DELAYED FORMATION OF CHROMOSOME ABERRATIONS IN MOUSE PACHYTENE SPERMATOCYTES TREATED WITH TRIETHYLENEMELAMINE (TEM)

DELAYED FORMATION OF CHROMOSOME ABERRATIONS IN MOUSE PACHYTENE SPERMATOCYTES TREATED WITH TRIETHYLENEMELAMINE (TEM) W. M. GENEROSO,* M. KRISHNA,+,2 R. E. SOTOMAYOR+ AND N. L. A. CACHEIRO* +Biology Division, Oak Ridge National Laboratory and +University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Manuscript received July 21, 1976 Revised copy received October 6, 1976 ABSTRACT Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis-metaphase 1 stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis-metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed--a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis. UALITATIVELY, there is generally no difference in variolus germ-cell Q stages of mice between measurable chromosome breakage effects of ionizing radiations and of the many mutagenic alkylating chemicals. Dominant lethal mutations, heritable translocations, heritable inversions, sex-chromosome loss, and various types of cytologically detectable aberrations are end points that are common for both ionizing radiations and clastogenic alkylating agents. Questions then arise about the nature of events that lead to the formation of chemically induced chromosomal aberrations in mouse germ cells, and about the similarity of mechanisms involved to those for radiation. Both X rays and TEM induce dominant lethal mutations in pachytene spermatocytes of mice. There is strong evidence that dominant lethal mutations induced Research jointly sponsored by the National Center for Toxicological Research and de U. S. Energy Research and and Development Administration under contract with Union Carbide Corporation. a Postdoctoral investigator supported by Subcontract No. 3322 from de Biology Division of Oak Ridge National Laboratory to the University of Tenliessee. Genetics 85: 65-72 January, 1977

66 w. M. GENEROSO et al. in this germ-cell stage by X rays arise from induced chromosome breakage. OAKBERG and DIMINNO (1960) found an increased number of anaphase I and anaphase I1 cells with bridges, and WENNSTROM (1971), MCGAUGHEY and CHANG (1973), and TSUCHIDA and UCHIDA (1975) found increased frequency of structural aberrations in diakinesis-metaphase I cells following gamma- or X-ray treatment of primary spermatocytes. No such information was available for TEM. Nor was there information on the inducibility by any mutagen of heritable reciprocal translocations in meiotic spermatocytes of mice. Accordingly, the present study was conducted to determine the nature of TEM-induced dominact lethal mutations in pachytene spermatocytes by cytological examination of diakinesis-metaphase I cells and by scoring for transmissible translocations. A comparative study of the effects of TEM and X rays revealed contrasting mechanisms between the two agents in the formation of chromosome aberrations. MATERIALS AND METHODS (101 x C3H)F, hybrid male mice, approximately 12 weeks old, were treated intraperitoneally with 2mg/kg of TEM or exposed to 400 R of acute X rays (partial body irradiation). Control mice were given comparable volumes of Hanks balanced salt solution. In the dominant lethal study, analysis was done on 10- to 12-week-old (C3H x C57BL)F1 females mated on days 25 to 28 after treatment of males. In the heritable translocation study, (SEC x C57BL)F, females mated to TEM-treated males during this posttreatment period were allowed to go to term. Female progeny were discarded, while the males were tested for translocation heterozygosity at maturity (see GENEROSO et al. 1974 for procedure). Two groups of mice were killed for analysis of diakinesis-metaphase I spermatocytes. The first group was composed of only TEM-treated males, while the second group included TEMand X-ray-treated and control males. Two males per treatment were killed daily at intervals 3 to 7 days postreatment. The two testes of each male were prepared separately, using the air-dry technique of EVANS, BRECKON and FORD (1964.). Twenty-five cells were scored per testis. It is assumed that TEM had no effect on the progression of spermatogenesis and that the cells analyzed at diakinesis-metaphase I stage are the same as those studied in the dominant lethal and heritable translocation experiments at the time of treatment. RESULTS Data presented in Table 1 show that the doses of TEM and X rays used generally cause comparable reductions in pregnancy rate and number of living embryos. That dominact lethal mutations were induced by the two agents is clearly demonstrated by the high frequencies of dead implantations. In the case of X rays, it is known that killing of spermatocytes, in addition to dominant TABLE 1 Comparable dominant lethal effects of X ray and TEM in pachyiene spermatocytes Number of Pregnancy Number of Number of living Dead Tie ilnieut female; mated rate (%) Implants (avg) embryos (avg) implants (%I Control 76 92 10.2 9.5 6 TEM, 2 mg/kg 82 37 3.9 2.3 40 X ray, 400 R 31 29 4.0 1.3 67

