Masaru Nakamura

Masatoshi Mita

Yuki Takeshige

Springer - Publisher Connector

115
 Abstract  As in lower vertebrates, starfi sh oocyte maturation and ovulation are induced 
by a hormonal substance. Gonad-stimulating substance (GSS) is the fi rst mediator in 
inducing these maturation and ovulation. GSS secreted from the nervous system acts 
on the ovary to produce 1-methyladenine (1-MeAde) as a maturation- inducing hor-
mone. 1-MeAde induces germinal vesicle breakdown (GVBD) and  follicular envelope 
breakdown (FEBD), releasing the oocyte from the ovaries, that is, spawning. Recently, 
GSS was purifi ed from the radial nerves of the starfi sh  Asterina pectinifera and its 
chemical structure was determined to be a relaxin-like peptide. To further elucidate the 
physiological roles of GSS on oocyte maturation and ovulation, this study examined 
the effect of synthetic GSS on 1-MeAde production in intact ovaries, folliculated 
oocytes, isolated follicle cells, and spawned  ovaries containing follicle cells of the 
starfi sh (sea star)  A. pectinifera . Spawning was induced by synthetic GSS as a dose-
dependent manner. However, a high concentration of GSS failed to induce spawning. 
1-MeAde production in an ovarian fragment also declined at a high concentration of 
GSS. Similar dual effects of GSS were observed in folliculated oocytes. In contrast, 
1-MeAde production in isolated follicle cells and spawned ovaries did not decrease at 
a high concentration of GSS. Interestingly, egg jelly inhibited GSS-induced 1-MeAde 
production in follicle cells. These results may suggest that egg jelly disturbs GSS action 
on 1-MeAde production and spawning in ovaries. 
 Keywords  1-Methyladenine •  Egg jelly •  Gonad-stimulating substance •  Spawning 
•  Starfi sh 
 Chapter 10 
 Effect of Relaxin-Like Gonad-Stimulating 
Substance on Gamete Shedding and 
1-Methyladenine Production in Starfi sh 
Ovaries 
 Masatoshi  Mita ,  Yuki  Takeshige , and  Masaru  Nakamura 
 M.  Mita (*) •  Y.  Takeshige 
 Department of Biology, Faculty of Education ,  Tokyo Gakugei University , 
 Tokyo  184-8501 ,  Japan 
 e-mail: bio-mita@u-gakugei.ac.jp 
 M.  Nakamura 
 General Research Center, Churashima Foundation ,  Ishikawa 888 ,  Motobu-cho, 
Kunigami- gun ,  Okinawa  905-0206 ,  Japan 
H. Sawada et al. (eds.), Sexual Reproduction in Animals and Plants, 
DOI 10.1007/978-4-431-54589-7_10, © The Author(s) 2014
116
10.1  Introduction 
 In most starfi sh, fertilization occurs in seawater, outside the female’s body. A ripe 
ovary contains a huge number of fully grown oocytes of almost equal size. Each 
oocyte still possesses a large nucleus (germinal vesicle), which is arrested in late 
prophase of the fi rst maturation division. The oocyte is surrounded by a single 
 follicular layer. The follicles adhere fi rmly to each other and to the ovarian 
wall (Kanatani and Shirai  1969 ). Resumption of meiosis in immature oocytes and 
spawning from the ovary are induced by 1-methyladenine (1-MeAde) (Kanatani 
et al.  1969 ). 1-MeAde is produced by ovarian follicle cells on stimulation by 
gonad- stimulating substance (GSS) released from radial nerves (Kanatani  1985 ). 
However, the action of 1-MeAde is indirect; it acts on the oocyte membrane 
 receptor (Yoshikuni et al.  1988 ; Tadenuma et al.  1992 ) to activate a cytoplasmic 
maturation or M-phase promoting factor (MPF) (Okumura et al.  2002 ; Hiraoka 
et al.  2004 ), which is the direct trigger of germinal vesicle breakdown (GVBD) 
(Kishimoto and Kanatani  1976 ) and follicular envelope breakdown (FEBD) 
(Kishimoto et al.  1984 ). Once FEBD occurs, the denuded oocytes become freely 
movable within the ovary and are forced out by contraction of the  ovarian wall 
(Shirai et al.  1981 ). 
