Starfish oocyte maturation and reduction of disulfide-bond on oocyte surface

STARFISH

OOCYTE

MATURATION

DISULFIDE-BOND T. KISHIMOTO, Laboratory

of Physiology,

MARILYN

AND REDUCTION

ON OOCYTE

OF

SURFACE

L. CAYER,’ and H. KANATANI

Ocean Research Institute,

University

of Tokyo, Nakano,

Tokyo 164, Japan

SUMMARY Disultide-reducing agents such as DTT and 2,3-dimercapto-I-propanol (J3AL) are known to induce starfish oocyte maturation [16]. However, microinjection of DTT into the immature oocytes of the starfish, Asterina pectimfera, failed to induce oocyte maturation, suggesting that the substance acts on the oocyte surface from the outside and indirectly induces oocyte maturation. Microinjection of the cytoplasm taken from the DTT-treated oocytes into immature oocytes was found to induce oocyte maturation, indicating that the cytoplasmic factor triggering the germinal vesicle breakdown and subsequent meiotic process is produced under the influence of DTT, as well as I-methyladenine (I-MeAde). The pattern of changes m maturation-inducing capacity of the maturing cytoplasm of the DTT-treated oocytes was similar to that of the I-MeAde-treated oocytes. Morphological changes in the oocyte surface and the vitelline coat of the DTT-treated oocytes were similar to those in the I-MeAde-treated oocytes. The action of DTT in inducing maturation is considered to be the same as that of I-MeAde. Sulfhydryl content of oocyte-cortex protein was found to increase after the treatment with I-MeAde, suggesting that the action of I-MeAde in inducing oocyte maturation is to be ascribed to its action of reducing disulfide bond.

Maturation of starfish oocytes is induced in the ovaries by a hormonal peptide, gonad-stimulating substance (GSS), released from the nervous tissue [l-4]. GSS acts on the follicle cells around the oocytes [5, 63 to produce 1-methyladenine (lMeAde), a natural trigger of germinal vesicle breakdown [7-111. The site of action of I-MeAde is the oocyte surface [12, 131, where it renders the oocyte fertilizable [ 141 and also produces and releases a factor into the cytoplasm which induces germinal vesicle breakdown (GVBD) and the meiotic divisions [ 151.On the other hand, disulfidereducing agents such as dithiothreitol

(DTT) and 2,3-dimercapto-1-propanol (BAL) have recently been found to induce maturation of Asterina pectini’era oocytes in vitro just as 1-MeAde does [ 161. Some experiments concerning the reduction of disultide bond were conducted in the present study in an attempt to obtain a clue to elucidate the role of disulfide-reduction in inducing starfish oocyte maturation. MATERIALS

AND METHODS

The materials used were the starfishes, Asterina pectinifera and Asterias amurensis. The animals were collected during their breeding season, and kept in laboratory aquaria supplied with circulating cold sea water at the Ocean Research Institute, University of Tokyo. Asterina pectinifera was used for microinjection experiments and electron microscopic observations. The ’ Present address: Papanicolaou Cancer Research In- oocytes of this species generally do not undergo sponstitute at Miami, 1155NW 14th str., Miami, FL 33123, taneous maturation when isolated in sea water. Ovaries were isolated and washed in artificial sea water, after USA.

Exp Cd Res 101 (1976)

SuljAydryl groups and oocyte maturation which they were torn with forceps to release the oocytes. The isolated oocytes were washed thoroughly with sea water. Asterias amurensis was used for the determination of sulthydryl content in oocyte cortices.

Reagents The sea water used was modified Van? Hoffs artificial sea water (ASW) [17] and calcium-free sea water (CaFSWJ r171. I-MeAde (Sinma Chemical Co.) was dissolved m-deionized and distilled water (DDW) at a concentration of 10m3M, and kept in a deepfreezer as a stock solution. Dithiothreitol (DTT) (Nakarai Chemicals. Kvoto) was dissolved at the time of use in DDW at ‘concentrations of 0.1 M, 1.0 M and 10 M. and in ASW at lo-* M. 5.5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and glutathione (reduced type) (GSH) were purchased from Wako Pure Chemical Industries.

