Turning on of Activities in Unfertilized Sea Urchin Eggs

Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4469-4473, November 1975 Cell Biology Turning on of activities in unfertilized sea urchin eggs: Correlation with changes of the surface (fertilization/derepression/cell surface) DANIEL MAZIA, GERALD SCHATTEN, AND RICHARD STEINHARDT Department of Zoology, University of California, Berkeley, Calif. 94720 Contributed by Daniel Mazia, August 20, 1975

ABSTRACT Unfertilized sea urchin eggs exposed to low NaOH to pH 9-9.2. The latter method is convenient for con- concentrations of ammonia enter into a number of activities trolling and calculating the concentration of the active com- which normally appear after fertilization. It is shown that ponent, NH3, at a constant pH. the effects are attributable to ammonia, rather than to NH4+ Observation of Chromosomes. The methods are de- ions or elevated ]H. The same effects are obtained by expo- scribed by Mazia (5). sure to isotonic urea and to glycerol at very low ionic used have been de- strengths. All treatments which produce these changes (such Membrane Potential. The techniques as the turning on of chromosome replication and condensa- scribed earlier (7). tion in unfertilized eggs) also bring about changes of the Scanning Electron Microscopy. After treatments, eggs outer cell surface which are visible in the scanning electron were glued to polylysine-coated glass plates (8) and fixed microscope. The most striking indicator is the elongation of with 5% glutaraldehyde in 80% sea water (pH 8.2). The the microvilli which cover the surface of the unfertilized egg. samples were dehydrated in ethanol, dried at the critical The changes of the surface are interpreted as the dissociation and coated with platinum-carbon. of a component from the outer surface layer. This component point in Freon, is not the "vitelline" sheet as defined morphologically or by Protamine-Coated Glass Fibers. The glass fibers were the ability of the egg to form a fertilization membrane upon prepared by grinding Whatman glass paper (GF/A) in a insemination. It is proposed further that this component is a mortar with a small amount of water. The fibers, a few mi- peripheral component of the plasma membrane, whose re- crometers thick and of variable (about 20-200 /m) length, moval modifies the membrane functionally and leads to the were then washed four to five times in sea water to remove derepression of various processes within the egg. unadsorbed protamine. An earlier series of publications describes the effects of am- RESULTS AND INTERPRETATIONS monia in initiating, in unfertilized sea urchin eggs, a number of events which normally begin after fertilization. They in- 1. The ammonia effects clude: polarization of the membrane by the development of (a) Changes in the Outer Cell Surface. With the scanning K+-conductance (1); stimulation of protein synthesis (2); electron microscope (SEM), it is possible to see changes in turning on of DNA synthesis (3) leading to the condensation the outer aspect of the egg which will be correlated consis- of the replicated chromosomes (4*, 5); the polyadenylylation tently with the ammonia effects. The surface of the normal of cytoplasmic messenger RNA (6). The effects are not unfertilized egg is densely papillated with low microvilli, equivalent to parthenogenetic activation; the "early" events and the vitelline sheet, the precursor to the fertilization coat, of activation are bypassed (1, 2) and the egg does not divide overlies the plasma membrane. Fig. 1 shows the surface of or develop. normal unfertilized eggs of L. pictus. Fig. 2 shows the sur- The present work describes other treatments which arouse faces after a treatment with NH3-sea water just sufficient to these activities in unfertilized eggs. It will be shown that all produce the ammonia effects. The conspicuous change of the effective treatments modify the surface of the egg visi- surface is the elongation and disarrayal of the microvilli, as bly. Their action will be interpreted as the dissociation of a though they have been released from some ordering re- peripheral component of the membrane from the outer sur- straint. This visible change of the surface will be seen with face. all treatments which have the same effects as does NH3-sea water. MATERIALS AND METHODS (b) Variables of the Effects of Ammonia-Sea Water. An Eggs. The gametes of the sea urchins Lytechinus pictus aqueous solution of NH40H contains NH4+ and OH- ions and Strongylocentrotus purpuratus were used. The jelly and NH3 molecules. The effects of NH3-sea water depend coats were removed from the eggs by a brief (about 1 