EARLY DEVELOPMENT OF THE SEA URCHIN
97
(1960b), 'in the absence of adequate electron microscopic observations
it is difficult to be sure that the isolated fractions are really identical with
the pre-existing intracellular granules ; rupture of nuclei or mitochondria,
as well as aggregation of smaller cytoplasmic particles, are difficult to
avoid completely'. In general, it seems unwarranted from the study of a
certain process in a homogenate to draw immediate conclusions as to the
conditions prevailing in the intact living cell. Even under the best
possible conditions that may not cause any significant alteration in the
individual subcellular components, the process of homogenization no
doubt brings about a reshuffling of the cell components themselves.
Thus structures or substances which in the living cells were separate
may come into contact, giving rise to reactions that, although they
cannot be considered as unnatural, may not have taken place in the
particular physiological condition of the cell when it was homogenized.
The risk of artifacts arising from autolytic processes is also very serious
(Kavanau, 1958) and this is especially true when handling stages in
which proteolytic enzymes are highly active such as, for example, in the
stages immediately following fertilization. It may be further added that
it is known that the incorporation of amino-acids into the proteins of
homogenates, supplied with all the necessary metabolites, is considerably lower than that of the whole cells. In homogenates of sea
urchin eggs and embryos the incorporation of
14
C-leucine is indeed one
hundred times lower than that obtained when the labelled amino-acid is
presented to the intact embryo (Giudice, 1962b).
Turning now to the specific case of the sea urchin egg, the first point
to be kept in mind when working with homogenates is the risk of
contamination with sea-water. The deleterious effect of sea-water
seems to be largely due to its Ca
2 + content. The addition of small
amounts of Ca
2 + to a homogenate of sea urchin eggs (Hultin, 1950a, b)
causes a transient outburst of oxygen consumption and a gel-like
transformation of the homogenate. Furthermore small amounts of seawater added to a mitochondrial preparation cause drastic uncoupling of
the oxidative phosphorylation and it has been proved that Ca
2 + is
responsible for this reaction (Aiello and Maggio, 1961). Ca
2+ is in fact
known to be an uncoupler of oxidative phosphorylation (see Lehninger,
1949; Lehninger et al., 1954). The effect of homogenization in different
media on the respiration of the homogenates of unfertilized and fertilized
sea urchin eggs is clearly shown by the recent experiments of Gonse
(1960). Therefore, when carrying out experiments involving as a first
step the homogenization of sea urchin eggs or embryos, great care must
be taken to make sure that the amount of contaminating sea-water is
below the 'toxic' level. Taking advantage of the fact that Na
+
is
practically absent in the cytoplasm of the Echinoderm egg (Rothschild
97
(1960b), 'in the absence of adequate electron microscopic observations
it is difficult to be sure that the isolated fractions are really identical with
the pre-existing intracellular granules ; rupture of nuclei or mitochondria,
as well as aggregation of smaller cytoplasmic particles, are difficult to
avoid completely'. In general, it seems unwarranted from the study of a
certain process in a homogenate to draw immediate conclusions as to the
conditions prevailing in the intact living cell. Even under the best
possible conditions that may not cause any significant alteration in the
individual subcellular components, the process of homogenization no
doubt brings about a reshuffling of the cell components themselves.
Thus structures or substances which in the living cells were separate
may come into contact, giving rise to reactions that, although they
cannot be considered as unnatural, may not have taken place in the
particular physiological condition of the cell when it was homogenized.
The risk of artifacts arising from autolytic processes is also very serious
(Kavanau, 1958) and this is especially true when handling stages in
which proteolytic enzymes are highly active such as, for example, in the
stages immediately following fertilization. It may be further added that
it is known that the incorporation of amino-acids into the proteins of
homogenates, supplied with all the necessary metabolites, is considerably lower than that of the whole cells. In homogenates of sea
urchin eggs and embryos the incorporation of
14
C-leucine is indeed one
hundred times lower than that obtained when the labelled amino-acid is
presented to the intact embryo (Giudice, 1962b).
Turning now to the specific case of the sea urchin egg, the first point
to be kept in mind when working with homogenates is the risk of
contamination with sea-water. The deleterious effect of sea-water
seems to be largely due to its Ca
2 + content. The addition of small
amounts of Ca
2 + to a homogenate of sea urchin eggs (Hultin, 1950a, b)
causes a transient outburst of oxygen consumption and a gel-like
transformation of the homogenate. Furthermore small amounts of seawater added to a mitochondrial preparation cause drastic uncoupling of
the oxidative phosphorylation and it has been proved that Ca
2 + is
responsible for this reaction (Aiello and Maggio, 1961). Ca
2+ is in fact
known to be an uncoupler of oxidative phosphorylation (see Lehninger,
1949; Lehninger et al., 1954). The effect of homogenization in different
media on the respiration of the homogenates of unfertilized and fertilized
sea urchin eggs is clearly shown by the recent experiments of Gonse
(1960). Therefore, when carrying out experiments involving as a first
step the homogenization of sea urchin eggs or embryos, great care must
be taken to make sure that the amount of contaminating sea-water is
below the 'toxic' level. Taking advantage of the fact that Na
+
is
practically absent in the cytoplasm of the Echinoderm egg (Rothschild