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Characterization and action of meiotic maturation inhibitors in starfish ovary
CHARACTERIZATION MATURATION
of Agricultural Japan.
ACTION
INHlBITORS
S. IKEGAMI.’ ‘Department
AND
IN STARFISH
Y. KAMlYA’* Chemistry.
Nakano-ku.
and
University
and ‘Wc-etrn Research
OF MEIOTIC
Insrilure, Tokyo
OVARY
H. SHIKAI”
of‘ Tokyo.
Bunkyo-ku.
tinii~ersir~
of ?okp.
l’ok~o
113.
164, Jupan
SUMMAKY Mechanical release of oocytes from the ovary of the starfish Asreria.\ omurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the mn:uration of Asferias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapabic ot inhibiting oocyte maturation. The ovarian extract inhibited the productlon of I-methyladenine (I-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence IMA-induced maturation of the mcyte with or without follicle cells. It can be concluded from the present results that the role of tge ovarian extract containing steroid glycosides is to arrest “spontaneous” production of I-MA in follicle cells. The suppression can he overcome by the action of a gonadotropic peptide hormone released from the nerve tiszuc.
In many starfish species, fully grown oocytes undergo “spontaneous” meiotic maturation when they are isolated manually in normal sea water [I]. The occurrence of substances which inhibit “spontaneous” oocyte maturation has been known in the starfish ovary [2-4]. Heilbrunn et al. [2] reported that the maturation inhibitors were heparin-like substances on the basis of several chemical experiments carried out on crude ovarian extracts. Later, lkegami & Tamura [4] in a preliminary report concluded that the maturation inhibitors were steroid glycosides rather than heparin-like substances based on their partition coefficient between n-butanol and water and by gel filtration experiments. * Present Research.
address: Institute of Physical and Chemical Wako City, Saitama 35 I, Japan.
On the other hand. the maturation of oocytes in the starfish ovary is induced by I-methyladenine ( I -MA) which is produced in follicle cells under the influence of a peptide designated as gonad-stimulating substance (GSS) released from the nerve tissue [5, 61. In calcium-free sea water (CaFSW) the follicle cells are detached from oocytes and the germinal vesicle remains intact in the oocyte [6,7]. When oocytes denuded of follicle cells in CaFSW are subsequently incubated with isolated follicle cells in normai sea water, they undergo germinai vesicie breakdown [6-8]. Thus, it was suggested that “spontaneous” oocyte maturation i3 caused by the production of I-MA in the absence of GSS in follicle cells Mhen they are removed from the inhibitory intraovarian environment [7]. It is not uniike!y.
234
Ikegami,
Kamiya and Shirai
Table 1. Purification steps and specific activity of ovarian extract from Asterias amurensis Preparation
Weight
Spec. act.
Lyophilized ova@’ Acetone powder 20 % Acetone extract 80 % Acetone extract Butanol extract
1 5256 1 400 423 98 3.2
30’ 10 0.25
a Obtained from 240 females. b Weight in (g). c Dose (mg/ml) required for 50% inhibition of “spontaneous” maturation in Asterias oocytes surrounded by follicle cells.
Biological
assays
The maturation-inhibitory action of the extract obtained at each purification step was assayed with isolated oocytes. Oocytes were tom from an ovarian fragment with forceps and immediately transferred into a small Petri dish containing ASW with a measured amount of the test sample. Oocytes prepared in this manner were surrounded by follicle cells. The number of immature oocytes still possessing intact germinal vesicle was counted 1 h after initiation of the treatment. The control specimens consisted of oocytes kept in CaFSW, whereby the germinal vesicle remained intact and in ASW where the germinal vesicle disintegrated. The methods for obtainine isolated follicle cells bv treatment with CaFSW havelbeen described elsewhere [7]. The amount of I-MA in test solutions was bioassayed with isolated oocytes freed from follicle cells according to the method of Shirai [7].
Preparations
of ovaries and oocytes
therefore, that the maturation inhibitors act for extraction Ovaries were excised in ASW, washed three times on the follicle cell to block “spontaneous” with ASW and then lyophilized. The maturation inproduction of l-MA. However, the site of hibitors were extracted from the lvoohilized material. action of the matureuion inhibitors remains Oocytes were dejellied and denuded of follicle . . cells in the following way. Ovaries were washed three trmes to be established. with ASW, rinsed and then induced to spawn by addiIn the present report, chemical properties tion of isotonic KCl. Intensive discharge of oocytes immediately after the treatment. Oocytes of the maturation inhibitors obtained from resulted were filtered through cheese cloth and were dejellied the ovary ofAsterias amurensis and the site by treatment with ASW (pH 5.0) in the usual way. After thorough washing with ASW. the dejellied of their action have been described. oocytes were lyophihzed. Prevaration
MATERIALS
AND METHODS
The starhsh,Asterias amurensis, for the present study were collected at Tokyo Bay and kept in a laboratory aquarium supplied with circulating sea water.
