Harold H. Lee

Effects of metals on sea urchin development: A rapid bioassay☆

Abstract Embryos of sea urchins show a differential response to Cd, Cu, Cr, Ni and Zn. The responses are also stage-specific. The different morphological effects of various metals reflect the differentiation of the early embryonic cells. Since fertilization can be inhibited in the presence of metals, especially Cu, the ability of the gametes to undergo fertilization can then be used as a biological indicator for a rapid assay, 10–20 min., of marine pollutants. Since embryo-genesis up to plutei stage requires only 20 h, this duration is also short compared to other systems utilizing embryonic or larval forms.

INTERACTIONS OF 1-METHYLADENINE AND THE SURFACE OF STARFISH OOCYTE

Three metabolic inhibitors, mycostatin, concanavalin A (Con A) and cytochalasin B (CB) were used to study the interactions between l‐methyladenine (1‐MA) and the starfish oocyte surface leading to germinal vesicle breakdown (GVB). Mycostatin and Con A had no obvious effects on GVB. CB did not inhibit, but did delay GVB. This delaying effect was interpreted as having multiple 1‐MA reactive “sites” on the surface. The results also suggested that not all of them were needed to react with 1‐MA to bring about GVB.

MEMBRANE REACTIONS DURING FERTILIZATION AND 1‐METHYLADENINE STIMULATION IN IMMATURE STARFISH OOCYTES

Immature oocytes from starfishes were obtained during the breeding and non‐breeding seasons. The oocytes were “fertilized” without previous induction of maturation by 1‐methyladenine (1‐MA) or germinal vesicle breakdown (GVB). Elevation of the fertilization membrane (FM) was observed with eggs obtained during the breeding season, but not observed with eggs obtained during the non‐breeding season. Abnormal development was observed when these “fertilized” eggs with a FM were subsequently treated with 1‐MA. The results of these experiments indicated that the elevation of FM was independent of GVB or the mixing of nuclear and cytoplasmic components.

Reaggregation and reorganization of juvenile chicken testicular cellsin vitro

Abstract A culture system has been developed which maintains the characteristics of the androgen-producing cells together with fibroblastic cells dissociated from juvenile chicken testes. The behavior of these cells, including their ability to reaggregate in vitro and reorganize into distinct patterns in monolayer cultures, indicates that the maintenance of a stable arrangement of homotypic cells is achieved by intercellular materials holding the cells in discrete groupings.

A simplified and effective method for freezing and storage of chick embryo fibroblasts at −79°C*

Summary A simple method has been worked out forfreezing and storage of chick embryo fibroblastic cells. Cell suspensions were shelf-frozen at −75°C to −79°C, stored in the growth medium, and supplemented with 10% dimethyl sulfoxide in polypropylene tubes. More than 90% of the cells which were stored for 220 days were still viable after thawing.

Differential response of marine organisms to certain metal and agrichemical pollutants

Oocyte maturation of the starfish, fertilization and embryogenesis of sea urchins, and the development of amphioxus and brine shrimps were used to assay the effects of several common metals and agrichemicals frequently found in marine environments. While brine shrimp embryos were tolerant to metals and agrichemicals used here, sea urchins and amphioxus showed a differential response to the common metal pollutants. Starfish oocyte maturation process was affected by agrichemicals. The results show that no one single organism, or its embryonic form, or a particular stage of development, can be used as the indicator for a particular pollutant. However, the use of lower forms of marine organisms can be useful collectively for environmental investigations and the management of waste disposal.

Incorporation of [3H]glucosamine to the surface component of testicular cultures and the differential response of steroidogenic and fibroblastic cells in vitro to cytochalasin B

SummaryIt has been demonstrated that glucosamine-containing components of the surfaces of various types of cultured cells can be isolated by trypsin treatment under conditions in which the cells remain viable. By using this method, we found that the surface-associated materials of testicular cells in vitro may be stable once formed. Cytochalasin B inhibits reversibly the incorporation of [3H]glucosamine into these macromolecules, whereas ctinomycin D only slightly reduces the uptake. There is also a differential response of the steroidogenic versus the fibroblastic cell populations to cytochalasin B treatment, the steroidogenic cells being much more sensitive to cytochalasin B than the fibroblastic cells. Based upon the present studies, and those of others, we hypothesized that during histogenesis in vitro there may exist more than one synthetic process for cell surface components. Stability of tissue structures may require a stable factor (s), which may differ from the aggregating factor.

Effects of inhibitors on 1-methyladenine induced maturation of starfish oocytes

Abstract1-methladenine (1-MA) induces starfish oocytes maturation via surface reaction followed by the appearance of a cytoplasmic maturation factor which in turn induces germinal vesicle breakdown (GVBD) to resume meiosis. Cellular mechanisms involved in GVBD were investigated by microinjection of metabolic inhibitors. Colchicine (Co) inhibited maturation, cytochalasin-B (CB) delayed GVBD and actinomycin-D-(Act-D) and puromycin (Pu) had no effect. It appears that the microtubule and the microfilament systems are associated with the nuclear membrane dissolution during the process of oocyte maturation of starfish.

UPTAKE AND LOCALIZATION OF 3H‐1‐METHYLADENINE BY THE GONADAL EPITHELIAL CELLS OF SEA URCHINS IN VITRO

The meiotic inducing hormone, 1‐methyladenine, first isolated from starfish has been implicated to play some role in the gamete maturation of a number of marine invertebrates. However, there have been controversial and sometimes opposite conclusions due to the fact that there is no direct bioassay system other than in starfish.