AMER. ZOOL., 15:493-505 (1975).
Maturation-Inducing Substances in Asteroid and Echinoid Oocytes
HARUO KANATANI
Laboratory ofPhysiology, Ocean Research Institute, University of Tokyo, Tokyo 164, fapan
SYNOPSIS. In starfish a hormonal peptide, gonad-stimulating substance (GSS), which is
released from nervous tissue, acts on the gonad to produce a maturation-inducing substance
(MIS), an inducer of oocyte maturation and spawning. MIS is 1-methyladenine (1 -MA). This
substance acts on the surface of the oocyte. Cytoplasmic maturation as revealed by fertilizability is also induced by 1-MA. The amount of 1-MA can be determined very accurately with
a bioassay method using isolated oocytes. In 1-MA formation, GSS seems to enhance the
methylation of some compound which contains a purine nucleus at its N1 site. The methyl
donor is probably S-adenosylmethionine. 1-Methylated precursor seems to be transformed
to 1-methyl AMP and then hydrolyzed into 1-methyladenosine and phosphate by phosphomonoesterase. 1-Methyladenosine is finally split into 1-MA and ribose by
1-methyladenosine ribohydrolase. So-called spontaneous maturation of oocytes isolated in
sea water is due to the action of 1-MA produced in follicle cells even in the absence of GSS.
1-MA is present in echinoid gonads and seems to act as MIS also in these animals. Disulfidereducing agents such as dithiothreitol and 2,3-dimercapto-l-propanol induce starfish oocyte maturation. On the other hand, sulfhydryl reagents such as/>-chloromercuribenzoate,
iodoacetamide, and iV-ethylmaleimide suppressed 1-MA-induced oocyte maturation. Since
Concanavalin A acts on the follicle cells to produce 1-MA, the action of this substance seems
to be quite similar to that of GSS.
INTRODUCTION
In starfish, oocytes which are still in the
ovary usually have a single large germinal
vesicle. In other words, the first maturation
division of oocytes within an ovary is arrested at a late stage of prophase. At the
time of spawning, these oocytes begin to
resume the maturation process, resulting in
the breakdown of the germinal vesicle and
the formation of polar bodies.
Since starfishes are plentiful and relatively simple in body structure, especially
with respect to the reproductive system,
they provide good material for biochemical
and physiological studies of reproduction.
Furthermore, small fragments of isolated
ovary can be simply kept in sea water for
hours without losing their physiological acThis research was supported in part by Grants-inaid from the Ministry of Education of Japan and the
Toray Science Foundation.
The author is indebted to Dr. J. C. Dan for reading
the manuscript.
Full address of the author is: Laboratory of Physiology, Ocean Research Institute, University of Tokyo,
Minamidai 1-15-1, Nakano-ku, Tokyo 164, Japan.
tivity, thus providing suitable material for
in vitro experiments. In fact, several
hundred bioassays can be performed with
either ovarian fragments or isolated oocytes obtained from a single female, and
this eliminates individual differences.
In spite of these advantages, few significant investigations into the mechanism
of oocyte maturation and ovuladon in the
starfish were performed until some 10
years ago. This seems to be attributed to a
lack of knowledge with regard to the endocrinology of this animal, since reproductive phenomena such as oocyte maturation
and ovulation are considered to be controlled by hormonal mechanisms in invertebrates as well as in vertebrates.
In 1959, Chaet and McConnaughy reported that water extract of the radial
nerves of the starfish induced spawning
when injected into a ripe starfish. The active substance present in the extract was
called gamete-shedding substance, GSS
(Chaet, 1964). This finding opened the way
to the study of the hormonal mechanisms
of starfish reproduction and led to a
number of investigations along this line on
oocyte maturation and ovulation in this
493
494
HARUO KANATANI
form (for literature see Kanatani, 1973). neither a breakdown product of GSS nor a
GSS is released into the coelomic cavity peptide since it was not destroyed by proimmediately (within 1 hr) before spawning teolytic enzymes and since GSS treated with
occurs (Kanatani and Shirai, 1969), and such enzymes completely lost its activity.
acts directly on the gonads (Kanatani, 1964; This substance is heat stable, and is a dialyzChaet, 1966a, 1967). So far the presence of able small molecule insoluble in organic
GSS has been shown in the radial nerves of solvents such as ether, petroleum ether,
about 30 starfish species and it is believed benzene, or acetone, although it is soluble
that the substance is of general occurrence in 95% ethanol. When gel-filtrated on a
in asteroids, although some species spec- Sephadex column (G-15 or G-25), it is
ificity among different starfishes has been eluted after the ordinary seawater salts
reported (see Chaet, 19666; Kanatani, such as sodium chloride, indicating that it
1973). With respect to the chemical nature has the property of adsorbing weakly to the
of GSS, the substance was purified from dextran gel.
