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Germinal vesicle migration and dissolution in Rana pipiens oocytes: effect of steroids and microtubule poisons
Cell Differentiation, 20 (1987) 239 251 Elsevier Scientific Publishers Ireland, Ltd.
239
C D F 00413
Germinal vesicle migration and dissolution in Rana pipiens oocytes: effect of steroids and microtubule poisons Charles A. L e s s m a n Department ~[ Biolow. St. Franci.~ Xat,ier Unit,er~itv. A nti~onish, Nm,a Scotia, ('anada (Accepted 6 October 1986l
Germinal vesicle migration (GVM) as evidenced by the appearance of the germinal vesicle at the animal pole surface was induced by nocodazole and demecolcine (colcemid). Nocodazole significantly lowered the progesterone EDs0 for germinal vesicle dissolution (GVD). Both demecolcine and nocodazole enhanced centrifugation-induced GVM (i.e., lowered ooplasmic viscoelasticity) after 6-h incubation, and both potentiated the effect of progesterone in this assay. Estradiol, by contrast, inhibited GVM induced by demecolcine in both follicle-enclosed and denuded oocytes. Estradiol was also found to inhibit the normal enhancement of centrifugation-induced GVM by demecolcine or progesterone. Taxol was found to have effects that were generally opposite to those of demecolcine and nocodazole. Taxol inhibited centrifugation-induced GVM either alone or in the presence of progesterone, in addition, taxol significantly increased the progesterone EDs0 for GVD induction. Taken together the available data support the hypothesis that microtubules play a role in maintaining the internal position of the germinal vesicle in the prematuration oocyte and that changes occur in the oocyte cytoskeleton during maturation.
Meiosis; Meiotic maturation; Taxol; Colcemid; Nocodazole; Estradiol; Cytoskeleton; Tubulin
! ntroduction During oocyte maturation in the amphibian
Rana pipiens, the chromatin of the germinal vesicle (GV) must be moved from a position well within the oocyte to a position very close to the animal pole surface in order to produce the unequal division leading to polar body formation. Migration of the GV during oocyte maturation in amphibians was described by Makino (1934) in a
Correspondence address: Dr. Charles A. Lessman, Box 30, St. Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 1C0.
classic study, and has been described more recently by Brachet et al. (1970) and Lessman et al. (1986). In fact, the process of moving the GV or its contents must occur in virtually all animal oocytes, since the production of polar bodies seems to be a universal phenomenon. How does the GV or its contents move during oocyte maturation? One possibility is that cytoskeletal elements actively move the contents of the GV as an intact unit, or as components after GV breakdown or dissolution (GVD), to the oocyte surface. Another possibility would also involve cytoskeletal elements but only in a passive way, such that the GV is restrained or held in position by the cytoskeleton, and during maturation this restraint is
0045-6039/87/$03.50 ~' 1987 Elsevier Scientific Publishers Ireland, Ltd.
240 released. In this second possibility, the active force might come from other factors, for example changes in ooplasmic density causing the GV to float toward the surface. A third possibility, which combines the first two, would involve a reorganization of the cytoskeleton so that elements stabilizing the position of the GV would change along with activation of others involved in the migration of the GV or its contents. All of the possibilities given here are contingent on the presence of cytoskeletal elements in oocytes. The amphibian oocyte does indeed possess a cytoskeleton (Wylie et al., 1985). Recent evidence indicates that microtubules are present in immature, fully grown amphibian oocytes (Heidemann et al., 1985; Huchon et al., 1985; Jessus et al., 1986) and confirms earlier reports (Dumont and Wallace, 1972; Colman et al., 1981). Nevertheless, for some time it was presumed that immature Xenopus oocytes did not contain microtubules since basal body or centrosome injection (Heidemann and Kirschner, 1975; Maller et al., 1976; Heidemann and Kirschner, 1978); and taxol treatment (Heidemann and Gallas, 1980; Huchon and Ozon, 1985; Jessus et al., 1986) failed to induce detectable levels of microtubules in spite of high tubulin pools. A recent paper (Jessus et al., 1985) using [35S] methionine labeled oocytes reported that taxol did induce tubulin assembly in immature oocyte extracts in vitro. In a previous paper from this laboratory (Lessman et al., 1986), demecolcine (colcemid), a potent inhibitor of tubulin polymerization (Dustin, 1984), was found to induce GV migration (GVM) in R. pipiens oocytes and to significantly reduce the centrifugal force required to move the GV. These results suggest that microtubules are involved with GV positioning within the oocyte and support the second and third possibilities given above. The present paper puts forth additional evidence in support of this hypothesis which includes use of another microtubule destabilizing agent, nocodazole, and the microtubule stabilizing agent, taxol (Schiff et al., 1979) which has antimitotic activity (Wani et al., 1971). Two steroids, progesterone and estradiol, known to affect meiotic maturation in Rana oocytes in opposing ways (Lin and Schuetz, 1983) were also tested for effects on GV positioning.
Materials and Methods
Animals Adult female leopard frogs (R. pipiens) were obtained from Lemberger Assoc. (Oshkosh, WI) and held in artificial hibernation at 4°C in trays containing 10% amphibian Ringer's. Ringer's solution was changed in the trays at least three times per week. Just before use, animals were stunned by a blow to the head and killed immediately by decapitation. The spinal cord was pithed, and the ovaries were dissected and placed in cold Ringer's.
Follicle and oocyte preparation Type I follicles (intact, fully grown follicles) as defined by Schuetz and Lessman (1982) were dissected from other ovarian tissue with watchmaker forceps. When type IV follicles (denuded oocytes without follicle cells; Schuetz and Lessman, 1982) were required, the follicle wall was removed by microdissection from type I follicles, and the follicle cells were removed by shaking for 2 h in Ca2+-free Ringer's (Lessman and Schuetz, 1981). The resulting denuded oocytes were rinsed several times with normal Ringer's and allowed to incubate for at least 1 h in Cae+-containing Ringer's (Lessman and Schuetz, 1981) prior to use. Type I and IV follicles were inspected, using a stereoscope, for damage and, in the case of type IV, the absence of follicle cells. Groups of 20 follicles were assigned randomly to treatment wells containing 2 ml Ringer's in a 24-well culture dish (CoStar no. 3524). Incubations were carried out in darkness at 22°C in a thermostatically controlled incubator).
Assay for germinal vesicle dissolution and migration Meiosis reinitiation was scored after fixing the follicles by brief boiling or by hot trichloroacetic acid (15%, 60°C). The presence or absence of the oocyte nucleus or GV was determined by dissection; absence of the otherwise conspicuous GV is defined as GV breakdown or dissolution (GVD) and denotes meiosis reinitiation. Prior to dissection, follicles were also scored for the presence of a GV at the animal pole surface (Lessman et al., 1986), oocytes containing such externally visible GVs were scored as having undergone GV migra-
241 tion ( G V M ) (Fig. 1). In those oocytes scored as positive for G V M , the presence of the presumed G V was verified during dissection at the time of G V D assay (Fig. 1). Often the GV would only be partially visible with some pigmented cortical cytoplasm remaining above the G V (Fig. 1). In some experiments, the presence of a transient white spot in the animal pole was scored; subsequent dissection indicated that the presence of the white spot correlated perfectly with the absence of an intact GV.
Centrifugation-induced GVM assay Details of this assay have been described previously (Lessman et al., 1986). Briefly, after incubation, follicles were placed at the interphase between Ringer's and 40% Ficoll (Sigma Chem. Co.) in Ringer's and centrifuged for 5 min at 0, 35, 250, 500 or 1 000 × g. Immediately after centrifugation, the cells were fixed in hot 15% trichloroacetic acid and scored for G V M and G V D as described above. The G V M scored after centrifugation appeared
similar to that induced by nocodazole or demecolcine (Fig. 1 and Lessman et al., 1986) with the exception that centrifuged cells exhibiting G V M tended to have less pigmented cytoplasm trapped above the GV, especially at the higher centrifugal forces.
Histological preparations Follicles were fixed in Bouin's fluid, dehydrated in an alcohol series, cleared in xylene and embedded in paraffin. Serial sections were cut at 10 txm, and the resulting sections were stained with Delafield's hematoxylin and congo red.
Chemicals Progesterone and estradiol-17/~ were obtained from Steraloids Inc. and were dissolved in steroid vehicle (propylene g l y c o l / E t O H , 1 : 1). Demecolcine (N-desacetyl-N-methylcolchicine; colcemid) was obtained from Sigma Chem. Co. and was also dissolved in steroid vehicle. N o c o d a z o l e was obtained from Aldrich Chem. Co. and was dissolved
A
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Fig. 1. A typical follicle-enclosed oocyte with GVM elicited by 48-h incubation with nocodazole (0.5 ~g/ml, 1.7 I~M); the follicle has been fixed with trichloroacetic acid (15% in Ringer's) at 60°C which increases the visibility of the otherwise clear GV. (A) Micrograph of an entire follicle, the GV is present at the oocyte surface and extends between the arrow tips. Bar = 500 t~m. (B) Same follicle as in (A) but hemisected parallel to the animal-vegetal axis showing GV at animal pole surface, small arrows indicate areas where the GV is externally visible from the oocyte surface. Bar = 500 /~m.
242
in dimethyl sulfoxide (DMSO) and steroid vehicle (1 : 1). Taxol (lot FB1046) was a gift of Dr. Surfness ( N I H ) and was dissolved in D M S O just prior to use. The final concentration of vehicle in the incubation medium was < 0.1%; control oocytes also received the appropriate vehicle.
Statistical analysis Quantal data such as that generated by G V M and G V D scoring were analyzed by a paired test of proportions based on binomials (Downie and Heath, 1965). Significance was assigned at P < 0.05 and was indicated by superscripts associated with data.