min) on the concentration of the unionized NH3. Epel et al. (2) treatment with sea water acidified to pH 4. The full account found that chromosome condensation could be turned on of experimental results given below applies to eggs of L. even at pH 8 in sea water containing a 5 mM or higher total pictus. The basic findings on the ammonia effects, including concentration of ammonium salt. In the present work, the the surface changes, have been confirmed with eggs of S. same experiment was done at pH 9.2, adding various purpuratus. amounts of NH4Cl to sea water and titrating with NaOH. At Solutions. "NH3-sea water" was prepared in two ways. this pH, sea water containing 0.5 mM NH4Cl was fully ef- One was the titration of sea water to pH 9-9.2 with fective (in 20 min) in turning on chromosome condensation; NH40H. The other was the addition of known amounts of 0.2 mM NH4Cl was insufficient. The concentration of NH3 NH4Cl (0.5-5 mM final concentration) and titration with required to produce the described effects, calculated on the basis of pK = 4.75, is 0.2-0.3 mM. Abbreviation: SEM, scanning electron microscope. NaOH does not have the * This early report of the turning on of chromosome condensation Sea water brought to pH 9 with by sea water containing ammonia has been overlooked in the effects of sea water titrated to the same pH with NH40H. more recent publications. NaOH-sea water at pH 9 could not turn on chromosome 4469 Downloaded by guest on October 2, 2021 4470 Cell Biology: Mazia et al. Proc. Nat. Acad. Sci. USA 72 (1975)

FIG. 1. (left) Surface of normal unfertilized egg of Lytechinus pictus. A regular array of short microvilli is seen, with the thin vitelline sheet draped over them. (X10,000). FIG. 2. (right) Surface of unfertilized egg of L. pictus after a 15 min exposure to ammonia sea water at pH 9.2. This treatment is suffi- cient to produce the ammonia effects described in the text. Microvilli are longer and somewhat disarrayed. Strands of material interconnect- ing microvilli are probably remnants of the vitelline sheet (X10,000).

condensation even with an exposure of 1 hr. Nor did the ing 0.1 mM Ca and adjusted to pH 8 with NaOH. After a NaOH-sea water affect the aspect of the surface of the egg wash in the urea solution to remove residual sea water, the as observed with the SEM; the low profile of the microvilli eggs are exposed for 2-3 min, then returned to sea water. was preserved. The increase in membrane potential sets in rapidly (Fig. 3). An analog of NH40H, ethylamine, has been found to be The chromosome condensation is seen by around 90 min effective in turning on chromosome replication and conden- after the treatment with urea. The surface changes, seen by sation in Lytechinus eggs. The solution used was 5 mM eth- SEM as the extension and disarrayal of microvilli, are con- ylamine hydrochloride in sea water, titrated to pH 9.2 with spicuous. Na2CO3, with a 15 min exposure. The same effects have been obtained with isotonic (1 M) solutions of glycerol and of glucose. The non-electrolyte so- 2. Treatments with non-electrolytes; "ammonia lutions contained 0.1 mM CaCl2 and 1 mM Na added as effects" without ammonia? Na2CO3; they were adjusted to pH 8. Again the eggs were The set of changes which have been described as effects of quickly washed in the medium before the exposures of 5-10 ammonia-sea water can be produced by brief exposure of min. The chromosome cycle was turned on; condensed repli- unfertilized eggs to isotonic solutions of non-electrolytes con- cated chromosomes were seen in all the eggs by 90 min. The taining very low concentrations of electrolytes. modification of the outer surface is pronounced after these In older literature, it was recognized that such media at- brief treatments with non-electrolyte media (Fig. 4); the vi- tack the outer surface layers, at least to the extent of remov- telline sheet is removed completely, and the microvilli are ing the vitelline sheet (9). quite extended and are spaced irregularly. Sea urchins washed thoroughly in isotonic non-electrolyte These results with non-electrolyte solutions undermine the solutions undergo spontaneous lysis. Addition of CaCl2 to 0.1 earlier (1, 2) interpretation of the ammonia effects as conse- mM will stabilize them. quences of the penetration of ammonia into the cell, acting Lytechinus eggs are exposed briefly to 1 M urea, contain- through changes in internal pH. 3. Mechanical removal of surface components 0 In the results presented so far, all the treatments which (.) caused unfertilized eggs to undergo certain changes typical - i 20 of fertilized eggs could be interpreted as the removal of 0

