J. Embryol exp. Morph. Vol. 63, pp. 267-283, 1981
Printed in Great Britain © Company of Biologists Limited 1981
267
The effect of delay on the expression
of the t6/t6 genotype
By MARY NADIJCKA, 1 MARIE MORRIS 1 AND
NINA HILLMAN 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
In vivo, f6//6 embryos are developmental^ arrested between gestation days 5-5 (short-eggcylinder stage) and 6-75 (long-egg-cylinder stage). In the present series of studies we used both
in vivo and in vitro blastocyst delay followed by in vitro outgrowth to determine whether the
/8/>6 lethality is time- or stage-specific. The results show that the t6/tG genome is expressed
differently in vivo and in vitro and that the in vitro expression of the homozygous /6 genome
differs with different methods of effecting developmental delay. Although delay increases the
life span of t6/t° embryos it does not alter the stage of lethality. One method used to effect
delay (ovariectomy) causes the tG/t6 embryos to remain as blastocysts for a significantly longer
period of time than their wild-type littermates when placed into outgrowth medium. This
distinction provides a unique method for obtaining a sample composed entirely of t6/t6
embryos at a stage prior to the lethal period.
INTRODUCTION
Chromosome 17 in the mouse contains a series of recessive t mutations, many
of which are homozygous lethal. These lethal mutations (tL) can be placed into
six complementation groups based on their ability to partially complement each
other. Each complementation group is named in accordance with a designated
prototype / mutation which has a specific syndrome of phenotypic expressions.
The members of each complementation group, in a homozygous condition,
(tL/tL), exhibit a similar syndrome of abnormal development and the same
embryological period of lethality, and in a heterozygous condition, {T[tL, + /tL),
exhibit a similar frequency of transmission of /^-bearing spermatozoa (for review see Bennett, 1975 and Sherman & Wudl, 1977).
In utero, homozygosity for the t6 mutation, a member of the t° complementation group, is lethal to the embryo during egg-cylinder formation, an event
which occurs between gestation days 5-5 and 6-75. Most t6/t6 embryos die
during the short-egg-cylinder stage; however, 10% develop to the elongatedegg-cylinder stage. By day 7, all of the mutant embryos exhibit varying degrees
1
Authors' Address: Department of Biology, Temple University, Philadelphia, Pennsylvania 19122, U.S.A.
268
M. NADIJCKA, M. MORRIS AND N. HILLMAN
of pyknosis and are being resorbed. These embryos can be distinguished at the
ultrastructural level from their phenotypically wild-type littermates prior to
their phenolethal period. For example, as early as the late blastocyst substages,
the homozygous mutant embryos begin to accumulate cytoplasmic lipid droplets. During egg-cylinder formation, the /6 homozygotes can also be identified by
the presence of crystalline inclusions in their mitochondria and by disorganized
entodermal cells. Based on criteria of gross morphology, however, the homozygous mutants cannot be distinguished prior to death (Nadijcka & Hillman,
1975).
Recently, Wudl, Sherman & Hillman (1977) and Sherman & Wudl (1977)
compared the in vitro development of t^/t6 blastocyst embryos and their wildtype counterparts. These investigators noted that the outgrowths of mutant
blastocyst embryos are distinguishable by two phenotypic characteristics: first,
the trophoblast cells of mutant outgrowths do not transform to giant cells and
second, the inner cell mass cells of the mutant outgrowths 'die' and/or 'disappear' soon after the mutant blastocysts attach to the substratum and outgrow.
Again, the te/t& embryos are not distinguishable until they are developmentally
arrested.
