Significance of structural chromosome aberrations in human sperm: analysis of induced aberrations

Summary A significant increase in the incidence of structural chromosome anomalies has been observed in the sperm of patients treated with radio and/or chemotherapy for different types of cancer when analyzed by the interspecific fertilization of hamster eggs. The analysis of these aberrations shows that while in controls only 9.4% of structural abnormalities are of the stable type, in treated patients this figure increases to 39.3%, thus indicating that the anomalies have not been produced during the fertilization of the hamster egg. However, it is possible that part, or even most, of the breaks appear as a result of a reduced repair capacity of sperm chromosomes in the cytoplasm of the hamster egg.     

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.     

An analysis of structural aberrations in human sperm chromosomes

We have analyzed structural aberrations in 5,000 sperm chromosome complements obtained from 20 men over a 5-yr period by fusion of human sperm with hamster eggs. Detailed data are presented on 366 abnormal cells with 379 analyzable breakpoints. The frequency of cells with structural aberrations ranged from 1.9% to 14.5% among donors; this interindividual variability was statistically significant (p less than 0.0001). In contrast, repeat samples from individual men showed no significant variation over time. The number of sperm chromosome sets processed per hamster egg had no effect on the frequency with which structural aberrations occurred, nor were sperm chromosome abnormalities altered by varying capacitation or culture conditions. The spectrum of structural aberrations observed in human sperm chromosomes and a chi-square analysis of breakpoints based on DNA content are presented. Although human sperm chromosome abnormalities were visualized with a cross-species system, we believe that they represent an inherent, biologically significant phenomenon.     

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.     

Chromosomal analysis in mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-type aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.     

The effect of methylated oxypurines on the size of newly-synthesized DNA and on the production of chromosome aberrations after UV irradiation in Chinese hamster cells.

A comparison has been made, in Chinese hamster cells, of the ability of various methylated oxypurines to inhibit post-replication repair of DNA after UV irradiation and to potentiate UV-induced chromosome aberrations. DNA synthesized in UV-irradiated cells contains gaps, which are subsequently sealed by a process termed post-replication repair. In rodent cells this process is inhibited by caffeine and its analogues. This has been quantitated by measuring the molecular weight of the DNA synthesized in UV-irradiated cells during a 4-h pulse-labelling period in the presence or absence of inhibitors--the lower molecular weight the greater the inhibition. Eight methylated oxypurines were tested; caffeine and chlorocaffeine were always the most potent inhibitors, tetramethyluric acid was inactive, and the other five derivatives (methoxycaffeine, ethoxycaffeine, paraxanthine, theobromine and theophylline) had intermediate effects. Measurements of the potentiation of UV-induced chromosome aberrations showed that treatments with caffeine or chlorocaffeine again had the greatest effects, tetramethyluric acid and also theophylline had no potentiating activity, and methoxycaffeine was intermediate. This correlation between effects at the molecular and cytological levels is consistent with the hypothesis that the inhibition of post-replication repair by methylated oxypurines gives rise to the increased production of chromosome aberrations.     

A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.

Superovulated hamster oocytes were cryopreserved and thawed according to our carefully designed procedures. More than 90% (92 +/- 4%) of oocytes survived freezing and thawing. They were proven to be well conserved, showing excellent performance comparable to freshly ovulated oocytes in the human sperm penetration test (proportion of penetrated ova: 94.7% vs. 93.6%) and human sperm chromosome analysis (proportion of metaphasic ova: 81.8% vs. 83.6%). There was no statistically significant difference in the incidences of sperm chromosome aberrations between assays using fresh and frozen-thawed oocytes. In addition, there was no statistically significant increase of aberrations in female pronuclear (hamster) chromosomes. This freezing-thawing method was found to be reliable, yielding viable hamster oocytes of high quality.     

