The phenotypic expression of a t6/t6/t6 genotype
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/ . Embryol. exp. Morph. Vol. 69, pp. 107-113, 1982 JQ7
Printed in Great Britain © Company of Biologists Limited 1982
The phenotypic expression of a t6/t6/t6 genotype
By JAMES McGRATH 1 AND NINA HILLMAN
Department of Biology, Temple University, Philadelphia, Pa. 19122, U.S.A.
SUMMARY
In vivo, embryos which are homozygous for the te mutation die during egg cylinder de-
velopment (gestation days 5-5-6-5). In vitro, these mutant embryos can be distinguished from
their wild-type littermates by their developmental arrest and by the failure of their tropho-
blast cells to transform to giant cells. We have investigated the nature of this lethality by
constructing triploid embryos with varying combinations of the t6 mutant chromosome. The
phenotypes of outgrowths from these triploid embryos were examined and compared with
the phenotypes of outgrowths from + / + , +// 6 , and t6/t6 embryos. The data show 1) that
+ // 6 // 6 embryos are phenotypically wild-type, while t6ft6/t6 embryos are phenotyplcally
mutant and 2) that t6/t6/t6 and t6/te embryos are developmental^ arrested at the same stage
of outgrowth.
INTRODUCTION
6
Homozygosity for the / mutation, one of a series of recessive mutations of
the complex T region located on chromosome 17, is lethal to mouse embryos
between gestation days 5-5 and 6-5. Homozygous mutant embryos can be
distinguished as early as the late blastocyst substages (gestation days 4-5-5-25)
by morphological abnormalities at the ultrastructural level (Nadijcka & Hill-
man, 1975). In vitro outgrowths from t*/t6 blastocyst embryos, developing from
either in vivo (Wudl, Sherman & Hillman, 1977; Wudl & Sherman, 1978) or
in vitro (McGrath & Hillman, 1980) fertilized ova, are morphologically dis-
tinguishable from the outgrowths of their wild-type littermates at the light
microscope level. The mutant outgrowths are developmentally arrested oil the
third day of outgrowth (o.D. 3; Nadijcka, Morris & Hillman, 1981) before
polyploidization of the trophoblast nuclear DNA is complete (Wudl & Sherman,
1978). The trophoblast cells of the phenotypically wild-type embryos continue
to polyploidize and complete their transformation to giant cells by o.t>. 4.
Because of the developmental airest of mutant outgrowths, the nuclei of their
trophoblast cells remain significantly smaller in volume and contain less DNA
than the giant cell nuclei of their wild-type counterparts (Wudl & Sherman,
1978). This developmental arrest of the mutant outgrowths occurs prior to their
death and degeneration (Wudl et al. 1977; Wudl & Sherman, 1978; Nadijcka
eta!. 1981).
1
Author's address: The Wistar Institute of Anatomy and Biology, 36th and Spruce Sts.,
Philadelphia, Pa. 19104, U.S.A.108 J. McGRATH A N D N. HILLMAN
In the current studies we have analysed /6-induced embryonic lethality by
constructing +/t*/t6 and t6/t6/t6 embryos. The results show that +/t6/t*
embryos are phenotypically wild-type while t*/t6/te embryos are phenotypically
mutant. Outgrowths from the latter embryos display the same temporal syn-
drome of developmental arrest and death as te/t6 outgrowths. These findings
support the hypothesis that the lethality of embryos homozygous for specific
recessive lethal t mutations is stage-specific (Nadijcka et al. 1981; McGrath &
Hillman, 1981) and suggest that the lethal effect is caused by the mutant allele
coding for a product which is less active than the wild-type product.
MATERIALS AND METHODS
Ova were recovered from +/t6 and T/+ hybrid females which had been
obtained by crossing C57BL/6J females with Tjf males. The F x offspring can
be classified according to genotype using a phenotypic criterion: + /tG animals
have tails of normal lengths; T/+ animals are short-tailed. The females were
injected intraperitoneally with pregnant mare serum gonadotrophin (Gestyl,
Organon: 5 i.u.) followed 48 h later by an injection of human chorionic gonado-
trophin (Pregnyl, Organon: 5 i.u.) (Edwards & Gates, 1959). At 12-13 h after
the HCG injection the females were killed and their oviducts excised and placed
in drops of modified Tyrode's medium (Fraser & Drury, 1975) under silicone
oil. The cumulus masses containing the ova were removed from the ampullary
regions of the oviducts and twice transferred to fresh drops of medium (0-2 ml)
under silicone oil. Ova from + ft% females were used to produce experimental
diploid and triploid embryos and ova from Tj + females, to produce control
triploid embryos.
Spermatozoa were obtained from the caudae epididymides and vasa deferentia
excised from F x + ft* males. Gametes from the same male were used for the
in vitro fertilization of both the experimental and control ova. To achieve
optimal levels of fertilization, the spermatozoa were first incubated in a 1 ml
drop of modified Tyrode's medium under silicone oil for 2 h (Niemierko &
Komar, 1976). The spermatozoa were then diluted (1:3) with modified Tyrode's
medium and 10 fi\ aliquots of the diluted suspension was added to 0-2 ml drops
of medium containing the ova. The insemination dishes were placed into an
anaerobic jar and gassed for 20 min with 5 % O2, 5 % CO2 and 90 % N 2 at
37 °C. The jar was then sealed and the gametes allowed to co-incubate for 2 h.
