DEVELOPMENTAL

46,40-48 (1975)

BIOLOGY

Changes

of Uridine

Permeability Tunicate

during

the Maturation

of

Eggs

CHARLES C. LAMBERT Department Fullerton,

of Biological

California

Science and Institute for Molecular Biology, California State University-Fullerton, 92634, and Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington 98250 Accepted

April

15, 1975

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [*H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 &f show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (- 1%) and Na+-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis. INTRODUCTION

Fertilization initiates a program of biochemical and functional changes in the egg which ultimately leads to the formation of a new individual. Early changes in 0, consumption (Epel, 1964, for review), permeability to phosphate (Litchfield and Whiteley, 1959; Whiteley and Chambers, 1966), amino acids (Epel, 1972), uridine (Piatigorsky and Whiteley, 1965), cytidine (Mitchison and Cummins, 1966) and potassium (Steinhardt, Shen and Mazia, 1971) have been studied in sea urchin eggs allowing the construction of a time table of biochemical changes in early development (Epel et al., 1969). Rothschild (1956) has called attention to the comparative rarity of organisms which, like sea urchins, complete meiosis within the ovary prior to fertilization, and he has reviewed the sparse comparative works dealing with the early developmental changes of organisms which are fertilized at different points of the maturation process. What is needed to understand more fully 40 Copyright 0 All rights of

1975 by Academic

reproductionin

Press, Inc. any form reserved.

the significance of differences between different forms are more detailed comparative studies using forms that differ in the state of the nucleus at fertilization. Tunicate eggs which are fertilized at first meiotic metaphase are excellent material for such studies. The eggs are slightly larger than those of the sea urchin and they develop free in the sea water, similar to urchin eggs. The eggs of many species can be obtained in large quantities and are just as convenient as urchin eggs for physiological and biochemical studies. Tunicate eggs exhibit an increase in respiratory rate at fertilization (RunnStrom, 1930; Tyler and Humason, 1937; Minganti, 1957; Lentini, 1961; D’Anna 1973). The increase in respiration is accompanied by a fall in ATP level (D’Anna, 1969; Spada, unpublished) an increase in ADP content (Spada, unpublished) and increases in cytochrome oxidase (D’Anna, 1966) and succinic dehydrogenase activities (D’Anna, 1973). Monroy and his colleagues have found surface changes in

CHARLES C. LAMBERT

Uridine Permeability

in Tunicate Eggs

41

tunicate eggs that coincide with meiotic curred in selected cultures grown beyond 3 events. Susceptibility to sodium taurocho- days. Uptake of [3H]uridine. For the timelate-induced lysis increases dramatically course studies, duplicate or triplicate at first polar body production (Monroy, lo-ml samples were withdrawn from the 1954), while the binding of concanavalin A increases with the completion of matura- stock culture with a wide-mouth pipet, tion (Monroy et al., 1973; O’Dell et al., placed in conical centrifuge tubes, cen1974). These studies suggest that profound trifuged by hand, and the sea water withchanges in the structure of the plasma drawn to 1.0 ml. [5-3H]uridine (New Engmembrane may be cued to meiotic events. land Nuclear, 25.7 Ci/mmole) was added to To investigate further the cell surface 2.5 PCilml and the eggs agitated for 5 min changes in the early development of the by gentle bubbling of air. Uridine was tunicate, I have studied changes in applied by the same basic method for the substrate concentration and inhibitor studpermeability to uridine following fertilization. The time course of changes in ies except that the uridine concentration permeability, the effect of substrate con- varied within and between experiments centration on permeability and the effects and labeling was continued for 10 min. Following exposure to [3H]uridine, the of inhibitors on permeability are included eggs were washed five or six times with in this report. ice-cold Millipore-filtered sea water containing 0.25 mg/ml of streptomycin sulfate MATERIALS AND METHODS and either dissolved in NCS (Nuclear ChiAscidia callosa, a fairly large enterogonid cago) and counted in toluene-based scintilascidian which is abundant on floating lation fluid or extracted with 0.4 N HClO, docks on San Juan Island, was used for this for 1 hr and counted in Aquasol (New study. England Nuclear). Since incorporation of Culture method. Gametes were obtained uridine into acid-precipitable compounds by surgical removal from the gonoducts is negligible during the duration of the and grown as previously described (Lamexperiments reported here (Lambert, 1971) bert, 1971) with minor changes. Eggs from both methods gave equivalent results. A lo-30 individuals were incubated in 300 Nuclear Chicago scintillation counter was rg/ml of trypsin (Sigma III) for 1 hr at used to determine radioactivity. 8-14°C and washed five times with 100 ml of Millipore-filtered sea water. The enzyme RESULTS treatment is necessary to obtain synchronous development; apparently there is a Time Course proteinaceous inhibitor of fertilization in Ascidia callosa exhibits two periods of the oviducts of some individuals. The eggs amoeboid deformation which correspond to (2-3 packed ml) were then placed in 1 liter first and second polar body production at 5 of Millipore-filtered sea water and gently and 40 min after fertilization. The unfertilstirred to maintain suspension. At the ized egg is very permeable to uridine (Fig. appropriate time, the eggs were fertilized 1) with a sharp decline being evident and streptomycin sulfate added to 0.25 beginning a few minutes after fertilization, mg/ml. Cultures were raised at constant coincident with first polar body formation, temperatures between 9 and 14°C. Cleav- reaching a minimum about 40 min after age and normal development occurred in at fertilization. Following this, there is a least 95% of the eggs in all cultures used. sharp increase in permeability at the time Metamorphosis and normal growth oc- of second polar body production with a

