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Cytogenetic effects of hydrostatic pressure treatment to suppress the first cleavage of salmon embryos
Aquaculture, 110 (1993) 51-59 Elsevier Science Publishers B.V.. Amsterdam
51
AQUA 30023
Cytogenetic effects of hydrostatic pressure treatment to suppress the first cleavage of salmon embryos Fumio Yamazakia and John Goodierb “Faculty of Fisheries, Hokkaido University,Hakodate, Japan bDepartment OfBiochemistry, Memorial Universityof St. Johns’, Nfld..,Canada (Accepted 10 June 1992)
ABSTRACT Yamazaki, F. and Goodier, J.. 1993. Cytogenetic effects of hydrostatic pressure treatment to suppress the first cleavage of salmon embryos. Aquaculture, 110: 5 l-59. Tetraploids in chum and masu salmon were induced by suppressing the first cleavage by hydrostatic pressure of 680 kg/cm’. Ploidy level was scored by counting nucleoli using silver-stained preparations. True tetraploids were induced in some chum embryos treated at 13.2 h after fertilization; however, aneuploids or polyploid mosaics were also found. These showed abnormal embryogenesis. Chromosomes isolated from the nucleus or precociously moving to the centrosome were observed in the dividing cells. Chromosome bridges between two centrosomes were also observed, as well as chromosome fragments, rings and gaps. Even if damages induced by mitotic suppression with hydrostatic pressure treatment is minor at the first cleavage, it will be amplified through rapid cell cycles.
INTRODUCTION
Triploids or gynogenetic diploids have been successfully induced in various species by suppressing the second meiotic division with temperature shock or hydrostatic pressure treatment (Thorgaard, 1983 ) . These techniques have been applied by fish culturists to produce populations of sterile triploid female or all-female fish. Another useful technique utilizes suppression of the first cleavage by hydrostatic pressure. When irradiated sperm or eggs are used for fertilization, mitotically suppressed gynogenetic or androgenetic diploids are created which are considered to be homozygous at all loci (Parson and Thorgaard, 1985; Onozato, 1990). Inbred diploid or tetraploid fish induced in this manner are useful for genetic research. However, the survival of fertilized eggs treated with hydrostatic pressure is sometimes very low: less than Correspondence to: F. Yamazaki, Japan.
0044-8486/93/$06.00
Faculty of Fisheries,
Hokkaido
0 1993 Elsevier Science Publishers
University,
Hakodate
B.V. All rights reserved.
04 1,
52
F. YAMAZAKI AND J. CiOODIER
10% at hatching when compared with those undergoing suppression of the second meiotic division. Incidence of physical abnormalities also tends to be higher among tetraploid than among triploid embryos (Thorgaard, 1983 ). We supposed that low survival following suppression of the first cleavage would be caused by chromosome damage. This study aimed to explore success rates of tetraploid induction and to clarify chromosome damage affecting the viability of embryos treated with hydrostatic pressure at the first cleavage. MATERIAL
AND METHODS
Gametes used in the experiments were from chum salmon, Oncorhynchus keta, and masu salmon, 0. masou. Those of chum salmon were collected at the hatchery of the Kamiiso Fishermen’s Cooperative, about 6 km from the Faculty of Fisheries, Hokkaido University. The eggs were collected by abdominal incision and sperm by squeezing, and brought to the Faculty. A small number of fertilized eggs (about 200 each in three groups) was held at 8 ‘C and subjected to 680 kg/cm2 pressure for 7 min at 12.7, 13.2, and 13.7 h after fertilization. The same number of eggs without treatment served as control. The masu salmon eggs and sperm were collected at Mori Branch of the Hokkaido Fish Hatchery, and transported 40 km to the Faculty. Fertilized eggs were treated as above at 4.75, 5.75, 6.5,7.0, 7.3, 8 and 9 h after fertilization. About 200 eggs were used in each group. The pressure-treated and control groups in chum and masu salmon were incubated at 8 to 10°C in a recirculating water system. Chromosome preparations were made by the chopping method (Yamazaki et al., 198 1) during the lirst to third week after fertilization. The slides were silverstained to reveal nucleoli by the method of Gold ( 1984). Staining chum and masu salmon chromosome preparations with silver nitrate permitted di-
Fig. 1. Chum salmon embryos at 2 1 days after fertilization. A: normal control; B, C: abnormal embryos from hydrostatic pressure-treated group. Fig. 2. Normal diploid chum salmon cells having two nucleoli stained with silver nitrate. Fig. 3. Tetraploid chum salmon cells having mainly four nucleoli. These cells were stained with silver nitrate after hydrostatic ,pressure treatment at the first cleavage. Fig. 4. Typical tetraploid chum salmon cell having four nucleoli induced by hydrostatic pressure treatment at the first cleavage. Fig. 5. Aneuploid or mosaic chum cells having various numbers of nucleoli. This figure was taken from a hypertetraploid individual, embryo no. 9 in Table 1. Fig. 6. Masu salmon chromosome preparation showing chromosome bridge at anaphase following hydrostatic pressure treatment at 8 h after fertilization. Preparations were made at 72 h. Fig. 7. Lagging chromosomes in the chromosome bridge at early telophase found in masu salmon chromosome preparation following hydrostatic pressure treatment.
