Induction of maturation in small Xenopus laevis oocytes

DEVELOPMENTAL

BIOLOGY

121,111-118

(1987)

Induction of Maturation

in Small Xenopus hew’s Oocytes

MARK A. TAYLORAND L. DENNISSMITH Dtqnw-tment of Biological

Sciences, Purdue

Received June 6,

University,

West Lafayette,

Indiana

47907

1986;accepted in revised fwm November 24, 1986

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; 4100 pm in diameter) do not. The defect which prevents i’50to 1050-pm oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed. o 1987 Academic press, IX 1NTR:ODUCTION

Full-grown (stage 6) Xenopus laevis oocytes, arrested in prophase I of meiosis, can be induced to mature in vitro with progesterone (see reviews by Smith, 1981; Maller, 1985). The most obvious morphological event associated with oocyte maturation is the breakdown of the oocyte nucleus (germinal vesicle). Stage 6 Xenopus oocytes also undergo several biochemical changes in response to progesterone. T:hese changes include a decrease in the level of CAMP (Maller et ab, 1979; Cicirelli and Smith, 1985), an increase in protein synthesis (Wasserman et al, 1982; Taylor et aZ., 1985a), an increase in total protein phosphorylation (Maller et al., 1977, 1979; Kawata et al., submitted), and the activation of the maturation-promoting factor (MPF)’ (Schorderet-Slatkine and Drury, 1973; Reynhout and Smith, 1974; Wu and Gerhart, 1980). Growing Xenw oocytes (stages 3-5) do not normally resume meiosis and exhibit few if any morphological or biochemical changes when exposed to progesterone. Only Reynhout et al. (1975), Kofoid et al. (1979), and Wasserman et ah (1986) have so far obtained a significant percentage of maturation in small oocytes (cl100 pm in diameter). Reynhout et al. (1975) observed that in oocytes from HCG-stimulated flemales the competence for a maturation response in vitro to progesterone extends to late stage 4 oocytes (about 900 pm in diameter). Wasserman et al. (1985) and Kofoid et al. (1979) noted that

1 Abbreviations used: MPF, maturation-promoting germinal vesicle breakdown.

factor;

GVBD,

in the absence of extracellular potassium, small oocytes (>850 pm in diameter) can respond to progesterone and undergo nuclear membrane dissolution. Kofoid et al. (1979), however, also observed that incubation in potassium-free media allows the induction of oocyte maturation by agents that do not normally induce maturation. The exact nature of the block that prevents small oocytes (stages 3-5) from responding to progesterone is not known. Sadler and Maller (1983) have reported that both stage 4 and stage 6 oocytes possess membrane steroid receptors that are apparently linked to adenylate cyclase. The Kd for binding of steroid to the receptor is the same in growing and full-grown oocytes. Moreover, the coupling between receptors and cyclase does not appear to be deficient, since the IC& for inhibition of adenylate cyclase by progesterone is identical in stage 4 and stage 6 oocytes (Sadler and Maller, 1983). The inability of the heat-stable protein kinase inhibitor to induce maturation in stage 4 oocytes also implies that the defect is not solely at the level of adenylate cyclase (Sadler and Maller, 1983). Furthermore, it may be inferred that the block prevents the activation of MPF, since small oocytes undergo germinal vesicle breakdown when microinjected with MPF (Hanocq-Quertier et al,, 1976; Sadler and Maller, 1983; Wasserman et al., 1984). Consequently, the defect that prevents small oocytes from responding to progesterone appears to occur subsequent to the CAMP decrease but prior to the activation of MPF. In the current study, we confirm that small oocytes (cl100 pm in diameter) are normally unresponsive to progesterone, but undergo germinal vesicle breakdown

111

0012-1606/8’7 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

112

DEVELOPMENTAL BIOLOGY

when microinjected with MPF. More importantly, we report that small oocytes, ranging from 750 to 1050 pm in diameter, injected with cytoplasm taken from a stage 6 oocyte can respond to progesterone stimulation and undergo germinal vesicle breakdown. GVBD in such small oocytes occurs on a timetable similar to that exhibited by stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in the rate of protein synthesis as well as the activation of MPF. These results are discussed with respect to the idea that a cytoplasmic factor(s) may be responsible for the competence to respond to progesterone. The possible identity and role of the cytoplasmic factor(s) is discussed in relation to the development of maturation competence and the regulation of maternal mRNA translation. MATERIALS

AND

METHODS

Animals and Collection of Oocytes Large adult X laevis females were purchased from South Africa (Snake Farm, Fish Hoek, Cape Province), Michigan (Kelly Evans, Ann Arbor, 716 Northside), and Wisconsin (Nasco, Fort Atkinson). These animals were maintained as described by Webb et al. (1975). Ovaries were surgically removed from hypothermically anesthetized females. Dumont (1972) stage 3,4,5, and 6 oocytes were manually defolliculated with matchmaker’s forceps and cultured in OR-2 medium (Wallace et al., 1973). Oocyte diameters were measured with an ocular micrometer.

