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[An embryological study of mouse ear using the whole embryo culture technique].

Mouse embryos were explanted at 11.5 days of gestation and cultured for periods of 6, 12, 18, 24, 30, 36, 42 and 48 hours using the whole embryo culture system. A continuous flow of gas (5% CO2+95%O2) was maintained through the culture bottles during the culture periods. The cultured embryos were morphologically and histologically examined at the end of the designated periods. The growth of the embryos was depressed after about 36 hours, and differentiation was retarded as the culture time increased. General development had ceased almost completely by 42-48 hours. At 18-36 hours, the external auditory canal and pinna, which are not developed in 11.5 day embryos, could be observed clearly on the surface of the head. After 42 hours, the facial features including the external ear were similar to those of 13 day in vivo embryos. Serial sections of the ear region of cultured embryos showed that development of the inner and middle ear structures up to 42 hours was the same as that seen from 11.5 to 13 days in vivo. In the inner ear, the otocyst had developed into the vestibule, cochlea and semicircular canals by 12-30 hours. In the middle ear, the stapedial anlage appeared and its circular shape became clear between 0 and 12 hours. Subsequently, the medial portion of the stapedial anlage grew until it was adjacent to the developing otic capsule, and the anlages of other ossicles were also recognizable after 18 hours. Differentiation of the horizontal portion of the facial nerve was noted at the same time that stapedial anlage and otic capsule were developing.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effects of oxygen concentration on embryonic development in rats: a light and electron microscopic study using whole-embryo culture techniques.

By using a whole-embryo culture technique (New 1978), the effects of oxygen concentration (5%, 20% and 95% oxygen) on embryonic development in the rat were investigated by light and electron microscopy. The best embryonic development occurred when the 9.5-day-old embryos were cultured for 24 h with 5% oxygen, and the 10.5-day-old embryos with 20% oxygen (optimum oxygen concentration). When the 9.5- and 10.5-day-old embryos were cultured for 24 h with too little or too much oxygen, retardation of the embryonic growth and abnormal development was observed. Using light microscopy, numerous degenerating cells, exhibiting granular deposits in the cytoplasm, were seen, but the distribution of the degenerating cells was quite different between the two groups. With electron microscopy, the most striking feature of the degenerating cells in the embryos cultured with too little oxygen, was the extreme swelling of the mitochondria without any morphological alterations of the nucleus or the other cell organelles. On the other hand, the characteristic feature of the degenerating cells in the embryos exposed to too much oxygen, was the formation of phagolysosomes in the cytoplasm. Morphological alterations of the nucleus or mitochondria were not evident. In the present study, the possible teratogenic mechanism of too much or too little oxygen in the whole-embryo culture of the rat embryo is discussed.

Animals↗

Chick-embryo culture techniques employed at Karnatak University in Dharwad, India, for studying cellular and molecular aspects of morphogenesis.

The objective of the courses which this syllabus describes is to expose developmental biologists to embryo culture and embryo manipulation techniques and applications in quantitative analyses. The laboratory program complements classroom teaching by exposure to both inductive and deductive methodologies. Developmental biology teaching requires good background in cell biology, molecular biology and genetics. Developmental biology research requires computer literacy and an aptitude for quantitative methodology and graphics.

Animals↗

Embryotoxic effects of sodium arsenite and sodium arsenate on mouse embryos in culture.

Embryotoxic effects of two inorganic arsenic compounds, sodium arsenite (Asi) and sodium arsenate (Asa), on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with three to five somites exposed to 1-40 microM Asi or to 10-400 microM Asa were cultured for 48 hours and their development was compared with that of control embryos. Asi proved to be teratogenic between 3 and 4 microM and embryolethal at higher concentrations; Asa had similar activity but at concentrations ten times higher than for Asi. Both compounds produced a growth retardation and a similar pattern of defects. Growth retardation was indicated by a statistically significant reduction in crown-rump length, head length, and yolk sac diameter. Abnormal embryos were characterized by hypoplasia of the prosencephalon with open neural tube, hydropericardium, somite abnormalities, and failure of development of limb buds and sensory placodes. These results confirm that both Asa and Asi are embryotoxic compounds and that the Asi activity occurs at concentrations ten times lower than for Asa. Our results suggest that in humans both of these compounds may be involved in part of "unaccountable" early abortions and malformations claimed to be due to the toxicity of heavy metals.