Chemicals The sea water used was Vant’ Hoffs artificial sea water (ASW) with the following composition: 2.7% NaCl, 0.07% KCI, 0.1% CaCI,, 0.34% MgCl, and 0.21% MgSO+ The ASW was adjusted to pH 8.0-8.3 with borate buffer (final concentration. 0.05 M) unless otherwise stated. CaFSW was prepared by omitting CaCI, in the ASW formula. Water and organic solvents were redistilled in ah-glass apparatus. I-MA was purchased from Sigma Chemical Co. Authentic asterosaponins A (ASA) and B (ASB) were prepared from Asferias amurensis collected during the nonbreading season (summer) according to the method described elsewhere [9, IO]. GSS used in the present studv was oreoared from lvoohilized radial nerves of As&as arhr>nsis and it -c&responds to the active fraction purified by gel filtration on the first Sephadex G-SOcolumn [l I]. E.rp Cell Res 103 (1976)
of the butanol extract
An acetone powder prepared from the lyophilized ovaries or oocytes was extracted with 20% aqueous acetone. To the extract anhydrous acetone was added to make an 80% acetone solution. The mixture was left standing overnight in the cold and filtered to remove the precipitates. The filtrate was concentrated in vacua and the aqueous residue was adjusted to pH 8.0 with dilute sodium carbonate solution. The aqueous solution was extracted with an equal volume ofn-butanol. The aqueous layer was separated, acidified to DH 3.6 with dilute sulfuric acid and re-extracted with an equal volume of n-butanol. The butanol layer was seoarated and concentrated in vacua to drvness. The residue was dissolved in distilled water and dialvsed overnight against distilled water. The retentate was lyophilized to yield an amorphous powder, which was designated as butanol extract.
Chromatography Thin layer chromatography (tic) of the butanol extract was carried out by use of silica gel HF*,, plates of 0.38 mm thickness and a solvent system. chloroformmethanol-acetic acid-water (60 : 20 : 10: 4, v/v). In some experiments. the plates were sprayed with
0 &OSE 1 D-GALACTOSE
D-XYLOSED-G:UCrJSE-
C-QUINOk'OSE-ED-l?LOjE ?-O-METHYL-D-GLUCOSE
(D-OUINOVOSE)
A
B
Fig. 1. Structures of ovarian asterosaponins ‘4 (A) and
I3 (B), and a Stichop~s japonicus saponin (C). R= CH,CO, CH$Z(OH)CH2COCH2CH(CH3)2, CH&=
CHCOCH,CH(CH,),, CH&HCH,COCH,CH(CM,;,, CH,CHCH,COCH=C(CH,),, CH3CHCHeCHOHCHzCH(CH&, etc.
effective purification was not accomplished by the gel filtration. Therefore, further separation of the butanol extract was carried out by preparative tic. Bioassay of the methanol extract from the developed plates revealed that the active fractions corresponded to the phosphotungustic acidreactive zones at RI values 0.1 and 0.05. Chemical analysis of the active fractions showed that the fractions at Rf values 0. I and 0.05 contain steroid glycosides, ASA and ASB, respectively (fig. 1). On the assumption that the butane: extract is distributed evenly in the ovary, the RESULTS concentration of the maturation inhibitors is fsofatiovl and chemical characterization around 2.1 mglg of dry ovary; this amount of the maturation inhibitors in is sufficient to inhibit the “spontaneous” the ovary oocyte maturation in vitro (tables 112). A summary of the inhibitory activity of the The ASA fraction consists of I moie of steroid sulfate and 2 moles each of a-fucose ovarian extract on “spontaneous” oocyte maturation at each purification step is pre- and o-quinovose (6deoxy-n-glucose) [12], sented in table 1. The butanol extract was while the ASB fraction contains 1 mole each applied onto a Sephadex G-50 column ac- of steroid sulfate: o-xyiose, n-galactose and cording to the procedure described in previ- n-fucose, and 2 moles of o-quinovose [l2], ous reports [4, 121. The elution profile ASA and ASB were originally defined as showed a characteristic dispersion of the glycosides containing a steroid with the [ 141.However7 biological activity. This suggests that as- molecular formula C22H3-203 sociation of the active substances occurs in our later studies [9] have shown that such various degrees in aqueous media and that steroids are undetectable in the hydrolysate phosphotungstic acid reagent [12] to make the presence of steroid glycosides visible. Each active fraction on the plates was extracted with methanol and applied onto a column (1.4X 12 cm) of DEAE-cellulose (acetate form). The column was washed with chloroform-methanol (2 : 1, v/v) and then developed with chloroform-methanol (2 : 1, v/v) containing 5% 0.5 M LiCl (fraction size, 10 ml; flow rate, 50 ml/h). The fractions containing sugar residues which were identified with phenol-sulfuric acid reagent [ 13, 141were combined and partitioned with an equal volume of water. The upper layer was separated and evaporated in vacua to yield a powder. Since Li+ ions induced germinal vesicle breakdown in starfish oocytes Ll.51, the powder was dissolved in distilled water and dialysed against ASW to remove Li’ ions from the active acidic materials. Then, the retentate was bioassayed.
236
Ikegami, Kamiya and Shirai
Table 2. Effect of purified materials from Asterias on “spontaneous” maturation in Asterias oocyte
Effect of the ovarian butanoi extract on l-MA-induced oocyte maturation The effect of the ovarian butanol extract on Preparation Spec. act. l-MA-induced oocyte maturation was next Butanol extract from ovary 0.25” examined. Asterosaponin A fraction from ovary 0.28 (1) Isolated oocytes with follicle cells Asterosaponin B fraction from ovary 0.20 Butanol extract from oocyte >l.OO were placed in ASW containing the ovarian butanol extract at a concentration of 850 a Dose (mglml) required for 50% inhibition of “spontaneous” maturation in Aslerias oocytes surrounded pg/ml. The extract suppressed effectively by follicle cells. the “spontaneous” maturation of the oocyte (8 % maturation). The addition of l-MA at a final concentration of 5~ lo-’ M induced of the glycosides obtained from the same 100% oocyte maturation in the presence of starfish. Therefore, we propose to call the ovarian butanol extract. steroid glycosides which are present in (2) Oocytes were treated with CaFSW to starfishes asterosaponins, regardless of the remove follicle cells and were transferred chemical structure of steroids constituting directly from CaFSW to ASW containing the glycosides. These steroid glycosides are the ovarian butanol extract (750 pg/ml) tonot homogeneous [9] and the aglycones constituting these saponins are closely re- gether with l-MA at various concentralated steroids with a characteristic ring tions. Germinal vesicle breakdown was obstructure of a 3P,6a-diol system [9]. Fur- served after 1 h. As demonstrated in table 3, ther separations of each fraction into homo- the ovarian butanol extract at 750 pg/ml did genous compounds were hampered since not inhibit the l-MA-induced oocyte mamethods to achieve separation of the close- turation. The present results suggest that the ly related steroid glycosides on the basis of ovarian butanol extract is not toxic to the molecular difference in aglycones are not available. Therefore, it is not yet known oocyte even at a high concentration and which molecules are responsible for the that oocyte maturation is induced by the maturation inhibitory activity in the steroid application of l-MA, irrespective of the presence or absence of the ovarian saglycoside fractions. ponins .
