Wilhelm Roux' Arehiv 166, 226--235 (1971) �9 by Springer-Verlag 1971

Effects of Mycostatin in Sea Urchin Development*

MARY LEE BARBER Biology Department, San Fernando Valley State College, Northridge, California 91324

Received May 8 / August 1, 1970

Summary. Sea urchin development is inhibited in the presence of mycostatin, a fungi- cidal antibiotic believed to alter cell membranes. Pretreatment of eggs inhibits fertilization in L. pictus, but not in S. purpuratus. The doses resulting in abnormal development in S. purpuratus increase as the treatment is started later in development. All tissues are sensi- tive to mycostatin at high concentrations, but the endodermal and mesenchyme derivatives are most sensitive at lower concentrations. The results suggest the heterogeneity of cell mem- branes and also indicate that membranes change with time of development.

Introduction

The physical and chemical properties of embryo surfaces change during development and differentiation (Curtis, 1967). Changes in cell surfaces are asso- ciated with fertilization (Tyler et al., 1959), cleavage, (Wolpert, 1966; F ry et al., 1970), morphogenetic movements (Ito]tfreter, 1968), tissue interaction (McLough- lin, 1963), intercellular communication (Loewenstein, 1968) and control of growth and division (Rubin, 1966; Sachs, 1967). These changes involve collectively the alteration of surface coats, permeability, mechanical properties, cell adhesiveness, and motility. Thus, specific adult tissue types differ from one another and from undifferentiated cells in morphological specializations for adhesiveness, moti- lity, and selective permeabili ty (Whittaker, 1968). Membranes from all cells are complex systems consisting of varying ratios of phospholipids, proteins, sterols (Van Deenen, 1968; Whittaker, 1968; Wallach etal., 1968; Korn, 1969) and enzymes (De-Th6, 1968; Korn, 1969). Development of the diversity of these constituents, and of their arrangement, required to explain the intracellular locali- zation and specificity of membrane function (De-Th6, 1968), must be an important par t of cell differentiation. There is little information about the interactions between proteins and lipids in embryonic membranes, or in fully differentiated adult cells. Such information will obviously be necessary to understand the transitions which occur in membranes during development. One approach to the problem of examining membrane differentiation is to experimentally alter these membranes to determine how developmental patterns are altered.

The present s tudy follows the effect of the fungicide, mycostat in on deve- lopmental patterns of sea urchin embryos. Mycostatin is a large amphipathic polyene antibiotic which affects cell permeability in certain organisms (Kinsky, 1967). I t is known to preferentially penetrate and bind to synthetic membranes containing sterols (Demel et al., 1965). Mycostatin injected beneath a lipid mono-

* This work was supported by San Fernando Valley State College Foundation Grants No 4.267.01, No 4.268.05.

Effects of Myeostatin in Sea Urchin Development 227

layer conta in ing cholesterol produces a pressure change in t he monolayer a t cons tan t a rea and a change in surface area a t cons tan t pressure. Since these changes are much grea ter t han in monolayers of o ther l ipids wi thou t cholesterol, pene t r a t ion of the ant ib io t ic into the mono]ayer wi th specific in te rac t ion wi th sterols is suggested. Al te ra t ion b y mycos t a t i n of ion pe rmeab i l i t y of syn the t ic l ip id spherules, which was far more effective when sterols were pre incorpora ted , was demons t r a t ed b y release f rom them of anions or glucose (Sessa et al., 1967; Weissman et al., 1967). Mycos ta t in (10 -5 - - 10 -6 M) preferen t ia l ly a l ters the electri- cal propert ies , and re la t ive ionic pe rmeab i l i ty of syn the t ic th in l ipid membranes conta ining cholesterol, bu t does no t a l ter those proper t ies in membranes dep le ted of cholesterol (Andreoli et al., 1968). The ra te of pe rmeab i l i t y a l t e ra t ion in Neurospora by mycos t a t i n is not dup l i ca ted or modif ied b y agents such as eye lohe~mide , iodoace tamide and other g rowth and metabol ic inhibi tors (Kinsky, 1967).

