Analysis of radiation-induced micronuclei in two-cell human-hamster embryos using telomeric and centromeric FISH probes
Department de Biologia Cel.lular i Fisiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain. Cytogenetics and cell genetics
02/1996; 74(1-2):102-6. DOI: 10.1159/000134392
Simultaneous, fluorescent in situ hybridization using a centromeric human alpha satellite DNA probe and a telomeric DNA probe was used to analyze the chromosome content of micronuclei induced in two-cell human-hamster embryos by in vitro gamma-ray irradiation of human spermatozoa. In unirradiated samples, about 26% of micronuclei were centromere positive, indicating that both structural chromosome aberrations and numerical changes are involved in the spontaneous production of micronuclei. After exposure of spermatozoa to radiation, a significant increase in the number of micronuclei was found. About 77% of induced micronuclei contained only telomeric signals suggesting that they originated from acentric fragments. However, both centromere-positive and centromere-negative micronuclei increased with radiation dose. These results are consistent with the well known clastogenic effect of ionizing radiation and with its weak aneugenic effect.
Available from: Maitane Barasoain
- "For FISH analysis, to identify centromeres, 50 MN for each individual and type of culture on coded slides in a blind study were analyzed for the presence of a fluorescent signal. MN were classified as centromere-negative (CN − ) or centromerepositive (CN + ) by considering the presence of a hybridization signal in a MN as a direct indication of the presence of a centromere . Among CN + MN, signals of different intensities were found due to variable amounts of alpha-satellite DNA on different human chromosomes . "
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ABSTRACT: Angiotensin II receptor blockers (ARBs) are a new class of drugs for the treatment of hypertension. In this study, we studied the potential genotoxic effects of five ARBs in vivo and in vitro in human peripheral blood lymphocytes (PBLs) by means of the cytokinesis-block micronucleous (CBMN) assay in combination with fluorescence in situ hybridization (FISH) with a centromeric probe. The nuclear division index (NDI) was used as a measure of cytotoxicity. We also analyzed the association between sex, age, duration of treatment and MN formation. The in vivo study was carried out in 55 hypertensive patients. The in vitro study was performed in 10 control individuals by adding the drugs to the culture medium at a final concentration similar to the levels found in plasma in patients. Our results showed a significant increase in the frequencies of MN and binucleated cells with MN (BNMN) in vivo and especially in vitro. We observed variability in the mean frequency of MN and BNMN among the five drugs analyzed. In vivo, patients treated with Candesartan, Telmisartan and Valsartan showed a statistical significant increase in these parameters, while Olmesartan showed the highest effect in vitro. We also found that the drugs inhibit the NDI in vitro and that Eprosartan, Olmesartan and Telmisartan are the ARBs studied with the highest effect in decreasing the proliferation of the cells. FISH analysis revealed no significant difference between patients and controls in the frequency of centromeric signals. A slight variability, without statistical significance, in the frequency of micronuclei with a centromere signal (CN+MN) was found among the different ARBs analyzed, ruling out an aneugenic potential. When accounting for risk factors, we found that in patients there is a positive correlation between MN, BNMN and sex and a negative correlation with duration of treatment.
Available from: Hossein Mozdarani
- "Therefore, in this study we have investigated the fertilization rate and outcome of irradiated sperm with gamma radiation, after injection into golden hamster oocyte. We didn’t use human oocytes because of ethical issues, and on the other hand the variety of studies showed that human sperm would penetrate to hamster oocyte and form pronuclei and 2 cell embryos (23-25). In this study we showed that human-hamster embryos can progress to 8 cells. "
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ABSTRACT: Background: Irradiation is one of the major causes of induced sperm DNA damage. Various studies suggested a relation between sperm DNA damage and fertilization rate after intra-cytoplasmic sperm injection (ICSI).
Objective: In this study, fertilization rate and premature chromosome condensation (PCC) formation after ICSI of hamster oocytes with irradiated sperms from normal and oligosperm individuals was investigated.
Materials and Methods: Human sperms were classified according to counts to normal and oligosperm. Ten samples were used for each group. Golden hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. From retrieved oocytes, 468 were in metaphase II. Control and 4 Gy gamma irradiated sperms were then injected into oocytes. After pronuclei formation in injected oocytes and formation of 8 cells embryos, slides were prepared using Tarkowskie's standard air-drying technique. The frequency of embryos and PCC were analyzed using 1000× microscope after staining in 5% Giemsa.
Results: The extent of embryo development in oocytes injected by irradiated sperms was lower than those injected by non-irradiated sperms (p=0.0001). The frequency of PCC in failed fertilized oocytes was significantly higher in oligosperms (46%) compared with normal ones (0%), but there was no significant difference between irradiated and non-irradiated samples in each group (p=0.12).
Conclusion: The results showed that irradiation of sperms might influence the fertilization outcome possibly due to sperm DNA damage. One possible cause of precluding oocytes from fertilization in oligosperm individuals might be the formation of PCC.
Available from: Immaculada Ponsa
- "Although the micronucleus test can be used to evaluate the residual lesions left after occupational or accidental exposure to radiation, this test cannot be used to estimate the dose received at the testicular level due to the different radiosensitivities of spermatogenic cells. Since micronuclei may contain acentric fragments  as well as centric fragments or whole chromosomes with damaged centromeres , and even groups of chromosomes that produce large micronuclei , we decided to carry out a FISH study with telomeric and centromeric probes for all human chromosomes to characterize the chromosomal content of micronuclei . The results showed that over 75% of micronuclei were centromere-negative, indicating that they originated from acentric fragments. "
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