HYDROSTATIC PREWJRE
53
TREATMENT OF SALMON EMBRYOS
-_-_
--
1
54
F. YAMAZAKI AND J. GOODIER
rect determination of ploidy level through chromosome and nucleoli counting (Phillips et al., 1986). Embryos of both masu and chum salmon were sampled periodically and fixed with Bouin’s solution for histological examination of gross morphology and chromosome kinetics. RESULTS
Some chum salmon embryos in the 13.2- and 13.7-h groups appeared normal and survived until 30 days after fertilization when the experiment was terminated (Fig. IA). No eggs of masu salmon from the 6.5- to 8-h groups survived more than 10 days after fertilization. Abnormal embryonic development was evident in all groups of hydrostatic pressure treatment. The appearance of abnormal embryos in both species varied greatly, from mere cell masses lacking any normal embryonic differentiation, to small-eyed embryos with stunted or twisted bodies (Fig. 1B,C ) . The results of nucleoli counting in chum salmon experiments are listed in Table 1. The cells of control chum salmon embryos had one or two nucleoli: those with two nucleoli averaged 88% of the cells examined (Fig. 2). The group treated with hydrostatic pressure at 12.7 h contained diploid and hyperdiploid individuals only. Three individuals in the 13.2-h group having over 80% of their cells with four nucleoli were probably true tetraploids (Table 2, Figs. 3,4). However, more individuals were polyploid mosaics or aneuploid, based on the number of nucleoli (Table 1) and chromosomes (Table 2). In fact, the majority of cells of one hypertetraploid embryo (Table 1, 13.2 h, no. 9 ) displayed 5 to 7 nucleoli (Fig. 5 ) . In the chromosome preparations from supposed polyploid embryos, chromosome bridges between two centrosomes, probably caused by abnormally formed dicentric chromosomes, were clearly observed (Fig. 6 ). Chromo-
Fig. 8. Paraffin section of chum salmon cell showing a chromosome precociously moving to pole at metaphase following hydrostatic pressure treatment. Sample was taken at 4 days after the treatment. Fig. 9. Isolated chromosome in the cytoplasm at anaphase of chum salmon cell sampled at 4 days after pressure treatment. Fig. 10. Isolated chromosome in the cytoplasm far from the nucleus in chum salmon cell sampled at 4 days after pressure treatment. Fig. 11. Chromosome spread from chum salmon embryo after hydrostatic pressure treatment at the first cleavage, 13.2 h after fertilization. Chromosome gap is shown by an arrow. Fig. 12. Chromosome gap (arrow) found in the chromosome spread from chum salmon embryo after pressure treatment at the first cleavage ( 13.2 h). Fig. 13. Many chromosome fragments (f ) and fused chromosomes caused by inter-chromosome exchange (fc) are found in chromosome spread from masu salmon embryo following pressure treatment at 8 h after fertilization. Fig. 14. Ring chromosomes in chromosome spread from masu salmon embryo after pressure treatment. Fig. 15. Chromosome spread showing ring chromosomes caused by intra-arm exchange (r) and interarm exchange ( re).