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-60 UREA| SEA WATER 2 4 6 8 0 12 MINUTES FIG. 3. Development of K+ ion conductance in L. pictus eggs after a 2-3 min exposure to 1 M urea containing sea water at 100- fold dilution. The microelectrode was inserted 6 min after the egg was returned to sea water. The initial values of membrane poten- tial are probably low while the punctured membrane is healing around the electrode. The healing process is completed and the membrane potential is recorded at its maximum value of more than 60 mV by 10 min after the end of the urea treatment. The re- FIG. 4. Surface of unfertilized L. pictus egg after a 5 min sponse to external K+, which follows prediction for a potassium treatment with 1 M glycerol containing 0.1 mM CaCl2, 1 mM diffusion potential, is not included in the figure. Normal Unf. (bro- Na2CO3 at pH 9.2. The microvilli are quite elongated and the cell ken line) indicates the potential of normal unfertilized egg. surface between them appears smooth (X10,000). Downloaded by guest on October 2, 2021 Cell Biology: Mazia et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4471 mV or more, just as do eggs treated with NH4OH or urea. They entered the chromosome cycle and continued to repeat it, as is shown in Fig. 5c. The success of the mechanical "peeling" of the outer sur- face of the Lytechinus egg could not be repeated with the eggs of S. purpuratus. Neither the visible effect on the sur- face nor the activation of post-fertilization activities could be obtained with fibers coated with protamine. 4. Components of the outer surface; removal of the vitelline sheet The vitelline sheet is the recognized structure overlying the plasma membrane of the unfertilized egg. It is defined by its function as the precursor to the fertilization membrane and has been described in electron microscopic studies (ref. 10; earlier literature summarized in ref. 11). Various methods have been used to remove it in order to obtain fertilized eggs without fertilization coats. One such method employs di- thiothreitol (12). In the present work, we have treated eggs of both L. pictus and S. purpuratus with dithiothreitol in sea water at concentrations of 5-20 mM and at pH levels up to 9. The vitelline sheets are removed; membranes do not el- evate at fertilization. The removal of the vitelline sheet with dithiothreitol is not followed by the changes observed after treatment with ammonia or urea. The membrane potential is not changed; the chromosome cycle is not turned on. With the SEM, we observe (Fig. 6a) that the microvilli retain their regular ar- rangement and low profile, as though still restrained. If the exposure to dithiothreitol is followed by exposure to NH3-sea water (or to the non-electrolyte medium) the microvilli ex- tend (Fig. 6b). The changes typical of the ammonia effect follow. Comparing Fig. 6a and 6b, it is seen that some mate- rial remains on the outer surface after the dithiothreitol treatment and is then removed by the NH3-sea water. It is the removal of this layer which, we propose, is responsible for the changes in the outer surface which lead to the turn- ing on of various processes in the unfertilized egg. There is no correlation between the removal of the vitel- line sheet and the turning on of activities in the egg. Non- electrolyte solutions remove the vitelline sheet rapidly. Am- FIG. 5. L. pictus eggs were shaken for 5 min after the attach- monia removes it slowly and the eggs will still raise fertiliza- ment of protamine-coated glass fibers to their surfaces. (a) Low coats when inseminated after the minimum effective power view of the surface of an egg fixed immediately after the tion above treatment. Some glass fibers remain. The torn and peeled- treatment with ammonia. Ethylamine produces the ammo- back outer surface is seen (X1000). (b) At higher magnification, nia effects with still less impairment of the vitelline sheet. In greatly elongated microvilli are seen in a region from which the Fig. 7 one sees the surface of the egg through the vitelline surface layer has been torn away. Around this area one sees rem- sheet; the microvilli have elongated but are pressed flat nants of the outer surface layer, detached and crumpled (X10,000). against the cell surface. (c) An egg fixed 6 hr later for cytological observations. The treat- ment has turned on chromosome replication and a number of cy- cles have been completed without mitosis (5), giving a highly poly- 5. Summary of results ploid nucleus (X1000). Unfertilized sea urchin eggs exposed to NH3-sea water are incited to enter a number of changes which normally follow components of the outer