In Vitro Analysis of Pre- and Early Postimplantation Development of Lethal Yellow (AWAY) Mouse Embryos LINDA L. JOHNSON AND NELS H. GRANHOLM Department of Biology, South Dakota State Uniuersity, Brookings, South Dakota 57007
ABSTRACT
Preimplantation embryos from matings between yellow heterozygous (AYIa) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control m a t ings (AYIa Q x ala d,ala ? x Ayla d,ala ? x ala d). Most embryos were a t the 8-cell stage a t recovery; however fewer embryos from the experimental cross had developed to t h e 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P < 0.01). The yellow (AYIa) uterus did not contribute (P = 0.05) t o delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying t h e class of AylAy mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (101106) of t h e morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (101398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24196) of embryos t h a t developed successfully to t h e blastocyst stage failed to hatch from t h e zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.
When two heterozygous yellow (AYIa) mice are mated, approximately 25% of the embryos are homozygous yellow (Ay/Ay) and lethal a t implantation. In utero, AYIAY embryos die as partially implanted blastocysts (Ibsen and Steigleder, '17; Kirkham, '19; Robertson, '42; Eaton and Green, '63; Eaton, '68). Pedersen ('74) reported t h a t AylAy embryos can be identified in vitro after the 8-cell stage by t h e presence of excluded blastomeres and subsequently die without hatching from t h e zona pellucida. Cinematographic observations have indicated t h a t AY expression may cause a developmental lag of AylAy embryos as early as the 2- t o 4-cell stage (Pedersen and Spindle, '76). In addition to AY-caused problems in embryos, there is also evidence t h a t uteri of yellow heterozygotes (AYIa) may inherently be poorer environments for developing embryos than non-yellow uteri (Ibsen and Steigleder, '17; Kirkham, '19; Robertson, '42; Wolff and J. EXP. ZOOL. (1978)204: 381-390.
Bartke, '66; Cizadlo e t al., '75). Adult yellow heterozygotes have also been shown to possess a number of abnormalities including obesity, susceptibility to certain cancers, reproductive inefficiency in females, and others (reviewed by Cizadlo, '76). Thus yellow embryos (AYIAY and AYla) developing in yellow uteri may well be deleteriously affected by their genome as well as factors within the uterine milieu. Recognizing the importance of controlling these variables, Ibsen and Steigleder ('17) were the first investigators to utilize four crosses (1experimental and 3 controls) in their study on causes of death of homozygous yellow embryos. The present study was undertaken to pinpoint t h e AYIAY phenocritical period and characterize the generation of lethal yellow embryos in vitro in order ultimately to discover basic biochemical alterations specified by AY. Our precise objectives were to: (1)define morphological characteristics that will identify
381
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LINDA L. JOHNSON AND NELS H. GRANHOLM
the class of AYIAY homozygotes in preimplantation embryo culture, (2) discover t h e extent of the phenocritical period of AY homozygotes, (3) test the background variability and abnormalities occurring in three control crosses a s compared to the experimental cross, (4) assess t h e role of yellow (AYla) versus black (ala) uteri by comparing numbers and condition of embryos upon recovery and subsequent culture, and (5) quantitatively confirm and extend the observations of Pedersen ('74) and Pedersen and Spindle ('76). MATERIALS AND METHODS
Source of mice and embryo recovery Mice were derived from C57BLIGJ-AYIa and -ala stock obtained from the Jackson Laboratory, Bar Harbor, Maine. One Ay/a or ala male was housed with three to four AYIa or a/a females. Three control (9 AYIa x 0' a / a , 0 a / a x d AYIa, and ala x ala) and one experimental cross (AYIa x AYla) were used. All females were examined for the presence of a vaginal plug a t 8:00 A.M. daily. Females having vaginal plugs were assumed to have copulated a t the midpoint of the dark cycle (0200) and hours post coitum (h.p.c.) were calculated from t h a t time. Embryos were flushed (Rafferty, '70) from female reproductive tracts at 56 to 60 h.p.c., rinsed through two changes of Brinster's BMOC-3 Medium (GIBCO, Grand Island, New York), and transferred to 35-mm plastic culture dishes (Falcon) containing 2.0 ml of BMOC-3. Embryos were scored for developmental stage and morphology using phase-contrast microscopy. Scoring criteria a r e comprehensively outlined in Johnson ('77). Embryo culture Embryos were cultured for 44 to 48 hours in Brinster's BMOC-3 Medium. At 104 h.p.c. embryos were transferred to dishes containing Eagle's Minimum Essential Medium (GIBCO) with 10% Fetal Calf Serum (GIBCO) and cultured for a n additional 54 hours; this medium allows embryos to attach to t h e substrate and trophoblast cells to outgrow. Embryos were observed, scored a t 6- and 18hour intervals, and photographs of normal and abnormal embryos were taken throughout the 5-day culture period (plates 1, 2). Embryos were scored as arrested if they were 24 hours behind normal staging. Embryos were scored as abnormal if any blastomere was disintegrating, if the blastomeres were of unequal
size, if t h e embryo was fragmenting or granular, if the embryo was shrunken within the zona pellucida, or if the cells constituting the embryo did not form a n integrated structure (Pedersen, '74). Embryos were also scored for hatching and outgrowth success. Data were statistically evaluated using x 2 analysis and analysis of variance. RESULTS