HERITABLE TRANSLOCATIONS IN SPERMATOCYTES 67 lethality, contributes to the reduction in litter size (OAKBERG and DIMINNO 1960). Similarly, the reductions in number of living embryos and pregnancy rate after treatment with 2 mg/kg of TEM are partially attributable to killing of pachytene spermatocytes. The rate of fertilization (as judged by the presence of at least one pronucleus) in females mated 25 to 28 days after "EM treatment of males was 42%, compared with 84% for controls (total eggs examined were 188 and 103, respectively). Of the 247 male progeny tested for heritable translocations, 13 (5%) were translocation heterozygotes. The rate of 5% is significantly higher (P < 0.01) than the spontaneous rate of 0.091% (4 translocations in 4392 mice tested) pooled from previous experiments (GENEROSO et al., in press). Of the 13 translocation heterozygotes, all confirmed cytologically, IO were partially sterile and 3 were completely sterile. Results of the cytological analysis of diakinesis-metaphase I spermatocytes are shown in Table 2. It is clear from these data that although a very high frequency TABLE 2 Cytological analysis of diakinesis-metaphase I spermatocytes Posttreatment Number of Number of cells Number of cells Cells with Treatment interval (days) cells scored with fragments with exchanges aberrations (%) Control 2.5 1 0 0 0 0 3.5 100 1 0 1 4.5 50 0 0 0 5.5 100 0 0 0 6.5 100 0 - _. 0-0 - 450 1 0 0.2 X ray, 400 R 2.5 100 44 32 68 3.5 100 39 46 72 4.5 50 17 30 78 5.5 100 45 51 84 6.5 100 44 52 - - 81-450 189 211 76.4 TEM, 2 mg/kg 2.5 100 2 0 2 3.5 10'0 0 1 1 4.5 50 3 1 8 5.5 100 1 0 1 6.5 100 2 0 - - - 2-450 8 2 2.2 TEM, 2 mg/kg* 2.5 100 2 0 2 3.5 100 1 0 1 4.5 100 0 0 0 5.5 10'0 1 0 1 6.5 100 0 0 0 - - - - 500 4 0 0.8 * Replicate of the above TEM study.

68 w. M. GENEROSO et al. of cells treated with X rays at pachytene stage had fragments and/or interchanges, only a small increase in aberration was found with TEM. DISCUSSION The results show that TEM and X rays are alike in the sense that chromosome breakage resulted from treatment of pachytene spermatocytes. In the case of X rays, chromosome breakage was manifested by dominant lethal mutations and by the presence of chromosome aberrations in diakinesis-metaphase I spermatocytes. In the case of TEM, dominant lethal mutations were also clearly induced, but analysis of diakinesis-metaphase I spermatocytes did not give proof that chromosome breakage was the underlying cause. The frequency of observed abcrrations was much too low to account for all the lethals. The clear-cut proof that breakage was induced came rom data on heritable translocations. The very low frequency of aberrations in diakinesis-metaphase I stage and the recovery of heritable translocations in the "EM study provide the first evidence, for any chemical mutagen, that the formation of the bulk of breaks in pachytene spermatocytes of mice is delayed, a marked contrast to the more immediate formation of X-ray-induced breakage. Such a difference between X rays and a chemical mutagen is also evident in mouse dictyate oocytes. SEARLE and BEECHEY ( 1974) observed a high incidence of aberrations when X-irradiated dictyate oocytes were analyzed in metaphase I. BREWEN and PAYNE (in press) on the other hand, using methyl methanesulfonate (MMS), did not find a significant increase over controls in the number of aberrations found in metaphase I. However, they found a marked increase in aberrations when first-cleavage metaphase cells were scored, which indicates that formation of breakage in MMS-treated dictyate oocytes is also delayed, as ir! the present results with pachytene spermatocytes treated with TEM. Similar persistence of TEM-induced premutational lesions in the mouse postmeiotic male germ cells was suggested to be the reason for the premature condensation of certain regions of chromosomes and for the presence of chromatid-type aberrations in the second and third cleavage divisions (MATTER and JAEGER 1975). However, direct evidence for this reasoning is lacking. The present study. indeed, shows that a reasonably high frequency of reciprocal translocations was recovered after treatment of meiotic spermatocytes with a chemical mutagen. This finding is significant in view of the observation from X-ray studies on mouse spermatogonia stem cells that strongly indicates that, for a hitherto unknown reason, the transmission of balanced exchanges is markedly lower than that expected on the basis of the frequency of multivalent association at diakinesjs-metaphase I (BREWEN, PRESTON and GENEROSO 1974; FORD et al. 1969; GENEROSO, CAIN and HUFF 1974). Because of this and the fact that the interchanges induced by X rays in pachytene spermatocytes are of the chromatid type, it is expected that, relative to dominant lethals, the frequency at which reciprocal translocations are recovered among progeny from treated pachytene spermatocytes is lower for X rays than for TEM. On the ather hand,

