Molecular and Cellular Endocrinology, 9 (1977) 91-100 0 Elsevier/North-Holland Scientific Publishers, Ltd.
PRIMARY ACTION OF STEROID HORMONE AT THE SURFACE OF AMPHIBIAN OOCYTE IN THE INDUCTION OF GERMINAL VESICLE BREAKDOWN
K. ISHIKAWA *, Y. HANAOKA, Y. KONDO and K. IMAI Institute of Endocrinology, Received
Gunma University, Maebashi, Gunma 371, Japan
10 May 1977; accepted
12 July
1977
An insoluble steroid derivative was prepared by the coupling of desoxycorticosterone with aminoethylated agarose beads. When the naked full-grown Xenopus oocytes were incubated in contact with the steroid-bound agarose beads, the dissociation of oocyte nuclei or the initial step in the maturation of amphibian oocyte was induced in 30-100% of total oocytes. The induction did not occur in the oocytes covered with follicle cells and when the naked oocytes were placed apart from the steroid-bound agarose beads in the medium. The above findings confirmed that the oocyte surface was a primary site for this particular steroid action, eliminating the possibility of participation of free steroid artifactually released from the agarose. The 105,OOOg supernatant fraction of oocytes showed no sign of the presence of steroid receptor. This was not inconsistent with the assumption mentioned above. Keywords:
oocyte
surface;
germinal
vesicle
breakdown;
desoxycorticosterone;
insol-
uble steroid; amphibia; Xenopus Iaevis.
It has been generally accepted as a mechanism of steroid hormone action that the steroid is taken up by the target cell and bound to the cytoplasmic receptor protein which carries the hormone to the nucleus (Jensen and Desombre, 1973; O’Malley and Means, 1974). However, biological studies on the oocyte maturation processes in an amphibian, Rana pipiens, have suggested that another mechanism of steroid action works for this particular event. As is generally known, in amphibian oocytes the disappearance of the large nuclear architecture called germinal vesicle breakdown (GVBD) is the first sign of oocyte maturation which follows meiosis and ovulation. Smith and Ecker (1971) and Masui and Markert (1971) found that progesterone induced GVBD in vitro only when the steroid was introduced into a reaction medium, while progesterone injected directly into the oocytes failed to induce GVBD. They have suggested that the steroid hormone acts primarily on the surface of the oocyte. On the other hand, Schorderet-Slatkine * Present Shizuoka
address: Biological 422, Japan.
Institute,
Faculty
91
of Science,
Shizuoka
University,
Oya
836,
92
K. Ishikawa
et al.
(1972) found that the injection of hydrocortisone induced GVBD of Xe?roplls Zuevis. In addition, Ozon and Belle (1973) reported that labeled progesterone could be incorporated into Xelzopus oocyte and bound to melanosomes. They have suggested that Xenopus oocyte maturation, in contrast with Rana oocyte, is induced by the intracellular action of the steroid. However, Baltus et al. (1973) reported that the steroid-bound melanosomes were not effective in the induction of GVBD when the activated melanosomes were injected into untreated oocytes. Although most findings to data favor the hypothetical action of the steroid hormone at the surface of the frog oocyte, no direct evidence has been provided. The present study provides direct evidence for the steroid hormone action at the oocyte surface, using the technique in which an insoluble steroid derivative is used as an inducer for GVBD.
MATERIALS
AND METHODS
Chemicals
[ 1 ,2,6,7(n)-3H]Progesterone (84 Ci/mmol) which was more than 97% pure was obtained from New England Nuclear Corp., Sepharose 4B from Pharmacia (Uppsala, Sweden), ovine luteinizing hormone from the National Institutes of Health (NIH-LH-S 13). All other reagents were reagent grade. Animals atzd tissues
Amphibian ovaries were obtained from sexually mature female Xe?zopus laevis bred and fed at 20°C in our laboratory until use. Female Rhode Island Red chicks obtained from a commercial source were given daily subcutaneous injections of 5 mg diethylstilbestrol in 0.5 ml sesame oil for 6 days beginning at day 12 of life, in order to obtain a stimulated and enlarged oviduct. Removal of follicle cells
Frog ovarian fragments were immersed in double Steinberg’s solution (Masui, 1967) containing 10 mM dithiothreitol for l-2 h at 2O”C, in order to dissolve the vitelline membrane existing between the follicle cells and the oocyte surface. After this treatment the follicle cells were easily removed manually with fine watchmaker’s forceps under a dissecting microscope. The naked oocyte lost responsiveness to gonadotropin, as shown in table 1, column (2). Therefore, the degree of response to gonadotropin of the treated oocytes may serve as a measure of incompleteness of the removal of follicle cells. Determination
of the rate of GVBD
The naked oocytes or the ovarian fragments (in the form of oocytes covered with follicle cells) were incubated with or without the agents to be tested in small vials containing 4 ml of double Steinberg’s solution for 16 h at 20°C. After incuba-
Progesterolle actiorzat the surface of amphibian oocyte
93
tion the oocytes were fixed by boiling for 10 min and dissected under a dissecting microscope to see whether the germinal vesicles had been broken down or not (Masui, 1967). The rate of GVBD was expressed as a percentage of the number of oocytes in which GVBD occurred, taking the total number of oocytes as 100%. Incubation
of oocytes with insoluble steroid
The naked oocytes were incubated with 50-500 mg of the insoluble steroid desoxycorticosterone derivative bound to agarose in the same condition as described above. The agarose beads stayed at the bottom of the vials and the oocytes were buried in the accumulation of beads. In some control experiments, in order to avoid contact between the oocytes and the insoluble steroid, the oocytes were put in a nylon net basket which was placed in the medium at a distance of several millir’neters above the accumulation of agarose beads. Coupling of desoxycorticosterone
to agarose
Preparation of desoxycorticosterone bound to agarose was performed by a slight modification of the method of Porath et al. (1973), using Sepharose 4B as agarose beads. Descxycorticosterone was succinylated by the procedure described by Erlanger et al. (1967) and purified by recrystallization. Aminoethylated Sepharose (AE-agarose) was prepared by the cyanogen bromide procedure and coupled with desoxycorticosterone hemisuccinate with the aid of ‘water-soluble carbodiimide’. The coupling reaction proceeded for 16 h at room temperature and pH 4.8 with constant and gentle shaking. The substituted agarose (about 5 g wet weight) was washed, while packed in a column, extending over 2 days with 5 liters of 80% methanol in Steinberg’s solution. Following this, it was suspended using a shaker in chloroform-Steinberg’s solution (1 : 1, v/v), and then washed repeatedly (2 days) with 5 liters of total volume after removal of the chloroform phase. Finally, the derivative was washed (2 days) with 5 liters of Steinberg’s solution. The washed product (DOC-agarose) contained about 2 ymol of desoxycorticosterone per gram of packed wet DOC-agarose. Preparation of 105,OOOg supernatant fractions
The naked oocytes (270 oocytes) were homogenized manually with 1 ml of 0.01 M Tris-HCl containing 15 mM EDTA (pH 7.4) in a Potter-Elvehjem teflon homogenizer. In contrast to the case of somatic cells, not only the oocyte’cell membrane but also the oocyte nuclear membrane was broken by this treatment, because of the large size of the nuclei. The homogenate was centrifuged at SSOg and the pellet was removed. The supernatant was then centrifuged at 105,OOOg for 60 min at 1°C. This supernatant fraction, containing nucleoplasmic proteins in addition to cytoplasmic ones, was used to study the binding of progesterone. Chicks were sacrificed and the oviducts were removed and thoroughly rinsed with saline. Two oviducts weighing 1.8 g were homogenized with 5 ml of the medium in a metal blade homogenizer (Polytron, Kinematica GmbH, Lucerne, Switzerland). The ,lOS,OOOg super-
