| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Laboratory of Differentiation and Radiobiology of the Gonads (R.L., H.C., C.P., C.L., R.H., V.R.-F.), Unit of Gametogenesis and Genotoxicity, Unité Mixte de Recherche-S 566, Université Paris7-Denis Diderot, CEA, DSV/IRCM/SCSR/LDRG, and INSERM, Unité 566, F-92265, Fontenay aux Roses, France; and Service de Gynécologie-Obstétrique (R.F.), Hôpital A. Béclère, and INSERM, Unité 782, F-92141 Clamart, France
Address all correspondence and requests for reprints to: Virginie Rouiller-Fabre (Professor), Unit of Gametogenesis and Genotoxicity, LDRG/SCSR/IRCM/DSV, CEA, BP6, F-92265, Fontenay aux Roses, France. E-mail: virginie.rouiller-fabre{at}cea.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Objectives: We investigated the effect of 
-irradiation on the functions of human fetal testis during the first trimester of gestation by using an organ culture system. Then we focused on the role of the p53 pathway in the observed effects.
Results: Germ cells were highly sensitive to irradiation even at doses as low as 0.1 and 0.2 Gy. Indeed, for these doses, one third of germ cells died by apoptosis. Other germ cells were blocked in their cycle, but no repair seemed to occur, and longer culture with the highest dose used showed that they were destined to die. Sertoli cells were less affected, although their proliferation and the level of anti-Müllerian hormone were reduced. Irradiation had no effect on testosterone secretion or on the expression of steroidogenic enzymes by Leydig cells. After irradiation, p53 phosphorylated on serine 15 was detected from 1–24 h in all cell types. This activation of p53 was accompanied by an increase in mRNA levels of proapoptotic factors Bax and Puma, whereas that of antiapoptotic Bcl-2 remained unchanged. P21, which is responsible for cell cycle arrest, was also up-regulated 6, 30, and 72 h after irradiation. Finally, when we added pifithrin-
, a specific inhibitor of p53 functions, a significant decrease in irradiation-induced apoptosis in both germ and Sertoli cells was observed, indicating the involvement of the p53 pathway in irradiation-induced apoptosis.
Conclusions: This study demonstrated here for the first time the great sensitivity of human fetal germ cells to genotoxic stress caused by ionizing radiation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several studies have described an increasing frequency of male reproductive disorders in humans, such as a decline in fertility (8), increased incidence of testicular cancer (9), cryptorchidism, and hypospadia (10, 11, 12). It has been suggested that these alterations are symptoms of a single syndrome called the testicular dysgenesis syndrome (10, 13). Several studies in rodents have shown that testicular dysgenesis syndrome could be caused by an exposure to endocrine disruptors during fetal life (11, 13). However, the effects of genotoxic agents, such as ionizing radiation, have never been assessed on the development of human fetal testis.
During development, the effects of irradiation exposure are dependent on dose and age, and sterility is the main consequence (14). In rats, we have previously shown that a dose of 1.5 Gy delivered during the quiescent period of gonocytes led to a total and irreversible loss of fertility at adulthood (15). In mice, germ cells were more sensitive to X-irradiation in the fetal testis than in adult testis (16). Until now, no study has been conducted in the human fetus, even though the number of germ cells formed during fetal life is essential for adult fertility. In the present study, we used the organ culture system we developed previously (7) to analyze the effects of various doses of -rays on the cellular and functional development of germ, Sertoli, and Leydig cells. We also focused on one key DNA damage checkpoint pathway, the p53 pathway, to explain these effects.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human fetal testes were obtained from pregnant women referred to the Department of Obstetrics and Gynecology at the Antoine Béclère Hospital, Clamart (France), for legally induced abortion in the first trimester of pregnancy, i.e. from the sixth until the 12th week of gestation, as previously described (7). The Antoine Béclère Ethics Committee approved this study. The sex of the fetus was determined by the morphology of the gonads, and the fetal age was evaluated by measuring the length of limbs and feet (17). We found testes within the abortive material in only 12% of cases.
