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Hyperthermia-induced embryonic aneuploidy in mice
.I. Th,,rmol Bhdoor Vol. 4. pp. 179 to ISl 4 Pergamon Press Ltd 1979. Prinled in Great Brilain
0306-4565'79'0104 0179502.00/0
HYPERTHERMIA-INDUCED EMBRYONIC ANEUPLOIDY IN MICE C. L. CHRISMAN and D. P. DOOLITTLE Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A.
(Receired 5 May 1978: rerised 25 May 1978: accepted 5 November 1978) Abstract--Chromosomal aneuploidy, as an effect of elevated ambient temperature during early gestation, was studied in 650 eight-day embryos from 60 females of two different mouse stocks. Exposure of treatment females to 34 C and 500,, relative humidity during days 5 and 6 of gestation caused an increase in embryo chromosomal hyperploidy. No embryo sex interaction with the percent hyperploidy was found.
INTRODUCTION The incidence of embryonic mortality as well as of congenital defects in several m a m m a l i a n species has been s h o w n to increase following exposure of pregnant females to increased ambient temperature (Ulberg, 1958; Edwards, 1969, 1974; Bellve, 1973; Garrard et al., 1974). Causative mechanisms have not been thoroughly researched. Ulberg & Burfening (1967) demonstrated that direct embryonic changes were more likely the cause of embryo decline than an indirect embryonic effect due to uterine changes. Edwards et al. (1974) found nuclear clumping and mitotic delay in guinea-pig embryos subjected to a short period of maternal hyperthermia on day 21 of gestation. This might suggest mitotic spindle apparatus changes such as the microtubule damage found by Zeuthen (1972) in heat stressed sea-urchin • embryos. The o b j e c t i v e of the present study was to determine if embryonic numerical c h r o m o s o m e aberrations were generated following exposure of pregnant female mice to high ambient temperature after implantation.
MATERIALS AND METHODS Females from two random bred stocks of mice (Mus musculus), "J'" and "ICR" were randomly divided into treatment and control groups. Primaparous J females were derived from generations 20-34 of random mating from an original four way cross of the inbred lines LP/J, SJL/J, BALB/cJ, and C57BL/6J, Virgin ICR females were derived from the stock Breeder: Ha (ICR) selected for large litter size developed at the Institute of Cancer Research, Philadelphia (Hauschka & Mirand, 1973). The J females were mated to the same males that sired their first litters, whereas the ICR females were mated to adult ICR males of proven fertility. Day one of gestation was determined by presence of the copulatory plug. All animals were maintained in a controlled environmental chamber at 2 1 C with a relative humidity of 50-700~,. On the morning of the fifth day of gestation, treatment females were moved to an environmental chamber maintained at 34 C and 50°,i relative humidity for a 48 h period, then returned to the control chamber. All animals had free access to feed and water. On the eighth day of gestation, all females were killed by cervical dislocation and their reproductive tracts were removed. Ovulation rate
was estimated by a count of corpora lutea (CL), and the number of decidua and number of recovered embryos recorded. All embryos were processed for chromosome analysis following minor modifications to a procedure established by Wroblewska & Dyban (1969). Two slides were prepared from each embryo, stained with Giemsa and coded to prevent observer bias. An attempt was made to count the chromosomes of ten intact, well-spread metaphase nuclei; however a few embryos presented less than ten countable cells in metaphase. Counts were made of chromosomes of metaphase nuclei which were well-stained, evenly-spread and not mechanically broken. Metaphase nuclei with widely spread chromosomes or with chromosomes not contained within a circular group were not counted. An observer made five chromosome counts from each metaphase nucleus selected at 1000 x : if all five counts did not agree, the count was begun again until agreement was reached. Independent of the chromosome counts, a second observer sexed each embryo, finding at least five nuclei that exhibited either the darkly stained heteropyknotic X chromosome of the female or the darkly stained Y chromosome of the male. In some cases, the Y chromosome of the male was not darkly stained but was evident because of its morphology. Although exact chromosome counts were recorded including hypo- and hyper-diploid (<40 or >40 chromosomes, respectively), normal cells with 40 chromosomes and polyploid cells, the statistical analyses were performed on data including only diploid and hyperploid cells. The data were expressed as the raw percentage hyperploidy and transformed to the arcsin of the square root of the raw percent hyperploidy (Li, 1964) to approximate a normal distribution. A correction factor, the Bartlett correction, of 1/4n (where n is the number of observations per embryo) was used for embryos with 0". hyperploidy (Walker & Lev, 1953). The data were then assembled as dam means and the main effects of treatment and stock were tested for significance by analysis of variance based on a 2 × 2 factorial design. To determine if there was an effect of sex on percent hyperploidy the embryo data were analyzed with a least squares analysis of variance (Harvey, 19601 with embryo sex nested within stock-treatment combinations. RESULTS AND DISCUSSION Reproductive performances of the four groups of females as measured during the eighth day of gestation are summarized in Table 1. C o r p o r a lutea 179
C. L. CHRISMANand D. P. DOOLITTL£
180
Table 1. Reproductive performance of females in the control and hot environments Stock/Treatment
No. of females
Mean no. C.L. ~ S.E.
