In vivo, t6/t6 embryos are developmentally arrested between gestation days 5·5 (short-egg-cylinder 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 r°/7c lethality is time- or stage-specific. The results show that the t6/t6 genome is expressed differently in vivo and in vitro and that the in vitro expression of the homozygous t6 genome differs with different methods of effecting developmental delay. Although delay increases the life span of t6/t6 embryos it does not alter the stage of lethality. One method used to effect delay (ovariectomy) causes the t6/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.

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 t 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 tL -bearing spermatozoa (for review see Bennett, 1975 and Sherman & Wudl, 1977).

In utero, homozygosity for the t6 mutation, a member of the t0 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 elongated-egg-cylinder stage. By day 7, all of the mutant embryos exhibit varying degrees 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 t6 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 t6/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 t6/t6 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 t6/t6 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 t6/t6 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 t6/t6 embryos from their phenotypically wild-type littermates prior to develop mental arrest and death. A preliminary report on a portion of this work was previously published (Dizio & Hillman, 1978).

To obtain the experimental and control embryos, males from an inbred T/t6 stock were mated to BALB/c ( + / + ) females. The offspring ( + / t6 and T/ + ) from this cross are phenotypically distinguishable from each other. The + / t6 offspring have tails of normal length while the T/ + offspring have short tails. The + / t6 offspring were mated inter se to obtain the experimental litters. These litters contain + / +, + (both phenotypically wild-type) and t6/t6 mutant embryos. The T/+ female offspring were mated to + /t6 males to obtain the control litters. These litters contain only viable genotypes. The 22 + /t6 males used in these studies were tested for their transmission frequencies of the t6 - bearing 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 t6/t6 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 × 30 mm Falcon tissue culture dishes at 37°C in an atmosphere of 5% CO2,95% 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 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 χ 2 test.

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. 15). 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).

Fig. 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, × 200.

Fig. 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, × 200.

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 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 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 t6/t6 embryos based on the averaged +/ t6 male transmission frequencies. These observations suggest that the developmentally arrested experimental embryos were homozygous for the i6 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 t6/t6 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 t6/t6 embryos developing in vitro (Wudl et al. 1977), the developmentally arrested outgrowths were scored as t6/t6 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.

Table 1

Development of non-diapaused blastocyst embryos transferred to outgrowth medium

Development of non-diapaused blastocyst embryos transferred to outgrowth medium
Development of non-diapaused blastocyst embryos transferred to outgrowth medium
Fig. 6

An experimental embryo on O.D. 5. This embryo was developmentally arrested at Stage IV and was scored as a t6/t6 embryo. Note the disorganized ICM. × 200. Fig. 7. A photomicrograph of the t6/t6 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 1CM cells are present, × 200.

Fig. 6

An experimental embryo on O.D. 5. This embryo was developmentally arrested at Stage IV and was scored as a t6/t6 embryo. Note the disorganized ICM. × 200. Fig. 7. A photomicrograph of the t6/t6 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 1CM cells are present, × 200.

Fig. 7

Fig. 7. A photomicrograph of the t6/t6 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, × 200

Fig. 7

Fig. 7. A photomicrograph of the t6/t6 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, × 200

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 the experimental 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 t6/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 t6/t6 embryos in this experiment.

Table 2

Development of Stage I embryos transferred to complete medium after a 5-day diapause in vitro

Development of Stage I embryos transferred to complete medium after a 5-day diapause in vitro
Development of Stage I embryos transferred to complete medium after a 5-day diapause in vitro
Fig. 8

An outgrowth from a t6/t6 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, × 200.

Fig. 8

An outgrowth from a t6/t6 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, × 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 in vitro delayed, wild-type embryos did not. × 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 in vitro delayed, wild-type embryos did not. × 200.

Fig. 10

A wild-type embryo, delayed in vitro, on O.D. 7. Note the dispersal of the ICM cells, × 200.

Fig. 10

A wild-type embryo, delayed in vitro, on O.D. 7. Note the dispersal of the ICM cells, × 200.

Fig. 11

A wild-type littermate of the embryo shown in Fig. 10 on O.D. 7. × 200.