Embryos at recovery At 56-60 h.p.c. 638 embryos from 93 females were recovered from oviducts, observed, and scored for stage and condition of embryonic development (table 1). An analysis of variance revealed t h a t mean numbers of embryos per female were not different among the four crosses (computed F value = 0.32). The number of embryos judged to be morphologically abnormal a t recovery between t h e four groups is also presented in table 1. The experimental cross (AYIa x AY/a) yielded 7.5% (16/212) abnormal embryos, while control groups had 8.8% (13/148), 3.5'%,(6/169), and 5.5% (61109) abnormal embryos respectively. A chi square test of all four groups revealed no differences in the number of morphologically abnormal embryos (computed x 2 = 4.3). However, a chi square comparison of abnormal embryos between th.e yellow female by black male and the reciprocal cross yielded a computed x' value of 3.8 which falls into the P = 0.05-0.10 range (0.05 < P < 0.10). In order to compare developmentally retarded embryos between groups, developmental stages a t recovery (table 1) were mathematically pooled into two categories for comparison, i.e., embryos less than or equal to the 4-cell stage and embryos greater than or equal to the 8-cell stage. Fewer embryos from the experimental group (P < 0.01) had reached the 8-cell stage a t 56-60 h.p.c. than embryos of control crosses (computed x 2 = 22.9). Differences in developmental stages of embryos among t h e three control groups were not significant (computed x' = 5.3). Culture of embryos Following their recovery at 56-60 h.p.c., 575 embryos from 77 females were cultured for 98 to 102 hours until 158 h.p.c. (6.6 days). Table 2 provides data on the developmental progress of embryos from the four crosses. In order to statistically test for differences in developmental success between experimental and control groups, contingency tables were deter-
383
I N VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS TABLE 1
AnalysLs of embryos at recovery (56-60 hpc) ~~
Parental genotype
Total number of females'
Total number of embryos
Average number of embryosJ
k> abnormal at flushing
AYIax AYIa
31
212
6.8
AYlax ala
22
148
6.7
25
169
6.8
7.5 (161212) 8.8 (13i148) 3.5 (61169) 5.5 (61109)
9
d
ala X A y l a ala X ala I
J
15
109
7.3
Stage a t flushing
2-cell
4-cell
8-cell
5 2.3%'
57 26.9% 25 16.9X 16 9.5% 20 18.3%
139 65.5X 122 82.4% 147 87.0% 86 78.9'%,
0 0.0% 1 0.6% 1 0.9%
Blavto~ cyst
Morula
4 1.9% 1 0.7'%,
7 3.3'X 0
o.o'%, 3 1.8'%,
2
1.2% 2 1.8X
0
o.o'%,
All females were nulliparous and < 100 days of age. The percentage figure represents the number of embryos in t h a t class divided by the total number of embryos for that cross Standard error was 2 0.32 lor AYIa X AYIa. r 0.42 for AYIaY x d a d , ? 0.31 for alar x AYIa,! and % 0.46 for ala x a/a
TABLE 2
Development of embryos in culture (56-158 h.p.c.1 Parent genotype
Total number
V
females
Total number of embryos
AYIa x AYIa
16
113
AY/a x ala
17
117
alax AY/a
23
172
alax aia
21
173
of
1
d
b
x,
Y;.
developed to morula ' A
developed to blastocyst
hatched from zona pellucida
outgrown
93.8 (1061113) 82.9 (971117) 86.0 (148i172) 88.4 (153/173)
85.0 (961113) 81.2 (951117) 84.3 (1451172) 85.5 (1481173)
63.7 (721113) 72.6 (851117) 75.0 (1291172) 79.2 (1371173)
61.1 (691113) 65.8 (771117) 72.7 (1251172) 77.5 11341173)
'
4,
'
Percent flgure in each category represents the percent of the total number of embryos Ln each cross. Computed 2' value = 1.0, critical x' values for P = 0.05 and 3 degrees of freedom is 7 8;P = 0.05-0.10 Computed x z value = 1.1;nonsignificant Computed x' value = 8.6; P < 0.05. Computed x' value = 10 5; P < 0.05.
mined based on t h e ratio of successful to unsuccessful development, and chi square analyses were conducted for each developmental stage (footnotes 2-5, table 2). Chi square analyses using t h e total number of embryos from each cross revealed t h a t no differences at t h e P = 0.05 level existed between groups in their ability to successfully develop to morulae and blastocysts (footnotes 2 and 3, table 2). In contrast, when comparing development to blastocysts following successful morula formation (rather than total number of embryos from each cross as presented in table 21, significant differences were observed; fewer experimental embryos (90.6%) were able to complete development from morulae to blastocysts (x2 = 11.0; P = 0.0100.025) t h a n controls (97.976, 98.0%, and 96.7% respectively). Similarly significant differences (P < 0.05) were observed in t h e hatch-
ing success of experimental versus control embryos (footnote 4, table 2). Of the embryos from Ay/a x Ayla matings which successfully developed to blastocyst stages only 75.0% (72/ 96) hatched from the zona pellucida. In control crosses t h e number of embryos t h a t hatched after normal blastocyst development were 89.5% (85/95), 89.0% (129/145), and 92.6% (1371148) in yellow female x black male, black female x yellow male, and black female x black male crosses, respectively. As a result of hatching failure in the AY/a X AyIa cross, fewer experimental embryos outgrew (P < 0.05) than did control embryos (footnote 5, table 2). In t h e experimental group, 95.8% (69/ 72) of the embryos t h a t hatched from the zona pellucida successfully outgrew. Of t h e embryos which hatched in control groups, 90.6% (771851, 96.9% (12511291, and 97.8% (134/137) respectively formed outgrowths.