Results
Effects of demecolcine, estradiol, and progesterone Demecolcine (colcemid) was found to induce GV migration (GVM) in a dose-related manner (Table I) in Rana follicle-enclosed oocytes which confirmed earlier findings (Lessman et al., 1986). G V M continued to occur in the interval between 24- and 48-h incubation indicating the relatively slow nature of demecolcine-induced G V M (Table I). The lowest dose of demecolcine failed to elicit a significant effect compared to vehicle controls at either 24- or 48-h incubation. This was in contrast to our previous finding (Lessman et al., 1986) that
demecolcine at 0.1 /~g/ml was moderately effective; the difference may be due to interanimal variation. It should be noted that the near maximal GVM induced by demecolcine (10/xg/ml, 27 /~M) at 24 h coincided with the appearance of a white spot in the animal pole of progesteronetreated cells (Table I), indicating a similar time course for both events. Although progesterone did not induce significant G V M scored as an intact GV at the oocyte surface, there were significant numbers of oocytes at 24 h which exhibited a white spot at the animal pole (Table I). The transient white spot has been associated with remnants of the GV at the oocyte surface and has been used by some researchers to score meiosis reinitiation or G V dissolution (GVD). Thus progesterone was responsible for movement of some G V material, if not the entire GV, to the oocyte surface. An earlier study (Masui, 1972) reported that Rana oocytes in which the GV was displaced to the vegetal pole by centrifugation, underwent G V M toward the animal pole after progesterone treatment. In order to obtain additional baseline information regarding G V M induced by progesterone, serial sections of follicles incubated with 1 /~g/ml progesterone for 0, 16.5, 18, 24, 32 and 48 h were prepared from three different females. Control oocytes throughout the incubation period had spherical GVs well below the oocyte surface; the nucleoli were quite distinct in all control GVs
TABLEI E f f e c t o f e s t r a d i o l - l ~ ondemecolcine(colcemid) induced GVM infollicle-enclosedoocytesof Treatment
Estradiol (/x g / m l ) 0.0
1.0
0.0
22 * 5 5 6 -
61 38 4 5 2
GVM (24 h) Demecolcine 10 ~ g / m l (27~M) Demecolcine 1 # g / m l (2.7 p,M) Demecolcine 0.1 t~g/ml (0.27/~M) Control (vehicle only) Progesterone 1 p,g/ml
R.p~iens
56 (0) 10 (0) 3 (0) 1 (0) 4 (29)
1.0
GVM (48 h)
0.0
1.0
GVD (48 h) 32 * 18 * 6 6
1 0 0 0 80
0 0 0 0 -
GVM data presented as number of oocytes with a germinal vesicle at their animal pole surface of 100 oocytes tested per treatment. Twenty follicles from each of five females were used per treatment. Numbers in parentheses represent number of oocytes with white spot at animal pole. GVD data presented as number of oocytes without a GV upon dissection of 100 oocytes tested per treatment. Incubation conditions: 22°C, 2 ml Ringer's containing 20 follicles per well. Data pairs were tested for statistical differences using a test of proportions based on binomials (Downie and Heath, 1965). An asterisk indicates a significant difference at P < 0.05 from demecolcine alone.
243
(Fig. 2A). By 16.5 h incubation, the GVs of progesterone-treated oocytes began to exhibit marked changes which resemble those reported during Xenopus oocyte GVD (Brachet et al., 1970; Huchon et al., 1981, 1985; Jessus et al., 1986). The GVs became flattened and were closer to the oocyte surface (i.e., they exhibited partial GVM); they contained fibrillar material, especially at the basal side, and the nucleoli disappeared (Fig. 2B). Since the GV could be isolated by dissection at
this stage, GVB by strict definition had not occurred. At 18-h incubation with progesterone, the nuclear envelope disappeared, the fibrillar material of the disrupted GV radiated from a dense band of material toward the oocyte surface (Fig. 2C). G V D had occurred by definition at 18-h incubation, since the GV at this stage could not be dissected and isolated from the oocyte. However, since pigmented cytoplasm remains between the GV remnants and the oocyte surface, even though
D
Fig. 2. A series of micrographs depicting meiotic maturation induced by progesterone in R. pipiens follicle-enclosed oocytes in paraffin sections (10 p,m thick). (A) Control follicle after 48-h incubation, GV remains spherical and well below the oocyte surface, numerous nucleoli (n) are visible; bar = 100 /~m. (B) Follicle after 16.5-h incubation with progesterone (1 p,g/lnl, 3.2 /~M), (iV contents are fibrillar especially at basal or vegetal side and nucleoli are indistinct; magnification same as (A). The GV has moved closer to the oocyte surface. (C) Follicle after 18-h incubation with progesterone (1 ~ g / m l ) , nuclear envelope no longer visible thus by definition GVD has occurred. The area of the GV is occupied by fibrillar material radiating from a dense band of material; the GV contents extend nearly to the oocyte surface, separated only by a thin layer of pigmented cortical cytoplasm: magnification same as (A). (D) Follicle after 48-h incubation with progesterone (1 ~ g / m l ) showing a metaphase spindle (s) at oocyte surface (unstained section); bar = 50 /~m.
244 the G V has moved closer to the surface (i.e., partial GVM), scorable G V M as defined earlier would not be detected. Beginning at this time a transient white spot could be seen in the animal pole surface of whole oocytes (Tables I and II). By 48 h a metaphase spindle was visible at the oocyte surface, thus during the 48-h incubation period partial G V M had occurred along with G V D in response to progesterone (Fig. 2D). Nevertheless, since G V D occurred prior to complete G V M defined as the G V visible at the oocyte surface, it was not surprising that progesterone did not elicit scorable GVM. Estradiol (1 / l g / m l , 3.6 /zM) was found to significantly inhibit G V M induced by demecolcine at b o t h 24- and 48-h incubation (Table I). Only progesterone elicited significant G V D in this experiment, neither demecolcine nor estradiol were active alone in inducing G V D . In order to determine whether the inhibitory effect of estradiol on demecolcine-induced G V M was related to the follicle wall, the experiment was repeated using d e n u d e d oocytes (type IV follicles; Schuetz and Lessman, 1982) which lack follicle wall components. Estradiol significantly inhibited demecolcine-induced G V M at 48-h incubation in the absence of the follicle wall (Table II). The response to demecolcine was slower and of less magnitude than in the previous experiment; in addition, the white spot was present in some progesterone-treated cells at 48 h. Thus both events appeared to follow the same retarded time course. This m a y be due to inter-animal differences or could be attributable to the presence or absence of the follicle wall, in any case the results of both experiments (Tables I and II) are qualitatively the same, i.e., estradiol inhibited demecolcine-induced G V M in the presence or absence of the follicle wall. In order to directly test the effect of estradiol on ooplasmic viscoelasticity changes, the procedure of oocyte centrifugation (Merriam, 1971; Masui, 1972; Lessman et al., 1986) was used. Previous work had established that progesterone and demecolcine were both capable of enhancing centrifugation-induced G V M (Lessman et al., 1986); the present findings confirm this earlier report (Fig. 3). Estradiol alone (1 /~g/ml) had no effect on centrifugation-induced GVM, but did significantly inhibit demecolcine's usual enhance-
TABLE II Effect of estradiol-17,8 on demecolcine (colcemid) induced GVM in denuded oocytes of R. pipiens Treatment Demecolcine 10 8g/ml (27~M) Demecolcine 10 t~g/ml plus estradiol 1/~g/ml (3.6~M) Progesterone 1/xg/ml (3.2 /LM) Estradiol 1 #g/ml (3.6#M) Control (vehicle only)
GVM
GVD
24 h
48 h
48 h
14 (0)
48 (0)
0
6 (0)
"18 (0)
1
0 (24)
0 (12)
90
1 (0) 1 (0)
1 (0) 1 (0)
0 0
GVM data presented as number of oocytes with externally visible GV of 100 tested per treatment. Numbers in parentheses indicate oocytes with a white spot in animal pole. Twenty oocytes from each of five females were used per treatment. GVD data presented as number of oocytes lacking a GV upon dissection of 100 tested per treatment. Incubation conditions: 22°C, 20 follicles per well containing 2 ml Ringer's. GVM data pairs for demecolcine were statistically compared using a test of proportions based on binomials (Downie and Heath, 1965). The asterisk denotes the estradiol-demecolcine treatment which is significantly lower than the respective control (demecolcine alone) at P < 0.05.
ment of centrifugation-induced G V M (Fig. 3). A similar experiment was carried out to determine whether estradiol had any effect on the progesterone-induced changes observed in centrifugation-induced GVM. The results indicated that estradiol (1 t~g/ml) significantly inhibited the usually observed enhancement of centrifugation-induced G V M by progesterone at 4-h incubation (Fig. 4). Masui (1972) had previously shown that R a n a oocytes subjected to centrifugal forces of 2 0 0 - 4 0 0 x g remained capable of responding to progesterone. Since somewhat higher forces were used in this study, the responsiveness of oocytes to progesterone was tested after centrifugation at 1 000 x g for 5 min. Over 75% of the centrifuged oocytes had G V M before incubation; after 24-h incubation G V M remained high in the vehicle control group (i.e., no evidence of G V recoil to original position), while G V D was near maximal in the progesterone treated group (data not shown). This latter finding along with that of Masui (1972)
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Fig. 3. Effect of cytoskeleton-active agents on centrifugationinduced G V M in Rana follicle-enclosed oocytes at 6-h incubation. Data presented as number of follicles containing oocytcs with germinal vesicles visible at the animal pole after hot trichloroacetic acid fixation of 30 follicles tested per treatment (replicated with three females, 10 follicles per female): a total of 1500 oocytes were used in this experiment. Treatments: VC, vehicle control ( E t O H / p r o p y l e n e glycol/DMSO, 1.5 : 1.5 : 3); D, demecolcine 10 ~ g / m l (27 p,M); E, estradiol-17fl 1 ~tg/ml (3.6 ~M): D + E , demecolcine 10 ' a g / m l and estradiol 17fl 1 ~tg/ml; P, progesterone 1 t*g/ml (3.2 'aM); D + P, demecolcine 10 p~g/ml and progesterone 1 ' a g / m l ; N, nocodazole 0.5 ~tg/ml (1.7 /*M): N + P , nocodazole 0.5 p.g/ml and progesterone 1 ' a g / m l ; T, taxol 10 ,ag/ml (12 ~M): T + P , taxol 10 , a g / m l and progesterone 1 ,ag/ml. Unccntrifuged follicles which were placed on Ficoll cushions after treatment were without G V M and are indicated by D; @ . . . . . . • = 35 × g; .... ~=250×g; + .... + =500×g; • I=1000 × g. Arrows denote significant difference from respective vehicle control (i.e., same centrifugation speed) at P < 0.05 using a test of proportions based on binomials (Downie and Heath, 1965). Follicles were centrifuged for 5 min at the interface between 40% Ficoll and amphibian Ringer's using a modification of a technique described by Masui (1972).