Asterias amurensis and identified as a heat- On the basis of such knowledge as to the
stable polypeptide consisting of about 22 properties of MIS, its purification and
amino acids, with a molecular weight of chemical identification were carried out
about 2100 (Kanatani etal., 1971). Purified with Asterias amurensis (Kanatani et al.,
GSS is active in inducing spawning at a con- 1969; Kanatani, 1972). A total of 20 kg of
centration of 0.0096 //.g/ml.
fresh ovarian fragments obtained from
In 1967 an important finding was inde- about 3000 females were incubated in 100
pendently made by Schuetz and Biggers in liters of artificial sea water (ASW) containthe United States and Kanatani and Shirai ing GSS for 6 hr to make MIS in the
in Japan, indicating that GSS acts on the medium. The sample was centrifuged, and
ovary to produce a second substance which its supernatant was concentrated and
by itself brings about oocyte maturation washed with chloroform and ether. After
and spawning (Kanatani and Shirai, 1970). removal of the excess seawater salts, MIS
We called this second substance the was purified by fractionation on Sephadex
maturation-inducing substance (MIS) G-15 columns of various sizes and a CM(Kanatani and Shirai, 1972). So far the Sephadex C-25 column. The amount of
physiological significance of GSS thus ap- purified MIS finally obtained was 8.5 mg.
pears to be its action in producing MIS, a This sample induced 97% maturation of
direct trigger of oocyte maturation and isolated oocytes of Asterina pectinifera at a
spawning (Kanatani, 1969a), in the follicle concentration of 0.012 /ng/ml.
cells (Hirai and Kanatani, 1971; Hirai et al.,
Since the ultraviolet absorption charac1973). On the basis of this fact we have teristics of the purified MIS led us to asproposed that the so-called gamete- sume that the substance was one of the nushedding substance should be called cleic acid bases, the ultraviolet absorption
"gonad-stimulating substance" (GSS) spectra were measured at various pH's.
(Kanatani, 1969a). The present paper deals The data obtained were quite similar to
with some recent investigations carried out those reported for 1-methyladenine
mainly in my laboratory with respect to the (1-MA) (Fig. 1) (Brookes and Lawley,
mechanism of oocyte maturation in relation to MIS, as well as with a brief survey of
those on the chemical nature and action of
MIS.
CHEMICAL NATURE OF MIS
The following properties
elucidated at the time of
(Schuetz and Biggers, 1967;
Shirai, 1967, 1970; Schuetz,
of MIS were
its discovery
Kanatani and
1969). MIS is
(B)
FIG. 1. Structure of (A) 1-methyladenine and (B)
1-methylhypoxan thine.
MATURATION-INDUCING SUBSTANCES IN ECHINODERMS
1960). When 1-MA was synthesized and
compared with the purified MIS, the ultraviolet absorption spectra at different
pH's, infrared absorption spectrum, high
resolution mass spectrum, and melting
point (301°-303°C) were identical. Furthermore, synthetic 1-MA had a strong capacity to induce oocyte maturation, and its ,
effective dose was the same as that of the
purified MIS. It was therefore concluded
that the MIS of Asterias amurensis is 1-MA.
Since synthetic 1-MA also induced
spawning of isolated ovarian fragments in a
variety of starfishes, it was proved that the
substance is responsible for both oocyte
maturation and ovulation in starfishes
(Kanatani, 19696; Stevens, 1970).
The effect of various adenine derivatives
on spawning and oocyte maturation was
next investigated in order to determine the
chemical structural requirements for the
induction of these phenomena. Among 29
compounds tested, 1-MA, 1-ethyladenine
(1-EA), 1-methyladenosine (1-MAR), and
1-methyladenosine monophosphate (1MAMP) are effective in inducing spawning
and oocyte maturation when assayed with
ovarian fragments (Kanatani and Shirai,
1971; Schuetz, 1971; Shirai and Kanatani,
1973). 1-MA is most effective and 1-EA has
x
/z its effectiveness. However, when assayed
with isolated oocytes, maturation is induced
only by 1-MA and 1-EA. Since 1-methylhypoxanthine (Fig. 1) has no effect in inducing oocyte maturation, the presence of
a short alkyl radical, such as those of methyl
or ethyl, at Nl site and an imino radical at
the C6 site of the purine nucleus appears to
be important for inducing oocyte maturation and spawning in starfishes. The reason
why 1-MAR and 1-MAMP fail to induce
maturation in isolated oocytes will be discussed below.
PHYSIOLOGICAL ACTION OF 1-MA
A comparative study has shown that
more than 20 starfish species respond to
1-MA, and there is no case in which 1-MA
fails to induce spawning and oocyte maturation in the starfishes so far tested.
Therefore, it is reasonable to assume that
1-MA is a general MIS in starfishes (see
495
Kanatani, 1973; Komatsu, personal communication).
When fully grown oocytes of Asterinapectinifera and Patiria miniata, which do not
undergo spontaneous maturation when
isolated in sea water, are placed in sea water
containing 1-MA, their germinal vesicles
usually begin to break down after 20 to 30
min. Breakdown of the follicles also occurs
almost simultaneously. Polar bodies are
formed later and the eggs begin to develop
normally upon insemination. However,
1-MA acts only on the fully grown oocytes;
small, young oocytes fail to respond to its
maturation-inducing activity (Kanatani,
19696).
At least three different sites of action of
1-MA are considered according to its various functions. These are related to induction of (i) spawning, (ii) a special spawning
posture or spawning movements consisting
of rhythmic waves of circular contraction of
the arms from the tips to the proximal
parts, which apparently push the gonads
and assist in expelling the gametes (Kanatani and Shirai, 1972), and (iii) oocyte maturation. For inducing spawning of isolated
ovarian fragments, 1-MA acts on the follicles, presumably by dissolving the cementing substance and causing their breakdown
so that the oocytes within an ovary become
freely movable and are forced out by the
contraction of the ovarian wall (Kanatani,
1969a). The same results are caused by
deficiency of divalent cations such as calcium and magnesium in sea water (Kanatani, 1964; Kanatani and Shirai, 1969;
Schuetz and Biggers, 1968). Also 1-MA
seems to act on some higher center of the
nervous system to induce the spawning
movements, since this activity is considered
to be highly coordinated (Kanatani, 1970).