Meam no. d e c i d u a ± S.E.
Mean no. recovered embryos ± S.E.
Sex ratio (% males)
J/control
15
12.6 ± 0.54
12.2 ± 0.45
11.5 ± 0.52
50.0
J/heat
15
13.2 ± 0.65
12.1 ± 0.69
11.4 ± 0.76
49.7
ICR/control
15
ii.0 ± 1.24
ii.i ~ 1.06
10.3 ± 0 . 8 2
48.7
ICR/heat
15
12.2 ± 0.72
II.i t 0.88
10.3 ± 0.01
58.7
counts, number of decidua and number of recovered embryos did not significantly differ between stocks or treatments. A total of 655 embryos were recovered, of which 651 presented 5696 cells for chromosome counts. The raw mean hyperploidy percentages are shown in Table 2. A factorial analysis of variance testing transformed percent hyperploidy indicated that no stock difference existed. However, embryos from heat treated dams had a significantly greater rate of hyperploidy than controls (P < 0.02). The stock x treatment interaction was not significant. Polyploid cells (3n and 4n) were encountered only in the control litters (14 and 8 in the J and ICR controls, respectively). The absence of polyploid cells in the large number of treatment embryo cells observed could be interpreted as meaning that cells with a large number of extra chromosomes were unable to survive the stress conditions or that such cells were not entering the mitotic cycle. Sex ratios in each group are reported in Table 1. The sex ratios in both the J control and heat litters and the ICR control litters fit an expected 1:1 ratio quite well. However, the ICR heat group sex ratio differed significantly from both the ICR control and hypothesized 1:1 ratio (X2- 6.22 and X2= 4.7, respectively, P < 0.05). Because of this altered sex ratio in the ICR heat litters, there was a suggestion that the higher percentage of males was involved in the increased hyperploidy observed in this group. A least squares analysis of variance with embryo sex nested within stock-treatment combinations demonstrated that no sex effect was present (P > 0.05). Therefore it was concluded that the excess males in the ICR heat litters were not solely responsible for the increased hyperploidy.
Since maternal body temperature was not measured in this study, the increase in aneuploidy cannot be concluded to have been directly influenced by uterine hyperthermia. Bellve (1973) exposed ICR mice to 34.5°C and 65% relative humidity and recorded a 2°C increase in rectal temperature after 24 h. Rectal temperature measurements in this laboratory have not confirmed such an increase. It appears that the handling of each mouse and insertion of the rectal probe is sufficient to cause body temperature to rise in some mice. The relationship between chromosome aberrations and embryonic mortality and congenital abnormalities has been well established in humans (Hamerton, 1971). Although not nearly so well documented in animals, chromosome abnormalities are thought to be important in causing a pOrtion of the impaired fertility observed in polytocous species (McFeely, 1967; Vickers, 1969; Wroblewska, 1971; MartinDeLeon et al., 1973). Acknowledgements--The technical assistance of Gail Hillis and Jane Hatmaker is appreciated. This study is published as Indiana Agricultural Experiment Station Journal Paper No. 6297.