Although there have been numerous studies which delimit the in vivo and
in vitro phenolethal period for several tL homozygous mutant embryos, there
have been no definitive studies to determine whether the tL/tL genotype is lethal
at a specific stage of development or at a specific time postfertilization. One way
to distinguish between these possibilities is to increase the length of time an
embryo remains in a specific developmental stage prior to reaching the stage
at which it normally dies. Since uterine t6/t6 embryos display their first phenotypic aberration at the late blastocyst substages (Nadijcka & Hillman, 1975),
we increased the length of this developmental stage to determine if this extension
alters the stage of homozygous lethality. There are various in vivo and in vitro
techniques which can be used to increase the length of time a mouse embryo
remains as a blastocyst. In the current series of experiments we have used two
of these techniques; delayed implantation effected by ovariectomy (Dickson,
1969) and maintenance of blastocyst embryos in outgrowth medium lacking
serum (Mintz, 1964; Cole & Paul, 1965). The use of these techniques has
enabled us to distinguish between time-specific and stage-specific lethality. The
results show first, that the tG/tG genome is expressed differently in vivo and in
vitro; second, that the in vitro syndrome of lethal expression differs with different methods of effecting blastocyst delay (diapause); and third, that while the
lethal expression of the t6/t6 genome can be delayed temporally, the stage of
lethality can not be altered. One method used to effect diapause (ovariectomy)
causes f6//6 embryos to remain as blastocysts for a significantly longer period
of time than their wild-type littermates following transfer to outgrowth medium.
This latter observation provides us with a unique method for distinguishing
Z6//6 embryos from their phenotypically wild-type littermates prior to develop-
The effect of delay on tG/tG embryos
269
mental arrest and death. A preliminary report on a portion of this work was
previously published (Dizio & Hillman, 1978).
MATERIALS AND METHODS
To obtain the experimental and control embryos, males from an inbred Tft6
stock were mated to BALB/c ( + / + ) females. The offspring ( + /t6 and T/ + )
from this cross are phenotypically distinguishable from each other. The + /tG
offspring have tails of normal length while the TJ + offspring have short tails.
The + / / 6 offspring were mated inter se to obtain the experimental litters. These
litters contain +/ + , +/t* (both phenotypically wild-type) and t^/t6 mutant
embryos. The T/+ female offspring were mated to +/f 6 males to obtain the
control litters. These litters contain only viable genotypes. The 22 + /? 6 males
used in these studies were tested for their transmission frequencies of the tQbearing spermatozoa in normal matings prior to being used for the current
series of studies (McGrath & Hillman, 1980). The averaged transmission
frequency of the mutation was found to be 0-80. Thus, the expected incidence of
/ G // 6 embryos from the experimental crosses was 40 %.
One hundred control, and 110 experimental females, were used for these
studies. The females were time-ovulated by intraperitoneal injections of 2-5 i.u.
pregnant mare serum gonadotrophin (Gestyl, Organon) followed 45-48 h later
by 2-5 i.u. human chorionic gonadotrophin (Pregnyl, Organon) (Edwards &
Gates, 1959). Immediately after the second injection, each female was placed
with a single male overnight and checked the following morning for a copulation
plug (Gestation Day 0). One half of the experimental and of the control pregnant females were sacrificed by cervical dislocation 24 h later and 2-cell embryos
flushed from their oviducts. These embryos were allowed to develop to the late
blastocyst substages in embryo culture medium (Goldstein, Spindle & Pedersen,
1975). These zonaless blastocysts, both experimental and control, were then
transferred either to modified Eagle's medium supplemented with 10% heat
inactivated (56 °C, 30 min) fetal calf serum (a medium which allows blastocysts
to attach and outgrow; Spindle & Pedersen, 1973), or to unsupplemented modified Eagle's medium. Embryos kept in the unsupplemented medium for five days
remained as free-floating blastocysts. After five days, these blastocysts were transferred to medium containing fetal calf serum to promote embryo attachment
and outgrowth. All of the hatched blastocyst embryos were cultured in 15 x 30
mm Falcon tissue culture dishes at 37 °C in an atmosphere of 5 % CO 2 ,95 % air.
The embryos were observed with a Zeiss-inverted phase-contrast microscope
and the extent of trophoblastic outgrowth, giant-cell formation, and inner cell
mass (ICM) development of each embryo scored on each subsequent day following blastocyst transfer to the supplemented modified Eagle's medium (outgrowth
medium).