HYDROSTATIC PRESSURE TREATMENT OF SALMON EMBRYOS
55
somes were pulled toward both centrosomes and fragmented, while the fragments remained isolated between the two daughter nuclei at telophase (Fig. 7). Paraffin sections of the pressure-treated embryos also showed abnormal cell divisions. Some tigures of the cell divisions suggested that certain chromosomes move precociously to the polar region at metaphase (Fig. 8). Some fragmented chromosomes were found in cytoplasmic regions far from the spindle ( Fig. 9 ) or nuclear areas (Fig. 10 ) . Morphologically abnormal chromosomes were also evident in the nuclear plates of apparently aneuploid embryos. Chromosome gaps were frequently
17 17
17
1 2 3 4 5 6 7
1 2 3 4 5 6 7 8 9
1
2 3
4
Pressuretreated at 12.7 h
Pressuretreated at 13.2 h
Pressure-
treated at 13.7 h
17
15
15 15 18 15 31 31 15
21 19 21 21 19 21 19
12 samples
Control
Age (day)
Embryo number
Groups
91 91 87 86 85 21 12
9 8 12 14 15 2 1 1 1
1 o-2
22 25
7 6 19
1 2
35
22
12 11 19 29 30 11 22 44 2
76 14
3
4
1
1 1 2 1
88 86-90
11 8-14
1 4 1 6 7 40 33 18 3
2
1
Number of nucleoli per cell
42
70 69
71
86 85 80 62 62 46 42 36 5
9 52
4
8 8 E
1
?1 S $
$ .?
3
8
1
30
7
g
44
6
pressure at 12.7, 13.2 and 13.7
z
2
13
2
4
1 1
21
5
Percent frequency of cells with specific number of nucleoli of chum salmon embryos which were treated with hydrostatic h after fertilization
TABLE 1
1 2
1 3 4 6
Control
Pressuretreated atl2.7h
Pressure- I treated 2 at 13.7 h 3 4
Pressure- 1 treated 7 at 13.2 h
Embryo number
Groups
6
1
1
3 3
1
2 1
1 2 1 112310
I
2
~70
3
number
1
I 63 92
61
1
1 1
2
2
1
1
2
1
13
12
2
3
1
1
1 1
1
1
2
1
1
1
1 1 3 2
4 6
7
18 19
Total number 70 71 72 73 74 75 76 77 78 79 82 83 99 111 117 118 135 141 145 146 147 148 149 153 ofcells counted
Chromosome
Distribution of chromosome number of chum salmon embryos following hydrostatic pressure treatment
TABLE 2
58
F. YAMAZAKI AND J. GOODIER
observed (Figs. 11, 12 ) and chromosome fragments were numerous in nuclear plates (Fig. 13 ) . Long chromosomes caused by fusion of two mosomes or chromosome exchange were also found (Fig. 13 ) . Ring mosomes with or without centromeres were observed (Figs. 14, 15 ); might have been caused by intra- or inter-arm exchange within a single mosome (Fig. 15 ).
some chrochrothese chro-
DISCUSSlON
In the present study, ploidy level of experimental embryos was simply identified by counting the number of nucleoli per cell, as suggested by Phillips et al. ( 1986) for salmonids. We have shown that most diploid cells in chum and masu salmon have two nucleoli. Suppression of the first cleavage with hydrostatic pressure treatment induced tetraploids having four nucleoli and polyploid mosaics having a variable number of nucleoli. Most embryos were aneuploid or polyploid mosaic in chromosome constitution, and were morphologically abnormal and died during embryogenesis. In fact, in some cases, no embryonic differentiation beyond simple cell mass formation was observed after hydrostatic pressure treatment. Cytological investigation revealed chromosomes stranded in the cytoplasmic regions, and both morphologically abnormal chromosomes and chromosome bridges were observed in dividing cells. Even minor or small damages to chromosomes at the first cleavage might sometimes cause serious changes such as terminal deletion, exchange type aberration, inter- or intra-arm exchange or inter-chromosome exchanges through rapid cell cycles. These changes would amplify the damage to the chromosomes. We observed many chromosome fragments, ring chromosomes and also long fused chromosomes suggesting frequent occurrence of the terminal deletion, arm exchange within a single chromosome and intra-chromosome exchanges. Chromosome bridges observed at anaphase also suggest the occurrence of dicentric chromosomes and chromosomes with “sticky” ends due to breakage. Such chromosome bridges would result in chromosome fragmentation at