384
LINDA L. JOHNSON AND NELS H. GRANHOLM TABLE 3
Characterization of abnormal embryos from recovery through five days of culture (56-158 h.p.c.1 Parent genotype Y
d
AYIax AYIa
AYlax ala alax AYla a l a x ala
Total number of abnormal embryos I
38.9% (441113) 34.2% (401117) 27.3% (471172) 22.5% (391173)
stage
&cell stage
Morula stage
Blastocyst stage
Post blastocyst stage ’’
4.5x (2144) 10.0% (4140) 14.9% (7147) 10.3% (4139)
9.1% (4/44) 17.5% (7140) 23.4% (11147) 23.1% (9/39)
2.3% (11441 25.0% (10/40) 12.8% (6147) 20.5% (8/39)
22.1% (10144) 2.5% (11401 8.5% (4147) 10.3% (4139)
61.4% (27/44) 45.0% (18140) 40.4% (191471 35.9% (14139)
2-4 cell
’ Represents the number of embryos (from table 1) that were abnormal at recovery (56 h.p.c.1 or became abnormal during the four day culture penod (from table 2). Represents the stage at which embryos were first judged abnormal. Computed x’ value = 2.7; nonsignificant. Computed x’ value = 3.9; nonsignificant. Computed x z = value = 9.9; P < 0.05. DComputedx 2 value = 9.4; P < 0.05. ’ Computed x’ value = 6.4; P = 0.05-0.10. Table 3 summarizes data on abnormal embryos from experimental and control crosses according to the developmental stage at which they were first judged abnormal. A chi square test indicates t h a t the total number of abnormal embryos in the experimental cross was greater ( x 2 = 10.5, P < 0.05) than in control matings. In order to statistically test for differences in the generation of stage-specific abnormalities, chi square analyses of contingency tables were conducted (footnotes 3-7, table 3 ) . No differences were found in the numbers of embryos judged abnormal in experimental and control crosses a t either the 2- to 4-cell or 8-cell stages. However, fewer embryos from Ayla x Ayla crosses were first judged abnormal a t t h e morula stage (P < 0.05) than control embryos at t h a t stage. The number of embryos first revealing abnormalities at t h e blastocyst stage was higher in the experimental (P < 0.05) than control crosses. Also abnormalities which first appeared during postblastocyst development, although not statistically different (at P = 0.05) between groups, were more frequent in embryos from experimental matings (P = 0.05-0.10). The same types of abnormalities were present among embryos from all matings, with no unique morphological characteristics identifying A y l A y mutants through blastocyst development. Stages of normal development a r e observed in plate 1. Abnormalities included blastomere disintegration (plate 2, fig. c), blastomere exclusion (plate 2, figs. d,e), granularity (plate 2, fig. a), unequally sized blas-
tomeres and fragmented or vesiculated embryos (plate 2, figs. b,g). In t h e experimental cross 15.9%of abnormal embryos had excluded blastomeres. However blastomere exclusion was evident in 17.5%of abnormal embryos from the yellow female control cross and in 10.6% of abnormal embryos from the black female x yellow male cross. Interestingly, no embryos from black x black crosses were observed to have excluded blastomeres. The greatest incidence of abnormal development in experimental embryos was hatching failure. In this instance embryos t h a t had formed blastocysts failed to hatch and instead collapsed within the zona pellucida. Unhatched embryos became disorganized; inner cell mass and trophoblast cells were indistinguishable (plate 2, fig. f). Control and experimental cleavage stage embryos with excluded blastomeres would often develop into small morulae and blastocysts with t h e excluded cells hanging on the periphery (plate 2, figs. d,e). However, blastocysts in all crosses with excluded cells were unable to hatch from the zona pellucida. DISCUSSION
Since no differences in mean number of embryos per female were observed in the four crosses tested (table 11, apparently Ay homozygotes are present and viable a t 56-60 h.p.c. However, significantly fewer experimental embryos (P < 0.01) had cleaved to 8cell stages a t recovery, and there exists a class of developmentally lagging embryos (pre-
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS
sumably AYlAy) within experimental (AY/a x Ayla) populations. No corresponding developmental lag was observed in embryos from t h e yellow female by black male cross; accordingly, delayed development is evidently due to factors inherent in the yellow homozygote and not to the yellow uterus. In addition, since no differences were observed in the number of morphologically abnormal embryos at recovery (56-60 h.p.c.1 between experimental and control groups, factors causing retardation in presumed AY homozygotes do not grossly (morphologically) disrupt development prior to recovery. Data reported in this study thus quantitatively document the observation of Pedersen and Spindle ('76) who reported t h a t effects of AY homozygosity occur during early cleavage stages. Furthermore, although t h e yellow uterine environment may adversely affect development (Wolff and Bartke, '66; Cizadlo e t al., '751, embryos within P AYIa x 8 a/a matings were not developmentally delayed by the yellow uterus up to t h e time of recovery. Even though some experimental embryos were lagging in development at recovery, they were a s capable of developing to morula stages as were control embryos. Although some develop to blastocysts, the normality of presumed AY homozygotes is questionable. Pedersen and Spindle ('76) report t h a t presumed AYIAY blastocysts possess about half a s many blastomeres as non-mutant littermates (33 & 5 for AYIAY, n = 15 embryos versus 60 t 4 for AY/a and ala, n = 36 embryos). Also, histological analyses of embryos within uterine tracts at 105 h.p.c. revealed t h a t 24 embryos from yellow by yellow matings averaged 30.2 -+ 2.4 ICM nuclei, whereas 22 embryos from nonyellow female by yellow male crosses P a / a x d AYIa) averaged 41.7 -t 4.5 ICM nuclei, a significant (P < 0.05) difference (Cizadlo and Granholm, '77). Our d a t a showed t h a t 25.0% (24196) of t h e blastocysts from experimental crosses failed to hatch from their zonae. Similarly, Pedersen ('74) reported a 26.9% hatching failure and considered t h e class of unhatched blastocysts to be homozygous yellow mutants. In the present study 9.5% (371388) of t h e pooled controls t h a t developed to blastocysts failed to hatch; therefore, 25.0% minus 9.5% or 15.5% (15196) of t h e blastocysts within experimental crosses were presumably AY homozygotes. After successfully hatching from zonae, embryos from all four crosses were equally capable of grow-
385