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Fig. 4. Effect of estradiol (1 tzg/ml, 3.7/~M) on progesterone (1 ~ g / m l , 3.2 ,aM) enhancement of centrifugation-induccd GVM. C, control (vehicle only): P, progesterone: E, estradiol: B, both steroids added simultaneously. Follicles were incubated for 4 h after steroid addition. Rana follicle-enclosed oocytes (each of three females contributed 20 follicles per treatment) were placed at the interphase of 40% Ficoll and Ringer's and centrifuged in a swinging bucket rotor for 5 rain. Follicles were immediately fixed in hot (60°C) trichloroacetic acid and scored for the presence of a germinal vesicle visible at the animal pole surface without dissection. Asterisk denotes statistically significant difference from other treatment groups at that centrifugal force using the test of proportions (Downie and Heath, 1965).
mecolcine treatment (Fig. 1 and Lessman et al., 1986). G V M elicited by both drugs was always to the animal pole regardless of the orientation of the oocyte to gravity. Nocodazole was somewhat more active (EDs0 = 0.7/.tM) than demecolcine (EDs0 =
T A B L E 11I
indicates that centrifugation at the forces used does not disorganize the oocyte to the point of loss of hormone responsiveness. Effects of nocodazole
If demecolcine was acting on microtubules to elicit G V M then other agents affecting microtubules should exhibit effects on GVM. Thus nocodazole which also acts to destabilize microtubules (Dustin, 1984) was tested for GVM-inducing activity. The results show a dose-dependent effect on G V M (Table III); increasing doses of nocodazole (0.05-5 /.tg/ml) were associated with increased GVM, but nocodazole alone did not elicit GVD. N o c o d a z o l e induced G V M which was indistinguishable from that obtained with de-
Effect of nocodazole on GVM and G V D in follicle-enclosed oocytes of R. pipiens Treatment Nocodazole 5 ' a g / m l (17'aM) Nocodazole 0.5 p,g/ml (1.7'aM) Nocodazole 0.05 g g / m l (0.17/.tM) Control (vehicle only)
GVM (24 h)
GVM (48 h)
GVD (48 h)
41 (4-12)
56 (6-16)
0
30(2-11)
44(1-13)
0
1 (0-1) 0
3 (0-1) 0
0 0
G V M data presented as percentage of oocytes with a germinal vesicle visible externally (80 oocytes at 24 h and 100 oocytes at 48 h were tested per treatment). Twenty follicles from each of 4 5 females were used per treatment: numbers in parentheses arc ranges of response (out of 20 follicles tested) for individual females. G V D data presented as the number of oocytes which lack a (iV upon dissection of 100 tested per treatment.
246 0.9/~M) in eliciting GVM. Nocodazole (0.5/~g/ml, 1.7 /xM) induced G V M in over 50% of oocytes containing a GV (Table IV) in the presence of progesterone (0-0.7 ~ g / m l ) . Increasing doses of progesterone were associated with increased G V D at 48-h incubation indicating that oocytes remained viable and capable of responding to progesterone in the presence of nocodazole. C o l m a n et al. (1981) reported that 10 ~ M nocodazole was toxic to Xenopus oocytes. By contrast, Elinson (1985) found that Xenopus eggs treated with nocodazole (10/~g/ml, 33/~M) could be activated and showed cortical contractions, suggesting low toxicity even at a relatively high dose. In any case, species differences and the doses used may account for any toxicity observed in previous studies. The relatively low dose of 1.7 /~M nocodazole (0.5 /xg/ml) significantly decreased the progesterone EDs0 (Table IV) from 0.23 # g / m l (95% confidence limits = 0.20 0.27) to 0.13 ~ g / m l (95% confidence limits = 0.10-0.16) calculated by the S p e a r m a n - K a r b e r procedure (Brown, 1970). This enhanced progesterone activity further indicated the absence of toxic effects by this dose of
TABLE IV The effect of nocodazole and several doses of progesterone on GVM and GVD in follicle-enclosed oocytes of R. pipiens Progesterone ( ~ g/ml) 1.0 (3.2 gM) 0.7 0.1 0.07 0.01 0.007 0 Control
Nocodazole (/Lg/ml) 0.0 0.5 GVM
GVD
GVM
GVD
0/11 0/30 6/88 1/93 2/100 0/100 1/100
89 70 12 7 0 0 0
3/14 "9/15 *48/73 *54/79 *55/94 "51/99 "51/99
86 **85 **27 *'21 **6 1 1
GVM data presented as number of oocytes with a germinal vesicle visible externally (numerator) of the total number with intact germinal vesicles (denominator) after 48-h incubation. GVD data are presented as the number of oocytes which lack a germinal vesicle upon dissection of 100 tested per treatment. Twenty follicles from each of five females were used per treatment. Both types of data were collected after 48-h incubation at 22° C. Asterisks indicate significant differences at P < 0.05 with and without nocodazole using a test of proportions based on binomials (Downie and Heath, 1965).
nocodazole. Although there does not appear to be an enhancement of G V M by the combination of nocodazole and progesterone, it should be pointed out that increasing G V D may mask any additive or synergistic effects. In order to test the possibility that nocodazole and progesterone act synergistically on GVM, centrifugation was used at 6-h incubation to induce G V M without the complication of G V D . The results indicated that at 35 × g, and especially at 250 × g, nocodazole and progesterone acted synergistically to enhance centrifugation-induced G V M (Fig. 3). As in the case of demecolcine and progesterone, nocodazole enhanced G V M induced by centrifugation after short term incubation (i.e., 6 h) before G V M or G V D would otherwise have occurred. These results indicate that nocodazole, as well as demecolcine and progesterone, decrease the apparent viscoelastic properties of ooplasm thus allowing the GV to be moved more readily.
Effects of taxol Taxol has been demonstrated to stabilize microtubules and p r o m o t e tubulin assembly into microtubules (Schiff et al., 1979; H e i d e m a n n and Gallas, 1980). Taxol (10 # g / m l , 12 /_tM) significantly increased the EDs0 of progesterone-induced G V D (Fig. 5) from 0.15 ~ g / m l (95% confidence l i m i t s = 0 . 1 3 - 0 . 1 8 ) to 0.22 ~ g / m l (95% confidence limits = 0.18-0.26). In addition, taxol-progesterone treated oocytes which underwent G V D showed a distinctive streaked or 'starburst' pattern especially visible in the animal pole (Fig. 6A). Such oocytes when sectioned contained m a n y extensive cytaster arrays (Fig. 6B). Oocytes which did not undergo G V D under the influence of both agents or those treated with taxol alone did not exhibit the cytasters (data not shown). Nevertheless, it could not be ruled out that additional microtubules, not visible at the light microscope level, were induced by taxol. Thus cytaster formation was dependent u p o n G V D and t a x o l - p r o gesterone treatment. Taxol was not toxic since it shifted the progesterone dose-response curve to the right (i.e., significantly increased the EDso for progesterone-induced GVD), rather than producing total inhibition of progesterone action. It should also be noted that taxol alone (10 ~ g / m l )
247
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Fig. 5. Effect of taxol on the progesterone dose-response curve for germinal vesicle dissolution (GVD). Ranafollicle-enclosed oocytes (each of 5 females contributed 20 follicles per treatment) were incubated in wells containing 2 ml Ringer's, doses of progesterone (0.007, 0.01, 0.07, 0.1, 0.7, 1.0 ~ g / m l ) , in the presence (m) or absence (D) of taxol (10 ~ g / m l , 12 p,M) for 48 h at 22°C.
13
did not elicit GVM at either 24 or 48-h incubation (data not shown). To test the effect of taxol on viscoelasticity of ooplasm, centrifugation was used. Taxol (10 ~g/ml, 6-h incubation) was found to significantly inhibit centrifugation-induced GVM compared to vehicle controls and also significantly inhibited progesterone (1 /~g/ml) enhanced GVM induced by centrifugation (Fig. 3). This striking result suggests that taxol increases the apparent viscoelastic properties of the ooplasm and stabilizes the position of the GV within the oocyte.
Discussion
During amphibian oocyte meiotic maturation, chromatin must be transported close to the oocyte surface to permit normal polar body formation; some oocytes apparently accomplish this transport, at least in part, by GVM. A logical question one might ask relates to the mechanism and regulation of GVM, or, perhaps an even more basic question may be asked, what is responsible for positioning the GV inside the oocyte? It seems probable that the cytoskeleton, which plays such an important role in general cell organization in other cell types, would also be involved in oocyte organization. Cell nuclei generally are associated with cytoskeletal components which presumably anchor the nuclei in position (Franke, 1974). Unfortunately, the oocyte cytoskeleton has not been well studied and thus the relationship between the oocyte nucleus and the cytoskeleton is largely unexplored except for a few pioneering studies (Godsave et al., 1984a, b; Huchon et al., 1985; Jessus et al., 1986). This paper has investigated the possible relationship between GVM and the cytoskeleton, especially microtubules, by testing the hypothesis that cytoskeleton-active drugs and Fig. 6. Morphological effect of combined taxol (10 ~ g / m l , 12 /xM) and progesterone (1 /~g/ml, 3.2 ~M) on follicles after 48-h incubation. (A) Whole follicles containing oocytes which have undergone GVD, numerous cytasters (c) are visible as light-colored "starbursts" or streaks; bar = 500 A~m.(B) Paraffin section of one of the follicles in (A) showing large cytasters (c) near the oocyte surface, each 10 ALtosection typically contained several cytasters concentrated at the animal pole; bar = 100 p~m.
