Lack of heat-shock response in preovulatory mouse oocytes

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Lack of Heat-Shock

Response in Preovulatory

ARMANDO&RCI,* *Dipartiwwnto

ARTUROBEVILACQUA,**'ANDFRANCO

Mouse Oocytes MANG1A-t

di Scienze e Tecnologie Biwmediche e di Biometria, University of L’Aquila, Italy, and tfipartimento di Istobgia ed Embriobgia gonerale, University Lo Sapienza of Rome, Italy Received August 18, 1986; accepted in revised fm

di Psicologia and Istituto

March 26, 1987

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43’C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of ‘IO-kDa heat-shock cognate protein (hsc70) and of 89- and llO-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43°C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsci’0 and hsp89 of granulosa cells. 0 1987 Academic Press, Inc INTRODUCTION

Eucaryotic cells respond to a wide variety of stresses, including heat shock, by expression of specific genes and synthesis of their translation products, named heatshock proteins (hsps) (see Burdon, 1986; Lindquist, 1986, for reviews). Although the functions of the hsps are still a matter of debate (Pelham, 1986; Schlesinger, 1986), it is commonly accepted that a shock-induced synthesis of these proteins protects cells from heat-elicited damages. In contrast to differentiated somatic tissues, early vertebrate embryos are sensitive to hyperthermia (Bell&, 19’72; Heikkila et aZ., 1985), and this is apparently related to the absence of an inducible synthesis of hsps at early developmental stages. Heat sensitivity of early embryos is overcome at later stages, when the ability to respond to a stress by hsp synthesis is finally developed (Wittig et al, 1983; Morange et al., 1984; Heikkila et al., 1985; Nickells and Browder, 1985). These findings on early vertebrate embryos raise the question whether the lack of a response to heating (hs response) is inherited from oogenesis. This seems to be the case of amphibians, whose oocytes do not synthesize hsps in response to heat shock (King and Davis, 1987). In mammals, including humans, oocyte meiotic maturation, ovulation, and fertilization are sensitive to hyperthermia (Waites, 1976; Hirao and Yanagimachi, 1978; Baumgartner and Chrisman, 1981; Lenz et al, 1983), and this indirectly suggests that mammalian female germ cells may lack the machinery for cell protection from 1 Present address: Department Michigan, Ann Arbor, Michigan 0012-X06/87 Copyright All rights

of Human 48109.

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

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stresses. We asked the question whether preovulatory mouse oocytes are sensitive to heat and show here that these cells lack an inducible hs response and constitutively synthesize 70- and 89-kDa polypeptides resembling typical 70-kDa heat-shock cognate protein (hsc70) and hsp89 of mouse L cells (Lowe and Moran, 1984). MATERIALS

AND

METHODS

Animals Forty- to sixty-day-old, random bred Swiss-CD1 female mice (Charles River Italia) were primed by 5 IU pregnant mare serum gonadotropin (PMSG) and used as donors of preovulatory dictyate oocytes and of granulosa cells 46-48 hr later. Oocytes Oocytes were collected by pricking large Graafian follicles with a needle and isolated from surrounding granulosa cells in the presence of egg culture medium N16 (Whittingham, 1971) buffered with Hepes and containing 2 mg/ml bovine serum albumin (BSA) (MH). Routinely, dictyate nuclear configuration was actively maintained during oocyte isolation, heat-shock, and labeling procedures by addition of 200 fidibutyryl cyclic AMP (Cho et ak, 1974) to MH (MH-dBcAMP) and N16 (N16dBcAMP). For experimental treatments isolated oocytes were transferred to 0.5 ml MH or MH-dBcAMP in a plastic tube and incubated at the appropriate temperature for the appropriate time in a water bath. Oocytes were then collected from the tube, allowed to recover in

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HS Response in Mouse Preavulatmy

N16 or NlG-dBcAMP at 37°C for 15 min, and eventually labeled in N16 or NlG-dBcAMP containing 2 mCi/ml [35S]methionine as reported under Results. Oocyte incubations in N16 were performed in a drop of medium under paraffin oil, in the presence of an atmosphere of 5% COZ, 95% air. Labeled oocytes were washed in phosphate-buffered saline (PBS) containing 2 mg/ml polyvinylpyrrolidone and processed for determinations of labeled amino acid uptake and incorporation into total protein or for one- (SDS-PAGE according to Laemmli, 1970) and two-dimensional electrophoresis (O’Farrell, 1975). For determinations of amino acid uptake and incorporation into total protein fraction, oocytes were heat shocked as described before and incubated in the presence of 20 PALM[?S]methionine at 37°C for 3 hr. Radiolabeled proteins were precipitated by 10% (final concentration) trichloroacetic acid (TCA) in the presence of 0.05% BSA as a carrier, heated at 95°C for 10 min, collected on GFA filters (Whatman), washed with 5% TCA and 70% ethanol, and dissolved by Nuclear Chicago Solubilizer (Amersham) in a scintillation vial. Radioactivity was measured in a standard PPO-toluene cocktail by a Packard Model 4530 liquid scintillation counter. Determinations of [35S]methionine uptake were performed as previously described (Colonna and Mangia, 1983). In all experiments control cells were processed similarly to treated cells, but constantly maintained at 37’C.