94
K. Ishikawa et al.
natant fraction of oviducts has no reason for the significant contamination with nucleoplasmic proteins and is denoted as the ‘cytosol fraction’. The subsequent procedures were the same as that for frog oocytes. Analysis of receptor in the 105,OOOg supernatant fraction 0.2 ml of the supernatant containing 4 mg of protein was incubated overnight at 2°C with 7 ng of [ 1 ,2,6,7(n)-3H]progesterone. After the incubation, free steroid molecules were removed by gel filtration of the incubation mixture using Sephadex G-25 (4 ml column). 0.2 ml of excluded fraction was applied to 4.6 ml of S-20% sucrose density gradient in 0.01 M Tris-HCl buffer containing 15 mM EDTA (pH 7.4) in the presence of 0.3 M KC1 and centrifuged for 16 h at 1°C at 45,000 rev/min in a Hitachi RPS 65T rotor. 37 fractions were collected by piercing the bottom of the centrifuge tube. Bovine serum albumin (BSA) was spun down under the same conditions as a reference standard. Radioactivity counting Radioactivity was counted with an Aloka liquid scintillation spectrometer with 20-40% efficiency, using a mixture containing 5.5 g of 2,5-diphenyloxazole and 100 mg of p-bis-[2-(5-phenyloxazoyl)] benzene in 30% toluene solution of a detergent, Nissan Nonion NS-2 10. Protein assay Protein was analyzed by the method of UV absorption at 280 nm.
of Lowry et al. (1951) or by measurement
RESULTS Induction of germinal vesicle breakdown of the oocytes by DOCugarose When oocytes were incubated with DOC-agarose the rate of GVBD increased strikingly, reaching almost 100% in some cases. As shown in table 1, the DOCagarose was effective as low as 50 mg/4 ml, corresponding to about lo-’ M steroid. On the other hand, the rate of GVBD could be influenced by the physiological state of the oocyte and therefore varied considerably from experiment to experiment. This was reflected also in the value for untreated oocytes, as shown in column (1) of the table. Nevertheless, the treated oocytes always showed definitely higher values than those of untreated ones. When AE-agarose was substituted for DOCagarose the rate of GVBD stayed within the control range (O-12%) (column (5)). This result indicated that the activity of DOC-agarose was due to the presence of DOC; in other words, DOC was active in producing GVBD of oocytes even in its insoluble form. The rate of GVBD induced by DOC-agarose, however, was lower in most cases than that induced by free DOC, as shown in column (4). This may be explained by the fact that only a small amount of the bound steroid was able to make contact
Progesterone
action at the surface of amphibian oocyte
95
with the oocyte surface under the experimental conditions. In contrast with the above findings, when the oocytes were covered with the follicle cells, DOC-agarose failed to induce GVBD (O-S%) (column (3)), possibly because of the protection of the oocyte surface by the follicle cells. This suggested that the attachment of DOC molecules to the surface of the oocytes was necessary in inducing GVBD, although the incorporation of the steroid was not inevitable. Table 1 also shows the results of other control experiments. When the activity of insoluble steroid is to be determined, we must be careful regarding the possible contamination of DOC-agarose preparation with unreacted steroid molecules which are highly potent. In order to check this possibility, the final wash of DOC-agarose preparation was used as the incubation medium. As shown in column (6), the final wash did not have any appreciable activity in inducing oocyte maturation. The results indicated that the washing of DOC-agarose was sufficient to eliminate uncoupled DOC-hemisuccinate. However, even if the washing was complete, there was also a possibility that the steroid molecules were dissociated from the conjugate during incubation. To check this, the reaction medium was reused after removing the incubated oocytes, for the incubation of the fresh naked oocytes. As shown in column (7), the used medium caused scarcely any induction of GVBD (O-14%), indicating very little or no dissociation of active steroid molecules from the conjugate during incubation. As already mentioned, DOC-agarose hardly reacted with oocytes covered with follicle cells. This fact also provided evidence for the stability of the bound steroid, because if the bound steroid molecules were once liberated, the free DOC molecules should penetrate into the follicle cells and induce GVBD (cf. column (4)). Column (8) shows the results of the experiment in which oocytes were separated from DOC-agarose with a nylon net basket placed in a double Steinberg’s solution and incubated. This artificial barrier disturbed the contact with DOC-agarose. The rates of GVBD in the incubation, 5-13%, were much lower than that for DOCagarose in normal conditions. Absence of receptor activity in the 105,OOOgsupernatantfraction of the frog oocyte If the primary site of steroid action in inducing oocyte maturation exists on the cell surface, we may assume the absence of an intracellular receptor system in the frog oocyte. In relation to this, it had been reported that the enucleated amphibian oocyte responded to progesterone treatment as did the nucleated oocyte (Smith et al., 1968), and that the pigment granules in the cytoplasm could bind a steroid hormone (Ozon and Belle, 1973) but was not confirmed to be an active receptor for inducing oocyte maturation (Baltus et al., 1973). Therefore, the nuclei and the insoluble fraction of cytoplasm could be excluded from the subjects of preliminary searching for the intracellular receptor with any role for the induction of GVBD. Based on this consideration, the 105,OOOg supernatant fraction containing cytoplasmic as well as nucleoplasmic soluble proteins was analyzed in terms of its binding characteristics to progesterone. The 105,OOOg supernatant fraction was incubated
96
K. Ishikawa etal.
Table 1 Effect of DOC-agarose an the germinal ______ ~.~_~._. . - .--. _. ---. Experiment
vesicle breakdown
(“0) of oocytes
-..