Animals↗

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture.

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.

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Sterile filtered paraffin oil supports in vitro developmental competence in bovine embryos comparable to co-culture.

PURPOSE: To investigate whether sterile filtered light paraffin oil (SPO) overlaying is superior to washed light mineral oil (WMO) in supporting the in vitro developmental competence of bovine follicular oocytes. In addition, the effects of the two types of oil overlaying were compared with oil overlaying plus co-culture (CC) on bovine embryo development in vitro. METHODS: Bovine follicular oocytes retrieved from abattoir-derived ovary were in vitro matured, fertilized and cultured in 50 microL drops overlayed with WMO or SPO and were subsequently evaluated for development rates. In second experiment, day 2 embryos grown under WMO overlaying were further cultured for 6 days in the presence (WMO+CC and SPO+CC) or absence of adult ear skin fibroblast-based co-culture system overlaid with WMO or SPO. Blastocysts from each group were evaluated for total nuclei number or were further cultured for 48 h to evaluate post-hatching development. RESULTS: SPO overlaying resulted in significant higher (p < 0.05) development rate to morula (44.8% versus 30.6%) and blastocyst (32.8% versus 21.7%) than WMO. Also, treatment of the day 2 embryo cultures with SPO overlaying or oil plus CC (WMO+CC or SPO+CC groups) reached significantly higher development rates from the morula stage compared to embryo cultures treated with the WMO overlaying (p < 0.05). However, the development rates of the SPO treatment group (morula: 72.7%; blastocyst: 53.1%) were slightly high compared to development of the culture treated with WMO+CC (69.6 and 50.4%, respectively). This similar developmental competence pattern was also observed in cell number and embryo hatching rate. CONCLUSION: SPO overlaying is superior to WMO and WMO+CC in supporting in vitro development of bovine embryos. The development rates are further enhanced when embryos are cultured in co-culture system overlaid with SPO. Thus, these data suggest that overlaying oil can significantly influence the pre-implantation embryo development in vitro.

Animals↗

Manipulation of canine fertility using in vitro culture techniques.

Highlighting the important aspects of canine reproductive physiology, this review documents developments made with in vitro culture of canine gametes, and indicates how these techniques can be further exploited. In vitro culture of gametes and embryos has been achieved in many mammalian species, allowing the development of reproductive technology. Despite numerous investigations in other species, only a small number have been performed in dogs. Further studies are required to establish the differences between canine reproductive physiology and that of other mammals. Ultimately, these studies may lead to the development of a gamete salvage technique incorporating oocyte, spermatozoa and embryo culture, and cryopreservation to allow genetically important material from Canidae to be saved. In dogs, the oocyte is ovulated when immature rather than mature, as in other species, and the survival rate of spermatozoa is greater within the female tract. By incorporating knowledge of canine reproductive physiology and work on in vitro culture in other mammals, some important achievements have been made. Sperm capacitation, induction of the acrosome reaction, maturation of canine oocytes and fertilization have all been achieved in vitro. Embryo culture has proven more difficult, with only two studies reporting success. Cryopreservation of spermatozoa has been perfected and is used routinely when transporting semen for artificial insemination. However, improvement in embryo culture techniques and development of oocyte and embryo freezing are required. This work in the domestic model may lead to development of a gamete-banking programme to safeguard the future of endangered canine species.

Acrosome Reaction↗

Dynamic in vivo imaging of mammalian hematovascular development using whole embryo culture.

The yolk sac is the initial site of hematopoiesis in the mammalian embryo. As the embryo develops, blood vessels form around primitive erythroblasts to connect the yolk sac to the embryo, delivering newly formed blood cells to the embryonic circulation. The limited accessibility of the mammalian embryo has made it difficult to study the dynamic changes in cellular development during the formation of the early hematovascular system. Therefore, we have developed a culture system for studying early hematopoiesis, vasculogenesis. and angiogenesis in the mouse embryo. Early embryos (E7.5-E9.5) can be grown on the microscope stage to study the dynamics as vessels form and circulation begins. In addition, this mouse embryo culture system provides an excellent model for understanding the interplay between flow dynamics and cellular development.