After co-incubation, the ova were removed and washed through 4 drops of
medium (Whitten, 1971). Half of the ova were allowed to continue incubation
in Whitten's medium for 3-5 h in the anaerobic chamber to obtain diploid
embryos. During this time the second polar body was formed. The remaining
half of the ova were placed into modified Whitten's medium (Abramczuk,
Solter & Koprowski, 1977) supplemented with 10/tg of cytochalasin B/ml of
medium and incubated in the anaerobic chamber for 5 h (to obtain triploidt6 trip hid embryos 109
embryos). The supplemented medium was prepared by diluting a stock solution
of 1 ml dimethyl sulphoxide (Sigma) containing 1000/*g cytochalasin B (CB)
with modified Whitten's medium (1:100, v/v). After the 3-5 h incubation in
Whitten's medium, all of the putative diploid embryos were transferred to
Whitten's medium containing CB for a 5 h period to determine the effect of
CB-treatment on subsequent development.
Following the 5 h incubation in CB-supplemented medium, randomly selected
experimental and control zygotes were removed from culture and prepared for
light microscopic analysis (Toyoda & Chang, 1974). These zygotes were scored
for their number of pronuclei, the presence of a spermatozoan tail(s) within
the ooplasm, and the formation of a second polar body. The remainder of the
fertilized ova were allowed to develop in vitro until they reached the blastocyst
stage. At this stage, randomly selected embryos were collected, placed on slides,
and prepared for chromosome counts according to the technique of Tarkow$ki
(1966). The remainder of the blastocyst embryos were placed into outgrowth
medium (Modified Eagle's medium, Spindle & Pedersen, 1973) and cultured
for an additional 6 days as previously described (McGrath & Hillman, 1980).
Since blastocysts developing from in vitro fertilized and CB-treated ova exhibit
a reduced ability to hatch from their zonae pellucidae (McGrath & Hillman,
unpublished observations) the zonae were mechanically removed with a small
bore pipette. Embryo outgrowths were observed on O.D. 2 thiough 6 with an
inverted microscope (Zeiss) equipped with phase contrast optics.
In order to quantitate the te mutant embryo phenotype, outgrowths were
photographed on O.D. 5. From these photographs the trophoblastic cell nuclear
diameters were measured according to the method of Wudl & Sherman (1978).
The nuclear diameter values of all of the nuclei measured in a single embryo
were averaged and the mean nuclear diameter for all of the blastocyst out-
growths in a single phenotypic class (i.e. mutant v. non-mutant) determined.
The number of mutant and wild-type blastocyst outgrowths were compared
using a x2 contingency test. Nuclear diameter values were compared using the
Student Mest.
RESULTS AND DISCUSSION
To determine the effectiveness of the cytochalasin B treatment in producing
triploid embryos, experimental and control zygotes were examined for triploidy
immediately after CB treatment and compared with diploid zygotes. The data
from these studies (Table 1) show that of the 109 fertilized ova which were not
CB-treated during second polar body formation, 91 % contained two pronuclei
and 9 %, three pronuclei. Of the 449 fertilized control and experimental zygotes
which were CB-treated during second polar body abstriction, 91 % contained
three pronuclei. The remainder contained either two or four pronuclei. These
results support the previous finding that CB-treatment can effectively suppress
the abstriction of the second polar body and produce triploid embryos if the110 J. McGRATH AND N. HILLMAN
Table 1. Light microscopic analysis of 1-cell-staged embryos
Unfertilized ova
Con-
densed Fertilized ova
• p i
MV» o f Ar>ti
Clll UII1U
genotype ova somes vated* 2n 3n 4n
Diploid + // 6 175 53 13 99 (91 %) 10(9%) 0
series t
Triploid + // 6 414 115 31 7 246 15
series % r/+ 246 52 13 10 162 9
Total 660 167 44 17(4%) 408 (91 %) 24(5%)
* Ova were scored as activated when they contained either one or two pronuclei and no
spermatozoan tail.
t Embryos were placed into CB medium subsequent to extrusion and stabilization of the
second polar body.
t Embryos were placed into CB medium during second polar body formation.
treatment occurs immediately following fertilization (Niemierko & Komar,
1976; Fraser, 1977).
The effect of the triploid genotype on subsequent development was assayed
by comparing the percentages of 2-cell-staged diploid and triploid embryos
which developed to the blastocyst stage. The frequency of cavitation did not differ
significantly among the three groups (227/290 or 78 % of the diploids, 305/378
or 81 % of the triploid control embryos, and 525/664 or 79% of the experi-
mental triploid embryos), indicating that triploidy is not deleterious during
preimplantation development. Chromosome counts from 53 putative triploid
blastocyst-staged embryos showed that 43 embryos (81 %) possessed the triploid
genome (60 ± 2 chromosomes). (The remaining ten embryos were diploid (five),
tetraploid (three) and diploid/tetraploid mosaics (two).) These results support
earlier conclusions that the triploid chromosome complement does not ad-
versely affect the preimplantation development of the mouse embryo (Beatty &
Fischberg, 1951; Fischberg & Beatty, 1952).
