A M . ZOOLOGIST, 6:263-271 (1966).
The Gamete-Shedding Substances of Starfishes:
A Physiological-Biochemical Study
ALFRED B.
CHAET*
Scripps Institution of Oceanography, University of California, San Diego,
La Jolla, California and the Marine Biological Laboratory, Woods Hole, Mass.
SYNOPSIS. A shedding substance, found in the radial nerves of 14 species of starfish,
induced the release of gametes from intact animals as well as from whole or fragmented
gonads. The shedding substance was not sex-specific, being present in the radial nerves
of both males and females throughout the year, and, in general, was not species-specific.
This neurosecretory-like polypeptide appeared to function by stimulating ovarian
muscle to contract, and was calcium-dependent: it also stimulated the maturation of
immature eggs. The shedding substance has been purified and its amino acid composition investigated.
A second physiologically-active material, termed "shedhibin" because it inhibited
shedding activity, was also found in the radial nerves of ripe sea stars. In the presence
of shedhibin, normally-adequate quantities of shedding substance would not cause the
release of gametes from isolated ovarian fragments. It appeared that although the level
of shedding substance was constant throughout the year, the level of shedhibin fluctuated, possibly controlling the natural release of gametes from sea stars. The precise
chemical nature of shedhibin has yet to be determined.
The experiments to be described evolved and a search into the biochemical nature
over the past four years in two directions. of the shedding substance (Chaet, 1966).
The original problem started with the reflection that most investigators who had
METHOD OF RELEASE OF GAMETES
used marine eggs (or sperm) for physioExtracts were prepared from the radial
logical, developmental, or biochemical studies had neglected the sea stars—easily pro- nerves of the starfish, Asterias forbesi (Chaet
cured, abundant, large, hardy animals— and McConnaughy, 1959). The nerves were
possibly because mature, synchronized gam- easily dissected by removing the aboral
etes were not released following injections surface of the animal after making a lateral
of potassium chloride or on electric stimu- - incision along both sides of each ray. After
lation, as in the case of sea urchins and a slight incision had been made along the
sand dollars. Thus, we set out to develop ambulacral groove, the left and right halves
a reliable method for obtaining fertile eggs of the oral portion of each ray were sepaand/or sperm from various species of sea rated, thus exposing the relatively large,
stars by injecting neural extracts (Chaet, intact, radial nerve. By this technique, one
could remove the nerves from 25-50 animals
1964a).
The second problem was approached within a few hours. In the original experiwith the knowledge that nerves not only ments, extracts were prepared by suspendtransmit biological impulses, but that some ing nerves in a tube of sea water (one nerve
are specialized to produce chemical secre- per ml) and heating to 76°C for two mintions, i.e., neurohormones. This problem utes. Following centrifugation, the superresolved itself into a study of extracts of natant was injected into the coelomic cavstarfish radial nerves, with especial empha- ity of starfish (0.15 ml/g). A second method
sis on the physiological mechanisms by involved lysing the nerves in distilled water
which neural extracts, containing shedding (one nerve per ml), and when salts had
substances, caused the release of gametes been added to the supernatant to make it
isotonic, it was injected into sea stars. The
most convenient and satisfactory technique
* National Institutes of Health Special Fellow.
for preparing neural extracts, however, was
Present address: University of West Florida, Pensato pool large numbers of isolated iced
cola, Fla.
(263)
264
ALFRED B. CHAET
s sw
HELIASTER KUBINIJII
70 MIN
I
2
13
14
3
4
5
6
EIG. 1. Male Heliaster kubinijii 70 minutes after
injection of 2 ml of a 5 mg % solution of radial
nerve extract from Asterias forbesi. Note copious
quantities of sperm (s) issuing from the 44 gonopores.
FIG. 2. Method of micro-assay of shedding substance by use of the 96-hole transparent spot plate.
Each depression contained one ovarian fragment of
about 5-8 fingers and was immersed in 0.05 ml of
the solution to be assayed. Note shedding in frac-
7
8
9
10
II
12
tions 1317 only, each fraction in quadruplicate.
