- Email: [email protected]
Inhibition of lymphocyte peripolesis and cytotoxic action in vitro by antilymphocyte serum (ALS)
136
P. Biberfeld et al. INHIBITION OF LYMPHOCYTE PERIPOLESIS AND CY TOTOXIC IN VITRO BY ANTILYMPHOCYTE (AL9
ACTION SERUM
P. BIBERFELD, G. HOLM and P. PERLMANN, Department of Pathology at Sabbatsberg Hospital, Karolinska Institutet Medical School, and Department of Immunology, Wenner-Gren Institute, Stockholm, Sweden
Fig. 1. Rabbit ova after 4 h exposure to medium containing ferric sulfate instead of ferrous sulfate.
ferrous complexes are also known to be important for the cell division process of plant root tips (e.g. [5]) and similar substances could conceivably have .a role in the early mitotic activity of animal zygotes. In other experiments, rabbit blastocysts that had been isolated from the uterus on the 5th day post coitum were also maintained in vitro in Fe2+-free medium, or Fe3+-substituted medium. These embryos were collapsing by 6 h of culture while the controls in full medium continued to grow for at least 24 h. Thus, both of the preimplantation stages (i.e., cleavage and blastocyst) of rabbit embryogenesis demonstrate a distinct requirement for the ferrous ion. This research was supported by AEC contract AT (1 l-l)1597 and NIH grant DE-00198.
REFERENCES 1. 2. 3. 4. 5.
Daniel, J C, J embryo1 exptl morph01 13 (1965) 89. - Exptl cell res 47 (1967) 619. Ham, R G, Exptl cell res 28 (1962) 489. - Proc natl acad sci 53 (1965) 288. Amoore, J E, J cell biol 13 (1962) 373.
Received August 21, 1968 Exptl Cell Res 54
In the presence of phytohaemagglutinin (PHA), lymphocytes will aggregate to monolayers of tissue culture cells and move around the surface of these in a fashion consistent with that described as peripolesis [l]. This is followed by a cytotoxic action of the lymphocytes on the tissue culture cells, as evidenced by the detachment of the latter from the substratum and by the accumulation of damaged or dead cells in the culture medium [ 11. The observations presented below indicate that in the presence of heat inactivated antilymphocytic serum (ALS) in the culture medium, lymphocytes are agglutinated and seem to form stable aggregates. Peripolesis does not occur and the cytotoxic action of lymphocytes is inhibited. Materials
and methods
Chang liver cells (established cell strain, human origin) were grown as monolayers in plastic petri dishes (NUNCLONB, NUNC A/S, Roskilde, Denmark). Lymphocyte suspensions from peripheral human blood were prepared as described previously 121. lo6 lymphocytes in 0.1 ml of culture medium were added in 2 drops to the petri dishes and allowed to sediment onto the target cells in the middle of the monolayer for 30 min, whereafter 5 ~1 stock solution of phytohaemagglutinin (PHA-M, Difco Lab., Detroit, Mich.) per ml culture medium, and ALS to a final concentration of 1: 10, were added. The petri dishes were incubated at 37°C in an atmosphere of 95 % CO, and 5 % air and were observed under a phase microscope for signs of cytotoxic effect, such as plaque formation. The degree of plaque formation was graded from 0 to 3 + (1 + = moderate clearance of monolayer; 3 + =disappearance of the majority of the target cells within the central part of the monolayer). For light and electron microscopy the cells were fixed and dehydrated in situ; the monolayers were detached from the petri dishes with epoxy propane and the lymphocyte-covered areas were embedded separately as described elsewhere 131. The supernatant medium from the petri dishes was centrifuged and embedded in the same way as the monolayer cells. Antilymphocyte serum (ALS) was obtained from a rabbit (RS 183) and a horse (HS) [9] both injected several times with suspensions of partially purified lymphocytes from human peripheral blood and thoracic duct lymph. The sera were unactivated for 30 min at 56°C and stored frozen until used.
Inhibition
of lymphocyte peripo~e$~~
137
Table 1
Expt
Donor
I
B. G.
II
c. P.