REFERENCES BELLVEA. T. (1973) Development of mouse embryos with abnormalities induced by parental heat stress. J. Reprod. Fert. 35, 393-403. EDWARDSM. J. (1969) Congenital defects in guinea pigs: fetal resorptions, abortions and malformations following induced hyperthermia during early gestation. Teratology 2, 313-328.
Table 2. Hyperploidy of cells from embryos in control and hot environments* Control
Across Treatments
Heat
hyperploldy ± S.E.
No. embryos
No. cells analyzed
% hyperploldy ± S.E.
No. embryos
No. cells analyzed
% hyperploldy ± S.E.
J
170
1535
2.23 ± 0.626
171
1507
2.87 ± 0.512
2.55 ± 0.404
ICR
154
1352
2.06 + 0.768
155
1302
4.48 ± 1.105
3.27 ± 0.673
Across stocks
324
2887
2.15 ± 0.495
326
2809
3.68 ± 0.609
2.91 ± 0.402
Stock
* From raw data, not transformed.
Hyperthermia-induced embryonic aneuploidy in mice EDWARDS M. J. (1974) The effects of hyperthermia on pregnancy and prenatal development. In Experimental Embryology and Teratology, (Edited by WOOLLAM D. H. M. & MORRiSS G. M.) Elek Science, London. EDWARDS M. J., MULLEY R., RING S. 8/. WANNER R. A. (1974) Mitotic cell death and delay of mitotic activity in guinea-pig embryos following brief maternal hypertermia, d. Emhryol. exp. Morph. 32, 593~i02. GARRARD G., HARRISON G. A. & WEINER J. S. (1974) Reproduction and survival of mice at 23: and 32°C. J. Reprod. Fert. 37, 287-298. HAMERTON J. L. (1971) Human Cytogenetics, Vol. II. Academic Press, New York. HARVEY W. R. (1960) Least-squares analysis of data with unequal subclass numbers. USDA ARS 20. HAUSCHKA T. S. & M1RAND E. J. (1973) The Breeder: Ha (ICR) Swiss mouse, a multi-purpose stock selected for fecundity. Roswell Park Memorial Institute 75th Anniversary Volume. Alan R. Liss, N.Y. LI J. C. R. (1964) Statistical Inference I. p. 505. Edwards Brothers, Ann Arbor, Michigan. MCFEELY R. A. (1967) Chromosome abnormalities in early embryos of the pig. J. Reprod. Fert. 13, 579-581. MARTIN-DE LEON P. A., SHANER E. L. & GAMMAL E. G. (1973) Chromosome abnormalities in rabbit blastocysts resulting from spermatozoa aged in the male tract. Fert. Steril. 24, 212-219.
181
ULBERG L. C. (1958) The effects of climate on animal performance. The influence of high temperature upon reproduction. J. Hered. 49, 62-64. ULBERG L. C. & BURFENING P. J. (1967) Embryo death resulting from adverse environment on spermatozoa or ova. J. Anita. Sci. 26, 571-577. VICKERS A. D. (1969) Delayed fertilization and chromosomal anomalies in mouse embryos. J. Reprod. Fen. 20, 69-76. WALKER H. M. & LEV J. (1953) Statistical Inference," p. 424. Henry Holt, New York. WROBLEWSKA J. (1971) Developmental anomaly in the mouse associated with triploidy. Cytoqenetics 10, 199-207. WROBLEWSKAJ. & DYBAN A. P. (1969) Chromosome preparations from mouse embryos during early organogenesis: dissociation after fixation, followed by air drying. Stain Technol. 44, 147-150. ZEUTHEN E. (1972) Inhibition of chromosome separation in cleaving Psammechinus eggs by elevated temperature. Expl Cell Res. 72. 337-344.
