Microvillar elongation following parthenogenetic activation of sea urchin eggs

Experimental Cell Research 159 (1985) 211-223

Microvillar Elongation Following Parthenogenetic Activation of Sea Urchin Eggs

WILLIAM BYRD* and B. W. BELISLE Department of Obstetrics and Gynecology, Southwestern Medical School, Dallas, TX 75235,

and Department of Microbiology, Uniformed Services, University of the Health Sciences, Bethesda, MD 20814, USA

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4C1 treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microviUi was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while re- addition of NH4C1 caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4CI is pH- and calcium- dependent and is similar to that observed during normal fertilization. © 1985 Academic Press, Inc.

Fertilization of the sea urchin egg results in a dramatic reorganization of the cell surface, as well as metabolic activation [1, 2]. Prior to fertilization the egg surface is covered by an even array of short microvilli (approx. 0.2 ~tm) lacking any discernible organized microfilament structures [3-8]. Within seconds after sperm-egg fusion, cortical granule exocytosis occurs, resulting in the formation of a new mosaic membrane comprised of the egg plasma membrane plus added cortical granule membrane [3, 5, 7, 9]. Concomitant with cortical granule discharge microvilli elongate to approx. 1 ~tm and microfilament bundles form within the microvilli [9-15]. These microfilaments bundles lie parallel to the long axis of the microvilli, and are unidirectionally polarized towards the cell center [15].

Treatment of unfertilized sea urchin eggs with parthenogenetic agents, such as ammonium chloride, initiates a number of events normally associated with fertilization [1, 9, 16]. These agents act by inducing an elevation of cytoplasmic pH, but do not increase intracellular free calcium, as does normal fertilization [17]. Ammonia activation has been reported to stimulate microvillar elongation of Lytechinus pictus eggs [16], but does not induce cortical granule release [18, 19]. Begg & Rebhun [12] using isolated egg cortices of Strongylocentrotus purpuratus

* To whom offprint requests should be sent. Address: Department of Obstetrics and Gynecology, Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75235, USA.

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

0014-4827/85 $03.00

212 Byrd and Belisle

eggs found that microvillar elongation and microfilament bundle formation could be induced in vitro. Actin polymerization in vitro was dependent upon alkalinization of the isolation medium, similar to the alkalinization of the egg cytoplasmic pH seen during fertilization. On the basis of this work and that of Mazia et al. [16] it can be hypothesized that increased cytoplasmic pH which accompanies fertilization or parthenogenic activation can induce microvillar elongation and microfilament formation. However, more recent work by Carron & Longo [8] and Begg et al. [20] suggests that alkalization of the intracellular pH of the egg by ammonia alone is not sufficient to induce microvillar elongation and that calcium plays a major role in the regulation of actin polymerization in sea urchin eggs.

There appears to be a relationship between cytoplasmic alkalinization, the cortical reaction, calcium release and microvillar elongation during fertilization and egg activation. The discharge of cortical granules during fertilization is linked to an increase in cytoplasmic free calcium [1, 21, 22]. Treatment of sea urchin eggs with calcium ionophore A23187 in sodium-free sea water also induces a cortical reaction and a transient increase in intracellular free calcium, but no cytoplasmic alkalization [23, 24]. Arbacia punctulata eggs treated with A23187 in sodium-free sea water form a loose network of microvilti, but no microfilament bundles form within these microvilli [8, 12]. Subsequent alkalinization of ionophore-treated eggs in sodium-free sea water with ammonia or the addition of sodium induces bundle formation. This evidence suggests that both increases in free calcium as well as cytoplasmic pH regulate the formation of microvilli and microfilament bundles at fertilization.

The present study has examined in greater detail the relationship between calcium, cytoplasmic pH and microvillar formation in parthenogenetically activated sea urchin eggs. Results indicated that microvillar elongation and microfilament bundle formation occurred when sea urchin eggs were treated with ammonium chloride, and that this process was dependent upon calcium influx into the egg.