III
P. B.
ALS
No. of cultures
R138 HS R 138 R138
Development of plaques (time of observation, h) 16
24
48
2 3 3 1 3
I Not examined
+ -
+ + -’ -
+ + -
+‘+ -
2 1
I!z -
+t -
Not examined -
70-80
+ 2 + -
The culture medium from monolayers which underwent plaque formation contained (1) results of 3 experiments in regard to the devel- Chang cells with various signs of cell dama,ge, opment of plaques in Chang cell monolayers, (2) a few undamaged Chang cells, and (3) normal incubated with lymphocytes and PHA, and the lymphocytes and lymphocytes with signs of effect of ALS on plaque formation. In the pres- PHA induced stimulation. The lymphocytes were ence of PHA, clearance was observed within the either single or aggregated to each other but first 10 to 24 h in the area covered with lymphoonly rarely attached to damaged Chang cells. cytes. The time of appearance of definite plaques In cultures incubated with lymphocytes, P varied in different experiments, probably reflectand ALS, the number of single lymphocytes ating differences in target cell density and local tached to Chang cells was greatly reduced and lymphocyte concentration. In all monolayers lymphocytes were only rarely seen to penetrate treated with both PHA and lymphocytes, plaque the monolayers. The agglutination caused by the formation was scored 3 + within 48 h. addition of ALS was different from that observed The addition of ALS at the same time as in the presence of PHA only. In the presence of PHA and lymphocytes, completely prevented ALS almost the entire surfaces of the aggluthe appearance of plaques in all experiments. tinated lymphocytes adhered to each other, When ALS was added to cultures, which already which gave a dense tissue-like appearance to the showed signs of clearance, further plaque formacell aggregates (figs 1 b, 2). These aggregates tion was arrested. seemed to be rather stable and rested on the Light and electron microscopic observations. surface of Chang cells (fig. 16). Lymphocyte peripolesis-as previously described Large areas of the cell surface of lym~bocytes [l]-was seen in all cultures incubated with from ALS-treated cultures were often seen to PHA. From 3 h onward, increasing numbers of be coated with a fuzzy material (fig. lc), which lymphocytes were seen to penetrate the monowas also present in the space between the plasma layers and to appear under the Chang cells. membranes, of aggregated lymphocytes. This By the time a monolayer showed signs of clear- material was not seen on lyrnp~o~yt~s in culance, most Chang cells were surrounded and tures without ALS. often partially detached by lymphocytes (fig. 1 a). Most aggregates contained a few damaged or The PHA induced agglutination of lymphocytes dead lymphocytes or cell debris, stuck to the to Chang cells was relatively firm, since the surface of the healthy cells. Dead or damaged travelling lymphocytes were not detached by Chang cells were usually also seen in the medium of the AU-treated cultures. In these culvigo.urous shaking before fixation. Results Plaque formation (PF). Table 1 summarizes the
138
P. Bibevfeld et al.
Fig. 1. (a, b) Chang cells and Ivmuhocvtes from monolaver cultures incubated for 43 h with PHA (a)._. PHA (b) and ALS. Note dense aggregation of lymphocytes and basophilic granulation of Chang cells in the presence of ALS. x 1200. (c) Electron micrograph of area of lymphocyte plasma membrane covered with a fuzzy layer of moderately electron dense material; same culture as in b. x 140,000.
tures, many Chang cells contained large amounts of basophilic granules (fig. 1b). In the electron microscope these seemed to correspond to lysosomal structures of endocytic or autophagic origin. Comments The prevention of plaque formation by ALS described here is in agreement with observation on the effect of ALS on PHA-induced plaque formation in other lymphocyte target cell sysExptl
Cell Res 54
terns [4]. ALS has also been shown to inhibit the cytotoxic effect of lymphocytes on 51Cr-labelled Chang cells in suspension,appearing under several experimental conditions [5, 61. Previous observations have indicated that peripolesis of lymphocytes on and underneath Chang cell monolayers is a characteristic pattern of cell interrelationship of cultures, in which plaque formation is induced by the addition of PHA [I]. As shown here, the presenceof ALS in such cultures inhibits both plaque formation
Inhibition
of lymphocyte peuipolesis
139
Fig. 2. Electron micrograph of a dense tissue-like aggregate of lymphocytes; same culture as in fig. 1 h. :16000.
and peripolesis and leads to the formation of seemingly stable lymphocyte aggregates. Trapping of cytotoxic (effector) cells may thus be responsible for the suppressionof plaque formation. The fuzzy layer seenon the cell membrane of ALS-treated lymphocytes probably contains antigen-antibody deposits, since antibodies have been shown by immunofluorescence to attach to the surface of ALS-treated cells [7]. It is possible that this surface coating also contributes to the suppression of lymphocyte cytotoxicity
in vitro, by inducing membrane alterations or blocking membrane structures (‘“blind-folding”) necessary for the effectuation of the cytotoxic reaction [g].