VOLUME 121, 1987

the vitelline membrane, rotation of the egg, alteration of the pigment distribution pattern, or the appearance of abortive cleavage furrows.

Measurement of Protein Synthesis Stage 4 and stage 6 oocytes were injected with 15 nl of L-[3H]leucine (55 Ci/mmole, Schwarz & Mann) concentrated to deliver 50 pmoles of leucine. At various times after injection, groups of oocytes were processed as described by Wasserman et ab (1982). Rates of protein synthesis were calculated from the linear portion of the slope of incorporated radioactivity and the expanded leucine pool size, and assuming that Xenopus oocyte proteins are 10% by weight leucine (Wasserman et al, 1982).

Cytoplasm Transfers Transfers of cytoplasm (2-40 nl) from one oocyte to another were usually performed under mineral oil to minimize the possibility that dilution of cytoplasmic factors might occur. Injected oocytes were removed from the mineral oil and placed in Gurdon’s media (90 mM KzP04, 10 mM NaCl, 1 mM MgSOJ for 15-30 min to promote healing. Oocytes were then put in OR-2 for steroid responsiveness testing. The time that elapsed between the injection of cytoplasm and the application of the steroid generally did not exceed 30 min. In some cases, injections of cytoplasm or MPF (cytoplasm from a mature oocyte) were performed in OR-2.

Cycloheximide Treatment Maturation Response Responsiveness to in vitro steroid treatment was measured after continuous exposure of the oocytes to progesterone (10 pg/ml) in OR-2 medium. The criterion for maturation was the absence of a nucleus as determined by manual dissection after fixation of the oocytes in 0.5 N perchloric acid. Maturation in stage 6 oocytes was also scored by the appearance of a white spot on the pigmented animal pole. Analysis for GVBD was usually performed 6-10 hr after steroid administration. In some cases, the presence or the absence of a germinal vesicle was confirmed by viewing sections (10 pm) of stage 4-6 oocytes that were fixed in Smith’s fixative, dehydrated through an ethanol series, and embedded in paraffin.

Activaticm Criteria Activation was induced by pricking eggs with a glass needle. The criteria of activation included elevation of

Stage 4 oocytes were microinjected with 20 ng of cycloheximide and cultured for 60 min in OR-2 containing 50 pg/ml cycloheximide. Assuming a water volume of 0.2 pl/oocyte, the internal concentration of cycloheximide was 100 pg/ml. This concentration is sufficient to inhibit protein synthesis by >95% (Wasserman and Smith, 1978a). RESULTS

Progesterone Treatment The data in Fig. 1 demonstrate a correlation between increased oocyte diameter and the ability to respond to progesterone in vitro. None of the oocytes smaller than 1000 pm in diameter responded to progesterone and less than 5% of the oocytes with diameters between 1000 and 1050 pm underwent GVBD in response to steroid. Of the oocytes with a diameter of 1100 pm, 35% underwent GVBD. The largest percentage of GVBD (95% ) was ob-

TAYLOR AND SMITH

Maturation

601

60 .

!it

Q

20

I’

1

.L; 500

600

113

0ocyte.s

ment. While 95% of the stage 6 oocytes induced to mature with MPF could be activated, no similarly treated stage 3 or stage 4 oocytes had activation capacity (Table 1). Comparable findings on the activatability of stage 3 and stage 4 oocytes were reported by Hanocq-Quertier et al. (1976). These observations suggest that activation capacity as well as progesterone responsiveness is acquired between stage 4 and stage 6 of oogenesis.

8 2

in Small Xenopus

I’

700

600

OOCYTE

900

:d---,,,

1000

DIAMETER

1100

1200

1300

1400

(MM)

FIG. 1. Relationship between oocyte diameter and competence to undergo nuclear membrane dissolution in response to progesterone treatment (10 pg/ml). Each point represents the mean of results obtained using two different females. The percentage GVBD was scored by dissection after 0.5 N HCIO,, fixation. (0 --- 0) Oocytes exposed to progesterone; (0 - 0) oocytes cultured in OR-2 alone.

served in oocytes 1250 pm or larger in diameter. All the oocytes (>llOO pm in diatmeter) that underwent GVBD were activatable (see also Table 1). Moreover, the induction of maturation with progesterone in full-grown stage 6 oocytes resulted in a twofold increase in the absolute rate of protein synthesis (Table 2). By contrast, stage 4 oocytes (about 800 pm in diameter) exhibited a basal protein synthetic rate half that of stage 6 oocytes and did not show a synthetic increase in response to progesterone (Table 2). Thus, it appears that small oocytes undergo few if any morphological or biochemical changes in response to p:rogesterone. This conclusion is in agreement with the findings of Hanocq-Quertier et al. (1976), Sadler and Mailer (1983), and Wasserman et al. (1984).

