Heat-shock response in Xenopus oocytes during meiotic maturation and activation

Cell Differentiation, 16 (1985) 161-168 Elsevier Scientific Publishers Ireland, Ltd.

161

CDF 00295

Heat-shock response in Xenopus oocytes during meiotic maturation and activation Elyane Baltus and Jacqueline H a n o c q - Q u e r t i e r Dbpartement de Biologic Molbeulaire, Universitb libre de Bruxelles, 67, rue des Chevaux, 1640 Rhode St. Genbse, Belgium (Accepted 12 December 1984)

After a 60 min heat-shock at 36°C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36°C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight. heat-shock; meiotic maturation; activation

Introduction

In response to various stresses, in particular to a rapid and transitory increase in temperature (heat-shock), eucaryotic cells generally display major changes in their pattern of protein synthesis: a large decrease in overall protein synthesis is observed, together with the conspicuous synthesis of a small set of heat-shock or 'stress' proteins which are believed to protect the cells from the stress (Adams and Rinne, 1982). While the structure of certain heat-shock genes (lngolia et al., 1980), as well as the cellular localization of some heat-shock proteins (Velasquez and Lindquist, 1984) have already been elucidated, the function of these proteins in cell survival remains unexplained. In most cells, the appearance of heat-shock proteins results from the transcription of previously silent genes, but Xenopus oocytes are excep-

tions to this rule: although they can survive a heat-shock and synthesize a major heat-shock protein of molecular weight (Mr) approx. 70000 (hsp70), this synthesis does not result from a new transcription, but from translation of a maternal m R N A stored in the cytoplasm: this protein is synthesized in heated enucleated oocytes as well (Bienz and Gurdon, 1982). Preliminary results have shown that the great majority of Xenopus laevis fertilized eggs, when submitted to a 30 min heat-shock at 36°C during cleavage, do not withstand this treatment; it is only after the mid-blastula transition that the embryos become resistant again to a heat-shock. However, Brachet (1957) showed that the development of gastrulae recovering from a heat-shock is, according to the temperature used, either arrested or slightly delayed and followed by developmental abnormalities. The molecular basis for this developmental con-

0045-6039/85/$03.30 ~ 1985 Elsevier Scientific Publishers Ireland, Ltd.

162 trol of the heat-shock response can be found in recent results of Bienz (1984), who showed that the hsp70 mRNA is undetectable during early cleavage of Xenopus eggs and reappears at gastrulation. This situation is reminiscent of that prevailing during embryonic development of sea-urchins (Roccheri et al., 1981), Drosophila (Graziosi et al., 1983), and mammals (Morange et aI., 1984). The purpose of the present study was to find out whether the loss of the capacity to make hsp70 takes place during one of the main steps preliminary to fertilization, i.e. maturation and activation. Maturation (meiotic division) includes a series of physiological and biochemical changes (Masui and Clarke, 1979) leading to the formation of the maturation promoting factor, the breakdown of the nuclear membrane (GVBD), and the expulsion of the first polar body; activation, which is normally triggered by the entrance of the spermatozoon, leads to the elimination of the second polar body and the reconstitution of the female pronucleus. We also studied the effects of an inhibitor of protein synthesis, cycloheximide, on heat-shock protein synthesis in full-grown oocytes.

Materials and Methods

Oo~Tte preparation Xenopus laevis females were obtained from Cape Town (Snake Farm). Full-grown stage VI (Dumont, 1972) oocytes were isolated by manual dissection with watchmaker's forceps and were maintained in modified Barth solution (MBS) (88 mM NaC1, 1 mM KC1, 2.4 mM N a H C O 3, 0.82 mM MgSO 4, 0.33 mM Ca(NO3) 2, 0.41 mM CaCI 2 and 10 mM HEPES at pH 7.4). Germinal vesicle breakdown (GVBD) in oocytes treated with progesterone (10 /zg/ml MBS) was scored by observing the white spot which appears at the animal pole and by dissection of a few boiled oocytes. Activation of in vitro matured oocytes was obtained by a 5 rain treatment of the oocytes with the A 23187 calcium ionophore at 20 ~ g / m l MBS; it was prepared from a stock solution (5 m g / m l dimethylsulfoxide). In some experiments, the

oocytes were pretreated with cycloheximide (10 # g / m l MBS) during 1 h, kept in the inhibitor during the heat-shock and finally washed in MBS for 20 rain.