248 modulators of oocyte meiosis in Rana (i.e., progesterone and estradiol) might affect GV position within the oocyte. The present results have confirmed an earlier report that demecolcine induces GVM in Rana oocytes and that both demecolcine and progesterone enhance centrifugation-induced GVM (Lessman et al., 1986). In addition, estradiol was found to inhibit both types of GVM suggesting that this steroid may have a stabilizing effect on the viscoelastic properties of ooplasm. The present data did not indicate an estradiol-related change in GVM induced by centrifugation after estradiol treatment alone, but did show a resistance to changes in viscoelasticity normally observed with demecolcine or progesterone co-treatment. Estrogens have been implicated in yolk production and deposition (Wallace, 1985), it may be that the inhibitory GVM effects observed are in some way related to the participation of the cortical cytoskeleton in yolk uptake. It has been demonstrated previously that agents which disrupt cytoskeleton integrity such as colchicine and vinblastine also inhibit movement of proteins either into or out of the oocyte (Dumont and Wallace, 1972; Colman et al., 1981). It is important to note that steroids which elicit GVD also inhibit protein uptake into oocytes before GVD occurs (Schuetz et al., 1974) and induce changes in the oocyte surface (Schuetz, 1972). The data presented here indicate that progesterone has effects opposite to those of estradiol. Similarly, it has been reported that estradiol inhibits meiosis reinitiation by progesterone in denuded Rana oocytes (Lin and Schuetz, 1983). It may be that the inhibitory effects reported are due in part to the inhibitory effects of estradiol on GVM. The finding that estradiol inhibits the changes normally associated with centrifugationinduced GVM caused by progesterone supports this idea. The findings that nocodazole enhances GVM and centrifugation-induced GVM as well as increasing progesterone-induced GVD, strengthen the argument that microtubules are involved with GV positioning within the oocyte. Clearly, if two different compounds such as demecolcine and nocodazole have similar actions, it seems less likely that the observed effects are due to non-specific
drug actions. This is supported by work with other species. Colman et al. (1981) found that 2 mM colchicine elicited GVM in Xenopus oocytes, and Habibi and Lessman (1986a) found that demecolcine elicited GVM in goldfish (Carassius) oocytes. A further convincing piece of evidence is the effect of taxol or inhibiting centrifugation-induced GVM, with or without progesterone, and the suppression of progesterone-induced GVD. Thus three agents, all known to be active in destabilizing (nocodazole and demecolcine) or stabilizing (taxol) microtubules have consistent effects in this system. A fourth agent, vinblastine which is also a microtubule destabilizer, has been shown to have effects similar to those of nocodazole and demecolcine in the goldfish oocyte system (C.A.L., unpublished data) and in Xenopus oocytes (Colman et al., 1981). Morphological evidence is now needed to further elucidate the role of microtubules in oocyte maturation. Recent reports on oocyte cytoskeleton indicate a growing interest in this area (Wylie et al., 1985; Kuriyama et al., 1986). Microtubules do exist in fully grown amphibian oocytes before maturation (Dumont and Wallace, 1972; Colman et al., 1981; Heidemann et al., 1985; Huchon et al., 1985; Jessus et al., 1986); however, all agree that they exist in small numbers. Contrary to earlier reports (Heidemann and Gallas, 1980) that taxol did not induce tubulin polymerization in immature amphibian oocytes, recent evidence using sensitive methods indicate that some taxolinduced assembly of microtubules does occur in Xenopus oocytes (Heidemann et al., 1985) and Xenopus oocyte extracts (Jessus et al., 1985). Difficulties with morphological studies center on the ability to quantify microtubules and detect changes in the polymerized versus soluble tubulin within the oocyte. There appears to be consensus that tubulin is readily polymerized only after GVD (Heidemann and Kirschner, 1978; Jessus et al., 1984; Huchon and Ozon, 1985), and that a ~ansient microtubule-containing structure appears at the site of initial nuclear membrane rupture (Brachet et al., 1970; Huchon et al., 1981, 1985; Jessus et al., 1986). Therefore, the situation during maturation may be similar to that reported during early Rana egg cleavage in which polymeric tubulin showed changes with the cell cycle,
249 alternating between high levels of soluble and polymeric tubulin (Elinson, 1985). This would be consistent with the changes observed in EDs0 values for progesterone-induced GVD (i.e., increased by taxol and decreased by nocodazole). Thus it is hypothesized that microtubules present in the prematuration oocyte are depolarized to allow for partial GVM and that during GVD microtubules are again produced. Microtubules appearing at GVD might be involved with continued movement of nuclear components to the oocyte surface as suggested by Huchon et al. (1981, 1985) and Jessus et al. (1986). This hypothesis is supported by work on starfish oocytes by Schroeder and Otto (1984), since they report that fluorescently labeled microtubules are markedly reduced 25 rain after 1-methyl adenine treatment to induce GVD. Recently, cyclical changes in microtubules related to meiosis have been reported in Spisula oocytes (Kuriyama et al., 1986). Additional support for the concept that progesterone alters the viscoelastic properties of amphibian ooplasm comes from a study by Merriam (1971) in which Xenopus oocytes were tested for 'stratifiability' by centrifugation at 1 500-8 000 × g for 2 min. Progesterone increases stratifiability after 2-h incubation, well before G V D was observed. The results of this earlier study support the idea that progesterone affects the general organization of the oocyte, which presumably includes GV positioning and the cytoskeleton, soon after application. Antimitotic drugs such as demecolcine have been found to affect intermediate filaments associated with nuclei in somatic cells (Lin, 1981). Gerbil fibroma cells, treated with 1 /~M demecolcine for 24 h and stained with fluorescein-labelled antibodies to intermediate filaments, show characteristic 'capping'; of fibers which coil tightly around the nucleus (Lin, 1981). It is not clear whether the intermediate filament 'capping' is due to a direct action of demecolcine on the filaments or is a result of microtubule disruption. In view of these findings in somatic cells, it is tempting to speculate that the effect of demecolcine on oocyte GVM may be due to changes in intermediate filament arrangement as well as microtubule disruption. Intermediate filaments such as cytokeratin and vimentin have been described in the Xenopus
oocyte (Franz et al., 1983; Godsave et al., 1984a, b; Wylie et al., 1985). Microtubule poisons such as the ones used in the present study may have non-specific or even toxic effects especially at high doses (Dustin, 1984). No evidence of toxic effects was obtained in this study since progesterone continued to elicit GVD in the presence of the microtubule poisons. In an earlier study (Lessman et al., 1986), electrophysiological measurements of Rana oocyte membrane potential and resistance after demecolcine treatment (270/xM) indicated no significant change in membrane function, thus indicating the absence of toxic effects even at a relatively high dose of demecolcine. Although the consistent effects obtained with the three microtubule poisons used strongly indicate microtubule-specific action, definitive demonstration that microtubules are being specifically affected must come from future morphological studies. It seems unlikely that depolymerization of microtubules alone can account for the active migration of the GV and yet two agents which destabilize microtubules, i.e. nocodazole and demecolcine, result in GVM. Thus it may be necessary to consider the possibility that separate active agents exist, such as microfilaments, which are responsible for the migration. In studies of GVM in the goldfish oocyte, cytochalasin B was found to inhibit steroid-induced GVM (Habibi and Lessman, 1985). The mitochondrial poison, 2,4-dinitrophenol was also found to inhibit GVM in goldfish oocytes (Habibi and Lessman, 1986b), suggesting that GVM is an energy-dependent event. The energy requirement would be consistent with the idea that microfilaments were involved. Furthermore, the maturation-inducing substance, 1-methyl adenine, produced marked changes in the actincontaining cytoskeleton of the maturing starfish oocyte (Schroeder, 1985). Thus, it may be postulated that during maturation, microtubules destabilize while microfilaments become increasingly active with the net result being GVM. Clearly, morphological studies are required to elucidate the relationship of the cytoskeleton to GVM. Studies which explore several types of cytoskeletal elements (i.e., microtubules, microfilaments and intermediate filaments) with respect to GV positioning would be particularly welcome.
250
Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada (operating grant A8397). I acknowledge a gift of taxol from Dr. Matthew Suffness of the National Cancer Institute, NIH. I thank Barbara Pluta and Debra Henke for technical assistance and Anne-Louise MacDonald for animal care. The curve-fitting programs Allfit and Grafit used to generate Fig. 5 were a gift of the Biomedical Computing Technology Information Center, Nashville, TN.
References Brachet, J., F. Hanocq and P. Van Gansen: A cytochemical and uhrastructural analysis of in vitro maturation in amphibian oocytes. Dev. Biol. 21, 157-195 (1970). Brown, B.W.: Quantal response assays, In: Statistics in Endocrinology, eds. J.W. McArthur and T. Colton (MIT Press, Cambridge) pp. 129-143 (1970). Colman, A., J. Morser, C. Lane, J. Besley, C. Wylie and G. Valle: Fate of secreto~ proteins trapped in oocytes of Xenopus laevis by disruption of the cytoskeleton or by imbalanced subunit synthesis. J. Cell Biol. 91, 770-780 (1981). Downie, N.M. and R.W. Heath: Basic Statistical Mcthods, 2nd eds. (Harper and Row, New York) pp. 146-153 (1965). Dumont, J.N. and R.A. Wallace: The effects of vinblastine on isolated Xenopus oocytes. J. Cell Biol. 53, 605-610 (1972). Dustin, P.: Microtubule poisons. In: Microtubules (SpringerVerlag, New York) pp. 171-233 (1984). Elinson, R.P.: Changes in levels of polymeric tubulin associated with activation and dorsoventral polarization of the frog egg. Dev. Biol. 109, 224-233 (1985). Franke, W.W.: Structure, biochemistry., and function of the nuclear envelope. Int. Rev. Cytol. (suppl. 4), 71-236 (1974). Franz, J.K., L. Gall, M.A. Williams, B. Picheral and W.W. Franke: Intermediate-size filaments in a germ cell: Expression of cytokeratins in oocytes and eggs of the frog Xenopus. Proc. Natl. Acad. Sci. USA 80, 6254-6258 (1983). Godsave, S.F., B.H. Anderton, J. Heasman and C.C. Wylie: Oocytes and early embryos of Xenopus laet,is contain intermediate filaments which react with anti-mammalian vimentin antibodies. J. Embryol. Exp. Morphol. 83,169-187 (1984a). Godsave, S.F., C.C. Wylie, E.B. Lane and B.H. Anderton: Intermediate filaments in the Xenopus oocyte: The appearance and distribution of cytokeratin-containing filaments. J. Embryol. Exp. Morphol. 83, 157 167 (1984b). Habibi, H.R. and C.A. Lessman: Effects of cytochalasin B on steroid-induced oocyte meiosis, and centrifugally-induced
nuclear movement in goldfish (Carassius auratus). Can. J. Biochem. Cell Biol. 63, 743-751 (1985). Habibi, H.R. and C.A. Lessman: Effect of demecolcine (colcemid) on goldfish oocyte meiosis in vitro. Gamete Res. 13, 103-114 (1986a). Habibi, H.R. and C.A. Lessman: A study of goldfish oocyte meiosis in vitro: effects of 2,4-dinitrophenol and adenosine5-triphosphate. Fish Physiol. Biochem. (in press) (1986b). Heidcmann, S.R. and P.T. Gallas: The effect of taxol on living eggs of Xenopus laet,is'. De,,,. Biol. 80, 489-494 (1980). tteidemann, S.R. and M.W. Kirschner: Aster formation in eggs of Xenopus laet,is. Induction by basal bodies. J. Cell Biol. 67, 105-117 (1975). Heidemann, S.R. and M.W. Kirschner: Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation. J. Exp. Zool. 204, 431-444 (1978). Hcidemann, SR., M.A. Hamborg, J.E. Balasz and S. Lindley: Microtubules in immature oocytes of Xenopus laevis. J. Cell Sci. 77, 129-141 (1985). Huchon, D. and R. Ozon: Microtubules during germinal vesicle breakdown (GVBD) of Xenopus oocytes: effect of Ca 2+ ionophore A-23187 and taxol. Repro& Nutr. Dev. 25,465-479 (1985). Huchon, D., N. Crozet, N. Cantenot and R. Ozon: Germinal vesiclc breakdown in the Xenopus laevis oocyte: description of a transient microtubular structure. Rcprod. Nutr. Dev. 21,135 148 (1981). Huchon, D., C. Jessus and R. Ozon: hnmunofluoresccnce distribution of microtubules during the first meiotic cell division of the Xenopus [aet#s oocyte. C.R. Acad. Sc. Paris Ser. III 12, 463-465 (1985). Jcssus, C., D. Huchon, E. Fricderich, J. Francon and R. Ozon: Interaction between rat brain microtubule associated proteins (MAPs) and free ribosomes from Xenopus oocyte: a possible mechanism for the in ovo distribution of MAPs. Cell Differ. 14, 295-30l (1984). Jessus, C., C. ThiNer and R. Ozon: Identification of microtubule-associated proteins (MAPs) in Xenopus oocyte. FEBS Lett. 192, 135 140 (1985). Jcssus, C., D. Huchon and R. Ozon: Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study. Biol. Cell 56, 113-120 (1986). Kuriyama, R., G.(}. Borisy and Y. Masui: Microtubule cycles in oocytes of the surf clam, Spisula solidissima: .An immunofluorescence study. Dcv. Biol. 114, 151-160 (1986). Lessman, C.A. and A.W. Schuetz: Role of follicle wall in meiosis reinitiation induced by insulin in Rana pipiens oocytes. Am. J. Physiol. 241, E51-E56 (1981). Lessman, C.A., W.S. Marshall and H.R. Habibi: Movement and dissolution of the nucleus (germinal vesicle) during Rana oocyte meiosis: Effect of demecolcine (colcemid) and ccntrifugation. Gamete Res. 14, 11-23 (1986). Lin, J.J.C.: Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc. Natl. Acad. Sci. USA 78, 2335-2339 (1981).