FIG. 3. Eight ovarian fragments from two different
Paliria miniata (1 and 2) immersed for 15 minutes
in 0.05 ml of 10 mg % shedding substance containing shedhibin (SS/SH) or sea water (SW). Note
spontaneous shedding by fragment 1 when in sea
water, but not in shedding substance containing
shedhibin. Fragment 2 subsequently shed in shedding substance, but not in sea water.
THE GAMETE-SHEDDING SUBSTANCE(S) OF SEA STARS
nerves, which were then frozen, dried, and
pulverized. For preparing active extracts,
aliquots of this powder were suspended in
sea water (5 mg %). When extracts of
radial nerves, prepared by any of the above
three methods, were injected into the coelomic cavity of the intact starfish, they
induced the release of gametes. Figure 1
illustrates the release of copious amounts
of sperm from Heliaster kubinijii after such
an injection. Frequently, 10 to 20 minutes
after injection, but prior to the release of
gametes, some sea stars raised the disc area
while their distal tube feet remained adherent to the substratum, but gametes were
not released until some 30 minutes after
injection. In the case of Asterias jorbesi,
this lag period was quite precise and usually did not extend beyond 35 minutes. Once
gametes began to leave the gonads, they
continued to stream out for 15-60 minutes,
depending upon the quantity contained in
the ovary (or testis). It should be noted
that, in essentially all of the species of sea
stars studied, an injection into the coelomic
cavity of only one ray stimulated all ovaries
(10 in most species studied, but more than
40 in Pycnopodia helinnllioides and Heliaster kubinijii) to shed their eggs.
Injections of extracts prepared from other
portions of the starfish, such as tube feet,
digestive tract, aboral or oral surfaces,
hepatic caeca, gonads, etc., failed to induce
the release of eggs or sperm from recipient
starfish; only extracts prepared from the
radial nerves were effective. The unknown
physiologically-active factor present in these
extracts was called the shedding substance.
It was found in the nerves of both male
and female donor starfish and was not sexspecific; that is, extracts from either sex
induced the release of gametes from both
male and female starfish. The eggs released
by this in vivo technique, as well as by the
utilization of ovarian fragments in vitro
(to be described below), were washed once
in fresh sea water, and exhibited a high
degree (90-100%) of breakdown of the
germinal vesicle, indicating completion of
maturation. Further, shed eggs fertilized
with sperm obtained by the same technique
developed elevated fertilization membranes,
265
and the cells proceeded to divide (Chaet and
Musick, 1960). Thus, the eggs and sperm
obtained after injection of the shedding
substance (or by exposure to it, in the case
of the in vitro method) were highly fertile
and could be used as physiologically suitable experimental material (Chaet and
McConnaughy, 1959; Chaet and Musick,
I960; Chaet and Rose, 1961; Chaet, 1946«,
b, and c).
PHYSIOLOGICAL PHENOMENA
Once satisfied that the technique of obtaining eggs from sea stars was sufficiently
reliable (Chaet, 1966), we directed our attention toward the possibility that the shedding substance was a neurosecretory-like
product. Were neurosecretory granules present in the radial nerve, and if so, did a
correlation exist between the presence of
shedding substance and neurosecretory
granules? It was first determined, by longitudinally stripping the nerve (enzymatically and/or mechanically) into two or
three layers from which extracts were prepared and assayed. It was concluded that
only the most ventral layer contained physiologically-active shedding substance, and
that it was found along the entire length
(proximal to distal) of the nerve (Uter,
]966).
When the shedding substance had been
located physiologically in the ventral portion of the radial nerve, a search for neurosecretory granules was undertaken, using
the following neurosecretory stains: paraldehyde-fuchsin, performic acid-alcian blue,
chromalum - hematoxylin - phloxine, and
azan. Three areas within the radial nerve,
where the shedding substance was physiologically located, stained positively with
all four neurosecretory stains. Preliminary
evidence suggested that the nerves of an
extremely ripe animal contained two distinct sizes of granules (1 p. and 2 /x). The
physiological significance of the 2 ^ granules is still obscure (Imlay and Chaet, 1965).