MPF Injection The inability of growing oocytes (cl100 pm in diameter) to resume meiosis in response to progesterone may be due to a defect in their ability to activate MPF. In this regard, several investigators have shown that small oocytes as well as full-grown oocytes undergo GVBD when injected with MPF ‘(Hanocq-Quertier, 1976; Sadler and Maller, 1983; Wasserman et al., 1984). The data in Table 1 clearly confirm this observation. We found that approximately 65, 74, and 87% of stage 3, stage 4, and stage 6 oocytes, respectively, underwent GVBD in response to MPF injection. Furthermore, both stage 4 and stage 6 oocytes increased their rates of protein synthesis twofold after MPF injection (Table 2). Thus, growing oocytes possess the potential to undergo GVBD and increase protein synthesis, but appear to be defective in the ability to activate MPF in response to steroid treat-

TABLE 1 MATURATION RESPONSEOF STAGE 3-6 OOCYTES Oocyte stage” and treatment* Stage 6 + OR-2 Stage 6 + progesterone Stage 6 + 20 nl of Stage 6 MPF Stage 6 + 30 nl of Stage 4 MPF Stage 5 + OR-2 Stage 5 + progesterone Stage 5 t stage 6 cytoplasm + OR-2 Stage 5 + stage 6 cytoplasm + progesterone Stage 4 + OR-2 Stage 4 + progesterone Stage 4 + 20 nl of MPF Stage 4 + cycloheximide + 20 nl of MPF Stage 4 + stage 6 cytoplasm + OR-2 Stage 4 + stage 6 cytoplasm + progesterone Stage 4 + stage 6 cytoplasm + cycloheximide + progesterone Stage 4 + stage 4 cytoplasm + progesterone Stage 4 (700 pm) + stage 6 cytoplasm + progesterone Stage 3 + OR-2 Stage 3 + progesterone Stage 3 + 10 nl of MPF

-

Percentage GVBD”

Percentage activation

0.0 k 0.0 (16)d 87.1 f 8.3 (16)

100.0 f 0.0 (3)

7.5 (7)

95.0 + 4.1 (3)

67.5 k 10.4 (4)

90.0 * 8.2 (3)

87.1 *

0.0LO,O](2) 0.0LO,O](2)

-

0.0LO,o](s) 80.0 [80, 801 (2) 0.0 + 0.0 (16) 0.0 + 0.0 (16)

0.0LO,O](2)

74.3 f

0.0 f 0.0 (3)

7.8 (7)

75.0 [70, 801 (2)

-

0.0 LO,O](2)

2.8 (14)

0.0 2k0.0 (3)

77.2 + 8.4 (14)

0.0 + 0.0 (3)

4.0 f

0.0 PI 01(Z)

-

0.0P, O](2)

-

2.5 LO,5lC4

0.0 LO,O](2) 0.0[O,01(Z) 65.0 [60, 701 (2)

0.0[O,01(Z) 0.0 LO,01(Z)

’ Where not specified, stage 3,4,5, and 6 oocytes averaged 500,822, 1050, and 1237 pm in diameter, respectively. *Where not specified, 20 or 30 nl of cytoplasm was microinjected. ‘Values listed are means f SE; where only two experiments were performed, the individual determinations are given in brackets. d Numbers in parentheses refer to numbers of experiments on separate females (lo-20 oocytes were used for each treatment per female).