Dithiothreitol induces sperm nuclear decondensation and protects against chromosome damage during male pronuclear formation in hybrid zygotes between Chinese hamster spermatozoa and Syrian hamster oocytes

The present study was undertaken to investigate whether a time lag in sperm nuclear decondensation and male pronuclear formation in the course of development of eggs is associated with any occurrence of structural chromosome aberrations in male genomes of hybrid zygotes between Chinese hamster spermatozoa and zona-free Syrian hamster oocytes. Shortly after insemination, hybrid zygotes were treated with dithiothreitol (DTT) at different concentrations (0.1-10.0 mM) for 30 min to reduce protamine disulphide (S-S) bonds and thereby accelerate sperm nuclear decondensation and male pronuclear formation. The incidence of sperm nuclear decondensation and male pronuclear formation increased with increasing DTT concentrations, indicating that a reduction in S-S bonds effectively induces these cytological events. Chromosomes of male genomes in hybrid zygotes generated by treatment with 1.0 mM, 2.5 mM and 10.0 mM DTT were analysed at the first cleavage metaphase. Incidence of structural chromosome aberrations in each treatment was 34.5%, 27.1% and 24.7%, respectively. There was a significant difference between the incidences with 1.0 mM and 10.0 mM DTT treatment. As the time lag in nuclear decondensation and male pronuclear formation was greatest in the 1.0 mM treatment condition, followed in order by 2.5 mM and 10.0 mM, it is suggested that the lag in sperm nuclear development behind egg development is responsible for structural chromosome aberrations in male genomes of hybrid zygotes.     

Cytogenetic effect of irradiation and subsequent cytosine arabinoside and hydroxyurea treatment on Chinese hamster cells in the G1 phase of the cell cycle

A two-hour treatment of Chinese hamster cells at the G1 stage of the cell cycle with arabinoside cytosine combined with hydroxyurea after X-irradiation (50-300 cGy) produced a 2- to 4-fold increase in the frequency of chromosome aberrations. The mitotic selection method was used to synchronize the cells. The potentiating effect of the inhibitors, that was estimated by the yield of centric exchanges, decreased with increasing radiation dose. It is suggested that DNA repair processes determining a linear component of the dose-response curve are modified within the dose-range under study.     

Investigations on the frequency of chromosome aberrations in bone marrow cells of Chinese hamsters after simultaneous application of caffeine and cyclophosphamide

Summary The potentiating effect of caffeine (1,3,7-trimethylxanthine) on chemically induced chromosome aberrations was studied in bone marrow cells of chinese hamsters, exposed to the alkylating agent cyclophosphamide.Four experimental series were performed: In the first two tests caffeine (200 mg/kg) or cyclophosphamide (40 mg/kg), respectively, were administered. A third and fourth test was performed with caffeine plus cyclophosphamide (200+40 mg/kg and 35+40 mg/kg, respectively) simultaneously.Aberrations induced by cyclophosphamide (40 mg/kg) were strongly potentiated by simultaneous application of caffeine (200 mg/kg) not only additively but even synergistically. This increase of aberrations cannot be found after injection of the lower dose of caffeine (35 mg/kg).     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.     

Chromosome analysis of human spermatozoa following in vitro exposure to cyclophosphamide, benzo(a)pyrene and N-nitrosodimethylamine in the presence of rat liver S9

For the purpose of assessing mutagenic effects (clastogenicity) of metabolites derived from chemical mutagens/carcinogens on human sperm chromosomes, spermatozoa were exposed in vitro to cyclophosphamide (CP), benzo(a)pyrene (BP) or N-nitrosodimethylamine (NDMA) for 2h in the presence or absence of rat liver S9, a metabolic activator of these chemicals. After in vitro fertilization between human spermatozoa and zona-free hamster oocytes, chromosome complements of sperm origin were analyzed cytogenetically.In the absence of S9, none of three chemicals (20 microg/ml CP, 200 microg/ml BP and 20mg/ml NDMA) caused a significant increase in spermatozoa with structural chromosome aberrations (8.6, 10.0 and 7.5%), as compared with their matched controls (10.9, 11.0 and 8.5%). In the presence of S9, however, a significant increase in chromosomally abnormal spermatozoa was observed in CP (37.1%, P < 0.001) and BP (31.0%, P < 0.001), indicating that enzymatic activation of CP and BP induced chromosomal abnormalities in human sperm. In contrast, NDMA did not induce chromosome aberrations in human spermatozoa by S9 treatment, although positive results have been observed in somatic cells. The present results on in vitro clastogenicity of CP, BP and NDMA are consistent with the results in previous in vivo studies with murine spermatozoa. Our S9/human sperm chromosome assay seems to be useful for estimation of hereditary risk of chemicals in human. Because most chemicals need metabolic activation to bind to DNA.     

