©1992 S. Karger AG. Basel 0301-0171 /92/0594-0284S2.75/0
Cytogenel Cell Genet 59:284-287 ( 1992)
Transmission of gametes with normal or translocation chromosomes in male and female single-sex mouse chimeras P. de Boer, F.A. van der Hoeven, and J.M.G.M. Scholler Department of Genetics. Wageningen Agricultural University. Wageningen (The Netherlands)
When feral mice with Robertsonian translocations are crossed with chromosomally normal laboratory mice, both the homozygous and heterozygous male offspring may show a depression in testis weight and sperm count, as in carriers of Rb(l 1.13)4Bnr (Cattanach and Moseley, 1973), or may have normal or subnormal fertility, as evidenced by carriers of Rb(4.6)2Bnr(Mahadevaiah et al., 1990). The effect of heterozy gosity for Rb4Bnr on spermatogenesis correlates with an in creased number of meiotic figures at stage XII in the seminifer ous tubules (Nijhoff and de Boer, 1979) and prolongation of meiotic prophase in the male (Speed and de Boer. 1983). Males homozygous for Rb( 11.13)4Bnr T( 1:13)70H (hereafter referred to as RbT/RbT). a translocation involving Rb( 11.13)4Bnr. and the 131chromosome of T(1;13)70H (Table I and Fig. 1: see also Wessels-Kaalen et al.. 1986) likewise demonstrate an increase in the number of degenerating primary spermatocytes at diakinesis-metaphase 1and a reduction in testis weight, as well as sperm count. To further characterize the fertility of homozygous carriers of RbT/RbT. we produced chimeras by fusing embryos carrying this mutation with chromosomally normal embryos. By tagging the translocation with fu zzy (fz). we could follow the two compo nents of the germ line in liveborn offspring. As this translocation is an excellent cytological marker (note in Fig. 1 that the 13 arm of Rb4Bnr has been replaced by the long 131 marker chromo some of T70H), we could also estimate the ratio of RbT/RbT to +/+ cells in dividing spermatogonia, as well as in primary and secondary spermatocytes. Observations on the breeding behav ior of female chimeras with the same genetic constitution are also presented. Received 26 November 1990: revision accepied 15 July 1991. Request reprints from Dr. Peter de Boer. Department of Genetics. Wageningen Agri cultural University. Dreijenlaan 2.6703 HA Wageningen (The Netherlands).
in males, but decreased in two of the three female chimeras. Within males, there was generally good agreement between the proportions of translocation and nontranslocation germ cells from spermatogonial mitosis through the first and second meiotic division. In one male, this ratio was also reflected in the offspring. In the other two males, there was significant selection during hapiophase. from which both types of spermatozoa could benefit.
Materials and methods Homozygous RbT/RbT mice were bred by crossing T(I:13)70H with Rb(l 1.13)4Bnr (NijholT. 1981) mice and then intercrossing the combined translocation heterozygous offspring. The mice are maintained in a breeding system that prohibits inbreeding and ensures that each generation generates a sufficient inflow of Swiss +/+ genes so that the mutant and +/+ embry os used for fusion are of the same Swiss CpbSE(S) genetic background. In these experiments, RbT/RbT embryos of the 14th generation were used. The mice were between 2 and 4 mo of age when bred together. Females were superovulated by injecting pregnant mare serum and human chorionic gonadotropin and were mated with males of their own karyotype to produce donor embryos. Production o f chimeras Chimeras were produced by the eight-cell fusion technique (Mullen and Whitten. 1971). After overnight culture (Whittingham. 1971), fused embryos were nonsurgically transplanted into 2.5-d pseudopregnant recipients. The latter were 2- to 4-mo-oid F| females derived from a cross between LIII. a Chilton (UK) MRC Radiobiology Unit linkage testing stock, and Swiss ran dom-bred mice (van der Hoeven el al., 1991). Lymphocyte cultures were pre pared from orbital blood of liveborn mice (de Boer et al.. 1977). Chromo somes were C-banded according to the method of Sumner (1972) and scored for sex and chromosome mutations.