114

DEVELOPMENTAL

TABLE PROTEIN

SYNTHESIS

BIOLOGY

2

IN STAGE 4 AND STAGE 6 OOCYTES

Oocyte stage: injected material,* and incubation conditions

Protein synthesis (ng/hr/oocyte)

Stage 4 + distilled water + OR-2 Stage 4 + distilled water + progesterone Stage 4 + stage 6 cytoplasm + OR-2 Stage 4 + stage 6 cytoplasm + progesterone Stage 4 + MPF + OR-2d Stage 6 + distilled water + OR-2 Stage 6 + distilled water + progesteroned Stage 6 + MPF + OR-2d

rate”

8.5 (7.7, 9.3) 8.7 (8.0, 9.4) 9.0 (8.3, 9.7) 17.9 (16.1, 19.7) 19.7 17.3 (13.2, 21.4) 35.3 (33.0, 37.6) 43.7

a Stage 4 and stage 6 oocytes averaged 822 and 1237 pm in diameter, respectively. *All injections were in a volume of 20 nl. ‘Determined according to Taylor and Smith (1985). The values listed are means; the individual determinations are given in parentheses. d Data taken from Taylor et al. (1985a).

MPF Amplijcaticm Stage 6 Xenopus oocytes that receive an injection of MPF undergo GVBD and their cytoplasm acquires the ability to induce GVBD when injected into other stage 6 oocytes. Moreover, MPF activity is retained after sev-

VOLUME

121, 1987

eral serial transfers (Reynhout and Smith, 1974; Wasserman et al., 1984). This secondary development of MPF activity in recipient oocytes is referred to as MPF amplification. The data in Table 3 demonstrate that oocytes ‘750 pm or larger in diameter retain MPF activity after four serial transfers, while oocytes smaller than ‘750 pm in diameter do not. The latter observation argues convincingly that the percentage of oocytes undergoing GVBD after four serial transfers is not due simply to carryover of MPF activity from the original donor oocytes, since dilution of the original donor MPF activity increases with increasing recipient oocyte diameter. Therefore, it is reasonable to conclude that only oocytes with a diameter of 750 pm or larger have the ability to amplify MPF. The finding that oocytes smaller than 750 pm in diameter lack the capacity to amplify MPF has significant implications. It is generally thought that MPF amplification proceeds via the autocatalytic activation of a stored pool of inactive MPF. Wasserman and Masui (1975) and Gerhart et al. (1984) have demonstrated that protein synthesis is not required for MPF amplification in stage 6 oocytes. Similarly, protein synthesis does not appear to be necessary for the amplification reaction in stage 4 oocytes (>750 pm in diameter). Treatment of stage 4 oocytes with cycloheximide does not prevent either MPF-induced maturation (Tables 1 and 3) or MPF amplification (Table 3). Consequently, it is tempting to

TABLE 3 MPF AMPLIFICATION IN STAGE 4-6 OOCYTES~ Percentage Oocyte diameter Oocvte stage and treatment Stage 6 + Stage 5 + Stage 4 + Stage 4 + + OR-2 Stage 4 + + OR-2 Stage 4 + Stage 4 +

OR-2 OR-2 OR-2 stage 6 cytoplasm

GVBD”

(urn)*

Recipient Id

Recipient II”

Recipient III’

Recipient IVQ

1237 + 16 1055 + 15 822 f 10

97.5 (95.0, 100.0) 90.0 (90.0, 90.0) 90.0 (90.0, 90.0)

92.5 (90.0, 95.0) 90.0 (90.0, 90.0) 72.5 (70.0, 75.0)

92.5 (90.0, 95.0) 90.0 (90.0, 90.0) 62.5 (60.0, 65.0)

90.0 (90.0, 90.0) 85.0 (80.0, 90.0) 60.0 (55.0, 65.0)

822 f 10

90.0 (85.0, 95.0)

70.0 (65.0, 75.0)

60.0 (50.0, 70.0)

65.0 (60.0, 70.0)

822 5 10 750+ 6 700* 5

85.0 (80.0, 90.0) 82.5 (80.0, 85.0) 70.0 (65.0, 75.0)

70.0 (65.0, 75.0) 70.0 (70.0, 70.0) 5.0 (5.0, 5.0)

65.0 (60.0, 70.0) 60.0 (55.0, 65.0) 2.5 (0.0, 5.0)

60.0 (55.0, 65.0) 50.0 (45.0, 55.0) 0.0 (0.0,O.O)

cycloheximide OR-2 OR-2

0 Results from two experiments using different animals (20 oocytes of each size class per animal). * Means f SE of groups of 10 oocytes representative of the stage used. ‘The values listed are means; individual determinations are given in parentheses. d Recipient I oocytes of each size class were injected with cytoplasm taken from a progesterone-matured stage 6 donor oocyte (20 nl/oocyte). eRecipient II oocytes of each stage received cytoplasm taken from recipient I oocytes of the same stage. Cytoplasmic transfers were made at 3-hr intervals. *Recipient III oocytes received cytoplasm taken from recipient II oocytes of the same stage. B Recipient IV oocytes received cytoplasm taken from recipient III oocytes of the same stage.

