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Spermatid micronucleus analysis of aging effects in hamsters
DNAging
Genetic Instability and Aging
ELSEVIER
Mutation Research 316 (1996) 261-266
Spermatid micronucleus analysis of aging effects in hamsters 1 J.W. A l l e n
a,*,
B.W. Collins
a,
R.W. Setzer b
a Environmental Carcinogenesis Division, Mail Drop 68, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA b Research and Administrative Support Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
Abstract
Spermatid micronuclei (MN) from Armenian hamsters in different age groups were compared with regard to frequencies and kinetochore status (presence or absence) as determined with immunofluorescent staining. Six thousand cells analyzed from each of fifteen young animals (3 months) revealed a group mean frequency of 0.45 MN/1000 spermatids; kinetochore staining was uniformly negative. Six thousand cells scored from each of fifteen older animals (2 years) revealed a group mean frequency of 1.00 MN/1000 spermatids. Most of the MN in these animals were negative for kinetochore staining, although a significant representation of MN with positive kinetochore staining was also observed. The results indicate that frequencies of spermatid MN increase with advancing age, and suggest that the increase is due to significant elevations in both chromosome breakage and chromosome loss.
Keywords: Aging; Spermatid; Micronucleus; Kinetochore; Armenian hamster
I. Introduction Meiotic errors in chromosome distribution are believed to be responsible for high levels of aneuploidy at conception and for associated preg-
* Corresponding author. Tel.: 1 919 541 4778; Fax: 1 919 541 0694. 1 Although the research in this article has been supported by the United States Environmental Protection Agency, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
nancy loss and debilitating syndromes in liveborn (Ford, 1990; Burgoyne et al., 1991). A number of studies, including several involving sperm analysis, have indicated that chromosome loss is detectable with greater frequency than chromosome gain (Ford, 1990; reviewed in Burgoyne et al., 1991). This suggests that not all of the loss is due to nondisjunction wherein missegregation of nondisjoined chromosomes should result in equal numbers of monosomic and trisomic daughter cells. It has remained questionable as to whether the excess of ceils with missing chromosomes is attributable to technical artifact or to anaphase lag which constitutes a different pathway by which single, or nondisjoined, chromosomes may be-
0921-8734/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 9 2 1 - 8 7 3 4 ( 9 5 ) 0 0 0 3 2 - 1
262
J.W. Allen et al. / Mutation Research 316 (1996) 261-266
come lost (Bond and Chandley, 1983; Ford, 1990; Martin et al., 1991; Benet et al., 1992). Chromosome lagging at premeiotic/meiotic cell division may arise from dysfunctional spindles or kinetochores among other possible defects. There has been a long-standing interest in the effects of aging to increase frequencies of chromosome lag and aneuploidy (reviewed in Schneider, 1985 and Ford, 1990). Although numerous studies have presented evidence of elevated chromosome loss in lymphocytes of older individuals (reviewed in Schneider, 1985; Fenech and Morley, 1989; Hando et al., 1994), relatively little is known about the extent to which such effects are occurring in the germ-line. In experimental animals, most studies of agerelated chromosome loss in germ cells have relied upon chromosome counts at metaphase. An alternative approach for detecting potential chromosome loss is that of micronucleus (MN) analysis in which frequency measurements are combined with centromere detection, as determined with a kinetochore protein antibody (Fenech and Morley, 1989) or DNA sequence probe (Ford et al., 1988; Miller et al., 1991). Micronuclei with centromeres are considered to result from the nuclear loss of intact chromosomes that are displaced (e.g., by lagging) from the spindle. Micronuclei without evidence of centromeres likely represent chromosome fragments arising from earlier breakage events. The extent to which these different types of micronucleated spermatids may be selected against, be reincorporated into the nucleus/pronucleus, or lead to chromosome loss, is unknown. In the present study, a spermatid micronucleus assay with immunofluorescent detection of kinetochore signals was used to evaluate chromosome loss in young and old Armenian hamsters. The Armenian hamster was selected for this study of age effects because with earlier spermatocyte metaphase analysis (Allen and Gwaltney, 1985) we found that a modest increase in hyperploidy is detectable in older animals. It is of interest to determine if this animal model also detects age influences upon frequencies of chromosome lagging and MN formation. An increase in spermatid MN (of the whole chromosome type) would
support arguments that age-related excesses of chromosome loss in gametes result from increased frequencies of lagging.