Organ cultures
Testes were cultured on Millicell-CM Biopore membranes (pore size 0.4 µm; Millipore, Billerica, MA) in Ham F12/DMEM (1:1) (Life Technologies, Inc., Grand Island, NY) containing 80 µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO), as previously described (7). In some experiments, the culture lasted 8 d, and in this case, 100 ng/ml LH from human pituitary (
5000 IU/mg) (Sigma) was added to media to ensure proper development of the gonad. We measured the response to irradiation by comparing one testis exposed to a source of 137Cs (dose-rate 0.6 Gy·min–1) to the other testis from the same fetus used as a control (for 0.01 Gy, the source was 60Co; dose-rate 0.021 Gy·min–1). For cellular analyses, the whole explant was fixed for 2 h in Bouin’s fluid (except for phosphorylated p53, which needed fixation with buffered 4% formaldehyde at 4 C overnight) embedded in paraffin, and 5-µm sections were cut. In some cases, for the determination of kinetics, different pieces from the same testes were fixed at different times after irradiation. For the study of the role of p53, pifithrin- (PFT), an inhibitor of p53 functions, was added to the medium 8–12 h before irradiation until the end of the culture period. The concentration was of 1 µM to avoid cytotoxic effects (18, 19). In this case, a testis was divided in several pieces that were cultured 4 d with or without PFT and were irradiated or not at 0.5 Gy.
For gene expression, testes were placed in the RLT lysis buffer of RNeasy minikit (QIAGEN, Courtaboeuf, France) and for Western blotting in a lysis buffer.
Germ and Sertoli cell counting
We mounted serial sections on slides, removed the paraffin, and rehydrated the section. We then carried out immunohistochemical assays for anti-Müllerian hormone (AMH) as previously described (7) using an anti-AMH polyclonal antibody (1:2000) (generously provided by Dr. N. Di Clemente, INSERM U782, Clamart, France). The germ cells were identified as AMH-negative cells within the seminiferous cords, whereas the Sertoli cells were the AMH-positive cells. Peroxidase activity was visualized using 3,3'-diaminobenzidine as substrate. The counting was done as previously described and validated for rodents (3, 20) and human (7). Briefly, we counted germ and Sertoli cells in only one of 10 sections for the 6- to 7-wk-old fetuses and one of 20 sections for later stages but never less than 10 sections equidistantly distributed along the testis. All counts were done using Histolab analysis software (Microvision Instruments, Evry, France). We counted all germ cells on the section. For Sertoli cells, we counted the ones present in randomly selected fields of the section. We multiplied the sum of the values obtained for the observed sections of one testis by 10 or 20, respectively, to obtain a crude count of germ or Sertoli cells per testis. We then used the Abercrombie formula (21) to correct for any double counting due to single cells appearing in two successive sections, and so we obtained a true count. All counts were done blind.
Immunohistochemical staining for cleaved caspase-3, cleaved caspase-9, Ki67, and phospho-p53
Cleaved caspase-3 and caspase-9 were used as markers of apoptosis (22). Ki67 (nuclear antigen present only in cells in the cycle) (23) was used as a marker of germ cell proliferation. We also investigated the expression of p53 phosphorylated on serine 15. We mounted six sections on a single slide and heated the slide for 30 min in a permeabilization solution (0.05 M Tris, pH 10.6 for caspases; 0.01 M sodium acetate, pH 2 for Ki67; 0.01 M citrate, pH 6 for phospho-p53). The procedure was then the same as for detection of AMH, except that the primary antibody was the anti-cleaved caspase-3, anti-cleaved caspase-9, or anti-phospho-p53 (serine 15) antibody (for these three antibodies, 1:50) (Cell Signaling Technology, Beverly, MA). For Ki67, we used a monoclonal anti-Ki67 antibody (1:50) (Dako, Trappes, France) as previously described (7). Stained and unstained germ (for caspases-3 and -9 and Ki67) and Sertoli (for caspase-3 and Ki67) cells were counted in all six sections. For all immunohistochemical staining, negative controls were done by omitting the primary antibody.