The site of action of 1-MA in inducing oocyte maturation is known to be the surface
of the oocyte from the fact that 1-MA microinjected into single Asterina oocytes with
germinal vesicles failed to induce their
maturation, whereas a small amount of
1-MA (only 1/10 of the amount microinjected per oocyte) applied from the outside
invariably induced maturation (Kanatani
and Hiramoto, 1970).
With regard to the fertilizability of oo-
496
HARUO KANATANI
cytes in some marine invertebrates, as revealed by the elevation of the fertilization
membrane, earlier work has led to the belief that breakdown of the germinal vesicle
must occur before the oocytes can be fertilized (Delage, 1901; Chambers, 1921;
Wilson, 1928; Costello, 1940), that is, mixing of the germinal vesicle material with the
egg cytoplasm seemed to be a necessary
prerequisite for the acquisition of fertilizability. Hirai et al. (1971) have recently used
enucleation techniques to reinvestigate the
role of the germinal vesicle in cytoplasmic
maturation as induced by 1-MA in Asterina
pectinifera. Isolated oocytes from which the
germinal vesicles had been removed remained unchanged and failed to elevate a
fertilization membrane on insemination,
thus confirming the results of previous investigators such as Delage (1901) and
Chambers (1921). However, when such
enucleated oocytes were treated with 10~5 M
1-MA for about 30 min and then inseminated, 84% of them formed a fertilization
membrane, although continued observation revealed that these oocytes with fertilization membranes failed to develop further
except that some of them showed signs of
abortive cleavage. This suggests that mixing of the germinal vesicle material with
cytoplasm is required not for cytoplasmic
maturation but for cleavage and further
development. Therefore, it is highly probable that the trigger of oocyte maturation,
such as 1-MA, acts on the surface of the
oocyte to induce cytoplasmic maturation.
BIOCHEMICAL ASPECTS OF 1-MA FORMATION
Understanding the biochemical mechanism by which 1-MA is produced under the
influence of GSS is considered to be important not only for a better grasp of the mechanism of oocyte maturation and spawning
in asteroids, but also on a wider basis, to
elucidate the mode of action of hormonal
peptides in general from the point of view
of the biochemical aspects of endocrinologyAlthough the precise pathway of 1-MA
formation is not clear, some fragmentary
evidence has been obtained with respect to
its production. For these studies it is neces-
sary to establish a precise method to determine the amount of 1-MA in a given tissue
or reaction mixture. For this purpose, it has
been shown that a bioassay method with
isolated oocytes using authentic 1-MA as
the reference standard is very sensitive and
accurate (Shirai et al., 1972). The detailed
method has recently been described by
Shirai (1974). Figure 2 compares the data
for the amounts of 1-MA obtained by the
bioassay method with those obtained from
a spectrophotometric determination at 260
nm, showing that all points obtained from
S2h
S5
O o
9 a-
in
o
-Q
>•
~
2
A
6
8
10 12
1-MA concentration ()ag/ml)
(by weight)
FIG. 2. Concentration of 1-MA determined by bioassay (•) and by spectrophotometric assay at 260 nm
(O), showing the accuracy of these two assay methods.
Solid line represents the theoretical value. Various
concentrations of 1-MA in ASW were prepared (by
weight) and designated as A (1.49 /xg/ml), B (2.98
/tg/ml), C (4.92 Mg/ml), D (7.45 ^g/ml) and E (11.92
^tg/ml). In the photometric assay, the concentrations
of 1-MA in B, C, D and E solutions were calculated by
measuring the absorbance of each solution as compared with the absorbance of A by assuming that A
contains 1.49 /tg 1-MA/ml. In the bioassay, ASW was
added to these solutions (A-E) to give various dilutions. Isolated oocytes of Asterina pectinifera were
placed in the diluted solutions and breakdown of germinal vesicle (GVBD) was observed after 60 min.
Dose-response curves of these solutions were obtained
by plotting percentages of GVBD on semilogarithmic
section paper according to the degree of dilution on
the abscissa. From these curves the concentrations of
1-MA of the solutions B, C, D and E were determined
using the solution A as the reference standard. (From
Shirai, 1974).
MATURATION-INDUCING SUBSTANCES IN ECHINODERMS
the bioassay as well as from the spectrophotometric assay are in good agreement with the theoretical value. Furthermore, it should be noted that the bioassay
method is very sensitive since oocyte maturation in some females can be induced
with 1-MA at concentrations as low as 10~8
to 10~9 M. In such cases 0.0015 to 0.00015
fig/ml of 1-MA can be determined by this
method.