Key Word lndex--Hyperthermia; mouse embryo chromosomes; aneuploidy; hyperploidy; Mus musculus.
0306-4565'79'0104 0179502.00/0
HYPERTHERMIA-INDUCED EMBRYONIC ANEUPLOIDY IN MICE C. L. CHRISMAN and D. P. DOOLITTLE Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A.
(Receired 5 May 1978: rerised 25 May 1978: accepted 5 November 1978) Abstract--Chromosomal aneuploidy, as an effect of elevated ambient temperature during early gestation, was studied in 650 eight-day embryos from 60 females of two different mouse stocks. Exposure of treatment females to 34 C and 500,, relative humidity during days 5 and 6 of gestation caused an increase in embryo chromosomal hyperploidy. No embryo sex interaction with the percent hyperploidy was found.
INTRODUCTION The incidence of embryonic mortality as well as of congenital defects in several m a m m a l i a n species has been s h o w n to increase following exposure of pregnant females to increased ambient temperature (Ulberg, 1958; Edwards, 1969, 1974; Bellve, 1973; Garrard et al., 1974). Causative mechanisms have not been thoroughly researched. Ulberg & Burfening (1967) demonstrated that direct embryonic changes were more likely the cause of embryo decline than an indirect embryonic effect due to uterine changes. Edwards et al. (1974) found nuclear clumping and mitotic delay in guinea-pig embryos subjected to a short period of maternal hyperthermia on day 21 of gestation. This might suggest mitotic spindle apparatus changes such as the microtubule damage found by Zeuthen (1972) in heat stressed sea-urchin • embryos. The o b j e c t i v e of the present study was to determine if embryonic numerical c h r o m o s o m e aberrations were generated following exposure of pregnant female mice to high ambient temperature after implantation.
MATERIALS AND METHODS Females from two random bred stocks of mice (Mus musculus), "J'" and "ICR" were randomly divided into treatment and control groups. Primaparous J females were derived from generations 20-34 of random mating from an original four way cross of the inbred lines LP/J, SJL/J, BALB/cJ, and C57BL/6J, Virgin ICR females were derived from the stock Breeder: Ha (ICR) selected for large litter size developed at the Institute of Cancer Research, Philadelphia (Hauschka & Mirand, 1973). The J females were mated to the same males that sired their first litters, whereas the ICR females were mated to adult ICR males of proven fertility. Day one of gestation was determined by presence of the copulatory plug. All animals were maintained in a controlled environmental chamber at 2 1 C with a relative humidity of 50-700~,. On the morning of the fifth day of gestation, treatment females were moved to an environmental chamber maintained at 34 C and 50°,i relative humidity for a 48 h period, then returned to the control chamber. All animals had free access to feed and water. On the eighth day of gestation, all females were killed by cervical dislocation and their reproductive tracts were removed. Ovulation rate
was estimated by a count of corpora lutea (CL), and the number of decidua and number of recovered embryos recorded. All embryos were processed for chromosome analysis following minor modifications to a procedure established by Wroblewska & Dyban (1969). Two slides were prepared from each embryo, stained with Giemsa and coded to prevent observer bias. An attempt was made to count the chromosomes of ten intact, well-spread metaphase nuclei; however a few embryos presented less than ten countable cells in metaphase. Counts were made of chromosomes of metaphase nuclei which were well-stained, evenly-spread and not mechanically broken. Metaphase nuclei with widely spread chromosomes or with chromosomes not contained within a circular group were not counted. An observer made five chromosome counts from each metaphase nucleus selected at 1000 x : if all five counts did not agree, the count was begun again until agreement was reached. Independent of the chromosome counts, a second observer sexed each embryo, finding at least five nuclei that exhibited either the darkly stained heteropyknotic X chromosome of the female or the darkly stained Y chromosome of the male. In some cases, the Y chromosome of the male was not darkly stained but was evident because of its morphology. Although exact chromosome counts were recorded including hypo- and hyper-diploid (<40 or >40 chromosomes, respectively), normal cells with 40 chromosomes and polyploid cells, the statistical analyses were performed on data including only diploid and hyperploid cells. The data were expressed as the raw percentage hyperploidy and transformed to the arcsin of the square root of the raw percent hyperploidy (Li, 1964) to approximate a normal distribution. A correction factor, the Bartlett correction, of 1/4n (where n is the number of observations per embryo) was used for embryos with 0". hyperploidy (Walker & Lev, 1953). The data were then assembled as dam means and the main effects of treatment and stock were tested for significance by analysis of variance based on a 2 × 2 factorial design. To determine if there was an effect of sex on percent hyperploidy the embryo data were analyzed with a least squares analysis of variance (Harvey, 19601 with embryo sex nested within stock-treatment combinations. RESULTS AND DISCUSSION Reproductive performances of the four groups of females as measured during the eighth day of gestation are summarized in Table 1. C o r p o r a lutea 179
C. L. CHRISMANand D. P. DOOLITTL£
180
Table 1. Reproductive performance of females in the control and hot environments Stock/Treatment
No. of females
Mean no. C.L. ~ S.E.
