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/. Embryo/, exp. Morph. Vol. 33, 3, pp. 685-695, 1975 Printed in Great Britain 685 Ultrastructural studies of tw32/tw32 mouse embryos By NINA HILLMAN 1 AND RALPH HILLMAN 1 From the Department of Biology, Temple University, Philadelphia SUMMARY Homozygous t"' lt"' mouse embryos, obtained from both spontaneously ovulated and superovulated T+lt"'32 females mated to T+jtu'32 males, have a lethal period which extends from the 8-12 cell stage to the late morula stage. Most of the homozygous mutant embryos die at the early morula stage and are characterized by excessive amounts of cytoplasmic lipid, mitochondrial abnormalities, binucleated cells and nuclear lipid droplets. The excessive cytoplasmic lipid and nuclear lipid droplets distinguish 35-50% of the embryos (presumably f "32 homozygotes) from their litter-mates prior to the lethal period. The remainder of the distinguishing characteristics appear in the later (8-cell to late morula) tw32jtw3i embryos in frequencies high enough to be considered phenotypic expressions of the mutant genome. The present study indicates that the non-complementary tu'32 and t12 alleles are in fact separate T locus recessive alleles. 32 32 INTRODUCTION The complex TJocus is located on linkage group IX (chromosome 17) of the house mouse. The homozygous T\T genotype is lethal at gestation days 10-11 (Chesley, 1932) while the heterozygous genotype T+jT is viable but results in offspring with a short-tail phenotype. The dominant allele, T, and a series of recessive lethal alleles (tn) are maintained in balanced lethal lines (Tftn) which are tailless. Two of these recessive alleles, tw32 and t12, have been assigned to the same complementation group of the T locus (Bennett & Dunn, 1964). This assignment is a result of studies which show that the heterozygous twZ2/t12 embryos die at the late morula stage, as do the tw32 and the t12 homozygotes. Also, both tu'32 homozygotes and twS2/t12 heterozygotes show, at the light microscope level, the same syndrome of phenotypic expression as previously described for homozygous t12 embryos (Smith, 1956). A more recent study (Hillman, Hillman & Wileman, 1970) has shown, however, that the t12 homozygotes die over a range of preimplantation stages, death not being limited to the late morula stage; that the t12lt12 genotype is a cell lethal; and that the mutant embryos can be identified before developmental arrest and degenerative changes by the presence of both nuclear lipid droplets and nuclear fibrillo-granular bodies at each cleavage stage. In addition, t12/t12 embryos contain excessive cytoplasmic lipid droplets and, in the later cleavage 1 Authors'1 address: Department of Biology, Temple University, Philadelphia, Pennsylvania 19122, U.S.A. 43-2 686 N. HILLMAN AND R. HILLMAN stages, frequently contain binucleate cells. These characteristics are visible only at the ultrastructural level. Since tw3Z has been described as being the same as t12, the present study has been undertaken to determine if the tw32/tw32 genome elicited the same ultrastructural syndrome of phenotypic characteristics as described for the t12/t12 genome. Inasmuch as the ultrastructure of cleavage-stage wild-type embryos and mutant embryos (t12jt12) have been previously reported (Hillman & Tasca, 1969; Hillman et al. 1970), those organelles which are ultrastructurally similar in all embryos, at each cleavage stage, are not described. MATERIALS AND METHODS + w32 Heterozygous (T /t ) animals were obtained from 8-week-old BALB/cT+ homozygous females mated to T\tw32 males. (The original T/tw32 breeding pairs were obtained from Dr Dorothea Bennett.) Before mating inter se, the T+/tu'32 females from the parent cross were either superovulated [intraperitoneal injections of 10 i.u. of pregnant mare serum gonadotropin (PMS, Ayerst) followed 45 h later with 10 i.u. human chorionic gonadotropin (HCG, Organon)] or timed-ovulated (2-5 i.u. PMS followed 45 h later with 2-5 i.u. HCG). The superovulated mice averaged 32 2-cell embryos per litter whereas the time-ovulated mice averaged ten 2-cell embryos per litter. The superovulated females were separated at random into two groups. Two-cell