2. Methods
Male Armenian hamsters [Cricetulus migratorius; 2 N = 22 metacentric, submetacentric and subtelocentric chromosomes (Lavappa and Yerganian, 1970); life span of approximately 2.7 years (Yerganian et al., 1978)] were obtained from Cytogen Research and Development, West Roxbury, MA. They were housed at a U.S. EPA animal facility under laminar flow conditions with 15 cycles/h of biocleaned air and a 14h:10h light/dark cycle. Animals were fed Purina rodent chow and water ad libitum. The animals were housed and treated in accordance with approved guidelines (DHEW Guidelines for the Care and Use of Laboratory Animals), and approved project review (National Health and Environmental Effects Research Laboratory Animal Care and Use Committee, U.S. EPA). Spermatids were harvested from 15 3-month old and 15 2-year old hamsters using a suspension technique adapted from Tates et al. (1983) and L~ihdetie (1988). Both testes were excised from each animal, tunicas were removed, and the seminiferous tubules were gently minced in testis isolation medium (TIM; see Tates et al., 1983). The cell suspensions were incubated in 2 mg/ml collagenase type IA (Sigma Chemical, St. Louis, MO) for 15 min at 31-33°C in a shaking water bath (150 cycles/min). Trypsin (20 mg/ml; Gibco, Grand Island, NY) was added to each flask, and incubations continued for 10 min under the same conditions. The cell suspensions were then transferred to centrifuge tubes and washed 3 times with fresh TIM, centrifuging 5 min at 40 x g and 10°C between washes. Cells were filtered through 70/.~m nylon mesh, washed twice more with TIM, and placed on microscope slides. After the cells settled for 15 min, the slides were air dried and fixed in 100% methanol for 15 min. Slides were stored dessicated under nitrogen in a -20°C freezer until use. The kinetochore staining procedure was modi-
J. IV..Allen et aL / Mutation Research 316 (1996) 261-266
fied from that of Eastmond and Tucker (1989). Slides were rinsed for 15 min in 0.3% Tween 20 (Sigma Chemical) diluted in distilled water. Anti-kinetochore antibody (Chemicon International, Temecula, CA) diluted 1:15 with 0.1% Tween was added to the slides, which were incubated for 90 min in a 37°C humidified chamber. Following 3 rinses with 0.3% Tween, the slides were incubated for 60 min with fluorescein-conjugated goat anti-human antibody (Chemicon International; supplied as a 1:55 dilution) and again rinsed 3 times. Cells were counterstained with antifade solution (Johnson and Nogueira Araujo, 1981) containing 1.5 /zg/ml DAPI (4',6-diamidino-2-phenyl indole dihydrochloride, Sigma Chemical Co.) and topped with a coverglass. For each animal, six thousand round spermatids were scored for the presence of MN using simultaneous phase contrast and DAPI excitation. Each micronucleated spermatid was also observed with a fluorescein filter to determine the presence or absence of kinetochores in the MN. Only cells in which kinetochores were visible in the main nucleus were counted when scoring kinetochores in the MN. Occasional slides were rejected for overall poor antibody binding; however, on acceptable slides essentially all spermatid nuclei revealed kinetochore staining. Age differences in mean number of spermatid MN per animal were tested using one-sided t-tests on the log-transformed MN counts (plus 1/2), and P-values confirmed with random permutation t-tests (Randles and Wolfe, 1979).
263
No. MN mean (se) 3 months: 2.7 (0.36) 2 years: 6.0 (0.52)
¢,O
~
i
i
i
0
i
f
3 5 6 7 No. MN/Animal
i
8
9
Fig. 1. Distributions of the total number of spermatid M N per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
revealed from 3 to 9 micronucleated spermatids per 6000 cells to give a total of 90 MN observed; the group mean value was 1.0/1000 spermatids.