The skilled technical assistance of Miss Marianne Bj6rk and Mrs Brita-Maria Berg is gratcfully acknowledged. The horse antilymphocyte serum was obtained from Dr G. Moller, Inst. of Bacteriology. -_ Karolinska InstiWet, Stockholm, Sweden. Supported by grants from the Swedish National Association against Heart and Chest Diseases, the Lotten Bohman Foundation and grant no. B67-16X-I4805A from the Swedish Medical Research Council.
140
V. 1). Vacquier REFERENCES
1. Biberfeld, P, Helm, G & Perlmann, P, Exptl cell res. In press. 2. Holm, G & Perlmann, P, Immunol 12 (1967) 525. 3. Biberfeld, P, Proc Stand sot electron microscope (June 5, 1968). In press. 4. Iundgren, G, Collste, L & Miiller, G, Nature. In press. 5. Holm, G & Perlmann, P, Antibiot chemother 15 (1968) 21. 6. - Proc 2nd intern congr transplantation sot (New York 1968). In press. 7. Woodruff, M F A. Anderson, N F & Abaza, H M, Bristol symp lymphocyte immunol haemopoieses, p: 286 (1966). 8. Levey, R.H & Medawar, P B, Proc natl acad sci 56 (1966) 1130. Received September 16, 1968
THE ISOLATION AND PRELIMINARY ANALYSIS OF THE HYALINE LAYER SEA URCHIN EGGS V. D. VACQUIER,r of California,
Berkeley,
Department of Zoology, Calif. 94720, USA
OF
University
Within minutes of insemination of echinoderm eggs a clear coating several micra in thickness forms directly upon the exterior surface of the plasma membrane. This is the hyaline layer (fig. 1) and it is composed of material discharged onto the surface of the egg by the rupture of the cortical granules [3, 4, 81. After its formation, the hyaline layer remains as a rigid investing layer to which the blastomeres, produced during cleavage, are attached by surface villi. By virtue of the rigidity of the hyaline layer and the firm attachment of the blastomeres to its inner surface, the blastula is formed as a hollow ball of cells one cell in thickness [3, 4, lo]. Despite its importance, the hyaline layer has not been adequately characterised. Procedures have been reported for the isolation of the so-called cortex [9] and the plasma membrane [l] of sea urchin eggs. Therefore, it seems useful to describe the isolation of the hyaline layer since it is a prominent structure 1 Predoctoral fellow of the USPHS; fellowship no. 4-Fl-GM-30, 187-02. Present address: Laboratorio Internazionale di Genetica e Biofisica, Casella Postale 3061, 80100 Napoli, Italy. Exptl
Cell
Res 54
that is bound to the plasma membrane and hence to the cortex of the seaurchin egg.
Isolation of intact hyaline layers The procedure consists of digesting the fertilization membranes as they rise from the surface of the eggs before the formation of the hyaline layer followed by the lysis of the cells and the solubilization of the cytoplasmic material by a hypotonic detergent solution. Gametes of Strongylocentrotus purpuratus and S. franciscanus were obtained by injection of 0.5 M KCl. Six ml of thoroughly washed eggs were allowed to settle in the bottom of a 40 ml conical centrifuge tube. Five ml of fresh sperm suspensionin sea water was added, a stopwatch started and the tube inverted several times to insure thorough mixing. A drop of the suspension was placed on a microscope slide for immediate observation. When the fertilization membranesbegan to rise, 30 ml of pronase solution (Calbiochem, 1 mg/ml) in Ca-free seawater was added, the tube stoppered and inverted continuously at 1 cps. The time of pronase addition is critical: with S. purpuratus it must be between 35 and 55 set after sperm addition whereas with S. franciscanus 90 set is the upper limit. The fertilization membranes were completely digested after 2 min of exposure to pronase. The tube was placed in a hand centrifuge and spun very slowly through 15 revolutions of the hand crank (ratio 16: 1). The supernatant was removed by aspiration, 35 ml of Ca-free sea water added and the pellet of naked eggs resuspended by gently inverting the tube. The contents of the tube were poured into a Petri dish 14 cm in diameter that already contained 200 ml normal sea water. No more than 3.5 min had elapsed between insemination and the pouring of the pronase suspension into the Petri dish. The eggs settled on the bottom of the dish and formed a cohesive sheet due to the adhesiveness of the hyaline layers. The sheet of eggs was washed repeatedly in seawater to remove pronase and free spermatozoa. Hyaline layers were fully formed in 20 min. The sea water was removed by aspiration,
P. Biberfeld et al. INHIBITION OF LYMPHOCYTE PERIPOLESIS AND CY TOTOXIC IN VITRO BY ANTILYMPHOCYTE (AL9
ACTION SERUM
P. BIBERFELD, G. HOLM and P. PERLMANN, Department of Pathology at Sabbatsberg Hospital, Karolinska Institutet Medical School, and Department of Immunology, Wenner-Gren Institute, Stockholm, Sweden
Fig. 1. Rabbit ova after 4 h exposure to medium containing ferric sulfate instead of ferrous sulfate.