cytokinesis. Chromosome fragments without centromeres would eventually be eliminated in subsequent division cycles. Minor damage to chromosome structure caused by pressure treatment might remain as chromosome gaps. Chromosomal changes caused by hydrostatic pressure treatment at the first cleavage are similar to those induced by irradiation (Tonomura, 1975; Yamazaki, 198 1)) aging, or interspecific hybridization (Yamazaki et al., 1989). It is our experience that survival rates of gynogenetic homozygotes or tetraploids induced by suppression of the first cleavage by pressure are very low compared with those of gynogenetic diploids or triploids induced by suppression of the second meiotic division. This appears to be the case in chum salmon, masu salmon and rainbow trout, and is probably also true in other fish (Tabata, 199 1). Chourrout ( 1984) stated that low survival rate resulting
HYDROSTATIC PRESSURE TREATMENT OF SALMON EMBRYOS
59
from suppression of the first cleavage would probably not permit the maintenance of lines until reproductive age. Some of the embryological abnormalities could be explained by inbreeding depression arising from homozygous deleterious alleles. As Thorgaard ( 1983) suggested in his review, however, it is important to separate the effects of treatment from those of inbreeding. Our results show that hydrostatic pressure treatment at the first cleavage seriously affects chromosome constitution and morphology. Therefore, abnormalities found in hatched gynogenetic diploids or tetraploids induced by suppression of the first cleavage may be attributed to the genetic imbalance precipitated by chromosome damage. ACKNOWLEDGEMENTS
We thank T. Kanazawa, Manager of Kamiiso Fishermen’s Cooperative, and the staff of Oshima Branch of Hokkaido Salmon Hatchery, who kindly provided us with chum salmon eggs and milt. Thanks also to the staff of Mori Branch of Hokkaido Fish Hatchery who kindly provided masu salmon gametes. We are grateful to Dr. A. Goto, E. Yamaha, T. Lee, Dr. H.-F. Ma and graduate students in our laboratory, Hokkaido University. REFERENCES Chourrout, D.. 1984. Pressure-induced retention of second polar body and suppression of first cleavage in rainbow trout: production of all-triploids, all tetraploids and heterozygous and homozygous diploid gynogenesis. Aquaculture, 36: 1 1 l- 126. Gold. J.R., 1984. Silver staining and heteromorphism of chromosome nucleolus organizer regions in North American cyprinid fishes. Copeia, 1984 ( 1): 133-l 39. Onozato, H.. 1990. Androgenesis. Monthly J. Science (Kagaku Asashi), 3: 14- 19 (in Japanese). Parson, J.E. and Thorgaard, G.H., 1985. Production of androgenetic diploid rainbow trout. J. Hered.. 76: 177-181. Phillips, R.B., Zajicek, K.D., Ihssen, P.E. and Johnson, O., 1986. Application of silver staining to the identification of triploid fish cells. Aquaculture, 54: 3 13-3 19. Tabata, K., 1991. Application of chromosome manipulation in aquaculture in hirame, Purulichthys olivaceus. Bull. Hyogo Pref. Fish. Exp. Stn., 28: l-134 (in Japanese with English summary 1. Thorgaard, G.H., 1983. Chromosome set manipulation and sex control in fish. In: W.S. Hoar. D.J. Randal and E.M. Donaldson (Editors), Fish Physiology, Vol. IX. Part B. Academic Press, New York, NY, pp. 405-434. Tonomura, A., 1975. Atlas of chromosome abnormalities in human. Kodansha Scientific Press, Tokyo, 129 pp. (in Japanese). Yamazaki, F., 198 1. Chromosome variations in salmonids. Chromosomal aberration by overripening and irradiation. Kaiyo Kagaku, 13 ( 1): 7 l-80 (in Japanese). Yamazaki, F., Onozato, H and Arai, K., 198 1. The chopping method for obtaining permanent chromosome preparations from embryos of teleost fishes. Bull. Jpn. Sot. Sci. Fish., 47: 963. Yamazaki, F., Goodier, J. and Yamano, K., 1989. Chromosome aberration caused by aging and hybridization in charr, masu salmon and related salmonids. Physiol. Ecol. Jpn.. Spec. 1: 529-542.