Meam no. d e c i d u a ± S.E.
Mean no. recovered embryos ± S.E.
Sex ratio (% males)
J/control
15
12.6 ± 0.54
12.2 ± 0.45
11.5 ± 0.52
50.0
J/heat
15
13.2 ± 0.65
12.1 ± 0.69
11.4 ± 0.76
49.7
ICR/control
15
ii.0 ± 1.24
ii.i ~ 1.06
10.3 ± 0 . 8 2
48.7
ICR/heat
15
12.2 ± 0.72
II.i t 0.88
10.3 ± 0.01
58.7
counts, number of decidua and number of recovered embryos did not significantly differ between stocks or treatments. A total of 655 embryos were recovered, of which 651 presented 5696 cells for chromosome counts. The raw mean hyperploidy percentages are shown in Table 2. A factorial analysis of variance testing transformed percent hyperploidy indicated that no stock difference existed. However, embryos from heat treated dams had a significantly greater rate of hyperploidy than controls (P < 0.02). The stock x treatment interaction was not significant. Polyploid cells (3n and 4n) were encountered only in the control litters (14 and 8 in the J and ICR controls, respectively). The absence of polyploid cells in the large number of treatment embryo cells observed could be interpreted as meaning that cells with a large number of extra chromosomes were unable to survive the stress conditions or that such cells were not entering the mitotic cycle. Sex ratios in each group are reported in Table 1. The sex ratios in both the J control and heat litters and the ICR control litters fit an expected 1:1 ratio quite well. However, the ICR heat group sex ratio differed significantly from both the ICR control and hypothesized 1:1 ratio (X2- 6.22 and X2= 4.7, respectively, P < 0.05). Because of this altered sex ratio in the ICR heat litters, there was a suggestion that the higher percentage of males was involved in the increased hyperploidy observed in this group. A least squares analysis of variance with embryo sex nested within stock-treatment combinations demonstrated that no sex effect was present (P > 0.05). Therefore it was concluded that the excess males in the ICR heat litters were not solely responsible for the increased hyperploidy.
Since maternal body temperature was not measured in this study, the increase in aneuploidy cannot be concluded to have been directly influenced by uterine hyperthermia. Bellve (1973) exposed ICR mice to 34.5°C and 65% relative humidity and recorded a 2°C increase in rectal temperature after 24 h. Rectal temperature measurements in this laboratory have not confirmed such an increase. It appears that the handling of each mouse and insertion of the rectal probe is sufficient to cause body temperature to rise in some mice. The relationship between chromosome aberrations and embryonic mortality and congenital abnormalities has been well established in humans (Hamerton, 1971). Although not nearly so well documented in animals, chromosome abnormalities are thought to be important in causing a pOrtion of the impaired fertility observed in polytocous species (McFeely, 1967; Vickers, 1969; Wroblewska, 1971; MartinDeLeon et al., 1973). Acknowledgements--The technical assistance of Gail Hillis and Jane Hatmaker is appreciated. This study is published as Indiana Agricultural Experiment Station Journal Paper No. 6297.