TAYLOR

AND

SMITH

Maturation

in Small Xenops

115

Oocytes

100 -

speculate that the pool of inactive MPF begins to be synthesized and accumulated at about mid-stage 4 of oogenesis.

60 8

Cytoplasmic

Transfer

Data described above suggest that oocytes 750 pm or larger in diameter contain a stored pool of inactive MPF which can be activated in response to injected MPF. One explanation for the inability of small oocytes (750-1050 pm in diameter) to activate MPF in response to progesterone is that they are deficient in some required factor(s) which mediates the effect of progesterone. This hypothesis was tested directly by, first, injecting small oocytes with stage 6 oocyte cytoplasm, and then treating them with progesterone. Because of technical limitations regarding micropipette size and cytoplasmic viscosity, no oocyte less than 700 pm in diameter was injected with stage 6 oocyte cytoplasm. Table 1 presents specific cases in which stage 4 and stage 5 oocytes were treated in this manner. Of the stage 4 (-800 pm) and stage 5 oocytes injected with stage 6 cytoplasm, ‘77 and 80%, respectively, underwent GVBD in response to progesterone. The smallest oocytes responsive to these manipulations had diameters of 750 pm (65% GVBD). By contrast, 700-pm oocytes in.jected with stage 6 cytoplasm and treated with progesterone did not undergo GVBD (2.5%, Table 1). Control experiments showed that the percentage of GVBD obtained in growing oocytes (750-1050 pm in diameter) injected with sta.ge 6 cytoplasm and treated with progesterone was not due to an artifact resulting from the experimental manipulations. For example, stage 4 oocytes injected with stage 6 cytoplasm and cultured in OR-2 medium alone (no progesterone) did not exhibit a significant percentage of GVBD (Table 1). Also, GVBD was not observed in stage 4 oocytes injected with stage 4 cytoplasm and treated with progesterone (Table 1). Moreover, cycloheximide inhibition of the progesterone response of stage 4 oocytes injected with stage 6 cytoplasm (Table 1) argues against the possibility that a subthreshold amount of active MPF was in the transferred cytoplasm. Hence, it appears that the stage 6 oocytes contain a cytoplasmic factor(s) that is required to mediate the response to progesterone and that small oocytes are deficient in t.his factor. Nuclear membrane dissolution in stage 6 X laevis oocytes from unstimulated females in our laboratory normally takes place 5 to 7 hr following the start of progesterone treatment (Wasserman and Smith, 197813). Figure 2 depicts the time course of progesterone action on stage 4 oocytes (-800 pm in diameter) injected with

2

60-

5 8

40-

% 200 1

0 TIME

2 AFTER

3

4

PROGESTERONE

I 6

I 5

TREATMENT

I 7

1 6 (hr)

FIG. 2. Time course of progesterone action on stage 4 oocytes injected with 20 nl of full-grown oocyte cytoplasm (0 --- 0) and stage 6 oocytes (0 - 0). Progesterone was added at time zero. The percentage GVBD was scored by dissection after 0.5 N perchloric acid fixation. The data presented reflect the means of experiments performed on two separate females.

stage 6 cytoplasm. It can be seen that GVBD in such small oocytes occurs on a timetable similar to that exhibited by stage 6 oocytes exposed to progesterone (about 6.5 hr). Unlike full-grown oocytes, small oocytes (7501050 pm in diameter) injected with stage 6 cytoplasm do not acquire the ability to be activated. However, nuclear membrane dissolution in these small oocytes is accompanied by a twofold increase in protein synthesis (Table 2), increased protein phosphorylation (Kawata et al., submitted), and the activation of MPF on a time course similar to that observed in stage 6 oocytes exposed to steroid (Table 1). Comparable biochemical changes have been detected in stage 4 oocytes injected with MPF (Hanocq-Quertier et ah, 1976; Wasserman et al., 1984). Dose-Response and Time of Appearance Masui and Markert (1971) found that as little as 2 nl of cytoplasm from a mature oocyte (MPF) could induce GVBD in a recipient oocyte. We find that 5 nl of stage 6 oocyte cytoplasm is sufficient to enable a small oocyte to respond to progesterone. Further, the effect of stage 6 cytoplasm appears to be dose-dependent up to 20 nl of injected material (Fig. 3). At 20 nl or more of injected cytoplasm, the percentage of GVBD in small oocytes seems to approach a plateau value. To determine the stage of appearance of the cytoplasmic factor required for progesterone responsiveness, 30 nl of cytoplasm was withdrawn from oocytes having diameters ranging from 1000 to 1300 /Irn and transferred to stage 4 recipient oocytes. The data in Table 4 demonstrate that the cytoplasmic factor appears, or accumulates above a threshold value, after oocytes reach 1100 /*rn in diameter.

