92
ALFRED J . COULOMBRE
saminoglycans. During the early development of the rabbit a faint
metachromasia characterizes the entire stroma (Smelser and Ozanics,
1959). Similarly the stroma of the chick embryo is PAS + from the
earliest stages (O'Rahilly and Meyer, 1960). There is widespread agreement (van Walbeek et al., 1950; van den Hooff, 1951; Ghiani and
Bergamini, 1958a; Coulombre and Coulombre, 1958a) that intense metachromasis begins to appear at the older inner border of the stroma of
the chick embryo on about the 13th or 14th day of incubation and
spreads toward the anterior surface during the following week. The
same sequence has been observed in other forms (human, guinea pig,
rat, mouse, and rabbit: Aurell and Holmgren, 1953; human: Gemolotto
and Patrone, 1955; r a t : Alagna, 1954; Seo, 1955; rabbit: Smelser and
Ozanics, 1956, 1957,1959,1960).
In this connection the time of deposition of glycosaminoglycans
during development is informative. Smits (1957) showed that early in
the development of the stroma of cattle mucopolysaccharide (measured
as hexosamine) decreased, as it does in the development of most other
collagenous connective tissues of the body. Later, in contrast to most
other connective tissues, glucosamine increased progressively in concentration in the stroma. In rabbits Smelser and Ozanics (1957, 1959,
1960) demonstrated the uptake of S
35
into the sulfated polysaccharides
of the embryonic stroma. The uptake commences at a very early stage,
and only in the presence of an intact anterior epithelium. Anseth (1961b)
used Ecteola column fractionation of the glycosaminoglycans of the
embryonic stromas of chick and cattle. In the chick embryo he demonstrated a relative increase in the concentration of keratan sulfate beginning about the 14th day of incubation. This change corresponds in
time with the increase in intense metachromasia in the posterior layers
of the stroma that was noted above. I t also correlates with the time
when the cornea begins to become transparent (van den HoofF, 1951;
Coulombre and Coulombre, 1958a) and begins to lose water rapidly and
become thinner (Coulombre and Coulombre, 1958a) under the influence
of the pituitary-thyroid axis (Coulombre and Coulombre, 1959; and unpublished data) (see Fig. 2). The observed increase in metachromasia
which commences in the posterior layers is probably due, in part, to the
increased rate of synthesis of sulfated mucopolysaccharides. I t is also
possible that it is due, in part, to increased local concentration of acid
groups which is brought about by the compacting of stromal layers as
the cornea dehydrates and becomes thinner. In addition to its relevance
to stromal metachromasia and corneal transparency the mucopolysaccharide content of the stroma is importantly involved in the considerable water-binding capacity of this layer (Ashton, 1960; van
Walbeek, 1960), in the determination of its refractive index, and possibly
ALFRED J . COULOMBRE
saminoglycans. During the early development of the rabbit a faint
metachromasia characterizes the entire stroma (Smelser and Ozanics,
1959). Similarly the stroma of the chick embryo is PAS + from the
earliest stages (O'Rahilly and Meyer, 1960). There is widespread agreement (van Walbeek et al., 1950; van den Hooff, 1951; Ghiani and
Bergamini, 1958a; Coulombre and Coulombre, 1958a) that intense metachromasis begins to appear at the older inner border of the stroma of
the chick embryo on about the 13th or 14th day of incubation and
spreads toward the anterior surface during the following week. The
same sequence has been observed in other forms (human, guinea pig,
rat, mouse, and rabbit: Aurell and Holmgren, 1953; human: Gemolotto
and Patrone, 1955; r a t : Alagna, 1954; Seo, 1955; rabbit: Smelser and
Ozanics, 1956, 1957,1959,1960).
In this connection the time of deposition of glycosaminoglycans
during development is informative. Smits (1957) showed that early in
the development of the stroma of cattle mucopolysaccharide (measured
as hexosamine) decreased, as it does in the development of most other
collagenous connective tissues of the body. Later, in contrast to most
other connective tissues, glucosamine increased progressively in concentration in the stroma. In rabbits Smelser and Ozanics (1957, 1959,
1960) demonstrated the uptake of S
35
into the sulfated polysaccharides
of the embryonic stroma. The uptake commences at a very early stage,
and only in the presence of an intact anterior epithelium. Anseth (1961b)
used Ecteola column fractionation of the glycosaminoglycans of the
embryonic stromas of chick and cattle. In the chick embryo he demonstrated a relative increase in the concentration of keratan sulfate beginning about the 14th day of incubation. This change corresponds in
time with the increase in intense metachromasia in the posterior layers
of the stroma that was noted above. I t also correlates with the time
when the cornea begins to become transparent (van den HoofF, 1951;
Coulombre and Coulombre, 1958a) and begins to lose water rapidly and
become thinner (Coulombre and Coulombre, 1958a) under the influence
of the pituitary-thyroid axis (Coulombre and Coulombre, 1959; and unpublished data) (see Fig. 2). The observed increase in metachromasia
which commences in the posterior layers is probably due, in part, to the
increased rate of synthesis of sulfated mucopolysaccharides. I t is also
possible that it is due, in part, to increased local concentration of acid
groups which is brought about by the compacting of stromal layers as
the cornea dehydrates and becomes thinner. In addition to its relevance
to stromal metachromasia and corneal transparency the mucopolysaccharide content of the stroma is importantly involved in the considerable water-binding capacity of this layer (Ashton, 1960; van
Walbeek, 1960), in the determination of its refractive index, and possibly