Heat treatment and labelling of the oocytes with [-~SS]methionine Groups of 15 oocytes were preheated in Eppendorf tubes in 150 /~1 MBS in a waterbath at 36°C for 15 rain or left at 22°C for the same time: they then received variable amounts of [35S]methionine (Amersham, ca. 1220 Ci/mM). After a 45 rain treatment at either 36°C or 22°C with the precursor, the oocytes were washed with cold methionine in MBS, homogenized in 150 /~1 of cold medium (30 mM Tris, 250 mM sucrose, pH 7.4), and the homogenate was centrifuged at 1 2 0 0 0 × g for 10 rain. The amount of [35S]methionine taken up by the oocytes was estimated by liquid scintillation in 10 /~1 aliquots of the supernatant to which 3.5 ml of a mixture of Lumac LipoLuma-LumaSolve (9 : 1) had been added. The amount of [35S]methionine incorporated in the proteins was estimated by trichloroacetic acid precipitation of samples of the supernatant spotted on Whatman G F / C filters. Their radioactivity was estimated by liquid scintillation as above.

One-dimension sodium dode~)'l sulfate-polyacrylamide gel electrophoresis (SDS-PA GE) The supernatant of homogenized and centrifuged 35S-labelled oocytes was processed and electrophoresed according to Bienz and Gurdon (1982). ~4C-methylated proteins from Amersham were used as molecular weight markers. Fluorography of the gels was performed according to Chamberlain (1979), using a 30 rain soaking in 1 M sodium salicylate. After drying, the gels were exposed to preflashed Fuji X Ray film and stored at - 70°C before development.

Results

Lffect of a heat-shock on maturation Oocytes were incubated in Eppendorf tubes placed in a 36°C waterbath for periods varying

163

%

GVBD

100

._.___._..

o

0

N~"r

I 5

4

i1

•

I 6

I 7

HOURS Of pg TREATMENT

Fig. 1. Germinal vesicle breakdown after a heat-shock given at different times during progesterone treatment. Groups of 50 oocytes were treated with progesterone (10 / , g / m l ) at zero time. A 45 min heat-shock at 36°C was applied at zero time (0), 45 min (A), 2 h 15 min (m), and 3 h 30 min ( I ) after the addition of progesterone. A group of progesterone-treated oocytes was not submitted to a heat-shock (o). The percentage of GVBD was recorded at various intervals in each group. pg = progesterone.

from 0 to 60 min and then received a progesterone treatment. Whatever the length of the heat-shock, the treated oocytes were able to accomplish a normal maturation; however, the first maturation white spots appeared with a delay almost equivalent to the length of the heat-shock treatment. In other experiments, oocytes were first treated with progesterone; a 45 rain heat-shock at 36°C was then applied at different times after addition of the hormone. Control progesterone-treated oocytes maintained at 22°C accomplished 50% G V B D in 4 h 30 min. Fig. 1 shows that, whatever the time the heatshock was applied during the progesterone treatment, the oocytes accomplished maturation; however, G V B D was delayed in oocytes which were submitted to the heat-shock during the late stages of the maturation process, which correspond to the amplification of the maturation promoting factor. Do maturing oocytes synthesize heat-shock proteins when they are submitted to a heat-shock? To answer this question, oocytes were heatshocked at different times after the addition of

TABLE I Incorporation of [3SS]methionine in progesterone-treated oocytes submitted to a heat-shock Treatment prior to labelling

Temperature of labelling ( ° C)