116

DEVELOPMENTAL BIOLOGY

VOLUME 121, 1987

also contain a pool of inactive MPF (Table 3) which is activated by steroid after injection of stage 6 cytoplasm. Preliminary studies indicate that microinjection of progesterone directly into small oocytes previously injected with stage 6 cytoplasm does not result in GVBD. Thus, 60 it does not appear that progesterone interacts directly with the injected cytoplasmic material. Taken together, the data suggest that the injected component acts subsequent to the initial steroid/oocyte interaction and prior to MPF activation. In other words, the evidence presented here suggests that stage 6 oocytes contain a cytoplasmic component(s) which mediates the initial 0 5 10 15 20 25 30 35 40 AMOUNT OF STAGE 6 CYTOPLASM TRANSFERRED (nl) progesterone action. The cytoplasmic activity which mediates progesterone FIG. 3. Dose response curve relating the quantity of full-grown oocyte cytoplasm required to establish progesterone responsiveness in stage action first appears or accumulates above a threshold 4 oocytes. Stage 4 oocytes injected with stage 6 oocyte cytoplasm were valve in oocytes about 1100 pm in diameter (Table 4). In incubated with progesterone (10 fig/ml). The percentage GVBD was this context, it is interesting to note that this correlates scored by dissection 10 hr later. The data presented were obtained well with the time that responsiveness to progesterone from two different females; results are expressed as the means. occurs. Fortune (1983) has also found that follicle cells switch from synthesizing estrogens to making progesDISCUSSION terone when the enclosed oocyte reaches stage 5. Thus, the two events, component appearance and progesterone In the current study, we confirm that X laevis oocytes synthesis, seem to be temporally regulated. smaller than 1100 pm in diameter are normally unreThe nature of the cytoplasmic component present in sponsive to progesterone but undergo GVBD in response stage 6 oocyte cytoplasm which mediates progesterone to MPF injection. More importantly, we report that the action is not known. However, Wasserman et al. (1986) defect which prevents small oocytes (750-1050 pm in dihave proposed that removal of potassium from the exameter) from responding to progesterone in vitro can be tracellular environment puts small oocytes (>850 pm in overcome by microinjecting cytoplasm taken from a diameter) into a permissive state whereupon progesterstage 6 oocyte. Nuclear membrane dissolution in these one triggers nuclear membrane dissolution. In this resmall oocytes occurs on a timetable similar to that exgard, the component may alter the permeability of the hibited by stage 6 oocytes exposed to progesterone. plasma membrane to certain ions. In such a case, the Moreover, GVBD in these small oocytes is accompanied component might represent a protein that is present, by a twofold increase in protein synthesis, increased initially, in the cytoplasm of the stage 6 oocyte and, then, protein phosphorylation, and the activation of MPF. The translocates to the plasma membrane in response to small oocytes, however, are still not mature in the sense progesterone. that they lack the ability to be activated. The cytoplasmic activity that mediates progesterone It is generally agreed that the induction of oocyte action may not even be a single component since at least maturation by steroid hormones is initiated via an inthree processes are activated in stage 4 oocytes: interaction at or near the oocyte surface (reviews by Wasserman and Smith, 1978b; Baulieu et ak, 1978; Masui and Clarke, 1979; Maller, 1985). Specifically, progesterone is TABLE 4 believed to interact with a plasma membrane receptor APPEARANCE OF COMPETENCE FACTOR that is linked to adenylate cyclase. This results in deDonor oocyte diameter Percentage GVBD* in stage 4’ creased enzyme activity, lowering CAMP levels and alrecipient oocytes (wY tering CAMP-dependent kinase activities. This process, in turn, leads to the activation and amplification of MPF, 1000 0.0 (0, 0) although the nature of the intermediate step(s) between 1100 15.0 (10, 20) the initial surface interaction and MPF activation re1200 80.0 (70, 90) 1300 85.0 (75, 95) mains obscure. Small oocytes are reported to possess steroid recepa Donor cytoplasm (30 nl) was transferred to recipient oocytes. tors, but the density of receptors in stage 4 oocytes is *The values listed are means; individual determinations are given only 20% of that in stage 6 oocytes (Sadler and Maller, in parentheses. 1983). Stage 4 oocytes (750 pm or larger in diameter) ‘Stage 4 oocytes averaged 822 pm in diameter.