 Previous studies have shown that an inhibitor of spawning, “shedhibin,” is 
 contained in the nerve extract (Cheat  1966a ,  b ). However, crude GSS inhibits 
spawning at a high concentration (Kanatani and Shirai  1970 ). Recently, GSS was 
purifi ed from the radial nerves of the starfi sh  Asterina pectinifera and its chemical 
structure was determined to be a relaxin-like peptide (Mita et al.  2009 ). In this 
 chapter, hormonal action of GSS on spawning is described from the aspect of 
1-MeAde production within ovaries. 
10.2  Effect of GSS on Spawning in Ovarian Fragments 
 Starfi sh (the sea star  Asterina pectinifera ) were collected from several locations in 
Japan in the breeding season. An experiment was carried out to examine the effect 
of synthetic GSS on spawning. Ovarian fragments were placed in modifi ed van’t 
Hoff’s artifi cial seawater (ASW) at pH 8.2 containing synthetic GSS at various 
concentrations. After 1 h incubation at 20 °C, spawning was induced by GSS at 
concentrations exceeding 0.2 nM GSS (Fig.  10.1 ). The median effective concen-
tration (EC 50 ) was obtained with about 0.5 nM GSS. However, GSS-induced 
spawning decreased at concentrations above 10 nM, and above 20 nM GSS failed 
to induce spawning. These oocytes within the ovary were matured in a high 
 concentration of GSS. It appears that GSS at a high concentration acts in some 
toxic way on the  ovarian wall to inhibit its contraction, resulting in failure of 
spawning.
M. Mita et al.
117
10.3  Effect of GSS on 1-MeAde Production 
 Because GSS mediates oocyte maturation by acting on the ovary to produce 
1-MeAde (Kanatani et al.  1969 ), we tested the action of synthetic GSS on 1-MeAde 
production in the whole ovary. When ovarian fragments of  A. pectinifera were incu-
bated in ASW containing synthetic GSS at various concentrations, the 1-MeAde 
produced was found in the incubation medium. The amount of 1-MeAde produced 
increased as the GSS concentration was raised, and reached maximum at 2 nM GSS 
(Fig.  10.2a ). However, it declined at concentrations above 2 nM GSS. In contrast, 
GSS-induced 1-MeAde production in isolated follicle cells increased in a dose- 
dependent manner and reached a plateau at concentrations above 2 nM GSS 
(Fig.  10.2b ), which agrees with previous reports (Mita et al.  1987 ,  2009 ). Because 
GSS stimulates follicle cells as a target in starfi sh ovaries ( Hirai and Kanatani  1971 ; 
Hirai et al.  1973 ), it is possible that unknown matter within the ovaries affects GSS 
action on follicle cells to produce 1-MeAde.
 The ovaries in starfi sh  A. pectinifera are fi lled with immature oocytes, each 
 surrounded by an envelope consisting of follicle cells (Fig.  10.3a ). Following treat-
ment of ovary with 1-MeAde, not only GVBD but also FEBD occurs, and denuded 
maturing oocytes are spawned from the ovary. The follicle cells become clustered 
within the ovaries ( Mita and Nakamura  1994 ). After spawning, follicle cells exit 
the ovaries. Thus, we examined the effect of GSS on 1-MeAde production in the 
spawned ovaries. Following incubation of the spawned ovaries with GSS at various 
GSS content (-Log M)
10 9 8
Sp
aw
ni
ng
 a
ct
ivi
ty
+++
++
+
–
7
 Fig. 10.1  Effect of gonad-stimulating substance ( GSS ) on gamete shedding from ovaries in starfi sh 
 Asterina pectinifera . Ovarian fragments were placed in artifi cial seawater (ASW) containing GSS 
at concentrations indicated. After 1 h incubation at 20 °C, the sample was examined to determine 
whether spawning had occurred. (+++), spawning occurred and most oocytes matured; (++), about 
50 % oocytes matured; (+), a few oocytes were matured; and (−), no spawning occurred. Values are 
mean ± SEM of four separate assays using different animals 
 