Granulosa

Cells

Granulosa cells were obtained by pricking large Graafian follicles with a needle in MH. Cells were harvested by centrifugation and used immediately or after culturing in vitro at 37°C for 48-72 hr in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Granulosa cells were heat shocked at the appropriate temperature for the appropriate time in Hepes-buffered MEM supplemented with 3% FCS. In few experiments freshly collected and in vitro cultured granulosa cells were heat shocked and labeled in MHdBcAMP. After heat shock the incubation medium was substituted by methionine-free MEM supplemented with 200 &i/ml [35S]methionine, and cells were labeled at 37°C for 3 hr. Granulosa cells were then washed with PBS and finally processed for electrophoretic analysis of radiolabeled proteins. For determinations of amino acid incorporation into total protein, granulosa cells were labeled for 3 hr in the presence of 20 &I [35S]methionine and processed further as described above for oocytes. Control cells were treated similarly, but constantly maintained at 37°C. Determinations of granulosa cell protein were performed by a commercial protein assay (Bio-Rad).

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Peptide Mapping Peptide mapping was performed according to Cleveland et al. (1977) by Staphylococcus aureus V8 protease digestion of oocyte and granulosa cell polypeptides. Briefly, [35S]methionine-labeled proteins of oocyte and granulosa cells were fractionated by two-dimensional gel electrophoresis. The gel was dried without fixation and exposed to an X-ray film to localize radioactive protein spots. The area containing the polypeptide to be analyzed was then excised from the gel, rehydrated, and directly digested by 0.5 pg/slot V8 protease for 30 min at room temperature in the stacking gel of a SDS-PAGE. Chemicals PMSG (Folligon) was purchased from Intervet, [35S]methionine (sp act > 800 Ci/mmole) from DuPontNew England Nuclear, culture media from GIBCO Bioculture, and other chemicals from Sigma. RESULTS

Eflect of Heat Shocks on Oocyte Maturation The effect of heat on mouse oocyte viability was evaluated by determining the frequency of oocyte resumption of meiosis after different treatments. The fractions of oocytes matured to metaphase II after an incubation at 40-45°C for 5-90 min are reported in Fig. 1. Incubations at 40-42°C did not grossly affect oocyte morphology. In contrast, heating at 43°C distorted oocyte shape, depending on heat-shock duration. Distorted oocytes progressively recovered a normal round shape in 15-30 min at 37°C. An exposure as long as 90 min at 40-41’C did not significantly affect meiotic maturation of mouse oocytes. In contrast, an incubation at 42°C longer than 30 min progressively inhibited oocyte resumption of meiosis, treatment duration for inhibiting spontaneous resumption of meiosis in 50% oocytes (&) being 45-50 min. At 43°C IS0 was about 25 min. Incubations at 44 or 45°C as short as 15 and 10 min, respectively, completely blocked oocyte maturation. Oocytes that did not resume meiosis after a heat shock usually remained at the dictyate stage. Eflect of Heat Shocks on Mouse Oocyte and Cranulosa Cell Protein Synthesis To investigate whether mouse preovulatory oocytes displayed a hs response similar to that of somatic tissues, protein synthesis activity of shocked oocytes was compared with that of shocked granulosa cells. Labeled amino acid incorporation into total TCA-insoluble fraction during the jirst 3 hr after the shock. Results are reported in Fig. 2. When granulosa cell mono-

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FIG. 1. In vitro meiotic maturation of heat-shocked preovulatory mouse oocytes. Groups of 30-40 oocytes were isolated from granulosa cells in MH-dBcAMP and incubated at 3’7 or 40-45°C for lo-90 min in the same medium. In few experiments dBcAMP was omitted from incubation medium during heat shock, with similar results. At the end of heat treatment oocytes were extensively washed with plain medium to completely remove dBcAMP and allow resumption of meiosis, cultured in vitro for 20 hr in N16 at 3’7”C, and finally screened for meiotic maturation. Values represent the fractions of oocytes progressed to metaphase II after different heat shocks (mean f SEM of 4-10 independent experiments), taking maturation rates of control groups as 100. The experiment was discarded when the maturation rate of control oocytes was lower than 95% .Symbols represent different heat-shock temperatures; (0) 40°C; (0) 41’C; (0) 42°C (m) 43’C; (A) 44°C;

(A) 45°C.

layers were preliminarily incubated at 43°C up to 40 min, the level of [35S]methionine incorporation into total protein during the first 3 hr after the shock was not significantly modified with respect to the value found in unstressed cells. Similar results were obtained after an