TAYLOR AND SMITH

Maturation

creased protein synthesis, appearance of MPF activity, and increased protein phosphorylation. The latter events could be coupled since activation and amplification of MPF have been linked to changes in phosphorylation (Maller et ah, 1977, 1979; Wu and Gerhart, 1980). Phosphorylation of ribosom,al protein S6 also has been implicated in the regulation of protein synthesis (Nielsen et ab, 1982; Wasserman and Houle, 1984), especially in stage 6 oocytes in which some component of the translational machinery is limiting for protein synthesis (Laskey et ab, 1977; Richter and Smith, 1981). However, Taylor et al. (1985a) have presented other evidence which appears to eliminate a direct role of S6 phosphorylation in regulating the quantitative aspects of protein synthesis in growing oocytes. The increase in protein synthesis that occurs in small oocytes injected with stage 6 cytoplasm and exposed to progesterone is of particular interest. In stage 6 oocytes, the induction of maturattion results in at least a twofold increase in protein synthesis (Wasserman et al., 1982; Taylor et al., 1985a) and this increase involves the recruitment of maternal mRNA into polysomes as well as an increase in the putative limiting component (Smith, in press). In small oocytes, mRNA availability alone is limiting for translation (Taylor et ah, 198513). Microinjection of globin mRNA increases the level of protein synthesis in small stage 4 oocytes to that seen in stage 6 oocytes, but no higher (Taylor et al., 1985a,b). Hence, it appears that stage 4 o’ocytes contain an excess amount of the factor that limits translation in stage 6 oocytes relative to their protein synthesis needs, and that by stage 6 all of the factor is in use. Since the cytoplasmic activity which mediates progesterone action does not appear until stage 5 of oogenesis, it does not appear to be the component that limits translation in stage 6 oocytes. It is also reasonable to conclude that the increase in protein synthesis observed in stage 4 oocytes injected with stage 6 cytoplasm and exposed to progesterone as well as the increase seen in stage 4 oocytes after MPF injection involves sole1.y the recruitment of maternal mRNA into polysomes. Thus, investigations of the mechanism(s) which results in increased mRNA availability can now be approached in stage 4 oocytes without the complication of changes in the translational machinery which occur in stage 6 oocytes.

This work was supported by a grant from The National Institutes of Health (HD04299) awarded to L.D.S. We also thank Dr. Prema Michael for helping with the histology techniques. REFERENCES BAULIEU, E. E., GODEAU, F., SCHORDERET, M., and SCHORDERET-~LATKINE, S. (1978). Steroid-induced meiotic division in Xenopus laevis oocytes: Surface and calcium. Nature (London) 275,593-598.

in Small Xenopus

Oocytes

117

CICIRELLI, M. F., and SMITH, L. D. (1985). Cyclic AMP levels during the maturation of Xenopus oocytes. Dev. Biol. 108,254-258. DUMONT, J. N. (1972). Oogenesis in Xenopus la&s (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol 136,153-180. FORTUNE, J. (1983). Steroid production by Xenopus ovarian follicles at different developmental stages. Dev. Biol. 99,502-509. GERHART, J., Wu, M., and KIRSCHNER, M. (1984). Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus 1aevi.s oocytes and eggs. J. Cell Biol. 98.1247-1255. HANOCQ-QUERTIER, J., BALTUS, E., and BRACHET, J. (1976). Induction of maturation (meiosis) in small Xenopus luevis oocytes by injection of maturation promoting factor. Proc. Natl. Acad. Sci. USA 73,20282032. KAWATA, E., TAYLOR, M. A., and SMITH, L. D. Phosphorylation of a unique set of proteins accompanies meiotic maturation and mitosis in Xenopus laevis. Submitted. KOFOID, E. C., KNOUBER, D. C., and ALLENDE, J. E. (1979). Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions. Dev. Biol. 72,374-380. LASKEY, R. A., MILLS, A. D., GURDON, J. B., and PARKINGTON, G. A. (1977). Protein synthesis in oocytes of Xenopus Levis is not regulated by the supply of messenger RNA. Cell 11,345-351. MALLER, J. L. (1985). Regulation of amphibian oocyte maturation. Cell Difer. 16,211-221. MALLER, J., Wu, M., and GERHART, J. C. (1977). Changes in protein phosphorylation accompanying maturation of Xenqpus Levis oocytes. Dev. Biol. 58, 295-312. MALLER, J. L., BUTCHER, F., and KREBS, E. G. (1979). Early effect of progesterone on levels of cyclic adenosine 3’5’-monophosphate in Xewpus oocytes. J. BioL Chem. 254,579-582. MASUI, Y., and CLARKE, H. (1979). Oocyte maturation. Int. Rev. Cytol. 57,185-223. MASUI, Y., and MARKERT, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177.129-146. NIELSEN, P. J., THOMAS, G., and MALLER, J. L. (1982). Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes. Proc. Nat1 Acad. Sci. USA 79,2937-2941. REYNHOUT, J. K., and SMITH, L. D. (1974). Studies on the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone. Dev. Biol. 38,394-400. REYNHOUT, J. K., TADDEI, C., SMITH, L. D., and LAMARCA, M. J. (1975). Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time of previous HCG-induced ovulation. Dev. Biol. 44,375-379. RICHTER, J. D., and SMITH, L. D. (1981). Differential capacity for translation and lack of competition between mRNAs that segregate to free and membrane-bound polysomes. Cell 27,183-191. RICHTER, J. D., WASSERMAN, W. J., and SMITH, L. D. (1982). The mechanism for increased protein synthesis during Xenopus oocyte maturation. Dev. Biol. 89,159-167. SADLER, S. E., and MALLER, J. L. (1983). The development of competence for meiotic maturation during oogenesis in Xenopus Levis. Dev. Bid 98,165-172. SCHORDERET-SLATKINE, S., and DRURY, K. (1973). Progesterone-induced maturation in oocytes of Xaopus laevti: Appearance of a maturationpromoting factor in enucleated oocytes. CeU &&r. 2,247-254. SMITH, L. D. (1981). Frog oocytes and sea urchin eggs: Steroid hormones and sperm induce similar events. In “Fortschitte der Zoologie,” 26th ed., pp. 35-47. Verlag, Stuttgart/New York. SMITH, L. D. (1986). Regulation of translation during amphibian oogenesis and oocyte maturation. In “Symposium of the Society for Developmental Biology,” (J. G. Gall, Ed.) Pp 131-150. A. R. Liss, Inc., New York.

