We have compared the behaviour of normal and t6/ t6 embryos in PCMF, a suboptimal ‘delay’ medium which arrests normal development. Morphologically, the two types of embryos are indistinguishable in this medium. Although β -glucuronidase activity in embryos does not rise as quickly in delay medium as in cNCTC, a nutritive medium, the rate of increase is similar in t6/t6 and normal embryos. We conclude from these observations that the lethality of r®/r® embryos is not a consequence of their reaching a given absolute age. Together with previous studies, our data suggest that embryo lethality correlates more closely with metabolic state than with morphological stage.

Blastocysts maintained in PCMF are unable to give rise to trophoblast outgrowths but do so upon transfer into cNCTC medium. When a mixture of fourth-day t6/ t6 and normal embryos are transferred to cNCTC medium after lengthy pre-incubation periods in PCMF, trophoblast outgrowth is initiated from both types of embryos at approximately the same time. However, when embryos are removed from the genital tract on the second or third day of gestation, t6/ t6 embryos are slower to produce trophoblast outgrowths than are normal embryos upon transfer from PCMF to cNCTC medium. Although the reason for this differenial behaviour is not yet clear, it is hypothesized that some product(s) required for the outgrowth process is (are) more unstable in third-day t6/ t6 embryos than in normal third-day embryos or fourth-day t6/ t6 embryos.

Our ability to separate t6/ t6 from normal embryos by their delayed initiation of trophoblast outgrowth provides us with a convenient way to identify, and to isolate for analysis, enriched populations of homozygous mutant embryos prior to the time at which they show gross morphological abnormalities.

The t6 mutation is one of a series of recessive lethal mutations of the T complex in mice (see Sherman & Wudl, 1977). t6/ t6 embryos in utero show overt morphological abnormalities between the short egg-cylinder and the elongated egg-cylinder stages (sixth to seventh day of gestation). Characteristically t6/ t6 embryos are recognized at this time by aberrantly arranged ectodermal and endodermal cells which contain excessive cytoplasmic lipid and mitochondria with crystalline inclusions (Nadijcka & Hillman, 1975). Because this t mutation and others act during embryogenesis, they have been classified by some as developmental mutations (Gluecksohn-Waelsch & Erickson, 1970; Bennett, 1975).

It appears from recent studies that during early mammalian embryogenesis there are different thresholds for the expression of ‘development-related’ gene products; that is, by manipulating either the embryos or the formulation of their culture media, it is observed that some developmental markers are expressed whereas others are suppressed (see, for example, Johnson, Handyside & Braude, 1977; Braude, 1979; Sellens & Sherman, 1980; Schindler & Sherman, 1981); It is also evident that the appearance of some indicators of embryonic development is actually time-dependent rather than stage-dependent; these markers are detectable when the embryo reaches the appropriate absolute age regardless of the extent of overt development (Sellens & Sherman, 1980; Johnson, Pratt & Handyside, 1981; Van Blerkom, 1981). In view of this new information, it becomes pertinent in attempting to understand the nature of embryolethal mutations such as t6 to determine whether lethality is a consequence of developmental stage or absolute age.

Preliminary reports have been published which bear upon the question of whether the lethality of t6/ t6 embryos is age-dependent or stage-dependent (DiZio & Hillman, 1978; Sherman et al., 1981). In these experiments, t6/ t6 embryos, along with their normal counterparts, were blocked in their development at a stage morphologically resembling the blastocyst either by ovariectomy of the mother or by placement under suboptimal culture conditions (Sellens & Sherman, 1980). Under these conditions, t6/ t6 embryos seemed to remain viable and morphologically indistinguishable from their +/+ and t6/ t6 counterparts despite reaching the absolute age at which lethality would normally occur (DiZio & Hillman, 1978; Sherman et al. 1981). These same studies suggested that after the embryos were released from delay conditions, putative t6/ t6 embryos in some cases underwent implantation-related events more slowly than +/+ and t6/ t6 embryos (DiZio & Hillman, 1978; Sherman et al. 1981). Accordingly, in this article we have examined more extensively the behaviour of t6-mutant embryos during, and following release from, developmental delay.