surface layer. A test of this inter- fertilization. The effects of NH3-sea water are attributed to pretation would be the direct mechanical removal -of the ammonia (or unionized NH40H); they have also been ob- outer surface layer. This was possible with Lytechinus eggs tained with ethylammonium hydroxide. Similar results may by attaching protamine-coated glass fibers to the outer sur- be obtained with isotonic urea solutions and with isotonic face and shaking the egg suspension strongly on a mechani- glycerol solutions at minimal concentrations of ions. The ef- cal shaker. The fibers adhered strongly to the outer surface fects are correlated with a conspicuous change of the outer and the turbulence caused them to pull away, carrying with cell surface that can be seen with the SEM; one can predict them the surface components to which they were attached the behavior of the egg from observations of the surface or (Fig. 5). The effects of this mechanical peeling of regions of predict the appearance of the surface from the behavior of the outer surface are seen in Fig. 5 where microvilli extend the eggs. in regions where an outer sheet has been torn. We will interpret these effects as the result of the removal The membrane potential of Lytechinus eggs so treated of a component of the outer surface. The component is not was measured. The membrane developed a potential of -60 the vitelline sheet as defined morphologically or as the pre- Downloaded by guest on October 2, 2021 4472 Cell Biology: Mazia et al. Proc. Nat. Acad. Sci. USA 72 (1975)

FIG. 6. (a) Surface of an L. pictus egg after 15 min in sea water at pH 8.5 containing 20 mM dithiothreitol. The microvilli are low but no longer so closely and regularly arranged as in Fig. 1. On the surface between the microvilli one sees a sprinkling of small particles. After re- moval of the vitelline sheet with dithiothreitol the egg behaves as a normal unfertilized egg (X10,000). (b) After removal of the vitelline sheet with dithiothreitol eggs were treated with sea water adjusted to pH 9.2 with NH40H. The microvilli are much longer and are separated by areas of the cell surface which no longer show the small particles observed in (a) (X10,000). tilization for a long time. Fertilization is an extreme case. A rather dormant egg, equipped to do almost everything a cell can do, is aroused to activities of all kinds; it is vividly evi- dent that the changes start at the cell surface. The surface of the egg is very complex, but the complexity may represent only a more explicit display of features found in other cell surfaces. In interpreting the results, we adapt the concept (13) that the plasma membrane consists of integral components, built into the lipid bilayer, and peripheral components which play a part in the functions of the membrane but can be dis- sociated from the bilayer. The substances removed by our various treatments are peripheral components; their removal FIG. 7. Surface of an L. pictus egg treated with sea water con- changes the membrane. [We present here no evidence that taining 5 mM ethylamine and adjusted to pH 9.2 for 15 min. The they are proteins, but the removal of surface proteins by disarrayed and elongated microvilli are seen through the remain- NH3-sea water is shown by Johnson and Epel (14).] ing vitelline sheet (X10,000). In the interpretive diagram (Fig. 8), the integral compo- nents are shown as channels and the removal of the periph- eral components is imaged fancifully as the pulling of stop- cursor to the fertilization membrane. It is proposed that the pers. This is only a symbolic figuration of changes in per- component is situated below the vitelline sheet and on the meabilities which follow fertilization and which also follow plasma membrane. the ammonia effects. In both cases, an increase in K+-con- ductance ensues. There are increases in the transport of thy- DISCUSSION midine (3) and of amino acids (2), though these come more The interpretation of these findings appeals to fundamental slowly after the treatment with ammonia than they do after theses of membrane biology. An older one is that changes in fertilization. After fertilization, there is an increase in pas- the cell membrane can regulate changes in the functions of sive permeability to water (15) and to non-electrolytes (16). cells. A newer one is that changes on the outer surface of the The diagram would be misleading if taken to imply that the cell can modify the functions of the plasma membrane. changes in various permeability functions following dissocia- These propositions have been implicit in the analysis of fer- tion of the peripheral component are immediate.