REFERENCES BELLVEA. T. (1973) Development of mouse embryos with abnormalities induced by parental heat stress. J. Reprod. Fert. 35, 393-403. EDWARDSM. J. (1969) Congenital defects in guinea pigs: fetal resorptions, abortions and malformations following induced hyperthermia during early gestation. Teratology 2, 313-328.
Table 2. Hyperploidy of cells from embryos in control and hot environments* Control
Across Treatments
Heat
hyperploldy ± S.E.
No. embryos
No. cells analyzed
% hyperploldy ± S.E.
No. embryos
No. cells analyzed
% hyperploldy ± S.E.
J
170
1535
2.23 ± 0.626
171
1507
2.87 ± 0.512
2.55 ± 0.404
ICR
154
1352
2.06 + 0.768
155
1302
4.48 ± 1.105
3.27 ± 0.673
Across stocks
324
2887
2.15 ± 0.495
326
2809
3.68 ± 0.609
2.91 ± 0.402
Stock
* From raw data, not transformed.
Hyperthermia-induced embryonic aneuploidy in mice EDWARDS M. J. (1974) The effects of hyperthermia on pregnancy and prenatal development. In Experimental Embryology and Teratology, (Edited by WOOLLAM D. H. M. & MORRiSS G. M.) Elek Science, London. EDWARDS M. J., MULLEY R., RING S. 8/. WANNER R. A. (1974) Mitotic cell death and delay of mitotic activity in guinea-pig embryos following brief maternal hypertermia, d. Emhryol. exp. Morph. 32, 593~i02. GARRARD G., HARRISON G. A. & WEINER J. S. (1974) Reproduction and survival of mice at 23: and 32°C. J. Reprod. Fert. 37, 287-298. HAMERTON J. L. (1971) Human Cytogenetics, Vol. II. Academic Press, New York. HARVEY W. R. (1960) Least-squares analysis of data with unequal subclass numbers. USDA ARS 20. HAUSCHKA T. S. & M1RAND E. J. (1973) The Breeder: Ha (ICR) Swiss mouse, a multi-purpose stock selected for fecundity. Roswell Park Memorial Institute 75th Anniversary Volume. Alan R. Liss, N.Y. LI J. C. R. (1964) Statistical Inference I. p. 505. Edwards Brothers, Ann Arbor, Michigan. MCFEELY R. A. (1967) Chromosome abnormalities in early embryos of the pig. J. Reprod. Fert. 13, 579-581. MARTIN-DE LEON P. A., SHANER E. L. & GAMMAL E. G. (1973) Chromosome abnormalities in rabbit blastocysts resulting from spermatozoa aged in the male tract. Fert. Steril. 24, 212-219.
181
ULBERG L. C. (1958) The effects of climate on animal performance. The influence of high temperature upon reproduction. J. Hered. 49, 62-64. ULBERG L. C. & BURFENING P. J. (1967) Embryo death resulting from adverse environment on spermatozoa or ova. J. Anita. Sci. 26, 571-577. VICKERS A. D. (1969) Delayed fertilization and chromosomal anomalies in mouse embryos. J. Reprod. Fen. 20, 69-76. WALKER H. M. & LEV J. (1953) Statistical Inference," p. 424. Henry Holt, New York. WROBLEWSKA J. (1971) Developmental anomaly in the mouse associated with triploidy. Cytoqenetics 10, 199-207. WROBLEWSKAJ. & DYBAN A. P. (1969) Chromosome preparations from mouse embryos during early organogenesis: dissociation after fixation, followed by air drying. Stain Technol. 44, 147-150. ZEUTHEN E. (1972) Inhibition of chromosome separation in cleaving Psammechinus eggs by elevated temperature. Expl Cell Res. 72. 337-344.
Key Word lndex--Hyperthermia; mouse embryo chromosomes; aneuploidy; hyperploidy; Mus musculus.