embryos were flushed from the oviducts of females in the first group. Entire litters were separately placed into culture and allowed to develop to the desired developmental stage (2-cell, 4-cell, 8-12 cell, early and late morulae, and substage 1 and 2 blastocysts) before being processed for ultrastructural studies. From the second group of superovulated females entire litters were removed from the oviducts or uteri at specific developmental stages and processed for electron microscopy immediately. Litters from all timed ovulations were allowed to develop in vivo and were removed from the mother at specific cleavage stages. BALBlcT+HBALB/cT+ embryos developing either in vivo or in vitro served as controls. The T+/tw32 male exhibits a high transmission ratio for the tw32 allele. Bennett & Dunn (1964) have found the sperm transmission ratio of this allele to vary between 0-94 and 0-74 depending upon the T+/tu32 tested. The averaged transmission ratio is 0-84 tw32:O-l6 T+. The pooled litters from large numbers of T+/tw32 inter se matings should therefore contain approximately 40 % tw32 homozygotes. Our results (Table 1) correspond to those reported by Bennett and Dunn. The expected ratios of tw32/tw32 embryos have been found in the pooled litters, and the percentages of tw32 homozygous embryos obtained from either superovulated or timed-ovulated females do not differ significantly from those expected. Superovulation does not, therefore, alter the transmission ratio of the female. Entire litters of staged embryos, both those developing in vitro and those developing in vivo, were fixed in 3 % glutaraldehyde in 0-1 M-PO 4 buffer (pH 7-4) Studies o/t w32 /t vv32 embryos 687 Table 1. Stage of developmental arrest T+ltw32 x T Number T+/T+ x T+/T+ V Number Total embryos Arrested embryos Stage of arrest 2-cell 8-cell Early morula Late morula Blastocyst 1260 625 21 165 336 89 14 V /o /o 49-6 968 72 7-4 1-7 130 26-7 71 11 23 14 9 17 9 2-4 1-4 0-9 1-8 0-9 — for 1 h, washed in 0-1 M-PO4 buffer overnight, postfixed in 1 % osmium tetroxide (Millonig's, pH 7-3), dehydrated through a series of alcohols including absolute alcohol and embedded in Epon. Thin sections were placed on uncoated copper grids, stained with either lead citrate (Venable & Coggeshall, 1965), or lead citrate preceded by 2 % uranyl acetate (Watson, 1958) and examined with either a Zeiss 9 A or a Philips 300 electron microscope. RESULTS Seventy-two (7-4%) of the BALB\cT+\\BALB\cT+ embryos died during development, the greatest number at the two-cell stage. Approximately 50 % of the experimental embryos from heterozygous tw32 inter se matings died (Table 1), mostly between the 8-cell and late morula stages. No significant differences in either the time of lethality or percentage of embryos dying at specific stages were noted between embryos developing in vitro and those developing in vivo. Because cell number alone is not a valid criterion for determining embryonic age during cleavage stages (particularly during the early and late morula stages), both cell counts and ultrastructural examination have been used to determine the time of death of the developmentally arrested experimental embryos. A major criterion for determining embryonic age is the ultrastructural appearance and shape of the nucleolus (Hillman & Tasca, 1969). Based on nucleolar ultrastructure, the highest attrition of tw™ embryos occurs at the 8-cell and early morula stages, with most dying as early morulae. These developmentally arrested tlr32ltu™ embryos can be distinguished from their developmentally arrested, phenotypically wild-type, litter-mates and from correspondingly staged control embryos by additional ultrastructural changes. These changes, either singly or in combination, distinguish the lethal fM'32 homozygotes in 8-cell and older embryos. The increased attrition, therefore, of embryos from /u'32 inter se matings in stages later than the 8-cell stage is a function of the homozygous lethal genotype. 