ing out, i.e., 95.8% (69/72), 90.6%) (77/85), 96.9% (1251129), and 97.8% (1341137) respectively. Since 95.8% (69/72) of t h e embryos from experimental matings outgrew normally after hatching success, AY homozygotes were most likely left behind in culture as hatching failures. Pedersen ('74) reported t h a t AYIAY embryos could be identified as cleavage embryos, morulae, and blastocysts by the presence of excluded blastomeres. However, in the present study embryos with excluded blastomeres were common to control as well as experimental groups and did not represent a distinct class in the yellow heterozygous cross. If indeed AY homozygotes can be exclusively identified by the presence of excluded blastomeres, it was not evident in this study. Although homozygous AY expression may cause a developmental lag during early cleavage, there appears to be no distinct period after this time when AY homozygosity causes unique embryo abnormalities t h a t aid in the identification of AY homozygotes prior to arrest. The AY/AY phenocritical period in vitro may extend throughout preimplantation development, showing effects of AY expression at various times. I t does appear, however, t h a t homozygous yellow embryos are incapable of hatching from their zonae in vitro. I n our study hatching failure accounted for 15.5% of the 25.0%Mendelian expectation, and morulato-blastocyst failures accounted for another 6.9%. Thus AY homozygotes are probably defective at cleavage stages and throughout preimplantation development (Pedersen and Spindle, '76; Cizadlo and Granholm, '78). A significantly higher percentage (P < 0.05) of abnormal blastocysts were observed in experimental (22.7%)versus control (2.5'%1,8.5'%,, and 10.3%)crosses respectively (table 3). Mutants t h a t do survive to blastocysts are unable, in t h e i r presumably weakened condition, to hatch. In summary, although the basic biochemical alteration specified by AY remains unknown, data from this study indicate a developmental lag of some experimental embryos (P < 0.01) which may be due to early effects of homozygous AY expression. Since no comparable lagging embryos from yellow female by black male crosses were found, yellow uterine factors apparently do not contribute to the observed developmental retardation. Although developmentally retarded, present data indicate t h a t homozygous yellow
386
LINDA L. JOHNSON AND NELS H. GRANHOLM
embryos are capable of development to t h e blastocyst stage, but AYIAY blastocysts are apparently unhealthy and weakened. Moreover, our observations reveal that excluded blastomeres may not be totally reliable indicators of AY homozygosity. Finally, data presented here confirm the previous observation of Pedersen ('74) t h a t hatching failure is unique to AY homozygotes in vitro. ACKNOWLEDGMENTS
The authors wish to acknowledge financial support from the South Dakota State University Agricultural Experiment Station (SD 7371, National Institutes of Health (HD 086311, and t h e Lalor Foundation. This manuscript has been approved for publication as Journal Series No. 1506 by t h e Director, Agricultural Experiment Station, South Dakota State University. LITERATURE CITED Calarco, P. G., and R. A. Pedersen 1976 Ultrastructural observations of lethal yellow (AY/AV mouse embryos. J. Embryol. exp. Morph., 35: 73-80. Cizadlo, G. R. 1976 Physiologic and Embryologic Studies of the Yellow (AY/a) Mouse. Ph.D. Dissertation. South Dakota State University. Brookings, South Dakota. Cizadlo, G. R., and N. H. Granholm 1978 In uiuo develop-
ment of the lethal yellow (AYiAY) mouse embryo at 105 hourspost coifurn. Genetica, 48. Cizadlo, G. R., S . K. Hoffman and N. H. Granholm 1975 Mating characteristics and embryonic losses in the yellow (Aria) mouse. Proc. S. D. Acad. Sci.,54: 157-161. Eaton, G. J. 1968 Stimulation of trophoblastic giant cell differentiation in the homozygous yellow mouse embryo. Genetica, 39: 371-378. Eaton, G. J., and M. M. Green 1963 Giant cell differentiation and lethality of homozygous yellow mouse embryos. Genetica, 34: 155-161. Ibsen, H. L., and E. Steigleder 1917 Evidence for the death in utero of the homozygous yellow mouse. Am. Nat., 51: 740-752. Johnson, L. L. 1977 ln uitto development of pre- and early post-implantation lethal yellow (AY/AY) mouse em^ bryos. M.S. Thesis. South Dakota State University, Brookings, South Dakota. Kirkham, W. B. 1919 The fate of homozygous yellow mice. J. Exp. Zool., 28: 125-135. Pedersen, R. A. 1974 Development of lethal yellow (AY/ AY) mouse embryos in uitro. J. Exp. Zool., 188: 307-320. Pedersen, R. A., and A. I. Spindle 1976 Genetic effects of mammalian development during and after implantation. In: Embryogenesis in Mammals. I . Elliott and M. O'Connor, eds. Ciba Foundation Symposium, New York. Rafferty, K. A., J r . 1970 Methods in Experimental em^ bryology of t h e Mouse. Johns Hopkins Press, Baltimore, Maryland. Robertson, G. G. 1942 An analysis of the development of homozygous yellow mouse embryos. J. Exp. Zool., 89: 197-231. Wolff, G. L., and A. Bartke 1966 Decreased survival of embryos in yellow (Aria) female mice. J. Heredity,57: 14.17.
PLATE 1 EXPLANATION OF FIGURES
Normal embryo development Four-cell embryo from Aria 9 X a/a d mating, 56 h.p.c., showing the typical cruciform arrangement with two pairs of cells a t right angles to one another. The blastomeres are nearly spherical and each is in contact with the other three. x 540. Eight-cell embryo from AY/a 9 x a/a d mating, 56 h.p.c., showing spherical, distinct blastomeres. x 549. Morula with evident polar body from a/a ? X AY/a d mating, 86 h.p.c., showing relatively smooth surface contour and indistinct blastomeres. x 540. Blastocyst from a/a 9 X AY/a d mating, 104 h.p.c., showing the inner cell mass in the lower right corner and flattened trophoblast cells surrounding the periphery of the embryo, with a large blastocoelic cavity in t h e center. X 432. Blastocyst hatching from the zona pellucida, from AYia 9 x Aria d, 104 h.p.c., showing the cell pushing out in the area of the ruptured zona. x 344. Outgrowth from a/a ? X a/a d mating, 158 h.p.c., showing evident inner cell mass (ICM) atop flattened trophoblast cells. Trophoblast nuclei are large and the outgrowth has a fan-like appearance. X 149.
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS Linda L. Johnson and Nels H. Granholm
PLATE 1
387
PLATE 2 EXPLANATION OF FIGURES
Abnormal embryo development a
Granular 4-cell embryo from AYia 9 equal size. X 460.
X
a/a d mating, 56 h.p.c., showing dark granular blastomeres of un-
b Degenerate embryo from AY/a 9 X a/a d mating, 62 h.p.c., showing dense disorganized mass with large clear vesicles on the periphery contacting the zona pellucida. X 395. c Abnormal 8-cel embryo from a/a P X AY/a d mating, 56 h.p.c., showing disintegrating blastomeres (arrow) with indistinct cell boundaries. At least four blastomeres still maintain cellular integrity. X 459. d Abnormal morula from AY/a 9 X a/ad mating, 80 h.p.c., showing small granular morula with two large excluded blastomeres. Judging from their size, the blastomeres appear to have been excluded a t the 8-cell stage. X 450. e Abnormal blastocyst from aia? X AY/ad mating, 104 h.p.c., showing dark excluded blastomeres (arrow), and evident bulging of trophoblast cells on the periphery. X 457.
388
f
Collapsed blastocyst from a/a? X AYiad mating, 152 h.p.c. At 104 h.p.c. the blastocyst had a small blastocoelic cavity and was shrunken away from the zona pellucida. Now there is no evident cavity and the embryo is dense and disorganized. x 383.
g
Abnormal blastocyst from aia? X AYiad mating, 132 h.p.c., showing blunt oblong protrusions projected in the area of the rupturized zona pellucida. The blastocoelic cavity is evident but there is no organization within the embryo. X 410.
h
Small abnormal blastocyst from a/a9 X AY/ad mating, 128 h.p.c., showing excluded material on the periphery of the embryo and extending through a small rupture in the zona pellucida and outward. The blastocoelic cavity is evident. x 188.
i
Shrunken, disorganized blastocyst (presumed lethal mutant) from AY/aP X Arkad, 104 h.p.c., showing a normally ruptured zona pellucida. The blastocyst was previously expanded but then collapsed and failed to hatch. X 390.