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DITHIOTHREITOL NH3 OR NONELECTROLYTE FERTILIZED FIG. 8. A schematic interpretation of present results. At the outer surface of the normal unfertilized egg, peripheral components (P) of the plasma membrane are linked to integral components (I), regulating functions of the membrane; components P also are bonded to the vi- telline sheet (VS). Removal of the vitelline sheet with dithiothreitol does not affect the peripheral component. Treatments with ammonia- sea water or non-electrolyte solutions dissociate the components P from the membrane; changes which normally follow fertilization are turned on. In normal fertilization, the surface reactions dissociate components P from the membrane as well as elevating the vitelline sheet to form a fertilization coat (FC). Downloaded by guest on October 2, 2021 Cell Biology: Mazia et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4473 We may include the surface changes at normal fertiliza- This work has depended on support from the following grants: tion in the scheme shown in Fig. 8. The longest-known and U.S. Public Health Service GM 13882 to D.M.; National Science most obvious happening at fertilization is the separation of Foundation GB 42547 to R.S.; and National Science Foundation GB an outer layer from the surface of the egg, forming a fertil- 38359, which provided a scanning electron microscope to the Elec- ization coat (17). This has seemed to be a dispensable event, tron Microscope Laboratory of this institution. since fertilization proceeds normally without the formation of the fertilization coat after various treatments, as with di- 1. Steinhardt, R. A. & Mazia, D. (1973) Nature 241, 400-401. Now it is that normal fertilization does 2. Epel, D., Steinhardt, R., Humphreys, T. & Mazia, D. (1974) thiothreitol. proposed Dev. Biol. 40,245-255. not merely lift up an overlying and dispensable vitelline 3. Mazia, D. & Ruby, A. (1974) Exp. Cell Res. 85,167-172. sheet. It separates from the membrane an outer surface com- 4. Landauer, W. (1922) Roux Arch. Entwicklungsmech. Org. plex which is better regarded as a component of the mem- 52,1. brane of the unfertilized egg and which may be responsible 5. Mazia, D. (1974) Proc. Nat. Acad. Sci. USA 71, 690-693. for the repression of activities in that egg. The visible sign of 6. Wilt, F. H. & Mazia, D. (1974) Dev. Biol. 37,422-424. this dissociation, the elongation of the microvilli, has been 7. Steinhardt, R., Lundin, L. & Mazia, D. (1971) Proc. Nat. observed in normal fertilization (18). Acad. Sci. USA 68,2426-2430. In fertilization, the egg uses the complex gadgetry of the 8. Mazia, D., Schatten, G. & Sale, W. (1975) J. Cell Biol. 66, "cortical reactions"-fusion of secretory vesicles with the 198-200. 9. Moore, A. R. (1930) Protoplasma 9,9. membrane and discharge of their contents-to bring about 10. Anderson, E. (1968) J. Cell Biol. 37,514. effects which can be imitated by ammonia, etc. The imita- 11. Austin, E. R. (1968) Ultrastructure of Fertilization (Holt, tion is not a substitution. Fertilization and proper partheno- Rinehart & Winston, New York). genetic procedures motivate additional events which are all- 12. :pel, D., Weaver, A. M. & Mazia, D. (1970) Exp. Cell Res. 61, important for development. 64-68. It is quite in accord with current views about the cell sur- 13. Singer, S. J. (1974) Annu. Rev. Biochem. 43, 805. face that peripheral components of the membrane, on the 14. Johnson, J. D. & Epel, D. (1975) Proc. Nat. Acad. Sci. USA 72, outer surface, should be responsible for the properties of the 4474-4478. membrane of the unfertilized egg and for the repressed state 15. McCutcheon, M. & Lucke, B. (1932) J. Cell. Comp. Physiol. in of ammonia and of non- 2,11. of activity that egg. The effects 16. Stewart, D. R. & Jacobs, M. H. (1936) J. Cell. Comp. Physiol. electrolytes define conditions of the dissociation of the com- 7,333. ponents from the surface, using visible changes and the turn- 17. Fol, H. (1879) Recherches sur la Fgcondation et le Com- ing on of various processes as criteria. It should be possible to mencement de L'Henogenie chez divers Animaux (Henri identify the components and to reassociate them with the Georg, Geneva-Basle-Lyon). surface; an indication that this may be done is reported by 18. Tegner, M. J. (1975) Ph.D. Dissertation, University of Califor- Johnson and Epel (14). nia, San Diego. Downloaded by guest on October 2, 2021