Radiation- and chemically-induced chromosome aberrations in mouse oocytes: a comparison with effects in males.

Data from studies on radiation- and chemically-induced chromosome aberrations in mouse oocytes have been summarized. An attempt has been made to assess the relative sensitivity to mutagenic agents of female and male germ cells through comparison of observations from mutation studies of female and male mice. No unequivocal evidence of a mutagenic effect limited to a single sex could be found in the cytogenetic data, although differences in relative germ cell sensitivity could be inferred for ionizing radiation and some chemicals. However, the pattern of inter-sex variations was not consistent: for example, irradiation of dictyate oocytes yielded a lower rate of heritable chromosome translocations than the same dose to spermatogonia; in contrast, some chemicals, such as mitomycin C, yielded a larger incidence of chromosome anomalies after treatment of dictyate oocytes than spermatogonia. Overall, the limitations in quality and quantity of cytogenetic data, and the uncertainties associated with comparing information obtained in disparate assays, place severe constraints on the use of observations on induced chromosome aberrations to assess the relative sensitivities of female and male germ cells to environmental mutagens.     

DNA damage and repair in somatic and germ cells in vivo

Alkylation-induced germ cell mutagenesis in the mouse versus Drosophila is compared based on data from forward mutation assays (specific-locus tests in the mouse and in Drosophila and multiple-locus assays in the latter species) but not including assays for structural chromosome aberrations. To facilitate comparisons between mouse and Drosophila, forward mutation test results have been grouped into three categories. Representatives of the first category are MMS (methyl methanesulfonate) and EO (ethylene oxide), alkylating agents with a high s value which predominantly react with ring nitrogens in DNA. ENU (N-ethyl-N-nitrosourea), MNU (N-methyl-N-nitrosourea), PRC (procarbazine), DEN (N-nitrosodiethylamine), and DMN (N-nitrosodimethylamine) belong to the second category. These agents have in common a considerable ability for modification at oxygens in DNA. Cross-linking agents (melphalan, chlorambucil, hexamethylphosphoramide) from the third category.The most unexpected, but encouraging outcome of this study is the identification of common features for three vastly different experimental indicators of genotoxicity: hereditary damage in Drosophila males, genetic damage in male mice, and tumors (TD₅₀ estimates) in rodents. Based on the above three category classification scheme the following tentative conclusions are drawn. Monofunctional agents belonging to category 1, typified by MMS and EO, display genotoxic effects in male germ cell stages that have passed meiotic division. This phenomenon seems to be the consequence of a repair deficiency during spermiogenesis for a period of 3–4 days in Drosophila and 14 days in the mouse. We suggest that the reason for the high resistance of premeiotic stages, and the generally high TD₅₀ estimates observed for this class in rodents, is the efficient error-free repair of N-alkylation damage. If we accept this hypothesis, then the increased carcinogenic potential in rodents, seen when comparing category 2 (ENU-type mutagens) to category 1 (MMS-type mutagens), along with the ability of category 2 genotoxins to induce genetic damage in premeiotic stages, must presumably be due to their enhanced ability for alkylations at oxygens in DNA; it is this property that actually distinguishes the two groups from each other. In contrast to category 1, examination of class 2 genotoxins (ENU and DEN) in premeiotic cells of Drosophila gave no indication for a significant role of germinal selection, and also removal by DNA repair was less dramatic compared to MMS. Thus category 2 mutagens are expected to display activity in a wide range of both post- and premeiotic germ cell stages. A number of these agents have been demonstrated to be among the most potent carcinogens in rodents. In terms of both hereditary damage and the initiation of cancers (low TD₅₀), cross-linking agents (category 3) comprise a considerable genotoxic hazard. Doubling doses for the mouse SLT have been determined for four cross-linking agents not requiring metabolic conversion and in all four cases the doubling doses for these agents were lower than those for MMS, DES and EMS. In support of this conclusion, two of 10 genotoxic agents, for which data on chromosomal aberrations were available for both somatic cells and germ cells in mice, were cross-linking agents and again the doubling dose estimates are lower than for monofunctional agents. Four cross-linking agents induced mutations in stem cell spermatogonia indicating that this type of agent can be active in a wide range of germ cell stages.Quite in contrast to what is generally observed in unicellular systems and in mammalian cells in culture, both cross-linking agents and MMS-type mutagens (high s value) predominantly produce deletion mutations in postmeiotic male germ cell stages. This is the uniform picture found for both Drosophila and the mouse. It is concluded that in vitro systems, in contrast to Drosophila germ cells, fail to predict this very intriguing feature of mouse germ line mutagenesis. In addition to their potential for induction of deletions and other rearrangements, cross-linking agents are among the most efficient inducers of mitotic recombination in Drosophila. Thus there are several mechanisms by which cross-linking agents may cause loss of heterozygosity for long stretches of DNA sequences, leading to expression of recessive genes. Since a substantial portion of agents used in the chemotherapy of cancers have cross-linking potential, the potential hazards of hereditary damage and cancers associated with this class of genotoxins should, in our opinion, receive more attention than they have in the past.     