Collection of embryos

Embryos in the experimental cross were obtained by mating +/ t6 mice inter se (for information concerning the generation of +/ t6 mice, see Wudl, Sherman & Hillman, 1977 and Wudl & Sherman, 1978). Because of transmission frequency distortion, the expected genotypes of experimental cross embryos were as follows: t6/ t6, 40%; +/ t6, 50%, + / +, 10% (Wudl et al. 1977). Similarly, the distribution of control cross ( + /+ x + /t6) embryos was 80% + /t6 and 20% + / + . Embryos were collected on the second (2*cell), third (4-to 8-cell) and fourth (late morulae to mid blastocyst) day of pregnancy from superovulated females (Runner & Palm, 1953). Oviducts and/or uteri were flushed with preimplantation culture medium (PCM; Goldstein, Spindle & Pedersen, 1975) on the second and third days or phosphate-buffered Saline (solution A of Dulbecco and Vogt) on the fourth day. The day of observation of the sperm plug is considered the first day of pregnancy.

Culture of embryos

Embryos were cultured either in PCMF or cNCTC medium. PCMF, a medium which does not permit trophoblast outgrowth and maintains embryos in a state resembling delay in vivo, consists of PCM (made with dialyzed bovine serum albumin) supplemented with dialyzed fetuin at a final concentration in the medium of 0-05% (Sellens & Sherman, 1980). PCMF is also referred to in the text as ‘delay medium’. In some experiments, second- or third-day embryos were cultured through preimplantation stages in PCM instead of PCMF. cNCTC, a medium supporting trophoblast outgrowth, consists of NCTC-109 medium (Microbiological Associates, Bethesda, Md.) supplemented with 10% heat-inactivated fetal calf serum and antibiotics (Sherman, 1976). Embryos were cultured in microtitre dishes (Microtest I, Type 3034, Falcon, Oxnard, Calif.). Periods of culture in the various media are indicated in the text.

Scoring for trophoblast outgrowth and statistical analyses

Embryos in cNCTC medium were scored for trophoblast outgrowth at least twice daily by inspection with an inverted microscope equipped with phasecontrast optics. Outgrowth was considered to have occurred when at least one cell with its nucleus could be seen flattened on the substratum. Four days after transfer to cNCTC medium, homozygous mutant (t6/ t6) embryo outgrowths were distinguished from those of normal phenotype (,+ /t6 and + / + ) by a paucity of ICM cells and by possession of trophoblast cells with smaller nuclei, less prominent nucleoli and birefringent cytoplasmic inclusions (Fig. 1).

Fig. 1

Comparison of morphologies of normal and t6/ t6 embryos. Embryos were removed from uteri on the third day of gestation and cultured in PCMF for six days prior to transfer to cNCTC medium. (A) Morphology of an embryo (later phenotyped as t6/ t6) approximately 48 h after transfer to cNCTC medium. (B) Morphology of a wild-type embryo after the same period of culture. (C) Morphology of a t6/t6 embryo approximately 96 h after transfer to cNCTC medium. (D) Morphology of a wild-type embryo after the same period of culture. Note that the t6/t6 outgrowth in (C) has lost its inner cell mass; trophoblast nuclei are substantially smaller than those in (D) and nucleoli are less prominent. Also, several cells in the mutant embryo outgrowth contain highly birefringent cytoplasmic droplets which are distinguishable by phase microscopy from the vacuoles seen in normal trophoblast cells shortly after the onset of outgrowth (B). All figures were photographed at the same magnification (scale marker in D = 50 μ m). Hoffmann modulation-contrast optics were used in (A) and phase-contrast optics were used in the other figures.

