Mutagenic effects of ionizing radiation on immature rat oocytes.

Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction.

Structural chromosomal aberrations, aneuploidy, and mosaicism in early cleavage mouse embryos derived from spermatozoa exposed to γ-rays.

PURPOSE: To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from γ-irradiated spermatozoa. MATERIALS AND METHODS: Mature males were exposed to 2 Gy or 4 Gy of ¹³7Cs γ-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos. In the chromosome preparations from two-cell and four-cell embryos, the separation of the sister blastomeres was precluded by treatment of the embryos with concanavalin A. The incidence of embryos with structural chromosomal aberrations, aneuploidy, or mosaicism was estimated. The fates of the different types of γ-ray-induced structural chromosomal aberrations were also investigated in those embryos. RESULTS: The exposure of spermatozoa to 2 Gy or 4 Gy γ-rays caused structural chromosomal aberrations in 25.9% and 35.7% of the resultant one-cell embryos, respectively. At two-cell embryonic stage, the incidence of structural chromosomal aberrations was 17.4% in the 2 Gy group and 27.1% in the 4 Gy group. At the four-cell embryonic stage, although the incidence of control embryos with structural chromosomal aberrations was considerably high, the net incidence of embryos with radiation-induced structural chromosomal aberrations was similar to that at the one-cell stage. The incidence of aneuploidy was high in two-cell and four-cell embryos after both doses of γ-rays. The incidence of mosaicism increased significantly in dose- and embryonic-stage-dependent manners. Anaphase lag, and the degeneration and non-disjunction of the aberrant chromosomes were frequently observed in aneuploid and mosaic embryos. CONCLUSIONS: Mouse sperm DNA is highly vulnerable to γ-rays. The structural chromosomal aberrations of sperm origin are unstable in their behaviour and structure during cleavage, and therefore cause secondary aneuploidy and mosaicism in the early cleavage embryos.

Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse.

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.

How long do parthenogenetically activated mouse oocytes maintain the ability to accept sperm nuclei as a genetic partner?

PURPOSE: Cytogenetic risk of intracytoplasmic sperm injection (ICSI) after artificial oocyte activation (post-activation ICSI) was evaluated in the mouse. METHODS: Mouse zygotes were produced by ICSI into eggs at various intervals after parthenogenetic exposure to strontium (Sr) for 30 min. Male pronucleus formation and the chromosome constitution were studied. RESULTS: Sperm nuclei injected into oocytes within 1 h after Sr exposure (from early through mid-telophase) transformed normally into male pronuclei, and the number of chromosome aberrations did not significantly increase in the resultant zygotes. When sperm nuclei were injected into eggs at intervals beyond 1 h after Sr exposure (from late telophase through the G1 pronuclear stage), the rate of male pronucleus formation was significantly reduced. The incidence of chromosome aberrations increased with time between oocyte activation and ICSI. CONCLUSIONS: ICSI into oocytes within 1 h after parthenogenetic activation produces cytogenetically competent embryos in the mouse.

Chromosome analysis of mouse zygotes after injecting oocytes with spermatozoa treated in vitro with green tea catechin, (-)-epigallocatechin gallate (EGCG).

The cytogenetic effects of (-)-epigallocatechin gallate (EGCG) on mouse spermatozoa were studied in vitro using an intracytoplasmic sperm injection (ICSI) technique. Spermatozoa were collected by the swim-up method and treated with EGCG at 1 microM and 10 microM. When motile, EGCG-treated spermatozoa were injected into oocytes, structural chromosome aberrations (SCAs) at the first cleavage metaphase did not increase significantly. However, a majority of immotile spermatozoa treated with 10 microM EGCG had the following abnormalities: pronuclear arrest (11% of activated oocytes), degenerated sperm chromatin (chromosome) mass (30% of activated oocytes) and occurrence of structural chromosome aberrations (57% of analyzed metaphases). The incidence of these abnormalities suggests that immotile spermatozoa were susceptible to EGCG, and that the damage of sperm chromatin was accelerated in immotile spermatozoa by 10 microM EGCG treatment.

Chromosome analysis of mouse one-cell androgenones derived from a sperm nucleus exposed to topoisomerase II inhibitors at pre- and post-fertilization stages.

Mouse spermatozoa and androgenetic one-cell embryos (androgenones) at various developmental stages were exposed to etoposide (1 microM), a topoisomerase II (topo II) poison, or to either of two catalytic inhibitors: ICRF-193 (10 microM) or merbarone (50 microM), for 2 h in order to study the clastogenic effects of these drugs on remodeled sperm chromatin. None of the drugs induced structural chromosome aberrations in condensed chromatin of spermatozoa. However, etoposide and merbarone exerted strong clastogenic actions on remodeled chromatin of androgenones. Expanding chromatin was most sensitive to both of these drugs at the time of pronuclear formation, as nearly 100% of androgenones exposed at this stage displayed structural chromosome aberrations. ICRF-193 did not affect sperm chromatin at all remodeling stages. A majority of the aberrations induced by etoposide and merbarone were of the chromosome-type. Chromosome exchanges, including translocation, dicentric, and ring chromosomes, preferentially appeared following exposure at the early stages of chromatin remodeling. Thus, despite their different modes of topo II inhibition, etoposide and merbarone showed similar clastogenic actions on remodeled sperm chromatin. These results suggest that the formation of transient DNA cleavage, mediated by ooplasmic topo II, accompanies the remodeling. The present findings provide insight into the mechanisms by which structural aberrations are generated in paternal chromosomes.

