induction of heteroploidy in gallus domesticus
TRANSCRIPT
263
Mutation Research, 69 (1980) 263--273 © Elsevier/North-Holland Biomedical Press
INDUCTION OF HETEROPLOIDY IN Gallus domesticus
NANCY WANG * and ROBERT N. SHOFFNER
Department of Genetics and Cell Biology and Animal Science, University of Minnesota, St. Paul, MN 55108 (U.S.A.)
(Received 10 May 1979) (Revision received 25 September 1979) (Accepted 3 October 1979)
Summary
Intraperitoneal colcemid injection at the dose level of 0.37 mg/kg applied 3--4 h before the first oviposition produces a high frequency of triploids among the second oviposition eggs laid the first day after colcemid treatment. The triploids, mostly 3A-ZZW type, appear to be as developmentally compatible as contemporary diploids at both embryonic and hatching level. No other kinds of heteroploids besides triploids were found at hatching time even though a high frequency was observed in 24-h embryos.
Polyploid animals are rare but occur in some species of fish (Cimino, 1972; Dingerkus, 1976; Uyeno'and Smith, 1972) and amphibians (Maxson, 1977). There are a number of barriers to the establishment of a polyploid animal pop- ulation including: (1) the extremely low probability of the sequential events required for the beginning of a polyploid group; (2) incompatibility of poly- ploidy and development; (3) numerical nondisjunction of chromosomes pro- ducing genetically imbalanced gametes; (4) random combination of the sex chromosomes in the heterogametic sex resulting in the production of intersex progeny, and (5) differential adaptability of polyploids compared with dip- loids.
In the chicken (Gallus domesticus) spontaneous triploids have been reported in both embryonic (Bloom, 1970, 1972; Miller et al., 1971; Mong et al., 1974) and post-hatch stages (Abdel-Hameed and Shoffner, 1971; Abdel-Hameed, 1972. Ohno et al., 1963; Shoffner, 1975; Shoffner et al., 1972) indicating that polyploidy is compatible with development in this organism. Spontaneous occurrence of triploidy was estimated to be in the range of 0.03--0.05%
* Present address: D e p a r t m e n t o f Laboratory Medic ine and Patho logy , Box 1 9 8 , Univers i ty o f Min- nesota, Minneapolis, MN 55455 (U.S.A.).
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(Shoffner et al., 1967) in adult chickens. 3 triploid types, 3A-ZZZ, 3A-ZZW and 3A-ZWW have been found in the early embryonic stages. However, no 3A- ZWW triploids have been found in post-hatched individuals. The 3A-ZZW trip- loid has an intersex phenotype with a range of gonadal development from almost complete maleness, two testes and no oviduct, to complete femaleness with a functional left ovary and oviduct (Abdel-Hameed and Shoffner, 1971). The 3A-ZZZ triploid males axe not different phenotypically from the 2A-ZZ diploid males; they do, however, produce malformed infertile spermatozoa (Shoffner et al., 1972).
Triploid chickens have been used in studies of sex determination (Abdel- Hameed and Shoffner, 1971), chromosomal pairing (Comings and Okada, 1971) and gene dosage compensation (Abdel-Hameed, 1972). Therefore, an effective method for induction of polyploidy would result in material highly useful for genetic and biological studies.
Triploidy may result from polyspermy, polygyny (two or more female pro- nuclei involved in fertilization) or from the union of a diploid gamete with a normal haploid one. The induction of a diploid gamete appears to be more feasible than either polyspermy or polygyny. A diploid gamete may be produced through chromosomal nondisjuction at metaphase 1 or 2 of meiosis or persis- tent tetraploidy arising in gonial cells. Chromosomal nondisjunction can be induced by alkaloid agents including colchicine and colcemid (Ciba) through the inhibition of meiotic spindle fiber formation (Borisy and Taylor, 1967).
This report describes effective methodology for the induction of diploid ova with colcemid. The diploid ova produced axe capable of fertilization by haploid spermatozoa and the resulting triploids axe developmentally viable and appear to have no great disadvantage compared with diploid contemporaries.
Materials and methods
The circadian rhythm of oviposition, ovaluation and reduction division in the laying hen is illustrated in Fig. 1. According to Olsen and Fraps (1950), the first meiotic spindle forms about 3 h prior to ovulation. Ovulation in the chicken occurs approx. 20 min after oviposition of the previous egg. Therefore, by timing the laying cycle of the hen, it is possible to approximate the time of the first and the second spindle formation of oogenesis. If the spindle fiber inhibitor is introduced at the appropriate time, either the first or the second meiotic division will be suppressed resulting in the production of a diploid gamete. If suppressed in the first meiotic division, the gamete will be 2A-ZW; if in the second meiotic division, a 2A-ZZ or 2A-WW gamete will result. When these diploid ova are fertilized with the normal haploid spermatozoa, they give rise to 3A-ZZW, 3A-ZZZ or 3A-ZWW triploids, respectively, as illustrated in Fig. 2. The type of triploids produced, therefore, defines the stage of spindle fiber suppression.