Uptake cpm

Incorporation into proteins

MBS MBS

22 36

370 570 444015

90 740 13097

100 14.4

36 36 36 22 36 36

363 215 277773 480 854 376 857 409617 405 756

15150 11 299 23 821 146 822 23 215 26 327

16.7 12.4 26.2 161 .g 25.6 29

cpm

% of controls

Progesterone in MBS during: 1 30 60 120 120 180

min rain min min rain rain

Groups of 15 control oocytes and 15 progesterone-treated oocytes * were preincubated at either 22 or 36°C for 15 min in 150 ttl MBS and then further incubated for 45 min at the same temperatures after addition of 45 p.Ci [35S]methionine. The results are expressed as cpm/oocyte per 45 min. • 50% of the oocytes treated With progesterone accomplished maturation in 3 h 45 rain.

164

progesterone, and both the level and the pattern of protein synthesis were analyzed. Table I indicates that the heat-shock induced a strong decrease in methionine incorporation in the oocytes, even in those treated with progesterone; yet, in non-heated oocytes, the level of protein synthesis strongly increased during the course of maturation, in confirmation of previous results (Baltus et al., 1973).

a

b

c

d

One-dimension gel electrophoresis (Fig. 2) shows that a heat-shock increases the labelling of a single heat-shock protein, hsp70. This protein is synthesized whether the heat treatment is applied to control oocytes or to oocytes treated with progesterone for various lengths of time. It must be pointed out that progesterone treatment without a heat-shock does not affect the labelling of hsp70. The almost uniform accumulation of hsp70 in

e

f

g

h

Fig. 2. Pattern of protein synthesis during a heat-shock given to oocytes at different times of a progesterone treatment. Autoradiograph of a one-dimension SDS-polyacrylarnide gel. 50000 cpm of each of the samples described in Table I were loaded in each lane of the gel. (a) Untreated oocytes incubated with [35S]methionine at 22°C. (b) Oocytes treated for 120 min with progesterone and then incubated with [35SJmethionine at 22°C. (c) Untreated oocytes incubated with [35S]methionine at 36°C. (d h) Oocytes treated with progesterone during 1 min (d), 30 rain (e), 1 h (f), 2 h (g) and 3 h (h) and then incubated with [35S]methionine at 36°C. The arrow points to hsp70. Exposure time: 11 days.

165 r e s p o n s e t o a h e a t - s h o c k g i v e n at a n y p e r i o d o f t h e

s y n t h e s i s o f h s p 7 0 in r e s p o n s e to a h e a t - s h o c k

m a t u r a t i o n p r o c e s s w a s u n e x p e c t e d since, as s h o w n

d o e s n o t b y itself a l l o w a n i m m e d i a t e r e s u m p t i o n

in Fig. 1, a h e a t - s h o c k i n d u c e s a s e v e r e d e l a y in

o f m a t u r a t i o n in o o c y t e s w h i c h h a v e b e e n h e a t -

the

accomplishment

of maturation,

u n l e s s it is

a p p l i e d at t h e b e g i n n i n g o f t h e p r o g e s t e r o n e t r e a t ment.

It m u s t b e c o n c l u d e d

that

s h o c k e d during the late stages of the meiotic process.

the increased

TABLE II Incorporation of [35S]methionine into activated oocytes submitted to a heat-shock Temperature of labelling (°C)

Amount of [35S]methionine in incubation medium (#Ci)

Uptake (cpm)

Incorporation into proteins (cpm)

% Incorporation/ uptake

Control fullgrown oocytes

22 36

5 45

16387 122257

4968 3601

30 2.9

Activated oocytes

22 36

31 341

21 333 125 525

8 405 2 899

39.4 2.3

Groups of 15 full-grown oocytes and groups of 15 ionophore-treated oocytes were preincubated at either 22 or 36°C for 15 min in 150 /~1 MBS and then further incubated for 45 min at the same temperatures with [35S]rnethionine. The results are expressed as cpm/oocyte per 45 min.