The remainder of the experimental and control pregnant females were
270
M. NADIJCKA, M. MORRIS AND N. HILLMAN
anaesthesized on gestation day 2 (early morula stage) with an intraperitoneal
injection of 2 mg Nembutal and bilaterally ovariectomized to delay blastocyst
implantation. Following ovariectomy, the females were treated in one of two
ways. One group, (both experimental and control), was administered 1 mg
6-a-methyl-17-acetoxyprogesterone (Sigma) subcutaneously (Dickson, 1969).
The remaining females received no hormone supplement. Seven days after being
ovariectomized the females were sacrificed, their uteri removed, flushed, and
zonaless, elongated blastocysts collected (McLaren, 1968,1971). These embryos
had been free-floating, late substaged blastocysts for 5 days. This length of time
in diapause was chosen since the dormant blastocyst embryos should be synchronized in the time required for attachment and outgrowth after transfer to
outgrowth medium (Naeslund & Lundkvist, 1978). The blastocyst embryos,
upon recovery from the uteri, were placed into outgrowth medium. The subsequent culture and observations were the same as those outlined above for
zonaless blastocyst embryos. In this report we have designated the day of transfer of blastocyst embryos to outgrowth medium as outgrowth day 0 (O.D. 0). All
experiments were terminated on O.D. 15. Significant difference was determined
by a contingency x2 test.
RESULTS
Once placed into the outgrowth medium, zonaless wild-type blastocyst
embryos from both the control and experimental series attach and 'outgrow',
displaying a temporal and sequential series of distinct morphologies. This
sequence of attachment and outgrowth development can be arbitrarily divided
into five stages which occur at rather delimited times following the placement of
the blastocysts into outgrowth medium (Figs. 1-5). The stages and when they
occur are: I, free-floating blastocysts (O.D. 0); II, the embryos attach to the
substratum and the trophoblast cells begin to outgrow (O.D. 1-0-1-5); III, the
trophoblast outgrowths begin to increase in total area through cell division
(O.D. 2-0-2-5); IV, the nuclei of the trophoblast cells begin to increase in diameter, signifying the onset of polyploidization (Barlow & Sherman, 1972)
(O.D. 3-0-3-5); and V, the trophoblast cells transform into giant cells (O.D. 4).
A. Development of outgrowths from blastocyst embryos developing
from the 2-cell stage in vitro
1. Comparative subsequent development of non-diapaused control and experimental blastocyst embryos
Control and experimental embryos were allowed to develop in vitro from the
2-cell stage to the blastocyst stage. After hatching, the embryos were immediately transferred to outgrowth medium. All of the experimental and control
hatched blastocysts remained floating for 1-0-1-5 days following transfer. On the
second day of culture, the embryos attached to the substratum and began to
The effect of delay on tG/tG embryos
271
4
Figs. 1-5. A series of photomicrographs which show the progressive development of
a single wild-type blastocyst embryo advancing from Stage 1 to Stage V of development in outgrowth medium, x 200.
Table 1. Development of non-diapaused blastocyst embryos
transferred to outgrowth medium
Control
Experimental
x
450
516
429(95%)
428(100%)
417(97%)
417(100%)
499(97%)
499(100%)
495(99%)
303 (61 %)
Parental genotypes
Total number of Stage I
embryos transferred
Number advancing to:
Stage II
Stage 111
Stage IV
Stage V
outgrow (Stage II). The results (Table 1) show no significant differences between
the percentages of embryos from experimental and control crosses which
reached Stage II. Also, similar numbers of experimental and control embryos
progressed to Stage III, and subsequently to Stage IV. There were no differences
in the rate of developmental progression through these stages. The only significant difference between the experimental and control groups was the number
272
M. N A D I J C K A , M. MORRIS AND N. HILLMAN
%£••::
#
Fig. 6. An experimental embryo on O.D. 5. This embryo was developmentally arrested at Stage IV and was scored as a te/t6 embryo. Note the disorganized ICM. x 200.
Fig. 7. A photomicrograph of the f6//6 embryo shown in Fig. 6 on O.D. 10. Note that
the outgrowth has remained as a Stage IV outgrowth and that it is reduced in size.