Fig. 1

Comparison of morphologies of normal and t6/ t6 embryos. Embryos were removed from uteri on the third day of gestation and cultured in PCMF for six days prior to transfer to cNCTC medium. (A) Morphology of an embryo (later phenotyped as t6/ t6) approximately 48 h after transfer to cNCTC medium. (B) Morphology of a wild-type embryo after the same period of culture. (C) Morphology of a t6/t6 embryo approximately 96 h after transfer to cNCTC medium. (D) Morphology of a wild-type embryo after the same period of culture. Note that the t6/t6 outgrowth in (C) has lost its inner cell mass; trophoblast nuclei are substantially smaller than those in (D) and nucleoli are less prominent. Also, several cells in the mutant embryo outgrowth contain highly birefringent cytoplasmic droplets which are distinguishable by phase microscopy from the vacuoles seen in normal trophoblast cells shortly after the onset of outgrowth (B). All figures were photographed at the same magnification (scale marker in D = 50 μ m). Hoffmann modulation-contrast optics were used in (A) and phase-contrast optics were used in the other figures.

For statistical analyses, embryos were assigned ‘outgrowth times’. Since we could not establish the exact time at which embryo outgrowths began, the outgrowth time was considered arbitrarily to be the midpoint of the interval between the time at which outgrowth was first observed and the preceding scoring time (for example, an outgrowth time of 40 h was assigned to a blastocyst that had failed to outgrow by 30 h but had outgrown by 50 h). Comparisons were made between the mean outgrowth times for normal v. presumptive homozygous mutant embryos in a given population by a two-sample t test. Mean outgrowth times from experiments involving the same culture regimens were then averaged and denoted in Table 1. The differences in values are denoted in the same table. The significance of the values, when these were averaged from replicate experiments, was assessed by the use of a χ2 method for combining probability estimates [Σ-2 In P (Fisher, 1946)].

Table 1

Time required for trophoblast outgrowth following culture of embryos at different stages and in different media

Time required for trophoblast outgrowth following culture of embryos at different stages and in different media
Time required for trophoblast outgrowth following culture of embryos at different stages and in different media

Embryos failing to outgrow (approximately 5% of all cultured fourth-day embryos, 10% of all cultured third-day embryos and 25% of all cultured second-day embryos) were excluded from analysis since their phenotypes could not be determined.

β-Glucuronidase analyses

Embryos were assayed for β-glucuronidase activity by measuring the release of 4-methylumbelliferone from 4-methylumbelliferyl-β-D-glucuronic acid by the microfluorometric method described by Wudl & Sherman (1976).

Trophoblast outgrowth analyses with normal and t6/ t6 embryos

When fourth-day blastocysts from the experimental ( + /t6x + /t6) cross are placed in cNCTC medium, the for the outgrowth is approximately 40 h and no significant difference is observed in the outgrowth time of embryos which are subsequently phenotyped as either mutant (t6/t6) or normal ( + /t6 or + / + ) (Table 1). If blastocysts are placed in PCMF for three to six days, they may attach transiently to the culture dish, but outgrowth of trophoblast cells does not occur. Outgrowth does occur, however, if these blastocysts are subsequently transferred from delay medium to cNCTC medium (Table 1). The for outgrowth of trophoblast cells in cNCTC medium is shorter for embryos previously maintained in delay medium for three days than for six days, although in both cases the values are lower than those of embryos placed directly into cNCTC medium. Once again, there are no significant differences in the time required for trophoblast outgrowth from normal v. t6/t6 embryos (Fig. 2; Table 1).

Fig. 2

Typical trophoblast outgrowth curves for normal and presumptive t6/ t6 embryos. (A) A total of 122 blastocysts were removed from + /t6 females on the fourth day after mating with + /t6 males. The embryos were maintained in PCMF for three days. The embryos were then transferred to cNCTC medium and scored for the time of trophoblast outgrowth. After four days in cNCTC medium, the embryos were categorized as (◯) normals (+/+ or + /t6) or (●) homozygous mutants (t6/t6). (B) A similar experiment was carried out with 51 8-cell embryos removed from uteri on the third day of pregnancy and cultured in PCMF for four days prior to transfer to cNCTC medium. Symbols are as in (A).