ferrous complexes are also known to be important for the cell division process of plant root tips (e.g. [5]) and similar substances could conceivably have .a role in the early mitotic activity of animal zygotes. In other experiments, rabbit blastocysts that had been isolated from the uterus on the 5th day post coitum were also maintained in vitro in Fe2+-free medium, or Fe3+-substituted medium. These embryos were collapsing by 6 h of culture while the controls in full medium continued to grow for at least 24 h. Thus, both of the preimplantation stages (i.e., cleavage and blastocyst) of rabbit embryogenesis demonstrate a distinct requirement for the ferrous ion. This research was supported by AEC contract AT (1 l-l)1597 and NIH grant DE-00198.
REFERENCES 1. 2. 3. 4. 5.
Daniel, J C, J embryo1 exptl morph01 13 (1965) 89. - Exptl cell res 47 (1967) 619. Ham, R G, Exptl cell res 28 (1962) 489. - Proc natl acad sci 53 (1965) 288. Amoore, J E, J cell biol 13 (1962) 373.
Received August 21, 1968 Exptl Cell Res 54
In the presence of phytohaemagglutinin (PHA), lymphocytes will aggregate to monolayers of tissue culture cells and move around the surface of these in a fashion consistent with that described as peripolesis [l]. This is followed by a cytotoxic action of the lymphocytes on the tissue culture cells, as evidenced by the detachment of the latter from the substratum and by the accumulation of damaged or dead cells in the culture medium [ 11. The observations presented below indicate that in the presence of heat inactivated antilymphocytic serum (ALS) in the culture medium, lymphocytes are agglutinated and seem to form stable aggregates. Peripolesis does not occur and the cytotoxic action of lymphocytes is inhibited. Materials
and methods
Chang liver cells (established cell strain, human origin) were grown as monolayers in plastic petri dishes (NUNCLONB, NUNC A/S, Roskilde, Denmark). Lymphocyte suspensions from peripheral human blood were prepared as described previously 121. lo6 lymphocytes in 0.1 ml of culture medium were added in 2 drops to the petri dishes and allowed to sediment onto the target cells in the middle of the monolayer for 30 min, whereafter 5 ~1 stock solution of phytohaemagglutinin (PHA-M, Difco Lab., Detroit, Mich.) per ml culture medium, and ALS to a final concentration of 1: 10, were added. The petri dishes were incubated at 37°C in an atmosphere of 95 % CO, and 5 % air and were observed under a phase microscope for signs of cytotoxic effect, such as plaque formation. The degree of plaque formation was graded from 0 to 3 + (1 + = moderate clearance of monolayer; 3 + =disappearance of the majority of the target cells within the central part of the monolayer). For light and electron microscopy the cells were fixed and dehydrated in situ; the monolayers were detached from the petri dishes with epoxy propane and the lymphocyte-covered areas were embedded separately as described elsewhere 131. The supernatant medium from the petri dishes was centrifuged and embedded in the same way as the monolayer cells. Antilymphocyte serum (ALS) was obtained from a rabbit (RS 183) and a horse (HS) [9] both injected several times with suspensions of partially purified lymphocytes from human peripheral blood and thoracic duct lymph. The sera were unactivated for 30 min at 56°C and stored frozen until used.
Inhibition
of lymphocyte peripo~e$~~
137
Table 1
Expt
Donor
I
B. G.
II
c. P.