At this time no ICM cells are present, x 200.
of embryos which advanced from Stage IV to Stage V. In the control crosses,
100% of the outgrowths developed from Stage IV to Stage V. In the experimental crosses, however, only 61 % of the embryonic outgrowths progressed to
Stage V (Table 1; Fig. 6). The numbers of embryos advancing from Stage IV to
V in the control and experimental groups are significantly different. However, the
percentage of developmentally arrested embryos in the experimental group
(39%) is not significantly different from the expected percentage (40%) of
homozygous /6/f6 embryos based on the averaged +/t& male transmission
frequencies. These observations suggest that the developmentally arrested
experimental embryos were homozygous for the te mutation. Supporting this
hypothesis were the observations first, that the trophoblast cells of these developmentally arrested embryonic outgrowths did not transform into giant cells and
second, that while all of the putative * 6 /? 6 outgrowths had ICM's until they
reached Stage IV, most became disorganized (Fig. 6) and many had disappeared by O.D. 4. Since these phenotypic expressions are characteristic for
t*/tG embryos developing in vitro (Wudl et al. 1977), the developmentally
arrested outgrowths were scored as ? 6 // 6 embryos. Although all of the mutant
outgrowths remained viable for at least five additional days after reaching
Stage IV, a low percentage remained viable, although reduced in size, for an
additional six days. After reaching Stage IV, the total area of the mutant trophoblast outgrowth diminished, presumably because of both cell death and cell
dispersal (Fig. 7). All of the control embryo outgrowths and the phenotypically
wild-type experimental outgrowths contained well developed ICM's which were
retained until the experiments were terminated.
The effect of delay on tG/lQ embryos
273
Table 2. Development of Stage I embryos transferred to complete
medium after a 5-day diapause in vitro
Control
Parental genotypes
Total number of Stage I
embryos transferred
Number advancing to:
Stage II
Stage III
Stage IV
Stage V
T/+ $ x +//•<?
348
334(96%)
330(99%)
328(99%)
328(100%)
Experimental
+ // 6 $ X + //6<?
464
449(97%)
446(99%)
444(99%)
268 (60%)
2. Comparative subsequent development of in vitro diapaused control and experimental blastocyst embryos
Like the non-diapaused blastocysts, all of the in vitro diapaused embryos
remained unattached and suspended for 1 to 1-5 days after transfer to outgrowth
medium. After this time, both the control and experimental embryos attached
and began to outgrow. The subsequent trophoblast development of these
experimental and control embryos (Table 2) was the same, temporally, as that
reported above for the non-diapaused blastocysts. The trophoblast cells of all of
the control embryos and of 60 % of the experimental embryos advanced from
Stage IV to Stage V. Forty percent of the experimental embryos remained as
Stage IV outgrowths. These outgrowths, based on the phenotype of their
trophoblast cells, were classified as t6/t6 embryos. Both the control and experimental outgrowths differed from the above described non-delayed blastocyst
outgrowths in ICM presence and retention. Most of the embryos scored as
homozygous mutant contained no ICM cells (97 %) (Fig. 8) and of the few which
contained ICM's, these cells became dispersed by O.D. 4. Similarly, of theexperimental embryos scored as wild-type and of the control embryos, only 17 (5%)
contained ICM's (Fig. 9), and of these, most ICM cells were dispersing (Fig. 10)
or had disappeared (Fig. 11) by O.D. 7. Although those wild-type embryos which
contained ICM's maintained these cells longer than tG/t6 embryos, the fact that
the majority of both groups of embryos lacked these structures prevented the
presence of, absence of, or length of retention of, the ICM to be used for
scoring the tG/t6 embryos in this experiment.
274
M. NADIJCKA, M. MORRIS AND N. HILLMAN
11
The effect of delay on tG/tG embryos
275
Table 3. Day of attachment (Stage II) of blastocysts from
progesterone- and non-progesterone-treated ovariectomized females
Day of attachment
Parental
genotype
No
Progesterone
A
O.D. 1
O.D. 2
O.D. 3
A
•\
A
r
i
w.t.*
t6/t6
W.t.
/6/f6
W.t.