Localization of the maturation Table 3. Effect of ovarian asterosaponin A inhibitors in the ovary on I-methyladenine-induced maturation in In order to determine the site of production Asterias oocytes without follicle cells of the maturation inhibitors in the ovary, % Oocyte maturation butanol extract was prepared from the isolated oocytes. The specific biological ac- 1-Methyladenine Asterosaponin ASW control (750 pg/ml) tivity of the butanol extract from oocytes cont. (M) were markedly lower than that of the 1.0x 10-S 13.3+ 3.3” 14.02 3.0’ ovarian ASA and ASB (table 2). Therefore, 4.0x 10-g 40.5+ 14.4 43.5k13.7 99.5+ 0.5 100.0~ 0 the maturation inhibitors are localized out- 1.6x lo-* side the oocytes in the ovary. a Values are mean&SE. Exp Cell Res 103 (1976)
Starfish maturation inhibitors
239
Table 4. Effect of ovarian asterosaponin A on I-methyladenine production in Astertas follicle cells u
Fig. 2. Schematic representation of mechanism of oocyte maturation in the starfish, Asterias amurensis.
Effect of the ovarian ASA fraction on production of i-MA in isolated follicle cells Follicle cells were collected in ASW and suspended in either ASW or ASW containing the ovarian ASA fraction (750 pg/ml) for 1 h at 20°C (cell number, IO’ cells/ml). The incubation media were separated from follicle cells and assayed with isolated oocytes freed of follicle cells. As demonstrated in table 4, the ovarian ASA fraction inhibited production of l-MA in follicle cells. When isolated follicle cells were incubated in the presence of GSS (140 pg dried nerve/ml), the production of l-MA was enhanced (tables 4, 5). The addition of the ovarian ASA fraction (750 pug/ml) in the medium inhibited GSS-induced production of I-MA in nearly the same proportion as in the preceding experiment (tables 4, 5). These results show that the applied GSS enhanced the production of l-MA in the isolated follicle cells in the presence of the ovarian ASA fraction. DISCUSSION The results of the present study show that the ovarian butanol extract from Asterias amurensis inhibits “spontaneous” maturation in Asterias oocytes surrounded by follicle cells. Functionally active substances are localized outside the oocytes in the ovary. The site of action of the ovarian
Expt no.
ASW control
AsteroInhibition saponin .4 (%)
I 2 3 4 Mean
200 50 8.1 8.6
11” 11 3.1 4.1
48 79 62 52 6Ok6.9’
o The follicle cell number: lo7 cells/ml; concentration of ovarian asterosaponin A: 750 pg/ml; incubation for 1 hat20”C. * ng I-methyladenine equivalent per nl of suoematant. c Mean4S.E.
butanol extract is not the oocyte itself but the follicle cells. The ovarian butanol extract containing asterosaponins suppresses the “spontaneous” production of I-MA in the follicle cells until GSS is released from nerve cells and acts on follicle cells to induce production of I-MA. I-MA released from follicle cells acts directly on the oocyte to induce maturation [S]. The role of the substances participating in oocyte
Table 5. Effect of ovarian asterosaporain A. on gonad-stimulating substance (GSSjinduced production of l-methyladenine in Asterias follicle ceils n Expt no.
GSS control
Asterosaponin A-GSS
inhibition %
lb 2 3 4 Mean
2gc 55 9.0 9.0
14’ 15 4.1 5.4
50 73 55 40 54ir7.F”
u The foliicle cell number: lo7 cellslmi; ovarian asterosaponin A cont.: 750 pgiml: GSS .conc.: 140 ,~g dried nerve/ml: incubation for 1 h at 20°C. Ir The experiment number corresponds to that of table 4, i.e., in the same experiment number in rabies 4 and 5, follicle cells taken from the same ovary were used. c ng I-methyladenine equivalent per ml of stpematant. d MeantS.E.