251 Lin, Y.W.P, and A.W. Schuetz: In vitro estrogen modulation of pituitary and progesterone-induced oocyte maturation in Rana pipiens. J. Exp. Zool. 226, 281-291 (1983). Makino, S.: A cytological study on the maturation and fertilization of the egg of ttvnobius retardatus (an urodelan amphibian). J. Fac. Sci. Hokkaido Univ. Ser. VI Zool. 3, 115-167 (1934). Maller, J., D. Poccia, D. Nishioka, P. Kidd, J. Gerhart and H. Hartman: Spindle formation and cleavage of Xenopus eggs injected with centriole-containing fractions from sperm. Exp. Cell Res. 99, 285-294 (1976). Masui, Y.: Distribution of the cytoplasmic activity inducing germinal vesicle breakdown in frog oocytes. J. Exp. Zool. 179, 365-378 (1972). Merriam, R.W.: Progesterone-induced maturational events in oocytes of Xenopus laevis. II. Change in intracellular calcium and magnesium distribution at germinal vesicle breakdown. Exp. Cell Res. 68, 81-87 (1971). Schiff, P.B., J. Fant and S.B. Horwitz: Promotion of microtubule assembly in vitro by taxol. Nature 277, 665-667 (1979). Schroeder, T.E.: Cortical expressions of polarity in the starfish oocytc. Dev. Growth Differ. 27, 311-321 (1985). Schroeder, T.E. and J.J. Otto: Cyclic assembly disassembly of cortical microtubules during maturation and early development of starfish oocytes. Dev. Biol. 103,493-503 (1984).
Schuetz, A.W.: Induction of structural alterations in the preovulatory amphibian ovarian follicle by hormones. Biol. Reprod. 6, 67-77 (1972). Schuetz, A.W. and C.A. Lessman: Role of the surface epithelium and follicle wall in ovulation and progesterone production by frog (Rana pipiens) follicles. Differentiation 22, 79-84 (1982). Schuetz, A.W., R.A. Wallace and J.N. Dumont: Steroid inhibition of protein incorporation by isolated amphibian oocytes. J. Cell Biol. 61, 26-34 (1974). Wallace, R.A.: Vitellogenesis and oocyte growth in nonmammalian vertebrates, In: Development Biology, Vol. 1, ed. L.W. Browder (Plenum, New York) pp. 127-177 (1985). Wani, M.C., H.L. Taylor, M.E. Wall, P. Coggon and A.T. McPhail: Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93, 2325-2327 (1971). Wylie, C.C., D. Brown, S.F. Godsave, J. Quarmby and J. Heasman: The cytoskeleton of Xenopus oocytes and its role in development. J. Embryol. Exp. Morphol. (suppl.) 89, 1-15 (1985).
239
C D F 00413
Germinal vesicle migration and dissolution in Rana pipiens oocytes: effect of steroids and microtubule poisons Charles A. L e s s m a n Department ~[ Biolow. St. Franci.~ Xat,ier Unit,er~itv. A nti~onish, Nm,a Scotia, ('anada (Accepted 6 October 1986l
Germinal vesicle migration (GVM) as evidenced by the appearance of the germinal vesicle at the animal pole surface was induced by nocodazole and demecolcine (colcemid). Nocodazole significantly lowered the progesterone EDs0 for germinal vesicle dissolution (GVD). Both demecolcine and nocodazole enhanced centrifugation-induced GVM (i.e., lowered ooplasmic viscoelasticity) after 6-h incubation, and both potentiated the effect of progesterone in this assay. Estradiol, by contrast, inhibited GVM induced by demecolcine in both follicle-enclosed and denuded oocytes. Estradiol was also found to inhibit the normal enhancement of centrifugation-induced GVM by demecolcine or progesterone. Taxol was found to have effects that were generally opposite to those of demecolcine and nocodazole. Taxol inhibited centrifugation-induced GVM either alone or in the presence of progesterone, in addition, taxol significantly increased the progesterone EDs0 for GVD induction. Taken together the available data support the hypothesis that microtubules play a role in maintaining the internal position of the germinal vesicle in the prematuration oocyte and that changes occur in the oocyte cytoskeleton during maturation.
Meiosis; Meiotic maturation; Taxol; Colcemid; Nocodazole; Estradiol; Cytoskeleton; Tubulin
! ntroduction During oocyte maturation in the amphibian
Rana pipiens, the chromatin of the germinal vesicle (GV) must be moved from a position well within the oocyte to a position very close to the animal pole surface in order to produce the unequal division leading to polar body formation. Migration of the GV during oocyte maturation in amphibians was described by Makino (1934) in a
Correspondence address: Dr. Charles A. Lessman, Box 30, St. Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 1C0.
classic study, and has been described more recently by Brachet et al. (1970) and Lessman et al. (1986). In fact, the process of moving the GV or its contents must occur in virtually all animal oocytes, since the production of polar bodies seems to be a universal phenomenon. How does the GV or its contents move during oocyte maturation? One possibility is that cytoskeletal elements actively move the contents of the GV as an intact unit, or as components after GV breakdown or dissolution (GVD), to the oocyte surface. Another possibility would also involve cytoskeletal elements but only in a passive way, such that the GV is restrained or held in position by the cytoskeleton, and during maturation this restraint is
0045-6039/87/$03.50 ~' 1987 Elsevier Scientific Publishers Ireland, Ltd.
240 released. In this second possibility, the active force might come from other factors, for example changes in ooplasmic density causing the GV to float toward the surface. A third possibility, which combines the first two, would involve a reorganization of the cytoskeleton so that elements stabilizing the position of the GV would change along with activation of others involved in the migration of the GV or its contents. All of the possibilities given here are contingent on the presence of cytoskeletal elements in oocytes. The amphibian oocyte does indeed possess a cytoskeleton (Wylie et al., 1985). Recent evidence indicates that microtubules are present in immature, fully grown amphibian oocytes (Heidemann et al., 1985; Huchon et al., 1985; Jessus et al., 1986) and confirms earlier reports (Dumont and Wallace, 1972; Colman et al., 1981). Nevertheless, for some time it was presumed that immature Xenopus oocytes did not contain microtubules since basal body or centrosome injection (Heidemann and Kirschner, 1975; Maller et al., 1976; Heidemann and Kirschner, 1978); and taxol treatment (Heidemann and Gallas, 1980; Huchon and Ozon, 1985; Jessus et al., 1986) failed to induce detectable levels of microtubules in spite of high tubulin pools. A recent paper (Jessus et al., 1985) using [35S] methionine labeled oocytes reported that taxol did induce tubulin assembly in immature oocyte extracts in vitro. In a previous paper from this laboratory (Lessman et al., 1986), demecolcine (colcemid), a potent inhibitor of tubulin polymerization (Dustin, 1984), was found to induce GV migration (GVM) in R. pipiens oocytes and to significantly reduce the centrifugal force required to move the GV. These results suggest that microtubules are involved with GV positioning within the oocyte and support the second and third possibilities given above. The present paper puts forth additional evidence in support of this hypothesis which includes use of another microtubule destabilizing agent, nocodazole, and the microtubule stabilizing agent, taxol (Schiff et al., 1979) which has antimitotic activity (Wani et al., 1971). Two steroids, progesterone and estradiol, known to affect meiotic maturation in Rana oocytes in opposing ways (Lin and Schuetz, 1983) were also tested for effects on GV positioning.
Materials and Methods
Animals Adult female leopard frogs (R. pipiens) were obtained from Lemberger Assoc. (Oshkosh, WI) and held in artificial hibernation at 4°C in trays containing 10% amphibian Ringer's. Ringer's solution was changed in the trays at least three times per week. Just before use, animals were stunned by a blow to the head and killed immediately by decapitation. The spinal cord was pithed, and the ovaries were dissected and placed in cold Ringer's.
Follicle and oocyte preparation Type I follicles (intact, fully grown follicles) as defined by Schuetz and Lessman (1982) were dissected from other ovarian tissue with watchmaker forceps. When type IV follicles (denuded oocytes without follicle cells; Schuetz and Lessman, 1982) were required, the follicle wall was removed by microdissection from type I follicles, and the follicle cells were removed by shaking for 2 h in Ca2+-free Ringer's (Lessman and Schuetz, 1981). The resulting denuded oocytes were rinsed several times with normal Ringer's and allowed to incubate for at least 1 h in Cae+-containing Ringer's (Lessman and Schuetz, 1981) prior to use. Type I and IV follicles were inspected, using a stereoscope, for damage and, in the case of type IV, the absence of follicle cells. Groups of 20 follicles were assigned randomly to treatment wells containing 2 ml Ringer's in a 24-well culture dish (CoStar no. 3524). Incubations were carried out in darkness at 22°C in a thermostatically controlled incubator).