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation.

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris-HCl buffered solution supplemented with antioxidants.

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P < 0.001) and 2-4 days (P < 0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2-4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.

Chromosomal analysis of mouse spermatozoa following physical and chemical treatments that are effective in inactivating HIV.

Human immunodeficiency virus (HIV) can be inactivated by heating at 56 degrees C for 30 min, treating with 50% ethanol at room temperature for 10 min, or treating with 2% sodium hypochlorite solution (NaClO) at room temperature for 60 min. Using a mouse model, we evaluated the risk of generating chromosome damage in spermatozoa following these treatments. The spermatozoa were all dead after the treatments. Although 41.3% of oocytes injected with ethanol-treated spermatozoa successfully activated, none of the oocytes injected with heated or NaClO-treated spermatozoa activated. When artificial stimulation with strontium was used, the fertilization of oocytes with heated or ethanol-treated spermatozoa was completely rescued. Sperm nuclei treated with NaClO neither decondensed nor developed to a male pronucleus. The incidences of structural chromosome aberrations in 1-cell zygotes derived from the heated spermatozoa (45.6%) and ethanol-treated spermatozoa (91.2%) were significantly higher than those in the matched controls (5.5% and 10.5%, respectively). Further study is needed to develop a methodology for the protection of spermatozoa against chromosome damage or the separation of damaged spermatozoa before intracytoplasmic sperm injection.

Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa.

Previous studies on the clastogenic effects of mutagens on human sperm chromosomes were reviewed. A marked increase of structural chromosome aberrations (SCAs) has been reported in the spermatozoa irradiated in vitro with five kinds of ionizing radiation (137Cs gamma-, 60Co gamma-, X-, and 3H beta-rays and 252Cf neutrons). The micronucleus (MN) test with hybrid two-cell embryos generated from human sperm and hamster oocytes was shown to be useful as a simple and rapid method for assessing the effects of radiation. Radiosensitivity of human spermatozoa was highest, being followed by golden hamster, Chinese hamster and mouse spermatozoa. Chromosome-damaging effects were also found with some chemicals (bleomycin, daunomycin, methyl methanesulfonate, triethylenemelamine, neocarzinostatin, N-methyl-N'-nitro-N-nitorosoguanidine and mitomycin C (MMC)), but not with other chemicals (urethane, nitrobenzene, dioxin, cyclophosphamide (CP), benzo(a)pyrene (BP) and N-nitrosodimethylamine (NDMA)). The clastogenicity of chemical metabolites was confirmed for CP and BP, by using the S9-based metabolic activation system. The results of sperm chromosome analysis from cancer patients who had undergone radio- and/or chemotherapy were contradictory among investigators and further studies are necessary. The importance of mutagenicity testing with human spermatozoa is discussed.

Abnormal chromosome migration and chromosome aberrations in mouse oocytes during meiosis II in the presence of topoisomerase II inhibitor ICRF-193.

Using a mouse parthenogenetic system, effects of ICRF-193, a noncleavable complex-forming topoisomerase II inhibitor, on female meiosis II chromosomes and pronuclear chromosomes were studied. Eggs were exposed to the inhibitor (10 microM) at various times after parthenogenetic stimulation, and chromosomes of them were analyzed at the first cleavage metaphase. When eggs were exposed to the inhibitor during the period from metaphase II to anaphase II, a significant increase in incidences of structural chromosome aberrations (51.1% versus 1.3% in the control) and aneuploidy (30.3% versus 0.7% in the control) was found. Structural chromosome aberrations were observed in 10-20% of eggs following treatments during telophase II, but there was no increased incidence of aneuploidy in treatments during this meiotic stage. When pronuclear eggs at S phase were targeted by the inhibitor, no significant increase in chromosome aberrations was found.Interestingly, when chromatids moved to each pole during anaphase II in the presence of ICRF-193, most of them oriented their centromeres toward the spindle equator as if moving backwards. Moreover, lagging chromatids with the centromeres present were observed in more than 50% of treated eggs. However, chromosomal bridges that resulted from chromosome stickiness did not appear in any egg.These findings indicate that ICRF-193 can induce structural chromosome aberrations and aneuploidy in mouse secondary oocytes in meiotic stage-dependent manner. The induction of aneuploidy is due to disruption of the separation of sister centromeres at anaphase II. There appears to be mechanism(s) other than cleavable complex formation or chromosome stickiness behind the induction of structural chromosome aberrations by ICRF-193.