Follicle
Germinal
RliS
_.--
(1) None
_---
vesicle breakdown
~-(%I (number
(2)
(3)
Gonadotropin a
DOC-
of oocytcs)
agarose
--~ A
+
l(72) O(40)
33176) 31401
R
-t
O(65)
26(58)
7(441 [500 mg] 29(42) [400 mg]
O(37)[300mgl
14(50)
-
30(47)
7(45)
O(44)
[ 300 mgl
Z45)
c
f -
0(47) O(B)
40(15) 4(24)
_
50(22)1400mg] 46(13) 1300mg] 44(25)
D
+
-
W6) O(33)
40(25) 9(35)
8(38)[200mgl 59(34)[ZOOmg] 37(35) 3X34)
+
X39)
0(33)
83(36) 16(32)
ll(95)
-
16(51)
-
[ 150 mg]
[ 100 mg]
lOO(32)[400mg] 97(32)[300mg] 84(32)
i-
[2OO mg]
[ZOO mg]
_ 97(37) lOO(39) 92136)
[loo [50
mgl
msl [SOmgl
In experiments
A to F, ovarian fragments in the form of oocytes covered with follicle cells, or naked oocytes (indicated by + or -) were incubated in small vials containing 4 ml of double Steinberg’s solution for 16 h at 2O”C, with or without the agents to be tested. Each row represents a separate experiment conducted with different frogs on different days. Numbers in parentheses are the total number of oocytes examined. Numbers in brackets are the wet weight of
DOC-agaroseOI AE-agarose added to 4 ml of the reaction medium.
with [3H] progesterone as described in Materials and methods. After removal of the unreacted steroid, an aliquot of the labeled fraction was analyzed by sucrose density gradient centrifugation. The same amount of labeled protein of oviduct cytosol fraction obtained from estrogen-stimulated chicks was
Progesterone
(4) DOC (lo@
action at the surface of amphibian oocyte
(3 AE-agarose
(6) Final wash
(7) Medium used
(8) DOC-agarose with barrier
-
-
-
_ 5(38)
[SO0 mg]
-
14(38)
[300 mg]
M) b
43(61) 95(42)
O(43) [500 mg]
o(42)
57(46)
2(61)
1300 mg]
O(54)
75(44)
0(46)
[ 300 mg]
0(46)
43(21) 44(25)
O(13) 1400 mg] 6(16) [400 mg]
76(29) 94(35)
86(29) lOO(32)
-
003 OW)
O(15) 6(17) 5(19)
4(28) 4(28) 3(29)
O(34) [400 mg]
15(34) lS(34)
_
_
3(30) 3(33)
[ 200 mg]
12(34)
-
_
X36)
98(40)
_
91
17(35) lO(39) 8(39)
15(26) ll(27) 14(37) 9(32) 9(23)
a) In experiment A, ovine luteinizing hormone (200 Mg), and in experiments B-F, Xenopus laevis pituitary (one-half pituitary equivalent/vial) were used. b, In experiments A-C and E, desoxycorticosterone, and in experiments D and F, progesterone were used. Abbreviations: DOCagarose, desoxycorticosterone coupled to aminoethylated Sepharose; DOC, desoxycorticosterone; AEagarose, aminoethylated Sepharose.
subjected to the analysis as a reference control. Fig. 1 shows the distribution of radioactivity in the intracellular soluble proteins. No radioactivity peak was found in the pattern for Xenopus oocyte fraction, but in the case of chick oviduct a sharp peak was shown around a protein peak. The result showed that the oocyte superna-
98
K. Ishikawa et al.
b
8 BSA 4
0
20
40
0
20
40
Fraction number Fig. 1. Sucrose density gradient centrifugation of [ 3H]progesterone-binding components in the 105,OOOg supernatant fraction of frog oocytes (a) and oviducts of diethylstilbestrol-treated chicks (b). The labeled fraction was subjected to centrifugation at 45,000 rev/min in a Hitachi RPS 65T rotor for 16 h at 1°C in a 5-20s sucrose density gradient containing 0.3 M KCl. 37 fractions were collected by piercing the bottom of the centrifuge tube. The radioactivity and UV absorption at 280 nm of each fraction were measured and plotted. The arrow indicates the location of bovine serum albumin (BSA) sedimenting under the same conditions as a reference standard.
tant fraction had no receptor for progesterone, or at least that the content of steroid receptor per unit amount of soluble protein in the oocyte was much lower than that in the oviduct cytosol protein.