No. K- MN
~i]
mean (se) 3 months: 2.7 (0.36) 2 years: 5.1 (0.42)
3. Results and discussion
Relatively few MN were detected in the 15 animals and 90,000 ceils scored for each age group of hamsters (Figs. 1-3). However, the data clearly indicate that frequencies of spermatid MN increase with advancing age. In the younger age group, each animal revealed from 1 to 5 micronucleated spermatids per 6000 cells to give a total of 41 MN observed; the group mean value was 0.45/1000 spermatids. All of the MN recorded in young animals were of the kinetochore-negative ( K - ) type. In the older age group, each animal
o
0
1
2 3 4 5 6 No. K- MN/Animal
7
Fig. 2. Distributions and means of the number of K- spermatid MN per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
J. IV..Allen et al. / Mutation Research 316 (1996) 261-266
264
No. K+ MN tt3 mean (se)
3 months: 0.00 ( 0 ) 2 years: 0.87 (0.26) O ,rE <
O
0
II
I
--I
1 2 NO. K+ MN/Animal
3
Fig. 3. Distributions and means of the number of K + spermatid MN per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
Although most ( ~ 85%) of the MN recorded in older animals were of the K - type, frequencies of both K - and kinetochore-positive (K + ) types
were significantly increased (each at P < 0.001). As compared with the lack of detectable K + MN in young animals, roughly half of older animals revealed from one to three K + MN. All of the observed micronucleated spermatids contained a single MN, with one interesting exception. A single cell from an older animal revealed two K + MN which differed in their overall size, and in the size of their fluorescent kinetochore signals (Fig. 4). The larger MN with larger kinetochore signal may represent a chromosome or chromosomal bivalent lost at the first meiotic division, while the smaller MN with smaller kinetochore signal represents a chromatid lost at the first (after p r e m a t u r e chromatid separation) or second meiotic division. Alternatively, the larger MN may represent 'clustered' centromeres in condensing chromatin which has been reported to characterize the nucleus of mouse spermatids (del Mazo et al., 1986). Our findings of increased MN with age in the present study are consistent with similar observations reported in studies of human lymphocytes; MN frequencies are elevated in older individuals due to increases in both K + and K - types of
Fig. 4. (a) Armenian hamster spermatid with 2 micronuclei as seen with simultaneous phase contrast and DAPI. (b) The same cell as seen with a fluorescein filter, showing 1 kinetochore signal in each micronucleus (1000 x ).
J. W. Allen et al. / Mutation Research 316 (1996) 261-266
MN (Fenech and Morley, 1989; Odagiri et al., 1990). However, germ cells reveal multi-fold lower absolute numbers of MN. Meiotic processes regulating chromosome segregation (e.g. chiasma formation), as well as selection against micronucleated spermatogonia a n d / o r cells with lesions that would ultimately generate MN at spermatocyte divisions, may have influenced the results. The low baseline level of only K - type MN determined in spermatids of young hamsters is consistent with our observations from other studies of MN in mice (Collins et al., 1992; Allen et al., 1994). In normal young mice, we have usually detected MN baseline levels in the range of 0.52.0/1000 spermatids, with K + types infrequently observed. Kallio and lAhdetie (1993) have reported a mean frequency of 1.4 MN/1000 spermatids in young outbred Han:NMRI mice, and used fluorescence in situ hybridization and a major gamma satellite DNA probe to determine that 24% of the MN are positive for the presence of a centromere. This percentage of DNA probe positive MN is not necessarily inconsistent with our present data; we have conducted similar studies with the major gamma satellite.DNA probe and determined that DNA probe positive signals are detectable with greater frequency than K + signals in spermatid MN (R. Cannon et al., unpublished data). Recently, we have analyzed spermatid MN frequencies in young and old mice (Lowe et al., 1994; Lowe et al., 1995); age-related increases in only kinetochore-negative types of MN were detected. Sperm analysis from the same animals, using chromosome-specific probes and fluorescence in situ hybridization (studies of Lowe, Wyrobek and colleagues), revealed significant increases in hyperhaploidy. In both mice and hamsters (present study and Allen and Gwaltney, 1985), hyperploidy and MN increased with aging. A notable difference between the mice and hamsters pertains to our present evidence in the latter for an age-related increase in K + MN indicative of chromosome loss. Whether this discrepancy may be due to species differences in meiotic chromosomes (e.g. chiasma frequency/ distribution) or to the larger sample size in the present study is not clear.