1-MAR (1-methyladenosine) is effective
in inducing oocyte maturation as well as
spawning when applied to ovarian fragments, whereas it has no effect on isolated
oocytes, as described above. This indicates
the presence in the starfish ovary of an enzyme which splits 1-MAR into 1-MA and
ribose (Kanatani, 1970; Schuetz, 1970,
1971). The enzyme in question can be obtained as a precipitate at 0.45 saturation of
ammonium sulfate from the supernatant of
ovarian wall homogenate. Since this enzyme does not act to a significant extent on
adenosine, it seems to be not an ordinary
adenosine ribohydrolase (Wang, 1955), but
rather a specific enzyme that is called
1-methyladenosine ribohydrolase (1MARase) (Shirai and Kanatani, 1972). The
optimum pH of the enzyme is about 7.5 and
its molecular weight is about 96,000. Its
isoelectric point is pH 5.1 (Shirai and Kanatani, 1972). Isolated follicles, which produce 1-MA under the influence of GSS,
have recently been found to contain the
enzyme (Hirai et al., 1973). Further, since
GSS has no effect on the activity of
1-MARase, the hormonal peptide seems to
take part in some reaction other than this
step in the formation of 1-MA.
1-MAMP has the same effect as 1-MAR in
inducing oocyte maturation and spawning
when applied to isolated ovarian fragments; however, isolated oocytes fail to mature even in the presence of this substance
(Kanatani and Shirai, 1972; Shirai and
Kanatani, 1973). When ovarian fragments
or their homogenate are incubated with
1-MAMP, the supernatant of the incubation mixture acquires maturation-inducing
activity, suggesting that 1-MAMP also is decomposed to 1-MA through 1-MAR. This
idea is supported by the fact that
L-phenylalanine, known as a rather specific
497
inhibitor of phosphomonoesterases as
compared with other inhibitors of this
group of enzymes (Fishman et al., 1962;
Ghosh and Fishman, 1966) and considered
to inhibit the production of 1-MAR from
1-MAMP, inhibits the production of 1-MA
in the incubation mixture of GSS and ovarian tissue or follicle cells (Shirai, 1974).
To determine whether GSS acts to induce the production of 1-MA de novo or to
accelerate the breakdown of some precursor containing 1-MA, the following experiments were performed by Shirai (1972)
using a hydrolysis procedure which liberates 1-MA from the compounds containing
it, such as tRNA's. Asterina ovarian fragments were incubated in ASW (artificial sea
water) with or without GSS. Each sample
was divided into two parts, one of which was
hydrolyzed with 0.6 N hydrochloric acid at
100°C for 30 min. After centrifugation and
pH adjustment, the supernatants were diluted with artificial sea water and assayed
with isolated oocytes. The supernatant of
the ovarian homogenate without GSS and
without hydrolysis did not show any detectable activity. The amount of MIS (1-MA)
present in the hydrolysate of the incubation
mixture of ovary and GSS (0.28 /xg/ml)
corresponded to the sum of that present in
the hydrolysate of the incubation mixture
of ovary alone (0.13 ptg/ml) and that present in the incubation mixture of ovary and
GSS without hydrolysis (0.16 fJLg/ml). These
results demonstrate that 1-MA produced
under the influence of GSS is not a breakdown product of some compound(s)
preexisting in the ovary, such as tRNA's,
but is newly produced.
In this respect, it is of interest that the
addition of methionine, known as a methyl
donor for biological methylation, to the incubation mixture of ovary and GSS enhanced the production of MIS. In the absence of GSS, the addition of methionine
had no effect. The addition of ethionine to
the ovary-GSS system greatly reduced the
production of MIS. However, such reduction of MIS activity caused by the presence
of ethionine was no longer observed in the
presence of methionine at the same concentration (Shirai et al., 1972). When
methionine labeled with C14 at its methyl
498
HARUO KANATANI
"spontaneous maturation." Since the work
of Dalcq in 1924, it has been known that
calcium ion in sea water plays an important
role in inducing oocyte maturation, because isolated oocytes fail to mature in
calcium-free sea water (CaFSW) (Kanatani,
1964). Recently Cloud and Schuetz (1973)
have reported that calcium ion stimulated
the release of MIS from Asterias forbesi follicle cells which had been isolated in CaFSW.
Immature, fully grown oocytes in the absence of follicle cells do not mature even
when calcium is available. On the other
hand, Shirai (1974) has shown that when
follicle cells of Asterias amurensis are incubated in CaFSW for 1 hr at 20°C, production of 1-MA is markedly suppressed,
suggesting that the failure of spontaneous
maturation in CaFSW is due to the inhibition of 1-MA production in the follicle cells.
Further, Shirai (1974) has investigated
the effect of phenylalanine on spontaneous
maturation. Isolated oocytes with follicle
cells fail to mature when they are placed in
ASW containing 50 mM phenylalanine, the
follicles of phenylalanine-treated oocytes
showing a tendency to retain their morphological integrity. Thus, when Asterias
follicle cells were isolated by pipetting, collected in phenylalanine-ASW, and suspended in either ASW or ASW containing
50 mM phenylalanine for 1 hr at 20°C,
bioassay with isolated oocytes showed that
the amount of MIS produced in
phenylalanine-ASW is much less than that
in ASW, suggesting that phenylalanine
suppresses the production of 1-MA. This
finding was confirmed by the following experiment. Isolated follicles, incubated in
ASW containing phenylalanine or in ASW
alone for 1 hr, were heated in a boiling
water bath for 15 min and homogenized,
and the amount of 1-MA was determined
by bioassay. This procedure is thought to
extract all of the 1-MA contained in the
follicle cells. The data presented in Table 1
show that phenylalanine inhibits the production of 1-MA. The degree of decrease in
SPONTANEOUS OOCYTE MATURATION
the amount of 1-MA in the incubation mixture of follicles and phenylalanine is almost
In many starfishes, fully grown oocytes the same whether or not the follicle cells are
generally undergo maturation when they homogenized after incubation, indicating
are mechanically isolated in normal sea wa- that the 1-MA produced in the follicle cells
ter. This phenomenon has been called
radical was added to the incubation mixture of ovarian fragments and GSS-sea water, and the supernatant was fractionated
on a Sephadex G-15 column, an elution
peak containing the radioactivity was in
good agreement with those of the biological
activity and absorbance of authentic 1-MA
at 260 nm. Therefore, it is probable that
methionine is a methyl donor in 1-MA production. This was confirmed by thin layer
chromatography when the biologically active fractions were pooled and applied to an
Avicel SF plate after desalting. Both the
radioactivity, determined by a gasflow TLC
scanner, and the biological activity were
confined to a single spot corresponding to
that of 1-MA as detected by ultraviolet light
(254 nm). From these experiments it is clear
that the methyl radical of L-methionine is
incorporated into 1-MA synthesized in the
ovary under the influence of GSS (Shirai et
al., 1972).