Measurement of bromodeoxyuridine (BrdU) incorporation index
The procedure was as previously described (24). The BrdU incorporation index was obtained by a blind counting of stained and unstained germ or Sertoli cell nuclei in all six sections.
Double staining using VIP
After the first immunohistochemical staining (cleaved caspase-3, cleaved caspase-9, Ki67,BrdU, and phospho-p53) revealed with 3,3'-diaminobenzidine, the sections were washed in PBS for 10 min, processed for AMH immunohistochemical staining, as previously described, and visualized using VIP as a peroxidase substrate (Vector Laboratories, Burlingame, CA).
Testosterone RIA
We measured the testosterone secreted into the medium in duplicate by RIA as previously described (7).
RT and real-time PCR
We evaluated the expression of P450 cholesterol side-chain cleavage (P450SCC), P450 17-hydroxylase/C17–20 lyase (P450C17), AMH, p53, p21, Bax, Puma, and Bcl-2 in fetal testes by RT, followed by real time RT-PCR, as previously described (7). The primers and probes used were designed by Applied Biosystems (Courtaboeuf, France) (sequences not provided; P450scc, Hs00167984_m1; P450c17, Hs00164375_m1; AMH, Hs00174915_m1; p53, Hs00153340_m1; p21, Hs00355782_m1; Bax, Hs00180269_m1; Puma, Hs00248075_m1; Bcl-2, Hs00153350_m1; ß-actin, Hs99999903_m1). Reactions were carried out and fluorescence was detected using an ABI Prism 7000 apparatus (Applied Biosystems, Foster City, CA). Each sample was run in duplicate. Negative controls were run for every primer/probe combination. The measured amount of each cDNA was normalized using ß-actin.
Protein extraction and Western blotting
One testis was lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na4O7P2, 1 mM ß-glycerol phosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 µg/ml leupeptin. Seven micrograms of proteins in total cell lysates were resolved by SDS-PAGE, electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham), and probed with the AMH (same as for immunohistochemistry) and actin (Calbiochem) antibodies. Horseradish peroxidase-coupled antirabbit (Pierce, Rockford, IL) or antimouse antibodies were used to detect antigen-antibody interactions by enhanced chemiluminescence (Amersham). We measured the intensity of the bands by densitometry with the software Bio1D (Bio-Profil Vilber Lourmat, Marne-la-Vallée, France).
Statistical analysis
All values are expressed as means ± SEM. The significance of the differences between mean values for the irradiated and nonirradiated testes from the same fetus or pieces from the same testis was evaluated using Student’s paired t test. Student’s unpaired t test was used only to determine the significance of the differences between mean values of the irradiated testes cultured for 4 d and the irradiated testes cultured for 8 d.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Whatever the age of the fetus at explantation, irradiation had no effect on the organization of the testis, and the appearance of all cells was similar to that in the control (Fig. 1
, A and B). Irradiation induced a dramatic and dose-dependent reduction in the number of germ cells, whatever the dose up to 0.1 Gy (Fig. 1, A–C). In particular, the number of germ cells decreased from 18 x 103 ± 7.7 x 103 to 4.5 x 103 ± 1.6 x 103 cells per testis (from 100 to 27% in percentage of control) at a dose of 1.5 Gy. We then evaluated their apoptotic and mitotic indices. Irradiation significantly increased the percentage of cleaved caspase-3-positive germ cells in a dose-dependent manner up to 0.1 Gy (Fig. 1, D and E). For example, this percentage was increased from 3.3 ± 0.6% in the control to 25.8 ± 4.7% of the germ cells in the irradiated sample (from 100 to 772 ± 143%) at a dose of 1.5 Gy. The same trend was seen in the percentage of cleaved caspase-9-positive germ cells at a dose of 1.5 Gy (Fig. 1, F and G). Irradiation also significantly increased the number of cycling germ cells, detected by the immunostaining of Ki67, in a dose-dependent manner up to 0.1 Gy (Fig. 1, H and I). More precisely, the percentage of Ki67-positive germ cells increased from 35 ± 2.4 to 63 ± 6.6% (from 100 to 181 ± 19%) at a dose of 1.5 Gy. Surprisingly, the percentage of mitotic germ cells determined by the immunostaining of BrdU incorporation was not modified after 1.5 Gy irradiation, i.e. 27.3 ± 1.2% in the control and 28 ± 1.5% after irradiation (Fig. 1, J and K).