10 Effect of Relaxin-Like Gonad-Stimulating Substance on Gamete Shedding…
118
concentrations, 1-MeAde was produced in the incubation medium (Fig.  10.2c ). 
The production of 1-MeAde was markedly dependent on GSS concentration. 
A decline in 1-MeAde production was not observed at a high concentration of GSS, in 
agreement with the result obtained in isolated follicle cells. Thus, effects of a high 
concentration of GSS on 1-MeAde production in the spawned ovaries are different 
from those in the intact ovaries. To elucidate this, we used folliculated oocytes to 
examine GSS action on 1-MeAde production. The folliculated oocytes were pre-
pared as described previously (Mita  1985 ). GSS stimulated 1-MeAde production in 
folliculated oocytes (Fig.  10.2d ). The amount of 1-MeAde produced by folliculated 
oocytes increased in a dose-dependent manner of GSS, but it decreased slightly at a 
high concentration of GSS. This result suggests that oocytes or other related matter 
disrupt 1-MeAde production in follicle cells.
 On the other hand, it has been shown that  l -glutamate inhibits GSS-induced 
spawning in  A. pectinifera (Ikegami et al.  1967 ). Thus, we examined the effect of 
 l -glutamate on GSS-induced 1-MeAde production in follicle cells. When isolated 
follicle cells were incubated with GSS at concentrations of 2 and 20 nM in the 
10-10
GSS (M)
1-
M
eA
de
 p
ro
du
ct
io
n
(nm
ol/
15
 m
in/
0.4
 g 
of 
ov
ary
)
10-9
0.2
0.4
0
10-8
0.6
10-7 10-10 10-9 10-8 10-7
10-10 10-9 10-8 10-7 10-10 10-9 10-8 10-7
GSS (M)
1-
M
eA
de
 p
ro
du
ct
io
n
(nm
ol/
2 h
/2 
x 1
05
 o
o
cy
te
s)
0.2
1.0
0
1.2
0.8
0.6
0.4
GSS (M)
1-
M
eA
de
 p
ro
du
ct
io
n
(nm
ol/
2 h
/10
7  
ce
lls
)
0.2
1.0
0
1.2
0.8
0.6
0.4
GSS (M)
1-
M
eA
de
 p
ro
du
ct
io
n
(nm
ol/
15
 m
in/
ov
ary
 sp
aw
ne
d)
0.2
0.3
0
0.4
0.1
a b
c d
 Fig. 10.2  Effect of GSS on 1-methyladenine (1-MeAde) production by an ovarian fragment 
( a ), isolated follicle cells ( b ), folliculated oocytes ( c ), and spawned ovary ( d ) of starfi sh  A. pec-
tinifera . After incubation in ASW at 20 °C containing GSS at concentrations as indicated, 1-MeAde 
concentration was determined by biological assay. Values are mean ± SEM of four separate assays 
using different animals 
 