DISCUSSION Although many steroids such as progesterone, androgen and some corticoids induce the GVBD of oocytes in Xenopus laevis, progesterone and desoxycorticosterone are most effective in producing both GVBD and ovulation (Hanaoka et al., 1975). Thorton and Evennett (1969) have reported that the injection of human chorionic gonadotropin causes the ovarian follicle cells to release a progesteronelike hormone about 12 h before subsequent ovulation. This suggests that the follicle cells secrete progesterone which causes the maturation of oocytes. In the present study, desoxycorticosterone (DOC) instead of progesterone was used to obtain an insoluble steroid, since DOC was also active in inducing GVBD, at least in vitro, and the hydroxyl group at 21-C was ready for coupling reaction with agarose. In addition, the structure of the conjugated steroid was rather similar to progesterone, except that the methyl group at 21 -C of progesterone was replaced by aminoethylated agarose (AE-agarose). This might suggest that the conjugated desoxycorti-
Progesterone action at the surface of amphibian oocyte
99
costerone is closer to progesterone than to DOC in terms of its structural characteristics, and that the conjugated desoxycorticosterone might behave as progesterone for the oocyte stimulation. This is a reason for the use of progesterone to search the intracellular receptor which may participate in the steroid action to induce oocyte maturation, The agarose beads, ranging between 50 and 190 pm in diameter (Pharmacia Fine Chemicals leaflet, Pharmacia Fine Chemicals, Inc., Uppsala, Sweden), were much smaller than the oocytes (1.2-l .3 mm) used in the experiments. This led us to examine the possibility of pinocytotic penetration of the hormone into the oocyte even in its insoluble form. In fact, pinocytotic activity of frog oocyte has been found in young oocytes (Wallace et al., 1973). However, pinocytotic vesicles found in the young oocytes were not as large as the agarose beads, and full-grown oocytes which were ready for GVBD were found to have scarcely any pinocytotic activity. Therefore, the action of the insoluble steroid does not seem to be an intracellular action caused after pinocytotic incorporation. The incubation of progesterone with the 105,OOOg supernatant fraction of the oocytes did not produce an appreciable amount of bound steroid. The supernatant fraction contained the nucleoplasmic proteins in addition to the cytoplasmic ones. Therefore, it may be concluded that the oocyte has no soluble protein which reacts to progesterone. Although the insoluble constituents of the nuclei such as chromatin fraction might have some receptor activity for the steroid even if such receptor exists, it should not participate in the steroid induction of the maturation. As mentioned in previous sections, the steroids react with the enucleated oocytes to produce some cytoplasmic factor which in turn induces GVBD when it is injected into the nucleated oocyte. Concerning the insoluble fraction of the cytoplasm, it has recently been shown that progesterone binding sites exist in the pigment granules fraction (melanosomes) of Pleurodes waltlii oocytes (Ozon and Belle, 1973) and Xenopus Zaevis oocytes (Iacobelli et al., 1974; Belle et al., 1975). These binding sites were found to have high affinity and limited binding capacity for progesterone. Baltus et al. (1973), however, reported more recently that the binding of progesterone to the melanosomes was not sufficient to induce maturation. They injected normal melanosomes or progesterone-treated melanosomes into Xenopus oocytes. Both the normal and the treated melanosomes had no effect at all on GVBD. Summarizing, it seems likely that there is no intracellular receptor mechanism for preparing the steroid induction of GVBD in the full-grown oocytes of Xenopus.
All the findings mentioned above are consistent with the conclusion that the oocyte surface is the location of the primary reaction of the steroid hormone in the induction of GVBD. The only exception is a report that the injection of hydrocortisone causes GVBD (Schorderet-Slatkine, 1972). This might be due to the technical difficulty of avoiding the leaking of hormone when it was injected into the oocytes in vitro. If the steroid hormone acts at the surface of the oocyte, a second messenger sys-
100
K. Ishikawa et al.
tern may transport the hormonal information to the germinal vesicle. Although cyclic AMP is well known as a second messenger in peptide hormonal action (Sutherland et al., 1965) cyclic AMP or dibutyryl cyclic AMP failed to induce GVBD in Xenopus oocytes (Schorderet-Slatkine, 1972; Brachet et al., 1974). To search the second messenger system, it should be noted that in amphibia the cytoplasm of progesterone-treated oocytes could induce GVBD when the cytoplasm was injected into untreated oocytes (Smith and Ecker, 1971; Masui and Markert, 1971; Schorderet-Slatkine and Drury, 1973). Wasserman and Masui (1976) reported the characterization of this maturation-promoting factor. In relation to the above findings on amphibian oocytes, it is interesting to note that 1-methyladenine, which could induce GVBD in starfish oocytes, has been felt to act also at the surface of the oocyte (Kanatani and Hiramoto, 1970).
ACKNOWLEDGEMENT This investigation tion of Japan.
was supported
in part by a grant from the Ministry of Educa-
REFERENCES Balms, E., Brachet, J., HanocqQuertier, .J. and Hubert, E. (1973) Differentiation 1, 127-143. Belle. R., Schorderet-Slatkine, S., Drury, K.C. and Ozon, R. (1975) Gen. Comp. Endocrinol. 25,339-345. Brachet, J., Baltus, E., De Schutter, A., Hanocq, F., HanocqQuertier, J., Hubert, E., lacobelli, S. and Steinert, G. (1974) Mol. Cell. Biochem. 3, 189-205. Erlanger, B.F., Beiser, S.M., Borek, F., Edel, F. and Lieberman, S. (1967) In: Methods in Immunology and Immunochemistry, Vol. 1, Eds.: C.A. Williams and M.W. Chase (Academic Press, New York) p. 148. Hanaoka, Y., Ishikawa, K., Kondo, Y. and lmai, K. (1975) Unpublished data. lacobelli, S., Hanocq, J., Baltus, E. and Brachet, J. (1974) Differentiation 2,129-135. Jensen, E.V. and Desombre, E.R. (1973) Science 182,126-134. Kanatani, H. and Hiramoto, Y. (1970) Exp. Cell Res. 61,280-284. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J.Biol.Chem. 193,255276. Masui, Y. (1967) J. Exp. Zool. 166, 365-376. Masui, Y. and Markert, C.L. (1971) J. Exp. Zool. 177, 1299146. O’Malley, B.W. and Means, A.R. (1974) Science 183,610-620. Ozon, R. and Belle, R. (1973) Biochim. Biophys. Acta 320,588-593. Porath, J., Aspberg, K., Drevin, H. and Axen, R. (1973) J. Chromatogr. 86,53-56. Schorderet-Slatkine, S. (1972) Cell Differ. 1,179-189. Schorderet-Slatkine, S. and Drury, K.C. (1973) Cell Differ. 2,247-254. Smith, L.D. and Ecker, R.E. (1971) Dev. Biol. 25,232-247. Smith, L.D., Ecker, R.E. and Subtelny, S. (1968) Dev. Biol. 17,627-643. Sutherland, E.W., Oye, I. and Butcher, K.W. (1965) Recent Prog. Horm. Res. 21,623-642. Thorton, V.F. and Evennett, P.J. (1969) Gen. Comp. Endocrinol. 13,268-274. Wallace, R.A., Jared, D.W., Dumont, J.N. and Sega, M.W. (1973) J. Exp. Zool. 184,321-333. Wasserman, W.J. and Masui, Y. (1976) Science 191,1266-1268.