/6//6
100
100
264
163
T/+6 x +/t 6
115
111
+ // x +/t 6
328
199
* w.t. = wild-type
98
—
—
3
4
—
—
—
15
—
114
Treatment
Progesterone
Number
of
blastocysts
T/+ x + / / 6
+ // 6 x +/t 6
B. Development of outgrowths from in vivo diapaused control and
experimental blastocysts
1. Diapaused embryos from progesterone-treated, ovariectomized females
(Table 3).
One hundred, hatched, blastocyst embryos were collected from ovariectomized, progesterone-treated control females and placed into outgrowth medium
(Table 3). On O.D. 1 all of these blastocysts attached and began to outgrow.
Two hundred and sixty-four blastocysts were collected from progesteronetreated, experimental females. All except three blastocysts attached and began
to outgrow on O.D. 1. The remaining three attached and outgrew on O.D, 2.
The subsequent development of the trophoblast cells of both groups of control
and experimental embryos followed the temporal in vitro development of control
and experimental blastocysts described in the previous experiments. Ninetyeight (37 %) of the experimental outgrowths did not advance from Stage IV to
Stage V and were scored as homozygous mutant.
In this series, most te/tG embryos did not have ICM's. Some of the exceptional
6 G
t /t embryos had ICM's which were retained until O.D. 10 (Fig. 12). The ICM's
Fig. 8. An outgrowth from a tG/tG embryo on O.D. 4. This embryo had been delayed
in vitro at the blastocyst stage. This mutant embryo, like most of its genetic counterparts, contained no ICM cells, x 200.
Fig. 9. An outgrowth of a Stage IV embryo on O.D. 3. This embryo, a littermate of
the embryo shown in Fig. 8, advanced to Stage V on O.D. 4 and was scored as wildtype. Although this embryo contained an ICM, most /// vitro delayed, wild-type embryos did not. x 200.
Fig. 10. A wild-type embryo, delayed in vitro, on O.D. 7. Note the dispersal of
the ICM cells. x200.
Fig. 11. A wild-type littermate of the embryo shown in Fig. 10 on O.D. 7. x 200.
276
M. NADIJCKA, M. MORRIS AND N. HILLMAN
Fig. 12. A Stage IV t6/t* embryo on O.D. 10. This embryo had been delayed in a
progesterone-treated, ovariectomized female. Although this mutant embryo contained an ICM, most t6/t6 embryos did not. All of the wild-type embryos in this
experimental series contained ICM's. x 200.
Figs. 13-16. A series of photomicrographs which show the progressive development
of a t6ft6 embryo obtained from a non-hormone treated, ovariectomized female. All
of the / 6 // 6 embryos and wild-type embryos in this series of studies contained
ICM's. x200.
Fig. 13. The embryo on O.D. 0. Fig. 14. The embryo on O.D. 3. This embryo is
still free- floating and did not attach until later on O.D. 3. All embryos from this
series which were subsequently scored as wild-type attached on O.D. 1. Fig. 15. The
embryo at Stage II on O.D. 4. Fig. 16. The embryo on O.D. 8. This mutant embryo
reached Stage IV on O.D. 6.
of other tG/tQ outgrowths became disorganized and had either disappeared or
were being dispersed by O.D. 4. All of the wild-type experimental embryos and
control embryos contained ICM's which were retained until the experiment was
terminated.
2. Diapaused embryosfrom non-hormone treated ovariectomized females (Table 3).
Of the 115 hatched control blastocysts obtained from non-hormone treated,
ovariectomized control females, 111 attached and began to outgrow on O.D. 1
while the remaining 4 attached and outgrew on O.D. 2. One hundred and
twelve of these embryos progressed in a normal temporal sequence to Stage V.
A total of 328 blastocyst embryos was recovered from the non-hormone treated
experimental females. All of these blastocysts ultimately attached and outgrew.