Microinjection Microinjection was performed according to the method of Hiramoto [ 181.The volume of the microinjected materials was calculated by assuming the part of the micropipette where the meniscus moved to be the frustum of a cone [18]. In the microinjection experiments, recipient oocytes were placed in 80% ASW, since they are more tolerant to a large amount-injection in 80 % ASW than in normal ASW.

Electron microscopic observation Samples were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.8) containing 8.6% sucrose at 0,5, 15,30 and 60 min after treatment with DTT, postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.8) containing 8.6% sucrose, dehydrated in ethanol and embedded in Epon 812. They were sectioned with a Sorvall MT2 or LKB ultramicrotome and examined with JEM 7 or Philips 200 electron microscope.

Isolation of oocyte cortices Oocytes of Asterias amurensis usually undergo spontaneous maturation when isolated in sea water. However, in CaFSW they fail to mature. For obtaining isolated immature Asterias oocytes, ovaries were, therefore, isolated and washed several times with cold CaFSW for 1.5 h. These ovaries were cut into small fragments in cold CaFSW and soawnina was induced by-adding 0.7 M CaCI, and 1.d M KC‘i to make the final concentration of Ca2+ eauivalent to that in ASW and of K+, three times higher-than that of ASW. After filtrating with a sheet of gauze to remove ovarian fragments, isolated oocytes were collected by low speed centrifugation (1300 rpm, 1 min) and washed four times with CaFSW. Cortices of immature or I-MeAde-treated oocytes were isolated according to the method which Sakai [19] used with sea urchin eggs. That is, isolated oocytes were gently subjected to about 25 strokes of a hand-homogenizer in cold 0.1 M MgC&. To avoid excessive homogenization, the suspension was frequent-

105

ly examined under a microscope. The isolated cortices were sedimented by centrifugation at 1700 rpm for 3 min and washed 3 times with cold 0.1 M MgC12.

Determination of SH content in cortex-protein Isolated cortices were denatured by HCI-acetone according to the method of Sakai [19, 201, in which the amount of SH groups was determined on the basis of the weight of protein. DTNB, Ellman’s reagent, was used to measure the amount of SH groups[21]. DTNB was dissolved at lo-* M in 0.02 M Tris-HC] buffer (pH 7.0). This solution was filtered to remove insoluble materials and diluted 5 times with 0.1 M Tris-HCI buffer (pH 8.0) at the time of use. The general procedure for the determination of SH groups was as follows. Isolated and washed cortices susnended in 2.0 ml of DDW were denatured by adding 8.0 ml of acetone containing 0.1 N HCI in a glass-stoppered centrifuge tube andieft standing for 4% min. The precipitate of denatured cortices was collected by brief centrifugation (1300 rpm, 1 min) and washed with 100% acetone. Sometimes it was necessary to grind the precipitate into powder in a glass centrifuge tube. The precipitate was washed with a solution containing 0.05 M TrisHCl buffer (pH 8.0) and 50% acetone. After brief centrifugation, the precipitate of denatured cortices was suspended in a -glass-stoppered centrifuge tube containing 1.2 ml of 0.1 M Tris-HCl buffer (pH 8.0) mixed with 1.5 ml of acetone, and 0.3 ml of the DTNB solution described above was added immediatelv and mixed vigorously (final amounts: 3.0 ml of 0105 M Tris-HCI buffer (nH 8.0) and 50% acetone). The reaction mixture was- incubated at room temperature for 40 min with occasional stirring and then centrifuged at 3 500 rpm for 10 min at 15°C whereafter the optical density of 2-nitro-5-thiobenzoate anion in the supernatantwas measured at 412 nm, indicating the amount of SH groups. Measurement of the optical density was repeated more than three times for each sample until a fixed value was obtained. As the standard of SH amount, the optical density was measured on GSH which was dissolved in Tris-HC1 buffer-acetone and reacted with DTNB solution in the same manner as with the experimental solution. The amount of protein was determined according to Lowry et al. [22] with the precipitate of the reaction mixture, the supernatant of which was used for SH determination. The precipitate, with 0.3 g of urea added, was dissolved in 0.5 ml of 1.0 N NaOH and then diluted with DDW to a concentration of 0.1 N NaOH. Protein determination was performed three times for each sample, and the mean value was used as the protein amount.

