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 -j-40 -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.