Table I. Fertility and spermatogenesis data on male mice homozygous for RB( 11.13S)4Bnr T (l; l3)70Ha Karyotype
Testis weight
(mg)
+/+ RbT/RbT
122± 11(15) 94 ±12(11)
Epididvmal sperm countb
5I2± 125 (55) 248 94 (11)
Degenerating diakinesis-Ml figures Number
Percent
600 (3) 1,200(6)
4.7 30.3
a Data from Wessels-Kaalen et al. (1986); numbers of males are given in paren theses. b Actual hemocylometer counts.
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Abstract. Three male and four female mouse single-sex chi meras derived from fusions of Rb( 11.13)4Bnr T( 1; 13)70H ho mozygous embryos with +/+ embryos were caged with T(1;!3)70H homozygotes of the opposite sex and followed through their reproductive lifespans. Six animals (three males and three females) were germline chimeras. The f : gene was used as a marker for the T70H reciprocal translocation. The ratio of fz/fz to fz/+ offspring did not change with increasing age
285
Single-sex mouse chimeras
a
b
e
Fifi. I. Bone marrow mitosis (a), primary spermatocyte in diakincsis (b). and secondary spermatocyte (e) from a male mouse homozygous for Rb( 11. 13S)4Bnr T( 1; 13)70H. Large arrow denotes the large marker chromosome and bivalent; small arrow denotes the small marker chromosome and bivalent. Within the large marker chromosome, the double helerochromatic band surrounding the 11,13 centromere region is clearly visible.
Overall frequency Frequency per litter Frequency per reproduction Young Middle age Old
Male A
Male B
MalcC
0.12 i 0.03 (153) 0.12 ± 0.10 (14)
0.46+0.05(122) 0.48 rt 0.23 (16)
0.63 + 0.05(83) 0.71 ±0.28(12)
0.38(55) 0.58 (33) 0.47(34)
0.40(15) 0.71 (35) 0.64(33)
period 0.13(77) 0.17(41) 0.03 (35)
a Numbers of young and litters are given in parentheses. b Because of the presence of two females, litters sometimes coincided; these have been treated as one.
Reproduction and cytological analysis Each of the selected XY/XY chimeras was allowed to breed continuously with two T( I ; 13)70H/Tid.,/-//; females until sterility. When breeding stag nated. the females were replaced by two new ones. In addition, males that were not chimeras, as determined by coat characteristics and examination of cultured blood lymphocytes, were similarly bred. Female XX/XX chimeras were each caged with a T7ÜH homozygous male. The numbers of fz /f: and f:/+ offspring (the latter having glossy coats) were recorded. For each male chimera, chromosome preparations of spermatogonia and primary and secondary spermatocytes were made using a combination of the techniques of Evans et al. (1964) and Meredith (1969). Two of the three males were injected with colchicine (4 pg/g bodyweight) 3 h before sacrifice. After the tubules were macerated with curved forceps, the suspension of cells and tubular fragments was incubated in 0.8% sodium citrate supplemented with 250 gg/ml hyaluronidase (Sigma, type I-S from bovine testis) for 20 min at 37 °C. Following hypotonic treatment, tubular fragments and suspended cells were separated by brief centrifugation; the fragments were then processed according to Meredith (1969) and the cell suspension according to Evans et al. (1964). Chromosomes were C-banded following the method of Sumner ( 1972). A like number of cells were sampled from every slide made of the two testes from each male. Differences between cell stages and offspring with regard to the relative contributions of RbT/RbT and +/+ cells or gametes were analyzed by x2testing.
Table III. Percentages of translocation-carrying spermatogonial mitoses, diakinesis-metaphase I, and metaphase II mouse spermatocytes, and off spring in the chimeric male mice A-Ca Male
Mitosis
Meiosis 1
Meiosis 11
Offspring
Ab Bb C
10 % (748) 98 % (4!l) 4.2%(71)c
13.5% (200) 99.0% (200) 37.0%(200)c
5.4% (37) 83.3%(36)c 11.7 %(120)
11.8%(153) 45.9% (122) 62.7% (83)
3 Numbers of cells studied are given in parentheses. b Injected with colchicine. c Significantly different from expected values.