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS Linda L Johnson and Nels H. Granholm
PLATE 2
389
ABSTRACT
Preimplantation embryos from matings between yellow heterozygous (AYIa) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control m a t ings (AYIa Q x ala d,ala ? x Ayla d,ala ? x ala d). Most embryos were a t the 8-cell stage a t recovery; however fewer embryos from the experimental cross had developed to t h e 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P < 0.01). The yellow (AYIa) uterus did not contribute (P = 0.05) t o delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying t h e class of AylAy mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (101106) of t h e morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (101398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24196) of embryos t h a t developed successfully to t h e blastocyst stage failed to hatch from t h e zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.
When two heterozygous yellow (AYIa) mice are mated, approximately 25% of the embryos are homozygous yellow (Ay/Ay) and lethal a t implantation. In utero, AYIAY embryos die as partially implanted blastocysts (Ibsen and Steigleder, '17; Kirkham, '19; Robertson, '42; Eaton and Green, '63; Eaton, '68). Pedersen ('74) reported t h a t AylAy embryos can be identified in vitro after the 8-cell stage by t h e presence of excluded blastomeres and subsequently die without hatching from t h e zona pellucida. Cinematographic observations have indicated t h a t AY expression may cause a developmental lag of AylAy embryos as early as the 2- t o 4-cell stage (Pedersen and Spindle, '76). In addition to AY-caused problems in embryos, there is also evidence t h a t uteri of yellow heterozygotes (AYIa) may inherently be poorer environments for developing embryos than non-yellow uteri (Ibsen and Steigleder, '17; Kirkham, '19; Robertson, '42; Wolff and J. EXP. ZOOL. (1978)204: 381-390.
Bartke, '66; Cizadlo e t al., '75). Adult yellow heterozygotes have also been shown to possess a number of abnormalities including obesity, susceptibility to certain cancers, reproductive inefficiency in females, and others (reviewed by Cizadlo, '76). Thus yellow embryos (AYIAY and AYla) developing in yellow uteri may well be deleteriously affected by their genome as well as factors within the uterine milieu. Recognizing the importance of controlling these variables, Ibsen and Steigleder ('17) were the first investigators to utilize four crosses (1experimental and 3 controls) in their study on causes of death of homozygous yellow embryos. The present study was undertaken to pinpoint t h e AYIAY phenocritical period and characterize the generation of lethal yellow embryos in vitro in order ultimately to discover basic biochemical alterations specified by AY. Our precise objectives were to: (1)define morphological characteristics that will identify
381
382
LINDA L. JOHNSON AND NELS H. GRANHOLM
the class of AYIAY homozygotes in preimplantation embryo culture, (2) discover t h e extent of the phenocritical period of AY homozygotes, (3) test the background variability and abnormalities occurring in three control crosses a s compared to the experimental cross, (4) assess t h e role of yellow (AYla) versus black (ala) uteri by comparing numbers and condition of embryos upon recovery and subsequent culture, and (5) quantitatively confirm and extend the observations of Pedersen ('74) and Pedersen and Spindle ('76). MATERIALS AND METHODS
Source of mice and embryo recovery Mice were derived from C57BLIGJ-AYIa and -ala stock obtained from the Jackson Laboratory, Bar Harbor, Maine. One Ay/a or ala male was housed with three to four AYIa or a/a females. Three control (9 AYIa x 0' a / a , 0 a / a x d AYIa, and ala x ala) and one experimental cross (AYIa x AYla) were used. All females were examined for the presence of a vaginal plug a t 8:00 A.M. daily. Females having vaginal plugs were assumed to have copulated a t the midpoint of the dark cycle (0200) and hours post coitum (h.p.c.) were calculated from t h a t time. Embryos were flushed (Rafferty, '70) from female reproductive tracts at 56 to 60 h.p.c., rinsed through two changes of Brinster's BMOC-3 Medium (GIBCO, Grand Island, New York), and transferred to 35-mm plastic culture dishes (Falcon) containing 2.0 ml of BMOC-3. Embryos were scored for developmental stage and morphology using phase-contrast microscopy. Scoring criteria a r e comprehensively outlined in Johnson ('77). Embryo culture Embryos were cultured for 44 to 48 hours in Brinster's BMOC-3 Medium. At 104 h.p.c. embryos were transferred to dishes containing Eagle's Minimum Essential Medium (GIBCO) with 10% Fetal Calf Serum (GIBCO) and cultured for a n additional 54 hours; this medium allows embryos to attach to t h e substrate and trophoblast cells to outgrow. Embryos were observed, scored a t 6- and 18hour intervals, and photographs of normal and abnormal embryos were taken throughout the 5-day culture period (plates 1, 2). Embryos were scored as arrested if they were 24 hours behind normal staging. Embryos were scored as abnormal if any blastomere was disintegrating, if the blastomeres were of unequal
size, if t h e embryo was fragmenting or granular, if the embryo was shrunken within the zona pellucida, or if the cells constituting the embryo did not form a n integrated structure (Pedersen, '74). Embryos were also scored for hatching and outgrowth success. Data were statistically evaluated using x 2 analysis and analysis of variance. RESULTS