42

DEVELOPMENTAL

BIOLOGY

FIG. 1. Uptake of [*HI-uridine during early development. Approximately 2930 embryos were labeled for 5 min followed by six washes with ice-cold sea water. The vertical bars give the standard error of the mean (n = 3). PB, and PBr,, first and second polar bodies, respectively.

plateau level being reached about 3 hr after fertilization, shortly after first cleavage. The permeability to uridine of the unfertilized ovum is slightly higher than that of cleavage-stage embryos. The trypsin treatment which is required for synchronous cleavage in this species does not affect the microscopic appearance of the egg; the follicle cells and chorion appear unchanged after treatment. Permeability to uridine by the unfertilized egg is reduced by 50% after trypsinization.

VOLUME

46. 1975

substrateconcentmttcn GM) FIG. 2. The influence of substrate concentration on uridine uptake; low to intermediate concentrations. Embryos were labeled for 10 min with the indicated concentrations of uridine.

Thus strict concentration dependence is observed for uridine permeability in this tunicate. Calculations show that the eggs take up uridine to a concentration much greater than that present in an equivalent volume of sea water containing the isotope. This accumulation varied from several hundred- to several thousand-fold in different experiments. As an example, after 10 min in 0.195 pM uridine, each egg (1.81 x Substrate Concentration lo-” ml) contained 1.93 x 10-l’ pmoles of Because of the changes in uridine uridine while an equivalent volume of mepermeability following fertilization it was dium contained 3.53 x lo-l6 pmoles of necessary to examine the effects of chang- uridine giving a 5,400-fold amplification of ing uridine concentrations at three periods uridine content. of early development: unfertilized eggs, embryos that had reached minimal uridine Inhibitor Studies Although the substrate concentration permeability, and embryos that had achieved plateau level permeability. Uri- studies showed strict concentration dedine concentrations were varied from pendence at high and low uridine levels, 0.01-200 pM in different experiments. At there could still exist several mechanisms low to intermediate (Fig. 2) and high (Fig. for penetration of uridine. To examine the 3) uridine concentrations there appears to possibility of multiple mechanisms for perbe a linear dependence of permeability on meation several agents known to inhibit uridine concentration, with no evidence of other uptake systems were applied to the saturation occurring in any developmental eggs and embryos of A. callosa. These stage even at the highest concentrations. agents were used at the three develop-

CHARLES C. LAMBERT

Uridine Permeability

43

in Tunicate Eggs

a%-

8

/ Unfert.

0 28 8 20

Changes of uridine permeability during the maturation of tunicate eggs.

DEVELOPMENTAL 46,40-48 (1975) BIOLOGY Changes of Uridine Permeability Tunicate during the Maturation of Eggs CHARLES C. LAMBERT Department F...
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