51
AQUA 30023
Cytogenetic effects of hydrostatic pressure treatment to suppress the first cleavage of salmon embryos Fumio Yamazakia and John Goodierb “Faculty of Fisheries, Hokkaido University,Hakodate, Japan bDepartment OfBiochemistry, Memorial Universityof St. Johns’, Nfld..,Canada (Accepted 10 June 1992)
ABSTRACT Yamazaki, F. and Goodier, J.. 1993. Cytogenetic effects of hydrostatic pressure treatment to suppress the first cleavage of salmon embryos. Aquaculture, 110: 5 l-59. Tetraploids in chum and masu salmon were induced by suppressing the first cleavage by hydrostatic pressure of 680 kg/cm’. Ploidy level was scored by counting nucleoli using silver-stained preparations. True tetraploids were induced in some chum embryos treated at 13.2 h after fertilization; however, aneuploids or polyploid mosaics were also found. These showed abnormal embryogenesis. Chromosomes isolated from the nucleus or precociously moving to the centrosome were observed in the dividing cells. Chromosome bridges between two centrosomes were also observed, as well as chromosome fragments, rings and gaps. Even if damages induced by mitotic suppression with hydrostatic pressure treatment is minor at the first cleavage, it will be amplified through rapid cell cycles.
INTRODUCTION
Triploids or gynogenetic diploids have been successfully induced in various species by suppressing the second meiotic division with temperature shock or hydrostatic pressure treatment (Thorgaard, 1983 ) . These techniques have been applied by fish culturists to produce populations of sterile triploid female or all-female fish. Another useful technique utilizes suppression of the first cleavage by hydrostatic pressure. When irradiated sperm or eggs are used for fertilization, mitotically suppressed gynogenetic or androgenetic diploids are created which are considered to be homozygous at all loci (Parson and Thorgaard, 1985; Onozato, 1990). Inbred diploid or tetraploid fish induced in this manner are useful for genetic research. However, the survival of fertilized eggs treated with hydrostatic pressure is sometimes very low: less than Correspondence to: F. Yamazaki, Japan.
0044-8486/93/$06.00
Faculty of Fisheries,
Hokkaido
0 1993 Elsevier Science Publishers
University,
Hakodate
B.V. All rights reserved.
04 1,
52
F. YAMAZAKI AND J. CiOODIER
10% at hatching when compared with those undergoing suppression of the second meiotic division. Incidence of physical abnormalities also tends to be higher among tetraploid than among triploid embryos (Thorgaard, 1983 ). We supposed that low survival following suppression of the first cleavage would be caused by chromosome damage. This study aimed to explore success rates of tetraploid induction and to clarify chromosome damage affecting the viability of embryos treated with hydrostatic pressure at the first cleavage. MATERIAL
AND METHODS
Gametes used in the experiments were from chum salmon, Oncorhynchus keta, and masu salmon, 0. masou. Those of chum salmon were collected at the hatchery of the Kamiiso Fishermen’s Cooperative, about 6 km from the Faculty of Fisheries, Hokkaido University. The eggs were collected by abdominal incision and sperm by squeezing, and brought to the Faculty. A small number of fertilized eggs (about 200 each in three groups) was held at 8 ‘C and subjected to 680 kg/cm2 pressure for 7 min at 12.7, 13.2, and 13.7 h after fertilization. The same number of eggs without treatment served as control. The masu salmon eggs and sperm were collected at Mori Branch of the Hokkaido Fish Hatchery, and transported 40 km to the Faculty. Fertilized eggs were treated as above at 4.75, 5.75, 6.5,7.0, 7.3, 8 and 9 h after fertilization. About 200 eggs were used in each group. The pressure-treated and control groups in chum and masu salmon were incubated at 8 to 10°C in a recirculating water system. Chromosome preparations were made by the chopping method (Yamazaki et al., 198 1) during the lirst to third week after fertilization. The slides were silverstained to reveal nucleoli by the method of Gold ( 1984). Staining chum and masu salmon chromosome preparations with silver nitrate permitted di-
Fig. 1. Chum salmon embryos at 2 1 days after fertilization. A: normal control; B, C: abnormal embryos from hydrostatic pressure-treated group. Fig. 2. Normal diploid chum salmon cells having two nucleoli stained with silver nitrate. Fig. 3. Tetraploid chum salmon cells having mainly four nucleoli. These cells were stained with silver nitrate after hydrostatic ,pressure treatment at the first cleavage. Fig. 4. Typical tetraploid chum salmon cell having four nucleoli induced by hydrostatic pressure treatment at the first cleavage. Fig. 5. Aneuploid or mosaic chum cells having various numbers of nucleoli. This figure was taken from a hypertetraploid individual, embryo no. 9 in Table 1. Fig. 6. Masu salmon chromosome preparation showing chromosome bridge at anaphase following hydrostatic pressure treatment at 8 h after fertilization. Preparations were made at 72 h. Fig. 7. Lagging chromosomes in the chromosome bridge at early telophase found in masu salmon chromosome preparation following hydrostatic pressure treatment.