RESULTS Effect of injected DTT on oocyte maturation When applied externally to isolated oocytes, DTT is effective at concentrations Exp Cell Res JOJ (1976)

106

Kishimoto, Gayer and Kanutuni

Table I. Eflect of injected DTT on the breakdorcw of the germinal r’esiclr in Asterinu pectinifera oocytr -

Injected DTT Cont. W)

Vol. (PI) IO

3.3x

10-d

5

1.0x lo-:' 3.3x 10-Z 6.6X 10-S 1.0x 10-Z 1.6x 1O-2 2.5x IO-' 3.3x 10-Z 6.6x 10-Z I .0x 10-l 3.3x 10-l

5

1.0

IO 20 30 50

75 100 20 30

110

around lop2 M in inducing oocyte maturation (not effective at concentrations lower than 10m3M or higher than 10-l M) [16]. DTT dissolved in DDW at 0.1 M, 1.O M or 10 M was microinjected into isolated immature oocytes to give final concentrations of 3.3~ 10e4to 3.3X 10-l M in each oocyte. The injected DTT solution was observed to disperse readily into the oocyte cytoplasm. The final concentration of injected DTT within the oocytes was calculated from the amount of injected material divided by the oocyte volume (about 3000 pl). The data presented in table 1 clearly show that the injected DTT always failed to induce oocyte maturation. The germinal vesicle of the recipient oocytes remained intact when observed even after 2 h. These recipient oocytes invariably underwent maturation when they were subsequently transferred into 80% ASW containing 10d6 M 1-MeAde. Furthermore, oocytes taken from the same ovaries invariably underwent maturation when treated externally with 10mzM DTT. These results indicate that the site where DTT acts to induce oocyte maturation is the surface of the oocyte. Exp Cd

Res

No. of examples

30

0. I

10.0

Final cont. in oocyte (M)

101 (1976)

8

5 : 5 5 6 6 6

Breakdown of germinal vesicle 0 0 0 0 0 0 0 0 0 0 0

Microinjection into immature oocytes of cytoplasm taken from DTT-treated oocytes Since DTT injected directly into immature oocytes failed to induce maturation even at a dose which could induce maturation when applied externally, DTT was considered to act on the oocyte surface to produce a factor in the cytoplasm which induces GVBD and meiotic divisions. The cytoplasm taken from DTT-treated oocytes undergoing maturation was therefore microinjected into immature oocytes. Donor oocytes were treated with lo+ M DTT for 30 min and washed with ASW. About 220 pl (about l/15-1/20 of the oocyte volume) of cytoplasm was removed from the donor oocytes at 20 min intervals after the commencement of DTT treatment and injected into immature oocytes having intact germinal vesicles. In oocytes taken from the same ovary and treated with DTT in the same way, GVBD occurred within 2545 min, and the first and second polar bodies were discharged around 80 min and 120 min, respectively, after DTT administration.

Suljhydryl groups and oocyte maturation

Fig. 1. Abscissa: time after commencement of DTT treatment (min); ordinate: oocyte maturation (%). (O), Number of injected oocytes. Changes in the maturation-inducing capacity of the cytoplasm of the DTT-treated oocyte during the course of maturation in Asterina pectinifera.

107

electron microscopy. Oocytes from a single female were used for each experiment. The isolated oocytes of Asterina pectinifera were washed in ASW and then placed in ASW containing lo-* M DTT. The percent maturation, as measured by GVEID, was 99 % after 30 min incubation in DTT. Electron microscopy revealed the following: the surfaces of the control oocytes (isolated and incubated in ASW) are thrown up into irregular microvilli. The vitelline coat is visible as a rather homogeneous amorphous material surrounding and covering the microvilli (fig. 2a). After 5 min of DTT treatment, the vitelline coat appears slightly condensed and somewhat flocculent. The microvilli and subsurface organelles show no significant changes (fig. 2b). After 15 min of DTT treatment, the vitelline coat has condensed further into a coarsely granular layer surrounding the microvilli (fig. 2~). At 30 or 60 min, the germinal vesicles have broken down and the oocytes appear to be maturing. The vitelline coat is a thinner, coarsely granular layer on the oocyte surface. The microvilli appear to have retracted and the oocyte surface is smoother (fig. 2d).