Fig. 11

A wild-type littermate of the embryo shown in Fig. 10 on O.D. 7. × 200.

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 progesterone-treated, 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.

Table 3

Day of attachment (Stage II) of blastocysts from progesterone- and non-progesterone-treated ovariectomized females

Day of attachment (Stage II) of blastocysts from progesterone- and non-progesterone-treated ovariectomized females
Day of attachment (Stage II) of blastocysts from progesterone- and non-progesterone-treated ovariectomized females

In this series, most t6/ t6 embryos did not have ICM’s. Some of the exceptional t6/t6 embryos had ICM’s which were retained until O.D. 10 (Fig. 12). The ICM’s of other t6/ t6 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.

Fig. 12

A Stage IV t6/ t6 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. × 200.

Fig. 12

A Stage IV t6/ t6 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. × 200.

Fig. 13-16

A series of photomicrographs which show the progressive development of a t6/ t6 embryo obtained from a non-hormone treated, ovariectomized female. All of the t6/ t6 embryos and wild-type embryos in this series of studies contained ICM’s. × 200. 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.

Fig. 13-16

A series of photomicrographs which show the progressive development of a t6/ t6 embryo obtained from a non-hormone treated, ovariectomized female. All of the t6/ t6 embryos and wild-type embryos in this series of studies contained ICM’s. × 200. 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.

2 Diapaused embryos from 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 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 Tate attaching’ blastocysts ultimately expressed the mutant phenotype. Unlike the mutant embryos obtained from progesterone-treated females, however, the mutant embryos in this series contained ICM’s which were retained until the experiment was terminated (Fig. 16). Although the t6/ 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 t6/ t6 blastocysts and from t6/ t6 blastocysts diapaused in ovari-ectomized, progesterone-treated females.

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, 1966 a, 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 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 t6/ t6 embryos develop at the same rate as their + / t6 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 non-diapaused and diapaused embryo outgrowths scored as t6/ t6, 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/ t6 embryos, although delayed in their attachment, did develop at the same rate as control embryos and as all other t6/ t6 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/ t6 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 t6/ t6 embryos from wildtype embryos developing in vitro, but also distinguishes them from t6/ t6 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 t6/ t6 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 z6/z° embryos contain ICM’s which develop into egg cylinders (Nadijcka & Hillman, 1975) while t6/ t6 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 t6/ t6 embryos suggest that either the genetic background or the external environment determines the presence or absence of the ICM in t6/ t6 embryos. Our present results also show that t6/ t6 embryos do not always contain ICM’s. These structures are present in the outgrowths of t6/ t6 embryos diapaused in nôn-hormone-treated ovariectomized females and in the mutant outgrowths from t6/ t6 embryos not diapaused in vitro. They are absent in the outgrowths of mutant embryos diapaused in progesterone-treated ovariectomized females and in most t6/ t6 embryos diapaused in vitro. Since all of the groups of t6/ 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 t6/ t6 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 t6/ 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 t6/ t6 embryos. However, because of the difference in the extent of development attained by the presumptive giant cells of t6/ t6 embryos in vivo and in vitro, a study is now in progress in which genetic markers are being used to score the t6/ t6 embryos developing in vitro. If the embryos scored as t6/ 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, 1973 a, 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).

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 t6/ t6 embryos, with one exception, respond to the cessation of diapause and activation in the same way as their wild-type counterparts. The exceptional t6/ t6 embryos obtained from non-hormone-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 non-hormone-treated, ovariectomized females, differs from that of hormone-treated animals and that this difference has a greater effect on t6/ t6 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 t6/ t6 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 t6/ t6 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 t6/ t6 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 t6/ t6 embryos can first be distinguished ultrastructurally from their littermates (Nadijcka & Hillman, 1975). Prior to the late blastocyst substages, there are no ultrastructural differences between t6/ t6 embryos and their phenotypically wild-type littermates. Beginning at the late blastocyst substages, t6/ 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/ tw32 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 t6/ 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 t6/ t6 late blastocyst embryos display aberrant rates of metabolism which interact with certain environments to extend their life 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 t6/ t6 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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