Results Chimera production The chimeras described in this study resulted from 40 fused pairs of embryos transplanted into five recipients. Nineteen pups were born, and 16 were weaned and karyotyped for sex and the presence of the translocation marker chromosomes. Ten pups were classified as chimeras based on an examination of 50 or 100 mitoses. Five of these mice were also coat-structure chi meras; the others were flat-coated. Of the 10 chimeric mice, 7 were single-sex chimeras (three XY/XY and four XX/XX) and 3 were X Y/XX (including two previously reported males [van der Hoeven et al., 1986] and one female). Six of the seven single-sex chimeras (three females and three males) were also germ-cell chimeras. Lifetime production ofyoung from X Y / X Y chimeras To analyze any shift in ratio of the two germ-line compo nents with age. males that had their first litter at 2 mo of age
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Table II. Frequencies of fz/fz offspring per male mouse chimera (litters summed), per average litter, and per reproductive period (age of male)
de Boer/van der Hoeven/Scholler
286
Table IV. Litter size and transmission data on female single-sex chimeric mice
A B C
Number of litters
h
8 7
Percentage of offspring from RbT/RbT component at weaning (n)
Litter size (± SD)
10.64 ±3.78 9.50±5.26 11.43 ±2.23
Total
During first 7 mo
From 8 mo to sterility
13.9 % ( 108) 48.0% (73) 9.2% (76)
22.0% (59) 55.2 % (58) 10.4% (48)
4.1% (49) 20.0% (15) 7.1% (28)
Table V. Average size of the first three litters at birth of female single-sex chimeric, nonchimeric fusions, and nonchimeric control mice Female karyotype
Mated with
Number
Litter size
+/+/RbT/RbT RbT/RbT/0» +/+/0 t/+ RbT/RbT
T70H/T70H
4 4 4 7 10
11.75 ±3.02 11.00 ±2.56 11.58 ± 2.97 10.76 ±2.64 9.70 ±1.95
*
T70H/T70H RbT/RbT
Three of these were from an earlier experimental series.
were allowed to breed until sterility at 10-12 mo. Lifetime pro duction was arbitrarily divided into three periods: young, mid dle-age, and old. Table II gives the overall frequency of RbT offspring (fz/fz), the frequency for each period of life, and the mean frequency per litter. Chi-square analysis showed no signif icant trend in the ratio of RbT to +/+ offspring over the repro ductive lifespan of any of the males. For male A, however, there seemed to be a loss of RbT/RbT-derived offspring near the end of its reproductive lifespan. As shown in Table II. the variance in per-litter frequency of fz /fz young is much larger than the overall variance. This indication of between-litter variance will be discussed later. Cell composition o f spermatogonia At 12-13 mo of age, the males were killed and their spermatogenic cells karyotyped. Males A and B were injected with colchicine (4 pg/g bodyweight) 3 h before sacrifice. Figure I shows the presence of the RbT marker chromosome and biva lents in spermatogonial mitoses and primary and secondary spermatocytes. For each male, 200 diakinesis-metaphase I cells were scored. Table 111 gives the percentages of translocationcarrying spermatogonial mitoses, MI and Mil cells, and off spring (from Table II). It can be seen that the use of a spindle poison strongly changes the chromosomal composition of the dividing cells. The percentages found at the spermatogonial level were very little “balanced” (i.e., the proportions were much closer to 0:100 than to 50:50). When colchicine was used, these percentages persisted into diakinesis-metaphase I, with no remarkable change in the much smaller secondary spermatocyte samples. For male A, germ-cell frequencies were reflected in the off spring. but for male B. there was a dramatic shift during haplophase favoring wild-type gametes. When colchicine was omitted (male C), the increased cell death among RbT/RbT primary spermatocytes (Table I, with
X2
9.00 6.28 0.23
P
Cytogenel Cell Genet 59:284-287 ( 1992)