118

DEVELOPMENTAL BIOLOGY

TAYLOR, M. A., ROBINSON, K. R., and SMITH, L. D. (1985a). Intracellular pH and ribosomal protein S6 phosphorylation: Role in translational control in Xenopus oocytes. J. Embryol. Exp. Morphol. 89 (Suppl.), 35-51. TAYLOR, M. A., JOHNSON, A. D., and SMITH, L. D. (1985b). Growing Xenopus oocytes have spare translational capacity. Proc. Natl Acad. Sci USA 82,6586-6589. TAYLOR, M. A., and SMITH, L. D. (1985). Quantitative changes in protein synthesis during oogenesis in Xenopus la&s. De-v. BioL 110, 230237. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian oocytes. III. Optimum incubation conditions. J. Exp. Zool l&321-334. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian oocytes. III. Optimum incubation conditions. J. Exp. Zool. 18,321-334. WASSERMAN, W. J., and HOULE, J. G. (1984). The regulation of ribosomal protein S6 phosphorylation in Xenopus oocytes: A potential role for intracellular pH. Dev. BioL 101,436-445. WASSERMAN, W. J., HOULE, J. G., and SAMUEL, D. (1984). The maturation response of stage IV, V, VI Xenopus oocytes to progesterone stimulation in vitro. Dev. Biol 105,315-324.

VOLUME 121, 198’7 WASSERMAN, W. J., and MASUI, Y. (1975). Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exp. Cell Res. 91, 381-388. WASSERMAN, W. J., PENNA, M. J., and HOLJLE, J. G. (1986). The regulation of Xenopus laevis oocyte maturation. In “Symposium of the Society for Developmental Biology,” (J. G. Gall, Ed.) Pp 111-130. A. R. Liss, Inc., New York.

WASSERMAN, W. J., RICHTER, J. D., and SMITH, L. D. (1982). Protein synthesis during maturation promoting factor and progesteroneinduced maturation in Xenopus oocytes. Dev. BioL 89, 152-158. WASSERMAN, W. J., and SMITH, L. D. (1978a). The cyclic behavior of a cytoplasmic factor controlling nuclear membrane breakdown. J. CeU Biol. 78, R15-R22. WASSERMAN, W. J., and SMITH, L. I;. (1978b). Oocyte maturation: Nonmammalian vertebrates. 1n “The Vertebrate Ovary” (R. E. Jones, Ed.), pp. 443-468. Plenum, New York. WEBB, A. C., LAMARCA, M. J., and SMITH, L. D. (1975). Synthesis of mitochondrial RNA by full-grown and maturing oocytes of Rana piyiens and Xenopus laevis. Dev. Biol 45,44-51. WV, M., and GERHART, J. C. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laemis. Dev. BioL 79.465-477.