Relative Rates at Which Dominant-Lethal Mutations and Heritable Translocations Are Induced by Alkylating Chemicals in Postmeiotic Male Germ Cells of Mice

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.     

Serial analysis of chromosomal aberrations and proliferation kinetics of mouse spermatogonia after single or fractionated X-ray exposures

Dose-fractionation studies on translocation induction in stem-cell spermatogonia of mice, as measured by spermatocyte analysis many cell generations after irradiation, revealed that a small conditioning dose of X-rays sensitizes the stem cells to the induction of translocations by a second dose 24 h later (Van Buul and Léonard, 1974, 1980). To find out whether such sensitization effects also occur at other spermatogonial stages, a comparison was made of the effects of single (50, 100 and 150 rad) and fractionated (100 + 50 rad, with 24 h in between) doses of X-rays on the induction of chromosomal aberrations in spermatogonia by analysing spermatogonial metaphases shortly after irradiation at multiple sampling times (0–48 h; every 4 h). In addition, the kinetics of spermatogonial proliferation was studied by using, in vivo, a BrdU chromosome-labelling procedure. The recorded frequencies of chromosomal aberrations did not indicate any sensitization effect of dose fractionation. It is concluded that the sensitization effects, as observed for chromosomal aberrations in male premeiotic germ cells, are characteristic for the stem-cell spermatogonia and do not occur in the more differentiated spermatogonia.     

A386G transition in DAZL gene is not associated with spermatogenic failure in Tamil Nadu, South India

The DAZ-like (DAZL) gene located on the short arm of autosomal chromosome 3 (3p24), an essential master gene for the premeiotic development of male and female germ cells, is the father of the Y-chromosome DAZ gene cluster and encodes for RNA-binding proteins. Reported instances of positive association of DAZL gene mutations with infertility in men have been found in a Taiwanese population but not in Caucasians. There is no study from Tamil Nadu, South India, to demonstrate the role of DAZL gene in male infertility; we, therefore, analyzed a total of 287 men, including 147 infertile and 140 normozoospermic fertile controls from rural areas of Tamil Nadu, South India, to assess the phenotypic effect of DAZL mutations in this region of the world. Interestingly, all our samples showed absence of the A386G (T54A) mutation that was found to be associated with spermatogenic failure in the Taiwanese population. Therefore, we suggest that the A386G (T54A) mutation is not associated with male infertility in Tamil Nadu, South India.     

DNA single-strand breaks,double-strand breaks,and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. ¹³⁷Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methane sulfonate, ethyl methane sulfonate, ethyl nitrosurea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency.This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.Abbreviations DBCP dibromochlorpropane - DSB(s) DNA double-strand break(s) - EDB ethylene dibromide - EMS ethyl methane sulfonate - ENU ethyl nitrosurea - MC mitomycin C - MMS methyl methane sulfonate - SDS sodium dodecyl sulfate - SSB (s) DNA single-strand break(s) - TEM triethylene melamine - UDS unscheduled DNA synthesis     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.     

Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium

Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant cells largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.     

