The role of Trp53 in the mouse embryonic response to DNA damage.

Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

Xenopus tropicalis peroxidasin gene is expressed within the developing neural tube and pronephric kidney.

Peroxidasin, originally identified in Drosophila, is a member of the myeloperoxidase family with a novel domain structure. It is proposed that peroxidasin is secreted and has functions associated with stabilization of the extracellular matrix. We report the identification of the Xenopus tropicalis orthologue of the peroxidasin gene. We show that the predicted protein sequence of Xenopus peroxidasin shows high sequence identity with the human orthologue and that the exon structure is highly conserved between the two species. We describe the first detailed developmental expression pattern for peroxidasin in a vertebrate species. Maternal expression of Xtpxn is localized to the animal hemisphere where it persists through early cleavage stages. Initial zygotic Xtpxn expression is detected in the developing neural tube and becomes localized to the hindbrain and midbrain. Xtpxn is expressed in the primordium of the pronephric kidney and expression persists in the pronephric tubules and duct throughout development. Potential roles for peroxidasin during early vertebrate development are discussed.

Uptake, localization, and dosimetry of 111in and 201tl in human testes.

UNLABELLED: This study concerns the testicular uptake and dosimetry of Auger electron-emitting radionuclides that are used during routine diagnostic nuclear medicine procedures. To consider the possible effects of these radionuclides on spermatogenic cells, a study has been undertaken to obtain in vivo data for quantification of (111)In chloride and (201)Tl chloride uptake into the human testis after intravenous administration. Values have been determined for uptake into the testis as a whole and to the seminiferous tubules where the germ cells are located. METHODS: Data were obtained from patients with prostate cancer who opted for orchidectomy to effect hormone suppression. Patients were administered intravenously 1.5 MBq of either (111)In chloride or (201)Tl chloride at 24 or 48 h before orchidectomy. Upon removal, the testes were analyzed to assess uptake of radionuclide. Conventional dosimetry has been used to estimate testicular radiation doses using our values of percentage uptake. RESULTS: Uptake of both (111)In chloride and (201)Tl chloride into the testes was seen at a level above that explained by simple homogeneous distribution of the radionuclide throughout the body; the testes as a whole demonstrated increased uptake by factors of 3.56 and 4.01 compared with nonspecific uptake for (111)In and (201)Tl, respectively, at 24 h after administration. Both radionuclides gained access to the seminiferous tubules. CONCLUSION: The results obtained indicate that the values of testicular radiation doses quoted by the International Commission on Radiological Protection for (111)In might be too low by a factor of 4, whereas those for (201)Tl might be too high by a factor of 4. No data were obtained for uptake by individual germ cells within the testis and, therefore, no consideration of dosimetry at the cellular level was possible. However, it has been demonstrated that uptake of diagnostic Auger electron-emitting radionuclides by male germ cells within the testis is possible after intravenous administration.

Inhibition of gonadotropin-induced oviposition and ovarian steroidogenesis in the African clawed frog (Xenopus laevis) by the pesticide methoxychlor.

Concern over the role of environmental toxicants in amphibian population declines has highlighted the need to develop more comprehensive ecotoxicological test methods for this at-risk group. With continued interest in environmental endocrine disrupters (EDs), and the paucity of data pertaining to endocrine disrupting effects in amphibia, such tests should incorporate reproductive and endocrine endpoints. We investigated the effects of in vivo exposure to the pesticide methoxychlor (MXC) on reproductive and endocrine function in adult female African clawed frogs, (Xenopus laevis). Frogs were exposed to MXC (0.5-500 microg/l) in tank water throughout a cycle of oogenesis stimulated by exogenous gonadotropins. Gonadotropin-induced oviposition was delayed, and reduced numbers of unfertilizable eggs of increased size were oviposited by frogs exposed to 500 microg/l MXC. Reduced egg output was mirrored by increased gonado-somatic index in MXC-treated frogs. Post-oviposition, plasma sex steroid profiles were altered in MXC-exposed frogs as estradiol/progesterone and estradiol/testosterone ratios were elevated. Ex vivo synthesis of progesterone by ovarian explants was significantly reduced for frogs exposed to MXC> or = 0.5 microg/l. Additionally, plasma vitellogenin concentrations were significantly depressed in frogs exposed to 500 microg/l MXC. These data indicate that reproductive and endocrine dysfunction can occur in adult amphibia exposed to high concentrations of an environmental toxin with endocrine disrupting activity. Such effects may be indicative of the potential for adverse effects on amphibian wildlife exposed to environmental EDs.

Sperm DNA damage and cancer treatment.

Cancer treatments are well known to adversely affect male fertility. Reduction of sperm output arises from the cytotoxic effects of chemo- or radiotherapy upon the spermatogenic epithelium. However, if the epithelium survives there is a hazard to reproduction as the treatments are also mutagenic. The presence of DNA damage in the male genome is shown in animal experiments where there is transgenerational expression as a variety of effects ranging from miscarriage to carcinogenesis. The application of DNA damage methodology to sperm provides the opportunity for direct assessment. The Comet and Chromatin structure assays (SCSA) measure DNA damage by different principles, however, conclusions arising from the data are similar. DNA damage is present in sperm from fertile and infertile men and there is some association with infertility. Both assays detect sperm DNA damage after in vivo treatment with genotoxic agents. In a man treated with chemotherapy for cancer there was increased and persistent DNA damage in sperm. This information is consistent with the generation of human genetic diseases after conception with sperm carrying high loads of DNA damage. Whilst studies have not supported any association between paternal cytotoxic cancer therapy and genetic disease in their children, it would be unwise to discount these observations. The institution of better surveillance of genetic disease in the offspring of men surviving cytotoxic therapies may provide more robust risk assessment.

Germ cell and dose-dependent DNA damage measured by the comet assay in murine spermatozoaa after testicular X-irradiation.

The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25-4 Gy X-rays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.