Transmission of gametes with normal or translocation chromosomes in male and female single-sex mouse chimeras P. de Boer, F.A. van der Hoeven, and J.M.G.M. Scholler Department of Genetics. Wageningen Agricultural University. Wageningen (The Netherlands)
When feral mice with Robertsonian translocations are crossed with chromosomally normal laboratory mice, both the homozygous and heterozygous male offspring may show a depression in testis weight and sperm count, as in carriers of Rb(l 1.13)4Bnr (Cattanach and Moseley, 1973), or may have normal or subnormal fertility, as evidenced by carriers of Rb(4.6)2Bnr(Mahadevaiah et al., 1990). The effect of heterozy gosity for Rb4Bnr on spermatogenesis correlates with an in creased number of meiotic figures at stage XII in the seminifer ous tubules (Nijhoff and de Boer, 1979) and prolongation of meiotic prophase in the male (Speed and de Boer. 1983). Males homozygous for Rb( 11.13)4Bnr T( 1:13)70H (hereafter referred to as RbT/RbT). a translocation involving Rb( 11.13)4Bnr. and the 131chromosome of T(1;13)70H (Table I and Fig. 1: see also Wessels-Kaalen et al.. 1986) likewise demonstrate an increase in the number of degenerating primary spermatocytes at diakinesis-metaphase 1and a reduction in testis weight, as well as sperm count. To further characterize the fertility of homozygous carriers of RbT/RbT. we produced chimeras by fusing embryos carrying this mutation with chromosomally normal embryos. By tagging the translocation with fu zzy (fz). we could follow the two compo nents of the germ line in liveborn offspring. As this translocation is an excellent cytological marker (note in Fig. 1 that the 13 arm of Rb4Bnr has been replaced by the long 131 marker chromo some of T70H), we could also estimate the ratio of RbT/RbT to +/+ cells in dividing spermatogonia, as well as in primary and secondary spermatocytes. Observations on the breeding behav ior of female chimeras with the same genetic constitution are also presented. Received 26 November 1990: revision accepied 15 July 1991. Request reprints from Dr. Peter de Boer. Department of Genetics. Wageningen Agri cultural University. Dreijenlaan 2.6703 HA Wageningen (The Netherlands).
in males, but decreased in two of the three female chimeras. Within males, there was generally good agreement between the proportions of translocation and nontranslocation germ cells from spermatogonial mitosis through the first and second meiotic division. In one male, this ratio was also reflected in the offspring. In the other two males, there was significant selection during hapiophase. from which both types of spermatozoa could benefit.
Materials and methods Homozygous RbT/RbT mice were bred by crossing T(I:13)70H with Rb(l 1.13)4Bnr (NijholT. 1981) mice and then intercrossing the combined translocation heterozygous offspring. The mice are maintained in a breeding system that prohibits inbreeding and ensures that each generation generates a sufficient inflow of Swiss +/+ genes so that the mutant and +/+ embry os used for fusion are of the same Swiss CpbSE(S) genetic background. In these experiments, RbT/RbT embryos of the 14th generation were used. The mice were between 2 and 4 mo of age when bred together. Females were superovulated by injecting pregnant mare serum and human chorionic gonadotropin and were mated with males of their own karyotype to produce donor embryos. Production o f chimeras Chimeras were produced by the eight-cell fusion technique (Mullen and Whitten. 1971). After overnight culture (Whittingham. 1971), fused embryos were nonsurgically transplanted into 2.5-d pseudopregnant recipients. The latter were 2- to 4-mo-oid F| females derived from a cross between LIII. a Chilton (UK) MRC Radiobiology Unit linkage testing stock, and Swiss ran dom-bred mice (van der Hoeven el al., 1991). Lymphocyte cultures were pre pared from orbital blood of liveborn mice (de Boer et al.. 1977). Chromo somes were C-banded according to the method of Sumner (1972) and scored for sex and chromosome mutations.