Fig. 2

Typical trophoblast outgrowth curves for normal and presumptive t6/ t6 embryos. (A) A total of 122 blastocysts were removed from + /t6 females on the fourth day after mating with + /t6 males. The embryos were maintained in PCMF for three days. The embryos were then transferred to cNCTC medium and scored for the time of trophoblast outgrowth. After four days in cNCTC medium, the embryos were categorized as (◯) normals (+/+ or + /t6) or (●) homozygous mutants (t6/t6). (B) A similar experiment was carried out with 51 8-cell embryos removed from uteri on the third day of pregnancy and cultured in PCMF for four days prior to transfer to cNCTC medium. Symbols are as in (A).

Third-day embryos do not develop well when placed directly into cNCTC medium (unpublished observations). If they are placed in PCM (or PCMF) for 24 h, they reach the late morula or early blastocyst stage, after which they can develop satisfactorily upon transfer to cNCTC medium. The values for trophoblast outgrowth from such normal and t6/t6 embryos are similar and in each case greater than those of fourth-day embryos (Table 1). If embryos are maintained in delay medium for two days, that is until they reach the late blastocyst stage, the values for trophoblast outgrowth are shorter than those of fourth-day blastocysts placed directly into cNCTC medium (Table 1). There is a progressively increasing difference in the time required for trophoblast outgrowth between normal and t6/t6 embryos with longer periods of culture in PCMF prior to transfer to cNCTC (Table 1 and Fig. 2). Some embryos (presumably t6/t6) fail to outgrow after more than 180 h in cNCTC following a 6-day pre-incubation period in PCMF: during incubation in cNCTC they retain the morphology of expanded blastocysts (Fig. 1).

When embryos removed from oviducts at the 2-cell stage are cultured for three days in PCM or PCMF, they reach the late blastocyst stage. Upon transfer to cNCTC medium, normal embryos give rise to trophoblast outgrowths in less than 30 h. Trophoblast cells from homozygous t6-mutant embryos are significantly slower to outgrow than those from normal embryos. A similar result is observed when 2-cell embryos are maintained in delay medium for six days prior to transfer to cNCTC.

Table 1 illustrates that of the total of 1552 embryos subjected to analysis in these experiments, 40% were phenotyped as t6/ t6 embryos, consistent with the proportion expected from transmission frequency studies with our stocks of t6 -bearing animals (Wudl et al. 1977).

β-Glucuronidase analyses of normal and t6/ t6 embryos

We have previously measured β-glucuronidase activities of individual normal and t6-mutant embryos cultured in cNCTC medium (Wudl & Sherman, 1978). Since we could not distinguish morphologically between normal and t6/ t6 embryos at early stages, we reasoned that so long as t6-mutant embryos were keeping pace with normals, the ratio of enzyme activities in the upper 60% group (proportion of normals) to the low 40% group (proportion of mutants) would remain approximately constant. The data obtained (Wudl & Sherman, 1978), replotted in Fig. 3, indicate that β-glucuronidase activity in the low 40% group of embryos fails to rise as rapidly as the upper 60% group beyond the first day of culture. The upper 60%: low 40% enzyme activity ratio rises steadily from 2·0 after one day of culture to 8·6 after five days (Wudl & Sherman, 1978). Figure 3 also illustrates that when experimental cross embryos are maintained in PCMF, the average values of β-glucuronidase activity after 3·5 days of culture for the entire population are very close to those observed for the low 40% of embryos cultured in cNCTC medium. Furthermore, the β-glucuronidase levels reached by embryos after five days of culture in PCMF are the Same whether the embryos are obtained from experimental or control crosses (Fig. 3), despite the fact that the former population contains 40% t6-homozygous mutant embryos whereas the latter population contains none.

