EARLY DEVELOPMENT OF THE SEA URCHIN
99
personal communication). On the other hand much higher sucrose
concentrations are required for the isolation of the intact pigment
granules (Monroy and De Nicola, 1952). This may be an indication that
pigment granules are more sensitive to the lowering of osmotic pressure
than other cell components. Indeed the osmotic pressure of a 0-44 M
sucrose solution is considerably lower than that of sea-water. On the
other hand, Giudice (1962b) has shown that cells isolated from various
stages of sea urchin embryos, from blastula onwards, are able to carry
out incorporation of labelled amino-acids into their proteins almost as
well in 0-44 M sucrose as in Ca-free artificial sea-water.
There are plenty of examples that show how a relatively slight change
in the composition of the homogenizing solution may cause considerable
changes in the apparent localization of enzymes (see reviews by De Duve
and Berthet, 1954; Allfrey, 1959). Therefore, in a rapidly developing,
and hence changing, system such as the sea urchin egg the reverse
situation may also be true, i.e., that the same method employed for
homogenization and the same procedure of differential centrifugation
may give rise to appreciably different results in the different stages of
development, differently affecting the various subcellular components.
For this reason reports claiming changes in the localization of enzymes
from one cell component to another in the course of development must
at present be considered, to put it mildly, with a critical eye.
It is known that unfertilized sea urchin eggs are easily broken by
gentle homogenization. After fertilization, however, homogenization
becomes increasingly difficult and the further development proceeds
the more difficult it is to achieve a 'good' homogenization. Although
the possibility that once differentiation has begun (i.e., at about the
mesenchyme blastula) some of the cells may be more resistant than
others must be considered ; the mere fact that the volume of the cells of
the blastula and gastrula are respectively 300 and 350 times smaller
than that of the unfertilized egg explains the great difficulty in breaking
up these cells.
Experiments in progress in our Laboratory (Vittorelli, unpublished)
give clear evidence of this. If unfertilized eggs and blastulae of
Paracentrotus
are homogenized in 0-44 M sucrose in citrate buffer
containing EDTA, in the former roughly 70 per cent of the mitochondria
(as estimated by the percentage distribution of ATPase and cytochrome
oxidase among the different subcellular fractions) is found in the
mitochondrial fraction whereas in the blastulae about 70 per cent is
sedimented with the 1,000^ fraction. Evidently the great majority of
the cells were not broken by the homogenization or only insufficiently so.
Far better results are obtained if the blastulae are washed before
homogenization in a sucrose solution containing EDTA. In this case the
99
personal communication). On the other hand much higher sucrose
concentrations are required for the isolation of the intact pigment
granules (Monroy and De Nicola, 1952). This may be an indication that
pigment granules are more sensitive to the lowering of osmotic pressure
than other cell components. Indeed the osmotic pressure of a 0-44 M
sucrose solution is considerably lower than that of sea-water. On the
other hand, Giudice (1962b) has shown that cells isolated from various
stages of sea urchin embryos, from blastula onwards, are able to carry
out incorporation of labelled amino-acids into their proteins almost as
well in 0-44 M sucrose as in Ca-free artificial sea-water.
There are plenty of examples that show how a relatively slight change
in the composition of the homogenizing solution may cause considerable
changes in the apparent localization of enzymes (see reviews by De Duve
and Berthet, 1954; Allfrey, 1959). Therefore, in a rapidly developing,
and hence changing, system such as the sea urchin egg the reverse
situation may also be true, i.e., that the same method employed for
homogenization and the same procedure of differential centrifugation
may give rise to appreciably different results in the different stages of
development, differently affecting the various subcellular components.
For this reason reports claiming changes in the localization of enzymes
from one cell component to another in the course of development must
at present be considered, to put it mildly, with a critical eye.
It is known that unfertilized sea urchin eggs are easily broken by
gentle homogenization. After fertilization, however, homogenization
becomes increasingly difficult and the further development proceeds
the more difficult it is to achieve a 'good' homogenization. Although
the possibility that once differentiation has begun (i.e., at about the
mesenchyme blastula) some of the cells may be more resistant than
others must be considered ; the mere fact that the volume of the cells of
the blastula and gastrula are respectively 300 and 350 times smaller
than that of the unfertilized egg explains the great difficulty in breaking
up these cells.
Experiments in progress in our Laboratory (Vittorelli, unpublished)
give clear evidence of this. If unfertilized eggs and blastulae of
Paracentrotus
are homogenized in 0-44 M sucrose in citrate buffer
containing EDTA, in the former roughly 70 per cent of the mitochondria
(as estimated by the percentage distribution of ATPase and cytochrome
oxidase among the different subcellular fractions) is found in the
mitochondrial fraction whereas in the blastulae about 70 per cent is
sedimented with the 1,000^ fraction. Evidently the great majority of
the cells were not broken by the homogenization or only insufficiently so.
Far better results are obtained if the blastulae are washed before
homogenization in a sucrose solution containing EDTA. In this case the