Animals↗

[Co-culture of embryos: influencing factors and mechanisms of action].

In comparison with their in vivo counterparts, the in vitro produced mammalian embryos had markedly lower rates of morula/blastocyst development and pregnancy after transfer to the recipients. Things became even worse in the cloned embryos. This necessitates improvement of the embryo culture system. Co-culture of embryos with different types of somatic cells was found beneficial for embryo development in vitro and many studies have been conducted in this area in recent years. In this paper, recent developments and the authors' own work in studies of co-culture of early mammalian embryos with somatic cells were reviewed, with emphasis on the effects of cell type, stage of estrous cycle and number of passages of somatic cells and supplement of serum on embryo development, and the mechanisms by which co-culture promote embryo development. The recent developments are summarized as follows: 1. Somatic cells of both homogeneous and heterogeneous origins can be used for co-culture of mammalian embryos, with similar developmental rates. 2. Supplementation of animal serum at appropriate concentrations improved the somatic cell growth and consequently the development of embryos in co-culture. 3. The estrous cycle stages of oviduct epithelial cells used for co-culture had no effect on the development of embryos. 4. Over-passaging of somatic cells reduced their efficiency in promoting development of the co-cultured embryos. In conclusion, studies have shown that co-culture overcame the block of embryo development in vitro and improved embryo quality with increased rates of implantation and pregnancy, but many problems remain to be solved on its influencing factors and mechanisms of action.

Animals↗

In vitro development of zinc-deficient and replete rat embryos.

The development of the zinc-deficient rat embryo has been studied in vitro using embryo culture techniques. Normal 9.5 day embryos cultured for 48 h in serum obtained from zinc-deficient rats grew and developed to the same extent as those cultured in zinc-replete serum. Embryos from dams which had been fed a zinc-deficient diet since mating were also removed for culture. Such zinc-deficient embryos fell into two broad morphological categories. One group appeared identical to the normal embryos, while the others had apparently normal visceral yolk sacs but small embryonic poles and retarded or abnormal embryonic development. Culture of the first group in either zinc-deficient or replete serum produced morphologically normal embryos; however, those which appeared abnormal at day 9.5 were grossly malformed after 48 h incubation in either sera. When embryos were cultured in the presence of 65Zinc, the most severely affected zinc-deficient embryos accumulated as much zinc as the zinc-replete and apparently unaffected zinc-deficient embryos, indicating that the malformations do not arise from an inability of the embryo or yolk sac to accumulate zinc from the surrounding fluid. The results from these studies suggest that the teratogenic effects of zinc deficiency cannot be induced by direct culture of zinc-replete embryos in zinc-deficient serum. Furthermore, it would appear that maternal zinc deficiency can exert its teratological influence prior to day 9.5 of gestation and that these effects are not readily reversible.

Abnormalities, Multiple↗

Genetic determinants of teratogen-induced abnormal development in mouse and rat embryos in vitro.

The response of an embryo to a teratogenic treatment is often critically dependent on its genetic makeup. However, in conventional in vivo studies of gene-teratogen interactions it may be difficult to distinguish between the effects of genes that are carried by the embryo and those that are carried by the mother. It is likewise not easy to determine whether an observed interaction is between a particular gene and the parent compound administered, or whether it is with a metabolite that has been generated by the maternal system. The use of whole rodent embryo culture offers certain advantages in the study of gene-teratogen interactions. Not only can the effects of metabolism and the maternal genotype be more carefully controlled, but the stage of development at which embryos of different genotypes are exposed can be matched. Rodent whole embryo culture has been used to a limited extent to study interactions between single gene mutations and teratogenic treatments, variations in responses of different strains to teratogens, as well as species differences in response to teratogens. These studies point to the need to precisely control the stage of development at the time of treatment in order to be able to make valid comparisons. But, even more important, they highlight the versatility of the whole embryo culture technique, and underscore the need for its wider use in evaluating the relative contribution of genes and environment to abnormal embryonic development.

Animals↗

Establishment and characterization of new human embryonic stem cell lines.

