ANIMALIZATION AND VEGETALIZATION
179
the larvae as the two reduction gradients of Child, the Janus green and
the methylene blue gradients. The reduction of tetrazolium blue
coupled with the oxidation of the TPN is observed at all stages of
development, in normal, animalized, or vegetalized eggs, in the presence
of an appropriate substrate, citrate or glucose-6-phosphate. These
reductions are independent of the addition of TPN. The reduction
coupled with the oxidation of DPNH is observed before hatching only
when DPN is added ; after the hatching, the gastrulae and the vegetalized
larvae reduce tetrazolium blue independent of the addition of DPN.
At hatching the TPN- and DPN-dependent reductions are more rapid
in animalized larvae than in normal or vegetalized larvae. These
observations indicate a difference between the availability of TPN and
DPN. TPN is available at all the stages of development, while DPN is
available only after hatching, when the entomesoderm is forming. The
change of availability of DPN seems then to have some relation to the
differentiation of the entomesoderm.
Many enzymatic reactions are concerned with the reductions of DPN
and TPN. DPN is reduced to DPNH by some enzymatic reactions of
glycolysis and the tricarboxylic acid cycle. TPN is reduced to TPNH
during the two first steps of the hexosemonophosphate shunt. According
to Bäckström (1959d) the activity of the hexosemonophosphate shunt
and the gradients of reduction of Child and Hörstadius correspond to
each other.
TPN dependent systems however exist apart from the hexosemonophosphate shunt and their activity must interfere in the reduction of
tetrazolium blue. This is the case with isocitric dehydrogenase, an
enzyme of the tricarboxylic acid cycle. Finally it must be pointed out
that the microsomes are probably not completely devoid of oxidizing
activity; for example the microsomes of liver that contain a DPNcytochrome reductase and a cytochrome b5 (Strittmatter and Ball,
1954; Bailie and Morton, 1955). A microsomal TPNH—neotetrazolium
diaphorase has been found in guinea-pig kidney and rat liver (Williams
et ah, 1959). Since nothing is known about the enzymes of the microsomes
in sea urchin eggs, the respective role of these metabolic systems in the
reducing gradients is difficult to appreciate. The study of the effects of
diverse inhibitors on the formation of reduction gradients is likely to
give useful indications in this respect.
The reduction gradients may also result from the unequal distribution
of reducing substances. Among these, ascorbic acid and the SH-containing substances are specially interesting.
The content and distribution of ascorbic acid in sea urchin embryos
has been studied by Bäckström (1956, 1957) in normal and in vegetalized
or animalized embryos. Ascorbic acid exists either in a bound form
179
the larvae as the two reduction gradients of Child, the Janus green and
the methylene blue gradients. The reduction of tetrazolium blue
coupled with the oxidation of the TPN is observed at all stages of
development, in normal, animalized, or vegetalized eggs, in the presence
of an appropriate substrate, citrate or glucose-6-phosphate. These
reductions are independent of the addition of TPN. The reduction
coupled with the oxidation of DPNH is observed before hatching only
when DPN is added ; after the hatching, the gastrulae and the vegetalized
larvae reduce tetrazolium blue independent of the addition of DPN.
At hatching the TPN- and DPN-dependent reductions are more rapid
in animalized larvae than in normal or vegetalized larvae. These
observations indicate a difference between the availability of TPN and
DPN. TPN is available at all the stages of development, while DPN is
available only after hatching, when the entomesoderm is forming. The
change of availability of DPN seems then to have some relation to the
differentiation of the entomesoderm.
Many enzymatic reactions are concerned with the reductions of DPN
and TPN. DPN is reduced to DPNH by some enzymatic reactions of
glycolysis and the tricarboxylic acid cycle. TPN is reduced to TPNH
during the two first steps of the hexosemonophosphate shunt. According
to Bäckström (1959d) the activity of the hexosemonophosphate shunt
and the gradients of reduction of Child and Hörstadius correspond to
each other.
TPN dependent systems however exist apart from the hexosemonophosphate shunt and their activity must interfere in the reduction of
tetrazolium blue. This is the case with isocitric dehydrogenase, an
enzyme of the tricarboxylic acid cycle. Finally it must be pointed out
that the microsomes are probably not completely devoid of oxidizing
activity; for example the microsomes of liver that contain a DPNcytochrome reductase and a cytochrome b5 (Strittmatter and Ball,
1954; Bailie and Morton, 1955). A microsomal TPNH—neotetrazolium
diaphorase has been found in guinea-pig kidney and rat liver (Williams
et ah, 1959). Since nothing is known about the enzymes of the microsomes
in sea urchin eggs, the respective role of these metabolic systems in the
reducing gradients is difficult to appreciate. The study of the effects of
diverse inhibitors on the formation of reduction gradients is likely to
give useful indications in this respect.
The reduction gradients may also result from the unequal distribution
of reducing substances. Among these, ascorbic acid and the SH-containing substances are specially interesting.
The content and distribution of ascorbic acid in sea urchin embryos
has been studied by Bäckström (1956, 1957) in normal and in vegetalized
or animalized embryos. Ascorbic acid exists either in a bound form