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Why not artificial wombs?
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[Biomedicine and procreation: what will the future hold?].
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Would artificial wombs produce more harm than good?
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Transplanting and deep-freezing cows' eggs.
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Reproduction and the test tube baby.
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Studies on ectopic liver utilizing hepatocyte transplantation into the rat spleen.
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[Prospective study of human fertilization in vitro. Various technical and ethical aspects].
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[Latent differentiation in the endoderm of the young chick embryo].
Initiation and localization of the capacity of self-differentiation in the 1-2 day old chick embryo were studied by means of culturing endoderm fragments, wrapped in a piece of viteline membrane and completely deprived of mesenchyme.
Culture of early somite mouse embryos during organogenesis.
Early somite (2-4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum and Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.
Latent effects on in vitro development following cytochalasin B treatment of 8-cell mouse embryos.
Eight-cell mouse embryos when treated with 4.0 microgram/ml cytochalasin B (CB) in vitro undergo a reversible developmental arrest. Upon rinsing of embryos and subsequent culture in control medium, normal morphogenetic processes such as compaction of 8-cell embryos, cavitation, and post-blastocyst attachment and outgrowth are restored. However, the effects of CB on mouse embryos are not completely reversible; latent post-blastocyst defects become increasingly more prevalent as CB treatment duration increases. The present study was conducted to quantitatively determine latent effects of CB on post-blastocyst embryos by comparing their ability to attach and to sustain the growth and differentiation of ICM and trophoblast tissues. Groups of 8-cell embryos were cultured in Brinster's BMOC-3 medium containing 4.0 microgram/ml cytochalasin B for 6, 12, 18, and 24 h. Following treatment, embryos were rinsed and cultured until 190 h post coitum (h.p.c.) in Eagle's MEM/10% fetal calf serum modified to contain optimal levels of essential amino acids. Blastocysts generally attached to the surface of the plastic substratum by 120 h.p.c. At selected time periods after attachment (130, 160, and 190 h.p.c.), embryos were scored for outgrowth size, ICM size, extent of peripheral hyaloplasmic fan, and number of trophoblast nuclei per outgrowth. Analyses of variance (ANOVAs) were conducted for each of the four parameters listed above. Rates of attachment were analyzed by chi2 test. Results show that the treatments affect (P less than 0.01) embryo attachment, number of trophoblast nuclei per outgrowth, hyaloplasmic fan production, and ICM growth in a duration-dependent manner. Interestingly, since treatment effects on outgrowth areas are nonsignificant apparently CB does not significantly change total outgrowth area. But CB treatment does cause abnormal fan production and decreased trophoblast nuclei numbers. However, trophoblast cells are apparently more resistant than ICM to CB as is evident by the high incidence of trophoblast outgrowths devoid of ICM. CB (4.0 microgram/ml) treatments at 8-cell stages for relatively short durations (6 and 12 h) induce latent effects on post-blastocyst embryos. Finally, there exists a definite 4.0 microgram/ml CB duration response over the 68-190 h.p.c. observation interval.
Properties of extra-embryonic ectoderm isolated from postimplantation mouse embryos.
Extra-embryonic ectoderm isolated from the mouse embryo as late as 81/2 days post coitum can form cells with the morphological characteristics of trophoblast giant cells both in ectopic sites and in vitro. This similarity to the properties of ectoplacental cone tissue provides further support for the postulated common origin of both tissues from the trophectoderm of the blastocyst.
Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos.
Methods are described for preparing and analyzing single preimplantation mouse embryos for a variety of metabolites and cofactors (glucose-6-P, fructose-6-P, fructose-1,6-bisphosphate, ATP, AMP, Pi, citrate, isocitrate, alpha-ketoglutarate, and malate). Oil-well and enzymatic cycling techniques are combined to provide the sensitivity needed to measure the amounts present (10(-12) to 1o(-15) moles). After experimental treatment, embryos are collected on glass slides and freeze-dried. They can then be stored indefinitely under vacuum at -25 degrees C without deterioration. With these procedures, the embryos were collected at successive stages of development and subjected to starvation and refeeding with glucose, pyruvate or both. The results confirm the existence of a block at early stages at the P-fructokinase step. This may be due to inhibition by the very high citrate levels present. The data suggest that glycolysis is turned on late in preimplantation development by the rise in fructose-6-P, a deinhibitor of P-fructokinase. In the citrate cycle, no step between citrate and alpha-ketoglutarate is rate-limiting, but a step between alpha-ketaglutarate and malate appears to impede the flux at early embryonic stages.
[A hidden explosion: reproduction and babies in test tubes].
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Development of the rat conceptus in vitro and associated changed in components of culture medium.