MATERIALS AND METHODS

Gamete Collection and Handling Strongylocentrotus purpuratus and Lytechinus pictus were collected in the San Diego bay area;

Arbacia punctulata and Lytechinus variegatus were collected by permit from St. Andrews State Park, Panama City, Fla. Sea urchins were induced to spawn by intracoelomic injection of 0.53 M KC1. Gametes were gathered, washed and dejellied in artificial seawater (ASW) (Marine Biological Labora- tory Formula VI) according to previously described procedures [25]. All experiments were carried out at 17°C.

Microvillar Elongation To examine the effects of ammonium chloride (NH4CI) induction on microvillar elongation and

microfilament formation in unfertilized eggs, eggs were incubated in 10 mM NH4CI (pH 8.0) in ASW with periodic bubbling for 30 min to 4 h. Acid release and pH of the ASW were monitored after addition of NH4C1 and the pH of the seawater was titrated to pH 8.0 in all cases after ammonia addition. Eggs incubated in calcium-free seawater (OCaSw) with 2 mM EGTA and l0 mM NH4CI

Exp Cell Res I59 (1985)

Microvillar reorganization following NH4Cl treatment 213

were first washed three times with OCaSw containing 2 mM EGTA and then activated by the addition of NH4C1 to a final concentration of 10 mM NH4C1 (pH 8.0). Eggs were also activated in ASW- containing NH4C1 and lanthanum chloride (0.5-5 mM). Samples were taken at periodic intervals and prepared for scanning (SEM) and transmission electron microscopy (TEM).

Electron Microscopy Eggs were fixed in 2.5% glutaraldehyde-0.1 M phosphate buffer (pH 7.3) made isotonic to

seawater with sucrose for 60 min at 4°C and rinsed several times with 0.1 M phosphate-buffered- isotonic sucrose according to Byrd & Perry [25]. Eggs were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2 for 30 min on ice and stained with 1% uranyl acetate for 3 h. Samples were dehydrated with a graded series of ethanol and acetone. Samples processed for SEM were critical point-dried with carbon dioxide, sputter-coated with gold-palladium and viewed on a Hitachi S-500 SEM. Samples for TEM were embedded in Epon-araldite. Thin silver-gray sections were post- stained with uranyl acetate and lead citrate before examination on a Jeol 100C TEM.

Isolation o f Egg Cortices To demonstrate the presence of microfilaments in microvilli of activated and unactivated eggs,

cortices were isolated using the technique of Begg & Rebhun [12]. Eggs were washed once in Ca, Mg, Na-free ASW (0.5 M choline chloride, 27 mM KC1, 2 mM EDTA, pH 7.8 [8] and then in isolation medium at 4°C. The isolation medium consisted of 0.35 M glycine, 0.1 M PIPES buffer, 5 mM EGTA, 5 mM MgCI2, 1 mM dithiothreitol, 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 0.1 mg/ml soybean trypsin inhibitor at pH 6.5. Washed eggs were resuspended to a final concentration of 5 % and homogenized with a Dounce glass homogenizer. Cortices were washed three times in isolation medium at 600 g for 5 min and processed for TEM. Isolated cortices were fixed in 1% glutaraldehyde (0.4 % tannic acid was added to the fixative immediately before use). Cortices were then processed as described in TEM.

45Calcium Uptake

Dejellied eggs from S. purpuratus and A. punctulata (10%; v : v) were incubated in (1) ASW; (2) 10 mM NH4CI (pH 8.0); (3) ASW with 0.5-2.5 mM lanthanum chloride; or (4) 10 mM NH4C1 with 0.5-2.5 mM lanthanum chloride. At indicated times 2 ml aliquots of eggs were incubated for 10 min with 0.83 IxCi of 45Ca (CaC12 from ICN Pharmaceuticals, Inc.) with constant bubbling. Samples were then washed three times with cold zero-calcium ASW. Pellets collected by centrifugation were digested in 1.0 ml of Protosol (New England Nuclear) overnight and counted in Bray solution in a liquid scintillation counter.