Fig. 3

β-Glucuronidase activities in normal and t6-mutant embryos following culture in delay medium or cNCTC medium. Enzyme activities for fourth-day embryos cultured in cNCTC medium are taken from Wudl & Sherman (1978); the highest 60% values (◯) and lowest 40% values (●) are averaged and plotted separately. A, Average enzyme activities of the total population of embryos obtained by crossing +/ t6 females with +/ t6 males and culturing fourth-day embryos in PCMF medium. □, Average enzyme activities of the total population of embryos obtained by crossing + /+ (SWR/J) females with + /t6 males and culturing fourth-day embryos in PCMF. Each point is averaged from at least 10 embryos.

Fig. 3

β-Glucuronidase activities in normal and t6-mutant embryos following culture in delay medium or cNCTC medium. Enzyme activities for fourth-day embryos cultured in cNCTC medium are taken from Wudl & Sherman (1978); the highest 60% values (◯) and lowest 40% values (●) are averaged and plotted separately. A, Average enzyme activities of the total population of embryos obtained by crossing +/ t6 females with +/ t6 males and culturing fourth-day embryos in PCMF medium. □, Average enzyme activities of the total population of embryos obtained by crossing + /+ (SWR/J) females with + /t6 males and culturing fourth-day embryos in PCMF. Each point is averaged from at least 10 embryos.

In Table 2, average β-glucuronidase levels are given for embryos from the experimental cross cultured in PCMF from the third or the fourth days of gestation. Enzyme levels of cultured third-day embryos, even after four days, fail to reach those of fourth-day embryos after only one day in vitro. When enzyme activities are separated into the high 60% and low 40% categories and averaged, the ratios between the values obtained, unlike those for experimental cross embryos in cNCTC medium (Wudl & Sherman, 1978), remain similar throughout the culture periods whether embryos are obtained for culture on the third or the fourth day of gestation (Table 2).

Table 2

β-Glucuronidase activities of embryos placed into culture on the third or fourth gestation days

β-Glucuronidase activities of embryos placed into culture on the third or fourth gestation days
β-Glucuronidase activities of embryos placed into culture on the third or fourth gestation days

In previous studies with t12-, t6- and tw5-mutant embryos, we have demonstrated that the embryos degenerate in vitro at times which are in reasonable agreement with the embryolethal periods in utero (Wudl & Sherman, 1976, 1978; Wudl et al. 1977; Sherman & Wudl, 1977). In the present investigation, we have maintained embryos in delay medium for prolonged intervals such that after release from delay, the absolute age of the embryos is greater than that at which t6/ t6 embryos would normally die. Based upon the proportion of embryo outgrowths typed as tt6/ t6 in these studies (Table 1), we can state that virtually all of the t6-mutant embryos have remained viable through, and beyond, the delay period. We conclude, therefore, that the lethal period for t6/t6 embryos in culture, as in utero (DiZio & Hillman, 1978; Nadijcka, Morris & Hillman, 1981), is not predicated upon the absolute age of the embryos.

Is it then valid to conclude that the embryolethal period for t mutations is stage-specific? Traditionally, the stage of an embryo is based upon morphologic observation. We do not believe that the death of homozygous t-mutant embryos is related to morphologic stage. This is because many embryos fail to recapitulate morphologic development in cNCTC medium beyond the early egg-cylinder stage; whereas cultures of wild-type embryos generally remain viable despite this limitation, presumptive t6/ t6 and t6/ t6 embryos, as mentioned, degenerate in vitro near the times expected from their behaviour in utero (Wudl & Sherman, 1976, 1978; Wudl et al. 1977).