M. Mita et al.
119
absence or presence of 10 mM  l -glutamate, 1-MeAde production occurred regardless 
of presence of  l -glutamate (Table  10.1 ). This result suggests that  l -glutamate has 
no effect on GSS-induced 1-MeAde production in follicle cells.
10.4  Effect of Egg Jelly on GSS-Induced 1-MeAde 
Production 
 Although the follicle cells are covered with the oocyte surface (Fig.  10.3a ), there is an 
interspace between the follicular envelope and oocyte surface (Fig.  10.3b ). It has been 
shown that egg jelly is distributed in this interspace (Shirai et al.  1981 ). Thus, we exam-
ined whether the egg jelly affects GSS-induced 1-MeAde production by follicle cells. 
 Fig. 10.3  Cross sections 
of ovary in starfi sh 
 A. pectinifera .  (a) Light 
micrograph shows 
folliculated oocytes.  Bar 
50 μm. Association of 
follicle cells with oocyte 
( b ) was observed by 
ultramicroscopy.  Bars 1 mm. 
 Ct cortex,  EJ egg jelly,  Fc 
follicle cells,  GV germinal 
vesicle,  Oc oocyte 
 
10 Effect of Relaxin-Like Gonad-Stimulating Substance on Gamete Shedding…
120
Egg jelly was prepared by acidifying a 20 % defolliculated oocyte suspension in ASW 
with HCl to pH 5.5 for 4–5 min. The oocytes were removed by centrifugation at 500  g 
for 5 min, and the supernatant was adjusted to pH 8.2 with NaOH. Undissolved matter 
was removed by centrifugation at 10,000  g for 30 min at 4 °C. The concentration of egg-
jelly solution was estimated by carbohydrate determination using the method of Dische 
and Shettles ( 1951 ) with  d -fucose as the standard. When isolated follicle cells were 
incubation with 10 nM GSS in the presence of egg jelly at various concentrations, 
1-MeAde production decreased gradually as the egg-jelly concentration was raised 
(Fig.  10.4 ), suggesting that the egg jelly inhibits GSS-induced 1-MeAde production in 
follicle cells. The decrease in 1-MeAde production at a high concentration of GSS may 
be caused by the egg jelly. However, it is unexpected that egg jelly has an inhibitory 
effect on GSS action to stimulate 1-MeAde production within the ovaries. It has also 
been shown that egg jelly acts directly to induce contraction of the ovarian walls (Shirai 
et al.  1981 ). Further studies on ovarian wall contraction should provide useful insights 
into the hormonal control of ovulation and spawning of starfi sh.
0.8
1.0
0
0
Egg-jelly (mg fucose-equiv./ml)
1-
M
eA
de
 p
ro
du
ct
io
n
(nm
ol/
2 h
/10
7  
ce
lls
)
0.4 0.6
1.2
0.8
0.6
0.4
0.2
0.2
 Fig. 10.4  Effect of egg jelly 
on GSS-induced 1-MeAde 
production by isolated follicle 
cells in starfi sh  A. pectinifera . 
After 2 h incubation at 20 °C 
with 10 nM GSS in the 
presence of egg jelly at 
concentrations indicated, 
1-MeAde concentration was 
determined by biological 
assay. Values are mean ± SEM 
of three separate assays using 
different animals 
 Table 10.1  Effect of  l -glutamate on gonad-stimulating substance (GSS)-induced 
1-methyladenine (1-MeAde) production in isolated follicle cells of the starfi sh 
 Asterina pectinifera 
 Condition 
 1-MeAde production 
(nmoles/2 h/10 7 cells) 
 Control  Undetectable 
 l -Glutamate (10 mM)  Undetectable 
 GSS (2 nM)  0.92 ± 0.11 
 GSS (20 nM)  1.08 ± 0.17 
 GSS (2 nM) +  l -glutamate (10 mM)  0.88 ± 0.15 
 GSS (20 nM) +  l -glutamate (10 mM)  0.98 ± 0.15 
 After 2 h incubation of follicle cells with 2 or 10 nM GSS in the absence or presence 
of 10 mM  l -glutamate, 1-MeAde concentration was determined by biological assay 
 Values are mean ± SEM of three separate assays using different animals 
 