265
That most MN were of the K - type is in keeping with various lines of evidence suggesting that DNA changes and declining repair (which may be disposed to clastogenesis) can occur with aging (Esposito et al., 1989; Singh et al., 1990; Slagboom, 1990). However, it is also known that centromeres can become inactive (e.g. in dicentrics) and reveal a reduction or absence of reactivity with anti-kinetochore antibodies and other markers of centromere function (reviewed in del Mazo et al., 1986 and in Fenech and Morley, 1989). As pointed out by Fenech and Morley (1989), these centromere-inactive chromosomes may consequently be lost at cell division to form MN. Perhaps X-chromosome inactivation in spermatogenesis (reviewed in Handel, 1987) and loss accounts for some of the K - MN we observed in spermatids. This is similar to the speculation by Hando et al. (1994) that K - micronuclei in lymphocytes of older women may predominantly represent the inactive X. The question of whether some spermatid K - MN may, in fact, represent whole chromosomes could be studied further in mice wherein DNA probes to centromere regions and specific chromosomes are more readily available. Questions remain regarding the fate of micronucleated spermatids. However, our data in the hamster support the interpretation that chromosome lagging may account for at least some of the excess hypoploidy often observed with male gamete analysis.
References Allen, J.W. and C.W. Gwaltney (1985) Investigation of possible age effects on meiotic chromosomal recombination and segregation in Armenian hamster spermatocytes, Cytobios, 43, 225-232. Allen, J.W., B.W. Collins and P.A. Evansky (1994) Spermatid micronucleus analyses of trichloroethylene and chloral hydrate effects in mice, Mutation Res., 323, 81-88. Benet, J., A. Genesc~, J. Navarro, J. Egozcue and C. Templado (1992) Cytogenetic studies in motile sperm from normal men, Hum. Genet., 89, 176-180. Bond, D.J. and A.C. Chandley (1983) Aneuploidy, Oxford Monographs on Medical Genetics, No. 11, Oxford University Press, New York.
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Burgoyne, P.S., K. Holland and R. Stephens (1991) Incidence of numerical chromosome anomalies in human pregnancy estimation from induced and spontaneous abortion data, Hum. Repro., 6, 555-565. Collins, B.W., D.R. Howard and J.W. Allen (1992) Kinetochore-staining of spermatid micronuclei: studies of mice treated with X-radiation or acrylamide, Mutation Res., 281,287-294. del Mazo, J., M.J. Martin-Sempere, L. Kremer and J. Avila (1986) Centromere pattern in different mouse seminiferous tubule cells, Cytogenet. Cell Genet., 43, 201-206. Eastmond, D.A. and J.D. Tucker (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen., 13, 34-43. Esposito, D., G. Fassina, P. Szabo, P. De Angelis, L. Rodgers, M. Weksler and M. Siniscalco (1989) Chromosomes of older humans are more prone to aminopterine-induced breakage, Proc. Natl. Acad. Sci. USA, 86, 1302-1306. Fenech, M. and A.A. Morley (1989) Kinetochore detection in micronuclei: an alternative method for measuring chromosome loss, Mutagenesis, 4, 98-104. Ford, J.H. (1990) Aneuploidy in humans, in: A. Kappas (Ed.), Mechanisms of Environmental Mutagenesis-Carcinogenesis, Plenum, New York, pp. 97-114. Ford, J.H., C.J. Schultz and A.T. Correll (1988) Chromosome elimination in micronuclei: a common cause of hypoploidy, Am. J. Hum. Genet., 43, 733-740. Handel, M.A. (1987) Genetic control of spermatogenesis in mice, in: W. Hennig (Ed.), Results and Problems in Cell Differentiation, Volume 15: Spermatogenesis Genetic Aspects, Springer-Verlag, New York, pp. 1-62. Hando, J.C., J. Nath and J.D. Tucker (1994) Sex chromosomes, micronuclei and aging in women, Chromosoma, 103, 186-192. Johnson, G.D. and G,M. de C. Nogueira Araujo (1981) A simple method of reducing the fading of immunofluorescence during microscopy, J. Immunol. Methods, 43, 349-350. Kallio, M. and J. L~ihdetie (1993) Analysis of micronuclei induced in mouse early spermatids by mitomycin C, vinblastine sulfate or etoposide using fluorescence in situ hybridization, Mutagenesis, 8, 561-567. L~ihdetie, J. (1988) Induction and survival of micronuclei in rat spermatids, Comparison of two meiotic micronucleus techniques using cyclophosphamide, Mutation Res., 203, 47-53.