HERITABLE TRANSLOCATIONS IN SPERMATOCYTES 69 it seems likely that the reason heritable translocations were recovered at a frequemy of 5% after TEM treatment of pachytene spermatocytes was that the formation of aberrations was delayed-thus the mechanism may be similar to that of TEM in certain postmeiotic stages. We are presently studying the transmission of X-ray-induced translocations in pachytene spermatocytes. The question which brings into focus the mechanism of aberration formation is, at what point after TEM treatment of pachytene spermatocytes does formation of aberrations actually occur? It is reasonable to assume that the formation of aberrations is initiated by the TEM alkylation of DNA. Following this reaction, does the actual aberration formation occur as a consequence of normal DNA synthesis, as originally suggested by EVANS and SCOTT (1964) for maleic hydrazide effects in Vicia faba, or does it occur independently of DNA synthesis, through the action of various enzyme mechanisms? With respect to the latter possibility, two points need to be stated. First, an intervening round of DNA synthesis does not appear to be necessarily a prerequisite for the formation of exchanges. This is borne out by the high frequencies of exchange aberrations found in diakinesis-metaphase I spermatocytes following X-ray treatment of pachytene spermatocytes (Table 2) and the increase in the number of chromatid exchanges at the first mitosis of 5-bromodeoxyuridine-treated and G,-illuminated cells that were fixed at early periods (when there were very few S cells). Second, the formation of UV- or 5-bromodeoxyuridine-induced chromatid deletions in G,, X, and isochromatid deletions in GI, XI and G,, X, V,,B Chinese hamster tissue culture cells was postulated to be mediated by a single-strand nuclease and by this enzyme. plus a recombination or postreplication repair mechanisms respectively (GRIGGS and BENDER 1973; BENDER, BEDFORD and MITCHELL 1973; BENDER, GRIGGS and WALKER 1973). In other words, does the conversion into aberrations of the TEM premutational lesion placed in pachytene DNA occur during the postmeitoic stages, or after fertilization but before pronuclear DNA synthesis, or during pronuclear DNA synthesis? More research is needed before w2 can satisfactorily answer this important question, but we can speculate on the possibilities. The possibility that the formation of aberrations occurs sometime during spermiogenesis through the action of various repair enzymes, and not through the operation of postfertilization pronuclear DNA synthesis, is favored by the following arguments: First, it is widely known that the formation of aberrations induced by alkylating chemicals in various somatic cells is dependent upon DNA synthesis, and the aberrations observed at first mitosis are of the chromatid type. If such is the case in male meiotic and certair? postmeiotic stages, the great majority of sterile and partially sterile male progeny. classified as translocation heterozygotes, from male parents treated postmeiotically with either TEM or ethyl methanesulfonate (EMS), are expected to be mosaics. In our previous TEM and EMS studies (GENEROSO et al. 1974; GENEROSO et al., in press), in which male progeny from treated early spermatozoa or midspermatids were tested for translocation heterozygosity, we found no evidence of gonadal mosaicism. In one futile attempt to