Assay for germinal vesicle dissolution and migration Meiosis reinitiation was scored after fixing the follicles by brief boiling or by hot trichloroacetic acid (15%, 60°C). The presence or absence of the oocyte nucleus or GV was determined by dissection; absence of the otherwise conspicuous GV is defined as GV breakdown or dissolution (GVD) and denotes meiosis reinitiation. Prior to dissection, follicles were also scored for the presence of a GV at the animal pole surface (Lessman et al., 1986), oocytes containing such externally visible GVs were scored as having undergone GV migra-
241 tion ( G V M ) (Fig. 1). In those oocytes scored as positive for G V M , the presence of the presumed G V was verified during dissection at the time of G V D assay (Fig. 1). Often the GV would only be partially visible with some pigmented cortical cytoplasm remaining above the G V (Fig. 1). In some experiments, the presence of a transient white spot in the animal pole was scored; subsequent dissection indicated that the presence of the white spot correlated perfectly with the absence of an intact GV.
Centrifugation-induced GVM assay Details of this assay have been described previously (Lessman et al., 1986). Briefly, after incubation, follicles were placed at the interphase between Ringer's and 40% Ficoll (Sigma Chem. Co.) in Ringer's and centrifuged for 5 min at 0, 35, 250, 500 or 1 000 × g. Immediately after centrifugation, the cells were fixed in hot 15% trichloroacetic acid and scored for G V M and G V D as described above. The G V M scored after centrifugation appeared
similar to that induced by nocodazole or demecolcine (Fig. 1 and Lessman et al., 1986) with the exception that centrifuged cells exhibiting G V M tended to have less pigmented cytoplasm trapped above the GV, especially at the higher centrifugal forces.
Histological preparations Follicles were fixed in Bouin's fluid, dehydrated in an alcohol series, cleared in xylene and embedded in paraffin. Serial sections were cut at 10 txm, and the resulting sections were stained with Delafield's hematoxylin and congo red.
Chemicals Progesterone and estradiol-17/~ were obtained from Steraloids Inc. and were dissolved in steroid vehicle (propylene g l y c o l / E t O H , 1 : 1). Demecolcine (N-desacetyl-N-methylcolchicine; colcemid) was obtained from Sigma Chem. Co. and was also dissolved in steroid vehicle. N o c o d a z o l e was obtained from Aldrich Chem. Co. and was dissolved
A
~
iiiiiii
Fig. 1. A typical follicle-enclosed oocyte with GVM elicited by 48-h incubation with nocodazole (0.5 ~g/ml, 1.7 I~M); the follicle has been fixed with trichloroacetic acid (15% in Ringer's) at 60°C which increases the visibility of the otherwise clear GV. (A) Micrograph of an entire follicle, the GV is present at the oocyte surface and extends between the arrow tips. Bar = 500 t~m. (B) Same follicle as in (A) but hemisected parallel to the animal-vegetal axis showing GV at animal pole surface, small arrows indicate areas where the GV is externally visible from the oocyte surface. Bar = 500 /~m.
242
in dimethyl sulfoxide (DMSO) and steroid vehicle (1 : 1). Taxol (lot FB1046) was a gift of Dr. Surfness ( N I H ) and was dissolved in D M S O just prior to use. The final concentration of vehicle in the incubation medium was < 0.1%; control oocytes also received the appropriate vehicle.
Statistical analysis Quantal data such as that generated by G V M and G V D scoring were analyzed by a paired test of proportions based on binomials (Downie and Heath, 1965). Significance was assigned at P < 0.05 and was indicated by superscripts associated with data.
Results
Effects of demecolcine, estradiol, and progesterone Demecolcine (colcemid) was found to induce GV migration (GVM) in a dose-related manner (Table I) in Rana follicle-enclosed oocytes which confirmed earlier findings (Lessman et al., 1986). G V M continued to occur in the interval between 24- and 48-h incubation indicating the relatively slow nature of demecolcine-induced G V M (Table I). The lowest dose of demecolcine failed to elicit a significant effect compared to vehicle controls at either 24- or 48-h incubation. This was in contrast to our previous finding (Lessman et al., 1986) that
demecolcine at 0.1 /~g/ml was moderately effective; the difference may be due to interanimal variation. It should be noted that the near maximal GVM induced by demecolcine (10/xg/ml, 27 /~M) at 24 h coincided with the appearance of a white spot in the animal pole of progesteronetreated cells (Table I), indicating a similar time course for both events. Although progesterone did not induce significant G V M scored as an intact GV at the oocyte surface, there were significant numbers of oocytes at 24 h which exhibited a white spot at the animal pole (Table I). The transient white spot has been associated with remnants of the GV at the oocyte surface and has been used by some researchers to score meiosis reinitiation or G V dissolution (GVD). Thus progesterone was responsible for movement of some G V material, if not the entire GV, to the oocyte surface. An earlier study (Masui, 1972) reported that Rana oocytes in which the GV was displaced to the vegetal pole by centrifugation, underwent G V M toward the animal pole after progesterone treatment. In order to obtain additional baseline information regarding G V M induced by progesterone, serial sections of follicles incubated with 1 /~g/ml progesterone for 0, 16.5, 18, 24, 32 and 48 h were prepared from three different females. Control oocytes throughout the incubation period had spherical GVs well below the oocyte surface; the nucleoli were quite distinct in all control GVs
TABLEI E f f e c t o f e s t r a d i o l - l ~ ondemecolcine(colcemid) induced GVM infollicle-enclosedoocytesof Treatment
Estradiol (/x g / m l ) 0.0
1.0
0.0
22 * 5 5 6 -
61 38 4 5 2
GVM (24 h) Demecolcine 10 ~ g / m l (27~M) Demecolcine 1 # g / m l (2.7 p,M) Demecolcine 0.1 t~g/ml (0.27/~M) Control (vehicle only) Progesterone 1 p,g/ml
R.p~iens
56 (0) 10 (0) 3 (0) 1 (0) 4 (29)
1.0
GVM (48 h)
0.0
1.0
GVD (48 h) 32 * 18 * 6 6
1 0 0 0 80
0 0 0 0 -
GVM data presented as number of oocytes with a germinal vesicle at their animal pole surface of 100 oocytes tested per treatment. Twenty follicles from each of five females were used per treatment. Numbers in parentheses represent number of oocytes with white spot at animal pole. GVD data presented as number of oocytes without a GV upon dissection of 100 oocytes tested per treatment. Incubation conditions: 22°C, 2 ml Ringer's containing 20 follicles per well. Data pairs were tested for statistical differences using a test of proportions based on binomials (Downie and Heath, 1965). An asterisk indicates a significant difference at P < 0.05 from demecolcine alone.
243
(Fig. 2A). By 16.5 h incubation, the GVs of progesterone-treated oocytes began to exhibit marked changes which resemble those reported during Xenopus oocyte GVD (Brachet et al., 1970; Huchon et al., 1981, 1985; Jessus et al., 1986). The GVs became flattened and were closer to the oocyte surface (i.e., they exhibited partial GVM); they contained fibrillar material, especially at the basal side, and the nucleoli disappeared (Fig. 2B). Since the GV could be isolated by dissection at
this stage, GVB by strict definition had not occurred. At 18-h incubation with progesterone, the nuclear envelope disappeared, the fibrillar material of the disrupted GV radiated from a dense band of material toward the oocyte surface (Fig. 2C). G V D had occurred by definition at 18-h incubation, since the GV at this stage could not be dissected and isolated from the oocyte. However, since pigmented cytoplasm remains between the GV remnants and the oocyte surface, even though
D
Fig. 2. A series of micrographs depicting meiotic maturation induced by progesterone in R. pipiens follicle-enclosed oocytes in paraffin sections (10 p,m thick). (A) Control follicle after 48-h incubation, GV remains spherical and well below the oocyte surface, numerous nucleoli (n) are visible; bar = 100 /~m. (B) Follicle after 16.5-h incubation with progesterone (1 p,g/lnl, 3.2 /~M), (iV contents are fibrillar especially at basal or vegetal side and nucleoli are indistinct; magnification same as (A). The GV has moved closer to the oocyte surface. (C) Follicle after 18-h incubation with progesterone (1 ~ g / m l ) , nuclear envelope no longer visible thus by definition GVD has occurred. The area of the GV is occupied by fibrillar material radiating from a dense band of material; the GV contents extend nearly to the oocyte surface, separated only by a thin layer of pigmented cortical cytoplasm: magnification same as (A). (D) Follicle after 48-h incubation with progesterone (1 ~ g / m l ) showing a metaphase spindle (s) at oocyte surface (unstained section); bar = 50 /~m.
244 the G V has moved closer to the surface (i.e., partial GVM), scorable G V M as defined earlier would not be detected. Beginning at this time a transient white spot could be seen in the animal pole surface of whole oocytes (Tables I and II). By 48 h a metaphase spindle was visible at the oocyte surface, thus during the 48-h incubation period partial G V M had occurred along with G V D in response to progesterone (Fig. 2D). Nevertheless, since G V D occurred prior to complete G V M defined as the G V visible at the oocyte surface, it was not surprising that progesterone did not elicit scorable GVM. Estradiol (1 / l g / m l , 3.6 /zM) was found to significantly inhibit G V M induced by demecolcine at b o t h 24- and 48-h incubation (Table I). Only progesterone elicited significant G V D in this experiment, neither demecolcine nor estradiol were active alone in inducing G V D . In order to determine whether the inhibitory effect of estradiol on demecolcine-induced G V M was related to the follicle wall, the experiment was repeated using d e n u d e d oocytes (type IV follicles; Schuetz and Lessman, 1982) which lack follicle wall components. Estradiol significantly inhibited demecolcine-induced G V M at 48-h incubation in the absence of the follicle wall (Table II). The response to demecolcine was slower and of less magnitude than in the previous experiment; in addition, the white spot was present in some progesterone-treated cells at 48 h. Thus both events appeared to follow the same retarded time course. This m a y be due to inter-animal differences or could be attributable to the presence or absence of the follicle wall, in any case the results of both experiments (Tables I and II) are qualitatively the same, i.e., estradiol inhibited demecolcine-induced G V M in the presence or absence of the follicle wall. In order to directly test the effect of estradiol on ooplasmic viscoelasticity changes, the procedure of oocyte centrifugation (Merriam, 1971; Masui, 1972; Lessman et al., 1986) was used. Previous work had established that progesterone and demecolcine were both capable of enhancing centrifugation-induced G V M (Lessman et al., 1986); the present findings confirm this earlier report (Fig. 3). Estradiol alone (1 /~g/ml) had no effect on centrifugation-induced GVM, but did significantly inhibit demecolcine's usual enhance-
TABLE II Effect of estradiol-17,8 on demecolcine (colcemid) induced GVM in denuded oocytes of R. pipiens Treatment Demecolcine 10 8g/ml (27~M) Demecolcine 10 t~g/ml plus estradiol 1/~g/ml (3.6~M) Progesterone 1/xg/ml (3.2 /LM) Estradiol 1 #g/ml (3.6#M) Control (vehicle only)
GVM
GVD
24 h
48 h
48 h
14 (0)
48 (0)
0
6 (0)
"18 (0)
1
0 (24)
0 (12)
90
1 (0) 1 (0)
1 (0) 1 (0)
0 0
GVM data presented as number of oocytes with externally visible GV of 100 tested per treatment. Numbers in parentheses indicate oocytes with a white spot in animal pole. Twenty oocytes from each of five females were used per treatment. GVD data presented as number of oocytes lacking a GV upon dissection of 100 tested per treatment. Incubation conditions: 22°C, 20 follicles per well containing 2 ml Ringer's. GVM data pairs for demecolcine were statistically compared using a test of proportions based on binomials (Downie and Heath, 1965). The asterisk denotes the estradiol-demecolcine treatment which is significantly lower than the respective control (demecolcine alone) at P < 0.05.