Lavappa, K.S. and G. Yerganian (1970) Spermatogonial and meiotic chromosomes of the Armenian hamster, Cricetulus migratorius, Exp. Cell Res., 61, 159-172. Lowe, X., J. Baulch, L. Quintana, B. Collins, J. Allen, M. Ramsey, J. Breneman, J. Tucker, N. Holland and A. Wyrobek (1994) Molecular detection of chromosomal abnormalities in germ and somatic cells of aged mice, Environ. Mol. Mutagen., 23 (Suppl. 23), 40. Lowe, X., B. Collins, J. Allen, N. Holland, J. Breneman, M. van Beek, J. Bishop and A. Wyrobek (1995) Aneuploidies and micronuclei in the germ cells of male mice of advanced age, Mutation Res., 338, 59-76. Martin, R.H., E. Ko and A. Rademaker (1991) Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes, Am. J. Med. Genet., 39, 321-331. Miller, B.M., H. Zitzelsberger, H.-U. Weier and I.-D. Adler (1991) Classification of micronuclei in murine erythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with a biotinylated satellite DNA, Mutagenesis, 6, 297-302. Odagiri, Y., J.L. Dempsey and A.A. Morley (1990) Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique, Mutation Res., 237, 147-152. Randles, R.H. and D.A. Wolfe (1979) Introduction to the Theory of Nonparametric Statistics, Wiley and Sons, New York. Schneider, E.L. (1985) Cytogenetics of aging, in: C.E. Finch and E.L. Schneider (Eds.), Handbook of the Biology of Aging, Van Nostrand Reinhold, New York, pp. 357-373. Singh, N.P., D.B. Danner, R.R. Tice, L. Brant and E.L. Schneider (1990) DNA damage and repair with age in individual human lymphocytes, Mutation Res., 237, 123130. Slagboom, P.E. (1990) The aging genome: determinant or target? Report of the EURAGE Meeting on Genomic Instability and Aging, Nerja (Spain), 6-8 October 1989, Mutation Res., 237, 183-187. Tates, A.D., A.J.J. Dietrich, N. de Vogel, I. Neuteboom and A. Bos (1983) A micronucleus method for detection of meiotic micronuclei in male germ cells of mammals, Mutation Res., 121, 131-138. Yerganian, G., H.J. Gagnon and A. Battaglino (1978) Animal model of human disease: Angioimmunoblastic lymphadenopathy, immunoblastic sarcoma of B cells, Am. J. Pathol., 91,209-212.
Genetic Instability and Aging
ELSEVIER
Mutation Research 316 (1996) 261-266
Spermatid micronucleus analysis of aging effects in hamsters 1 J.W. A l l e n
a,*,
B.W. Collins
a,
R.W. Setzer b
a Environmental Carcinogenesis Division, Mail Drop 68, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA b Research and Administrative Support Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
Abstract
Spermatid micronuclei (MN) from Armenian hamsters in different age groups were compared with regard to frequencies and kinetochore status (presence or absence) as determined with immunofluorescent staining. Six thousand cells analyzed from each of fifteen young animals (3 months) revealed a group mean frequency of 0.45 MN/1000 spermatids; kinetochore staining was uniformly negative. Six thousand cells scored from each of fifteen older animals (2 years) revealed a group mean frequency of 1.00 MN/1000 spermatids. Most of the MN in these animals were negative for kinetochore staining, although a significant representation of MN with positive kinetochore staining was also observed. The results indicate that frequencies of spermatid MN increase with advancing age, and suggest that the increase is due to significant elevations in both chromosome breakage and chromosome loss.