Embryos at recovery At 56-60 h.p.c. 638 embryos from 93 females were recovered from oviducts, observed, and scored for stage and condition of embryonic development (table 1). An analysis of variance revealed t h a t mean numbers of embryos per female were not different among the four crosses (computed F value = 0.32). The number of embryos judged to be morphologically abnormal a t recovery between t h e four groups is also presented in table 1. The experimental cross (AYIa x AY/a) yielded 7.5% (16/212) abnormal embryos, while control groups had 8.8% (13/148), 3.5'%,(6/169), and 5.5% (61109) abnormal embryos respectively. A chi square test of all four groups revealed no differences in the number of morphologically abnormal embryos (computed x 2 = 4.3). However, a chi square comparison of abnormal embryos between th.e yellow female by black male and the reciprocal cross yielded a computed x' value of 3.8 which falls into the P = 0.05-0.10 range (0.05 < P < 0.10). In order to compare developmentally retarded embryos between groups, developmental stages a t recovery (table 1) were mathematically pooled into two categories for comparison, i.e., embryos less than or equal to the 4-cell stage and embryos greater than or equal to the 8-cell stage. Fewer embryos from the experimental group (P < 0.01) had reached the 8-cell stage a t 56-60 h.p.c. than embryos of control crosses (computed x 2 = 22.9). Differences in developmental stages of embryos among t h e three control groups were not significant (computed x' = 5.3). Culture of embryos Following their recovery at 56-60 h.p.c., 575 embryos from 77 females were cultured for 98 to 102 hours until 158 h.p.c. (6.6 days). Table 2 provides data on the developmental progress of embryos from the four crosses. In order to statistically test for differences in developmental success between experimental and control groups, contingency tables were deter-
383
I N VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS TABLE 1
AnalysLs of embryos at recovery (56-60 hpc) ~~
Parental genotype
Total number of females'
Total number of embryos
Average number of embryosJ
k> abnormal at flushing
AYIax AYIa
31
212
6.8
AYlax ala
22
148
6.7
25
169
6.8
7.5 (161212) 8.8 (13i148) 3.5 (61169) 5.5 (61109)
9
d
ala X A y l a ala X ala I
J
15
109
7.3
Stage a t flushing
2-cell
4-cell
8-cell
5 2.3%'
57 26.9% 25 16.9X 16 9.5% 20 18.3%
139 65.5X 122 82.4% 147 87.0% 86 78.9'%,
0 0.0% 1 0.6% 1 0.9%
Blavto~ cyst
Morula
4 1.9% 1 0.7'%,
7 3.3'X 0
o.o'%, 3 1.8'%,
2
1.2% 2 1.8X
0
o.o'%,
All females were nulliparous and < 100 days of age. The percentage figure represents the number of embryos in t h a t class divided by the total number of embryos for that cross Standard error was 2 0.32 lor AYIa X AYIa. r 0.42 for AYIaY x d a d , ? 0.31 for alar x AYIa,! and % 0.46 for ala x a/a
TABLE 2
Development of embryos in culture (56-158 h.p.c.1 Parent genotype
Total number
V
females
Total number of embryos
AYIa x AYIa
16
113
AY/a x ala
17
117
alax AY/a
23
172
alax aia
21
173
of
1
d
b
x,
Y;.
developed to morula ' A
developed to blastocyst
hatched from zona pellucida
outgrown
93.8 (1061113) 82.9 (971117) 86.0 (148i172) 88.4 (153/173)
85.0 (961113) 81.2 (951117) 84.3 (1451172) 85.5 (1481173)
63.7 (721113) 72.6 (851117) 75.0 (1291172) 79.2 (1371173)
61.1 (691113) 65.8 (771117) 72.7 (1251172) 77.5 11341173)
'
4,
'
Percent flgure in each category represents the percent of the total number of embryos Ln each cross. Computed 2' value = 1.0, critical x' values for P = 0.05 and 3 degrees of freedom is 7 8;P = 0.05-0.10 Computed x z value = 1.1;nonsignificant Computed x' value = 8.6; P < 0.05. Computed x' value = 10 5; P < 0.05.
mined based on t h e ratio of successful to unsuccessful development, and chi square analyses were conducted for each developmental stage (footnotes 2-5, table 2). Chi square analyses using t h e total number of embryos from each cross revealed t h a t no differences at t h e P = 0.05 level existed between groups in their ability to successfully develop to morulae and blastocysts (footnotes 2 and 3, table 2). In contrast, when comparing development to blastocysts following successful morula formation (rather than total number of embryos from each cross as presented in table 21, significant differences were observed; fewer experimental embryos (90.6%) were able to complete development from morulae to blastocysts (x2 = 11.0; P = 0.0100.025) t h a n controls (97.976, 98.0%, and 96.7% respectively). Similarly significant differences (P < 0.05) were observed in t h e hatch-
ing success of experimental versus control embryos (footnote 4, table 2). Of the embryos from Ay/a x Ayla matings which successfully developed to blastocyst stages only 75.0% (72/ 96) hatched from the zona pellucida. In control crosses t h e number of embryos t h a t hatched after normal blastocyst development were 89.5% (85/95), 89.0% (129/145), and 92.6% (1371148) in yellow female x black male, black female x yellow male, and black female x black male crosses, respectively. As a result of hatching failure in the AY/a X AyIa cross, fewer experimental embryos outgrew (P < 0.05) than did control embryos (footnote 5, table 2). In t h e experimental group, 95.8% (69/ 72) of the embryos t h a t hatched from the zona pellucida successfully outgrew. Of t h e embryos which hatched in control groups, 90.6% (771851, 96.9% (12511291, and 97.8% (134/137) respectively formed outgrowths.
384
LINDA L. JOHNSON AND NELS H. GRANHOLM TABLE 3
Characterization of abnormal embryos from recovery through five days of culture (56-158 h.p.c.1 Parent genotype Y
d
AYIax AYIa
AYlax ala alax AYla a l a x ala
Total number of abnormal embryos I
38.9% (441113) 34.2% (401117) 27.3% (471172) 22.5% (391173)
stage
&cell stage
Morula stage
Blastocyst stage
Post blastocyst stage ’’
4.5x (2144) 10.0% (4140) 14.9% (7147) 10.3% (4139)
9.1% (4/44) 17.5% (7140) 23.4% (11147) 23.1% (9/39)
2.3% (11441 25.0% (10/40) 12.8% (6147) 20.5% (8/39)
22.1% (10144) 2.5% (11401 8.5% (4147) 10.3% (4139)
61.4% (27/44) 45.0% (18140) 40.4% (191471 35.9% (14139)
2-4 cell
’ Represents the number of embryos (from table 1) that were abnormal at recovery (56 h.p.c.1 or became abnormal during the four day culture penod (from table 2). Represents the stage at which embryos were first judged abnormal. Computed x’ value = 2.7; nonsignificant. Computed x’ value = 3.9; nonsignificant. Computed x z = value = 9.9; P < 0.05. DComputedx 2 value = 9.4; P < 0.05. ’ Computed x’ value = 6.4; P = 0.05-0.10. Table 3 summarizes data on abnormal embryos from experimental and control crosses according to the developmental stage at which they were first judged abnormal. A chi square test indicates t h a t the total number of abnormal embryos in the experimental cross was greater ( x 2 = 10.5, P < 0.05) than in control matings. In order to statistically test for differences in the generation of stage-specific abnormalities, chi square analyses of contingency tables were conducted (footnotes 3-7, table 3 ) . No differences were found in the numbers of embryos judged abnormal in experimental and control crosses a t either the 2- to 4-cell or 8-cell stages. However, fewer embryos from Ayla x Ayla crosses were first judged abnormal a t t h e morula stage (P < 0.05) than control embryos at t h a t stage. The number of embryos first revealing abnormalities at t h e blastocyst stage was higher in the experimental (P < 0.05) than control crosses. Also abnormalities which first appeared during postblastocyst development, although not statistically different (at P = 0.05) between groups, were more frequent in embryos from experimental matings (P = 0.05-0.10). The same types of abnormalities were present among embryos from all matings, with no unique morphological characteristics identifying A y l A y mutants through blastocyst development. Stages of normal development a r e observed in plate 1. Abnormalities included blastomere disintegration (plate 2, fig. c), blastomere exclusion (plate 2, figs. d,e), granularity (plate 2, fig. a), unequally sized blas-
