James N. Dumont

Oogenesis in Xenopus laevis (Daudin)

SummaryThis study was designed to explore the relationship of estrogen, human chorionic gonadotropin (HCG), and food availability to endocytosis in developing oocytes. When estrogen alone is administered to an animal, large amounts of vitellogenin are synthesized by the liver and secreted into the circulatory system, where it accumulates. Under these conditions there is no evidence of endocytosis at the surface of the oocytes. Other studies have shown that following HCG injection into estrogen-treated animals, vitellogenin is removed from the circulation and the oocyte surface is highly contoured and displays endocytotic activity. Food deprivation has much the same effect on oocyte endocytosis as does estrogen. When animals are given HCG and subsequently starved for 20 days, developing oocytes show little endocytotic activity. We conclude that HCG acts to promote or stimulate endocytosis in developing oocytes while estrogen and/or starvation inhibits this process.

Frog Embryo Teratogenesis Assay: Xenopus (FETAX) — A Short-Term Assay Applicable to Complex Environmental Mixtures

There is no question that a rapid and inexpensive screening tool is needed to assess potential teratogenicity. The now classical Ames test (Ames, 1975) and other tests screen for potential mutagenicity and carcinogenicity. Although a number of relatively rapid bioassays, ranging from the invertebrates through lower vertebrates to mammals, as well as cell, organ, and embryo culture systems, have been used to examine teratogenicity, none has been adequately validated or widely accepted for routine use. The tier-testing approach, which uses rapid screens to detect potential hazard and indicate need for further testing, has been useful and expedient. Comparable tests, however, are not yet applicable for teratogenesis testing despite the fact that there are a number of methods available. Among these is one which we submit may be a valid model for preliminary assessment of potential teratogens. This model, referred to as FETAX (Frog Embryo Teratogenesis Assay: Xenopus), has been applied to examine the relative teratogenic risk of a variety of chemicals and complex mixtures. The mixtures we have tested come from coal-conversion and shale-oil technologies and their effects have been compared to those of similar materials derived from natural petroleums.

Dedifferentiation of iris epithelial cells

Abstract Electron and phase-contrast microscopic observations demonstrated that depigmentation of iris epithelial cells in vivo after lentectomy is preceded by alterations of cell shape and increases in microfilaments and microtubules in the periphery of the cell. Extensive cell processes are formed, with tips branching into fine strands that contain melanosomes. The macrophages invading the iris epithelium incorporate pieces of such strands, which are composed of cell membrane, cytoplasmic matrix, and melanosomes. Individual melanosomes are also taken up by macrophages. Some of the strands of iris cell processes seem to degenerate within the intercellular space. Thus, depigmentation of iris epithelial cells is accompanied by loss of a substantial part of the cell surface and cytoplasmic matrix of iris epithelial cells. Measurements of the absolute volume of whole iris epithelial cells, their cytoplasm, and their nuclei were conducted at various stages of depigmentation. These measurements reveal an extensive increase in the volume of the cytoplasm preceding activation of the cell surface and a significant reduction in cytoplasmic volume during the phase of extensive depigmentation. In the case of the nuclear volume, the extensive increase which occurs in parallel with that of the cytoplasmic volume is not followed by a significant change during the depigmentation phase.

Toxic and teratogenic effects of selected aromatic amines on embryos of the amphibian Xenopus laevis

Evaluations of the toxic and teratogenic effects of four aromatic amines, acridine, aniline, pyridine, and quinoline, have been made on amphibian (Xenopus laevis) embryos. For toxicity testing, the embryos were divided into three groups according to stage of development: Group I were mid-blastulae, Group II were tailbud embryos, and Group III were swimming larvae. Of the amines tested, acridine and quinoline were the most toxic, followed by aniline and pyridine. Ninety six hr LC50 values for acridine, quinoline, aniline, and pyridine were 4.5, 95, 150, and 1090 mg/L, respectively, for Group III larvae. Except for acridine, where embryos in all three groups were about equally affected, those in Group II were less sensitive to the other amines than those in Groups I and III. Group I embryos were exposed to all four amines to determine their teratogenicity. The amines were ranked in order of decreasing teratogenicity: acridine, quinoline, aniline, and pyridine having 96-hr EC50 values of 2.4, 29, 370, and 1200 mg/L, respectively. Electron microscopic examination of Group III larvae exposed to these amines reveals pathology of the spinal cord and musculature. This damage was correlated with immobility of the larvae. The uptake of acridine was followed in larvae (Group III) exposed to 5 mg/L and was found to reach a maximum level of about 85 mg/g wet weight in about one hr. Depuration kinetics were characterized by a rapid loss of 70% of the total acridine within 45 min. Acridine was undetectable after two hr. These data suggest that acridine, aniline, and quinoline have toxic and teratogenic effects at sufficiently low concentrations as to make them potential environmental hazards.

Structure-toxicity relationships of selected nitrogenous heterocyclic compounds.