Whether or not the smaller granules do
represent the shedding substance can be
determined only by isolating them and
determining whether they alone exhibit
physiological (shedding) activity.
2G6
ALFRED B. CHAET
Did the shedding reaction represent a
first-, second-, or third-order neuroendocrinological system as defined by Rothballer
(1957)? To answer this question, all 10
ovaries were carefully dissected from Asterias forbesi or Patiria miniata (Chaet and
Smith, 1962; Chaet, 1966). When the isolated ovaries were immersed in sea water,
no shedding occurred. However, when
sister ovaries were placed in sea water containing shedding substance (5 mg %), eggs
were released through the gonoduct into
the surrounding solution, indicating that
the reaction was a first-order system. Clearly, the shedding substance stimulated the
ovary, directly, and no other tissues of the
starfish were involved.
In attempting to explain the lag period
of about 30-35 minutes between treatment
with shedding substance and release of eggs,
we sought intermediate substances (Chaet
and Smith, 1962) by immersing ovaries in
shedding substance for 29 minutes. The
ovaries were then removed and fresh (sister)
ovaries exposed to the "used" solution of
shedding substance in which the 29-minute
ovary had been immersed. Fresh ovaries
were also immersed in extracts prepared
by homogenizing 29-minute ovaries. Various immersion times were tried, but in no
experiments were active intermediates ever
demonstrated; all fresh ovaries still exhibited a 30-minute lag period. Lag periods of approximately 30-50 minutes were
found in all 14 sea stars studied; however,
the time (30-35 minutes) was more predictable in Asterias forbesi than in any of
the other species.
When sister ovaries of equal size were
immersed (60-90 minutes) in shedding substance or sea water and then compared, it
was evident that ovaries bathed in shedding
substances became considerably smaller
than the controls after shedding their eggs.
Two alternative explanations came to
mind. It was possible that the ovary collapsed as a result of the elasticity of its
wall as the eggs were released. Or, the
shedding substance might stimulate the
ovarian wall to contract, thus expelling
the eggs. By electronically recording continuously the length of the ovary while
immersed in either control or experimental
solutions, it was demonstrated that the
ovary did indeed contract in the presence
of shedding substance. Furthermore, contraction began not more than two or three
minutes before eggs were first released, and
thus appeared to be responsible for expelling the eggs from the gonad (Chaet,
Andrews, and Smith, 1964).
Since we were dealing with contraction
of ovarian tissue, the significance of calcium
in this reaction was suspected (Weber,
1958). When an ovary was immersed for
two hours in calcium-free sea water containing shedding substance, eggs were not
released. However, if the ovary was then
placed in normal sea water (containing
calcium, but no shedding substance) eggs
were released within a few minutes (Kanatani, 1964; Mecklenburg and Chaet, 1964).
As a matter of fact, when the ovary was
placed in calcium-free sea water with sheding substance for only five minutes, and
then transferred to normal sea water, eggs
were shed. These experiments suggested
that the shedding phenomenon in sea stars
involved a two-step reaction, the first one
dependent upon a short exposure to shedding substance, the second reaction being
independent of shedding substance but
requiring calcium ions.
The next question proposed was whether
the shedding substance promoted the maturation of eggs. Sister ovaries or ovarian
fragments from Asterias forbesi or Patiria
miniata were isolated and immersed in
either sea water alone or in sea water containing shedding substance (5-10 mg %).
After 30-60 minutes, the ovaries were finely
minced and filtered through cheesecloth
which had been washed in sea water. The
eggs were washed three times and the per
cent of breakdown of germinal vesicles was
determined. Although all ovaries came
from the same animal, it was obvious
(Table 1) that relatively few eggs had matured in the ovary placed in sea water'-alone,
as compared to those ovaries stimulated
with shedding substance (11% vs. 97%).