5-adenosylmethionine, an active form of
methionine, as well as methionine, enhanced the production of 1-MA when incubated with follicle cells ofAsterias amurensis and GSS. These results suggest that
methionine, through its active form,
S-adenosylmethionine, acts as a donor of
the methyl group in the formation of 1-MA
in the isolated follicle cells (Shirai, 1973).
To sum up these results, although many
things remain to be solved with respect to
the biochemical pathway of 1-MA formation, the action of GSS seems to consist in an
enhancement of the methylation of some
compound which contains a purine nucleus
at its Nl site. Identification of the acceptor
of such a methyl radical should provide a
clue for understanding the mechanism of
GSS action in producing 1-MA. A methylated precursor seems to be transformed
into 1-MAMP, and this in turn is hydrolyzed into 1-MAR and phosphate by phosphomonoesterase. 1-MAR is finally split into 1-MA and ribose by 1-MARase.
499
MATURATION-INDUCING SUBSTANCES IN ECHINODERMS
TABLE 1. Effect of phenylalanine on production of 1-methyladenine in Asterias follicle cells.
/ig 1-methyladenine equivalent per
ml of supernatant
Exp. No.
ASW (control)
Phenylalanine-ASW
Inhibition
1
2
3
4
5
0.0008
0.0009
0.0004
0.0007
0.0007
0.0003
0.0002
0.0001
0.0002
0.0004
63%
78%
75%
71%
43%
Mean ± SE 66 ± 6%
is immediately released into the incubation
medium.
Further, when follicle cells obtained by
centrifugation after incubation in CaFSW
for 1 hr are resuspended in ASW, heated
and homogenized, the homogenate has no
maturation-inducing activity, indicating
that there is not a significant amount of
pooled 1-MA in the follicle cells. It seems
likely that calcium ions play an important
role in the synthesis of 1-MA in the follicle
cells exposed to sea water in this experiment and in the case of spontaneous maturation (Shirai, 1974). It can therefore be
concluded that both spontaneous and
natural maturations are induced by 1-MA
produced, in the first case, under the
influence of the ionic environment of sea
water, and in the second case, in response to
GSS. Recently Cloud and Schuetz (1973)
have also obtained evidence by isotopic experiments showing that isolated follicle
cells synthesize 1-MA.
MATURATION-INDUCING
SUBSTANCE
ECHINOID GONAD
IN
Although it has been well established that
1-MA is a general maturation-inducing
substance among starfishes (Kanatani,
1972, 1973), no such MIS has been so far
demonstrated in other classes of
echinoderms. This may be due to the technical difficulty of demonstrating such a substance in sea urchins, since the oocytes of
this group of animals undergo maturation
within the ovary one by one when they attain full size; there is no arrest of meiosis at
the prophase of the first maturation division as in starfishes. For this reason it is not
easy to obtain a large number of full-sized
sea urchin oocytes with germinal vesicles.
However, if a MIS which has the capacity to
induce maturation of starfish oocytes is present in sea urchins, the detection of such a
substance can be performed rather easily
by using starfish oocytes as assay material.
Some experiments along this line were performed in my laboratory (Kanatani, 1974).
When ovaries of the Japanese sea urchin,
Hemicentrotus pulcherrimus, were taken in
the height of the breeding season,
homogenized in deionized water, and centrifuged, the supernatant had the capacity
to induce maturation of isolated starfish
oocytes, suggesting that a substance which
induces maturation of starfish oocytes is
present in the sea urchin ovary. The presence of such a substance was also shown in
other sea urchins, Pseudocentrotus depressus
and Anthocidaris crassispina, and in the sand
dollars, Clypeaster japonicus and Peronella
japonica, when assayed with isolated oocytes
of Asterina pectinifera. Testes of these ani-
mals were also found to contain MIS.
The amount of MIS contained in the testis as well as in the ovary increases with the
growth of the gonads in Anthocidaris;
ovaries taken on May 3 had only a small
number of young oocytes attached to the
germinal epithelium and contained a very
small amount of MIS (0.004 /xg 1-MA
equivalent/gm of dry tissue), whereas those
of June 17 included mature ova and contained about 20 times this amount of MIS
(0.078 Mg/gm).