These membrane effects are though t to be the cause of m a n y o ther changes no ted in var ious organisms : a l tered ra tes of glycolysis, R N A synthesis , and induced enzyme p roduc t ion ; outf low of cy toplasmic cons t i tuents wi th loss of d r y weight in the fungi (reviewed b y Kinsky , 1967). Bac te r ia and viruses are no t affected b y this ant ib iot ic , p r e sumab ly because of the i r lack of membrane sterols (Kinsky et al., 1966). I n t issue cul tures of ve r t eb ra t e cells th is an t ib io t ic is used wi thou t t ox ic i ty a t the levels used in th is s t u d y (E. R. Squibb and Sons, Inc. , 1961).

This evidence suggests t h a t the an t ib io t ic causes membrane changes which lead to abnorma l ion select ivi ty , leakage of essential cons t i tuents and t h a t the t ox i c i t y is due to in te rac t ion with the sterol component of the membrane in affected orga- nisms (Kinsky, 1967). Al te ra t ions in the deve lopmenta l pa t t e rn s seen in this s tudy , due to the ant ib iot ic , are assumed to resul t f rom similar effects, a l though there is no di rec t proof t h a t mycos t a t i n is ac t ing on the cell membranes of sea urchin embryos in the same way. However , the resul ts s t rongly suggest t h a t i t does. Studies now in progress should t es t the level of effect of the drug on eggs more dea r ly .

Methods

Gametes were obtained from Lytechinus pictus and Ntrongylocentrotus purpuratus (supplied by Pacific Biomarine, Venice, California) by the injection of 1 ml 0.55 M KC1. L. l~ictus eggs and embryos were maintained at room temperature (22 ~ C) until after the post-fertili- zation washing, and then were kept on a very slowly moving reciprocal shaker at 13~ ~ C. Since this treatment led to clumping and abnormal development of S. purpuratus eggs and embryos, they were always kept at l0 ~ C or below, without agitation. Sperm were collected and stored in the cold as an undiluted paste. Eggs were collected in 10-100 ml sea water for up to one hour, filtered through silk bolting cloth and washed by decanting three changes of 30 mI sea water per ml eggs. Batches of 0.1 ml loosely packed eggs were fertilized with 0.1 ml freshly diluted sperm suspension (i : 100 in sea water) in 10 ml sea water with specified additives. L. pictus gametes were agitated for 10 rain on a rotating shaker (70 rev/min) to improve the percent fertilization in mycostatin, and S. 19urpuratus gametes were mixed gently by hand and left stationary for ten minutes, then observed. Batches with less than 95% fertilization in the control dishes or with contamination by ciliates were discarded. The sea water with excess sperm was decanted and fertilized embryos were transferred back to the dishes in fresh sea water. Eggs preineubated with mycostatin (E. t~. Squibb and Sons, Nystatin) were treated in plastic petri dishes, allowed to settle, and 0.1 ml eggs were ferti- lized as above. Washed embryos in plastic, covered petri dishes were kept for three days

228 M.L. Barber:

(L. pictus) or 14 days (S. purpuratus) in a dark incubator. The concentration of mycostatin was maintained the same throughout development, once added, unless otherwise stated. Dishes were exposed to light and room temperature only for short periods of observation and sampling. Early stages were photographed in the dishes using an inverted phase microscope. Later motile stages were placed live on slides with cover slips slowing down the movement, or fixed in trichloroacetic acid or glutaraldehyde and photographed with phase or dark field microscopes.

All sea water used was mitlipore filtered (type HA, 0.45 tz) adjusted to pH 7.8 with 0.001 IV[ tris when necessary, and contained 200 mg penicillin-G (Sigma Chemical Company) and 50 mg streptomycin sulfate (Sigma Chemical Company) per liter. Mycostatin (3000 units/rag) was suspended (by Waring blender) in this sea water and was stored in the dark at 4 ~ C in dark bottles, as it is unstable in the light, at warmer temperatures. All glassware was dry heat sterilized. Plastic ware was anproline (C. 1%. Bard, Inc.) sterilized.