R E S U L T S

Microvillar Elongation in Fertilized and Parthenogenetically Activated Eggs

The surface m o r p h o l o g y of four species of sea u rch ins was s tudied fol lowing

fer t i l iza t ion or NH4C1 ac t iva t ion by SEM. To visual ize pos t - fer t i l iza t ion events in

non-ac t iva ted eggs fol lowing i n s e m i n a t i o n , unfer t i l ized sea u rch in eggs were first

t rea ted wi th 0.01 M di th io thre i to l (DTT, p H 9.0) which removes mos t of the

vi tel l ine layer [26]. P r e t r e a t m e n t of eggs wi th D T T prevents fer t i l iza t ion m e m -

b r ane fo rma t ion fol lowing in semina t ion . The surface of an unfer t i l ized D T T -

t rea ted S. purpuratus egg is covered wi th n u m e r o u s evenly a r rayed microvi l la r

p ro jec t ions (fig. 1 A). Fer t i l i zed eggs i n s e m i n a t e d and fixed 15 min post-fer t i l iza-

t ion d isplayed n u m e r o u s e longa te surface microvi l l i (fig. 1 B). Unfe r t i l i zed eggs

i n c u b a t e d in NH4CI were no t t rea ted with D T T , s ince there was no cor t ical

Exp Cell Res 159 (1985)


Exp Cell Res t59 (1985)


reaction after activation which would obscure surface events. When S. purpuratus eggs were activated with 10 mM NH4C1 a gradual elongation of microvilli occurred during the first hours of incubation (fig. 1 C, D). Elongation of surface microvilli following extended treatment with NH4CI was also observed in three other species. Unactivated L. pictus eggs displayed typical shortened microvilli before activation (fig. 1 E) and elongated microvilli after 3 h of incubation in NH4C1 (fig. 1 F). Fig. 1 G shows the surface of an unactivated L. variegatus egg, while fig. 1 H reveals the surface of an egg after 3 h of NH4CI incubation. A. punctulata also demonstrated the characteristic shortened microvilli before NH4CI incubation (fig. l / ) and elongated microvilli after 4 h of incubation in NH4CI (fig. 1J). The response time of each species to NH4CI was variable; L. pictus and L. variegatus displayed substantial elongation of microvilli within 90 min of incubation (not shown), while S. purpuratus and A. punctulata required at least 3--4 h of incubation to induce microvillar elongation. The following sections will be concerned with the results obtained from S. purpuratus eggs, which are representative of observations made in the other species.

TEM of Surface Microvilli

To determine whether these elongated surface projections were due to micro° villar elongation in unactivated eggs we examined sections of whole eggs by TEM. The cortex of unfertilized eggs of S. purpuratus exhibited typical short microvilli, intact cortical granules and a thin vitelline layer (fig. 2A). Treatment of eggs for 210 min with 10 mM NH4C1 resulted in elongation of microvilli (fig. 2B), while the cortical granules remain intact. The presence of cortical granules in these preparations suggests that the microvillar elongation was not due to fertilization or spontaneous activation of the egg but rather the result of NH4CI treatment. In fig. 2 C microfilaments can be seen in the microvillar core. The vitelline layer as well as cortical granules are also present in fig. 2 C, D, again suggesting that fertilization or spontaneous activation of the eggs had not occurred.

Observations on Isolated Cortices

The organization of microviUar actin in activated eggs is more clearly demonstrated in isolated cortices. Eggs were treated with NH4C1 for 210 min and cortices isolated at pH 6.5 following the procedure of Begg & Rebhun [12] as modified by Carron & Longo [8]. Cortices isolated under these conditions had elongated microvilli containing microfilament cores (fig. 3A,B). Microfilament

Fig. 1. SEM of surfaces of unactivated, fertilized and ammonia-activated eggs. (A) Unfertilized S. purpuratus egg after DTT treatment. (B) Fertilized DTT-treated S. purpuratus egg approx. 15 min post insemination; (C, D) S. purpuratus egg treated with (C) 10 mM ammonia forlS0 min at pH 8.0; (D) ammonia for 240 min; (LD unactivated L. pictus egg; (F) L. pictus egg after 180 min of ammonia treatment; (G) unactivated L. variegatus egg. (H) L. variegatus egg after 180 rain ammonia treatment; (/) unactivated A. punctulata egg; (J) A. punctulata egg after 240 min ammonia treatment, x6400.