ment of centrifugation-induced G V M (Fig. 3). A similar experiment was carried out to determine whether estradiol had any effect on the progesterone-induced changes observed in centrifugation-induced GVM. The results indicated that estradiol (1 t~g/ml) significantly inhibited the usually observed enhancement of centrifugation-induced G V M by progesterone at 4-h incubation (Fig. 4). Masui (1972) had previously shown that R a n a oocytes subjected to centrifugal forces of 2 0 0 - 4 0 0 x g remained capable of responding to progesterone. Since somewhat higher forces were used in this study, the responsiveness of oocytes to progesterone was tested after centrifugation at 1 000 x g for 5 min. Over 75% of the centrifuged oocytes had G V M before incubation; after 24-h incubation G V M remained high in the vehicle control group (i.e., no evidence of G V recoil to original position), while G V D was near maximal in the progesterone treated group (data not shown). This latter finding along with that of Masui (1972)
245
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Fig. 3. Effect of cytoskeleton-active agents on centrifugationinduced G V M in Rana follicle-enclosed oocytes at 6-h incubation. Data presented as number of follicles containing oocytcs with germinal vesicles visible at the animal pole after hot trichloroacetic acid fixation of 30 follicles tested per treatment (replicated with three females, 10 follicles per female): a total of 1500 oocytes were used in this experiment. Treatments: VC, vehicle control ( E t O H / p r o p y l e n e glycol/DMSO, 1.5 : 1.5 : 3); D, demecolcine 10 ~ g / m l (27 p,M); E, estradiol-17fl 1 ~tg/ml (3.6 ~M): D + E , demecolcine 10 ' a g / m l and estradiol 17fl 1 ~tg/ml; P, progesterone 1 t*g/ml (3.2 'aM); D + P, demecolcine 10 p~g/ml and progesterone 1 ' a g / m l ; N, nocodazole 0.5 ~tg/ml (1.7 /*M): N + P , nocodazole 0.5 p.g/ml and progesterone 1 ' a g / m l ; T, taxol 10 ,ag/ml (12 ~M): T + P , taxol 10 , a g / m l and progesterone 1 ,ag/ml. Unccntrifuged follicles which were placed on Ficoll cushions after treatment were without G V M and are indicated by D; @ . . . . . . • = 35 × g; .... ~=250×g; + .... + =500×g; • I=1000 × g. Arrows denote significant difference from respective vehicle control (i.e., same centrifugation speed) at P < 0.05 using a test of proportions based on binomials (Downie and Heath, 1965). Follicles were centrifuged for 5 min at the interface between 40% Ficoll and amphibian Ringer's using a modification of a technique described by Masui (1972).
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Fig. 4. Effect of estradiol (1 tzg/ml, 3.7/~M) on progesterone (1 ~ g / m l , 3.2 ,aM) enhancement of centrifugation-induccd GVM. C, control (vehicle only): P, progesterone: E, estradiol: B, both steroids added simultaneously. Follicles were incubated for 4 h after steroid addition. Rana follicle-enclosed oocytes (each of three females contributed 20 follicles per treatment) were placed at the interphase of 40% Ficoll and Ringer's and centrifuged in a swinging bucket rotor for 5 rain. Follicles were immediately fixed in hot (60°C) trichloroacetic acid and scored for the presence of a germinal vesicle visible at the animal pole surface without dissection. Asterisk denotes statistically significant difference from other treatment groups at that centrifugal force using the test of proportions (Downie and Heath, 1965).
mecolcine treatment (Fig. 1 and Lessman et al., 1986). G V M elicited by both drugs was always to the animal pole regardless of the orientation of the oocyte to gravity. Nocodazole was somewhat more active (EDs0 = 0.7/.tM) than demecolcine (EDs0 =
T A B L E 11I
indicates that centrifugation at the forces used does not disorganize the oocyte to the point of loss of hormone responsiveness. Effects of nocodazole
If demecolcine was acting on microtubules to elicit G V M then other agents affecting microtubules should exhibit effects on GVM. Thus nocodazole which also acts to destabilize microtubules (Dustin, 1984) was tested for GVM-inducing activity. The results show a dose-dependent effect on G V M (Table III); increasing doses of nocodazole (0.05-5 /.tg/ml) were associated with increased GVM, but nocodazole alone did not elicit GVD. N o c o d a z o l e induced G V M which was indistinguishable from that obtained with de-
Effect of nocodazole on GVM and G V D in follicle-enclosed oocytes of R. pipiens Treatment Nocodazole 5 ' a g / m l (17'aM) Nocodazole 0.5 p,g/ml (1.7'aM) Nocodazole 0.05 g g / m l (0.17/.tM) Control (vehicle only)
GVM (24 h)
GVM (48 h)
GVD (48 h)
41 (4-12)
56 (6-16)
0
30(2-11)
44(1-13)
0
1 (0-1) 0
3 (0-1) 0
0 0
G V M data presented as percentage of oocytes with a germinal vesicle visible externally (80 oocytes at 24 h and 100 oocytes at 48 h were tested per treatment). Twenty follicles from each of 4 5 females were used per treatment: numbers in parentheses arc ranges of response (out of 20 follicles tested) for individual females. G V D data presented as the number of oocytes which lack a (iV upon dissection of 100 tested per treatment.
246 0.9/~M) in eliciting GVM. Nocodazole (0.5/~g/ml, 1.7 /xM) induced G V M in over 50% of oocytes containing a GV (Table IV) in the presence of progesterone (0-0.7 ~ g / m l ) . Increasing doses of progesterone were associated with increased G V D at 48-h incubation indicating that oocytes remained viable and capable of responding to progesterone in the presence of nocodazole. C o l m a n et al. (1981) reported that 10 ~ M nocodazole was toxic to Xenopus oocytes. By contrast, Elinson (1985) found that Xenopus eggs treated with nocodazole (10/~g/ml, 33/~M) could be activated and showed cortical contractions, suggesting low toxicity even at a relatively high dose. In any case, species differences and the doses used may account for any toxicity observed in previous studies. The relatively low dose of 1.7 /~M nocodazole (0.5 /xg/ml) significantly decreased the progesterone EDs0 (Table IV) from 0.23 # g / m l (95% confidence limits = 0.20 0.27) to 0.13 ~ g / m l (95% confidence limits = 0.10-0.16) calculated by the S p e a r m a n - K a r b e r procedure (Brown, 1970). This enhanced progesterone activity further indicated the absence of toxic effects by this dose of
TABLE IV The effect of nocodazole and several doses of progesterone on GVM and GVD in follicle-enclosed oocytes of R. pipiens Progesterone ( ~ g/ml) 1.0 (3.2 gM) 0.7 0.1 0.07 0.01 0.007 0 Control
Nocodazole (/Lg/ml) 0.0 0.5 GVM
GVD
GVM
GVD
0/11 0/30 6/88 1/93 2/100 0/100 1/100
89 70 12 7 0 0 0
3/14 "9/15 *48/73 *54/79 *55/94 "51/99 "51/99
86 **85 **27 *'21 **6 1 1
GVM data presented as number of oocytes with a germinal vesicle visible externally (numerator) of the total number with intact germinal vesicles (denominator) after 48-h incubation. GVD data are presented as the number of oocytes which lack a germinal vesicle upon dissection of 100 tested per treatment. Twenty follicles from each of five females were used per treatment. Both types of data were collected after 48-h incubation at 22° C. Asterisks indicate significant differences at P < 0.05 with and without nocodazole using a test of proportions based on binomials (Downie and Heath, 1965).
nocodazole. Although there does not appear to be an enhancement of G V M by the combination of nocodazole and progesterone, it should be pointed out that increasing G V D may mask any additive or synergistic effects. In order to test the possibility that nocodazole and progesterone act synergistically on GVM, centrifugation was used at 6-h incubation to induce G V M without the complication of G V D . The results indicated that at 35 × g, and especially at 250 × g, nocodazole and progesterone acted synergistically to enhance centrifugation-induced G V M (Fig. 3). As in the case of demecolcine and progesterone, nocodazole enhanced G V M induced by centrifugation after short term incubation (i.e., 6 h) before G V M or G V D would otherwise have occurred. These results indicate that nocodazole, as well as demecolcine and progesterone, decrease the apparent viscoelastic properties of ooplasm thus allowing the GV to be moved more readily.
Effects of taxol Taxol has been demonstrated to stabilize microtubules and p r o m o t e tubulin assembly into microtubules (Schiff et al., 1979; H e i d e m a n n and Gallas, 1980). Taxol (10 # g / m l , 12 /_tM) significantly increased the EDs0 of progesterone-induced G V D (Fig. 5) from 0.15 ~ g / m l (95% confidence l i m i t s = 0 . 1 3 - 0 . 1 8 ) to 0.22 ~ g / m l (95% confidence limits = 0.18-0.26). In addition, taxol-progesterone treated oocytes which underwent G V D showed a distinctive streaked or 'starburst' pattern especially visible in the animal pole (Fig. 6A). Such oocytes when sectioned contained m a n y extensive cytaster arrays (Fig. 6B). Oocytes which did not undergo G V D under the influence of both agents or those treated with taxol alone did not exhibit the cytasters (data not shown). Nevertheless, it could not be ruled out that additional microtubules, not visible at the light microscope level, were induced by taxol. Thus cytaster formation was dependent u p o n G V D and t a x o l - p r o gesterone treatment. Taxol was not toxic since it shifted the progesterone dose-response curve to the right (i.e., significantly increased the EDso for progesterone-induced GVD), rather than producing total inhibition of progesterone action. It should also be noted that taxol alone (10 ~ g / m l )
247
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Fig. 5. Effect of taxol on the progesterone dose-response curve for germinal vesicle dissolution (GVD). Ranafollicle-enclosed oocytes (each of 5 females contributed 20 follicles per treatment) were incubated in wells containing 2 ml Ringer's, doses of progesterone (0.007, 0.01, 0.07, 0.1, 0.7, 1.0 ~ g / m l ) , in the presence (m) or absence (D) of taxol (10 ~ g / m l , 12 p,M) for 48 h at 22°C.