Results

EHeet o /Mycostat in on StrongyIocentrotu8 purpuratus development

The sensi t iv i ty of embryos depends upon the t ime of t rea tment . Earlier stages are more sensitive t h a n later stages (Table 1). For example, 4 ~g/ml applied star- t ing before fert i l ization results in 90% arrested cleavage, whereas 8 ~g/ml are required for a similar response when applied after fertilization. Similar concentra- t ion dependence was no ted for all developmenta l defects examined.

Treatment Before Fertilization

Maintenance of eggs and embryos in mycos ta t in from a t ime prior to fertili- za t ion caused developmenta l defects a t the lowest concentra t ion (2 ~g/ml) tr ied (Table 1). The defects were no t due to immedia te cell death because pre incubat ion in mycos ta t in , with removal prior to ferti l ization, had no effect below 8 ~g/ml. At 2 ~g/ml decreased elevat ion of the fert i l ization membrane was noted together with abnormal cleavage (Fig. 2A). Most embryos were arrested a t a hatched, solid,

Table 1. The e//ect o/ time addition o/ mycostatin on S. purpuratus development. Numbers represent the lowest e//ective dose (~g/ml) o[ mycostatin giving a pronounced developmental de/ect when embryos were maintained in it starting at di//erent times. Concentrations o/33, 16, 8,

6, d, 2, 0 ~g/ml were applied at each stage

Time transferred to mycostatin

Developmental defect

arrested decreased reduced smaller reduced reduced cleavage hatching motility gut spicules arms 9O% 5O%

30 rain or more before 4 fertilization

At fertilization 6

After fertilization, 8 before or during early cleavage

After hatching (28 hrs after Fertilization)

2 4 2 2 2

4 4 4 2 2

6 6 4 4 4

16 6 6 15

Effects of Myeostatin in Sea Urchin Development 229

nontransparent stage by eleven days (Fig. 2B) but some became rounded and transparent, with spicules and pigment, but with reduced bilateral symmetry, reduced or no gut and no arm buds (Fig. 2 C). Embryos tended to clump together. At higher concentrations, cleavage furrows were incomplete in many cases, result- ing in embryos with one or two large blastomeres, surrounded by many small ones, and none hatched.

Treatment at Fertilization

Concentrations of 2 ~g/ml applied at fertilization (Fig. 2D) allowed more nor- mal development but did result in reduction of spicules and arm buds (Table 1). 4 ~zg/ml did not affect the elevation of a fertilization membrane but resulted in abnormal cleavage due to blebbing on the surface of many blastomeres, (Fig. 3A) and decreased the number of hatched embryos. Of those hatched, some were not transparent and showed little development (Fig. 3B, D), but others had reduced gut, arms, and spicules (Fig. 3 C, D). Higher concentrations prevented hatching or early development (Fig. 6B).

Treatment a]ter Fertilization

Concentrations of 2 ~g/ml applied after fertilization allowed normal develop- ment, but 4 ~g/ml resulted in smaller gut, reduced spicules and reduced arms (Table 1). Six ~g/ml resulted in reduced hatching, and many of those hatched were not transparent. In Fig. 4B, one embryo which is just hatching, one unhatched and one ciliated gastrula can be seen. Others were transparent but lacked arm buds, had reduced gut, and clumped together (Fig. 4 C). These deve- lopmental defects were not due to pH changes in the sea water. The pH of the dishes was checked after two days and varied from pH 7.52-7.75, but there was no correlation between lower pH and abnormal embryos.

Treatment alter Hatching

Concentrations of 6 ~zg/ml applied after hatching allowed fairly normal develop- ment (Fig. 4D, 5C) except for reduced spicules and smaller gut. Higher concen- trations {Fig. 5A and B) resulted in much smaller embryos that were less trans- parent, with shorter dorsalventral length, reduced spicules, and reduced gut without the visible ciliary movement inside seen in the controls or embryos cul- tured in lower concentrations of mycostatin. Motile cilia were seen on the embryo exterior, although treated embryos always moved more slowly. Proceeding from left to right in Fig. 5, more normal development is noted with the lower doses of mycostatin. Examples of embryos treated with 33 ~g/ml myeostatin after hatching are shown in Figs. 5A, 6A. These are two of the most advanced examples found. Most embryos retained the apical tuft (as seen in Fig. 6A) but had small or no spicules and no gut. Treatment of this species after hatching did not perma- nently immobilize the hatched embryos. They fell to the bottom of the dish after the first hour, but were swimming by the next day.