Comparison of chromosomal abnormalities in hamster egg and human sperm pronuclei

One thousand human sperm and hamster egg haploid karyotypes were analyzed at the pronuclear stage after in vitro penetration. The frequency of abnormalities in human sperm was 8.5%, with 5.2% aneuploidy and 3.3% structural abnormalities. The hamster egg complements had an abnormality rate of 3.8%, with 3.3% aneuploidy and 0.5% structural abnormalities. In both human and hamster complements, chromosome abnormalities were observed in all chromosome groups, demonstrating that all chromosomes are susceptible to nondisjunction, not just acrocentric or small chromosomes. There is an intriguing difference between the frequency of hyperhaploid and hypohaploid complements in human sperm and hamster eggs. In the human complements, 2.4% were hyperhaploid and 2.7% hypohaploid. This is very close to the theoretical 1 to 1 ratio expected from nondisjunction. The hamster egg complements had more hypohaploid (2.2%) than hyperhaploid (0.9%) complements, despite identical treatment. Higher rates of hypohaploidy are generally ascribed to artificial loss of chromosomes, but may in fact reflect a predisposition of oocytes to anaphase lag during meiosis. The frequency of abnormalities (both numerical and structural) is higher in human complements than in hamster. This may reflect an innate propensity for meiotic chromosome abnormalities in humans or may result from greater exposure of humans to mutagenic agents.     

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。     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

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.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

The role of ADP-ribosylation processes in the repair of cytogenetic damage in mammalian cells

Nicotinamide (NA) was shown to increase the yield of chromosome aberrations in irradiated Chinese hamster cells. The effect was observed with all doses used (1-4 Gy) and in all phases of the cell cycle; it was maximum as cells transferred from S to G1 phase. The modification of radiation-induced aberrations was more pronounced in the chromatid deletions and in exchanges. The combined action of NA and caffeine showed a synergism. It is assumed that NA inhibits reparation in a different way than caffeine does.     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multicolor fluorescence in situ hybridization

Both structural and numerical chromosome aberrations in sperm represent important categories of paternally transmitted genetic damage. Therefore, a new multiprobe fluorescence in situ hybridization (FISH) method, using DNA probes for three targets (centromere and telomere of chromosome 1, centromere of chromosome 8), was developed to detect human sperm carrying three types of chromosomal defects: (1) terminal duplications or deletions in chromosome 1p, (2) aneuploidy involving chromosomes 1 or 8, and (3) diploidy. Baseline frequencies were determined for three healthy donors who had been previously evaluated for sperm cytogenetics by the human-sperm/hamster-oocyte cytogenetic technique (hamster technique). Among ∼120 000 sperm analyzed by the new FISH method, the average baseline frequencies of sperm carrying telomeric duplications and deletions of 1p were 3.2 ± 1.9 and 2.9 ± 3.6 per 10⁴, respectively. Diploid sperm was found in an average frequency of 6.6 ± 4.0 per 10⁴. Average frequencies of disomic sperm for chromosomes 1 or 8 were 1.7 ± 2.2 and 1.9 ± 2.3 per 10⁴, respectively. Inter-individual differences were observed for deletions of 1p but not for the other sperm phenotypes. A good correlation was obtained between the frequencies of sperm with structural chromosome aberrations detected with the new assay and the frequency of sperm carrying premeiotic or meiotic cytogenetic damage detected with the hamster technique. The observed levels of numerical aberrations with the new FISH assay were within range of the baseline frequencies reported by the hamster technique. The newly developed FISH assay has promising applications in genetic, clinical, physiological and toxicological studies. Received: 26 February 1996 / Revised: 6 May 1996