D

m f b

Fig. 2. (A) Unactivated S. purpuratus egg exhibiting short microvilli lacking organized microfilaments. MV, Microvillus; CG, cortical granule; VL, vitelline layer. (B, C, D) Outer cortex of S. purpuratus eggs treated ammonia for 210 rain showing elongate microvilli with intact cortical granules and vitelline layer. MFB, Microfilament bundle; VL, vitelline layer; CG, cortical granules. (A) x17900; (B) x12500; (C, D) x32500.

meshworks such as those reported by Begg et al. [20] were not observed in these sections. Prolonged treatment in NH4CI resulted in increasingly fragile eggs, and the cortical isolation procedure may have disrupted these networks.

Reversibility of Microvillar Elongation

The integrity of microvillar elongation appeared to be dependent upon the continuous presence of the activating agent. When NH4CI was removed by washing after 3 h of incubation, the microvillar structure collapsed leaving short- stubby microvilli (fig. 4A) similar to that observed in unactivated eggs (see fig. 2A). The cortical granules remained intact and the vitelline layer was still apparent (fig. 4A). There were no apparent organized microfilament bundles in these shortened microvilli. Microvillar retraction was not immediate and 45-60 min were required for reversion to the unactivated appearance. SEM of these eggs which had NH4C1 removed (fig. 4B) revealed short surface microvilli,



A

?

Fig. 3. Cortices of ammonia-activated S. purpuratus eggs isolated at pH 6.5 in sodium-free medium. The elongated microfilaments contain microfilament bundles (MFB). (A) ×23 500; (B) ×50 500.

similar in appearance to unactivated-unfertilized controls (see fig. 1 A). Elonga- tion of microvilli could be induced a second time by re-incubating these eggs in NH4CI for another 60 min. Re-incubation in NHnC1 induced elongation of microvilli from the shortened stubs (fig. 4 C'). SEM confirmed that the surfaces of these eggs were covered with elongated microvilli (fig. 4 D). The vitelline layer and cortical granules were still present in these eggs after additional treatment.

Role of Calcium in Microvillar Elongation and Microfilament Formation

Activation with I0 mM NH4C1 stimulates a transient uptake of external calcium but no release of calcium from internal stores [27]. Other investigators [8, 20] have suggested that calcium release is required for microfilament formation. 45Ca uptake in NH4Cl-activated eggs was examined to determine if a net uptake of calcium might correlate temporally with microfilament and microvillar formation. In addition, the role of external calcium in inducing microfilament formation was studied using selective media to block extracellular calcium uptake.

Eggs from A. punctulata (fig. 5A) and S. purpuratus (fig. 5B) were incubated in ASW in the presence or absence of 10 mM NHaCI. At indicated times 2 ml aliquots of eggs (10%; v/v) were incubated for 10 min with 0.83 ~tCi of 45CAC12 with constant bubbling. After incubation, eggs were washed and total uptake of label determined. As seen in fig. 5A,B unactivated eggs did not incorporate appreciable amounts of 45Ca. However, when NHaC1 was included in the incubation medium, a 10-fold increase in 45Ca uptake occurred by 4 h of incubation in both species. This uptake was time-dependent with the maximum increase


218

A

Byrd and Belisle

C

Fig. 4. (A) Cortex of S. purpuratus treated with ammonia for 180 min, washed and transferred to ASW for 60 rain. CG, Cortical granule; VL, vitelline layer; MVS, microvillar stub. (B) SEM of an egg surface treated as in (A). (C) Cortex of egg treated with ammonia for 3 h, washed in ASW for 1 h and re- exposed to ammonia for 60 min. Elongation of microvilli from the microvillar subs. CG, Cortical granule; VL, vitelline layer. (D) SEM of the same group of eggs showing elongated microvilli. (A) x19500; (B, D) ×7500; (C) ×22500.