Treatment o/ Sperm

Normal eggs fertilized with sperm pre-treated with 33 tzg/ml mycostatin for 20 rain showed greatly reduced fertilization. Pretreatment of sperm for 1 or 10 rain

230 M.L. Barber:

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232 M.L. Barber:

Table 2. The e//ect o/ time o/ addition o/ 33 ~g/ml mycostatin on development o] L. pictus. Embryos were treated starting at di]/erent times: be]ore, during, or after /ertilization and alter

hatching

Time of treatment Most frequent Most advanced initiation developmental defect stage possible

Eggs preincubated only a unfertilized

20 min before fertilization unfertilized, arrested cleavage

At the time of fertilization hatched thick walled blastula, dissociation after hatch

Before and after fei% arrested cleavage morula ilization, not during

Between fertilization arrested cleavage morula and first cleavage

After hatching swimming stopped, solid blastula with disaggregation pigment cells

Sperm treated only Most normal, some plutcus exogastrulae

arrested cleavage

abnormal hatched blastula

blastula

a Eggs were preincubated but then placed in normal sea water.

had little effect. All myc0statin treated sperm were far less active, with a very small radius of movement of the sperm. They were clumped to a much greater extent than the normals by twenty minutes pretreatment.

The e//ect o] Mycostatin on Lytechinus Development

The results of treatment of Lytechinus with 33 ~g/ml mycostatin are summa- rized in Table 2. Eggs exposed to mycostatin from the time they were shed until addition of sperm three hours later were not fertilized. However, washing them in several changes of normal sea water before sperm addition resulted in a low percentage of elevated fertilization membranes, although none developed past early cleavage. Sperm suspensions in myeostatin sea water (33 ~g/ml) showed decreased mobility immediately and became immobile after 35 rain. They formed a latticework in the sea water as seen in dark field microscopy. To test whether the lack of fertilization of eggs in mycostatin was due to the sperm, normal eggs were fertilized with 0.1 ml sperm suspension made up in mycostatin (33 ~g/ml). All eggs were fertilized, although a few percent were arrested at cleavage, and a few percent exogastrulated. Exposure of the sperm, therefore, was not the pri- mary cause of lack of fertilization.

The sensitivity of Lytechinus embryos to mycostatin also depended on the time of addition, but a complete dose response was not studied for this species. Eggs placed in mycostatin 20 min prior to the time of sperm addition showed only a few percent fertilized, with those arrested at cleavage or blastula. Eggs main- rained in mycostatin from the time of sperm addition showed a few percent to one fourth unfertilized (in different batches). After one day, about one half

Effects of Mycostatin in Sea Urchin Development 233

of those fertilized were hatched, eiliated, slowly rotating abnormal blastulae with the remainder arrested in cleavage. After two days, the blastulae had almost all disaggregated, but a few slowly swimming ones remained along with the unaffected arrested cleavage stage embryos in their fertilization membranes. Treatment with myeostat in at the t ime of fertilization, without penicillin or streptomycin gave the same results. One half the concentration (16.6 ~g/ml) at fertilization did also. Preineubated eggs (20 min) fertilized in normal sea water and then transferred back to myeostatin, behaved the same way as embryos treated with myeostat in only after fertilization was complete. They showed good fertilization, but stopped deve- lopment a t cleavage or morula. Embryos allowed to develop normally for one day (hatched swimming blastula), then transferred to mycostatin, stopped swimming within one hour and rested on the bot tom of the dish, stopped development, and did not gastrulate. Red round pigment cells developed between the loose outer layer of disaggregating cells and the inner, larger more tightly bound cell mass.

Discussion

There are many effects from myeostat in in sea urchin eggs and embryos, according to the time of application. This agrees with the concept of changing membrane components and properties during development. There is a decrease in fertilizabflity of Lytechinus eggs, an affect on the system for elevation of the activation calyx, cleavage is arrested, the motil i ty of embryo cilia and sperm fla- gellae is reduced, cell adhesion is reduced, there is an increased stickiness of the whole embryo, particularly on the ventral surface, gastrulation is inhibited or altered resulting in a smaller gut, and an inhibition of skeleton and arm deve- lopment. All of these effects can be related to alteration of the selective perme- ability mechanisms or the molecular content or orientation within the cell surface.