A series of eleven nitrogen-containing heterocyclic compounds, which may be potentially associated with aqueous effluents from coal conversion technologies, were examined to determine the relationships between theirn-octanol/H2O partition coefficients, molecular weights, boiling points, and their toxicity monitored as reproduction impairment toTetrahymena pyriformis. Toxicity increases linearly with increased partition coefficient, molecular weight, and boiling point. All of these parameters increase with increased alkyl and ring addition.

Uptake, depuration, and distribution of selenium in Daphnia and its effects on survival and ultrastructure

Selenium is an important essential nutritional trace element which has been shown to provide protection against certain other metal poisoning. However, it is a suspected carcinogen and teratogen. The uptake, depuration, and toxicity of selenium inDaphnia pulex have been examined. The LC50 at 48 and 96 hr for juvenile animals is 0.6 mg/L and 0.1 mg/L respectively, and for adults it is 1.3 mg/L and 0.5 mg/L respectively. Uptake in adult unfed animals is rapid, reaching a maximum at about 12 hr, but depuration is slow. In fed animals, uptake is slower, reaching a maximum at 96 hr, but initial depuration is followed by a slower prolonged loss. Localization in cells is primarily in the cytoplasmic compartment although evidence is presented which suggests nucleolar localization. Ultrastructural damage is detected by 16 hr after exposure and is initially confined to the mitochondria. Dense deposits accumulate in the mitochondrial matrices. The nature of these deposits is unknown; they may represent a calcium-or phosphate-selenium complex. With time, the mitochondria degenerate. It is clear that relatively low concentrations of selenium are toxic to these aquatic organisms and render them incapable of survival in the natural environment. Concentrations higher than those lethal toDaphnia can be expected, at least in local areas, from the burning or conversion of fossil fuels.

Toxicity of selenium to developing Xenopus laevis embryos

Se in the form of sodium selenite is toxic to Xenopus laevis embryos and tadpoles continuously exposed to concentrations above 1 ppm. Concentrations of 2 ppm and above result in severe developmental abnormalities and increased mortality. Uptake and loss of radioactive Se from water are rapid, but depuration is not complete indicating that some Se can remain bound by the organism. The facts that Se is toxic at low levels to Xenopus embryos and tadpoles, can cause developmental abnormalities, and accumulates in tissues suggest that increased release of Se compounds into the environment poses a potential threat to aquatic organisms.

The ovary of Fundulus heteroclitus

Scanning and transmission electron microscopy, together with dissection and light microscopy, have produced heretofore unavailable structural detail of the ovary of Fundulus heteroclitus. Structural and functional interrelationships among developing follicles and other histological elements, particularly as they might relate to vascularization of follicles, oocyte development, and ovulation, are described and discussed. Mature eggs, ovulated into the ovarian lumen, accumulate in the posterior “ovisac” region of the ovary prior to oviposition. This “ovisac” region is thin‐walled and apparently nongerminal. The temporary retention of ovulated eggs permits cyclical oviposition even though oogenesis and ovulation are asynchronous. The histological differences between the ovisac and the anterior ovigerous of the ovary are described. The lumenal epithelium of the ovisac displays a localized population of unusual cells with long cytoplasmic extensions. The ultrastructure of these cells suggests that they might function in the transport of ovulated eggs into the oviduct and/or in secreting the substance (“jelly”) which forms the surface coat of extruded eggs.

Amino acid pools in developing oocytes of Xenopus laevis

Abstract The sizes of amino acid pools in developing oocytes at various stages have been determined for normal and for human chorionic gonadotropin (HCG)-stimulated Xenopus laevis. The total amino acid pool increases 20- to 30-fold in oocytes in stages II through V. This increase is commensurate with the increase in size of the oocyte. In addition, the pool is greater (5% in stage II and 29 to 38% in stages III through V) in oocytes from the HCG-stimulated frogs. Glutamic acid accounts for approximately 35–45% of the total amino acid pool in all stages of oocytes. In none of the stages, however, are there detectable amounts of either proline or cysteine.

Distribution of incorporated and synthesized protein among cell fractions of Xenopus oocytes

Abstract Developing oocytes of Xenopus laevis were isolated, pulsed for 10 minutes with either vitellogenin-3H, 32P or a mixture of l -leucine-3H and 32Pi, and subsequently incubated for various lengths of time in unlabeled medium. Homogenates were then prepared and centrifuged on 20–60% sucrose gradients. Vitellogenin-3H, 32P was found to associate initially with membranous material, but within 45 min more than half the label was associated with the yolk platelets. Since it takes at least 60–120 min for vitellogenin to be converted into lipovitellin and phosvitin, this transformation must occur within the platelets rather than at the oocyte surface or within pinosomes. Eighteen hours after a pulse with l -leucine-3H and 32Pi, neither the yolk nor the mitochondria fraction became significantly labeled; this indicates that the macromolecular components of these structures are not synthesized or phosphorylated by the oocyte to any great extent during vitellogenesis. Instead, various components within the membranous and “soluble” regions became labeled.