Keywords: Aging; Spermatid; Micronucleus; Kinetochore; Armenian hamster
I. Introduction Meiotic errors in chromosome distribution are believed to be responsible for high levels of aneuploidy at conception and for associated preg-
* Corresponding author. Tel.: 1 919 541 4778; Fax: 1 919 541 0694. 1 Although the research in this article has been supported by the United States Environmental Protection Agency, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
nancy loss and debilitating syndromes in liveborn (Ford, 1990; Burgoyne et al., 1991). A number of studies, including several involving sperm analysis, have indicated that chromosome loss is detectable with greater frequency than chromosome gain (Ford, 1990; reviewed in Burgoyne et al., 1991). This suggests that not all of the loss is due to nondisjunction wherein missegregation of nondisjoined chromosomes should result in equal numbers of monosomic and trisomic daughter cells. It has remained questionable as to whether the excess of ceils with missing chromosomes is attributable to technical artifact or to anaphase lag which constitutes a different pathway by which single, or nondisjoined, chromosomes may be-
0921-8734/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 9 2 1 - 8 7 3 4 ( 9 5 ) 0 0 0 3 2 - 1
262
J.W. Allen et al. / Mutation Research 316 (1996) 261-266
come lost (Bond and Chandley, 1983; Ford, 1990; Martin et al., 1991; Benet et al., 1992). Chromosome lagging at premeiotic/meiotic cell division may arise from dysfunctional spindles or kinetochores among other possible defects. There has been a long-standing interest in the effects of aging to increase frequencies of chromosome lag and aneuploidy (reviewed in Schneider, 1985 and Ford, 1990). Although numerous studies have presented evidence of elevated chromosome loss in lymphocytes of older individuals (reviewed in Schneider, 1985; Fenech and Morley, 1989; Hando et al., 1994), relatively little is known about the extent to which such effects are occurring in the germ-line. In experimental animals, most studies of agerelated chromosome loss in germ cells have relied upon chromosome counts at metaphase. An alternative approach for detecting potential chromosome loss is that of micronucleus (MN) analysis in which frequency measurements are combined with centromere detection, as determined with a kinetochore protein antibody (Fenech and Morley, 1989) or DNA sequence probe (Ford et al., 1988; Miller et al., 1991). Micronuclei with centromeres are considered to result from the nuclear loss of intact chromosomes that are displaced (e.g., by lagging) from the spindle. Micronuclei without evidence of centromeres likely represent chromosome fragments arising from earlier breakage events. The extent to which these different types of micronucleated spermatids may be selected against, be reincorporated into the nucleus/pronucleus, or lead to chromosome loss, is unknown. In the present study, a spermatid micronucleus assay with immunofluorescent detection of kinetochore signals was used to evaluate chromosome loss in young and old Armenian hamsters. The Armenian hamster was selected for this study of age effects because with earlier spermatocyte metaphase analysis (Allen and Gwaltney, 1985) we found that a modest increase in hyperploidy is detectable in older animals. It is of interest to determine if this animal model also detects age influences upon frequencies of chromosome lagging and MN formation. An increase in spermatid MN (of the whole chromosome type) would
support arguments that age-related excesses of chromosome loss in gametes result from increased frequencies of lagging.