Table I. Fertility and spermatogenesis data on male mice homozygous for RB( 11.13S)4Bnr T (l; l3)70Ha Karyotype
Testis weight
(mg)
+/+ RbT/RbT
122± 11(15) 94 ±12(11)
Epididvmal sperm countb
5I2± 125 (55) 248 94 (11)
Degenerating diakinesis-Ml figures Number
Percent
600 (3) 1,200(6)
4.7 30.3
a Data from Wessels-Kaalen et al. (1986); numbers of males are given in paren theses. b Actual hemocylometer counts.
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 8:27:30 AM
Abstract. Three male and four female mouse single-sex chi meras derived from fusions of Rb( 11.13)4Bnr T( 1; 13)70H ho mozygous embryos with +/+ embryos were caged with T(1;!3)70H homozygotes of the opposite sex and followed through their reproductive lifespans. Six animals (three males and three females) were germline chimeras. The f : gene was used as a marker for the T70H reciprocal translocation. The ratio of fz/fz to fz/+ offspring did not change with increasing age
285
Single-sex mouse chimeras
a
b
e
Fifi. I. Bone marrow mitosis (a), primary spermatocyte in diakincsis (b). and secondary spermatocyte (e) from a male mouse homozygous for Rb( 11. 13S)4Bnr T( 1; 13)70H. Large arrow denotes the large marker chromosome and bivalent; small arrow denotes the small marker chromosome and bivalent. Within the large marker chromosome, the double helerochromatic band surrounding the 11,13 centromere region is clearly visible.
Overall frequency Frequency per litter Frequency per reproduction Young Middle age Old
Male A
Male B
MalcC
0.12 i 0.03 (153) 0.12 ± 0.10 (14)
0.46+0.05(122) 0.48 rt 0.23 (16)
0.63 + 0.05(83) 0.71 ±0.28(12)
0.38(55) 0.58 (33) 0.47(34)
0.40(15) 0.71 (35) 0.64(33)
period 0.13(77) 0.17(41) 0.03 (35)
a Numbers of young and litters are given in parentheses. b Because of the presence of two females, litters sometimes coincided; these have been treated as one.
Reproduction and cytological analysis Each of the selected XY/XY chimeras was allowed to breed continuously with two T( I ; 13)70H/Tid.,/-//; females until sterility. When breeding stag nated. the females were replaced by two new ones. In addition, males that were not chimeras, as determined by coat characteristics and examination of cultured blood lymphocytes, were similarly bred. Female XX/XX chimeras were each caged with a T7ÜH homozygous male. The numbers of fz /f: and f:/+ offspring (the latter having glossy coats) were recorded. For each male chimera, chromosome preparations of spermatogonia and primary and secondary spermatocytes were made using a combination of the techniques of Evans et al. (1964) and Meredith (1969). Two of the three males were injected with colchicine (4 pg/g bodyweight) 3 h before sacrifice. After the tubules were macerated with curved forceps, the suspension of cells and tubular fragments was incubated in 0.8% sodium citrate supplemented with 250 gg/ml hyaluronidase (Sigma, type I-S from bovine testis) for 20 min at 37 °C. Following hypotonic treatment, tubular fragments and suspended cells were separated by brief centrifugation; the fragments were then processed according to Meredith (1969) and the cell suspension according to Evans et al. (1964). Chromosomes were C-banded following the method of Sumner ( 1972). A like number of cells were sampled from every slide made of the two testes from each male. Differences between cell stages and offspring with regard to the relative contributions of RbT/RbT and +/+ cells or gametes were analyzed by x2testing.
Table III. Percentages of translocation-carrying spermatogonial mitoses, diakinesis-metaphase I, and metaphase II mouse spermatocytes, and off spring in the chimeric male mice A-Ca Male
Mitosis
Meiosis 1
Meiosis 11
Offspring
Ab Bb C
10 % (748) 98 % (4!l) 4.2%(71)c
13.5% (200) 99.0% (200) 37.0%(200)c
5.4% (37) 83.3%(36)c 11.7 %(120)
11.8%(153) 45.9% (122) 62.7% (83)
3 Numbers of cells studied are given in parentheses. b Injected with colchicine. c Significantly different from expected values.
