Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes

To study the repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes, the potentiating effects of 3 well-known repair inhibitors, arabinofuranosyl cytosine (ara-C), 3-aminobenzamide (3AB) and caffeine, on the frequency of induced chromosome aberrations were examined in eggs fertilized with X-irradiated sperm or in eggs irradiated with X-rays at the mature oocyte stage immediately before fertilization. Gametic treatment, fertilization and embryo culture were carried out in vitro. Ara-C treatment was done only in the pre-DNA replication period, while treatment with 3AB and caffeine was continuous from fertilization to the first-cleavage metaphase. The induction of chromosome aberrations by exposing sperm or oocytes to X-rays was remarkably potentiated by post-treatment incubation in the presence of each of the 3 inhibitors. This result indicates the possibility that X-ray damage induced in sperm or oocytes is reparable in the fertilized eggs and that various types of repair processes are involved.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation

The induction of chromosome aberrations in eggs of mice fertilized with X-irradiated sperm was performed by using an in vitro fertilization technique. Capacitated mature sperm was irradiated with various doses of X-rays and cytological analysis of the first cleavage metaphase of in vitro fertilized eggs was made. The frequencies of chromosome aberrations increased exponentially with dose and the dose-response relationship for overall breaks fitted well to a quadratic equation. The chromosome aberrations were mainly chromosome-type (82.1%), and the majority of aberrations were fragments.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Repair of human sperm chromosome aberrations in the hamster egg

Summary In order to study the repair capacity of fertilized hamster eggs for the lesions present or induced in human sperm, we have examined the potentiating effect of caffeine, a DNA repair inhibitor, on the frequency and types of sperm chromosome aberrations. Sperm samples were donated by an individual treated with chemotherapy for a testicular cancer 3 years previously. Exposure of spermatozoa and inseminated oocytes to caffeine led to an increase of sperm chromosome aberrations, indicating that the damage to human sperm can be repaired in untreated hamster egg cytoplasm. The potentiating effect of caffeine was mainly reflected in an increase of unrejoined aberrations, indicating that the formation of chromosomal rearrangements is also inhibited. Since both chromatid-type and chromosome-type aberrations increase after treatment with caffeine, damage to human sperm can probably be repaired inside the hamster egg cytoplasm by pre and post-replication repair mechanisms.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. II. Chromosome aberrations induced in mature oocytes and fertilized eggs at the pronuclear stage following X-irradiation

Cytological analysis of the first-cleavage metaphase of eggs exposed to X-rays at the mature oocyte stage or the pronuclear stage 4 h after fertilization was performed using the in vitro fertilization technique. The frequency of chromosome aberrations in irradiated mature oocytes increased exponentially with dose, the dose-response relationship being best fitted to the linear-quadratic model. On the other hand, in eggs irradiated at the early pronuclear stage, the frequency increased linearly with dose and the dose-response relationship was best fitted to the linear model. The aberrations were mainly chromosome-type (mature oocytes: 86.0% and pronuclear stage: 88.5%) and the majority were fragments in both cases. Eggs in the early pronuclear stage were markedly more radiation-sensitive than mature oocytes. A comparison of the present results with the previous ones (Matsuda et al., 1985b) showed that the sensitivities to induction of chromosome aberrations were in the order: egg at early pronuclear stage (highest) greater than mature oocyte greater than mature sperm.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Chromosome aberrations induced by tritiated water or 60Co gamma-rays at early pronuclear stage in mouse eggs

The induction of chromosome aberrations in mouse eggs by exposure to HTO beta-particles and 60Co gamma-rays at the early pronuclear stage was examined at the first-cleavage metaphase by using an in vitro fertilization technique. Eggs at the pronuclear stage were exposed to beta-particles in a chemically defined medium containing tritiated water (HTO) for 2 h at 3-5 h after insemination. Other eggs at the same stage were exposed to gamma-rays from 60Co during the same period. The dose-response relationships for frequencies of chromosome aberrations per egg were fitted to a linear-quadratic model for HTO beta-particles, and to a linear model for 60Co gamma-rays. The chromosome aberrations were mainly chromosome-type, and the majority of all aberrations were fragments. RBE values of HTO beta-particles relative to 60Co gamma-rays and acute X-rays, which were estimated from the ratio of the linear regression coefficients over 0.05-Gy range, were 2.0 and 1.6, respectively.     