2. Methods
Male Armenian hamsters [Cricetulus migratorius; 2 N = 22 metacentric, submetacentric and subtelocentric chromosomes (Lavappa and Yerganian, 1970); life span of approximately 2.7 years (Yerganian et al., 1978)] were obtained from Cytogen Research and Development, West Roxbury, MA. They were housed at a U.S. EPA animal facility under laminar flow conditions with 15 cycles/h of biocleaned air and a 14h:10h light/dark cycle. Animals were fed Purina rodent chow and water ad libitum. The animals were housed and treated in accordance with approved guidelines (DHEW Guidelines for the Care and Use of Laboratory Animals), and approved project review (National Health and Environmental Effects Research Laboratory Animal Care and Use Committee, U.S. EPA). Spermatids were harvested from 15 3-month old and 15 2-year old hamsters using a suspension technique adapted from Tates et al. (1983) and L~ihdetie (1988). Both testes were excised from each animal, tunicas were removed, and the seminiferous tubules were gently minced in testis isolation medium (TIM; see Tates et al., 1983). The cell suspensions were incubated in 2 mg/ml collagenase type IA (Sigma Chemical, St. Louis, MO) for 15 min at 31-33°C in a shaking water bath (150 cycles/min). Trypsin (20 mg/ml; Gibco, Grand Island, NY) was added to each flask, and incubations continued for 10 min under the same conditions. The cell suspensions were then transferred to centrifuge tubes and washed 3 times with fresh TIM, centrifuging 5 min at 40 x g and 10°C between washes. Cells were filtered through 70/.~m nylon mesh, washed twice more with TIM, and placed on microscope slides. After the cells settled for 15 min, the slides were air dried and fixed in 100% methanol for 15 min. Slides were stored dessicated under nitrogen in a -20°C freezer until use. The kinetochore staining procedure was modi-
J. IV..Allen et aL / Mutation Research 316 (1996) 261-266
fied from that of Eastmond and Tucker (1989). Slides were rinsed for 15 min in 0.3% Tween 20 (Sigma Chemical) diluted in distilled water. Anti-kinetochore antibody (Chemicon International, Temecula, CA) diluted 1:15 with 0.1% Tween was added to the slides, which were incubated for 90 min in a 37°C humidified chamber. Following 3 rinses with 0.3% Tween, the slides were incubated for 60 min with fluorescein-conjugated goat anti-human antibody (Chemicon International; supplied as a 1:55 dilution) and again rinsed 3 times. Cells were counterstained with antifade solution (Johnson and Nogueira Araujo, 1981) containing 1.5 /zg/ml DAPI (4',6-diamidino-2-phenyl indole dihydrochloride, Sigma Chemical Co.) and topped with a coverglass. For each animal, six thousand round spermatids were scored for the presence of MN using simultaneous phase contrast and DAPI excitation. Each micronucleated spermatid was also observed with a fluorescein filter to determine the presence or absence of kinetochores in the MN. Only cells in which kinetochores were visible in the main nucleus were counted when scoring kinetochores in the MN. Occasional slides were rejected for overall poor antibody binding; however, on acceptable slides essentially all spermatid nuclei revealed kinetochore staining. Age differences in mean number of spermatid MN per animal were tested using one-sided t-tests on the log-transformed MN counts (plus 1/2), and P-values confirmed with random permutation t-tests (Randles and Wolfe, 1979).
263
No. MN mean (se) 3 months: 2.7 (0.36) 2 years: 6.0 (0.52)
¢,O
~
i
i
i
0
i
f
3 5 6 7 No. MN/Animal
i
8
9
Fig. 1. Distributions of the total number of spermatid M N per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
revealed from 3 to 9 micronucleated spermatids per 6000 cells to give a total of 90 MN observed; the group mean value was 1.0/1000 spermatids.
No. K- MN
~i]
mean (se) 3 months: 2.7 (0.36) 2 years: 5.1 (0.42)
3. Results and discussion
Relatively few MN were detected in the 15 animals and 90,000 ceils scored for each age group of hamsters (Figs. 1-3). However, the data clearly indicate that frequencies of spermatid MN increase with advancing age. In the younger age group, each animal revealed from 1 to 5 micronucleated spermatids per 6000 cells to give a total of 41 MN observed; the group mean value was 0.45/1000 spermatids. All of the MN recorded in young animals were of the kinetochore-negative ( K - ) type. In the older age group, each animal
o
0
1
2 3 4 5 6 No. K- MN/Animal
7
Fig. 2. Distributions and means of the number of K- spermatid MN per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
J. IV..Allen et al. / Mutation Research 316 (1996) 261-266
264
No. K+ MN tt3 mean (se)
3 months: 0.00 ( 0 ) 2 years: 0.87 (0.26) O ,rE <
O
0
II
I
--I
1 2 NO. K+ MN/Animal
3
Fig. 3. Distributions and means of the number of K + spermatid MN per animal in young (3 months) and old (2 years) hamsters. In each age group, 6000 round spermatids were analyzed from each of 15 hamsters. Mean and standard error values per animal (6000 cells) are also shown.