tomeres and fragmented or vesiculated embryos (plate 2, figs. b,g). In t h e experimental cross 15.9%of abnormal embryos had excluded blastomeres. However blastomere exclusion was evident in 17.5%of abnormal embryos from the yellow female control cross and in 10.6% of abnormal embryos from the black female x yellow male cross. Interestingly, no embryos from black x black crosses were observed to have excluded blastomeres. The greatest incidence of abnormal development in experimental embryos was hatching failure. In this instance embryos t h a t had formed blastocysts failed to hatch and instead collapsed within the zona pellucida. Unhatched embryos became disorganized; inner cell mass and trophoblast cells were indistinguishable (plate 2, fig. f). Control and experimental cleavage stage embryos with excluded blastomeres would often develop into small morulae and blastocysts with t h e excluded cells hanging on the periphery (plate 2, figs. d,e). However, blastocysts in all crosses with excluded cells were unable to hatch from the zona pellucida. DISCUSSION
Since no differences in mean number of embryos per female were observed in the four crosses tested (table 11, apparently Ay homozygotes are present and viable a t 56-60 h.p.c. However, significantly fewer experimental embryos (P < 0.01) had cleaved to 8cell stages a t recovery, and there exists a class of developmentally lagging embryos (pre-
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS
sumably AYlAy) within experimental (AY/a x Ayla) populations. No corresponding developmental lag was observed in embryos from t h e yellow female by black male cross; accordingly, delayed development is evidently due to factors inherent in the yellow homozygote and not to the yellow uterus. In addition, since no differences were observed in the number of morphologically abnormal embryos at recovery (56-60 h.p.c.1 between experimental and control groups, factors causing retardation in presumed AY homozygotes do not grossly (morphologically) disrupt development prior to recovery. Data reported in this study thus quantitatively document the observation of Pedersen and Spindle ('76) who reported t h a t effects of AY homozygosity occur during early cleavage stages. Furthermore, although t h e yellow uterine environment may adversely affect development (Wolff and Bartke, '66; Cizadlo e t al., '751, embryos within P AYIa x 8 a/a matings were not developmentally delayed by the yellow uterus up to t h e time of recovery. Even though some experimental embryos were lagging in development at recovery, they were a s capable of developing to morula stages as were control embryos. Although some develop to blastocysts, the normality of presumed AY homozygotes is questionable. Pedersen and Spindle ('76) report t h a t presumed AYIAY blastocysts possess about half a s many blastomeres as non-mutant littermates (33 & 5 for AYIAY, n = 15 embryos versus 60 t 4 for AY/a and ala, n = 36 embryos). Also, histological analyses of embryos within uterine tracts at 105 h.p.c. revealed t h a t 24 embryos from yellow by yellow matings averaged 30.2 -+ 2.4 ICM nuclei, whereas 22 embryos from nonyellow female by yellow male crosses P a / a x d AYIa) averaged 41.7 -t 4.5 ICM nuclei, a significant (P < 0.05) difference (Cizadlo and Granholm, '77). Our d a t a showed t h a t 25.0% (24196) of t h e blastocysts from experimental crosses failed to hatch from their zonae. Similarly, Pedersen ('74) reported a 26.9% hatching failure and considered t h e class of unhatched blastocysts to be homozygous yellow mutants. In the present study 9.5% (371388) of t h e pooled controls t h a t developed to blastocysts failed to hatch; therefore, 25.0% minus 9.5% or 15.5% (15196) of t h e blastocysts within experimental crosses were presumably AY homozygotes. After successfully hatching from zonae, embryos from all four crosses were equally capable of grow-
385
ing out, i.e., 95.8% (69/72), 90.6%) (77/85), 96.9% (1251129), and 97.8% (1341137) respectively. Since 95.8% (69/72) of t h e embryos from experimental matings outgrew normally after hatching success, AY homozygotes were most likely left behind in culture as hatching failures. Pedersen ('74) reported t h a t AYIAY embryos could be identified as cleavage embryos, morulae, and blastocysts by the presence of excluded blastomeres. However, in the present study embryos with excluded blastomeres were common to control as well as experimental groups and did not represent a distinct class in the yellow heterozygous cross. If indeed AY homozygotes can be exclusively identified by the presence of excluded blastomeres, it was not evident in this study. Although homozygous AY expression may cause a developmental lag during early cleavage, there appears to be no distinct period after this time when AY homozygosity causes unique embryo abnormalities t h a t aid in the identification of AY homozygotes prior to arrest. The AY/AY phenocritical period in vitro may extend throughout preimplantation development, showing effects of AY expression at various times. I t does appear, however, t h a t homozygous yellow embryos are incapable of hatching from their zonae in vitro. I n our study hatching failure accounted for 15.5% of the 25.0%Mendelian expectation, and morulato-blastocyst failures accounted for another 6.9%. Thus AY homozygotes are probably defective at cleavage stages and throughout preimplantation development (Pedersen and Spindle, '76; Cizadlo and Granholm, '78). A significantly higher percentage (P < 0.05) of abnormal blastocysts were observed in experimental (22.7%)versus control (2.5'%1,8.5'%,, and 10.3%)crosses respectively (table 3). Mutants t h a t do survive to blastocysts are unable, in t h e i r presumably weakened condition, to hatch. In summary, although the basic biochemical alteration specified by AY remains unknown, data from this study indicate a developmental lag of some experimental embryos (P < 0.01) which may be due to early effects of homozygous AY expression. Since no comparable lagging embryos from yellow female by black male crosses were found, yellow uterine factors apparently do not contribute to the observed developmental retardation. Although developmentally retarded, present data indicate t h a t homozygous yellow