Thus, the shedding substance promoted
maturation of eggs as well as contraction
of the ovary (Chaet, 1966; Chaet, Andrews,
267
T H E GAMETE-SHEDDING SUBSTANCE(S) OF SEA STARS
TABLE 1. Maturation of isolated eggs or eggs from ovarian fragments of Patiria miniata after immersion either in normal sea xuater (s.zu.) or in sea water containing 10 ing % of shedding substance from Patiria (sj.) for 60 minutes. The per cent of breakdown of germinal vesicles was
determined microscopically after isolating and washing the eggs.
Ovarian fragment
s.w.
s.s.
% Breakdown of germinal vesicles
11%
and Smith, 1964). It should also be pointed
out that this effect on maturation occurred
only when the eggs were in situ within the
intact ovarian tissue (fragments would
suffice). Isolated washed eggs, suspended
in either sea water alone or containing the
supernatant of ovarian filtrates, showed
minimal breakdown of germinal vesicles
when immersed in either sea water or shedding substance. Preliminary studies failed
to produce an extract from ovaries which
would induce isolated eggs to mature in
the presence of shedding substance, but
extracts from ovaries previously exposed
to shedding substance may possess such
activity. All attempts at physical separation of maturation activity from contractile
activity were unsuccessful, and it appeared
that both were due to the same shedding
substance.
97%
Eggs plus sea water
s.w.
s.s.
26%
15%
Eggs plus supernatant o£ ovarian
filtrate
s.w.
s.s.
23%
19%
pated lag period. Figure 2 illustrates a
typical micro-assay of 48 fractions run in
quadruplicate. Note the shedding activity
in fractions 13-17 only (controls, standards,
and remaining fractions did not appear in
this photograph). The assay could be quantitated by assigning an estimated intensityvalue of from 1 to 5 to each of the fragments. When a maximum number of eggs
was released from an ovarian fragment, it
was valued at number 5. The shedding
index of each solution assayed was calculated by dividing the observed quantity of
shedding by the theoretical maximum shed
(40 for a quadruplicate assay).
We turned next to determining some of
the chemical characteristics of the active
component of the shedding substance, using
the technique of microbiological assay for
detecting shedding activity (Chaet and
Rose, 1961; Chaet, 1964c; Chaet, 1966).
BIOCHEMICAL APPROACH
The shedding substance was found to be
Since injecting solutions into the coe- a heat-labile, dialyzable polypeptide, whose
lomic cavity of sea stars as an assay for activity was destroyed within 24 hours at
shedding substance required large volumes room temperature but persisted indefinitely
and many animals, it soon became evident when frozen. The shedding substances from
that a micro-assay was needed if one in- radial nerves of both Asterins forbesi and
tended to quantitate the reaction and iden- Patiria miniata have been substantially
tify the shedding substance. The following purified, using several techniques, particuprocedure was developed (Chaet, Andrews, larly gel-filtration over Sephadex. Figure 2
and Smith, 1964): Isolated ovaries from represents a series of fractions collected
any of 5 species of sea stars, were separated from a G-50 Sephadex column. When a
carefully into 20-30 fragments. These were relatively tall (58-60 cm) column of G-25
small enough to be immersed in 0.05 ml was used to fractionate the shedding subof test solution in depressions on a glass stance of Patiria (subsequently eluted with
spot plate, and the release of eggs observed. distilled water), two distinct peaks of shedBy this method, 200-500 assays could be ding activity were found. Their signifimade from the tissue of the same starfish, cance has yet to be determined; they may
thereby cancelling out much biological have been produced artificially by a comvariation. Ovarian fragments immersed in plexing of the shedding substance with
a 5 to 10 mg % solution of shedding sub- carriers of two different sizes. It is also
stance began to shed eggs alter the antici- possible that the shedding substance was
268
ALFRED B. CHAET
degraded and that both fractions remained
physiologically active. Since the shedding
substance of Paliria was slightly retarded
on G-25 Sephadex columns, it appeared to
have a molecular weight of less than
5,000. By use of the ninhydrin reaction
(Moore and Stein, 1954) before and after
hydrolysis of the shedding substance, with
data read at 570 niju, it was indicated that
the polypeptide contained some 10-15
amino acids.