III
P. B.
ALS
No. of cultures
R138 HS R 138 R138
Development of plaques (time of observation, h) 16
24
48
2 3 3 1 3
I Not examined
+ -
+ + -’ -
+ + -
+‘+ -
2 1
I!z -
+t -
Not examined -
70-80
+ 2 + -
The culture medium from monolayers which underwent plaque formation contained (1) results of 3 experiments in regard to the devel- Chang cells with various signs of cell dama,ge, opment of plaques in Chang cell monolayers, (2) a few undamaged Chang cells, and (3) normal incubated with lymphocytes and PHA, and the lymphocytes and lymphocytes with signs of effect of ALS on plaque formation. In the pres- PHA induced stimulation. The lymphocytes were ence of PHA, clearance was observed within the either single or aggregated to each other but first 10 to 24 h in the area covered with lymphoonly rarely attached to damaged Chang cells. cytes. The time of appearance of definite plaques In cultures incubated with lymphocytes, P varied in different experiments, probably reflectand ALS, the number of single lymphocytes ating differences in target cell density and local tached to Chang cells was greatly reduced and lymphocyte concentration. In all monolayers lymphocytes were only rarely seen to penetrate treated with both PHA and lymphocytes, plaque the monolayers. The agglutination caused by the formation was scored 3 + within 48 h. addition of ALS was different from that observed The addition of ALS at the same time as in the presence of PHA only. In the presence of PHA and lymphocytes, completely prevented ALS almost the entire surfaces of the aggluthe appearance of plaques in all experiments. tinated lymphocytes adhered to each other, When ALS was added to cultures, which already which gave a dense tissue-like appearance to the showed signs of clearance, further plaque formacell aggregates (figs 1 b, 2). These aggregates tion was arrested. seemed to be rather stable and rested on the Light and electron microscopic observations. surface of Chang cells (fig. 16). Lymphocyte peripolesis-as previously described Large areas of the cell surface of lym~bocytes [l]-was seen in all cultures incubated with from ALS-treated cultures were often seen to PHA. From 3 h onward, increasing numbers of be coated with a fuzzy material (fig. lc), which lymphocytes were seen to penetrate the monowas also present in the space between the plasma layers and to appear under the Chang cells. membranes, of aggregated lymphocytes. This By the time a monolayer showed signs of clear- material was not seen on lyrnp~o~yt~s in culance, most Chang cells were surrounded and tures without ALS. often partially detached by lymphocytes (fig. 1 a). Most aggregates contained a few damaged or The PHA induced agglutination of lymphocytes dead lymphocytes or cell debris, stuck to the to Chang cells was relatively firm, since the surface of the healthy cells. Dead or damaged travelling lymphocytes were not detached by Chang cells were usually also seen in the medium of the AU-treated cultures. In these culvigo.urous shaking before fixation. Results Plaque formation (PF). Table 1 summarizes the
138
P. Bibevfeld et al.
Fig. 1. (a, b) Chang cells and Ivmuhocvtes from monolaver cultures incubated for 43 h with PHA (a)._. PHA (b) and ALS. Note dense aggregation of lymphocytes and basophilic granulation of Chang cells in the presence of ALS. x 1200. (c) Electron micrograph of area of lymphocyte plasma membrane covered with a fuzzy layer of moderately electron dense material; same culture as in b. x 140,000.
tures, many Chang cells contained large amounts of basophilic granules (fig. 1b). In the electron microscope these seemed to correspond to lysosomal structures of endocytic or autophagic origin. Comments The prevention of plaque formation by ALS described here is in agreement with observation on the effect of ALS on PHA-induced plaque formation in other lymphocyte target cell sysExptl
Cell Res 54
terns [4]. ALS has also been shown to inhibit the cytotoxic effect of lymphocytes on 51Cr-labelled Chang cells in suspension,appearing under several experimental conditions [5, 61. Previous observations have indicated that peripolesis of lymphocytes on and underneath Chang cell monolayers is a characteristic pattern of cell interrelationship of cultures, in which plaque formation is induced by the addition of PHA [I]. As shown here, the presenceof ALS in such cultures inhibits both plaque formation
Inhibition
of lymphocyte peuipolesis
139
Fig. 2. Electron micrograph of a dense tissue-like aggregate of lymphocytes; same culture as in fig. 1 h. :16000.
and peripolesis and leads to the formation of seemingly stable lymphocyte aggregates. Trapping of cytotoxic (effector) cells may thus be responsible for the suppressionof plaque formation. The fuzzy layer seenon the cell membrane of ALS-treated lymphocytes probably contains antigen-antibody deposits, since antibodies have been shown by immunofluorescence to attach to the surface of ALS-treated cells [7]. It is possible that this surface coating also contributes to the suppression of lymphocyte cytotoxicity
in vitro, by inducing membrane alterations or blocking membrane structures (‘“blind-folding”) necessary for the effectuation of the cytotoxic reaction [g].