688 N. HILLMAN AND R. HILLMAN Studies o/t w 3 2 /t w 3 2 embryos 689 w32 Five ultrastructural characteristics distinguish homozygous t embryos from their phenotypically normal litter-mates as well as from control embryos. These characteristics are intranuclear lipid droplets, large clusters of cytoplasmic lipid droplets, mitochondrial variants, binucleated cells and single cell lethality. Both nuclear lipid droplets and excessive cytoplasmic lipid distinguish most of the developmentally arrested older embryos obtained from T+/tw32 inter se matings. In addition, 35-50 % of the viable 2-, 4- and 8-cell embryos obtained from T+/tw32 inter se matings also contain nuclear and excessive cytoplasmic lipid (Figs. 1, 2). Since the nuclear lipid droplets and large numbers of cytoplasmic lipid droplets are absent both in the remainder of the litter-mates and in correspondingly staged control embryos, it is presumed that these structures characterize the twS2/twS2 embryos at all cleavage stages. It has been noted that both the nuclear lipid droplets and the cytoplasmic lipid droplets are found in more cells and are more numerous as the embryos age (Fig. 3). The mitochondrial variants are found only in 8-cell, early and late morula ?""32 embryos. Although these variant mitochondria are not found in all embryos judged to be mutant because of their lipid inclusions, they do occur in a high enough frequency to be considered characteristic of the homozygous phenotype. In late 4-cell and 8-cell wild-type embryos, mitochondria are normally of two forms. They are either round or ovoid, have arc-shaped cristae and contain an electron-dense matrix; or they are elongate, have parallel cristae which traverse the mitochondrion, and contain a less dense matrix. Only round, electron-dense mitochondria are found in 2- and early 4-cell wild-type embryos (Hillman & Tasca, 1969). A large number of the twS2 8-cell, early morulae and late morulae embryos contain only rounded mitochondria with an electron-dense matrix (cf. Figs. 4, 5). Additionally, significant numbers of the cells of early and late morulae tw32 homozygotes also have mitochondria which contain crystalline inclusions (Fig. 6a, b). An additional characteristic of the twZ2ltw32 genotype is the presence of binucleated cells. Again, these cells are not found in all embryos presumed to be recessive homozygotes, but are found in a frequency high enough to be considered characteristic of the twS2ltwZ2 genotype. These binucleate cells are not found in mutant 2- and 4-cell embryos, but are found with increasing frequency in both viable and developmentally arrested older twS2 homozygotes. Serial sections of these cells show that the two nuclei are completely separated FIGURES 1 A N D 2 Fig. 1. A portion of a cell from an early 2-cell tK32/tu'32 embryo. Note the nuclear lipid droplets (arrows), x 15000. Fig. 2. A portion of a cell from an 8-cell r«32//"32 embryo. The nucleolus and cytoplasmic organelles appear ultrastructurally normal for this developmental stage. The nuclear lipid droplet (arrow) distinguishes this embryo as a homozygous f"'32 embryo, x 13000. N. HILLMAN AND R. HILLMAN »*i*5t* Fig. 3. A developmentally arrested twZ2/tw32 morula. Note the presence of the large lipid droplets in the cytoplasm. Similar cytoplasmic lipid droplets distinguish the homozygous tw32 embryos before arrest and cellular degeneration, x 5200. even though they are frequently juxtaposed. In the viable embryos, the binucleated cell or cells which undergo karyokinesis but not cytokinesis remain larger than the other blastomeres which continue to divide normally. In the binucleate cells there are generally two nucleoli, rarely three nucleoli, per nucleus. These nucleoli are ultrastructurally normal for the stage of development attained by the cell (Fig. 7). Although most twS2ltw*2 embryos die at the early morula stage, some stop development as early as the 8-cell stage while some continue until the late morula stage. When these embryos are examined as soon as they are found to be arrested, not all the cells are in the same state of degeneration. They may contain cells undergoing mitoses as well as cells in an advanced degenerative stage. Thus the tw32/tw32 genotype results in asynchronous cell death and can therefore be considered a cell lethal. With the exception of the phenotypic characteristics discussed above, the cellular organelles observed in viable tw32 