Although most ( ~ 85%) of the MN recorded in older animals were of the K - type, frequencies of both K - and kinetochore-positive (K + ) types
were significantly increased (each at P < 0.001). As compared with the lack of detectable K + MN in young animals, roughly half of older animals revealed from one to three K + MN. All of the observed micronucleated spermatids contained a single MN, with one interesting exception. A single cell from an older animal revealed two K + MN which differed in their overall size, and in the size of their fluorescent kinetochore signals (Fig. 4). The larger MN with larger kinetochore signal may represent a chromosome or chromosomal bivalent lost at the first meiotic division, while the smaller MN with smaller kinetochore signal represents a chromatid lost at the first (after p r e m a t u r e chromatid separation) or second meiotic division. Alternatively, the larger MN may represent 'clustered' centromeres in condensing chromatin which has been reported to characterize the nucleus of mouse spermatids (del Mazo et al., 1986). Our findings of increased MN with age in the present study are consistent with similar observations reported in studies of human lymphocytes; MN frequencies are elevated in older individuals due to increases in both K + and K - types of
Fig. 4. (a) Armenian hamster spermatid with 2 micronuclei as seen with simultaneous phase contrast and DAPI. (b) The same cell as seen with a fluorescein filter, showing 1 kinetochore signal in each micronucleus (1000 x ).
J. W. Allen et al. / Mutation Research 316 (1996) 261-266
MN (Fenech and Morley, 1989; Odagiri et al., 1990). However, germ cells reveal multi-fold lower absolute numbers of MN. Meiotic processes regulating chromosome segregation (e.g. chiasma formation), as well as selection against micronucleated spermatogonia a n d / o r cells with lesions that would ultimately generate MN at spermatocyte divisions, may have influenced the results. The low baseline level of only K - type MN determined in spermatids of young hamsters is consistent with our observations from other studies of MN in mice (Collins et al., 1992; Allen et al., 1994). In normal young mice, we have usually detected MN baseline levels in the range of 0.52.0/1000 spermatids, with K + types infrequently observed. Kallio and lAhdetie (1993) have reported a mean frequency of 1.4 MN/1000 spermatids in young outbred Han:NMRI mice, and used fluorescence in situ hybridization and a major gamma satellite DNA probe to determine that 24% of the MN are positive for the presence of a centromere. This percentage of DNA probe positive MN is not necessarily inconsistent with our present data; we have conducted similar studies with the major gamma satellite.DNA probe and determined that DNA probe positive signals are detectable with greater frequency than K + signals in spermatid MN (R. Cannon et al., unpublished data). Recently, we have analyzed spermatid MN frequencies in young and old mice (Lowe et al., 1994; Lowe et al., 1995); age-related increases in only kinetochore-negative types of MN were detected. Sperm analysis from the same animals, using chromosome-specific probes and fluorescence in situ hybridization (studies of Lowe, Wyrobek and colleagues), revealed significant increases in hyperhaploidy. In both mice and hamsters (present study and Allen and Gwaltney, 1985), hyperploidy and MN increased with aging. A notable difference between the mice and hamsters pertains to our present evidence in the latter for an age-related increase in K + MN indicative of chromosome loss. Whether this discrepancy may be due to species differences in meiotic chromosomes (e.g. chiasma frequency/ distribution) or to the larger sample size in the present study is not clear.
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That most MN were of the K - type is in keeping with various lines of evidence suggesting that DNA changes and declining repair (which may be disposed to clastogenesis) can occur with aging (Esposito et al., 1989; Singh et al., 1990; Slagboom, 1990). However, it is also known that centromeres can become inactive (e.g. in dicentrics) and reveal a reduction or absence of reactivity with anti-kinetochore antibodies and other markers of centromere function (reviewed in del Mazo et al., 1986 and in Fenech and Morley, 1989). As pointed out by Fenech and Morley (1989), these centromere-inactive chromosomes may consequently be lost at cell division to form MN. Perhaps X-chromosome inactivation in spermatogenesis (reviewed in Handel, 1987) and loss accounts for some of the K - MN we observed in spermatids. This is similar to the speculation by Hando et al. (1994) that K - micronuclei in lymphocytes of older women may predominantly represent the inactive X. The question of whether some spermatid K - MN may, in fact, represent whole chromosomes could be studied further in mice wherein DNA probes to centromere regions and specific chromosomes are more readily available. Questions remain regarding the fate of micronucleated spermatids. However, our data in the hamster support the interpretation that chromosome lagging may account for at least some of the excess hypoploidy often observed with male gamete analysis.
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