Human embryonic stem cells (hESC), with their ability to differentiate into all cell types in the human body, are likely to play a very important therapeutic role in a variety of neurodegenerative and life-threatening disorders in the near future. Although more than 120 different human embryonic stem cell lines have been reported worldwide, only a handful are currently available for researchers, which limits the number of studies that can be performed. This study reports the isolation, establishment and characterization of new human embryonic stem cell lines, as well as their differentiation potential into variety of somatic cell types. Blastocyst-stage embryos donated for research after assisted reproductive techniques were used for embryonic stem cell isolation. A total of 31 blastocysts were processed either for immunosurgery or direct culture methods for inner cell mass isolation. A total of nine primary stem cell colonies were isolated and of these, seven cell lines were further expanded and passaged. Established lines were characterized by their cellular and colony morphology, karyotypes and immunocytochemical properties. They were also successfully cryopreserved/thawed and showed similar growth and cellular properties upon thawing. When induced to differentiate in vitro, these cells formed a variety of somatic cell lineages including cells of endoderm, ectoderm and mesoderm origin. There is now an exponentially growing interest in stem cell biology as well as its therapeutic applications for life-threatening human diseases. However, limited availability of stem cell lines as well as financial or ethical limitations restrict the number of research projects. The establishment of new hESC lines may create additional potential sources for further worldwide and nationwide research on stem cells.

Animals↗

Investigations into mechanisms of amino acid supply to the rat embryo using whole-embryo culture.

The technique pioneered by D.A.T. New for the in vitro culture of early post-implantation rat embryos has been used to study nutritional mechanisms during early organogenesis. The results indicate that the principal route for amino acid supply to the 8.5- to 11.5-day embryo involves the endocytosis of proteins into cells of the visceral yolk sac endoderm, their digestion in lysosomes, and transmission of the amino acids to the growing embryo. Free amino acids constitute a comparatively unimportant source. Inhibition of either endocytosis or intralysosomal proteolysis diminishes amino acid supply to the embryo, and this can result in embryonic death or maldevelopment during organogenesis.

Amino Acids↗

Microinjection of cultured rat embryos: a new technique for studies in chemical dysmorphogenesis.

The utility of a new technique for exposure of cultured whole rat embryos to potential dysmorphogens was demonstrated with nitrosofluorene (NF), a cytotoxic and mutagenic metabolite of 2-acetylaminofluorene (AAF). At an initial concentration in the culture medium of 41 microM, NF produced a 100% incidence of defects in axial rotation with no significant effect on prosencephalic development, consistent with previous reports. This route of exposure was also associated with a significant decrease in yolk sac vasculature and protein content. However, when 2 to 20 ng of NF was microinjected directly into the amniotic space, the predominant malformation observed was prosencephalic hypoplasia. Injection of 10 ng NF resulted in approximately equivalent decreases in viability as 41 microM NF dissolved in the culture medium, but produced only a 41% incidence of rotation defects and a 27% incidence of open neural tubes in the rhombencephalic region. The protein content of injected conceptuses was significantly reduced in the embryo, but not in the visceral yolk sac. When 10 ng of NF was injected inside the yolk sac but outside the amnion, the incidence of abnormal rotation was increased to 75%, and the severity of prosencephalic hypoplasia as well as the incidence of neural tube abnormalities was attenuated. The protein content of both the embryo and yolk sac was significantly decreased relative to that of the controls. The data are consistent with the suggestion that NF elicits defects in axial rotation primarily via its effects on the visceral yolk sac and demonstrate the capacity of this technique to provide insights into mechanistic aspects of chemical dysmorphogenesis.

Animals↗

Use of embryo culture to study normal development and developmental mechanisms.

Mammalian embryo culture techniques have been used to study many aspects of embryonic development. The advantages and limitations of such studies as models for in vivo development are discussed by reference to the following specific examples: development of the paraxial mesoderm, regulation of growth, protein uptake and metabolism, and carbohydrate metabolism. Embryo culture techniques are useful for the study of morphogenesis and growth because the embryo is made accessible for manipulation and observation. Development in vivo and in vitro over equivalent periods can be compared. The limitations of the system have important implications for the interpretation of studies of embryonic metabolism. In vitro metabolic activity can be assessed by assays of the culture media and embryonic tissue at intervals throughout the culture. However, the sensitivity of the metabolic pathways to explantation remains unknown because of the technical difficulties involved in studying embryonic metabolism in vivo.

Animals↗