HYDROSTATIC PREWJRE
53
TREATMENT OF SALMON EMBRYOS
-_-_
--
1
54
F. YAMAZAKI AND J. GOODIER
rect determination of ploidy level through chromosome and nucleoli counting (Phillips et al., 1986). Embryos of both masu and chum salmon were sampled periodically and fixed with Bouin’s solution for histological examination of gross morphology and chromosome kinetics. RESULTS
Some chum salmon embryos in the 13.2- and 13.7-h groups appeared normal and survived until 30 days after fertilization when the experiment was terminated (Fig. IA). No eggs of masu salmon from the 6.5- to 8-h groups survived more than 10 days after fertilization. Abnormal embryonic development was evident in all groups of hydrostatic pressure treatment. The appearance of abnormal embryos in both species varied greatly, from mere cell masses lacking any normal embryonic differentiation, to small-eyed embryos with stunted or twisted bodies (Fig. 1B,C ) . The results of nucleoli counting in chum salmon experiments are listed in Table 1. The cells of control chum salmon embryos had one or two nucleoli: those with two nucleoli averaged 88% of the cells examined (Fig. 2). The group treated with hydrostatic pressure at 12.7 h contained diploid and hyperdiploid individuals only. Three individuals in the 13.2-h group having over 80% of their cells with four nucleoli were probably true tetraploids (Table 2, Figs. 3,4). However, more individuals were polyploid mosaics or aneuploid, based on the number of nucleoli (Table 1) and chromosomes (Table 2). In fact, the majority of cells of one hypertetraploid embryo (Table 1, 13.2 h, no. 9 ) displayed 5 to 7 nucleoli (Fig. 5 ) . In the chromosome preparations from supposed polyploid embryos, chromosome bridges between two centrosomes, probably caused by abnormally formed dicentric chromosomes, were clearly observed (Fig. 6 ). Chromo-
Fig. 8. Paraffin section of chum salmon cell showing a chromosome precociously moving to pole at metaphase following hydrostatic pressure treatment. Sample was taken at 4 days after the treatment. Fig. 9. Isolated chromosome in the cytoplasm at anaphase of chum salmon cell sampled at 4 days after pressure treatment. Fig. 10. Isolated chromosome in the cytoplasm far from the nucleus in chum salmon cell sampled at 4 days after pressure treatment. Fig. 11. Chromosome spread from chum salmon embryo after hydrostatic pressure treatment at the first cleavage, 13.2 h after fertilization. Chromosome gap is shown by an arrow. Fig. 12. Chromosome gap (arrow) found in the chromosome spread from chum salmon embryo after pressure treatment at the first cleavage ( 13.2 h). Fig. 13. Many chromosome fragments (f ) and fused chromosomes caused by inter-chromosome exchange (fc) are found in chromosome spread from masu salmon embryo following pressure treatment at 8 h after fertilization. Fig. 14. Ring chromosomes in chromosome spread from masu salmon embryo after pressure treatment. Fig. 15. Chromosome spread showing ring chromosomes caused by intra-arm exchange (r) and interarm exchange ( re).