238
Ikegami, Kamiya and Shirai
in ASW containing ovarian ASW and ASB at a high concentration for 1 h, followed by washing with ASW several times, they were arrested at the germinal vesicle stage Saponins in Stage of oocyte for more than 1 h. The addition of l-MA Class ovary development induced maturation in these oocytes. Echinoidea Absent Haploid Therefore, it is conceivable that the content Asteroidea Asterosaponins Germinal vesicle of saponins is high in the ovary ofAsterina stage Holothuroidea Holothurins Germinal vesicle and the washing of Asterina oocytes with stage ASW is unable to remove sufficient Crinoidea Present Germinal vesicle stage amounts of the saponins from the follicle cells. In fact, the content of saponins in Asterina ovary was five times higher than maturation in Asterias is schematically that found in Asterias ovary [15]. The demonstrated in fig. 2. physiological role of saponins in Asterina Yasumoto et al. [16] demonstrated that ovary in situ, however, remains to be clariASA and ASB are distributed in every part fied. The ubiquitous occurrence of saponins of Asterias amurensis in high concentrations during summer (non-breeding season), has been known in asteroids and holowhereas they are found only in gonads dur- thuroids [16]. ASA and ASB have been ing winter (breading season). Later, Ka- chemically studied extensively [9, 10, 12miya [17] found that aglycones in ASA and 14, 18, 191and shown to be complex steroid ASB obtained at the non-breeding season sulfate glycosides (fig. 1). On the other were highly heterogenous and Sa-pregn- hand, holothurian saponins are composed 9( 1l)-ene-3P,6a-diol-20-one (asterosapo- of highly oxygenated lanostanes and carbogenine I [9], 5a-cholesta-9( 11),20-diene-3/3,- hydrates, as was shown in fig. 1 [20]. The 6a, [ 181, Sa-cholest-9( 1l)-ene-3/?,6a,23& content of holothurian saponins, holotrio1 [ 191 and 5cu-cholesta-9(11),24-diene- thurins A and B, in ovaries of Holothuria 3&6a-diol-23-one [ 191 comprise the major leucospilota is exceptionally high compared aglycones in these ASA and ASB, whereas with other tissues at the breeding season ovarian ASA and ASB contain asterosapo- [21]. Saponins were not detected when a genine I as the major aglycone. However, sea lily was examined during the non-breedto date evidence is not available to show ing season [16]. On the other hand, sapowhether asterosapogenine I-3-sulfate-6- nins were found in the pinnule containing glycosides is responsible for the oocyte- fully grown ovaries, when they were colmaturation inhibitory activity of the ovarian lected just before oocyte maturation and extract, since further purification of the spawning [ 151. ASA and ASB fractions into homogenous Oocytes in sea urchin ovaries undergo compounds has not been achieved. maturation division long before spawning Contrary to the case of Asterias, “spon- occurs, contrary to oocytes in starfishes, taneous” maturation is rarely observed in sea cucumbers and sea lilies. Saponin-like oocytes of the starfish,Asterina pectinifera. activities were not found at all in extracts It is noteworthy that whenAsterias oocytes of echinoids [ 161. Therefore, the presence surrounded by follicle cells were suspended and absence of saponins in ovaries in Table 6. Correlation
of occurrence of saponins and stage of oocyte development in fully grown ovary in Echinodermata
Exp CeilRes 103 (1976)
Starfish maturation inhitx2or.s Echinodermata coincide with the stage of meiosis reached by the oocytes in fully grown ovaries (table 6). Whether holothurins and crinoid saponins act to arrest oocyte maturation in sea cucumbers and sea lilies, respectively, remains to be examined. Investigation is in progress to isolate the maturation inhibitors in a homogenous state from Asterias ovary and determine the structure of molecules which are concerned with the suppression of l-MA production in follicle cells. We thank Professors S. Tamura and H. Kanatani for encouragement and valuable advice and Dr S. S. Koide for reading the manuscript. Thanks are also due to staff members of Misaki Marine Biological Station of University of Tokyo in supplying the starfish used in the present study.
REFERENCES 1. 2. 3. 4.
Dalcq, A, Arch biol 34 (1924) 507. Heilbrunn, L V, Chaet, A B, Dunn, A & Wilson, W L, Biol bull 106 (1954) 158. Kanatani, H & Ohguri, M, Zoo1 mag 79 (1970) 58. Ikegami, S & Tamura, S, Experientia 29 (1973) 325.
239
5. Kanatani, H, Shirai, H, Nakanishi, K h Kurokawa, T, Nature 221 (1969) 273. 6. Hirai. S. Chida. K & Kanatani. H. Develooment. growth and differentiation I5 (1973) 21. 7. Shirai. H. Exo cell res 87 (1974) 3 1. 8. Cloud; J ‘& Schuetz, A We,Exp cell res 79 (1973) 446. 9. Ikegami, S, Kamiya, Y & Tamura. S. Agric biol them 36 (1972) 1777. 10. - Tetrahedron 29 (1973) 1807. 11. Kanatani, H. Ikegami, S, Shirai, H, Oide, H & Tamura, S, Development, growth and differentiation 13 (1971) I51. 12. Ikegami, S, Kamiya, Y & Tamura, S. Agric biol them 36 (1972) 2005. 13. Ikegami, S, Hirose, Y, Kamiya, Y & Tamura, S, Agric biol them 36 (1972) 2453. 14. Yasumoto, T & Hashimoto, Y, Agric biol them 3 1 (1967) 368. 15. Ikegami, S & Kamiya, Y. Unpublished data. 16. Yasumoto, T, Tanaka, M & Hashimoto. Y, Btl! Jap sot sci fish 32 (1966) 673. 17. Kamiya, Y, Ph D dissertation. Universitv oi‘ Tokyo (1975): 18. Sheikh. Y M. Tursch. B & Dierassi. C. 3 Am them soc94il972)3278.
-
19. Ikegami. S, Kamiya, Y & Tamura. S. Agric bioi chem37(1973)367. 20. Kitagawa, I, Sugawara, T & Yoshioka. 1. Tetrahedron letters (1974) 4111. 21. Matsuno, T & Ishida. T, Experientia 25 (1969) 1261. D^^ Received June 14, 1976 Accepted July 7, 1976
Exp Cell Res 103 ( 1976)
of Agricultural Japan.