PRIMARY ACTION OF STEROID HORMONE AT THE SURFACE OF AMPHIBIAN OOCYTE IN THE INDUCTION OF GERMINAL VESICLE BREAKDOWN
K. ISHIKAWA *, Y. HANAOKA, Y. KONDO and K. IMAI Institute of Endocrinology, Received
Gunma University, Maebashi, Gunma 371, Japan
10 May 1977; accepted
12 July
1977
An insoluble steroid derivative was prepared by the coupling of desoxycorticosterone with aminoethylated agarose beads. When the naked full-grown Xenopus oocytes were incubated in contact with the steroid-bound agarose beads, the dissociation of oocyte nuclei or the initial step in the maturation of amphibian oocyte was induced in 30-100% of total oocytes. The induction did not occur in the oocytes covered with follicle cells and when the naked oocytes were placed apart from the steroid-bound agarose beads in the medium. The above findings confirmed that the oocyte surface was a primary site for this particular steroid action, eliminating the possibility of participation of free steroid artifactually released from the agarose. The 105,OOOg supernatant fraction of oocytes showed no sign of the presence of steroid receptor. This was not inconsistent with the assumption mentioned above. Keywords:
oocyte
surface;
germinal
vesicle
breakdown;
desoxycorticosterone;
insol-
uble steroid; amphibia; Xenopus Iaevis.
It has been generally accepted as a mechanism of steroid hormone action that the steroid is taken up by the target cell and bound to the cytoplasmic receptor protein which carries the hormone to the nucleus (Jensen and Desombre, 1973; O’Malley and Means, 1974). However, biological studies on the oocyte maturation processes in an amphibian, Rana pipiens, have suggested that another mechanism of steroid action works for this particular event. As is generally known, in amphibian oocytes the disappearance of the large nuclear architecture called germinal vesicle breakdown (GVBD) is the first sign of oocyte maturation which follows meiosis and ovulation. Smith and Ecker (1971) and Masui and Markert (1971) found that progesterone induced GVBD in vitro only when the steroid was introduced into a reaction medium, while progesterone injected directly into the oocytes failed to induce GVBD. They have suggested that the steroid hormone acts primarily on the surface of the oocyte. On the other hand, Schorderet-Slatkine * Present Shizuoka
address: Biological 422, Japan.
Institute,
Faculty
91
of Science,
Shizuoka
University,
Oya
836,
92
K. Ishikawa
et al.
(1972) found that the injection of hydrocortisone induced GVBD of Xe?roplls Zuevis. In addition, Ozon and Belle (1973) reported that labeled progesterone could be incorporated into Xelzopus oocyte and bound to melanosomes. They have suggested that Xenopus oocyte maturation, in contrast with Rana oocyte, is induced by the intracellular action of the steroid. However, Baltus et al. (1973) reported that the steroid-bound melanosomes were not effective in the induction of GVBD when the activated melanosomes were injected into untreated oocytes. Although most findings to data favor the hypothetical action of the steroid hormone at the surface of the frog oocyte, no direct evidence has been provided. The present study provides direct evidence for the steroid hormone action at the oocyte surface, using the technique in which an insoluble steroid derivative is used as an inducer for GVBD.
MATERIALS
AND METHODS
Chemicals
[ 1 ,2,6,7(n)-3H]Progesterone (84 Ci/mmol) which was more than 97% pure was obtained from New England Nuclear Corp., Sepharose 4B from Pharmacia (Uppsala, Sweden), ovine luteinizing hormone from the National Institutes of Health (NIH-LH-S 13). All other reagents were reagent grade. Animals atzd tissues
Amphibian ovaries were obtained from sexually mature female Xe?zopus laevis bred and fed at 20°C in our laboratory until use. Female Rhode Island Red chicks obtained from a commercial source were given daily subcutaneous injections of 5 mg diethylstilbestrol in 0.5 ml sesame oil for 6 days beginning at day 12 of life, in order to obtain a stimulated and enlarged oviduct. Removal of follicle cells
Frog ovarian fragments were immersed in double Steinberg’s solution (Masui, 1967) containing 10 mM dithiothreitol for l-2 h at 2O”C, in order to dissolve the vitelline membrane existing between the follicle cells and the oocyte surface. After this treatment the follicle cells were easily removed manually with fine watchmaker’s forceps under a dissecting microscope. The naked oocyte lost responsiveness to gonadotropin, as shown in table 1, column (2). Therefore, the degree of response to gonadotropin of the treated oocytes may serve as a measure of incompleteness of the removal of follicle cells. Determination
of the rate of GVBD
The naked oocytes or the ovarian fragments (in the form of oocytes covered with follicle cells) were incubated with or without the agents to be tested in small vials containing 4 ml of double Steinberg’s solution for 16 h at 20°C. After incuba-
Progesterolle actiorzat the surface of amphibian oocyte
93
tion the oocytes were fixed by boiling for 10 min and dissected under a dissecting microscope to see whether the germinal vesicles had been broken down or not (Masui, 1967). The rate of GVBD was expressed as a percentage of the number of oocytes in which GVBD occurred, taking the total number of oocytes as 100%. Incubation
of oocytes with insoluble steroid
The naked oocytes were incubated with 50-500 mg of the insoluble steroid desoxycorticosterone derivative bound to agarose in the same condition as described above. The agarose beads stayed at the bottom of the vials and the oocytes were buried in the accumulation of beads. In some control experiments, in order to avoid contact between the oocytes and the insoluble steroid, the oocytes were put in a nylon net basket which was placed in the medium at a distance of several millir’neters above the accumulation of agarose beads. Coupling of desoxycorticosterone
to agarose
Preparation of desoxycorticosterone bound to agarose was performed by a slight modification of the method of Porath et al. (1973), using Sepharose 4B as agarose beads. Descxycorticosterone was succinylated by the procedure described by Erlanger et al. (1967) and purified by recrystallization. Aminoethylated Sepharose (AE-agarose) was prepared by the cyanogen bromide procedure and coupled with desoxycorticosterone hemisuccinate with the aid of ‘water-soluble carbodiimide’. The coupling reaction proceeded for 16 h at room temperature and pH 4.8 with constant and gentle shaking. The substituted agarose (about 5 g wet weight) was washed, while packed in a column, extending over 2 days with 5 liters of 80% methanol in Steinberg’s solution. Following this, it was suspended using a shaker in chloroform-Steinberg’s solution (1 : 1, v/v), and then washed repeatedly (2 days) with 5 liters of total volume after removal of the chloroform phase. Finally, the derivative was washed (2 days) with 5 liters of Steinberg’s solution. The washed product (DOC-agarose) contained about 2 ymol of desoxycorticosterone per gram of packed wet DOC-agarose. Preparation of 105,OOOg supernatant fractions
The naked oocytes (270 oocytes) were homogenized manually with 1 ml of 0.01 M Tris-HCl containing 15 mM EDTA (pH 7.4) in a Potter-Elvehjem teflon homogenizer. In contrast to the case of somatic cells, not only the oocyte’cell membrane but also the oocyte nuclear membrane was broken by this treatment, because of the large size of the nuclei. The homogenate was centrifuged at SSOg and the pellet was removed. The supernatant was then centrifuged at 105,OOOg for 60 min at 1°C. This supernatant fraction, containing nucleoplasmic proteins in addition to cytoplasmic ones, was used to study the binding of progesterone. Chicks were sacrificed and the oviducts were removed and thoroughly rinsed with saline. Two oviducts weighing 1.8 g were homogenized with 5 ml of the medium in a metal blade homogenizer (Polytron, Kinematica GmbH, Lucerne, Switzerland). The ,lOS,OOOg super-