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF)

Summary During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G₁-phase (G₁-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3–4% of all in vitro fertilized eggs.     

Protective effect of caffeine on chromosomal structures of mammalian cells irradiated with UV-rays]

The effect of caffeine (2mM) on the frequency of structural mutations induced by UV light (lambda = 265 nm at an incident dose of 40 erg/mm2) in the primary culture of mouse embryonic fibroblasts is studied. A half-hour treatment with caffeine of cells at the time of the first mitosis metaphase decreased approximately by 2 times the frequency of chromosome aberrations induced by UV light at the S stage and observed at the metaphase of this or the next C-mitosis. The frequency of both breaks and exchanges decreased as the result of caffeine treatment. The persistence of the protective effect of caffeine at the time of the second C-mitosis suggests that the observed decrease of the aberration rate is accompanied by the true reparation of pre-mutational lesions in chromosomes; the nature of the reparative process and the time when it takes place is as yet not clear. Caffeine did not decrease the frequency of spontaneous structural mutations.     

Sperm aging in the male after sexual rest: Contribution to chromosome anomalies

The chromosome complement was studied in first-cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3-day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33–35 h post-HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G-banded or Q-banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first-cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.     

Delayed sperm injection and fertilization in parthenogenetically activated insect egg (Athalia rosae,Hymenoptera)

Summary Mature eggs dissected from the ovary of unmated females of Athalia rosae ruficornis Jakovlev (Hymenoptera, Tenthredinidae) can be activated to develop (into haploid parthenogenetic males) simply by exposing them to distilled water. These eggs, which are primary oocytes arrested at the first meiotic metaphase, resume meiosis upon activation and reach the first meiotic telophase in 20 min. Mature eggs immediately upon dissection have previously been shown to complete karyogamy and develop as fertilized diploid females if injected with sperm. We show here that the eggs activated in water for 20 min have a much higher rate of successful fertilization if injected with sperm, and that the eggs activated for 40 min, upon sperm injection, though at a reduced frequency still develop as diploid fertilized females. Eggs left in water for 60 min, however, are no longer fertilized upon sperm injection and develop as haploid males.     

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.     

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.     

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.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

新制癌菌素和平阳霉素诱发蚕豆染色体畸变的比较研究

本文报道了新制癌菌素(NCS)能诱发植物染色体畸变,同时观察了利用咖啡因后处理对NCS、PYM诱发染色体畸变的影响,研究了PYM切断DNA断头的性质。结果表明,NCS切割DNA产生3'-羟基末端和3'-磷酸末端;咖啡因能封闭3'-羟基末端抑制DNA的修复,从而提高诱变频率。PYM加咖啡因后处理,其染色体畸变频率与PYM单独处理无明显差异。说明PYM切断DNA所得到的产物,不是3'-羟基末端,而是3'-磷酸末端。     

Evaluation of morpholine, 3-morpholinone, and N-substituted morpholines in the rat hepatocyte primary culture/DNA repair test

Although mature mammalian sperm are incapable of DNA repair, repair of damaged sperm DNA can occur after fertilization, as the sperm head decondenses and forms the male pronucleus. To quantify the cytogenetic effects of damage to sperm DNA we adapted the sister-chromatid exchange (SCE) test for use in early mouse embryos. After ultraviolet (UV) irradiation of sperm, eggs were fertilized in vitro and cultured for 2 cell cycles in medium containing fluorodeoxyuridine and bromodeoxyuridine; chromosomes were then prepared for SCE analysis. We found that UV-induced SCEs could be detected at the second cleavage division, and that eggs of different strains showed different frequencies of SCEs when fertilized by damaged sperm of a single strain. These results may indicate strain-specific differences in DNA repair of UV-induced DNA lesions by the early mouse embryo.