Fiber DNA studies of premeiotic mouse spermatogenesis

The technique of fiber DNA measurement was used to study the possibility that the lengthening of the DNA “S” phase previously reported for mouse premeiotic spermatogonia was due to a reduced number of initiation sites. The mean replicon size of neonatal mouse preleptotene cells was similar to sizes reported for adult mouse somatic cells. A slow rate of DNA chain growth was observed in all cells from day 1 through days 10–12 of age. It was felt that the meiotic entry in male mouse germ cells may involve a slower replication fork rate and other factors which increased the time between activation and replication of replicon families.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

c-Flip(L) is expressed in undifferentiated mouse male germ cells

Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-Flip(L) is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-Flip(L) was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-Flip(L) expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-Flip(L) was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia.     

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.     

Somatic cell mutagenicity in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages

With the intention of assessing the general performance, sensitivity and the underlying mechanisms of somatic cell mutagenicity assays in Drosophila, a study was undertaken to compare the effectiveness of 5 procarcinogens and 4 direct-acting agents in the white/white-coral eye mosaic assay (SMART) with their activity in early (premeiotic) male and female germ-cell stages, after exposure of Drosophila larvae. The outcome indicated a lack of agreement in the results from recessive lethal assays (SLRL) in comparison with the somatic mutation and recombination test (SMART). The procarcinogens 2-naphthylamine (NA), 3-methylcholanthrene (MC), 9,10-dimethylanthracene (DA) and 7,12-dimethylbenz[a]anthracene (DMBA), and the direct-acting mutagens bleomycin (BM), methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), were quite efficient in producing somatic recombination and mutations in white/white-coral larvae, as opposed to only weak effects in early germ-cell stages. 2-Acetylaminofluorene (2AAF) showed marginal effects in both germ cells and somatic tissue after exposure of female larvae, but was inactive in testis. The discrepancy in mutational response between somatic cells and premeiotic germ cells is most impressive for MMS and BM. There is sufficient evidence for attributing a good sized proportion of the encountered variation to efficient error-free DNA repair of premutational damage and to segregational elimination during meiosis of deleterious mutations: (1) The efficient point mutagen ENU was the but one agent producing high levels of viable genetic alterations in early germ cells and in somatic cells. A similar behaviour was previously described for diethylnitrosamine, which ethylates DNA in the same fashion as ENU. (2) In early germ-cell stages of mei-9L1 male larvae, MMS induced multiple mutations (putative clusters) at a low dose differing by a factor 20-40 from those needed to produce an equivalent response in repair-competent strains. This is consistent with the concept of an active excision repair in premeiotic cells. (3) In the case of EMS, next to DNA repair, germinal selection seems to restrict the realization of EMS-induced genetic damage in premeiotic cells. (4) Bleomycin-induced chromosome aberrations caused high mortality rates in males (hemizygous for an X-chromosome) but not in females. MMS and BM, agents known to show preference for chromosome aberration induction, produced 3-6-fold higher rates of somatic mutational events (SME) in female genotypes as compared with the other sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

We have generated a transgenic mouse line,Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia and is detected through preleptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF-a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells and zygotes of mice. II. Sensitivity of different postcopulation-precleavage stages to three alkylating chemicals.

The relative sensitivities of various postcopulation-precleabage and pronuclear stages to dominant-lethal effects of isopropyl methanesulfonate (IMS), ethyl methanesulfonate (EMS), and triethylenemelamine (TEM) were investigated. The pattern of sensitivity differed with the chemical. IMS was most effective when pronuclear formation was already completed and the majority of the zygotes were presumably undergoing DNA synthesis. EMS, on the other hand, induced its most pronounced effects when eggs in the course of second meiotic division and zygotes in early pronuclear stages were treated. The greatest effect of TEM was observed when zygotes were treated at the early pronuclear stage. EMS and TEM, in contrast to IMS, are similar to radiations in that zygotes undergoing DNA synthesis are more resistant to them than are the early pronuclear stages. In the case of IMS, effects induced in the most sensitive postcopulation-precleavage stage were 6 to 9 times greater than in the most sensitive precopulatory dictyate oocytes or male germ cells. On the other hand, in the case of EMS and TEM, the most sensitive precopulatory male germ cells, but not the dictyate oocytes, were more sensitive than the most sensitive postcopulation stages.