TABLE III Effect of cycloheximide on protein synthesis in oocytes submitted to a heat-shock at 36°C Batch

Pretreatment for 60 min

Treatment for 60 min at temperature (°C)

Time of labelling

Temperature of labelling (°C)

Uptake (cpm)

1 2 3 4 5 6

cyclo

36 36 36 36 36 (+cyclo)

during HS 20 mini 2 h ~ after HS 4h ] during HS

22 36 22 22 22 36

7 8 9

cyclo cyclo cyclo

36 ( + cyclo) 36 (+cyclo) 36 ( + cyclo)

20 min]after HS 2 h ~and removal 4 h ) o f cyclo

10

cyclo

22 ( + cyclo)

cyclo

22 (+ cyclo)

at the end of pretreatment 4 h after removal of cyclo

11

Incorporation into proteins cpm

% of controls

186521 164060 100097 141601 175 370 170110

19679 2478 7004 13928 26127 145

100 12.5 35.6 70.7 133 0.7

22 22 22

186765 113851 143 863

192 689 9638

1 3.5 49

22

184470

842

4.3

22

160893

17157

87.2

Groups of 15 control or cycloheximide-pretreated oocytes were left for 60 min at 22°C (1, 10, 11) or at 36°C (2-9) in 150 /xl MBS. Batch 1 received 70 t~l of [35S]methionine during 45 min at 22°C. In heat-shocked control (2-5) or cycloheximide-pretreated (6-9) oocytes, 70 p~l of [3SS]methionine were given during 45 rain, either during the heat-shock (2,6) or after a recovery period at 22°C (3-5 and 7-9). Batches 2 and 6 were left for 15 min at 36°C before addition of the isotope. In cycloheximide-pretreated oocytes at 22°C, 70 p.l of [35S]methionine were given to the oocytes either immediately after the pretreatment (10) or 4 h after the removal of the inhibitor (11). The results are expressed as cpm/oocyte per 45 min. cyclo = cycloheximide 10 ~g/ml; HS = heat-shock.

166

Do activated oocytes synthesize heat-shock proteins in response to a heat-shock?

The activation of matured oocytes with the A 23187 calcium-ionophore leads to vitelline membrane uplifting within about 5 min; it is followed by the appearance of irregular abortive furrows. A heat-shock was applied 10 min after the activating treatment; it did not prevent the abortive cleavages. Since activated oocytes display a much lower permeability to amino acids than full-grown

a

oocytes, the amounts of [35S]methionine given to the ionophore-treated oocytes had to be adjusted in order to obtain a sufficient darkening of the fluorograms. Table II shows that, in activated oocytes, the heat-shock led to a 94% inhibition of overall protein synthesis, similar to that observed in heated full-grown control oocytes (90%). Amongst the

a

b

c

d

b

Fig. 3. Pattern of protein synthesis after a heat-shock applied to activated oocytes. Autoradiograph of a one-dimension SDSpolyacrylamide gel. Equal amounts of the protein samples described in Table II were loaded in both lanes. (a) Activated oocytes incubated with [35S]methionine at 22°C. (b) Activated oocytes incubated with [35S]methionine at 36°C. The arrow points to hsp70.

Fig. 4. Pattern of protein synthesis in oocytes recovering from a combination of cycloheximide treatment and heat-shock at 36°C. Autoradiograph of a one-dimension SDS-polyacrylamide gel. 50000 cpm of the protein samples described in Table llI were loaded in each lane of the gel. (a) Untreated oocytes incubated with [35S]methionine at 22°C, (b) Oocytes submitted to a heat-shock at 36°C and allowed to recover during 4 h at 22°C before labelling with [35S]methionine. (c) Oocytes treated during 2 h at 22°C with cycloheximide and given [35S]methionine 4 h later. (d) Oocytes pretreated during 1 h at 22°C with cycloheximide, then submitted to a heat-shock at 36°C (in the presence of cycloheximide) and allowed to recover during 4 h at 22°C before labelling with [35S]methionine. Markers indicate molecular weights (kilodaltons). Thin arrow points to hsp83, thick arrow to hsp70. Exposure time: 6 days.