Of these, 199 embryos attached and outgrew on O.D. 1 and 15 on O.D. 2. All of
The effect of delay on t6/t6 embryos
277
the former embryos were phenotypically normal and all contained ICM's. All
of the latter embryos were later classified by phenotype as mutant. The remaining 114 embryos were delayed to O.D. 3 before attaching and outgrowing
(Figs. 13-15). All of these 'late attaching' blastocysts ultimately expressed the
mutant phenotype. Unlike the mutant embryos obtained from progesteronetreated females, however, the mutant embryos in this series contained ICM's
which were retained until the experiment was terminated (Fig. 16). Although the
tQ/t6 embryos were delayed in attachment, their rate of development subsequent
to attachment was the same as that of outgrowths from in vitro diapaused and
non-diapausing tG/t6 blastocysts and from f6//6 blastocysts diapaused in ovariectomized, progesterone-treated females.
DISCUSSION
Although there are strain differences in the rate of development of mouse
embryos (Mintz, 1964), the temporal sequence of development of 2-cell mouse
embryos of the same genetic background to the late blastocyst substages is the
same in vitro as in vivo (unpublished observations). Also, under most conditions,
there is a similar length of time (approximately one day) elapsing between the
time the embryos reach the late blastocyst substages and implant in the uterus
(Theiler, 1972; Witschi, 1972; Nadijcka & Hillman, 1974) or attach to the
substratum in vitro (Wiley & Pedersen, 1977; Hsu, 1979; current results).
(Mural trophoblast invasion and mural trophoblast attachment are presumed to
be analogous events (Bryson, 1964; Gwatkin, 1966a, b).) Following these events,
the temporal sequence of development between wild-type embryos in vitro and
those in vivo is not coincident. Divergence in the rate of development occurs
between the time of implantation/attachment and short-egg-cylinder formation.
In vivo, embryos reach the late blastocyst substages between gestation days
4-5 and 5-25 and implant and develop to the early-egg-cylinder stage between
gestation days 5-5 and 5-75 (Theiler, 1972; Witschi, 1972; Nadijcka & Hillman,
1975). In vitro the transition of the inner cell mass to the early egg cylinder
occurs two days, rather than one day, after embryo attachment (Wiley &
Pedersen, 1977, Hsu, 1979). The subsequent development from the early to the
elongated-egg-cylinder stage, however, occurs within one day both in vitro and
in vivo. Concomitant with the in vitro delay of ICM development to short egg
cylinder, there is a delay in the transformation of mural trophoblast cells to giant
cells. In vivo, this transformation begins during the late blastocyst substages
(Dickson, 1963, 1966; Nadijcka & Hillman, 1975) when blastocyst embryos
(presumably the presumptive giant cells), begin to accumulate DNA (Barlow,
Owen & Graham, 1972; Barlow & Sherman, 1972). Giant cells are readily observable in implanted, early-egg-cylinder-staged embryos (Nadijcka & Hillman,
1975). This transformation, in vitro, is not visually apparent until two days
after embryo attachment (Hsu, 1979; current results). Therefore, both the
278
M. NADIJCKA, M. MORRIS AND N. HILLMAN
transformation of trophoblast cells to giant cells and the development of the
inner cell mass to the early-egg-cylinder stage requires a longer period of time
in vitro than in vivo. Because of this lack of synchrony we have introduced a
system of staging to describe the extent of development attained by blastocyst
embryos in vitro.
In vivo, the * 6 // 6 embryos develop at the same rate as their + // 6 and + / +
littermates up to the stage of death - the early or elongated-egg-cylinder
stages (Nadijcka & Hillman, 1975). In the current series of studies, all of the nondiapaused and diapaused embryo outgrowths scored as te/te, except those
obtained from non-hormone treated, ovariectomized females, followed the same
sequence of attachment and subsequent in vitro trophoblast development as both
their phenotypically wild-type littermates and control embryos up to Stage IV.
The exceptional t6/^ embryos, although delayed in their attachment, did develop
at the same rate as control embryos and as all other t6fte embryos once attachment had taken place. These exceptional t6/t6 embryos were also developmentally
arrested at Stage IV. Thus, although blastocyst diapause can increase significantly
the total life span of the t6/te embryo prior to developmental arrest, it does not
alter the stage at which arrest occurs.
At Stage IV, all of the putative mutant embryos, like their wild-type counterparts, exhibit enlargement of the trophoblast cells; however, unlike the wild-type
outgrowths, giant cells do not form. (The observation that in vitro attachment
occurs in the absence of giant-cell transformation correlates with those by
Dickson & Araujo (1966) and Weitlauf & Kiessling (1980) who noted that giantcell transformation and in vivo implantation are not interdependent processes.)