|
After irradiation, testes were cultured for 7 more days, in the presence of LH to ensure proper development, as previously described (7). Irradiation had no effect on the organization of the testis, and the appearance of all cells was similar to that in the control (Fig. 2, A and B). The decrease in germ cells in the testis was dramatic: 1.2 x 103 ± 0.3 x 103 after 8 d (7 ± 1.6% in percentage of control), compared with 17 x 103± 3.6 x 103 in the control (Fig. 2C). The decrease was even greater than that observed 3 d after irradiation (P < 0.01 in the unpaired Student’s t test between the number of germ cells in the irradiated testis cultured for 3 and 7 d; Figs. 1C
and 2C). Irradiation still significantly increased from 2 ± 0.6 to 16 ± 3% (from 100 to 803 ± 65%) the percentage of cleaved caspase-3-positive germ cells 7 d after irradiation (Fig. 2D). Irradiation significantly increased from 42 ± 1.2 to 52 ± 3.2% (from 100 to 123 ± 8%) the number of cycling germ cells, detected by the immunostaining of Ki67 (Fig. 2E). Like 3 d after irradiation, the percentage of BrdU-positive germ cells was not modified (Fig. 2F).
|
We studied the ratio of apoptotic (cleaved caspase-3-positive, Fig. 3A) and cycling (Ki67-positive, Fig. 3B) germ cells at different times after a 1.5-Gy irradiation. Irradiation significantly increased from 4.8 ± 1.8 to 14.8 ± 5% (from 100 to 311 ± 105%) the percentage of cleaved caspase-3-positive germ cells from 6 h, and this effect was still apparent after 168 h. The percentage of Ki67-positive germ cells was increased as of 24 h after irradiation (from 39 ± 2.5 to 50 ± 3.5% and from 100 to 128 ± 9% in percentage of control) until 168 h.
|
Irradiation induced a smaller (compared with germ cells) but significant reduction in the number of Sertoli cells at 1.5 Gy (from 40.3 x 104± 10 x 104 to 25 x 104± 6.6 x 104 Sertoli cells and from 100 to 62 ± 16% in percentage of control) (Fig. 4A). This effect was characterized by an increase in the percentage of cleaved caspase-3-positive Sertoli cells (Fig. 4B). The proliferation of Sertoli cells was significantly reduced (Fig. 4, C and D). We then analyzed by real-time RT-PCR (Fig. 4E) and by Western blotting the effect of irradiation on AMH expression 3 d later (Fig. 4, F and G). Irradiation had no significant effect on the mRNA level of AMH but decreased the level of AMH protein.
|
Irradiation had no effect on testosterone production, whatever the dose (Fig. 5A). Moreover, after 96 h, we tested the response of testis to LH (100 ng/ml), and the irradiated testis responded in the same way as the control. For example, after 1.5-Gy irradiation, the testosterone production of the irradiated testis at d 3 was 4.1 ± 1.9 ng/testis·h, and after 3 h LH stimulation, it was 21 ± 10 ng/testis·h. We then analyzed by real-time RT-PCR the effect of irradiation on the levels of mRNA for P450SCC and P450C17 (Fig. 5B). Whatever the dose, irradiation had no significant effect on the mRNA level of these two steroidogenic enzymes.