167 proteins still synthesized during the heat-shock by the activated oocytes, there was a conspicuous hsp70, as shown in Fig. 3.

Effect of inhibition of protein synthesis during a heat-shock Is survival of the oocytes through a heat-shock always correlated with a heat-shock protein synthesis? In order to answer this question, we first observed that oocytes pretreated with cycloheximide can survive the heat-shock and even accomplish maturation in the presence of progesterone, provided that cycloheximide is washed out after the heat treatment. Since the cycloheximide treatment suppresses 99% of protein synthesis in oocytes submitted to a heat-shock at 36°C (Table III), it is unlikely that heat-shock proteins are made under these conditions during the heat treament; but they might be synthesized during the recovery period. Therefore, the ability of the oocytes to synthesize heat-shock proteins was studied at different times of recovery after the combined cycloheximide/heat-shock treatment. Table III shows that, following a heat-shock at 36°C for 1 h, the level of total protein synthesis had almost returned to normal after 2 h; in contrast, the recovery after 4 h was only 49% in cycloheximide-treated oocytes given the same heat-shock. Fig. 4 shows that a hsp70 is clearly visible after a 4 h recovery from a heat-shock, both in control and in cycloheximide-treated oocytes. Moreover, a second heat-shock protein of M r approx. 83000 (referred to as hsp83) appears in the oocytes submitted to a combined cycloheximide/heat-shock treatment.

Discussion

Our results show that, after a heat-shock at 36°C for 60 min, Xenopus laevis full-grown oocytes not only survive, but are able to accomplish maturation after treatment with progesterone. It has also been found that if a heat-shock is applied at any time during maturation, it induces a hsp70

similar to that already described (Bienz and Gurdon, 1982) in non-maturing oocytes submitted to the same heat-shock. During maturation, poly(A) + mRNAs undergo an important redistribution in their localization (Capco and Jeffery, 1982) and about 50% of them are lost during this process (Darnbrough and Ford, 1979). Our present results show that these changes do not affect the mobilization or translation of the hsp70 mRNA in response to a heat-shock given during maturation. In ionophore-activated oocytes, a significant hsp70 production is still observed in response to a heat-shock; these oocytes can make irregular cleavage furrows like the non-heated activated oocytes. When oocytes are given a cycloheximide treatment combined with a heat-shock, two heat-shock proteins, of M r approx. 70000 and 83000, are expressed during recovery. Thus, unmasking of the hsp70 mRNA takes place even in the absence of protein synthesis, and the oocytes can survive the heat treatment before accumulation of hsp70 takes place. The other heat-shock protein, hsp83, is not found in Xenopus oocytes (Bienz and Gurdon, 1982, and our present results) nor in Xenopus embryos or somatic cells (Bienz, 1984) submitted to a heat-shock alone. Our present experiments should establish whether the cycloheximide-induced synthesis of hsp83 results from new transcription or from unmasking of a preexisting mRNA. What might be the role of the heat-shock proteins in allowing the survival of heat-shocked oocytes? Among the heat-shock proteins, hspT0 is by far the most ubiquitous and the most abundant one found in all cell types studied so far. This protein is associated with the cytoskeleton (Schlesinger et al., 1982), and its role might be to maintain the structural organization of the cell after a heat-shock. Our own unpublished experiments show that a heat-shock applied to cleaving Xenopus eggs induces the fast disappearance of the furrows, possibly by altering the contractile ring; this is not an unlikely possibility, since Hughes and August (1982) have suggested that hsp70 mediates an association between the plasma membrane and the underlying cytoskeleton.

168

Our results also establish that the time where

Xenopus eggs lose their capacity to synthesize hsp70 must, at the earliest, be that of the reconstitution of a diploid nucleus (amphimixy) following fertilization.

Acknowledgments We are very grateful to Professor J. Brachet for his critical reading of our manuscript. E.B. is a Senior Research Associate of the National Fund for Scientific Research Belgium. J.H.Q. is Research Associate of the same fund.

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