The lack of giant-cell formation not only distinguishes / 6 / ? e embryos from wildtype embryos developing in vitro, but also distinguishes them from te/te embryos
developing and implanting in vivo. In the latter group, the trophoblast cells do
transform into giant cells (Nadijcka & Hillman, 1975). This distinction suggests
that the temporal expression of the te/te genome is determined, in part, by the
external environment and that the in vitro environment enhances, temporally,
the developmental arrest of the trophoblast cells. Also, in vivo, all of the f6//0
embryos contain ICM's which develop into egg cylinders (Nadijcka & Hillman,
1975) while f6/'6 embryos developing in vitro may (Sherman & Wudl, 1977;
Wudl, et al. 1977) or may not (Erickson & Pedersen, 1975) develop ICM's.
Although in both of these latter studies the embryos were allowed to develop to
the late blastocyst substages in utero, were removed and then placed into outgrowth medium, the genetic backgrounds of the t6/t6 embryos and the outgrowth media differed in the two studies. Since in these studies all of the control
embryo outgrowths and the phenotypically wild-type littermate outgrowths
contained ICM's which developed normally, the divergent descriptions of the
f6//6 embryos suggest that either the genetic background or the external environment determines the presence or absence of the ICM in f6//6 embryos. Our
present results also show that /6/?G embryos do not always contain ICM's. These
The effect of delay on tG/te embryos
6
6
279
structures are present in the outgrowths of ? // embryos diapaused in nonhormone-treated ovariectomized females and in the mutant outgrowths from
/ 6 // 6 embryos not diapaused in vitro. They are absent in the outgrowths of
mutant embryos diapaused in progesterone-treated ovariectomized females and
in most / 6 /7 6 embryos diapaused in vitro. Since all of the groups of t*/t6 embryos
had the same genetic diversity, and since the development of the ICM differed
depending upon environmental conditions, our findings suggest that variable
in vivo and in vitro external environments affect ICM as well as trophoblast
formation. The fact, however, that the majority of in vitro diapaused wild-type
embryos, identified by the transformation of their trophoblast cells to giant cells,
do not contain ICM cells should caution one from using the presence of,
development of, and/or the retention of the ICM as a unique characteristic
for scoring f6//6 embryos in vitro. This is supported by earlier studies which
showed that most t6/t6 embryos as well as most of their wild-type littermates
and wild-type counterparts developing from ova fertilized in vitro, lack an inner
cell mass (McGrath & Hillman, 1980). The sole microscopic criterion for
distinguishing t*/t6 embryos from their wild-type littermates in vitro should be,
therefore, the absence of trophoblast giant-cell transformation. This appears to
be a valid criterion since the frequency of embryos exhibiting this arrested
development is not significantly different from the expected frequency of t*/t6
embryos. However, because of the difference in the extent of development
attained by the presumptive giant cells of t°/te embryos in vivo and in vitro, a
study is now in progress in which genetic markers are being used to score the
Z6//6 embryos developing in vitro. If the embryos scored as t*/t6 on the basis of
genetic markers are also the embryos which lack trophoblast giant-cell transformation, the continued use of the present scoring system will be valid.
There have been numerous comparative studies between in vivo non-diapausing
and diapausing wild-type, blastocyst mouse embryos. In vivo diapause has been
effected either by ovariectomy (with or without progesterone treatment) or by
lactational delay. The results show that diapausing blastocyst embryos have less
protein synthesis (Weitlauf & Greenwald, 1971; Weitlauf, 1973a, 1974), less
CO2 production (Menke & McLaren, 1970; Menke, 1972; Torbit & Weitlauf,
1974), less nucleic acid synthesis (McLaren, 1968; Chavez & Van Blerkom, 1979;
Weitlauf & Kiessling, 1980) and less glycogen synthesis (Ozias & Weitlauf, 1971)
than control, non-diapausing blastocyst embryos. Bergstrom (1972) and
Naeslund, Lundkvist & Nilsson (1980) have found that wild-type blastocyst embryos delayed in vivo exhibit sequential ultrastructural changes following the
initiation of diapause. They show a progressive decrease in polyribosomes,
glycogen granules and rough endoplasmic reticulum. These morphological
changes reflect the decreased metabolic rates of the diapausing embryos.