94
K. Ishikawa et al.
natant fraction of oviducts has no reason for the significant contamination with nucleoplasmic proteins and is denoted as the ‘cytosol fraction’. The subsequent procedures were the same as that for frog oocytes. Analysis of receptor in the 105,OOOg supernatant fraction 0.2 ml of the supernatant containing 4 mg of protein was incubated overnight at 2°C with 7 ng of [ 1 ,2,6,7(n)-3H]progesterone. After the incubation, free steroid molecules were removed by gel filtration of the incubation mixture using Sephadex G-25 (4 ml column). 0.2 ml of excluded fraction was applied to 4.6 ml of S-20% sucrose density gradient in 0.01 M Tris-HCl buffer containing 15 mM EDTA (pH 7.4) in the presence of 0.3 M KC1 and centrifuged for 16 h at 1°C at 45,000 rev/min in a Hitachi RPS 65T rotor. 37 fractions were collected by piercing the bottom of the centrifuge tube. Bovine serum albumin (BSA) was spun down under the same conditions as a reference standard. Radioactivity counting Radioactivity was counted with an Aloka liquid scintillation spectrometer with 20-40% efficiency, using a mixture containing 5.5 g of 2,5-diphenyloxazole and 100 mg of p-bis-[2-(5-phenyloxazoyl)] benzene in 30% toluene solution of a detergent, Nissan Nonion NS-2 10. Protein assay Protein was analyzed by the method of UV absorption at 280 nm.
of Lowry et al. (1951) or by measurement
RESULTS Induction of germinal vesicle breakdown of the oocytes by DOCugarose When oocytes were incubated with DOC-agarose the rate of GVBD increased strikingly, reaching almost 100% in some cases. As shown in table 1, the DOCagarose was effective as low as 50 mg/4 ml, corresponding to about lo-’ M steroid. On the other hand, the rate of GVBD could be influenced by the physiological state of the oocyte and therefore varied considerably from experiment to experiment. This was reflected also in the value for untreated oocytes, as shown in column (1) of the table. Nevertheless, the treated oocytes always showed definitely higher values than those of untreated ones. When AE-agarose was substituted for DOCagarose the rate of GVBD stayed within the control range (O-12%) (column (5)). This result indicated that the activity of DOC-agarose was due to the presence of DOC; in other words, DOC was active in producing GVBD of oocytes even in its insoluble form. The rate of GVBD induced by DOC-agarose, however, was lower in most cases than that induced by free DOC, as shown in column (4). This may be explained by the fact that only a small amount of the bound steroid was able to make contact
Progesterone
action at the surface of amphibian oocyte
95
with the oocyte surface under the experimental conditions. In contrast with the above findings, when the oocytes were covered with the follicle cells, DOC-agarose failed to induce GVBD (O-S%) (column (3)), possibly because of the protection of the oocyte surface by the follicle cells. This suggested that the attachment of DOC molecules to the surface of the oocytes was necessary in inducing GVBD, although the incorporation of the steroid was not inevitable. Table 1 also shows the results of other control experiments. When the activity of insoluble steroid is to be determined, we must be careful regarding the possible contamination of DOC-agarose preparation with unreacted steroid molecules which are highly potent. In order to check this possibility, the final wash of DOC-agarose preparation was used as the incubation medium. As shown in column (6), the final wash did not have any appreciable activity in inducing oocyte maturation. The results indicated that the washing of DOC-agarose was sufficient to eliminate uncoupled DOC-hemisuccinate. However, even if the washing was complete, there was also a possibility that the steroid molecules were dissociated from the conjugate during incubation. To check this, the reaction medium was reused after removing the incubated oocytes, for the incubation of the fresh naked oocytes. As shown in column (7), the used medium caused scarcely any induction of GVBD (O-14%), indicating very little or no dissociation of active steroid molecules from the conjugate during incubation. As already mentioned, DOC-agarose hardly reacted with oocytes covered with follicle cells. This fact also provided evidence for the stability of the bound steroid, because if the bound steroid molecules were once liberated, the free DOC molecules should penetrate into the follicle cells and induce GVBD (cf. column (4)). Column (8) shows the results of the experiment in which oocytes were separated from DOC-agarose with a nylon net basket placed in a double Steinberg’s solution and incubated. This artificial barrier disturbed the contact with DOC-agarose. The rates of GVBD in the incubation, 5-13%, were much lower than that for DOCagarose in normal conditions. Absence of receptor activity in the 105,OOOgsupernatantfraction of the frog oocyte If the primary site of steroid action in inducing oocyte maturation exists on the cell surface, we may assume the absence of an intracellular receptor system in the frog oocyte. In relation to this, it had been reported that the enucleated amphibian oocyte responded to progesterone treatment as did the nucleated oocyte (Smith et al., 1968), and that the pigment granules in the cytoplasm could bind a steroid hormone (Ozon and Belle, 1973) but was not confirmed to be an active receptor for inducing oocyte maturation (Baltus et al., 1973). Therefore, the nuclei and the insoluble fraction of cytoplasm could be excluded from the subjects of preliminary searching for the intracellular receptor with any role for the induction of GVBD. Based on this consideration, the 105,OOOg supernatant fraction containing cytoplasmic as well as nucleoplasmic soluble proteins was analyzed in terms of its binding characteristics to progesterone. The 105,OOOg supernatant fraction was incubated
96
K. Ishikawa etal.
Table 1 Effect of DOC-agarose an the germinal ______ ~.~_~._. . - .--. _. ---. Experiment
vesicle breakdown
(“0) of oocytes
-..