White Leghorn females with an approx. 24-h laying cycle were used for the colcemid injection. To insure high fertility the hens were artificially insemi- nated twice a week with 0.1 ml pooled semen from Rhode Island Red males. The hens were palpated each morning for the presence of an egg in the uterus and then injected with phosphate buffered colcemid (0.05%) into the perito-
265
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neal cavity at a dose of 0.37 mg/kg body weight (Shoffner et al., 1967). Eggs were identified as to hen, date, and time of lay. In the initial experiment, eggs were incubated for 24 h in order to maximize identification of induced chro- mosomal changes. Eggs were removed from the incubator and colcemid (0.01 ml of 0.05% solution) was injected into the air cell above the embryo; this t reatment increased the frequency of metaphases in the embryo. Incubation was continued for an additional 30--45 min before excision of the embryos.
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The excised embryos were immediately transferred to a 0.45% (w/v) solution of sodium citrate for a 10--15 min hypotonic treatment. The material was then fixed in a 50% (v/v) acetic acid. Materials fixed in 50% acetic acid can be stored in a --20°C freezer until squash preparations are made for cytological observa- tions. All preparations were observed in the phase-contrast microscope for pre- liminary screening for heteroploidy. Permanent slides were made of all suspect, abnormal embryos. After CO2 freezing to allow removal of the coverslip, slides were immediately dehydrated in two changes of absolute alcohol and air dried. The air-dried slides were stained with Carbol fuchsin for further analysis of chromosomal composition.
In the second experiment, eggs were incubated to hatching time (21 days) and candled on Day 7 and 19, resp., to determine fertility, frequency of dl (embryos died before Day 7) and d2 (embryos died between Day 7 and Day 19). Chicks were identified at hatching, and feather pulp obtained for chro- mosomal analysis according to the method of Shoffner et al. (1967), except no colcemid was applied to accumulate metaphases. 5--7 primary wing feathers were plucked randomly and the semi-solid pulp from the feather's proximal end was removed and immediately transferred to a 0.45% (w/v) solution of sodium citrate for 15--20 min of hypotonic treatment. Material was then fixed in 50% acetic acid and stored a t - -20°C until cytologically examined for hetero- ploidy. Slides were checked under the phase-contrast microscope wi thout stain- ing.
All eggs were classified at first, second and post-second ovipositions accord- ing to the order of lay with respect to the colcemid injection. As shown in Fig. 1, only the second ovipositions after colcemid injection were expected to have their meiosis affected by colcemid. The first or post-second ovipositions, which entered meiosis either before or after the colcemid-effective period, were grouped together and used as controls. To determine chromosome composi- t ion, 20--25 metaphase spreads were analysed for each embryonic or feather pulp sample.
Results and discussion
The results obtained from the eggs incubated for 24 h are summarized in Ta- ble 1. The fertility of the second oviposition group was only 71% of the control suggesting a cytopathological effect which suppressed fertilization or zygotic development. 21 of the 104 embryos or 20.2% of the fertile eggs were found to be chromosomally abnormal. These included 3 haploids (A-Z) (Fig. 3a), 6 hap- loid/diploid mosaics (A-Z, 2A-ZZ), 5 haploid/diploid/triploid mosaics (A-Z/2A- ZZ/3A-ZZZ), 6 triploids (five 3A-ZZW and one 3A-ZWW) and an aneuploid (A-Z + 2') (Fig. 35).
The second ovipositions were then subdivided into two groups according to the date of lay. Day I Egg -- laid on the first day after the colcemid injection (or ovulated on the day of calcemid injection), and After Day I Egg -- laid later than the first day after colcemid injection. The decrease in fertility and increase in chromosomal abnormality of the second ovipositions were almost com- pletely contributed by the Day I eggs, as shown in Table 1. The fertility of Day I Egg is only 50.1% of the control while the fertility of After Day I Egg is
268
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270
close to the control level. Frequency of eggs with chromosomal abnormalities is 42.6% in Day I with only 1.8% (one haploid/diploid mosaic) in After Day I. 6 of the 47 fertile eggs or 12.8% embryos were found to be triploid in Day I. In contrast, no triploids were observed in either control or in After Day I. The nature and causation of the euploid mosaics are uncertain perhaps the haploid portion was contributed by sperm nuclei (Fechheimer et al., 1968; Snyder et al., 1975).
The data obtained at the early embryonic level clearly indicate that only those second ovipositions laid on the first day after colcemid treatment were affected by colcemid.