ACTION
INHlBITORS
S. IKEGAMI.’ ‘Department
AND
IN STARFISH
Y. KAMlYA’* Chemistry.
Nakano-ku.
and
University
and ‘Wc-etrn Research
OF MEIOTIC
Insrilure, Tokyo
OVARY
H. SHIKAI”
of‘ Tokyo.
Bunkyo-ku.
tinii~ersir~
of ?okp.
l’ok~o
113.
164, Jupan
SUMMAKY Mechanical release of oocytes from the ovary of the starfish Asreria.\ omurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the mn:uration of Asferias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapabic ot inhibiting oocyte maturation. The ovarian extract inhibited the productlon of I-methyladenine (I-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence IMA-induced maturation of the mcyte with or without follicle cells. It can be concluded from the present results that the role of tge ovarian extract containing steroid glycosides is to arrest “spontaneous” production of I-MA in follicle cells. The suppression can he overcome by the action of a gonadotropic peptide hormone released from the nerve tiszuc.
In many starfish species, fully grown oocytes undergo “spontaneous” meiotic maturation when they are isolated manually in normal sea water [I]. The occurrence of substances which inhibit “spontaneous” oocyte maturation has been known in the starfish ovary [2-4]. Heilbrunn et al. [2] reported that the maturation inhibitors were heparin-like substances on the basis of several chemical experiments carried out on crude ovarian extracts. Later, lkegami & Tamura [4] in a preliminary report concluded that the maturation inhibitors were steroid glycosides rather than heparin-like substances based on their partition coefficient between n-butanol and water and by gel filtration experiments. * Present Research.
address: Institute of Physical and Chemical Wako City, Saitama 35 I, Japan.
On the other hand. the maturation of oocytes in the starfish ovary is induced by I-methyladenine ( I -MA) which is produced in follicle cells under the influence of a peptide designated as gonad-stimulating substance (GSS) released from the nerve tissue [5, 61. In calcium-free sea water (CaFSW) the follicle cells are detached from oocytes and the germinal vesicle remains intact in the oocyte [6,7]. When oocytes denuded of follicle cells in CaFSW are subsequently incubated with isolated follicle cells in normai sea water, they undergo germinai vesicie breakdown [6-8]. Thus, it was suggested that “spontaneous” oocyte maturation i3 caused by the production of I-MA in the absence of GSS in follicle cells Mhen they are removed from the inhibitory intraovarian environment [7]. It is not uniike!y.
234
Ikegami,
Kamiya and Shirai
Table 1. Purification steps and specific activity of ovarian extract from Asterias amurensis Preparation
Weight
Spec. act.
Lyophilized ova@’ Acetone powder 20 % Acetone extract 80 % Acetone extract Butanol extract
1 5256 1 400 423 98 3.2
30’ 10 0.25
a Obtained from 240 females. b Weight in (g). c Dose (mg/ml) required for 50% inhibition of “spontaneous” maturation in Asterias oocytes surrounded by follicle cells.
Biological
assays
The maturation-inhibitory action of the extract obtained at each purification step was assayed with isolated oocytes. Oocytes were tom from an ovarian fragment with forceps and immediately transferred into a small Petri dish containing ASW with a measured amount of the test sample. Oocytes prepared in this manner were surrounded by follicle cells. The number of immature oocytes still possessing intact germinal vesicle was counted 1 h after initiation of the treatment. The control specimens consisted of oocytes kept in CaFSW, whereby the germinal vesicle remained intact and in ASW where the germinal vesicle disintegrated. The methods for obtainine isolated follicle cells bv treatment with CaFSW havelbeen described elsewhere [7]. The amount of I-MA in test solutions was bioassayed with isolated oocytes freed from follicle cells according to the method of Shirai [7].
Preparations
of ovaries and oocytes
therefore, that the maturation inhibitors act for extraction Ovaries were excised in ASW, washed three times on the follicle cell to block “spontaneous” with ASW and then lyophilized. The maturation inproduction of l-MA. However, the site of hibitors were extracted from the lvoohilized material. action of the matureuion inhibitors remains Oocytes were dejellied and denuded of follicle . . cells in the following way. Ovaries were washed three trmes to be established. with ASW, rinsed and then induced to spawn by addiIn the present report, chemical properties tion of isotonic KCl. Intensive discharge of oocytes immediately after the treatment. Oocytes of the maturation inhibitors obtained from resulted were filtered through cheese cloth and were dejellied the ovary ofAsterias amurensis and the site by treatment with ASW (pH 5.0) in the usual way. After thorough washing with ASW. the dejellied of their action have been described. oocytes were lyophihzed. Prevaration
MATERIALS
AND METHODS
The starhsh,Asterias amurensis, for the present study were collected at Tokyo Bay and kept in a laboratory aquarium supplied with circulating sea water.