M. Mita et al.
121
10.5  Conclusion 
 1.  The oocytes released from ovaries of starfi sh  Asterina (A.) pectinifera were induced 
by GSS in a dose-dependent manner. 
 2.  However, a high concentration of GSS failed to induce the spawning. 
 3.  1-MeAde production by ovarian fragments declined at a high concentration 
of GSS. 
 4.  Similar dual effects of GSS were observed in folliculated oocytes, whereas 
1-MeAde production in isolated follicle cells and spawned ovaries did not 
decrease at a high concentration of GSS. 
 5.  Because egg jelly inhibited GSS-induced 1-MeAde production in follicle cells, 
it may be possible that the egg jelly disturbs GSS action on 1-MeAde production 
and spawning in ovaries. 
 Open Access: This article is distributed under the terms of the Creative Commons Attribution 
Noncommercial License which permits any noncommercial use, distribution, and reproduction in 
any medium, provided the original author(s) and source are credited. 
 References 
 Cheat AB (1966a) Neurochemical control of gamete release in starfi sh. Biol Bull 130:43–58 
 Cheat AB (1966b) The gamete shedding substance of starfi shes: a physiological-biochemical 
study. Am Zool 6:263–271 
 Dische Z, Shettles LB (1951) A new spectrophotometric test for the detection of methyl pentose. 
J Biol Chem 192:579–582 
 Hirai S, Kanatani H (1971) Site of production of meiosis-inducing substance in ovary of starfi sh. 
Exp Cell Res 57:224–227 
 Hirai S, Chida K, Kanatani H (1973) Role of follicle cells in maturation of starfi sh oocytes. Dev. 
Growth & Differ 15:21–31 
 Hiraoka D, Hori-Oshima S, Fukuhara T, Tachibana K, Okumura E, Kishimoto T (2004) PDK1 is 
required for the hormonal signaling pathway leading to meiotic resumption in starfi sh oocytes. 
Dev Biol 276:330–336 
 Ikegami S, Tamura S, Kanatani H (1967) Starfi sh gonad: Action and chemical identifi cation of 
spawning inhibitor. Science 158:1052–1053 
 Kanatani H, Shirai H (1969) Mechanism of starfi sh spawning. II. Some aspects of action of a 
 neural substance obtained from radial nerve. Biol Bull 137:297–311 
 Kanatani H, Shirai H, Nakanishi K, Kurokawa T (1969) Isolation and identifi cation of meiosis- 
inducing substance in starfi sh,  Asterias amurensis . Nature (Lond) 221:273–274 
 Kanatani H, Shirai H (1970) Mechanism of starfi sh spawning. III. Properties and action of meiosis- 
inducing substance produced in gonad under infl uence of gonad-stimulating substance. Dev Growth 
Differ 12:119–140 
 Kanatani H (1985) Oocyte growth and maturation in starfi sh. In: Metz CB, Monroy A (eds) 
Biology of fertilization, vol 1. Academic, New York, pp 119–140 
 Kishimoto T, Kanatani H (1976) Cytoplasmic factor responsible for germinal vesicle breakdown 
meiotic maturation in starfi sh oocyte. Nature (Lond) 260:321–322 
 Kishimoto T, Usui N, Kanatani H (1984) Breakdown of starfi sh ovarian follicle induced by 
maturation- promoting factor. Dev Biol 101:28–34 
10 Effect of Relaxin-Like Gonad-Stimulating Substance on Gamete Shedding…
122
 Mita M (1985) Effect of cysteine and its derivatives on 1-methyladenine production by starfi sh 
follicle cells. Dev Growth Differ 27:563–572 
 Mita M, Ueta N, Nagahama Y (1987) Regulatory functions of cyclic adenosine 3',5'-monophos-
phate in 1-methyladenine production by starfi sh follicle cells. Biochem Biophys Res Commun 
147(1):8–12 
 Mita M, Nakamura M (1994) Infl uence of gonad-stimulating substance on 1-methyladenine level 
responsible for germinal vesicle breakdown and spawning in the starfi sh Asterina pectinifera. 
J Exp Zool 269:140–145 
 Mita M, Yoshikuni M, Ohno K, Shibata Y, Paul-Prasanth B, Pichayawasin S, Isobe M, Nagahama 
Y (2009) A relaxin-like peptide purifi ed from radial nerves induces oocyte maturation and 
ovulation in the starfi sh,  Asterina pectinifera . Proc Natl Acad Sci USA 106:9507–9512 
 Mita M, Yamamoto K, Nagahama Y (2011) Interaction of relaxin-like gonad-stimulating  substance 
with ovarian follicle cells of the starfi sh  Asterina pectinifera . Zoolog Sci 28:764–769 
 Okumura E, Fukuhara T, Yoshida H, Hanada Si S, Kozutsumi R, Mori M, Tachibana K, Kishimoto 
T (2002) Akt inhibits Myt1 in the signaling pathway that leads to meiotic G2/M-phase transition. 
Nat Cell Biol 4:111–116 
 Shirai H, Yoshimoto Y, Kanatani H (1981) Mechanism of starfi sh spawning. IV. Tension genera-
tion in the ovarian wall by 1-methyladenine at the time of spawning. Biol Bull 161:172–179 
 Tadenuma H, Takahashi K, Chiba K, Hoshi M, Katada T (1992) Properties of 1-methyladenine 
receptors in starfi sh oocyte membranes: involvement of pertussis toxin-sensitive GTP-binding 
protein in the receptor-mediated signal transduction. Biochem Biophys Res Commun 186: 
114–121 
 Yoshikuni M, Ishikawa K, Isobe M, Goto T, Nagahama Y (1988) Characterization of 
1- methyladenine binding in starfi sh oocyte corties. Proc Natl Acad Sci USA 85:1874–1877 
M. Mita et al.


Effect of Relaxin-Like Gonad-Stimulating Substance on Gamete Shedding and 1-Methyladenine Production in Starfish Ovaries

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Effect of Relaxin-Like Gonad-Stimulating Substance on Gamete Shedding and 1-Methyladenine Production in Starfish Ovaries

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