Results Chimera production The chimeras described in this study resulted from 40 fused pairs of embryos transplanted into five recipients. Nineteen pups were born, and 16 were weaned and karyotyped for sex and the presence of the translocation marker chromosomes. Ten pups were classified as chimeras based on an examination of 50 or 100 mitoses. Five of these mice were also coat-structure chi meras; the others were flat-coated. Of the 10 chimeric mice, 7 were single-sex chimeras (three XY/XY and four XX/XX) and 3 were X Y/XX (including two previously reported males [van der Hoeven et al., 1986] and one female). Six of the seven single-sex chimeras (three females and three males) were also germ-cell chimeras. Lifetime production ofyoung from X Y / X Y chimeras To analyze any shift in ratio of the two germ-line compo nents with age. males that had their first litter at 2 mo of age
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 8:27:30 AM
Table II. Frequencies of fz/fz offspring per male mouse chimera (litters summed), per average litter, and per reproductive period (age of male)
de Boer/van der Hoeven/Scholler
286
Table IV. Litter size and transmission data on female single-sex chimeric mice
A B C
Number of litters
h
8 7
Percentage of offspring from RbT/RbT component at weaning (n)
Litter size (± SD)
10.64 ±3.78 9.50±5.26 11.43 ±2.23
Total
During first 7 mo
From 8 mo to sterility
13.9 % ( 108) 48.0% (73) 9.2% (76)
22.0% (59) 55.2 % (58) 10.4% (48)
4.1% (49) 20.0% (15) 7.1% (28)
Table V. Average size of the first three litters at birth of female single-sex chimeric, nonchimeric fusions, and nonchimeric control mice Female karyotype
Mated with
Number
Litter size
+/+/RbT/RbT RbT/RbT/0» +/+/0 t/+ RbT/RbT
T70H/T70H
4 4 4 7 10
11.75 ±3.02 11.00 ±2.56 11.58 ± 2.97 10.76 ±2.64 9.70 ±1.95
*
T70H/T70H RbT/RbT
Three of these were from an earlier experimental series.
were allowed to breed until sterility at 10-12 mo. Lifetime pro duction was arbitrarily divided into three periods: young, mid dle-age, and old. Table II gives the overall frequency of RbT offspring (fz/fz), the frequency for each period of life, and the mean frequency per litter. Chi-square analysis showed no signif icant trend in the ratio of RbT to +/+ offspring over the repro ductive lifespan of any of the males. For male A, however, there seemed to be a loss of RbT/RbT-derived offspring near the end of its reproductive lifespan. As shown in Table II. the variance in per-litter frequency of fz /fz young is much larger than the overall variance. This indication of between-litter variance will be discussed later. Cell composition o f spermatogonia At 12-13 mo of age, the males were killed and their spermatogenic cells karyotyped. Males A and B were injected with colchicine (4 pg/g bodyweight) 3 h before sacrifice. Figure I shows the presence of the RbT marker chromosome and biva lents in spermatogonial mitoses and primary and secondary spermatocytes. For each male, 200 diakinesis-metaphase I cells were scored. Table 111 gives the percentages of translocationcarrying spermatogonial mitoses, MI and Mil cells, and off spring (from Table II). It can be seen that the use of a spindle poison strongly changes the chromosomal composition of the dividing cells. The percentages found at the spermatogonial level were very little “balanced” (i.e., the proportions were much closer to 0:100 than to 50:50). When colchicine was used, these percentages persisted into diakinesis-metaphase I, with no remarkable change in the much smaller secondary spermatocyte samples. For male A, germ-cell frequencies were reflected in the off spring. but for male B. there was a dramatic shift during haplophase favoring wild-type gametes. When colchicine was omitted (male C), the increased cell death among RbT/RbT primary spermatocytes (Table I, with
X2
9.00 6.28 0.23
P