Chemicals The sea water used was Vant’ Hoffs artificial sea water (ASW) with the following composition: 2.7% NaCl, 0.07% KCI, 0.1% CaCI,, 0.34% MgCl, and 0.21% MgSO+ The ASW was adjusted to pH 8.0-8.3 with borate buffer (final concentration. 0.05 M) unless otherwise stated. CaFSW was prepared by omitting CaCI, in the ASW formula. Water and organic solvents were redistilled in ah-glass apparatus. I-MA was purchased from Sigma Chemical Co. Authentic asterosaponins A (ASA) and B (ASB) were prepared from Asferias amurensis collected during the nonbreading season (summer) according to the method described elsewhere [9, IO]. GSS used in the present studv was oreoared from lvoohilized radial nerves of As&as arhr>nsis and it -c&responds to the active fraction purified by gel filtration on the first Sephadex G-SOcolumn [l I]. E.rp Cell Res 103 (1976)
of the butanol extract
An acetone powder prepared from the lyophilized ovaries or oocytes was extracted with 20% aqueous acetone. To the extract anhydrous acetone was added to make an 80% acetone solution. The mixture was left standing overnight in the cold and filtered to remove the precipitates. The filtrate was concentrated in vacua and the aqueous residue was adjusted to pH 8.0 with dilute sodium carbonate solution. The aqueous solution was extracted with an equal volume ofn-butanol. The aqueous layer was separated, acidified to DH 3.6 with dilute sulfuric acid and re-extracted with an equal volume of n-butanol. The butanol layer was seoarated and concentrated in vacua to drvness. The residue was dissolved in distilled water and dialvsed overnight against distilled water. The retentate was lyophilized to yield an amorphous powder, which was designated as butanol extract.
Chromatography Thin layer chromatography (tic) of the butanol extract was carried out by use of silica gel HF*,, plates of 0.38 mm thickness and a solvent system. chloroformmethanol-acetic acid-water (60 : 20 : 10: 4, v/v). In some experiments. the plates were sprayed with
0 &OSE 1 D-GALACTOSE
D-XYLOSED-G:UCrJSE-
C-QUINOk'OSE-ED-l?LOjE ?-O-METHYL-D-GLUCOSE
(D-OUINOVOSE)
A
B
Fig. 1. Structures of ovarian asterosaponins ‘4 (A) and
I3 (B), and a Stichop~s japonicus saponin (C). R= CH,CO, CH$Z(OH)CH2COCH2CH(CH3)2, CH&=
CHCOCH,CH(CH,),, CH&HCH,COCH,CH(CM,;,, CH,CHCH,COCH=C(CH,),, CH3CHCHeCHOHCHzCH(CH&, etc.
effective purification was not accomplished by the gel filtration. Therefore, further separation of the butanol extract was carried out by preparative tic. Bioassay of the methanol extract from the developed plates revealed that the active fractions corresponded to the phosphotungustic acidreactive zones at RI values 0.1 and 0.05. Chemical analysis of the active fractions showed that the fractions at Rf values 0. I and 0.05 contain steroid glycosides, ASA and ASB, respectively (fig. 1). On the assumption that the butane: extract is distributed evenly in the ovary, the RESULTS concentration of the maturation inhibitors is fsofatiovl and chemical characterization around 2.1 mglg of dry ovary; this amount of the maturation inhibitors in is sufficient to inhibit the “spontaneous” the ovary oocyte maturation in vitro (tables 112). A summary of the inhibitory activity of the The ASA fraction consists of I moie of steroid sulfate and 2 moles each of a-fucose ovarian extract on “spontaneous” oocyte maturation at each purification step is pre- and o-quinovose (6deoxy-n-glucose) [12], sented in table 1. The butanol extract was while the ASB fraction contains 1 mole each applied onto a Sephadex G-50 column ac- of steroid sulfate: o-xyiose, n-galactose and cording to the procedure described in previ- n-fucose, and 2 moles of o-quinovose [l2], ous reports [4, 121. The elution profile ASA and ASB were originally defined as showed a characteristic dispersion of the glycosides containing a steroid with the [ 141.However7 biological activity. This suggests that as- molecular formula C22H3-203 sociation of the active substances occurs in our later studies [9] have shown that such various degrees in aqueous media and that steroids are undetectable in the hydrolysate phosphotungstic acid reagent [12] to make the presence of steroid glycosides visible. Each active fraction on the plates was extracted with methanol and applied onto a column (1.4X 12 cm) of DEAE-cellulose (acetate form). The column was washed with chloroform-methanol (2 : 1, v/v) and then developed with chloroform-methanol (2 : 1, v/v) containing 5% 0.5 M LiCl (fraction size, 10 ml; flow rate, 50 ml/h). The fractions containing sugar residues which were identified with phenol-sulfuric acid reagent [ 13, 141were combined and partitioned with an equal volume of water. The upper layer was separated and evaporated in vacua to yield a powder. Since Li+ ions induced germinal vesicle breakdown in starfish oocytes Ll.51, the powder was dissolved in distilled water and dialysed against ASW to remove Li’ ions from the active acidic materials. Then, the retentate was bioassayed.
236
Ikegami, Kamiya and Shirai
Table 2. Effect of purified materials from Asterias on “spontaneous” maturation in Asterias oocyte
Effect of the ovarian butanoi extract on l-MA-induced oocyte maturation The effect of the ovarian butanol extract on Preparation Spec. act. l-MA-induced oocyte maturation was next Butanol extract from ovary 0.25” examined. Asterosaponin A fraction from ovary 0.28 (1) Isolated oocytes with follicle cells Asterosaponin B fraction from ovary 0.20 Butanol extract from oocyte >l.OO were placed in ASW containing the ovarian butanol extract at a concentration of 850 a Dose (mglml) required for 50% inhibition of “spontaneous” maturation in Aslerias oocytes surrounded pg/ml. The extract suppressed effectively by follicle cells. the “spontaneous” maturation of the oocyte (8 % maturation). The addition of l-MA at a final concentration of 5~ lo-’ M induced of the glycosides obtained from the same 100% oocyte maturation in the presence of starfish. Therefore, we propose to call the ovarian butanol extract. steroid glycosides which are present in (2) Oocytes were treated with CaFSW to starfishes asterosaponins, regardless of the remove follicle cells and were transferred chemical structure of steroids constituting directly from CaFSW to ASW containing the glycosides. These steroid glycosides are the ovarian butanol extract (750 pg/ml) tonot homogeneous [9] and the aglycones constituting these saponins are closely re- gether with l-MA at various concentralated steroids with a characteristic ring tions. Germinal vesicle breakdown was obstructure of a 3P,6a-diol system [9]. Fur- served after 1 h. As demonstrated in table 3, ther separations of each fraction into homo- the ovarian butanol extract at 750 pg/ml did genous compounds were hampered since not inhibit the l-MA-induced oocyte mamethods to achieve separation of the close- turation. The present results suggest that the ly related steroid glycosides on the basis of ovarian butanol extract is not toxic to the molecular difference in aglycones are not available. Therefore, it is not yet known oocyte even at a high concentration and which molecules are responsible for the that oocyte maturation is induced by the maturation inhibitory activity in the steroid application of l-MA, irrespective of the presence or absence of the ovarian saglycoside fractions. ponins .