Rat conceptuses of pregnancy day 11 (embryonic age approximately 10-5 days) with embryos within the yolk and amniotic sacs and polar allantoic placental tissues were cultured by the 'New system' utilizing homologous serum in 60 ml roller bottles with 20% O2, 5% CO2 and 75% N2 gas environment (five conceptuses/10 ml serum). Embryonic growth and development was assessed by external morphology and somite counts, and by measurement of DNA, RNA and protein contents. The embryonic growth was considerably retarded beyond 24 h when culture was extended to 48 h. During continuous culture of conceptuses for 48 h, the partial pressure of O2 in the medium decreased gradually with simultaneous increase of pCO2 and decline of pH values. Glucose level was depleted considerably and small quantities of urea, uric acid and creatinine accumulated in the medium. The concentrations of electrolytes (Na+, K+, Cl-) and total Ca and P, and lactic dehydrogenase activity in the medium increased during the latter part of the 48 h culture period. The growth and differentiation of embryos were significantly improved by transferring conceptuses to fresh serum medium with 40% O2, 5% CO2 and 55% N2 gas phase after 22-24 h culture and continuing the culture for an additional 24 h. Embryos grown in vitro for 48 h developed 39 somites and showed a tenfold increase in DNA content and a five- to seven-fold increase in RNA and protein contents, over initial values.
Determination of the embryonic origin of the mesencephalic nucleus of the trigeminal nerve in birds.
The precise site of origin and the cells of origin of the mesencephalic nucleus of the trigeminal nerve in birds have been studied by the method of growing embryonic fore-, mid- and hind brain grafts inside a millipore filter chamber filled with amniotic fluid, upon a host chorioallantoic membrane, and by the method of transplantation of cranial neural crest of the mesencephalon between embryo-donors and duck embryo-hosts. The growth and differentiation of grafts grown in the chambers resemble very closely those of normal embryos at corresponding stages of development. Data obtained from a total of 42 grafts representative of the three species of birds used in this study, show that the cells of the mesencephalic nucleus are present only in grafts of embryonic midbrain and totally absent in forebrain and hindbrain grafts. The presence of cells of the mesencephalic nucleus in midbrain grafts suggests that these neurons arise chiefly from the developing mesencephalon. Histological examination of such grafts has shown that these neurons are observed in the leptomeningeal tissue overlying the midbrain region and extend into the various laminae of the optic tecta. A progressive increase in the size of the cells from an immature state confined to the mesodermal tissue to fully mature neurons in the stratum griseum periventriculare of the optic tectum is observed. This is also indicative of a downward migration of these cells. In a few embryonic midbrain grafts mature cells clearly identifiable as belonging to the mesencephalic nucleus are observed in the leptomeningeal tissue and outside the external limiting membrane of the neural tube. These appear to be neurons which had failed to migrate. The resemblance of these latter neurons to those in the stratum griseum periventriculare of the optic tectum lend further support to the hypothesis that the neurons of the mesencephalic nucleus are derived from neural crest of the developing mesencephalon in birds. In interspecific transplantation experiments of cranial neural crest of the mesencephalon, the quail cells occur in the form of clusters and appear to migrate towards the ventricular surface independent of the migration of the cells of the tectum which takes place simultaneously. Three stages in the development of the grafted neural crest material of the quail are observed based on the perikaryal diameter of the cells, and the accumulation of Nissl material in the cytoplasm. The smallest cells are located more superficially near the pial surface, and the larger mature neurons are observed in the stratum griseum periventriculare of the optic tectum. All the layers of the optic tectum including the neuroepithelium on the experimental side are comprised entirely of duck cells...
Ultrastructural observations on the chorionic epithelium, parathyroid glands and bones from chick embryos developed in shell-less culture.
Three-day-old chick embryos were cultured in Petri dishes until they reached developmental stages 37 or 39 (Hamburger & Hamilton, 1951). The electron microscopical study of the chorio-allantoic membranes showed that, even in the absence of the shell and the corresponding calcium supply, 'capillary-covering' and 'villus-cavity' cells differentiated well. The parathyroid glands from the cultured embryos showed ultrastructural signs indicative of active synthesis and secretion of the parathyroid hormone. This correlates well with the significant hypocalcemia (5.4 mg/100 ml) observed in these embryos. In all cultured embryos mineralization of bones was greatly reduced as shown by alizarin bulk staining and confirmed by histological and electron microscopical analysis. The ultrastructural characteristics of osteoblasts and osteocytes, as well as of the bone matrix, appeared normal. The defect in mineralization appeared thus to be due to a deficiency in the availability of calcium and not to a delay in bone differentiation. This implies that the yolk sac appears to lack calcium regulatory capacity since it cannot compensate for the absence of shell by increasing its own contribution in order to assure an adequate mineralization of the embryonic bones.
Changes in responsiveness of preimplantation mouse embryos to serum.
Changes in uptake of radioactive uridine and its incorporation into RNA were determined in preimplantation mouse embryos, from the 2-cell to the blastocyst stage, as a measure of their responsiveness to extracellular conditions. Two media were tested, one contained serum and the other contained bovine serum albumen as a control. An increase in the acid-soluble pool occurred at the 8-cell stage and a marked increase in RNA synthesis occurred at the early blastocyst stage when the embryos were incubated with serum.