HYDROSTATIC PRESSURE TREATMENT OF SALMON EMBRYOS
55
somes were pulled toward both centrosomes and fragmented, while the fragments remained isolated between the two daughter nuclei at telophase (Fig. 7). Paraffin sections of the pressure-treated embryos also showed abnormal cell divisions. Some tigures of the cell divisions suggested that certain chromosomes move precociously to the polar region at metaphase (Fig. 8). Some fragmented chromosomes were found in cytoplasmic regions far from the spindle ( Fig. 9 ) or nuclear areas (Fig. 10 ) . Morphologically abnormal chromosomes were also evident in the nuclear plates of apparently aneuploid embryos. Chromosome gaps were frequently
17 17
17
1 2 3 4 5 6 7
1 2 3 4 5 6 7 8 9
1
2 3
4
Pressuretreated at 12.7 h
Pressuretreated at 13.2 h
Pressure-
treated at 13.7 h
17
15
15 15 18 15 31 31 15
21 19 21 21 19 21 19
12 samples
Control
Age (day)
Embryo number
Groups
91 91 87 86 85 21 12
9 8 12 14 15 2 1 1 1
1 o-2
22 25
7 6 19
1 2
35
22
12 11 19 29 30 11 22 44 2
76 14
3
4
1
1 1 2 1
88 86-90
11 8-14
1 4 1 6 7 40 33 18 3
2
1
Number of nucleoli per cell
42
70 69
71
86 85 80 62 62 46 42 36 5
9 52
4
8 8 E
1
?1 S $
$ .?
3
8
1
30
7
g
44
6
pressure at 12.7, 13.2 and 13.7
z
2
13
2
4
1 1
21
5
Percent frequency of cells with specific number of nucleoli of chum salmon embryos which were treated with hydrostatic h after fertilization
TABLE 1
1 2
1 3 4 6
Control
Pressuretreated atl2.7h
Pressure- I treated 2 at 13.7 h 3 4
Pressure- 1 treated 7 at 13.2 h
Embryo number
Groups
6
1
1
3 3
1
2 1
1 2 1 112310
I
2
~70
3
number
1
I 63 92
61
1
1 1
2
2
1
1
2
1
13
12
2
3
1
1
1 1
1
1
2
1
1
1
1 1 3 2
4 6
7
18 19
Total number 70 71 72 73 74 75 76 77 78 79 82 83 99 111 117 118 135 141 145 146 147 148 149 153 ofcells counted
Chromosome
Distribution of chromosome number of chum salmon embryos following hydrostatic pressure treatment
TABLE 2
58
F. YAMAZAKI AND J. GOODIER
observed (Figs. 11, 12 ) and chromosome fragments were numerous in nuclear plates (Fig. 13 ) . Long chromosomes caused by fusion of two mosomes or chromosome exchange were also found (Fig. 13 ) . Ring mosomes with or without centromeres were observed (Figs. 14, 15 ); might have been caused by intra- or inter-arm exchange within a single mosome (Fig. 15 ).
some chrochrothese chro-
DISCUSSlON
In the present study, ploidy level of experimental embryos was simply identified by counting the number of nucleoli per cell, as suggested by Phillips et al. ( 1986) for salmonids. We have shown that most diploid cells in chum and masu salmon have two nucleoli. Suppression of the first cleavage with hydrostatic pressure treatment induced tetraploids having four nucleoli and polyploid mosaics having a variable number of nucleoli. Most embryos were aneuploid or polyploid mosaic in chromosome constitution, and were morphologically abnormal and died during embryogenesis. In fact, in some cases, no embryonic differentiation beyond simple cell mass formation was observed after hydrostatic pressure treatment. Cytological investigation revealed chromosomes stranded in the cytoplasmic regions, and both morphologically abnormal chromosomes and chromosome bridges were observed in dividing cells. Even minor or small damages to chromosomes at the first cleavage might sometimes cause serious changes such as terminal deletion, exchange type aberration, inter- or intra-arm exchange or inter-chromosome exchanges through rapid cell cycles. These changes would amplify the damage to the chromosomes. We observed many chromosome fragments, ring chromosomes and also long fused chromosomes suggesting frequent occurrence of the terminal deletion, arm exchange within a single chromosome and intra-chromosome exchanges. Chromosome bridges observed at anaphase also suggest the occurrence of dicentric chromosomes and chromosomes with “sticky” ends due to breakage. Such chromosome bridges would result in chromosome fragmentation at