- Email: [email protected]
Methods for removal of the vitelline membrane of sea urchin eggs
Experimental Cell Research 61 (1970) 69-70
METHODS
FOR
REMOVAL
OF THE
SEA URCHIN II. Controlled
VITELLINE
MEMBRANE
OF
EGGS
Exposure to Trypsin to Eliminate Post-fertilization Clumping of Embryos D. EPEL
Department of Biological Sciences, Hopkins Marine Station of Stanford University, Pacific Grove, Calif. 93950, USA
SUMMARY A simple modification of the trypsin method for removing the vitelline membrane of sea urchin eggs yields membraneless eggs which do not clump appreciably after insemination. The modification entails minimal incubation in dilute trypsin (0.25 mg %). It is postulated that although this procedure functionally removes the vitelline membrane, patches of membrane remain on the surface and prevent adhesion between hyaline layers of adjacent cells.
A procedure for removing the vitelline membrane of sea urchin eggs, based on the sensitivity of the membrane to dithiothreitol, is described in the preceding paper [3]. This method is simple and rapid, but has the disadvantage that the eggs are temporarily sticky after fertilization and can form bothersome clumps under some conditions. I have found, and here report, that the widelyused trypsin procedure for removing membranes [l] can be modified to yield nonsticky eggs after insemination. This method has been developed with the eggs of Strongylocentrotus purpuratus and may also be applicable to eggs of other species. Principle of method Eggs are incubated in a low concentration of 2 x crystallized trypsin in sea water for the minimal time required to remove the vitelline membrane. As soon as the membranes are no longer apparent (as evidenced by failure to form a fertilization membrane upon insemination), the trypsin is removed from the eggs by dilution and washing.
Specific procedure Eggs are added to a trypsin solution in sea water (16°C) to a final concentration of 0.25 mg trypsin/ 100 ml sea water (2 x -crystallized trypsin, Worthington). A small aliquot (test aliquot) of the trypsinegg suspension is set aside and is periodically used to test for the presence of fertilization membranes. The remaining suspension is left undisturbed and allowed to settle by gravity (so that the trypsin can later be quickly removed by aspiration of the supernatant sea water). A sample is periodically (2 min intervals) taken from the test aliquot, inseminated, and assessed microscopically for the presence of fertilization membranes (membrane elevation becomes progressively smaller during the incubation in trypsin; thus, medium power microscopic examination may be necessary to assure the actual absence of the fertilization membrane). When the fertilization membrane no longer elevates in response to fertilization, the trypsin is immediately removed by aspiration of the supernatant sea water of the main egg suspension. Fresh sea water is added and the eggs are then washed three more times to completely remove the trypsin. Upon fertilization, these membrane-free eggs will develop normally and with little or no agglutination. With S. purpuratus eggs, the time required to remove the membranes is between 15-20 min at 16°C. I therefore begin to assessfor removal of membranes about 10 min after adding the trypsin. Exptl Cell Res 61
70 D. Epel Comments If membrane removal is incomplete, a nonvisible or “tight” membrane will form; blastomeres will not completely separate at cleavage, and a reduced blastocoel will result. Presence of this “tight” membrane, however, does not seem to affect p-1,3 glucanase excretion from the cortical granules [2] or with isolation of such organelles as the mitotic apparatus or cytasters [5]. Although I have not done extensive experiments, a highly purified trypsin seems to be a prerequisite for non-sticky eggs. Thus, a sample of one-time crystallized trypsin yielded sticky eggs after fertilization, whereas two-times crystallized trypsin, applied to a separate sample of eggs from the same female, yielded non-sticky eggs. Using the twotimes crystallized trypsin, I also find that longer incubation or exposure to higher trypsin concentrations yields sticky eggs upon fertilization. This agglutination is similar to that seen with membrane-free eggs obtained by other methods [3] and apparently results from the initial stickiness of the newlyformed hyaline layer.
moved only one of these components, whereas exposure to higher trypsin concentrations removed both components ([4], p. 224). Thus, in the method I have described, patches of the vitelline membrane may remain on the egg surface after the trypsin treatment. These mosaics may then effectively prevent contact between hyaline layers and hence prevent agglutination of adjacent cells, Irrespective of whether the membrane is completely removed by this procedure, the membrane appears to be functionally and microscopically (light-microscope) absent. For example, the permeability barrier normally imposed by the fertilization membrane is absent. Thus, following fertilization of trypsin-treated eggs the cortical granule enzyme ,i3-1,3 glucanase is excreted into the surrounding seawater [2]. Also, “tight” fertilization membranes are not present, since no fertilization-membrane-like structure is visible between adjacent blastomeres at the time of the first cell division. Supported by NSF grant GB-8002.