Purification of the MIS present in
Pseudocentrotus ovaries was carried out in
order to determine the nature of the substance. A lyophilized sample was extracted
500
HARUO KANATANI
with 85% ethanol and concentrated with a
rotary evaporator. This was diluted with
deionized water and washed with
chloroform and ether to remove lipids. The
water phase was dried and dissolved in a
small amount of 0.05 M borate buffer, pH
8.5. The sample was fractionated on a
Sephadex G-15 column equilibrated with
0.05 M borate buffer. The active fractions
were pooled and concentrated. The sample
dissolved in a small amount of 0.5 M
pyridine acetate buffer, pH 8.5, was next
applied to a Sephadex G-15 column
equilibrated with the same buffer. The active fractions were pooled and concentrated to dry ness. Thin layer chromatography of the purified sample on microcrystalline cellulose plate (Avicel SF), with or
without authentic 1-MA, revealed one distinct spot corresponding to that of 1-MA, as
detected by an ultraviolet light lamp (Fig.
3). When the location of the maturationinducing activity on the plate was investigated by extracting the active substance
from cellulose powder removed from the
plate, the biological activity was found to be
confined to this spot. Therefore, it is evident that the MIS present in Pseudocentrotus
ovary is 1-MA. Similar purification of the FIG. 3. Thin layer chromatography of maturationsubstance carried out with ovaries and inducing substance contained in ovary of the sea urtestes of Hemicentrotus and ovaries of An- chin, Pseudocentrotus depressus. Solvent system:
methanol/hydrochloric acid/water ( 7 : 2 : 1). Spots
thocidaris gave the same result.
were detected with ultraviolet light (254 nm). A, MIS
Next, the effect of 1-MA on oocyte mat- sample alone; B, MIS sample with authentic 1-MA; C,
uration in the isolated ovarian fragments of authentic 1-MA alone.
the sea urchin, Anthocidaris crassispina, were
tested. Females which had ovaries contain- present data. These results suggest that
ing 37 to 65% mature ova were selected. 1-MA produced in the sea urchin ovary
Ovarian fragments taken from such indi- induces oocyte maturation. However, it is
viduals were placed for 2 hr in sea water rather difficult to demonstrate its effect as
containing 1-MA at concentrations of 10~7 clearly as in starfishes since the immature
M, 10~6 M, and 10~5 M. Ovarian fragments oocytes within the sea urchin ovary do not
were also placed in sea water alone as con- seem to be as uniform as starfish oocytes in
trol. After treatment with 1-MA, ovarian their developmental stages and only fully
fragments were dissected and the percent- grown oocytes can respond to 1-MA. In
ages of mature ova were observed. As other words, in sea urchins, oocytes within
shown in Table 2,1-MA at concentration of the ovary are believed to undergo matura10~5 M showed significant maturation- tion one by one when they attain full size
inducing activity (P < 0.02), whereas 10~7 M under the influence of 1-MA already exist1-MA failed to induce oocyte maturation. ing in the ovary, whereas in starfishes most
Treatment with 10~6 M 1-MA caused some of the oocytes within an ovary develop to
increase in percentage of oocyte matura- full size and remain at the prophase of the
tion; however, the results were not sig- first maturation division. 1-MA is not presnificant so far as can be calculated from the ent in the ovary and these oocytes simul-
501
MATURATION-INDUCING SUBSTANCES IN ECHINODERMS
TABLE 2. Effect of 1-methyladenine on oocyte maturation in isolated ovarian fragments of Anthocidaris crassispina.
Exp. No.
1
2
3
4
5
6
Initial percentage
of oocyte maturation
Control
39
37
55
59
65
51
10
39
15
20
10
15
Mean + SE
18.2 ± 4.4
Increase in percentage of oocyte maturation
10-5M
10"6 M
taneously undergo maturation in response
to 1-MA produced in the follicle cells under
the influence of GSS at the time of spawning.
SUBSTANCES OTHER THAN 1-ALKYLADENINES INDUCING OOCYTE MATURATION
In order to understand the mechanism
of action of 1-MA it should be helpful to
find some substance capable of inducing
oocyte maturation and whose chemical action is already known.
Phytohemagglutinin M (PHA M, Difco)
1-MA and synthetic 1-ethyladenine were
the only substances found to induce starfish
oocyte maturation until Shida et al. (1972)
reported that phytohemagglutinin M
(Difco) also had maturation-inducing activity in isolated oocytes of Asterina pectinifera.
In order to determine the active substance
contained in the PHA M sample, Kanatani
and Kishimoto (1974) have fractionated the
PHA M on a Sephadex G-25 column. As
shown in Figure 4, the maturationinducing activity of the PHA M sample is
confined to fractions No. 37 to 39, which do
not contain any sugar components. Since
these fractions have the property of absorbing ultraviolet light at 260 nm, the small
peak showing absorption at 280 nm seems
to be a reflection of this property. It is to be
noted that the sodium chloride contained
in the original PHA M sample was eluted in
fractions No. 34 and 35 as checked by a
conductivity meter. When authentic 1-MA
solution containing sodium chloride was
fractionated on the same column under the
29
50
32
26
12
22
43
45
26
7
18
20
26.5 ± 6.1
28.5 ± 5.1
(0.01 < P < 0.02) (0.2 < P < 0.3)
10-'M
26
44
22
0
15
13
16.3 ± 5.5
(0.7 < P < 0.8)
same conditions, its elution pattern almost
coincided with that of the maturationinducing activity of the PHA M sample (Fig.