In preliminary experiments, a small
quantity of shedding substance from Asteritis forbesi was substantially purified by
preparing cold water extracts of dried
nerves, followed by extraction in 50% acetone (at 55°C for 5 minutes), fractionation
over CM and G-25 Sephadex columns, and
finally chromatography in butanol-H20acetic acid. After each step, biological activity was determined, and the active component followed. The shedding substance
was located on the chroma togram by elution and assay. The active fraction finally
eluted was hydrolyzed and assayed for
amino acids by electrophoresis chromatography. In this preliminary experiment,
10-11 different amino acids were detected,
but further analysis is needed before a
conclusive picture can be presented.
NATURAL SPAWNING
By what route did shedding substance
travel from the radial nerve of an intact
sea star to its gonad? Logically, it might
migrate from the ventral to the aboral portion of the radial nerve and, if secreted into
the water vascular system, could migrate
to the coelomic cavity and stimulate the
gonads directly. Three sets of observations
have a bearing on this question. First, only
the oral side of the nerve contained physiologically-active shedding substance or neurosecretory granules, and neither was found
to migrate toward the aboral surface.
(Unger, 1961, reported a migration of granules.) The second observation concerns
sexual stimulation by naturally-shedding
animals. A spawning starfish, whether male
or female, stimulates other animals within
the same tank to release their gametes. The
third observation concerns the ability of
tube feet to take up amino acids (Chaet
and Martin, 1966). Experiments in vivo
showed that amino acids in sea water surrounding the tube feet entered the coelomic
cavity of the starfish within two minutes
after exposure, and experiments in vitro
proved that isolated cannulated tube feet
transported amino acids across the wall into
the lumen. These data suggest that shedding substance is released from the oral
surface of the radial nerve (which is adjacent to sea water) into the sea water, and
if not diluted below a threshold level,
stimulates the release of gametes from its
neighbor. A threshold level of shedding
substance would be transported into the
coelomic cavity of the original starfish (in
the same way as amino acids) to stimulate
its gonad. This hypothesis is presently
under investigation.
The occurrence of the shedding substance
in 14 species of sea stars and its crossreactivity with ovaries of 8 species are
shown in Table 2 (cf. Chaet, 1966; Hartman and Chaet, 1962). Other investigators
have found shedding substance in four additional species (Unger, 1962; Kanatani
and Noumura, 1962; Noumura and Kanatani, 1962). Not all cross-reactions are
shown in Table 2 because of the difficulty
in obtaining certain species of sea stars in
a ripe condition. Aslropecten armalus,
Heliasler kubinijii, Pisaster ochraceus, Pisaslcr gignnleus, Aslerias forbesi and Paliria miniata each responded to the shedding substance of two or more different
species. Paliria and Aslropccl.cn did exhibit a degree of species specificity in that
they failed to respond to all shedding substances tested. No phylogenetic significance
can be attached to these findings until we
have further data. Of the eight species
studied, Henricia leviuscula and Olhelia
tenuispina were the only two whose gonads
did not respond to any shedding .substance.
It may be significant that the species in
question produce fewer but larger eggs
than found in the other six sea stars.
All attempts to demonstrate the shedding
substance in other invertebrate nerves have
proven unsuccessful.
T H E GAMETE-SHEDDING SUBSTANCE(S) OF SEA STARS
269
TABLE 2. Distribution of shedding substance and its cross-reactivity in different species of sea stars. Extracts
of radial nerves (5 and 10 nig %) were tested on ovarian fragments, with quadruplicate trials for both concentrations. ( + == shedding, — = failure to shed.)