occurring after 90 min. To block calcium uptake, eggs were incubated in 0.5-2.5 mM lanthanum chloride in the presence or absence of NH4CI. Lanthanum, which does not enter the cell, blocks transmembrane movement of calcium and displayed calcium from extracellular sites [28]. The uptake of 45Ca was dependent upon the concentration of lanthanum used. At a concentration of 2.5 mM lanthanum chloride, calcium uptake was the same as in unactivated control eggs, Decreasing the concentration of lanthanum resulted in elevated calcium uptake, Addition of lanthanum (2.5 mM) to unactivated eggs did not decrease the amount


c4

'o

X

(.~

A 20

Microvillar reorganization following NH4CI treatment 219

B/r.----m o.._.._.. o > < . - - S 8

3'0 6'0 9'0 ~"-~"~o

270 B /

60

40 =e~" "''-

3'o do 9'0 ~"~4o Time (minutes]

Fig. 5. 45Ca uptake in (A) A. punctulata; (B) S. purpuratus eggs. Each point repre- sents the total calcium uptake into eggs during a 30-min period. At indicated times, aliquots of eggs were incubated with 45Ca and then prepared for liquid scintillation as described in Materials and Methods. C), Unactivated, control eggs; O, eggs treated continuously with 10 mM NH4CI; eggs incubated with 10 mM ammonia and A, 0.5; II, 1.0; FI, 2.5 mM lanthanum chloride, pH 8.0.

of 45Ca uptake over untreated controls (data not shown). This suggested that the rather small amount of calcium uptake in lanthanum and untreated controls represented either surface binding, incomplete inhibition of calcium uptake or both.

The surface morphology of NH4CI activated S. purpuratus eggs incubated in lanthanum or in calcium-free ASW was examined using SEM and TEM. The surface of an unactivated S. purpuratus egg is shown in fig. 6A for comparison. Following 4 h of incubation in NH4C1 (fig. 6B) there was elongation of microvilli as shown prevously (fig. 1 D) which obscure the egg surface. Microvillar elongation was not observed in ammonia activated eggs continuously treated with 2.5 mM lanthanum (fig. 6C) or in 45Ca-ASW containing 2 mM EGTA, 10 mM NH4CI (fig. 6D). Instead a number of small irregular microvilli or projections were observed on the egg surface including surface areas free of any projections. The unusual amount of precipitate observed on the surface may represent lanthanum deposits or a fixation artifact. TEM of lanthanum-ammonia-treated eggs revealed a rather striking change in the egg cortex (fig. 6E). Cortical granules were still intact but had migrated into the interior of the egg resulting in a homogenous cortical matrix containing few, if any, organelles, and no visible surface microvilli in this section. Very few microvilli were seen by TEM.

DISCUSSION

Microvillar elongation and microfilament formation were induced in four species of sea urchin eggs using the parthenogenic agent NH4C1. These results support the early observation of Mazia etal . [16] that NH4OH induces microvillar elongation in L. pictus. Microvillar elongation was pH-sensitive, since removal of NH4CI caused a retraction of elongated microvilli along with the disorganization of microfilament bundles. Re-addition of NH4C1 to eggs which had under- gone microvillar retraction induced a second burst of elongation. The elongation of microvilli and microfilament bundle formation was dependent upon an influx of



Fig. 6. SEM of(A) S. purpuratus; egg surface; (B) after 240 min of incubation in ammonia, pH 8.0. (C, D) Egg incubated in (C) the continuous presence of 2.5 mM lanthanum chloride and 10 mM ammonia, pH 8.0 for 240 rain; (D) zero-calcium ASW with 2 mM EGTA with l0 mM ammonia for 240 min. (E) Cortex of an egg treated with 2.5 mM lanthanum chloride and 10 mM ammonia for 240 min. CG, Cortical granule. (A) x6400; (B, C, D) x12500; (E) x13800.