4). The active substance contained in the
PHA M sample was therefore thought to be
1-MA. This was confirmed by thin layer
chromatography, when a large amount of
PHA M sample was fractionated on a
Sephadex G-15 column and the active sample applied to a microcrystalline cellulose
plate (Avicel SF) with or without authentic
1-MA. The samples developed with a solvent system, isopropanol/hydrochloric
acid/water (65:16.7:18.3 by volume), gave a
faint spot corresponding to that of 1-MA,
together with other unidentified spots,
when examined with ultraviolet light (254
nm). The biological activity was confined to
the spot corresponding to that of 1-MA.
Furthermore, phytohemagglutinin P
(PHA P, Difco), a more-purified phytohemagglutinin, free from the polysaccharide moiety and approximately 50
times more potent than PHA M in its
hemagglutinating and leukocyte mitosisstimulating capacity, failed to induce oocyte
maturation of isolated oocytes of Asterina
pectinifera even at a high concentration (50
mg/ml). Therefore, it is concluded that the
active substance contained in PHA M of
Difco is 1-MA.
Disulfide-reducing agents
Quite recently we have found that
disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-lpropanol (BAL) induce maturation of isolated oocytes of Asterina pectinifera and Asterias amurensis (Kishimoto and Kanatani,
502
HARUO KANATANI
Fraction numb«r
treated ovarian fragments are transferred
to sea water, they begin to spawn, suggesting that DTT inhibits the contraction of the
ovarian wall and thus prevents spawning.
The process of oocyte maturation induced by DTT is quite similar to that induced by 1-MA with respect to the morphological changes and the time course of
the successive events. Other disulfidereducing agents, BAL and 2-mercaptoethanol, showed a similar effect in inducing
oocyte maturation.
In contrast to these agents, SH-blocking
agents such as p-chloromercuribenzoate
(PCMB) (5 x 10-" M), iodoacetamide
(IAM)(2.2 X 10-4M)andN-ethylmaleimide
(NEM) (10~4 M) inhibit the induction of oocyte maturation by 1-MA when isolated oocytes are pretreated with these substances.
However, the inhibitory effect of these SHreagents is eliminated by a subsequent
treatment with DTT or BAL, suggesting
that the reduction of disulfide on the oocyte
surface is important in inducing oocyte maturation in the starfish. Further analysis
along this line in regard to the oocyte surface, vitelline coat, and follicular envelope
seems likely to provide a successful way to a
better understanding of the mechanisms
of spawning, oocyte maturation, and fertilization.
FIG. 4. Chromatography of PHA M and authentic
1-methyladenine. PHA M (54.7 mg which contains
about 27 mg of NaCl) in 1 ml of 0.01 M acetate buffer
(pH 5.5) was fractionated on a Sephadex G-25 column
(2.5 x 39 cm) equilibrated with the same buffer containing 0.5 M sodium chloride. The eluant was the
same buffer containing 0.5 M sodium chloride. The
fraction size was 5 ml. Protein determination was directly performed on each fraction by UV absorption at
280 nm. For determination of the sugar components,
0.5 ml of 5% phenol and 2.5 ml of sulfuric acid were
added to 0.5 ml of each fraction or to the same volume
of diluted sample and the samples allowed to stand for
1 hr at 20°C. Absorbance at 490 nm was then measured. For assay of maturation-inducing activity, 0.05
ml of 0.2 M borate buffer (pH 8.5) containing 0.35%
KC1, 0.6% CaCU, and 2.3% MgCU was added to 0.2-ml
samples taken from each fraction. Isolated oocytes
were placed in the test solution and percentage of
breakdown of germinal vesicle was observed. Elution
pattern of authentic 1-MA from the same column obtained by measuring absorbance at 260 nm is also
shown for reference. (From Kanatani and Kishimoto,
Concanavalin A
1974).
1973, and unpublished). When isolated
oocytes with follicular envelopes ofAsterina
are placed in sea water containing DTT
(8.3 x 10- 3 M to 2.5 x 10-2 M), they undergo maturation and follicular disintegration. The period of DTT treatment required for the induction of oocyte maturation is very short (3 min at 10~2 M). On
insemination, the matured eggs form tight
fertilization membranes, cleave after 2 hr
and develop normally to bipinnaria larvae.
When ovarian fragments are treated with
DTT, germinal vesicle breakdown and follicular disintegration occur within the ovary, but no spawning is effected. Addition
of 1-MA (5 x 10~7 M) fails to induce spawning in such ovarian fragments in the presence of D T T . However, when DTT-
Although the maturation-inducing activity of the PHA M sample of Difco has been
ascribed to the contaminating 1-MA, as reported above, another phytohemagglutinin globulin, Concanavalin A (Con A) obtained from the Jack bean, has a capacity to
induce starfish oocyte maturation. Quite
recently Kubota and Kanatani (unpublished) have performed the following investigations with respect to the action of Con A
in inducing oocyte maturation in Asterina
pectinifera. When isolated oocytes were
placed in ASW containing Con A (Daiichi
Chemicals) at various concentrations, they
underwent maturation; Con A at a concentration of 0.25 mg/ml induced about
90% maturation (Fig. 5). Breakdown of the
follicular envelopes was also observed. In
order to test the possibility that the
MATURATION-INDUCING SUBSTANCES IN ECHINODERMS
503
cells obtained from 10,000 oocytes/ml of
ASW containing 5 mg of Con A). The incubated cell suspension was centrifuged at
3,000 rpm for 15 min. When the supernatant was assayed with isolated oocytes without
follicles, 100% maturation was obo-o oocyte with follicles
served, whereas follicle cells incubated in
• 50
sea water alone under the same conditions
• - • oocyte without follicles
failed to induce oocyte maturation. The
amount of MIS produced by the follicle
cells in the presence of a given concentration of Con A was proportional to the
number of follicle cells in the incubation
t
2
medium (Fig. 6). In order to determine
Concentration of ConA ( m g / m l )
FIG. 5. Effect of Con A on maturation of oocytes in whether the MIS produced in follicle cells
vitro in Asterina pectinifera. Isolated oocytes with or under the influence of Con A is 1-MA, the
without follicles were placed in ASW containing Con A active substance produced in an incubation
at various concentrations.