Localization of the maturation Table 3. Effect of ovarian asterosaponin A inhibitors in the ovary on I-methyladenine-induced maturation in In order to determine the site of production Asterias oocytes without follicle cells of the maturation inhibitors in the ovary, % Oocyte maturation butanol extract was prepared from the isolated oocytes. The specific biological ac- 1-Methyladenine Asterosaponin ASW control (750 pg/ml) tivity of the butanol extract from oocytes cont. (M) were markedly lower than that of the 1.0x 10-S 13.3+ 3.3” 14.02 3.0’ ovarian ASA and ASB (table 2). Therefore, 4.0x 10-g 40.5+ 14.4 43.5k13.7 99.5+ 0.5 100.0~ 0 the maturation inhibitors are localized out- 1.6x lo-* side the oocytes in the ovary. a Values are mean&SE. Exp Cell Res 103 (1976)
Starfish maturation inhibitors
239
Table 4. Effect of ovarian asterosaponin A on I-methyladenine production in Astertas follicle cells u
Fig. 2. Schematic representation of mechanism of oocyte maturation in the starfish, Asterias amurensis.
Effect of the ovarian ASA fraction on production of i-MA in isolated follicle cells Follicle cells were collected in ASW and suspended in either ASW or ASW containing the ovarian ASA fraction (750 pg/ml) for 1 h at 20°C (cell number, IO’ cells/ml). The incubation media were separated from follicle cells and assayed with isolated oocytes freed of follicle cells. As demonstrated in table 4, the ovarian ASA fraction inhibited production of l-MA in follicle cells. When isolated follicle cells were incubated in the presence of GSS (140 pg dried nerve/ml), the production of l-MA was enhanced (tables 4, 5). The addition of the ovarian ASA fraction (750 pug/ml) in the medium inhibited GSS-induced production of I-MA in nearly the same proportion as in the preceding experiment (tables 4, 5). These results show that the applied GSS enhanced the production of l-MA in the isolated follicle cells in the presence of the ovarian ASA fraction. DISCUSSION The results of the present study show that the ovarian butanol extract from Asterias amurensis inhibits “spontaneous” maturation in Asterias oocytes surrounded by follicle cells. Functionally active substances are localized outside the oocytes in the ovary. The site of action of the ovarian
Expt no.
ASW control
AsteroInhibition saponin .4 (%)
I 2 3 4 Mean
200 50 8.1 8.6
11” 11 3.1 4.1
48 79 62 52 6Ok6.9’
o The follicle cell number: lo7 cells/ml; concentration of ovarian asterosaponin A: 750 pg/ml; incubation for 1 hat20”C. * ng I-methyladenine equivalent per nl of suoematant. c Mean4S.E.
butanol extract is not the oocyte itself but the follicle cells. The ovarian butanol extract containing asterosaponins suppresses the “spontaneous” production of I-MA in the follicle cells until GSS is released from nerve cells and acts on follicle cells to induce production of I-MA. I-MA released from follicle cells acts directly on the oocyte to induce maturation [S]. The role of the substances participating in oocyte
Table 5. Effect of ovarian asterosaporain A. on gonad-stimulating substance (GSSjinduced production of l-methyladenine in Asterias follicle ceils n Expt no.
GSS control
Asterosaponin A-GSS
inhibition %
lb 2 3 4 Mean
2gc 55 9.0 9.0
14’ 15 4.1 5.4
50 73 55 40 54ir7.F”
u The foliicle cell number: lo7 cellslmi; ovarian asterosaponin A cont.: 750 pgiml: GSS .conc.: 140 ,~g dried nerve/ml: incubation for 1 h at 20°C. Ir The experiment number corresponds to that of table 4, i.e., in the same experiment number in rabies 4 and 5, follicle cells taken from the same ovary were used. c ng I-methyladenine equivalent per ml of stpematant. d MeantS.E.