70 w. M. GENEROSO et al. find gonadal mosaics, spermatogonial metaphases of three TEM-induced translocation heterozygotes were analyzed. That at least the great majority of translocation heterozygotes induced postmeiotically by TEM or EMS were not gonadal mosaics is also indicated by the very close similarity in reproductive performance between these traiislocation heterozygotes and those induced at the same staqes by X rays (GENEROSO et al., in press). About one-third of EMS-, TEM-, or X-ray-induced male translocation heterozygotes were completely sterile, and two-thirds were partially sterile. The average numbers of living embryos of females mated to EMS-, TEM-, and X-ray-induced partially sterile males and killed at inidpregnancy were 44, 43, and 44% that of females mated to normal males. If TEM- or EMS-induced translocation heterozygotes were mosaics, we would have expected relatively fewer complete steriles and relatively higher fertility among partial steriles induced by chemical mutagens than among those induced by X rays, because X-ray-induced translocations in male postmeiotic stages are almost certainly not mosaics. CACHEIRO, RUSSELL and SWART- OUT (1974, personal communication), who have been studying the cytogenetic nature of male sterility. have not found a single case of mosaicism in kidney cells of male sterile translocation heterozygotes induced in male postmeiotic stages by X-ray, TEM, EMS, isopropyl methanesulfonate, or cyclophosphamide. With the expectation that chromatid interchanges will result in gonadal mosaics if, indeed, premutational lesions placed in meiotic and certain postmeiotic stages are converted into aberrations during postfertilization DNA synthesis, one would expect lower frequencies of EMS- or TEM-kduced heritable translocations relative to X rays. Data actually show that in early spermatozoa or midspermatids, rates of 32 and 29% (which is about the highest frequency we found for X rays as well) were induced by EMS and TEM, respectively (GENEROSO et al. 1974; GENEROSO et al., in press). Furthermore, the relative frequencies of dominant lethals and heritable translocations for these stages appear to be the same for X rays as for the two chemicals. Secopd, LANG and ADLER (in press) observed a much higher frequency of transmitted translocations induced by MMS in the postmeiotic stages than would be expected from the frequency of chromatid interchanges observed by BREWEN et al. (1975). The latter investigators found only two cells (less than 1 %) with chromatid interchanges that may result in viable mosaics, while the former found 8-1 1 % translocation heterozygotes among 250 progeny tested. Since the dose used by LANG and ADLER produced at most 60% dominant lethals, the frequency of heritable translocations based on all conceptuses is calculated to be at least 3.2-4.5%. It should be noted that among zygotes with complete symmetrical chromatid interchanges we would expect the balanced combination to appear in only 50% of the two-cell eggs, if we assume random segregation. Thus, the frequency of first cleavage male pronuclear metaphases with complete symmetrical chromatid interchanges should be at least 6.4-9.0%. That the MMS-induced translocation heterozygotes are not mosaics is clearly demonstrated in LANG and ADLER'S study by the close similarity in the frequency of multivalents at diakinesis-metaphase I between the F, sons and their respec-

HERITABLE TRANSLOCATIONS IN SPERMATOCYTES 71 tive sires. There was only one exception in which there was considerable difference between the F, and the F, progeny, but this particular F, sire was not likely to be mosaic because he had a higher frequency of multivalents than his F, son. Thus, it is more likely that the translocation heterozygotes induced by MMS, like those induced by EMS, TEM, and X-ray, came from chromosome and not from chromatid interchanges--i.e., the formation of aberrations was completed prior to pronuclear DNA synthesis. It may be argued that if, indeed, the gonadal tissues arise from a single cell at any point during development (excluding the zygote itself), then no gonadal mosaicism would be expected. On the contrary, there is strong evidence which indicates that both the inner cell mass, from which all fetal cells arise, and the gonad are derived from few cells. At the embryo level, GARDNER (1975) recently reviewed the question of whether differentiation of blastomeres into trophoblast or inner cell mass depends on the segregation of cytoplasmic determinants present in the undivided egg (two distinct cytoplasmic regions in the egg segregate into the trophoblast and inner cell mass during cleavage) or on the interrelations of blastomeres during cleavage. Present data overwhelmingly favor the latter hypothesis. and it is thought that the decisive determinative events leading to differentiation of blastomeres into trophoblast or inner cell mass occur between the 8- and 16-cell stages. At the gonad level, RUSSELL (1964) concluded that there is no such thing as purity of germ line, or even an early separation of cell lineages in mammals. This conclusion was based on her studies with a large class of half-mutant animals (mosaics for mutation at any of five coat-color loci) in which both the germ line and the coat were a mixture of cell types. From the foregoing, the expectation that mosaics will be produced if the formation of aberrations occurs during pronuclear DNA synthesis seems unavoidable. Studies are now under way in our laboratory to obtain direct genetic evidence for the time of formation of aberrations after chemical treatment of various male germ-cell stages and to relate this genetic information to molecular mechanisms of aberration formation. LITERATURE CITED BENDER, M. A., J. S. BEDFORD and J. A. MITCHELL, 1973 Mechanisms of chromosomal aberration production. 11. Aberrations induced by 5-bromodeoxyuridine and visible light. Mutat. Res. 20: 403-416. BENDER, M. A., H. G. GRIGGS and P. L. WALKER, 1973 Mechanisms of chromosomal aberration production. I. Aberration induction by ultraviolet light. Mutat. Res. 20: 387-402. BREWEN, J. G. and H. S. PAYNE. Studies on chemically-induced dominant lethality: 11. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes. Mutat. Res., in press. BREWEN, J. G., H. S. PAYNE, K. P. JONES and R. J. PRESTON, 1975 Studies on chemicallyinduced dominant lethality: I. The cytogenetic basis of MMS-induced dominant lethality in postmeiotic male germ cells. Mutat. Res. 33: 239-250. BREWEN, J. G., R. J. PRESTON and W. M. GENEROSO, 1974 X-ray-induced translocations: Comparison between cytologically observed and genetically recovered frequencies. Biol. Div. Annu. Prog. Rep., June 30,1974,ORNL-4993, pp. 74-75.

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