REFERENCES
Mechanism The major factor resulting in non-sticky eggs with the above procedure could be the incomplete removal of the vitelline membrane by the controlled exposure to dilute trypsin. Thus, Runnstriim has noted that the vitelline membrane, as observed under the electron microscope, appears to be composed of two components. Dilute trypsin (0.001 %) re-
Exptl Cell Res 61
1. Berg, W E, Methods in developmental biology (ed F H Wilt & N K Wessels) p. 767. Crowell, New York (1967). 2. Eoel. D. Weaver. A M. Muchmore. A V & Schimke; R T, Science 163 (1969) 294. ’ 3. Eoel. D. Weaver. 66 (i97d) 64. ’ A M & Mazia. , D., Exotl _ cell res 4. Rurmstrom, J, Advances morphogenesis 5 (1966) 222. 5. Went, H. Personal communication (1970). Received January 16, 1970
METHODS
FOR
REMOVAL
OF THE
SEA URCHIN II. Controlled
VITELLINE
MEMBRANE
OF
EGGS
Exposure to Trypsin to Eliminate Post-fertilization Clumping of Embryos D. EPEL
Department of Biological Sciences, Hopkins Marine Station of Stanford University, Pacific Grove, Calif. 93950, USA
SUMMARY A simple modification of the trypsin method for removing the vitelline membrane of sea urchin eggs yields membraneless eggs which do not clump appreciably after insemination. The modification entails minimal incubation in dilute trypsin (0.25 mg %). It is postulated that although this procedure functionally removes the vitelline membrane, patches of membrane remain on the surface and prevent adhesion between hyaline layers of adjacent cells.
A procedure for removing the vitelline membrane of sea urchin eggs, based on the sensitivity of the membrane to dithiothreitol, is described in the preceding paper [3]. This method is simple and rapid, but has the disadvantage that the eggs are temporarily sticky after fertilization and can form bothersome clumps under some conditions. I have found, and here report, that the widelyused trypsin procedure for removing membranes [l] can be modified to yield nonsticky eggs after insemination. This method has been developed with the eggs of Strongylocentrotus purpuratus and may also be applicable to eggs of other species. Principle of method Eggs are incubated in a low concentration of 2 x crystallized trypsin in sea water for the minimal time required to remove the vitelline membrane. As soon as the membranes are no longer apparent (as evidenced by failure to form a fertilization membrane upon insemination), the trypsin is removed from the eggs by dilution and washing.
Specific procedure Eggs are added to a trypsin solution in sea water (16°C) to a final concentration of 0.25 mg trypsin/ 100 ml sea water (2 x -crystallized trypsin, Worthington). A small aliquot (test aliquot) of the trypsinegg suspension is set aside and is periodically used to test for the presence of fertilization membranes. The remaining suspension is left undisturbed and allowed to settle by gravity (so that the trypsin can later be quickly removed by aspiration of the supernatant sea water). A sample is periodically (2 min intervals) taken from the test aliquot, inseminated, and assessed microscopically for the presence of fertilization membranes (membrane elevation becomes progressively smaller during the incubation in trypsin; thus, medium power microscopic examination may be necessary to assure the actual absence of the fertilization membrane). When the fertilization membrane no longer elevates in response to fertilization, the trypsin is immediately removed by aspiration of the supernatant sea water of the main egg suspension. Fresh sea water is added and the eggs are then washed three more times to completely remove the trypsin. Upon fertilization, these membrane-free eggs will develop normally and with little or no agglutination. With S. purpuratus eggs, the time required to remove the membranes is between 15-20 min at 16°C. I therefore begin to assessfor removal of membranes about 10 min after adding the trypsin. Exptl Cell Res 61
70 D. Epel Comments If membrane removal is incomplete, a nonvisible or “tight” membrane will form; blastomeres will not completely separate at cleavage, and a reduced blastocoel will result. Presence of this “tight” membrane, however, does not seem to affect p-1,3 glucanase excretion from the cortical granules [2] or with isolation of such organelles as the mitotic apparatus or cytasters [5]. Although I have not done extensive experiments, a highly purified trypsin seems to be a prerequisite for non-sticky eggs. Thus, a sample of one-time crystallized trypsin yielded sticky eggs after fertilization, whereas two-times crystallized trypsin, applied to a separate sample of eggs from the same female, yielded non-sticky eggs. Using the twotimes crystallized trypsin, I also find that longer incubation or exposure to higher trypsin concentrations yields sticky eggs upon fertilization. This agglutination is similar to that seen with membrane-free eggs obtained by other methods [3] and apparently results from the initial stickiness of the newlyformed hyaline layer.
moved only one of these components, whereas exposure to higher trypsin concentrations removed both components ([4], p. 224). Thus, in the method I have described, patches of the vitelline membrane may remain on the egg surface after the trypsin treatment. These mosaics may then effectively prevent contact between hyaline layers and hence prevent agglutination of adjacent cells, Irrespective of whether the membrane is completely removed by this procedure, the membrane appears to be functionally and microscopically (light-microscope) absent. For example, the permeability barrier normally imposed by the fertilization membrane is absent. Thus, following fertilization of trypsin-treated eggs the cortical granule enzyme ,i3-1,3 glucanase is excreted into the surrounding seawater [2]. Also, “tight” fertilization membranes are not present, since no fertilization-membrane-like structure is visible between adjacent blastomeres at the time of the first cell division. Supported by NSF grant GB-8002.
REFERENCES
Mechanism The major factor resulting in non-sticky eggs with the above procedure could be the incomplete removal of the vitelline membrane by the controlled exposure to dilute trypsin. Thus, Runnstriim has noted that the vitelline membrane, as observed under the electron microscope, appears to be composed of two components. Dilute trypsin (0.001 %) re-
Exptl Cell Res 61
1. Berg, W E, Methods in developmental biology (ed F H Wilt & N K Wessels) p. 767. Crowell, New York (1967). 2. Eoel. D. Weaver. A M. Muchmore. A V & Schimke; R T, Science 163 (1969) 294. ’ 3. Eoel. D. Weaver. 66 (i97d) 64. ’ A M & Mazia. , D., Exotl _ cell res 4. Rurmstrom, J, Advances morphogenesis 5 (1966) 222. 5. Went, H. Personal communication (1970). Received January 16, 1970