The induction of triploidy was then studied at the hatching level to produce some live triploid chicks and to compare the developmental compatibility of diploids versus triploids. The results obtained at the hatching level are shown in Table 2. The fertility, percentage of dl (before Day 7) and d2 (between Day 7 and 19) embryonic deaths and hatchability of After Day I eggs were all similar to that of control. Triploidy was not found in either control or After Day I eggs. In contrast, the fertility and hatchability of Day I eggs has been decreased to only 49.2 and 27.1% resp., from that of the control. The percent of d2 embryonic deaths in Day I was only slightly higher than the control or After Day I. However, the percent of dl early embryonic death was tremendously increased in Day I as compared to either the control or After Day I. 5 of the 19 chicks (26.3%) of Day I were triploid. 4 were 3A-ZZW, one was 3A-ZZZ and all were hatched 1--2 days later than diploids sibs. The triploids were reared to adult stage. Blood cell DNA was determined by flow microfluorometry analysis
D
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Fig. 4. The D N A dis tr ibut ion o f diplo id and trfploid b l o o d cel ls d e t e r m i n e d b y the f l o w m i c r o f l u o r o - m e t r y ; the peak c h a n n e l pos i t i on o f the f o r m e r is 40 whi le the la t ter is 60.
271
according to the method of Krishan et al. (1975), and compared with that of diploids. A unimodal DNA distribution was obtained with the peak channel position at 60 in contrast to 40 for diploids (Fig. 4). This data coincides well with the cytological observations.
Phenotypically, baby triploid chicks are not different from the diploids (Fig. 4). The ratio of triploids to diploids at the hatching level was 5/14 (see Table 2) or 35.6%, while at the 24-h embryonic level was 6/27 (see Table 1) or 22.2%. The data suggest that the triploids, at least the 3A-ZZW type, appear to have no developmental disadvantage compared to diploids.
The percentage of non-triploid heteroploids observed in 24-h embryos was 14/47 (see Table 1) or 30% while none were found in the hatched chicks. It suggests that these non-triploid heteroploids must have died before hatch. As indicated in Table 2, the increase in embryonic death caused by colcemid was mainly at the dl stage (before Day 7) rather than at the d2 stage (between Day 7 and 19). Therefore, it can be concluded that developmental incompatibili ty of these embryos probably occurs in the first 7 days of incubation.
The time of colcemid injection which produced triploids is summarized with
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Fig. 5. Ca) Triploid Cleft) and diploid Cright baby chicks. (b) Chzornosomal cons t i tu t ion of the tr iploid c h i c k (3A-ZZW).
272
T A B L E 3
R E L A T I O N S H I P B E T W E E N TIME O F C O L C E M I D I N J E C T I O N A N D T Y P E O F I N D U C E D T R I P L O I D G A M E T E S
K i n d o f P o s t u l a t e d E x p e c t e d t i m e o f T ime o f c o l c e m i d N u m b e r o f t r i p l o i d y m e c h a n i s m o f m e i o t i c sp ind le i n j e c t i o n (h b e f o r e ind iv idua l s i n d u c e d i n d u c t i o n f o r m u l a t i o n (h 1s t l ay ing )
b e f o r e 1s t l a y i n g )
3 A - Z Z W S u p p r e s s i o n o f 1s t 3 4 .0 7 m e i o t i c sp ind le 3 .5 1 f o r m a t i o n 3 .0 1
3 A - Z Z Z S u p p r e s s i o n o f 2 n d 2 3 1 m e i o t i c sp ind le f o r m a t i o n
3 A - Z W W S u p p r e s s i o n o f 2 n d 2 3 1 m e i o t i c sp ind le f o r m a t i o n
respect to the time of the first oviposition (Table 3). Injections that produced triploids were all around 3--4 h before the first oviposition, which coincides well with the expected time of meiotic spindle fiber formation. Out of the 11 triploids induced at both embryonic and hatching level, 9 were of the 3A-ZZW type, one 3A-ZZZ and one 3A-ZWW type. The data suggest that colcemid is more efficient for induction of diploid ova through the suppression of the first meiotic spindle fiber formation than the second meiotic division. A possible explanation is that those colcemid injections which suppressed the second meiotic division also may have suppressed fertilization and/or subsequent embryonic divisions. As in mice (Zimmerman and Zimmerman, 1967) and in sea urchin eggs (McGaughey and Chang, 1965) colcemid has been reported to suppress fertilization and subsequent embryonic divisions. The significant decrease in fertility of the Day I eggs at both the embryonic (Table 1) and hatching (Table 2) levels supports this explanation.
Acknowledgements
The authors are deeply indebted to Drs. Charles R. Burnham, Ronald L. Phillips, Tingchung Wang and Alvin F. Weber for their valuable criticism on the manuscript and to Ruth King, Jack Otis, Paul Vogt and James O'Rourke for their technical assistance. We greatly appreciate the generosity of Dr. John R. Sheppard for providing the flow microfluorometer apparatus.
Scientific Journal Series No. 10 022 of the Minnesota Agricultural Experi- ment Station.
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