively follow the same evolution, and between Days 8 and 12 pp, the entire population is affected. The nature of the death signal involved is not yet elucidated. It may be related to the strong accumulation of p53 and its sudden translocation into the nucleus. Shaulsky and colleagues [47] have reported that in Balb/c 3T3 cells, p53 remained for about 3 h in the nucleus around the beginning of the S phase, and then accumulated in the cytoplasm. It was shown that p53-dependent apoptosis is particularly efficient at the G1- to S-phase transition [48]. Yin and colleagues [49] have reported the presence of p53 in the nuclear envelope of adult mouse germ cells. P53 may accumulate in the cytoplasm and in the nuclear envelope of arrested rat gonocytes, which may facilitate its translocation into the nucleus. If apoptosis shortly follows p53 translocation, it may explain why we could not detect it in the nucleus. Another possibility is that cytoplasmic p53 may signal apoptosis, as Blanco-Rodriguez and Martinez-Garcia [50] proposed for rat spermatids.
The unusual response of male gonocytes to radiation-induced damage may be related to the protection of genome integrity of the germ line. However, no similar mechanism seems to exist during female gametogenesis. Female germ cells undergo a long period of block at the dyctyate stage. During this period, p53 also accumulates in the cytoplasm [51], but this is not accompanied by an increased radiosensitivity.
In conclusion, this work provides some new data about the early testicular radiosensitivity of rodents, compared with published results in adult rodents. During the prenatal and neonatal periods of normal male rat, gonocytes display very unusual metabolic activities, cell cycling, and response to radiation exposure. P53 appears to be involved in radiosensitivity, particularly in terms of maintenance of cell cycle arrest and apoptosis. On the contrary, p21 may have a transitory antiapoptotic effect, via mitochondria. Study of genetically modified animals should improve our knowledge of the functions of the genes involved during this crucial period of gametogenesis.
ACKNOWLEDGMENTS
We are grateful to Dr. S. Magre (Université Paris 6, France) for the electron microscopy study. We are also grateful to Dr. E. May (CEA, Fontenay-aux-Roses, France) for the gift of anti-p53 (240) antibody and wild-type and p53 knockout mice. We thank Q. Chau and F. Trompier (IPSN/DPHD) for performing irradiation, J. Duvaleix and P. Flament (CEA) for the care of animals, and Dr. S. Gangloff (CEA) for a critical reading of the manuscript.
FOOTNOTES
1 This work was supported by Electricité de France and the Ministère de l'Education Nationale et de la Recherche Scientifique et Technique (France). 
2 Correspondence. FAX: 33 1 46 54 89 55; coffigny{at}dsvidf.cea.fr
Accepted: December 15, 2000.
Received: June 21, 2000.
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