|
To understand the mechanisms underlying the effect of irradiation in fetal testis, we studied the p53 pathway. We detected p53 phosphorylated on serine 15 in testis from 1 h after a 1.5-Gy irradiation, and this form of the protein was still present 24 h later (Fig. 6A). Positive germ, Sertoli, and Leydig cells were found 1, 3, and 24 h after irradiation, whereas the maximal number of positive cells was observed at 3 h. Very few germ and Sertoli cells and no Leydig cells were still stained 72 h after irradiation. We then measured by real-time RT-PCR the effect of 0.5-Gy irradiation on the mRNA levels of p53, Bax, Puma, Bcl-2, and p21 in whole testis 6, 30, or 72 h after irradiation (Fig. 6B). The mRNA levels of p53 and Bcl-2 were not modified, whatever the time after irradiation. However, the expression of Bax, Puma, and p21 was significantly increased from 6 h up to 72 h after irradiation. Finally, we measured the percentage of cleaved caspase-3-positive germ and Sertoli cells before and after a 0.5-Gy irradiation and with or without PFT (Fig. 6C). PFT (1 µM) induced a not significant increase in the apoptosis of germ and Sertoli cells in control conditions. When irradiated, germ and Sertoli cells displayed significantly less caspase-3-positive cells in presence of PFT. Indeed, for germ cells, irradiation increased cleaved caspase-3-positive cells from 1.18 ± 0.24 to 6.43 ± 0.89%, and when PFT was added, it increased only from 2.68 ± 0.5 to 4.85 ± 1.16%. For Sertoli cells, the increase was from 0.47 ± 0.03 to 1.57 ± 0.09% without PFT and from 0.68 ± 0.06 to 1.13 ± 0.15% with this p53 inhibitor.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-irradiation on the development of human fetal testis using the organ culture system we had previously developed and validated (7).
The natural sources of radiation are cosmic rays and radioactive substances occurring naturally in the earth itself and inside the human body. The average level of natural exposure is 2.5 mSv/yr (for -rays, 1 Sv is equivalent to 1 Gy), and it varies a lot around the globe, usually by a factor of about 3. However, in many locations, the level of natural radiation exceed average levels by a factor of 10 or 20. Human activities involving the use of radiation and radioactive substances that also cause radiation exposure in addition to natural exposure could contribute to developmental defects. The United Nations Scientific Committee on the Effects of Atomic Radiation, which assesses the global levels and effects of ionizing radiation, has defined low doses of ionizing radiation as under 0.2 Sv, thus 0.2 Gy for -rays (25).
The present study is the first conducted on the effects of ionizing radiation on human fetal testis. We chose to investigate the effects of low and medium doses to have the most relevant information for the assessment of the threat to human reproductive potency of fetal exposure to ionizing radiation.