As shown in fig. 1, cytoplasm taken from DTT-treated oocytes 40 min after DTT administration invariably brought about GVBD when injected into immature oocytes. In this case, GVBD occurred within 2wO min after microinjection, and breakdown of the follicular envelope was also observed at the same time. It was found that the GVBD-inducing capacity of the cytoplasm of DTT-treated oocytes decreased after more than 60 min subsequent to treatment: 69% at 60 min, and 31% at 80 min. Cytoplasm taken from the 120 min oocytes had no capacity to induce GVBD when injected into immature oocytes, although these recipient oocytes underwent matura- Changes in SH content of oocytetion normally when they were subsequently cortex protein during the course of transferred to 80 % ASW containing lo-” M I-MeAde-induced maturation I-MeAde. GVBD was never observed when DTT is well known as a powerful reducer the cytoplasm of untreated immature oo- of disultide bonds [23]. The foregoing seccytes was injected as control. tion of this study demonstrated that this reagent acts on the oocyte surface in causElectron microscopic observations ing maturation. Also, 1-MeAde, a natural on DTT-treated oocytes inducer of starfish oocyte maturation, acts Since the results of the experiments of the on the oocyte surface [12, 131. Then, the foregoing section showed that DTT acts on next experiment was conducted to deterthe oocyte surface to produce and release mine whether the reduction of protein-dia factor into the cytoplasm which in turn sulfide bonds in the cortical region of the triggers GVBD, the effect of DTT on the oocyte occurs in the process of I-MeAdeoocyte surface was studied by means of induced maturation. EXPCell Res 101 (1976)

108

Kishimoto, Cayer and Kanatani

Fig. 2. Electron micrographs of the surface ofAsferinu pecrinifera oocyte treated with DTT. (a) Control oocyte isolated in ASW. The vitelline coat is visible as a homogeneous layer surrounding and covering the microvilli; (b) oocyte after 5 min DTT treatment. The vitelline coat takes on a slightly condensed, flocculent appearance; (c) oocyte after I5 min DTT treatment.

The vitelline coat still surrounds and covers the microvilli but is coarsely granular in appearance; (4 oocyte after 60 min DTT treatment. The oocyte surface is smoother with fewer and shorter microvilli. The vitelline coat is granular in appearance and thinner. VC, vitelline coat; MV, microvilli. x23 000.

A suspension of isolated immature oocytes of Asterias amurensis was divided into four equal lots, each of which contained about 2 ml of oocytes precipitated after brief centrifugation (1300 t-pm, 1 min). Three of these lots were treated with 1O-5 M 1-MeAde, and the fourth was used for control as immature oocytes. At 10 min intervals after I-MeAde administration, one lot of oocytes was briefly homogenized and the cortices were isolated. The amount of protein-SH of these cortices was determined for each sample.

The sulfhydryl content of oocyte-cortex protein was found to have increased about 9% at 10 min after I-MeAde administration, and then decreased (fig. 3). These oocytes underwent GVBD within 15-25 min after commencement of the 1-MeAde treatment. In this study, the SH value was determined on oocyte-cortex material that was in a thoroughly denatured state. The amount of protein-SH in the cortex increased and reached a maximum before GVBD occurred. Furthermore, a chronological correlation was found to exist be-

Exp Cd RPS 101 (1976)