386
LINDA L. JOHNSON AND NELS H. GRANHOLM
embryos are capable of development to t h e blastocyst stage, but AYIAY blastocysts are apparently unhealthy and weakened. Moreover, our observations reveal that excluded blastomeres may not be totally reliable indicators of AY homozygosity. Finally, data presented here confirm the previous observation of Pedersen ('74) t h a t hatching failure is unique to AY homozygotes in vitro. ACKNOWLEDGMENTS
The authors wish to acknowledge financial support from the South Dakota State University Agricultural Experiment Station (SD 7371, National Institutes of Health (HD 086311, and t h e Lalor Foundation. This manuscript has been approved for publication as Journal Series No. 1506 by t h e Director, Agricultural Experiment Station, South Dakota State University. LITERATURE CITED Calarco, P. G., and R. A. Pedersen 1976 Ultrastructural observations of lethal yellow (AY/AV mouse embryos. J. Embryol. exp. Morph., 35: 73-80. Cizadlo, G. R. 1976 Physiologic and Embryologic Studies of the Yellow (AY/a) Mouse. Ph.D. Dissertation. South Dakota State University. Brookings, South Dakota. Cizadlo, G. R., and N. H. Granholm 1978 In uiuo develop-
ment of the lethal yellow (AYiAY) mouse embryo at 105 hourspost coifurn. Genetica, 48. Cizadlo, G. R., S . K. Hoffman and N. H. Granholm 1975 Mating characteristics and embryonic losses in the yellow (Aria) mouse. Proc. S. D. Acad. Sci.,54: 157-161. Eaton, G. J. 1968 Stimulation of trophoblastic giant cell differentiation in the homozygous yellow mouse embryo. Genetica, 39: 371-378. Eaton, G. J., and M. M. Green 1963 Giant cell differentiation and lethality of homozygous yellow mouse embryos. Genetica, 34: 155-161. Ibsen, H. L., and E. Steigleder 1917 Evidence for the death in utero of the homozygous yellow mouse. Am. Nat., 51: 740-752. Johnson, L. L. 1977 ln uitto development of pre- and early post-implantation lethal yellow (AY/AY) mouse em^ bryos. M.S. Thesis. South Dakota State University, Brookings, South Dakota. Kirkham, W. B. 1919 The fate of homozygous yellow mice. J. Exp. Zool., 28: 125-135. Pedersen, R. A. 1974 Development of lethal yellow (AY/ AY) mouse embryos in uitro. J. Exp. Zool., 188: 307-320. Pedersen, R. A., and A. I. Spindle 1976 Genetic effects of mammalian development during and after implantation. In: Embryogenesis in Mammals. I . Elliott and M. O'Connor, eds. Ciba Foundation Symposium, New York. Rafferty, K. A., J r . 1970 Methods in Experimental em^ bryology of t h e Mouse. Johns Hopkins Press, Baltimore, Maryland. Robertson, G. G. 1942 An analysis of the development of homozygous yellow mouse embryos. J. Exp. Zool., 89: 197-231. Wolff, G. L., and A. Bartke 1966 Decreased survival of embryos in yellow (Aria) female mice. J. Heredity,57: 14.17.
PLATE 1 EXPLANATION OF FIGURES
Normal embryo development Four-cell embryo from Aria 9 X a/a d mating, 56 h.p.c., showing the typical cruciform arrangement with two pairs of cells a t right angles to one another. The blastomeres are nearly spherical and each is in contact with the other three. x 540. Eight-cell embryo from AY/a 9 x a/a d mating, 56 h.p.c., showing spherical, distinct blastomeres. x 549. Morula with evident polar body from a/a ? X AY/a d mating, 86 h.p.c., showing relatively smooth surface contour and indistinct blastomeres. x 540. Blastocyst from a/a 9 X AY/a d mating, 104 h.p.c., showing the inner cell mass in the lower right corner and flattened trophoblast cells surrounding the periphery of the embryo, with a large blastocoelic cavity in t h e center. X 432. Blastocyst hatching from the zona pellucida, from AYia 9 x Aria d, 104 h.p.c., showing the cell pushing out in the area of the ruptured zona. x 344. Outgrowth from a/a ? X a/a d mating, 158 h.p.c., showing evident inner cell mass (ICM) atop flattened trophoblast cells. Trophoblast nuclei are large and the outgrowth has a fan-like appearance. X 149.
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS Linda L. Johnson and Nels H. Granholm
PLATE 1
387
PLATE 2 EXPLANATION OF FIGURES
Abnormal embryo development a
Granular 4-cell embryo from AYia 9 equal size. X 460.
X
a/a d mating, 56 h.p.c., showing dark granular blastomeres of un-
b Degenerate embryo from AY/a 9 X a/a d mating, 62 h.p.c., showing dense disorganized mass with large clear vesicles on the periphery contacting the zona pellucida. X 395. c Abnormal 8-cel embryo from a/a P X AY/a d mating, 56 h.p.c., showing disintegrating blastomeres (arrow) with indistinct cell boundaries. At least four blastomeres still maintain cellular integrity. X 459. d Abnormal morula from AY/a 9 X a/ad mating, 80 h.p.c., showing small granular morula with two large excluded blastomeres. Judging from their size, the blastomeres appear to have been excluded a t the 8-cell stage. X 450. e Abnormal blastocyst from aia? X AY/ad mating, 104 h.p.c., showing dark excluded blastomeres (arrow), and evident bulging of trophoblast cells on the periphery. X 457.
388
f
Collapsed blastocyst from a/a? X AYiad mating, 152 h.p.c. At 104 h.p.c. the blastocyst had a small blastocoelic cavity and was shrunken away from the zona pellucida. Now there is no evident cavity and the embryo is dense and disorganized. x 383.
g
Abnormal blastocyst from aia? X AYiad mating, 132 h.p.c., showing blunt oblong protrusions projected in the area of the rupturized zona pellucida. The blastocoelic cavity is evident but there is no organization within the embryo. X 410.
h
Small abnormal blastocyst from a/a9 X AY/ad mating, 128 h.p.c., showing excluded material on the periphery of the embryo and extending through a small rupture in the zona pellucida and outward. The blastocoelic cavity is evident. x 188.
i
Shrunken, disorganized blastocyst (presumed lethal mutant) from AY/aP X Arkad, 104 h.p.c., showing a normally ruptured zona pellucida. The blastocyst was previously expanded but then collapsed and failed to hatch. X 390.
IN VITRO DEVELOPMENT OF AYIAY MOUSE EMBRYOS Linda L Johnson and Nels H. Granholm
PLATE 2
389