Follicle
Germinal
RliS
_.--
(1) None
_---
vesicle breakdown
~-(%I (number
(2)
(3)
Gonadotropin a
DOC-
of oocytcs)
agarose
--~ A
+
l(72) O(40)
33176) 31401
R
-t
O(65)
26(58)
7(441 [500 mg] 29(42) [400 mg]
O(37)[300mgl
14(50)
-
30(47)
7(45)
O(44)
[ 300 mgl
Z45)
c
f -
0(47) O(B)
40(15) 4(24)
_
50(22)1400mg] 46(13) 1300mg] 44(25)
D
+
-
W6) O(33)
40(25) 9(35)
8(38)[200mgl 59(34)[ZOOmg] 37(35) 3X34)
+
X39)
0(33)
83(36) 16(32)
ll(95)
-
16(51)
-
[ 150 mg]
[ 100 mg]
lOO(32)[400mg] 97(32)[300mg] 84(32)
i-
[2OO mg]
[ZOO mg]
_ 97(37) lOO(39) 92136)
[loo [50
mgl
msl [SOmgl
In experiments
A to F, ovarian fragments in the form of oocytes covered with follicle cells, or naked oocytes (indicated by + or -) were incubated in small vials containing 4 ml of double Steinberg’s solution for 16 h at 2O”C, with or without the agents to be tested. Each row represents a separate experiment conducted with different frogs on different days. Numbers in parentheses are the total number of oocytes examined. Numbers in brackets are the wet weight of
DOC-agaroseOI AE-agarose added to 4 ml of the reaction medium.
with [3H] progesterone as described in Materials and methods. After removal of the unreacted steroid, an aliquot of the labeled fraction was analyzed by sucrose density gradient centrifugation. The same amount of labeled protein of oviduct cytosol fraction obtained from estrogen-stimulated chicks was
Progesterone
(4) DOC (lo@
action at the surface of amphibian oocyte
(3 AE-agarose
(6) Final wash
(7) Medium used
(8) DOC-agarose with barrier
-
-
-
_ 5(38)
[SO0 mg]
-
14(38)
[300 mg]
M) b
43(61) 95(42)
O(43) [500 mg]
o(42)
57(46)
2(61)
1300 mg]
O(54)
75(44)
0(46)
[ 300 mg]
0(46)
43(21) 44(25)
O(13) 1400 mg] 6(16) [400 mg]
76(29) 94(35)
86(29) lOO(32)
-
003 OW)
O(15) 6(17) 5(19)
4(28) 4(28) 3(29)
O(34) [400 mg]
15(34) lS(34)
_
_
3(30) 3(33)
[ 200 mg]
12(34)
-
_
X36)
98(40)
_
91
17(35) lO(39) 8(39)
15(26) ll(27) 14(37) 9(32) 9(23)
a) In experiment A, ovine luteinizing hormone (200 Mg), and in experiments B-F, Xenopus laevis pituitary (one-half pituitary equivalent/vial) were used. b, In experiments A-C and E, desoxycorticosterone, and in experiments D and F, progesterone were used. Abbreviations: DOCagarose, desoxycorticosterone coupled to aminoethylated Sepharose; DOC, desoxycorticosterone; AEagarose, aminoethylated Sepharose.
subjected to the analysis as a reference control. Fig. 1 shows the distribution of radioactivity in the intracellular soluble proteins. No radioactivity peak was found in the pattern for Xenopus oocyte fraction, but in the case of chick oviduct a sharp peak was shown around a protein peak. The result showed that the oocyte superna-
98
K. Ishikawa et al.
b
8 BSA 4
0
20
40
0
20
40
Fraction number Fig. 1. Sucrose density gradient centrifugation of [ 3H]progesterone-binding components in the 105,OOOg supernatant fraction of frog oocytes (a) and oviducts of diethylstilbestrol-treated chicks (b). The labeled fraction was subjected to centrifugation at 45,000 rev/min in a Hitachi RPS 65T rotor for 16 h at 1°C in a 5-20s sucrose density gradient containing 0.3 M KCl. 37 fractions were collected by piercing the bottom of the centrifuge tube. The radioactivity and UV absorption at 280 nm of each fraction were measured and plotted. The arrow indicates the location of bovine serum albumin (BSA) sedimenting under the same conditions as a reference standard.
tant fraction had no receptor for progesterone, or at least that the content of steroid receptor per unit amount of soluble protein in the oocyte was much lower than that in the oviduct cytosol protein.
