VITELLINE MEMBRANE AND CORTICAL PARTICLES 223
were studied with dark-ground illumination (Runnström et al, 1945).
In ripe, unfertilized eggs an outer membrane, the vitelline membrane,
is in very close contact with the plasma membrane. It is often difficult
to delimit them against each other in electron micrographs. In Clypeaster japonicus,
Endo (1961a) observed a double membrane, the
outer layer measuring about 30 Â and the inner layer about 60 Â. The
total thickness of the two membranes including an intermediate space
amounted to 150-200 Â.
Upon immersion in hypertonic sea water (20 ml of sea water plus
5-6 ml of 2.5 M NaCl) the vitelline membrane of the mature Psammechinus egg does not separate from the cytoplasmic surface. The cytoplasmic surface becomes wrinkled, but the vitelline membrane is so
closely attached to the cytoplasmic surface as to follow its form changes.
This is in keeping with the difficulty sometimes experienced in distinguishing the two membranes from each other in electron micrographs
(see also Afzelius, 1956, p. 277). A partial separation of the vitelline
membrane from the cytoplasmic surface occurs gradually in the eggs
exposed to the hypertonic medium. This process is much facilitated by
the presence of serum, serum albumin, or amino acids (Runnström et al.,
1946; Wicklund, 1954b). The high-potential dye porphyrindin stiffens
the vitelline membrane (Runnström, 1957; Runnström and Kriszat,
1957), but does not affect the cytoplasmic surface, at least not irreversibly. This observation was regarded as a proof of the existence of the
vitelline membrane that was questioned by Mitchison (1956). Now the
presence of a vitelline membrane in the mature unfertilized egg is beyond
doubt.
A characteristic property of the vitelline membrane in mature sea
urchin eggs is its sensitivity toward proteolytic enzymes. A criterion of
dissolution of the vitelline membrane used was the absence of a continuous fertilization membrane in eggs pretreated with proteolytic enzyme and then inseminated. Another criterion was the immersion of the
enzyme-treated eggs in a strongly hypotonic medium. In the presence of
a vitelline membrane, the eggs burst at one point, and the contents were
squeezed out to a varying extent through the hole. In the absence of
vitelline membrane the eggs swelled uniformly.
Runnström et al. (1943) showed that the vitelline membrane was sensitive to a Merck trypsin that also contains pancreatic enzymes other
than trypsin. It proved, however, also to be sensitive to crystalline trypsin (Runnström, 1948; Bohus-Jensen, 1953; Minganti, 1953; for further
references and for the effect of different trypsin concentrations, see also
Runnström, 1962).
Activated papain and chymotrypsin (Minganti, 1953) and active
were studied with dark-ground illumination (Runnström et al, 1945).
In ripe, unfertilized eggs an outer membrane, the vitelline membrane,
is in very close contact with the plasma membrane. It is often difficult
to delimit them against each other in electron micrographs. In Clypeaster japonicus,
Endo (1961a) observed a double membrane, the
outer layer measuring about 30 Â and the inner layer about 60 Â. The
total thickness of the two membranes including an intermediate space
amounted to 150-200 Â.
Upon immersion in hypertonic sea water (20 ml of sea water plus
5-6 ml of 2.5 M NaCl) the vitelline membrane of the mature Psammechinus egg does not separate from the cytoplasmic surface. The cytoplasmic surface becomes wrinkled, but the vitelline membrane is so
closely attached to the cytoplasmic surface as to follow its form changes.
This is in keeping with the difficulty sometimes experienced in distinguishing the two membranes from each other in electron micrographs
(see also Afzelius, 1956, p. 277). A partial separation of the vitelline
membrane from the cytoplasmic surface occurs gradually in the eggs
exposed to the hypertonic medium. This process is much facilitated by
the presence of serum, serum albumin, or amino acids (Runnström et al.,
1946; Wicklund, 1954b). The high-potential dye porphyrindin stiffens
the vitelline membrane (Runnström, 1957; Runnström and Kriszat,
1957), but does not affect the cytoplasmic surface, at least not irreversibly. This observation was regarded as a proof of the existence of the
vitelline membrane that was questioned by Mitchison (1956). Now the
presence of a vitelline membrane in the mature unfertilized egg is beyond
doubt.
A characteristic property of the vitelline membrane in mature sea
urchin eggs is its sensitivity toward proteolytic enzymes. A criterion of
dissolution of the vitelline membrane used was the absence of a continuous fertilization membrane in eggs pretreated with proteolytic enzyme and then inseminated. Another criterion was the immersion of the
enzyme-treated eggs in a strongly hypotonic medium. In the presence of
a vitelline membrane, the eggs burst at one point, and the contents were
squeezed out to a varying extent through the hole. In the absence of
vitelline membrane the eggs swelled uniformly.
Runnström et al. (1943) showed that the vitelline membrane was sensitive to a Merck trypsin that also contains pancreatic enzymes other
than trypsin. It proved, however, also to be sensitive to crystalline trypsin (Runnström, 1948; Bohus-Jensen, 1953; Minganti, 1953; for further
references and for the effect of different trypsin concentrations, see also
Runnström, 1962).
Activated papain and chymotrypsin (Minganti, 1953) and active