mixture of follicle cells and Con A in ASW
was purified in the following way. Follicle
maturation-inducing activity of Con A is cells obtained from about 1,830,000 oodue to contamination with 1-MA, ethanol cytes were incubated for 2 hr at 25°C in
was added to Con A dissolved in 0.5 M ASW containing Con A (follicle cells from
sodium chloride (25mg/ml) to a final con- 10,000 oocytes/ml of ASW containing 5 mg
centration of 85%. This sample was of Con A) and centrifuged. The supernahomogenized and centrifuged. The super- tant was washed with a half-volume of
natant and the precipitate were separately chloroform and the aqueous phase was
concentrated to dryness with a rotary concentrated. After removing most of the
evaporator. These samples were diluted se- inorganic seawater salts by adding 2 M
rially with ASW and assayed with isolated K2HPO4 and ethanol (final concentration,
oocytes with follicles. The maturation- 90%), the concentrated sample was fracinducing activity was found to be confined tionated on Sephadex G-15 columns
to the ethanol precipitate, suggesting that equilibrated with 0.05 M borate buffer, pH
the active substance is not 1-MA but Con A 8.5, and with 0.5 M pyridine acetate buffer,
itself, since 1-MA is known to be soluble in pH 8.5. The active fractions were pooled
85% ethanol. This was further confirmed
by heating Con A at 100°C for 20 min. After
0.05
the heat treatment a sample of Con A failed
to induce oocyte maturation; it is well
known that 1-MA is heat stable. On the
other hand, when isolated oocytes were
0D3
treated with CaFSW to remove the follicle
cells from them, and the denuded oocytes •5 <
were placed in ASW containing Con A, they
failed to undergo maturation (Fig. 5),
0.0!
suggesting that the action of Con A is indirect, and that the follicle cells are involved
10000
20000
30000
in its maturation-inducing activity, preRelative amount of follicle cells per ml
sumably by producing MIS under the
of incubation medium
influence of Con A. Therefore, production FIG. 6. Effect of follicle cell density on Con
of MIS in follicle cells in the presence of A-induced MIS production \n Asterina pectinifera. FolCon A was next investigated. Mechanically licle cells were incubated in ASW containing Con A (5
isolated follicle cells were incubated for 1 hr mg/ml) for 1 hr at 25°C. Relative amount of follicle
at 25°C in ASW containing Con A (follicle cells is expressed as the number of oocytes from which
KM
they were obtained.
504
HARUO KANATANI
and concentrated to dryness. Thin layer
chromatography of the purified sample on
a microcrystalline cellulose plate (Avicel SF)
with or without authentic 1-MA revealed
one distinct spot corresponding to that of
1-MA, as detected by an ultraviolet light
lamp. Therefore, it is concluded that Con A
acts on the follicle cells, causing them to
produce 1-MA just as does GSS, the gonad
stimulating hormone.
CONCLUDING REMARKS
Spawning and oocyte maturation in
starfishes are controlled by a hormonal
mechanism which consists of a chain of
reactions starting with the release of GSS
from the nervous system, presumably the
radial nerves and the circumoral nerve
ring, into the body fluid. The released GSS
reaches the body cavity where the gonads
are suspended. GSS acts on the follicle cells,
causing them to produce the second active
substance, the maturation-inducing substance (MIS), 1-MA. The 1-MA acts on the
surface of the oocyte to induce the cytoplasmic maturation which is a prerequisite
for acquisition of fertilizability, and
perhaps, since microinjection of 1-MA into
inner cytoplasm fails to induce germinal
vesicle breakdown, to produce in the cortex
of the oocyte a hypothetical third substance
which is responsible for breakdown of the
germinal vesicle. Although we have no evidence for the presence of such a substance
in starfish, recent investigations with frog
oocytes treated with progesterone, which is
known in frogs as a maturation-inducing
substance probably released under the
influence of pituitary gonadotropin, have
shown that a third substance is produced in
the cortical region which causes the breakdown of the germinal vesicle (Masui and
Markert, 1971; Masui, 1972). Itistherefore
reasonable to assume the presence of such a
cytoplasmic factor in starfishes. When this
factor causes the breakdown of the germinal vesicle, mixing of a nuclear factor(s)
with the cytoplasm occurs, resulting in providing some other factor(s) or conditions
necessary for cleavage and further development.
For further investigations on the
mechanism of oocyte maturation, the importance of the cell surface should be taken
into account. The production of 1-MA induced by Con A in follicle cells is considered to be a matter of the cell surface, since it
has been well established that the site of
action of Con A is the cell surface. We hope
that further investigations using Con A will
reveal the nature of the surface change related to the production of 1-MA under the
influence of GSS. Also further researches
on germinal vesicle breakdown should be
directed toward obtaining evidence showing the presence of the hypothetical third
substance.
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