cytokinesis. Chromosome fragments without centromeres would eventually be eliminated in subsequent division cycles. Minor damage to chromosome structure caused by pressure treatment might remain as chromosome gaps. Chromosomal changes caused by hydrostatic pressure treatment at the first cleavage are similar to those induced by irradiation (Tonomura, 1975; Yamazaki, 198 1)) aging, or interspecific hybridization (Yamazaki et al., 1989). It is our experience that survival rates of gynogenetic homozygotes or tetraploids induced by suppression of the first cleavage by pressure are very low compared with those of gynogenetic diploids or triploids induced by suppression of the second meiotic division. This appears to be the case in chum salmon, masu salmon and rainbow trout, and is probably also true in other fish (Tabata, 199 1). Chourrout ( 1984) stated that low survival rate resulting
HYDROSTATIC PRESSURE TREATMENT OF SALMON EMBRYOS
59
from suppression of the first cleavage would probably not permit the maintenance of lines until reproductive age. Some of the embryological abnormalities could be explained by inbreeding depression arising from homozygous deleterious alleles. As Thorgaard ( 1983) suggested in his review, however, it is important to separate the effects of treatment from those of inbreeding. Our results show that hydrostatic pressure treatment at the first cleavage seriously affects chromosome constitution and morphology. Therefore, abnormalities found in hatched gynogenetic diploids or tetraploids induced by suppression of the first cleavage may be attributed to the genetic imbalance precipitated by chromosome damage. ACKNOWLEDGEMENTS
We thank T. Kanazawa, Manager of Kamiiso Fishermen’s Cooperative, and the staff of Oshima Branch of Hokkaido Salmon Hatchery, who kindly provided us with chum salmon eggs and milt. Thanks also to the staff of Mori Branch of Hokkaido Fish Hatchery who kindly provided masu salmon gametes. We are grateful to Dr. A. Goto, E. Yamaha, T. Lee, Dr. H.-F. Ma and graduate students in our laboratory, Hokkaido University. REFERENCES Chourrout, D.. 1984. Pressure-induced retention of second polar body and suppression of first cleavage in rainbow trout: production of all-triploids, all tetraploids and heterozygous and homozygous diploid gynogenesis. Aquaculture, 36: 1 1 l- 126. Gold. J.R., 1984. Silver staining and heteromorphism of chromosome nucleolus organizer regions in North American cyprinid fishes. Copeia, 1984 ( 1): 133-l 39. Onozato, H.. 1990. Androgenesis. Monthly J. Science (Kagaku Asashi), 3: 14- 19 (in Japanese). Parson, J.E. and Thorgaard, G.H., 1985. Production of androgenetic diploid rainbow trout. J. Hered.. 76: 177-181. Phillips, R.B., Zajicek, K.D., Ihssen, P.E. and Johnson, O., 1986. Application of silver staining to the identification of triploid fish cells. Aquaculture, 54: 3 13-3 19. Tabata, K., 1991. Application of chromosome manipulation in aquaculture in hirame, Purulichthys olivaceus. Bull. Hyogo Pref. Fish. Exp. Stn., 28: l-134 (in Japanese with English summary 1. Thorgaard, G.H., 1983. Chromosome set manipulation and sex control in fish. In: W.S. Hoar. D.J. Randal and E.M. Donaldson (Editors), Fish Physiology, Vol. IX. Part B. Academic Press, New York, NY, pp. 405-434. Tonomura, A., 1975. Atlas of chromosome abnormalities in human. Kodansha Scientific Press, Tokyo, 129 pp. (in Japanese). Yamazaki, F., 198 1. Chromosome variations in salmonids. Chromosomal aberration by overripening and irradiation. Kaiyo Kagaku, 13 ( 1): 7 l-80 (in Japanese). Yamazaki, F., Onozato, H and Arai, K., 198 1. The chopping method for obtaining permanent chromosome preparations from embryos of teleost fishes. Bull. Jpn. Sot. Sci. Fish., 47: 963. Yamazaki, F., Goodier, J. and Yamano, K., 1989. Chromosome aberration caused by aging and hybridization in charr, masu salmon and related salmonids. Physiol. Ecol. Jpn.. Spec. 1: 529-542.