Sulfhydryl

groups and oocyte maturation

109

change appears to cause the cytoplasmic maturation at the oocyte surface which l&Orenders the oocyte fertilizable [ 141. 15.0. DTT mimics the action of 1-MeAde in ( p < 0.01 ) several ways. When it is applied to isolated 14.0 oocytes, their follicular envelopes are strip13.0 ped off and form small clumps in about 30 Imin. The oocyte surface undergoes changes 1 similar to those caused by 1-MeAde. The 1 0 io j0 i0 microvilli retract and the surface becomes Fig. 3. Abscissa: time after transfer into CaFSW con- flatter. The vitelline coat also becomes taining 10m5M I-MeAde (min); ordinate: nmole SH/mg thinner although the change in texture of protein. Changes in SH content of cortex protein during l- the vitelline coat is much more pronounced MeAde-induced oocyte maturation in Asterias amu- with DTT treatment than with exposure to rensis. Each point shows mean +S.E. of 6 experi1-MeAde. Some components of the coat are ments. probably lost or irreversibly altered by the treatment, since a normal fertilization memtween the maximal increase in SH content brane cannot be formed after DTT treatand the commencement of GVBD; that is, ment. Partial destruction of the vitelline the shorter the time was to reach the maxi- coat in sea urchins by DTT treatment has mum level of SH content, the earlier GVBD been reported [24, 251. Brief treatment with DTT (3 min) also causes oocyte maturation occurred. and renders the oocyte fertilizable; on insemination, the matured eggs form tight DISCUSSION fertilization membranes and develop norStarfish oocyte maturation normally occurs mally to bipinnaria larvae [ 161. The microunder the influence of 1-MeAde produced injection experiments in the present study by the follicle cells surrounding the oocyte. demonstrate that DTT, like 1-MeAde, acts This substance induces the breakdown of on the oocyte surface from the outside to the follicular envelope, the follicle cells produce the cytoplasmic factor which informing a small clump by about 20 min after duces GVBD. In addition, the changes in exposure to 1-MeAde [ 111. Morphological the maturation-inducing capacity of the changes also occur on the oocyte surface. cytoplasmic factor produced under the inThe vitelline coat becomes flat and thin fluence of DTT are quite similar to those demonstrated in the case of 1-MeAde treatand the number and length of the microvilli protruding into the coat decreases [14]. ment [ 151.The cytoplasmic factor produced GVBD is indirectly induced by 1-MeAde. under the influence of DTT is considered When microinjected into immature oocytes, to be identical to that produced by l-MeI-MeAde does not cause GVBD, but when Ade. The fact that the treatment with 1-MeAde applied to the oocyte it activates the surface and induces the production of another increases the amount of protein-SH in the substance(s), the cytoplasmic factor, which oocyte cortices before GVBD indicates that acts on the germinal vesicle, leading to the the reduction of disultide-bond in this recompletion of meiosis [12, 151. The surface gion is involved in the early step of oocyte 17D-

(pc0001)

(PC051

Exp Cell Res 101 (1976)

110

Kishimoto, Cuyer and Kanatani

maturation which is required for the subsequent events of this phenomena, suggesting the possibility that the effect of IMeAde in inducing starfish oocyte maturation is to be ascribed to a disulfide-reducing action, although the mechanism underlying such activity is still unknown. We thank Dr H. Sakai for valuable discussion, Dr Y. Hiramoto for guidance in the microinjection technique and Dr J. C. Dan for reading the manuscrid. This investigation was supported in-part by grants-m-aid from the Ministrv of Education and the Ford Foundation to H. K., and by a fellowship to M. L.C. during her stay at the Ocean Research Institute, University of Tokyo, from the Japan Society for the Promotion of Science.

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Exp CeNRes IO1 (1976)

7. Kanatani, H & Shirai, H, Nature 216 (1967) 284. 8. Schuetz, A W & Biggers, J D. Exp cell res 46 ( 1967)624. 9. Kanatani, H, Shirai, H. Nakanishi. K & Kurokawa, T, Nature 221 (1969) 273. IO. Kanatani, H, Exp cell res 57 (l%9) 333. Il. - Int rev cytol35 (1973) 253. 12. Kanatani, H-& Hiramoto, Y, Exp cell res 61 ( 1970) 280. 13. Doree, M & Guerrier. P, Exp cell res 96 (197.5) 2%. 14. Hirai, S, Kubota, J & Kanatani, H. Exp cell res 68 (1971) 137. 15. Kishimoto. T & Kanatani. H. Nature 260 (1976) _ 321. ’ 16. - Exp cell res 82 (1973) 2%. 17. Shirai, H, Development, growth and differentiation 15 (1973) 307. 18. Hiramoto, Y, Exp cell res 87 (1974) 403. 19. Sakai, H, J biophys biochem cytol8 (1960) 603. 20. - Anal biochem 26 (1%8) 269. 21. Ellman, Cl L, Arch biochem biophys 82 (1959) 70. 22. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 23. Cleland, W W, Biochemistry 4 (1964) 480. 24. Epel, D, Weaver, A M & Mazia, D, Exp cell res 61 (1970) 64. 25. Vacquier, V D, Tegner, M J & Epel, D, Nature 240 (1972) 352. Received March 2, 1976 Accepted March 12, 1976