13
did not elicit GVM at either 24 or 48-h incubation (data not shown). To test the effect of taxol on viscoelasticity of ooplasm, centrifugation was used. Taxol (10 ~g/ml, 6-h incubation) was found to significantly inhibit centrifugation-induced GVM compared to vehicle controls and also significantly inhibited progesterone (1 /~g/ml) enhanced GVM induced by centrifugation (Fig. 3). This striking result suggests that taxol increases the apparent viscoelastic properties of the ooplasm and stabilizes the position of the GV within the oocyte.
Discussion
During amphibian oocyte meiotic maturation, chromatin must be transported close to the oocyte surface to permit normal polar body formation; some oocytes apparently accomplish this transport, at least in part, by GVM. A logical question one might ask relates to the mechanism and regulation of GVM, or, perhaps an even more basic question may be asked, what is responsible for positioning the GV inside the oocyte? It seems probable that the cytoskeleton, which plays such an important role in general cell organization in other cell types, would also be involved in oocyte organization. Cell nuclei generally are associated with cytoskeletal components which presumably anchor the nuclei in position (Franke, 1974). Unfortunately, the oocyte cytoskeleton has not been well studied and thus the relationship between the oocyte nucleus and the cytoskeleton is largely unexplored except for a few pioneering studies (Godsave et al., 1984a, b; Huchon et al., 1985; Jessus et al., 1986). This paper has investigated the possible relationship between GVM and the cytoskeleton, especially microtubules, by testing the hypothesis that cytoskeleton-active drugs and Fig. 6. Morphological effect of combined taxol (10 ~ g / m l , 12 /xM) and progesterone (1 /~g/ml, 3.2 ~M) on follicles after 48-h incubation. (A) Whole follicles containing oocytes which have undergone GVD, numerous cytasters (c) are visible as light-colored "starbursts" or streaks; bar = 500 A~m.(B) Paraffin section of one of the follicles in (A) showing large cytasters (c) near the oocyte surface, each 10 ALtosection typically contained several cytasters concentrated at the animal pole; bar = 100 p~m.
248 modulators of oocyte meiosis in Rana (i.e., progesterone and estradiol) might affect GV position within the oocyte. The present results have confirmed an earlier report that demecolcine induces GVM in Rana oocytes and that both demecolcine and progesterone enhance centrifugation-induced GVM (Lessman et al., 1986). In addition, estradiol was found to inhibit both types of GVM suggesting that this steroid may have a stabilizing effect on the viscoelastic properties of ooplasm. The present data did not indicate an estradiol-related change in GVM induced by centrifugation after estradiol treatment alone, but did show a resistance to changes in viscoelasticity normally observed with demecolcine or progesterone co-treatment. Estrogens have been implicated in yolk production and deposition (Wallace, 1985), it may be that the inhibitory GVM effects observed are in some way related to the participation of the cortical cytoskeleton in yolk uptake. It has been demonstrated previously that agents which disrupt cytoskeleton integrity such as colchicine and vinblastine also inhibit movement of proteins either into or out of the oocyte (Dumont and Wallace, 1972; Colman et al., 1981). It is important to note that steroids which elicit GVD also inhibit protein uptake into oocytes before GVD occurs (Schuetz et al., 1974) and induce changes in the oocyte surface (Schuetz, 1972). The data presented here indicate that progesterone has effects opposite to those of estradiol. Similarly, it has been reported that estradiol inhibits meiosis reinitiation by progesterone in denuded Rana oocytes (Lin and Schuetz, 1983). It may be that the inhibitory effects reported are due in part to the inhibitory effects of estradiol on GVM. The finding that estradiol inhibits the changes normally associated with centrifugationinduced GVM caused by progesterone supports this idea. The findings that nocodazole enhances GVM and centrifugation-induced GVM as well as increasing progesterone-induced GVD, strengthen the argument that microtubules are involved with GV positioning within the oocyte. Clearly, if two different compounds such as demecolcine and nocodazole have similar actions, it seems less likely that the observed effects are due to non-specific
drug actions. This is supported by work with other species. Colman et al. (1981) found that 2 mM colchicine elicited GVM in Xenopus oocytes, and Habibi and Lessman (1986a) found that demecolcine elicited GVM in goldfish (Carassius) oocytes. A further convincing piece of evidence is the effect of taxol or inhibiting centrifugation-induced GVM, with or without progesterone, and the suppression of progesterone-induced GVD. Thus three agents, all known to be active in destabilizing (nocodazole and demecolcine) or stabilizing (taxol) microtubules have consistent effects in this system. A fourth agent, vinblastine which is also a microtubule destabilizer, has been shown to have effects similar to those of nocodazole and demecolcine in the goldfish oocyte system (C.A.L., unpublished data) and in Xenopus oocytes (Colman et al., 1981). Morphological evidence is now needed to further elucidate the role of microtubules in oocyte maturation. Recent reports on oocyte cytoskeleton indicate a growing interest in this area (Wylie et al., 1985; Kuriyama et al., 1986). Microtubules do exist in fully grown amphibian oocytes before maturation (Dumont and Wallace, 1972; Colman et al., 1981; Heidemann et al., 1985; Huchon et al., 1985; Jessus et al., 1986); however, all agree that they exist in small numbers. Contrary to earlier reports (Heidemann and Gallas, 1980) that taxol did not induce tubulin polymerization in immature amphibian oocytes, recent evidence using sensitive methods indicate that some taxolinduced assembly of microtubules does occur in Xenopus oocytes (Heidemann et al., 1985) and Xenopus oocyte extracts (Jessus et al., 1985). Difficulties with morphological studies center on the ability to quantify microtubules and detect changes in the polymerized versus soluble tubulin within the oocyte. There appears to be consensus that tubulin is readily polymerized only after GVD (Heidemann and Kirschner, 1978; Jessus et al., 1984; Huchon and Ozon, 1985), and that a ~ansient microtubule-containing structure appears at the site of initial nuclear membrane rupture (Brachet et al., 1970; Huchon et al., 1981, 1985; Jessus et al., 1986). Therefore, the situation during maturation may be similar to that reported during early Rana egg cleavage in which polymeric tubulin showed changes with the cell cycle,
249 alternating between high levels of soluble and polymeric tubulin (Elinson, 1985). This would be consistent with the changes observed in EDs0 values for progesterone-induced GVD (i.e., increased by taxol and decreased by nocodazole). Thus it is hypothesized that microtubules present in the prematuration oocyte are depolarized to allow for partial GVM and that during GVD microtubules are again produced. Microtubules appearing at GVD might be involved with continued movement of nuclear components to the oocyte surface as suggested by Huchon et al. (1981, 1985) and Jessus et al. (1986). This hypothesis is supported by work on starfish oocytes by Schroeder and Otto (1984), since they report that fluorescently labeled microtubules are markedly reduced 25 rain after 1-methyl adenine treatment to induce GVD. Recently, cyclical changes in microtubules related to meiosis have been reported in Spisula oocytes (Kuriyama et al., 1986). Additional support for the concept that progesterone alters the viscoelastic properties of amphibian ooplasm comes from a study by Merriam (1971) in which Xenopus oocytes were tested for 'stratifiability' by centrifugation at 1 500-8 000 × g for 2 min. Progesterone increases stratifiability after 2-h incubation, well before G V D was observed. The results of this earlier study support the idea that progesterone affects the general organization of the oocyte, which presumably includes GV positioning and the cytoskeleton, soon after application. Antimitotic drugs such as demecolcine have been found to affect intermediate filaments associated with nuclei in somatic cells (Lin, 1981). Gerbil fibroma cells, treated with 1 /~M demecolcine for 24 h and stained with fluorescein-labelled antibodies to intermediate filaments, show characteristic 'capping'; of fibers which coil tightly around the nucleus (Lin, 1981). It is not clear whether the intermediate filament 'capping' is due to a direct action of demecolcine on the filaments or is a result of microtubule disruption. In view of these findings in somatic cells, it is tempting to speculate that the effect of demecolcine on oocyte GVM may be due to changes in intermediate filament arrangement as well as microtubule disruption. Intermediate filaments such as cytokeratin and vimentin have been described in the Xenopus
oocyte (Franz et al., 1983; Godsave et al., 1984a, b; Wylie et al., 1985). Microtubule poisons such as the ones used in the present study may have non-specific or even toxic effects especially at high doses (Dustin, 1984). No evidence of toxic effects was obtained in this study since progesterone continued to elicit GVD in the presence of the microtubule poisons. In an earlier study (Lessman et al., 1986), electrophysiological measurements of Rana oocyte membrane potential and resistance after demecolcine treatment (270/xM) indicated no significant change in membrane function, thus indicating the absence of toxic effects even at a relatively high dose of demecolcine. Although the consistent effects obtained with the three microtubule poisons used strongly indicate microtubule-specific action, definitive demonstration that microtubules are being specifically affected must come from future morphological studies. It seems unlikely that depolymerization of microtubules alone can account for the active migration of the GV and yet two agents which destabilize microtubules, i.e. nocodazole and demecolcine, result in GVM. Thus it may be necessary to consider the possibility that separate active agents exist, such as microfilaments, which are responsible for the migration. In studies of GVM in the goldfish oocyte, cytochalasin B was found to inhibit steroid-induced GVM (Habibi and Lessman, 1985). The mitochondrial poison, 2,4-dinitrophenol was also found to inhibit GVM in goldfish oocytes (Habibi and Lessman, 1986b), suggesting that GVM is an energy-dependent event. The energy requirement would be consistent with the idea that microfilaments were involved. Furthermore, the maturation-inducing substance, 1-methyl adenine, produced marked changes in the actincontaining cytoskeleton of the maturing starfish oocyte (Schroeder, 1985). Thus, it may be postulated that during maturation, microtubules destabilize while microfilaments become increasingly active with the net result being GVM. Clearly, morphological studies are required to elucidate the relationship of the cytoskeleton to GVM. Studies which explore several types of cytoskeletal elements (i.e., microtubules, microfilaments and intermediate filaments) with respect to GV positioning would be particularly welcome.
250
Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada (operating grant A8397). I acknowledge a gift of taxol from Dr. Matthew Suffness of the National Cancer Institute, NIH. I thank Barbara Pluta and Debra Henke for technical assistance and Anne-Louise MacDonald for animal care. The curve-fitting programs Allfit and Grafit used to generate Fig. 5 were a gift of the Biomedical Computing Technology Information Center, Nashville, TN.
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