p63 expression pattern in foetal and neonatal gonocytes after irradiation and role in the resulting apoptosis by using p63 knockout mice.

PURPOSE: To investigate the role of p63, a member of the p53 family, in gonocyte apoptosis after radiation exposure. MATERIALS AND METHODS: Wild-type (WT) and p63 knock-out (KO) testes were exposed in vivo or in vitro to a 3 Gy dose of 137Cesium (137Cs) gamma-rays at day 18.5 post-conception (p.c.). p63 whole expression was studied in neonatal testes by immunohistochemistry, whereas TAp63 and DeltaNp63 isoforms were studied by Reverse-transcribed Polymerase Chain Reaction (RT-PCR). Gonocyte apoptosis was analysed by immunohistochemistry (cleaved caspase 3) and In Situ End labelling (ISEL). RESULTS: Such foetal irradiation leads to a strong increase of gonocyte apoptosis in newborns. It also induces the up-regulation of the TAp63alpha isoform and the down-regulation of the DeltaNp63alpha isoform. Moreover, in control p63KO testis, a significant increase in the number of gonocytes was associated with a strong reduction of their apoptosis compared with the control wild-type testis. Unexpectedly, after irradiation this increase of the number of apoptotic gonocytes was seen in p63KO testis, which was comparable to that in irradiated p63WT testis. CONCLUSION: We demonstrate that p63 is able to trigger gonocyte apoptosis in control testis but is not necessarily required in their radio-induced apoptosis.

The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.

Study of the gonocyte cell cycle in irradiated TP53 knockout mouse foetuses and newborns.

PURPOSE: To investigate the role of p53 in gonocyte cell-cycle arrest in rodents with or without radiation exposure. MATERIALS AND METHODS: Pregnant p53 (+/-) mice, mated with p53 (-/-) males, were exposed to (137)Cs gamma-rays at day 18 postcoitum (p.c.) at doses ranging from 1.5 Gy to 3 Gy. The gonocyte cell cycle was studied in p53 (-/-) male foetuses and newborns after BrdU incorporation and DAPI staining, and compared with those of p53 wild-type animals. RESULTS: The proliferation of gonocytes in wild-type mice is normally arrested between day 16 p.c. and birth, a period when p53 is strongly expressed in gonocytes; p53 high expression is prolonged in all gonocytes after 3 Gy irradiation. In p53 (-/-) mice, this period of gonocyte cell-cycle arrest is not modified, compared with wild-type mice. It is also prolonged after a 3 Gy exposure. CONCLUSION: Two hypotheses are proposed. Either p53 is not involved in the control of gonocyte cell-cycle arrest in control and irradiated mice, or its role is redundant in this process.

Status of p53, p21, mdm2, pRb proteins, and DNA methylation in gonocytes of control and gamma-irradiated rats during testicular development.

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.

High sensitivity of rat foetal germ cells to low dose-rate irradiation.

PURPOSE: To investigate the response of germ and Sertoli cells to gamma-irradiation at two distinct periods of testicular development in rat foetuses. MATERIALS AND METHODS: Pregnant rats were exposed to 60Co gamma-rays at days 15, 19 or 21 post-coitum (p.c.), at doses ranging from 0.1 to 1.5Gy, and at different dose-rates. Testicular weight, seminiferous tubule condition and the number of germ and Sertoli cells were measured at early and late times after irradiation. Apoptosis was studied by the ISEL method and p53 expression was studied by immunohistochemistry. RESULTS: At high dose-rates (> or = 3.3 Gy min(-1)), 1.5 Gy radiation at day 15 p.c. had a short-term effect on germ cell survival. A large proportion of these cells rapidly underwent p53-independent apoptosis. Apoptotic cells were strongly clustered. The remaining germ cells divided and differentiated normally leading to a majority of normal tubules in the adult testis. However, at low dose-rate (0.6mGy min(-1)), much greater depopulation of the seminiferous tubules occurred. When irradiation was given at day 19 p.c., the same dose had a delayed effect on germ cells, leading to sterility. Sertoli cells had a normal survival for irradiation at day 15 p.c. Their proliferation became higher in prepubescent testis compared with controls, when irradiation occurred at day 19 p.c. CONCLUSION: The position of gonocytes in the cell cycle at the time of irradiation seems to be a determining parameter for inducing gonocyte apoptosis. The strong effect of irradiation on germ cells at very low dose-rate and the appearance of clusters of apoptotic gonocytes may be a consequence of the syncitial organization of germ cells, favouring their cell synchronisation or the transmission of death signalling when they are in a radiosensitive period.