Exp Cell Res 159 (I985)

Microvillar reorganization following NH4CI treatment 221

external calcium. Incubation of eggs with NHaCI caused an increased uptake of 45Ca. When this uptake was blocked using either 2.5 mM lanthanum chloride or calcium-free 2 mM EGTA seawater, elongation and microfilament formation did not occur.

Several investigators have previously reported that incubation of sea urchin eggs in ammonia did not cause elongation of microvilli [8, 20, 29, 30]. The reported absence of microvillar elongation and microfilament formation may reflect the different incubation conditions used. The concentrations of ammonia used (10--40 mM) are the same as reported here; however, the incubation times were typically very short, 20-30 min. Our results showed that at least 3 h of incubation is required in most species before substantial elongation occurs. The time requirement for elongation is also dependent upon the species used. The two Lytechinus species (variegatus and pictus) were the most responsive to NH4CI treatment, while S. purpuratus and A. punctulata required a much longer incubation period.

It has been established that two intracellular ionic triggers (increased intracellular calcium and cytoplasmic alkalinization) are required for proper microvillar formation following fertilization [8, 12, 20]. In vitro preparations of isolated egg cortices could be induced to form microfilament bundles when the pH of the medium was raised from 6.5 to 7.3 [12]. This suggests that microfilament bundle formation is regulated by cytoplasmic alkalinization following fertilization. Whole eggs incubated in sodium-free sea water could be activated with the calcium ionophore A23187 which releases intracellular calcium but blocks cytoplasmic alkalinization [8, 20, 31]. This treatment resulted in cortical granule fusion, calcium release and elongation of long, flaccid microvilli. These microvilli did not contain organized microfilament bundles within the microvilli but rather irregular networks of actin. The addition of NaC1 or NH4CI resulted in alkalinization of the egg cytoplasm and the formation of core bundles of actin microfilaments. Alter- natively, cytoplasmic alkalinization can be induced without the release or influx of extracellular calcium. Treatment of eggs under these conditions did not support microvillar formation.

In this study, alkalinization of the egg cytoplasm by the parthenogenetic agent ammonia was not sufficient by itself to induce microvillar formation, but required the influx of extracellular calcium. While treatment of eggs with ammonia does not induce cortical granule discharge it apparently causes an increased influx of external calcium with prolonged incubation which paralleled microvillar elongation. Inhibition of calcium influx with lanthanum chloride or incubation in zero- calcium seawater with EGTA prevented microvillar formation in the presence of ammonia. Prolonged incubation in ammonia could affect eggs by making the plasma membrane more permeable to external calcium or activating calcium transport systems. Inhibition of calcium influx with lanthanum chloride or incubation in zero-calcium seawater with EGTA prevented microvillar formation in the presence of ammonia.



Elongated microvilli and microfilament cores are dependent upon the continuous presence of NH4CI. Removal of the activating agent resulted in a shortening of the microvilli and disorganization of the microfilament bundles. This was reversed by re-addition of NH4C1 and suggested a rather labile association of microfilament bundles in activated microvillar cores under these conditions. In contrast, Begg et al. [20] attempted to dissociate microvillar bundles in ionophore-activated or fertilized eggs by treatment with 10 mM acetate (pH 6.5). No disruption of rnicrovillar core bundles occurred, suggesting these bundles are not pH-sensitive. Similar alterations have been described in isolated brush border epithelial cell microvilli incubated in calcium concentrations greater than 10 - 6

M. The microvilli became shorter, vesicle-like blebs with disorganized microfilaments [32].

The generative force for elongation of microvilli in parthenogenetically activated eggs has yet to be established. Microfilament bundle formation is not a prerequisite for formation of microvilli during the normal fertilization process [8, 20]. This is in contrast to the formation of the acrosomal process in sperm, where elongation occurs with the appearnce of microfilament bundles and these bundles provide the force for elongation [20, 33]. Begg et al. [20] suggested that alkalinization does not provide the generative force, but rather provides a mechanism for generating a rigid actin support for microvilli. The collapse or retraction of microvilli after NH4C1 withdrawal and the elongation after NH4C1 re-addition suggests that pH-sensitive cytoskeletal elements play an important role in microvillar elongation. Further work is needed to determine if actin polymerization and assembly of microfilament bundles during parthenogenetic activation is the same as found during the normal fertilization process.