homozygotes do not differ from those observed either in their phenotypically normal litter-mates or in their wild-type counterparts. In all cases, the level of cellular ultrastructural differentiation is dependent upon the stage of development attained by either the entire embryo Studies o/t w 3 2 /t w 3 2 embryos 691 F I G U R E S 4 AND 5 Fig. 4. Wild-type morula. Note that the mitochondria are elongated and have transverse parallel cristae. x 26000. Fig. 5. Homozygous tu'32 early morula. These mitochondria are characteristic of the cells of the majority of 8-cell and older f""32 homozygous embryos. The matrix is condensed and the cristae aberrantly arranged. Compare this micrograph with Fig. 4. x 26 000. 692 N. HILLMAN AND R. HILLMAN Fig. 6. (a) Homozygous tw32 early morula. An additional distinguishing characteristic of older twZ2/tu'*2 embryos is the presence of crystals (arrows) in the mitochondria. The nucleus (N) of this cell appears to be breaking down, x 14000. (b) A higher magnification of two mitochondria containing crystals, from a tlc32/tu'32 early morula. x 32000. Fig. 7. A developmental^ arrested tw32/tK32 early morula. One of the cells is binucleate (nucleus: N). This cell and the other two cells are in advanced stages of degeneration, x 3400. Studies o/t w32 /t w32 embryos 693 or by its individual cells prior to developmental arrest. Differences in organelle structure, other than those described above, become apparent only in advanced stages of cellular degeneration. The stages of degeneration are the same as, and occur in the same sequential order as those already reported for t12jt12 embryos (Hillman et al. 1970). They are, therefore, not included in this report. DISCUSSION This paper is the second in a series describing the ultrastructural phenotype of homozygous lethal tn\tn mice. The first in the series described the ultrastructural changes found in developmental^ arrested t12/t12 embryos (Hillman et al. 1970). The homozygous t12/t12 and twS2ltwS2 genotypes are alike in that they result in (1) preimplantation lethality, (2) cell lethality, (3) the presence of nuclear lipid droplets and excessive cytoplasmic lipid, (4) the appearance of binucleate cells, and (5) the same chronology of degenerative changes. Embryos having the two homozygous mutant genotypes differ from each other in (1) their lethal periods, (2) their nuclear inclusions, and (3) the presence or absence of mitochondrial variants. Both homozygotes can be distinguished from their litter-mates as early as the 2-cell stage by the presence of nuclear lipid droplets and excessive cytoplasmic lipid. In addition, both homozygous mutant embryos often contain binucleate cells, especially in the later cleavage stages. Usually there is only one binucleate cell per embryo, but embryos of both genotypes may have as many as two or three, either peripherally or centrally located. They remain larger than the other cells, which continue to divide until individually arrested. The disproportionate cell size results in aberrantly shaped embryos. Since the ?12//12 and tw32ltwS2 genotypes are characterized by individual cell death, developmentally arrested embryos of both genotypes contain cells which are ultrastructurally normal as well as cells which are in advanced stages of degeneration. There are, as outlined above, certain phenotypic characteristics which distinguish the two homozygous mutant embryos from each other. It has been found, for example, that the ?12/?12 genotype can result in embryonic lethality as early as the 8-cell stage or as late as the early blastocyst stage. The majority of /12//12 embryos, however, are arrested as late morulae (Hillman et al. 1970). The present study shows that the twZ2ftwZ2 genotype also exhibits a range of lethal periods. This range is from the 8-cell to the late morula stage, and unlike tl2/t12 embryos, the highest percentage of tw32/tw32 attrition occurs at the early morula stage. It should be emphasized that a distinction between early and late morulae can only be achieved by an ultrastructural examination of mouse embryos. This, in turn, probably accounts for the difference in the timing of the lethal periods for twS2ltw*2 embryos as reported