Asterias
forbesi
Nerves from:
Asterias
forbesi
Asterias
vulgar is
Aslropectin
armatus
Heilastcr
kubinijii
Henricia
sanguinolenta
Henricia
leviuscula
Mediaster
aequalis
Ortha.sterias
hoehleri
Patiria
miniata
Pisaster
brevispinus
Pisaster
giganteus
Pisaster
ochraceus
Poraniopsis
inflata
Pycnopodia
helianlhoides
Astropectin
armatus
Heliaster
hubinifii
Ovaries from:
Henricia
Othelia
leviuscula tenuiipina
Paliria
miniata
Pisaster Pisaster
giganteus ochraceits
1
INHIBITION
Jt was evident during the early part of
these studies that ovarian fragments immersed in 5 mg % solutions of shedding
substance shed their gametes, and that, as
would be anticipated, lower concentrations
failed to reach a threshold needed for response and did not induce shedding. However, in some experiments, when sister
ovarian fragments were placed in 5 mg %,
15 mg %, 25 mg % or 100 mg % solutions
of the same shedding substance, only those
exposed to 5 mg % shed, the remaining
fragments failing to release many, or usually
any gametes. Thus, as the concentration
of shedding substance was increased, physiological shedding decreased. The extracts
in question were prepared from the radial
nerves of ripe starfish,
The above findings were explained by
the hypothesis that an inhibitor was present
in these extracts. It was present at a subthreshold concentration in 5 mg % solutions (where shedding substance was at a
threshold level), but in solutions, of 15 mg
% or higher, it rose to a threshold which
overrode the effects of the shedding substance. It was also hypothesized that the
nerves of unripe starfish were devoid of
this inhibitor since increasing concentrations of lyophilized nerve extracts from
unripe starfish continued to promote the
release of gametes.
The existence of such an inhibitor, called
shedhibin, was demonstrated in two ways.
In a small percentage of the starfish {Patiria miniata) dissected, ovarian fragments
spontaneously shed soon after having been
270
ALFRED B. CHAET
diced and placed in sea water-control solutions. It appeared that these ovaries had
already been triggered to shed by the sea
star and when placed in sea water, even
though shedding substance was not experimentally introduced, shedding proceeded.
Although these ovaries were not, of course,
used as an assay for shedding substance,
they were used to observe the presence of
shedhibin. When ovarian fragments (Figure 3) from the same ovary of such a spontaneously-shedding starfish (No. ]) were immersed in sea water, it was noted that shedding occurred spontaneously, but that fragments immersed in nerve extract containing shedhibin failed to shed spontaneously.
Since Figure 3 was taken some 15 minutes
after immersion, the ovarian fragments
from animal No. 2 (a normal, unstimulated
ovary) had not yet shed but began to do so
about 20 minutes after the photograph was
made. It was also noted that the level of
shedhibin was not sufficient to inhibit shedding permanently, and that shedding occurred in the two fragments from animal
No. 1 some 90 minutes later. Thus, shedhibin was present in the extract (prepared
from nerves of ripe Paliria miniata), and it
inhibited the release of eggs even though
the ovary had been stimulated to shed by
some external source, as was seen in the
sea water control. A more controlled and
satisfactory method of observing shedhibin
was realized in the following experiment.
Partially-purified (G-25 Sephadex) shedding substance, shown to be physiologically
active, was diluted with an equal volume
of the other (non-shedding) fractions eluted
during the same purification procedure.
When assayed on ovarian fragments, all
mixtures of purified shedding substance
plus unknown fractions caused shedding,
but those ovarian fragments placed in purified shedding substance plus shedhibin
failed to release their eggs (Chaet, 19646).
Thus, shedhibin was found only in those
nerve extracts which were prepared from
the nerves of ripe animals, but was not
detected in nerves of unripe sea stars.
It was surprising to find that the level of
shedding substance in the nerves of freshlycollected starfish
remained
constant
throughout the year (Chaet and Smith,
1962), and that there was no peak concentration just prior to natural shedding. On
the other hand, preliminary experiments
have shown that the level of shedhibin was
high when the animal was ripe, but that
after natural shedding had occurred, the
level of shedhibin in radial nerves was below assay. It is therefore possible that the
animal sheds naturally not due to fluctuation in shedding substance itself, but to
a fluctuation in shedhibin.