As embryogenesis proceeds, cells undergo shifts in their metabolic patterns (as reflected by alterations in metabolic requirements; see Sellens & Sherman, 1980) and, in the case of ICM derivatives, divide at increasingly rapid rates (see Snow, 1976). We have argued previously that cells from t6/ t6 embryos die because they possess a metabolic lesion and cannot, therefore, survive as metabolic demands increase to some particular level (Wudl & Sherman, 1978). In other words, the lethal period of t6/ t6 embryos would be related to a metabolic state as opposed to a morphologic stage. Our present findings reinforce this view. The data in Fig. 2 and Table 2 demonstrate that at least with respect to levels of β-glucuronidase activity, we can cause normal embryos to resemble t6/ t6 embryos by having their metabolism restricted through culture in suboptimal medium. Furthermore, the observation that t6/ t6 embryos do not possess more β-glucuronidase activity in cNCTC medium than in PCMF medium (Fig. 2) suggests that they are unable to respond in a positive manner to conditions supportive of an advanced metabolic state. In fact, these nutritive culture conditions lead to the marked negative response of t6/ t6 embryos (that is, death) which can be avoided for relatively long periods by culture in delay medium, which presumably protects the embryos by suppressing their progression to a more advanced metabolic state.

The data in Table 1 are complex, and an earlier explanation based on preliminary results with t6/ t6 embryos released from delay (Sherman et al. 1981) is likely to have been too simplistic. The present study and other recent ones (Sellens & Sherman, 1980; Sherman & Matthaei, 1980; Sherman et al. 1981; Schindler & Sherman, 1981) indicate that events leading to trophoblast outgrowth occur in at least two synthetic phases. Although we do not know which gene products are responsible for trophoblast outgrowth, the aforementioned studies allow us to make the following statements about the two phases: (a) the early phase occurs at or before the expanded blastocyst stage, whereas the later phase takes place within the period 20–40h before outgrowth; (b) early phase events can take place in nutrient or delay medium whereas the occurrence of the later phase, which appears to involve protein synthesis, requires nutrient medium; and (c) the later phase cannot take place until early phase events have been completed. These conclusions explain why normal embryos pre-incubated in PCMF require less time in cNCTC for outgrowth than do embryos placed directly into cNCTC medium (Table 1). On the other hand, the observation that the for normal embryos increases when pre-incubation in PCMF exceeds three days (Table 1) suggests that early phase products are eventually lost in suboptimal medium and must be replenished upon transfer to cNCTC medium prior to the onset of the later phase. If the latter proposal is correct, the longer periods required for outgrowth of t6/ t6 embryos relative to normal embryos following culture in PCMF from the third day (Table 1) can be attributed to more rapid destabilization of early phase products and/or a protracted period required for resynthesis of these products following transfer to cNCTC medium.

Why, then, is there no significant difference between the trophoblast outgrowth times when fourth-day normal and mutant embryos are cultured in PCMF for as long as six days prior to transfer to cNCTC (Table 1)? We can offer no ready explanation for this observation. However, from a comparison of β-glucuronidase activities between embryos placed into culture on the third or the fourth day of gestation (Table 2), it is clear that the latter embryos always contain substantially higher activities than the former during culture in PCMF. It is possible, therefore, that t6/ t6 embryos placed in delay medium from the fourth day possess other metabolic differences which render them more capable of stabilizing early phase products than t6/ t6 embryos cultured in PCMF from the third day. Finally, it should be stated that in preliminary results we have observed that embryos homozygous for other t6-complementation group mutations (t0,th20) show delayed trophoblast outgrowth times relative to normal embryos when culture is begun on the third or the fourth day of gestation (Sherman & Pai, unpublished observations). We are currently investigating possible reasons for this difference.

Our data suggest a convenient method for obtaining populations enriched for, or depleted of, t6/ t6 embryos. For example, after maintaining third-day embryos in delay medium for four days (Fig. 2), it should be possible, upon transfer to cNCTC medium, to obtain populations containing a large proportion of normal embryos (those outgrowing at early times) or of t6/ t6 embryos (those failing to outgrow after half or more of the embryos have already done so). We must stress that, for reasons described above as well as on the basis of ultrastructural studies (Nadijcka & Hillman, 1975), the homozygous mutants so obtained would not be expected to be completely normal. Nevertheless, they would be likely to contain a more restricted array of biochemical defects than they would when selected at later morphological stages on the basis of overt abnormalities; this might, therefore, provide us with a better opportunity than in the past to distinguish between the primary t6 lesion and secondary effects in molecular terms.

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