The concentrations which resulted in highly abnormal development affected ectodermal, mesodermal and endodermal structures ; the ectoderm did not spread and become transparent and the apical tuf t was retained abnormally, the mesen- chyme failed to form the extensive skeletal structures around the gut toward the oral arms, the gut was smaller and the oral invagination very shallow. All of these effects could result from faulty morphogenetic movements and the resulting lack of interee]hilar relationships necessary for normal development. Low concentra- tions, which allowed more normal development, seemed to preferentially affect the mesendoderm, while allowing ectodermal differentiation. These embryos look somewhat like the animMized embryos of other studies (Gustafson etal., 1952; t{Srstadius, 1953). Pigment cells seem to be particularly resistant to mycostat in effects.

The changes in sea urchin developmental patterns due to mycostat in are surprisingly similar to those resulting from varying doses and times of application of D-actinomycin in a similar concentration range (l~unnstrom etal., 1966; Guidicc et al., 1968). D-aetinomycin primarily affects the RNA synthetic mecha- nism in embryos (Gross et al., 1964). Mycostatin appears to primarily affect the membrane properties due to reaction with sterols of other systems tested, and pre- sumably therefore in these embryos. This similarity in abnormalities, but difference in target, suggests that a part of the information made in normal development,

234 M.L. Barber:

t h a t is necessary for d i f ferent ia t ion, is i n t i m a t e l y re la ted to the normal changes in cell membranes which occur a s the f irst mani fes ta t ion of different ia t ion. The two an t ib io t ics could then be in ter fer ing wi th the same mechanism a t two dif- ferent levels of control .

Molecular in te rac t ions in membranes and changes in t h e m depend upon in te rna l and ex te rna l condi t ions which affect pro te ins or l ipids. E x p e r i m e n t a l r emova l or a l t e ra t ion of e i ther the l ipids or the s t ruc tu ra l pro te ins of membranes al ters the enzymat i c act iv i t ies of m e m b r a n e - b o u n d enzymes, and the i n t eg r i t y and func- t ion of the m e m b r a n e as a whole (Barber et al., 1967; Kern , 1969). Membranes a l t e red b y fac tors in deve lopmen t would t hen a l te r secondar i ly the sys t em depen- den t on m e m b r a n e ac t iv i ty , inc luding celt morphology, me tabohsm, and t r a n s p o r t mechanisms.

The ac t inomye in would interfere wi th the p roduc t ion of new prote ins needed in membranes dur ing norma l deve lopment . The m y c o s t a t i n would complex wi th the l ipids, pe rhaps a l te r ing the sites of in te rac t ion wi th pro te ins of the membranes . Bo th the pro te ins and the lipids, and the p rope r in te rac t ion be tween the two are essent ia l to the i n t eg r i t y of m e m b r a n e s t ruc ture and funct ion.

The add i t i on of cer ta in ions or metabo l ic poisons (CuS04, ttgC12, KCN, LiCI, MgCI~, alcohol, ammonia) a t non-cy to ly t i c concent ra t ions (Child, 1941) can also resul t in abnormal i t i e s va ry ing wi th t ime a n d dose s imilar to those seen in these p resen t studies. I n these cases, m e m b r a n e proteins , enzymes, and resul t ing perme- ab i l i t y and cell me tabo l i sm m u s t also be affected. However , there is more evidence t h a t m y c o s t a t i n works d i rec t ly on the cell m e m b r a n e l ipids and the abnormal i - t ies resul t ing are due to a l te red membranes .

The resul ts wi th all of these agents suggest t h a t there is a he te rogene i ty of cell membranes in ea r ly embryos , so t h a t di f ferent ia l affects b y inhib i tors occur on cells of different regions. This he te rogene i ty changes wi th t ime in normal deve lopmen t and in terference wi th the normal p a t t e r n of change resul ts in abnor- mal deve lopment .

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Dr. Mary Lee Barber Department of Biology San Fernando Valley State College Northridge, California 91324: (U.S.A.)