238
Ikegami, Kamiya and Shirai
in ASW containing ovarian ASW and ASB at a high concentration for 1 h, followed by washing with ASW several times, they were arrested at the germinal vesicle stage Saponins in Stage of oocyte for more than 1 h. The addition of l-MA Class ovary development induced maturation in these oocytes. Echinoidea Absent Haploid Therefore, it is conceivable that the content Asteroidea Asterosaponins Germinal vesicle of saponins is high in the ovary ofAsterina stage Holothuroidea Holothurins Germinal vesicle and the washing of Asterina oocytes with stage ASW is unable to remove sufficient Crinoidea Present Germinal vesicle stage amounts of the saponins from the follicle cells. In fact, the content of saponins in Asterina ovary was five times higher than maturation in Asterias is schematically that found in Asterias ovary [15]. The demonstrated in fig. 2. physiological role of saponins in Asterina Yasumoto et al. [16] demonstrated that ovary in situ, however, remains to be clariASA and ASB are distributed in every part fied. The ubiquitous occurrence of saponins of Asterias amurensis in high concentrations during summer (non-breeding season), has been known in asteroids and holowhereas they are found only in gonads dur- thuroids [16]. ASA and ASB have been ing winter (breading season). Later, Ka- chemically studied extensively [9, 10, 12miya [17] found that aglycones in ASA and 14, 18, 191and shown to be complex steroid ASB obtained at the non-breeding season sulfate glycosides (fig. 1). On the other were highly heterogenous and Sa-pregn- hand, holothurian saponins are composed 9( 1l)-ene-3P,6a-diol-20-one (asterosapo- of highly oxygenated lanostanes and carbogenine I [9], 5a-cholesta-9( 11),20-diene-3/3,- hydrates, as was shown in fig. 1 [20]. The 6a, [ 181, Sa-cholest-9( 1l)-ene-3/?,6a,23& content of holothurian saponins, holotrio1 [ 191 and 5cu-cholesta-9(11),24-diene- thurins A and B, in ovaries of Holothuria 3&6a-diol-23-one [ 191 comprise the major leucospilota is exceptionally high compared aglycones in these ASA and ASB, whereas with other tissues at the breeding season ovarian ASA and ASB contain asterosapo- [21]. Saponins were not detected when a genine I as the major aglycone. However, sea lily was examined during the non-breedto date evidence is not available to show ing season [16]. On the other hand, sapowhether asterosapogenine I-3-sulfate-6- nins were found in the pinnule containing glycosides is responsible for the oocyte- fully grown ovaries, when they were colmaturation inhibitory activity of the ovarian lected just before oocyte maturation and extract, since further purification of the spawning [ 151. ASA and ASB fractions into homogenous Oocytes in sea urchin ovaries undergo compounds has not been achieved. maturation division long before spawning Contrary to the case of Asterias, “spon- occurs, contrary to oocytes in starfishes, taneous” maturation is rarely observed in sea cucumbers and sea lilies. Saponin-like oocytes of the starfish,Asterina pectinifera. activities were not found at all in extracts It is noteworthy that whenAsterias oocytes of echinoids [ 161. Therefore, the presence surrounded by follicle cells were suspended and absence of saponins in ovaries in Table 6. Correlation
of occurrence of saponins and stage of oocyte development in fully grown ovary in Echinodermata
Exp CeilRes 103 (1976)
Starfish maturation inhitx2or.s Echinodermata coincide with the stage of meiosis reached by the oocytes in fully grown ovaries (table 6). Whether holothurins and crinoid saponins act to arrest oocyte maturation in sea cucumbers and sea lilies, respectively, remains to be examined. Investigation is in progress to isolate the maturation inhibitors in a homogenous state from Asterias ovary and determine the structure of molecules which are concerned with the suppression of l-MA production in follicle cells. We thank Professors S. Tamura and H. Kanatani for encouragement and valuable advice and Dr S. S. Koide for reading the manuscript. Thanks are also due to staff members of Misaki Marine Biological Station of University of Tokyo in supplying the starfish used in the present study.
REFERENCES 1. 2. 3. 4.
Dalcq, A, Arch biol 34 (1924) 507. Heilbrunn, L V, Chaet, A B, Dunn, A & Wilson, W L, Biol bull 106 (1954) 158. Kanatani, H & Ohguri, M, Zoo1 mag 79 (1970) 58. Ikegami, S & Tamura, S, Experientia 29 (1973) 325.
239
5. Kanatani, H, Shirai, H, Nakanishi, K h Kurokawa, T, Nature 221 (1969) 273. 6. Hirai. S. Chida. K & Kanatani. H. Develooment. growth and differentiation I5 (1973) 21. 7. Shirai. H. Exo cell res 87 (1974) 3 1. 8. Cloud; J ‘& Schuetz, A We,Exp cell res 79 (1973) 446. 9. Ikegami, S, Kamiya, Y & Tamura. S. Agric biol them 36 (1972) 1777. 10. - Tetrahedron 29 (1973) 1807. 11. Kanatani, H. Ikegami, S, Shirai, H, Oide, H & Tamura, S, Development, growth and differentiation 13 (1971) I51. 12. Ikegami, S, Kamiya, Y & Tamura, S. Agric biol them 36 (1972) 2005. 13. Ikegami, S, Hirose, Y, Kamiya, Y & Tamura, S, Agric biol them 36 (1972) 2453. 14. Yasumoto, T & Hashimoto, Y, Agric biol them 3 1 (1967) 368. 15. Ikegami, S & Kamiya, Y. Unpublished data. 16. Yasumoto, T, Tanaka, M & Hashimoto. Y, Btl! Jap sot sci fish 32 (1966) 673. 17. Kamiya, Y, Ph D dissertation. Universitv oi‘ Tokyo (1975): 18. Sheikh. Y M. Tursch. B & Dierassi. C. 3 Am them soc94il972)3278.
-
19. Ikegami. S, Kamiya, Y & Tamura. S. Agric bioi chem37(1973)367. 20. Kitagawa, I, Sugawara, T & Yoshioka. 1. Tetrahedron letters (1974) 4111. 21. Matsuno, T & Ishida. T, Experientia 25 (1969) 1261. D^^ Received June 14, 1976 Accepted July 7, 1976
Exp Cell Res 103 ( 1976)