by Bennett & Dunn (1964) and that found in the present study. The additional morphological differences which distinguish tw32 homozygotes 694 N. HILLMAN AND R. HILLMAN 12 from t homozygotes can only be resolved at the ultrastructural level. For example, the presence of small nuclear fibrillo-granular bodies distinguish t12/t12 embryos from their phenotypically normal litter-mates as early as the 2-cell stage. These bodies are similar to nucleoli both in their structure and in their response to enzymic digestion. They are unlike nucleoli in that they remain unlabelled in the presence of [3H]uridine (Hillman et al. 1970). Such nuclear bodies are not found in twZ2 homozygotes and can therefore be used to distinguish the two genotypes. Finally, the mitochondria of t12/t12 embryos undergo the normal sequential development which has been described for mouse cleavage-stage embryos (Hillman & Tasca, 1969). Even in arrested t12/t12 embryos in advanced stages of degeneration, the mitochondria appear normal for the developmental stage attained by the embryo. These organelles maintain their structural integrity even in those cells which have degenerated to the stage of cellular separation and vacuolization. Conversely, the mitochondria of some tw32/tw32 embryos differ from those of their litter-mates as early as the 8-cell stage. In most mutant homozygotes, the mitochondria do not undergo the normal structural transition which typifies late 4- and early 8-cell mouse embryos. The continued presence of mitochondria which are similar to the single mitochondrial form found in 2- and early 4-cell wild-type embryos suggests that mitochondrial function, or cellular energy metabolism, may differ between the homozygous mutant embryos and their phenotypically and genetically wild-type counterparts. The fact that some mitochondria of twZ2/twZ2 embryos contain crystalline inclusions supports this hypothesis (Carafoli, Rossi & Lehninger, 1964; Greenawalt, Rossi & Lehninger, 1964; Lehninger, Carafoli & Rossi, 1967; Kotyk & Janacek, 1970; Trump, Croker & Mergner, 1971; Bonucci, Derenzini & Marinozzi, 1973). The two genotypes, therefore, elicit both similar and dissimilar phenotypic expressions. Although the two alleles are each, in a homozygous condition, preimplantation lethals, the evidence suggests that they are separate alleles, not a recurrence of the same allele. An ultimate method of testing the uniqueness of these two mutations is by backcrossing each allele with a single isogenic stock until they are both on the same isogenic background. Differences between the two could then be attributed solely to the homozygosity of the allele and not to the effect of a modifying genetic background. Such crosses have, however, generally led to either sterility or fetal and postfetal inviability. At this time, therefore, the expression of the two alleles must be examined on a heterogenic background. Under such conditions the alleles appear to be separate and distinct from each other. The ultrastructural characteristics suggest, however, that the primary effect of these two alleles results in the same or in closely associated developmental aberrations. The research was supported by U.S. Public Health Research Grant HD-00827. The authors would like to acknowledge the technical assistance of Marie Morris and Geraldine Wileman. Studies o/t w 3 2 /t w 3 2 embryos 695 REFERENCES BENNETT, D. & DUNN, L. C. (1964). Repeated occurrences in the mouse of lethal alleles of the same complementation group. Genetics 49, 949-958. BONUCCI, E., DERENZINI, M. & MARINOZZI, V. (1973). The organic-inorganic relationship in calcined mitochondria. /. Cell Biol. 59, 185-211. CARAFOLI, E., ROSSI, C. S. & LEHNINGER, A. L. (1964). Cation and anion balance during active accumulation of Ca ++ and Mg++ by isolated mitochondria. /. biol. Chem. 239,30553061. CHESLEY, P. (1932). Lethal action in the short-tailed mutation in the house mouse. Proc. Soc. exp. Biol. Med. 29, 437-438. GREENAWALT, J. W., ROSSI, C. S. & LEHNINGER, A. L. (1964). Effect of active accumulation of calcium and phosphate ions on the structure of rat liver mitochondria. /. Cell Biol. 23, 21-38. 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