The skilled technical assistance of Miss Marianne Bj6rk and Mrs Brita-Maria Berg is gratcfully acknowledged. The horse antilymphocyte serum was obtained from Dr G. Moller, Inst. of Bacteriology. -_ Karolinska InstiWet, Stockholm, Sweden. Supported by grants from the Swedish National Association against Heart and Chest Diseases, the Lotten Bohman Foundation and grant no. B67-16X-I4805A from the Swedish Medical Research Council.
140
V. 1). Vacquier REFERENCES
1. Biberfeld, P, Helm, G & Perlmann, P, Exptl cell res. In press. 2. Holm, G & Perlmann, P, Immunol 12 (1967) 525. 3. Biberfeld, P, Proc Stand sot electron microscope (June 5, 1968). In press. 4. Iundgren, G, Collste, L & Miiller, G, Nature. In press. 5. Holm, G & Perlmann, P, Antibiot chemother 15 (1968) 21. 6. - Proc 2nd intern congr transplantation sot (New York 1968). In press. 7. Woodruff, M F A. Anderson, N F & Abaza, H M, Bristol symp lymphocyte immunol haemopoieses, p: 286 (1966). 8. Levey, R.H & Medawar, P B, Proc natl acad sci 56 (1966) 1130. Received September 16, 1968
THE ISOLATION AND PRELIMINARY ANALYSIS OF THE HYALINE LAYER SEA URCHIN EGGS V. D. VACQUIER,r of California,
Berkeley,
Department of Zoology, Calif. 94720, USA
OF
University
Within minutes of insemination of echinoderm eggs a clear coating several micra in thickness forms directly upon the exterior surface of the plasma membrane. This is the hyaline layer (fig. 1) and it is composed of material discharged onto the surface of the egg by the rupture of the cortical granules [3, 4, 81. After its formation, the hyaline layer remains as a rigid investing layer to which the blastomeres, produced during cleavage, are attached by surface villi. By virtue of the rigidity of the hyaline layer and the firm attachment of the blastomeres to its inner surface, the blastula is formed as a hollow ball of cells one cell in thickness [3, 4, lo]. Despite its importance, the hyaline layer has not been adequately characterised. Procedures have been reported for the isolation of the so-called cortex [9] and the plasma membrane [l] of sea urchin eggs. Therefore, it seems useful to describe the isolation of the hyaline layer since it is a prominent structure 1 Predoctoral fellow of the USPHS; fellowship no. 4-Fl-GM-30, 187-02. Present address: Laboratorio Internazionale di Genetica e Biofisica, Casella Postale 3061, 80100 Napoli, Italy. Exptl
Cell
Res 54
that is bound to the plasma membrane and hence to the cortex of the seaurchin egg.
Isolation of intact hyaline layers The procedure consists of digesting the fertilization membranes as they rise from the surface of the eggs before the formation of the hyaline layer followed by the lysis of the cells and the solubilization of the cytoplasmic material by a hypotonic detergent solution. Gametes of Strongylocentrotus purpuratus and S. franciscanus were obtained by injection of 0.5 M KCl. Six ml of thoroughly washed eggs were allowed to settle in the bottom of a 40 ml conical centrifuge tube. Five ml of fresh sperm suspensionin sea water was added, a stopwatch started and the tube inverted several times to insure thorough mixing. A drop of the suspension was placed on a microscope slide for immediate observation. When the fertilization membranesbegan to rise, 30 ml of pronase solution (Calbiochem, 1 mg/ml) in Ca-free seawater was added, the tube stoppered and inverted continuously at 1 cps. The time of pronase addition is critical: with S. purpuratus it must be between 35 and 55 set after sperm addition whereas with S. franciscanus 90 set is the upper limit. The fertilization membranes were completely digested after 2 min of exposure to pronase. The tube was placed in a hand centrifuge and spun very slowly through 15 revolutions of the hand crank (ratio 16: 1). The supernatant was removed by aspiration, 35 ml of Ca-free sea water added and the pellet of naked eggs resuspended by gently inverting the tube. The contents of the tube were poured into a Petri dish 14 cm in diameter that already contained 200 ml normal sea water. No more than 3.5 min had elapsed between insemination and the pouring of the pronase suspension into the Petri dish. The eggs settled on the bottom of the dish and formed a cohesive sheet due to the adhesiveness of the hyaline layers. The sheet of eggs was washed repeatedly in seawater to remove pronase and free spermatozoa. Hyaline layers were fully formed in 20 min. The sea water was removed by aspiration,