The experiments on permeability and
inhibition might explain why different species of sea stars living side by side on a
piling do not shed at the same time and
thus risk cross-fertilization. Why does not
a population of spawning Pisnster ochraceus stimulate Pisnster giganteus to shed,
since they are in close proximity to one
another and since our studies have shown
that ovarian fragments of Pisnster giganteus
respond to shedding substance from Pisastcr ochraceus, and vice versa. Two possible
explanations are offered. Although both
species contained shedding substance and
both were moderately ripe, Pisnster giganteus did not shed when Pisaster ochraceus
did because of the presence of shedhibin in
Pisaster giganteus, which may have since
disappeared from Pisnster ochraceus. A
second possibility is that selective permeability, a type of membrane specificity,
controls the uptake of shedding substance
by the animal.
ACKNOWLEDGMENTS
The author expresses his sincere appreciation to
Dr. R. Doolittle for his advice and aid in the preliminary amino acid studies and to Dr. D. Fox for
his valuable suggestions and reading of this manuscript.
Supported in part by grants from the National
Institutes of Health and the National Science
Foundation.
REFERENCES
Chaet, A. B. 1964a. A mechanism for obtaining mature gametes from starfish. Biol. Bull. 126:8-13.
Chaet, A. B. 1964ft. Shedding substance and "shedhibin"—from the nerves of the starfish, Paliria
miniata. Am. Zoologist 4:407.
Chaet, A. B. 1964c. The shedding substance activity
of starfish nerves. Texas Repts. Biol. Med. 22:204.
T H E GAMETE-SHEDDING SUBSTANCE(S) OF SEA STARS
Chaet, A. B. 1966. Neurochemical control of gamete
release in starfish. Biol. Bull. 130:43-58.
Chaet, A. B., and E. C. Martin. 1966. Amino acid
uptake by sea star tube feet. Fed. Proc. (In press)
Chaet, A. B., and R. A. McConnaughy. 1959. Physiologic activity of nerve extracts. Biol. Bull. 117:
407-408.
Chaet, A. B., and R. S. Musick, Jr. 1960. A method
for obtaining gametes from Asterias forbesi. Biol.
Bull. 119:292.
Chaet, A. B., and R. A. Rose. 1961. Further studies
on the gamete "shedding substance" from radial
nerves. Biol. Bull. 121:385-386.
Chaet, A. B., and R. H. Smith. 1962. Role of
gamete-shedding substance from starfish nerves.
Am. Zoologist 2:511.
Chaet, A. B., P. M. Andrews, and R. H. Smith. 1964.
The shedding substance of starfish nerve—its
function and micro-assay. Fed. Proc. 23:204.
Hartman, H. B., and A. B. Chaet. 1962. Gamete
shedding with radial nerve extracts. Fed. Proc.
21:363.
Imlay, M. R., and A. B. Chaet. 1965. Microscopic
observations of gamete shedding substance in starfish radial nerves. Fed. Proc. 24:129.
Kanatani, H. 1964. Spawning of starfish: action of
gamete-shedding substance obtained from radial
271
nerves. Science 146:1177-1179.
Kanatani, H., and T. Noumura. 1962. On the nature
of active principles responsible for gamete-shedding in the radial nerves of starfishes. J. Fac. Sci.
U. Tokyo IV, 9:403-416.
Mecklenburg, T. A., and A. B. Chaet. 1964. Calcium
and the shedding substance of Patiria miniata.
Am. Zoologist 4:414.
Moore, S., and \V. H. Stein. 1954. A modified ninhydrin reagent for the photometric determination
of amino acids and related compounds. J. Biol.
Chem. 211:907-913.
Noumura, T., and H. Kanatani. 1962. Induction of
spawning by radial nerve extracts in some starfishes. J. Fac. Sci. U. Tokyo IV, 9:347-402.
Rothballer, A. B. 1957. Neuroendocrinology. Excerpta Med. Sect. Ill, ll:iii-xii.
Unger, H. 1962. Experimented und histologische
Untersuchungen uber Wirkfaktoren aus dem
Nervensystem von Asterias glacialis. Zool. Jahrb.
69:481-536.
Uter, A. 1966. Physiological location of shedding
substance in radial nerve complex of starfish
(Asterias forbesi). Thesis. The American University, Washington, D.C.
Weber, H. H. 1958. Motility of muscle and cells.
Harvard University Press, Cambridge, Mass.