Blastocyst embryos delayed in vitro also display low synthetic rates (Chavez &
Van Blerkom, 1979) and progressive ultrastructural changes which are similar to
those exhibited by blastocyst embryos diapaused in vivo (Naeslund et al 1980).
280
M. NADIJCKA, M. MORRIS AND N. HILLMAN
Overall, the studies suggest that in vivo and in vitro diapause have corresponding
effects on blastocyst embryos.
Cessation of blastocyst diapause can be effected either by estrogen treatment
in vivo (Yoshinaga & Adams, 1966; Humphrey, 1967) or by the transfer of
embryos to complete outgrowth medium (McLaren & Menke, 1971). Activation
of the diapaused embryos is accompanied by increased rates of metabolism and
macromolecular synthesis (McLaren, 1973; Weitlauf, 1973b; Torbit & Weitlauf,
1974; Van Blerkom & Brockway, 1975; Chavez & Van Blerkom, 1979) followed
by implantation in vivo or by attachment and outgrowth in vitro. Our studies
indicate that the various experimental groups of tG/t6 embryos, with one exception, respond to the cessation of diapause and activation in the same way as
their wild-type counterparts. The exceptional ? 6 // 6 embryos obtained from nonhormone-treated ovariectomized females are significantly delayed in their
attachment when transferred to outgrowth medium. The reason for this delay is
not apparent. However, it suggests that the uterine environment in nonhormone-treated, ovariectomized females, differs from that of hormone-treated
animals and that this difference has a greater effect on te/tG embryos than on
their wild-type counterparts. Since diapause is associated with a decrease
in metabolic rate and^ activation/attachment is associated with an increase in
metabolic rate, the delay in attachment of the te/te embryos from the untreated
females may be associated with either an aberrantly low metabolic rate during
diapause and/or a delay in the metabolic rate increase when these t*/te embryos
are placed into outgrowth medium. Although we cannot distinguish between
these alternatives, or exclude other reasons for the effected delay in attachment
without additional studies, the finding that the interruption of development of
f6//6 embryos at the blastocyst stage can modify the temporal expression of the
mutant genome's lethal phenotype supports our earlier observations that the
intital expression of the mutation occurs during the late blastocyst substages.
Our earlier studies showed that it is at these stages that the te/te embryos can
first be distinguished ultrastructurally from their littermates (Nadijcka &
Hillman, 1975). Prior to the late blastocyst substages, there are no ultrastructural
differences between tQ/t6 embryos and their phenotypically wild-type littermates.
Beginning at the late blastocyst substages, tG/t6 embryos can be identified ultrastructurally, by the presence of excessive lipid droplets. Excessive lipid also
distinguishes other tL/tL embryos from their littermates prior to death. Both
t12/t12 and tw32/jw32 embryos die at the morula stage and both can be distinguished
as early as the 2-cell stage. In these homozygous tL embryos the onset of structural abnormalities is associated with aberrant levels of intermediary metabolism (for review see Hillman, 1975). Our unreported studies also show that
te/t6 embryos have normal rates of ATP metabolism prior to the late blastocyst
substages when the rate becomes aberrant. These findings, together with our
current observations, suggest that t^/t6 late blastocyst embryos display aberrant
rates of metabolism which interact with certain environments to extend their life
The effect of delay on t6/t6 embryos
281
span by delaying their in vitro attachment and subsequent development. This
delay, does not alter the in vitro lethal phenotype but can be used to distinguish
the homozygous mutant embryos from their littermates at a stage prior to their
death. This distinction enables one, for the first time, to obtain and to undertake
experiments on pure populations of viable / 6 / ?6 embryos.
This research was supported by United States Public Health Service Grant HD 00827.
The authors would like to thank Geraldine Wileman for her technical assistance.
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{Received 6 November 1980, revised 5 January 1981)