The decrease in the number of germ cells after irradiation was extensive and was observed even with a dose as low as 0.1 Gy. Radiosensitivity of fetal germ cells was previously studied in rodents (15, 16, 26). It was observed that their radiosensitivity depended on the period of fetal development. In rodents, gonocytes proliferate until fetal d 18.5 (rats) or 15.5 (mice), then they enter synchronously in a quiescent period until the second or third postnatal day (rats) or birth (mouse) (3). When testes were irradiated during the fetal proliferative period, many gonocytes died rapidly, but the remaining ones then proliferated, and most tubules in the adult testis were normal. On the contrary, after a 1.5-Gy (rats) or 3-Gy (mice) exposure during the quiescent period, apoptosis was delayed but led to sterility of the adult (15, 26). It is clear that germ cells are the most sensitive during the quiescent period (14, 15, 16, 26). This notion remains puzzling because, in general, cell survival data show that maximum radiosensitivity occurs during G2/M phase, with resistance rising during S phase and peaking in the latter part of S phase (27). In our study, the loss of germ cells was continuous with a rapid increase of apoptotic index because 6 h after irradiation and 7 d later, very few of those remained and still had a very high apoptotic index and a mitotic index identical to that of the control. Hence, we could hypothesize that in our model, the remaining human fetal germ cells were destined to die. Thus, the pattern of human fetal germ cells to irradiation seemed to be a combination of the two response profiles observed in rodents. Few data are available on the proliferating activity of human fetal germ cells. We have two methods of assessing the proliferation of germ cells: immunostaining of Ki67, because Ki67 is expressed in cells that are in the cycle and not in G0, and immunostaining of BrdU incorporation, which identifies cells in S phase. In the control, Ki67 was detected in about 40% of germ cells and BrdU incorporation in about 27% of these cells, suggesting as previously described (7, 28) that fetal germ cells during the first trimester are mostly proliferating. However, we cannot exclude the fact that some germ cells are noncycling cells. After irradiation, there was no variation in the BrdU incorporation index after 3 or 7 d. But the Ki67 index increased from 24 h until the end of the experiment. This indicated that some germ cells were blocked in their cycle, because there were more cells in the cycle and the same number proliferating. Moreover, as the decrease in germ cell number continued after 3 d, the arrest seemed to lead to cell death with no repair of germ cells. Altogether, these results could explain the unique irradiation response profile observed for human fetal germ cells. Nevertheless, it is important to note that in the previous studies in rodents, the whole fetus body was irradiated in utero, whereas in our study, only the testis was exposed to radiation and cultured. In the adult, irradiation is also very damaging for germ cells. Temporary aspermia occurs for doses higher than 0.35 Gy and when the dose exceeds 2 Gy, spermatogenesis may resume only after several years. The most radiosensitive adult germ cells are the differentiating spermatogonia, whereas stem and meiotic germ cells are relatively radioresistant (29). Thus, human germ cells are highly sensitive to irradiation during the whole life.
Loss of germ cells was characterized by increased expression of cleaved caspase-3 and caspase-9. Caspase-3 is an effector caspase involved in both intrinsic and extrinsic pathways, whereas caspase-9 is an initiator that is associated with only the intrinsic pathway (30). Thus, we can assume that the intrinsic pathway of apoptosis was induced by irradiation in germ cells.
Sertoli cells were also sensitive because their number and the level of AMH expression were reduced but to a much lesser extent. Nevertheless, the level of AMH was referred to the level of actin; thus, the decrease of AMH, which was significant for proteins and only very slight for mRNA, could result from the decrease in Sertoli cell number. No cell cycle arrest was observed for Sertoli cells, because the BrdU staining and Ki67 staining matched, but they showed that the proliferative Sertoli cells disappeared after irradiation, thus indicating that Sertoli cells were more sensitive when they cycled. Like for rodents (15, 16), irradiation had no effect on Leydig cells because the steroidogenic function of Leydig cells was identical whatever the dose, and the level of expression of steroidogenic enzymes was unchanged.
One of the key proteins in checkpoint pathways is the tumor suppressor p53, which coordinates DNA repair with cell cycle progression and apoptosis (31). Irradiation induces both single- and double-strand DNA breaks, the latter generally being considered lethal if not repaired (27). A kinase, ataxia telangiectasia mutated, is located immediately downstream of the damage sensors (32). When specifically activated after double-strand DNA breaks, one of the actions of ataxia telangiectasia mutated is to phosphorylate p53 directly at serine 15, which stabilizes it (31). Thus, accumulated and activated p53 leads to cell cycle arrest that may facilitate DNA repair or lead to apoptosis or both. Our results are consistent with this because we found p53 phosphorylated at the serine 15 from 1 h until 24 h after irradiation, thus indicating a quick posttranscriptional activation of p53. As expected (31), no transcriptional activation of p53 was seen. Concerning cell cycle arrest, the G1 arrest triggered by irradiation occurs largely through the transactivation of p21 by activated p53, which inhibits G1 cyclin-dependent kinases and consequently prevents S-phase entry (31). P21 also participates in the G2 arrest (33). Because we found that p21 was widely overexpressed in the total testis from 6 h after irradiation until 72 h, we can hypothesize that p21 was responsible for the germ cell arrest.