The effect of the triploid chromosome complement on attachment and out-
growth in vitro was determined by comparing the percentage of triploid blasto-
cyst embryos with the percentage of their diploid counterparts which attached
and began to outgrow following transfer to outgrowth medium. The results
show that 93 % (207/223) of the diploid blastocysts and 84 % of the control
and experimental triploid blastocysts (189/223 and 404/482 respectively)
attached and began to outgrow. Overall, the triploid blastocyst embryos have
a decreased ability to form outgrowths in vitro when compared with diploid
embryos. Although the exact cause of this reduction is not known, it could
result from the fact that triploid blastocysts contain fewer cells than theirt6 triploid embryos 111
Table 2. Phenotypic expression of diploid and triploid te-mutant blastocyst
outgrowths
Triploid
Experimental Control Experimental
Cross + A6$x//6c? r/+$x +/t6a + // 6 ?X+// 6 c?
No. of non- 168 189 342
mutant outgrowths
No. of mutant 39 0 62
outgrowths
Total 207 189 404
diploid counterparts (Beatty & Fischberg, 1951; McGrath & Hillman, 1981).
However, the fact that control and experimental triploid blastocysts exhibited
an identical ability to begin outgrowth in vitro argues against the possibility
that the reduction is caused by the lethal expression of a specific genotype.
The blastocyst outgrowths were scored for phenotype on O.D. 4. Among the
207 experimental diploid outgrowths, 168 (81 %) exhibited the wild-type pheno-
type and 39 (19%), the mutant phenotype (Table 2). The in vitro transmission
frequency of the /6 mutation in these experiments was, therefore, 0-38. This low
in vitro transmission frequency of the t6 mutation agrees with our earlier report
which showed that this mutation is transmitted in vitro with a frequency
significantly less than the in vivo transmission frequency in normal matings but
not significantly different from the in vivo transmission frequency in delayed
matings (McGrath & Hillman, 1980).
Since spermatozoa from the same males were used to produce both diploid
and triploid embryos, the expected proportion of triploid genotypes was esti-
mated using the 0-38 transmission frequency of the t6 mutation. With this
frequency, the expected distribution of genotypes among the control triploid
embryos was T/T/ + , 0-31; T/T/t6, 0-19; + / + / + , 0-31; + / + /t\ 0-19. The
expected incidence of triploid genotypes among the experimental embryos was
+ / + / + , 0-31; +/ + /t«, 0-19; +/t*/t6, 0-31; and t«/t*/t\ 0-19. Data in
Table 2 show that all of the 189 control triploid blastocyst outgrowths exhibited
the wild-type phenotype. Among the 404 experimental triploid embryos, 342
were scored as wild type (85%) and 62 (15%) as mutant. Since the + / + / +
and + / + /t6 control triploid embryos are phenotypically wild type, the pheno-
typically mutant experimental embryos were scored as either t6/tG/te or f6//6/ + .
The percentage of morphologically abnormal experimental embryos is signific-
antly less than the expected percentage of t6/t*/+ embryos (15% v. 3 1 % ;
P < 0*05). Conversely, the observed percentage is not significantly different
from that expected for tG/t6/t* embryos (19%). For these reasons, and because
the developmentally arrested embryos display the same phenotype and ap-112 J. McGRATH AND N. HILLMAN
Table 3. Nuclear diameter of diploid and triploid
blastocyst outgrowths
Triploid
Diploid
Experimental Control Experimental
Cross + A6? x + ft« 005), or from the nuclear diameters of the
phenotypically wild-type outgrowths of the diploid experimental series. Since
the phenotypically wild-type population of experimental triploid embryos con-
tains the f6/'6/ + embryos, it can be concluded that the presence of two t6
mutant chromosomes in f6/'6/ + embryos is without phenotypic effect. The
averaged diameters of the experimental embryos scored as t6/t*/tG were not
significantly different from those of the t6/t6 embryos. Since the nuclear dia-
meters of the t*/t*/t* and tG/t* trophoblast cells are the same, and since the
diameter of a trophoblast nucleus can be directly correlated with its DNA
content (Barlow & Sherman, 1972), it can be concluded that a te/t*/t6 genotype
does not extend the in vitro development of the embryos beyond that attained by
a t6/te embryo. This observation underscores the stage-specific nature of certain
lethal t mutations (McGrath & Hillman, 1981; Nadijcka et al. 1981). It can bet6 triploid embryos 113
inferred from the present results that the lethal factor, L (Lyon & Mason, 1977),
of the t* mutation does not actively induce cell lethality (e.g. through the over-
production of its gene product). Rather, embryonic death would seem to result
from a lack of mutant allele product or from a product with activity less than
that of wild type.
This work was supported by U.S. Public Health Service Grants numbers HD00827 &nd
HD09753.
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(Received 18 June 1981, revised 23 October 1981)You can also read
