Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos.
BACKGROUND
Using vital dyes, we have previously shown that while hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death within specific tissues (e.g., neuroepithelium) of day 9 mouse embryos, cells of the heart are resistant to the cell death-inducing potential of these teratogens. Subsequent work has shown that teratogen-induced cell death is associated with activation of the mitochondrial apoptotic pathway, i.e., release of cytochrome c from mitochondria, activation/cleavage of procaspase-9, -3, and -2, inactivation of poly(ADP-ribose) polymerase, and internucleosomal fragmentation of DNA, whereas resistance to teratogen-induced cell death in the heart is associated with a failure to activate this pathway. Teratogen-induced activation of the mitochondrial apoptotic pathway is initiated between 2.5 and 5 hr after teratogens are added to the culture medium. Because both the heart and the surrounding yolk sac are essential to successful development of mouse embryos during early postimplantation mouse development, we hypothesized that cells of the yolk sac are also resistant to teratogen-induced cell death.


METHODS
To test our hypothesis, we cultured day 8.5 mouse conceptuses (embryo plus yolk sac) in whole embryo culture. On the morning of day 9, conceptuses were exposed to HS (43 degrees C for 15 min and then returned to 37 degrees C), 4CP (40 microM, 5-10 hr), or ST (0.5 microM 5-10 hr). At 5 and 10 hr after addition of teratogen, conceptuses were removed from culture and dissected into embryo and yolk sac. Activation of the mitochondrial apoptotic pathway was then assessed separately in embryos and yolk sacs using Western blot analysis to detect activation of procaspase-9, -3, and -2, enzyme assays to measure caspase-3-like activity, and immunohistochemistry to detect caspase-3 activation/cleavage in yolk sac cells.


RESULTS
Although Western blot analysis revealed that procaspase-9, -3, and -2 were activated/cleaved in the embryo as early as the 5-hr time point, activation/cleavage of these caspases could not be detected in the yolk sac at either the 5- or 10-hr time point. Using an enzyme assay, we determined that caspase-3-like activity in the yolk sac was induced 1.7-fold by HS, 4.4-fold by 4CP, and 3.3-fold by ST. This compares to the embryo in which caspase-3-like activity was induced 45-fold by HS, 26-fold by 4CP, and 45-fold by ST. Using an antibody specific for the active p17 subunit of caspase-3 and immunohistochemistry, we were able to detect a small number of yolk sac cells showing caspase-3 activation. Thus, the low-level induction of caspase-3-like activity in the yolk sac is in part related to activation/cleavage of procaspase-3.


CONCLUSIONS
Results presented indicate that cells of the extraembryonic yolk sac, like cells of the embryonic heart, are substantially more resistant to teratogen-induced activation of the mitochondrial apoptotic pathway and subsequent apoptosis compared to other embryonic tissues, particularly cells of the neuroepithelium.
Soleman
Donna
D
Birth Defects Research Laboratory, Division of Genetics and Development, Department of Pediatrics, University of Washington, Seattle, Washington 98195, USA.
Cornel
Leanne
L
Little
Sally A
SA
Mirkes
Philip E
PE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000633:toxicity; D004351:Drug Resistance; D004789:Enzyme Activation; D005260:Female; D005318:Fetal Heart / Q000187:drug effects / Q000648:ultrastructure; D005326:Fetal Proteins / Q000378:metabolism*; D005865:Gestational Age; D006257:Head / Q000196:embryology; D006358:Hot Temperature / Q000009:adverse effects*; D051379:Mice; D008928:Mitochondria / Q000502:physiology*; D009924:Organ Culture Techniques; D009928:Organ Specificity; D011247:Pregnancy; D019311:Staurosporine / Q000633:toxicity*; D013723:Teratogens / Q000633:toxicity*; D015017:Yolk Sac / Q000187:drug effects* / Q000648:ultrastructure
D005326:Fetal Proteins; D013723:Teratogens; D003520:Cyclophosphamide; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine; C011272:perfosfamide