The mechanisms by which p53 mediates apoptosis can generally be divided into two categories: transcription-independent and transcription-dependent. During this study, we focused on the transcription-dependent mechanism. We found that Puma and Bax, which are proapoptotic genes transactivated by p53, were greatly, quickly, and durably up-regulated after irradiation of human fetal testis, as in several other models (31, 34). They could be the key players in the tremendous and long-lasting apoptosis of germ cells and in the limited cell death of Sertoli cells. As expected, mRNA levels of Bcl-2, which is antiapoptotic and whose regulation by p53 is mostly transcription independent (34), remained unchanged after irradiation. Finally we used a specific inhibitor of p53, called PFT, that was identified by its capacity for rescuing mice from lethal genotoxic stress caused by -irradiation (18). The main function of PFT is to reduce the p53 transcription-dependent function (35). Because this function is implicated in p53-dependent apoptosis, we studied the effect of PFT on the increase of apoptosis induced by irradiation in germ and Sertoli cells. We found an increase in apoptosis after a 0.5-Gy irradiation, but PFT reduced significantly this phenomenon. This result confirmed the key role of p53 in the induction of apoptosis in germ and Sertoli cells by irradiation.
In conclusion, this study shows for the first time that human fetal germ cells are highly radiosensitive because their death was induced with doses as low as about 10–30 times the total average exposure level per year. Even if pregnant women are not exposed to such doses during gestation, we demonstrated here for the first time that genotoxic stress is very damaging for human fetal germ cells. Because people are also exposed to many other genotoxic agents via cigarette smoke (e.g. cadmium and benzene), water (e.g. heavy metals), and food (e.g. dioxins), it cannot be excluded that genotoxic stress could be implicated in the decrease of fertility and the increase of testicular cancer in the adult. Thus, it will be of the highest interest to test the effects of other genotoxic agents on human fetal testis development.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
R.L., H.C., C.P., C.L., R.H., and V.R.-F. have nothing to declare. R.F. received lecture fees from different pharmaceutical companies that are not directly related to the material being published.
First Published Online April 24, 2007
Abbreviations: AMH, Anti-Müllerian hormone; BrdU, bromodeoxyuridine; PFT, pifithrin-; P450C17, P450 17-hydroxylase/C17–20 lyase; P450SCC, P450 cholesterol side-chain cleavage.
Received December 1, 2006.
Accepted April 16, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
forms a sparingly soluble derivative via intramolecular cyclization under physiological conditions. Mol Pharm 2:462–474[CrossRef][Medline] can suppress heat shock and glucocorticoid signaling pathways. J Biol Chem 278:15465–15468This article has been cited by other articles:
 |
|
 |
|
  M.-J. Guerquin, C. Duquenne, H. Coffigny, V. Rouiller-Fabre, R. Lambrot, M. Bakalska, R. Frydman, R. Habert, and G. Livera Sex-specific differences in fetal germ cell apoptosis induced by ionizing radiation Hum. Reprod., March 1, 2009; 24(3): 670 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Forand, S Messiaen, R Habert, and J Bernardino-Sgherri Exposure of the mouse perinatal testis to radiation leads to hypospermia at sexual maturity Reproduction, March 1, 2009; 137(3): 487 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Jahnukainen, J. Ehmcke, M. Nurmio, and S. Schlatt Irradiation Causes Acute and Long-Term Spermatogonial Depletion in Cultured and Xenotransplanted Testicular Tissue from Juvenile Nonhuman Primates Endocrinology, November 1, 2007; 148(11): 5541 - 5548. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