We would like to thank Drs J. Boldt, D. Ochs and D. P. Wolf for comments and suggestions concerning this manuscript. The excellent secretarial assistance of S. Napier is gratefully acknowl- edged.

REFERENCES 1. Epel, D, Curr top dev biol 12 (1978) 185. 2. Vacquier, D, Dev biol 84 (1981) 1. 3. Eddy, E M & Shapiro, B M, J cell biol 71 (1976) 35. 4. Spiegel, E S & Spiegel, M, Exp cell res 109 (1977) 462. 5. Schroeder, T E, Dev biol 70 (1979) 306. 6. Longo, F J, Dev biol 74 (1980) 422. 7. Chandler, D E & Heuser, J, Dev biol 82 (1981) 393. 8. Carron, C P & Longo, F J, Dev biol 89 (1982) 128. 9. Schuel, H, Gamete res 1 (1978) 299.

10. Burgess, D R & Schroeder, T E, J cell biol 74 (1977) 1032. 11. Schroeder, T, Dev biol 64 (1978) 342. 12. Beg, g, D A & Rebhun, L I, J cell biol 83 (1979) 241. 13. Spudich, A & Spudich, J A, J cell biol 82 (1979) 212. 14. Kidd, P & Mazia, D, J ultrastruct res 70 (1980) 58. 15. Tilney, L G & Jaffe, L A, J cell biol 87 (1980) 771. 16. Mazia, D, Schatten, G & Steinhardt, R, Proc natl acad sci US 72 (1975) 4469.



17. Epel, D, The cell surface. Mediator of developmental processes (ed S Subtelny & N K Wesells) p. 169. Academic Press, New York (1980).

18. Steinhardt, R A & Mazia, D, Nature 241 (1973) 400. 19. Epel, D, Steinhardt, R, Humphreys, T & Mazia, D, Dev biol 40 (1974) 245. 20. Begg, D A, Rebhun, L I & Hyatt, H, J cell biol 93 (1982) 24. 21. Vacquier, V D, Dev biol 43 (1975) 62. 22. Jaffe, L A, Ann NY acad sci 339 (1980) 86. 23. Johnson, J D, Epel, D & Paul, M, Nature 262 (1976) 661. 24. Nishioka, D & McGwin, N F, J exp zool 212 (1980) 215. 25. Byrd, W & Perry, G, Exp cell res 126 (1980) 333. 26. Carroll, E J, Jr, Byrd, E W, Jr & Epel, D, Exp cell res 108 (1977) 365. 27. Zucker, R S, Steinhardt, R A & Winkler, M M, Dev biol 65 (1978) 285. 28. Godfraind-DeBecker, A, Int rev cytol 67 (1980) 141. 29. Nicotra, A & Arizzi, M, Int j invert reprod 1 (1979) 355, 30. Hylander, B L & Summers, R G, Dev biol 86 (1981) 1. 31. Rebhun, L I, Begg, D A & Fisher, G, Cell diff 11 (1982) 271. 32. Matsudaira, P T & Burgess, D R, J cell biol 92 (1982) 657. 33. Tilney, L G, Kiehart, D P, Sardet, C & Tilney, M, J cell biol 77 (1978) 536.

Received August 30, 1984 Revised version received January 25, 1985

Printed in Sweden

15-858337


Microvillar elongation following parthenogenetic activation of sea urchin eggs

Documents

Transcript of Microvillar elongation following parthenogenetic activation of sea urchin eggs