DISCUSSION Although many steroids such as progesterone, androgen and some corticoids induce the GVBD of oocytes in Xenopus laevis, progesterone and desoxycorticosterone are most effective in producing both GVBD and ovulation (Hanaoka et al., 1975). Thorton and Evennett (1969) have reported that the injection of human chorionic gonadotropin causes the ovarian follicle cells to release a progesteronelike hormone about 12 h before subsequent ovulation. This suggests that the follicle cells secrete progesterone which causes the maturation of oocytes. In the present study, desoxycorticosterone (DOC) instead of progesterone was used to obtain an insoluble steroid, since DOC was also active in inducing GVBD, at least in vitro, and the hydroxyl group at 21-C was ready for coupling reaction with agarose. In addition, the structure of the conjugated steroid was rather similar to progesterone, except that the methyl group at 21 -C of progesterone was replaced by aminoethylated agarose (AE-agarose). This might suggest that the conjugated desoxycorti-
Progesterone action at the surface of amphibian oocyte
99
costerone is closer to progesterone than to DOC in terms of its structural characteristics, and that the conjugated desoxycorticosterone might behave as progesterone for the oocyte stimulation. This is a reason for the use of progesterone to search the intracellular receptor which may participate in the steroid action to induce oocyte maturation, The agarose beads, ranging between 50 and 190 pm in diameter (Pharmacia Fine Chemicals leaflet, Pharmacia Fine Chemicals, Inc., Uppsala, Sweden), were much smaller than the oocytes (1.2-l .3 mm) used in the experiments. This led us to examine the possibility of pinocytotic penetration of the hormone into the oocyte even in its insoluble form. In fact, pinocytotic activity of frog oocyte has been found in young oocytes (Wallace et al., 1973). However, pinocytotic vesicles found in the young oocytes were not as large as the agarose beads, and full-grown oocytes which were ready for GVBD were found to have scarcely any pinocytotic activity. Therefore, the action of the insoluble steroid does not seem to be an intracellular action caused after pinocytotic incorporation. The incubation of progesterone with the 105,OOOg supernatant fraction of the oocytes did not produce an appreciable amount of bound steroid. The supernatant fraction contained the nucleoplasmic proteins in addition to the cytoplasmic ones. Therefore, it may be concluded that the oocyte has no soluble protein which reacts to progesterone. Although the insoluble constituents of the nuclei such as chromatin fraction might have some receptor activity for the steroid even if such receptor exists, it should not participate in the steroid induction of the maturation. As mentioned in previous sections, the steroids react with the enucleated oocytes to produce some cytoplasmic factor which in turn induces GVBD when it is injected into the nucleated oocyte. Concerning the insoluble fraction of the cytoplasm, it has recently been shown that progesterone binding sites exist in the pigment granules fraction (melanosomes) of Pleurodes waltlii oocytes (Ozon and Belle, 1973) and Xenopus Zaevis oocytes (Iacobelli et al., 1974; Belle et al., 1975). These binding sites were found to have high affinity and limited binding capacity for progesterone. Baltus et al. (1973), however, reported more recently that the binding of progesterone to the melanosomes was not sufficient to induce maturation. They injected normal melanosomes or progesterone-treated melanosomes into Xenopus oocytes. Both the normal and the treated melanosomes had no effect at all on GVBD. Summarizing, it seems likely that there is no intracellular receptor mechanism for preparing the steroid induction of GVBD in the full-grown oocytes of Xenopus.
All the findings mentioned above are consistent with the conclusion that the oocyte surface is the location of the primary reaction of the steroid hormone in the induction of GVBD. The only exception is a report that the injection of hydrocortisone causes GVBD (Schorderet-Slatkine, 1972). This might be due to the technical difficulty of avoiding the leaking of hormone when it was injected into the oocytes in vitro. If the steroid hormone acts at the surface of the oocyte, a second messenger sys-
100
K. Ishikawa et al.
tern may transport the hormonal information to the germinal vesicle. Although cyclic AMP is well known as a second messenger in peptide hormonal action (Sutherland et al., 1965) cyclic AMP or dibutyryl cyclic AMP failed to induce GVBD in Xenopus oocytes (Schorderet-Slatkine, 1972; Brachet et al., 1974). To search the second messenger system, it should be noted that in amphibia the cytoplasm of progesterone-treated oocytes could induce GVBD when the cytoplasm was injected into untreated oocytes (Smith and Ecker, 1971; Masui and Markert, 1971; Schorderet-Slatkine and Drury, 1973). Wasserman and Masui (1976) reported the characterization of this maturation-promoting factor. In relation to the above findings on amphibian oocytes, it is interesting to note that 1-methyladenine, which could induce GVBD in starfish oocytes, has been felt to act also at the surface of the oocyte (Kanatani and Hiramoto, 1970).
ACKNOWLEDGEMENT This investigation tion of Japan.
was supported
in part by a grant from the Ministry of Educa-
REFERENCES Balms, E., Brachet, J., HanocqQuertier, .J. and Hubert, E. (1973) Differentiation 1, 127-143. Belle. R., Schorderet-Slatkine, S., Drury, K.C. and Ozon, R. (1975) Gen. Comp. Endocrinol. 25,339-345. Brachet, J., Baltus, E., De Schutter, A., Hanocq, F., HanocqQuertier, J., Hubert, E., lacobelli, S. and Steinert, G. (1974) Mol. Cell. Biochem. 3, 189-205. Erlanger, B.F., Beiser, S.M., Borek, F., Edel, F. and Lieberman, S. (1967) In: Methods in Immunology and Immunochemistry, Vol. 1, Eds.: C.A. Williams and M.W. Chase (Academic Press, New York) p. 148. Hanaoka, Y., Ishikawa, K., Kondo, Y. and lmai, K. (1975) Unpublished data. lacobelli, S., Hanocq, J., Baltus, E. and Brachet, J. (1974) Differentiation 2,129-135. Jensen, E.V. and Desombre, E.R. (1973) Science 182,126-134. Kanatani, H. and Hiramoto, Y. (1970) Exp. Cell Res. 61,280-284. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J.Biol.Chem. 193,255276. Masui, Y. (1967) J. Exp. Zool. 166, 365-376. Masui, Y. and Markert, C.L. (1971) J. Exp. Zool. 177, 1299146. O’Malley, B.W. and Means, A.R. (1974) Science 183,610-620. Ozon, R. and Belle, R. (1973) Biochim. Biophys. Acta 320,588-593. Porath, J., Aspberg, K., Drevin, H. and Axen, R. (1973) J. Chromatogr. 86,53-56. Schorderet-Slatkine, S. (1972) Cell Differ. 1,179-189. Schorderet-Slatkine, S. and Drury, K.C. (1973) Cell Differ. 2,247-254. Smith, L.D. and Ecker, R.E. (1971) Dev. Biol. 25,232-247. Smith, L.D., Ecker, R.E. and Subtelny, S. (1968) Dev. Biol. 17,627-643. Sutherland, E.W., Oye, I. and Butcher, K.W. (1965) Recent Prog. Horm. Res. 21,623-642. Thorton, V.F. and Evennett, P.J. (1969) Gen. Comp. Endocrinol. 13,268-274. Wallace, R.A., Jared, D.W., Dumont, J.N. and Sega, M.W. (1973) J. Exp. Zool. 184,321-333. Wasserman, W.J. and Masui, Y. (1976) Science 191,1266-1268.