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incubation at 45°C for 10 min (not shown). The presence of dBcAMP in incubation and labeling media did not affect results. In contrast to findings on granulosa cells, protein synthesis of preovulatory mouse oocytes progressively decreased with increasing heat shocks (Fig. 2). In detail, oocyte incubations at 43°C for 10, 20, 30, and 40 min reduced labeled amino acid incorporation into total protein to about 71,37,26, and 16% of control values, respectively. Oocytes incubated at 45’C for 10 min displayed incorporation values of about 13% of control cells (not shown). Removal of dBcAMP from the external medium during heat shock and labeling procedures did not modify the effect of heating on these cells. To rule out the possibility that heat shock affected oocyte activity of amino acid uptake from the external medium, [35S]methionine entrance into preovulatory oocytes was measured after a heat shock (Fig. 3). Oocytes were heat shocked at 43°C for 30 min, allowed to recover their normal shape at 3’7°C for 15 min, incubated further at 37°C for 5 or 15 min in the presence of 20 PM r5S]methionine, rapidly washed in methionine-free medium, and finally processed for determination of total intracellular radioactivity. In these experiments TCAinsoluble radioactivity was negligible. Results show that heat shock did not significantly affect oocyte activity of methionine uptake from the external medium. Therefore observed changes of [35S]methionine incorporation in heat-shocked oocytes reflect an actual heat-elicited depression of protein synthesis in these cells. Qualitative

analysis

of hat

shock induced polypeptides.

The relationship between heat-shock duration and hsp induction in granulosa cells was investigated by SDS15r

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FIG. 2. Amino acid incorporation into total TCA-insoluble fraction of in vitro labeled mouse granulosa cells and preovulatory ooeytes. Granulosa cells and oocytes were incubated at 37°C or shocked at 43°C for lo-40 min and then radiolabeled with [3SS]methionine at 37°C for 3 hr, as described under Materials and Methods. Values (cpm/mg protein of granulosa cells (0); cpm/oocyte (U); mean f SEM of at least three independent determinations) represent the fractions of pS]methionine incorporation into total TCA-insoluble fractions found after different shock durations, taking incorporation values of control cells as 100.

of Incubation

(min)

FIG. 3. In vitro [8SS]methionine uptake by unstressed and heatshocked preovulatory mouse oocytes. Oocytes were incubated at 37°C (open bars) or heat-shocked at 43°C for 30 min (hatched bars) and then incubated in the presence of 20 PM [S6S]methionine for 5 or 15 min. Values (cpm/oocyte) represent means + SEM of nine determinations obtained in two independent experiments. Difference between uptakes of unstressed and heat-shocked oocytes: F(1/32) < 1, ns. Difference between incubation times: F(1/32) = 111.44, P < 0.001. Interaction: F(1/32) = 1.93, n.s. Values calculated by two-way ANOVA (2 x 2).

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PAGE (Fig. 4). Granulosa cells responded to incubations at 43°C of at least 20 min by synthesizing definite polypeptides (Fig. 4, lanes 3-5). A similar hsp-induction pattern was also elicited by an incubation at 45°C for 10 min (not shown). Heat-elicited polypeptide synthesis was then analyzed in detail by two-dimensional gel electrophoresis (Fig. 5). Major modifications were represented by the appearance of newly made polypeptides at 33and 6%kDa apparent MW and the enhanced synthesis of 89-, 70-, and llO-kDa polypeptides (Figs. 5A and 5B). Both freshly collected and in vitro cultured granulosa cells displayed similar hs responses, and the presence of dBcAMP during heat shock and labeling procedures did not affect results. Similar modifications of granulosa cell protein synthesis were also elicited by an exposure for 45 min to 100 PM Na arsenite, an agent capable of inducing some proteins similar to those induced by heat in various biological systems (Ashburner and Bonner, 1979; Johnston et ak, 1980; Li and Werb, 1982) (not shown). When proteins made by unstressed and heat-shocked preovulatory oocytes were analyzed by two-dimensional gel electrophoresis (Figs. 5C and 5D), it was apparent that different heat treatments did not induce any new or enhanced synthesis of hsps in these cells. An analysis of proteins synthesized by unstressed oocytes showed that these cells actively made 70- and 89kDa polypeptides resembling 70- and 89-kDa polypeptides of heat-shocked granulosa cells. In fact when

FIG. 4. Autoradiograms of radiolabeled proteins synthesized by mouse granulosa cells, before and after an incubation at 43°C for increasing times. Granulosa cells were prepared, incubated at 37 or 43°C and then labeled for 3 hr at 37°C as described under Materials and Methods. Equivalent amounts of TCA-precipitable radioactivity were loaded on each lane. Arrows indicate the positions of heat induced polypeptides. Numbers indicate molecular mass of standards (daltons X 1O-3). Lane 1: unstressed cells constantly maintained at 37’C. Lane 2: heat-shock duration, 10 min. Lane 3: heat-shock duration, 20 min. Lane 4: heat-shock duration, 30 min. Lane 5: heat-shock duration, 40 min.

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[35S]methionine-labeled proteins of unstressed oocytes and of heat-shocked granulosa cells were mixed together and then fractionated by two-dimensional gel electrophoresis, 70- and 89-kDa polypeptides of unstressed oocytes comigrated with heat-inducible corresponding polypeptides of granulosa cells (Fig. 6a). In addition, V8 protease digestion of 70-kDa polypeptides from unstressed oocytes and heat-shocked granulosa cells gave similar digestion patterns (Fig. 6B). Constitutive synthesis of 70- and 89-kDa polypeptides in mouse preovulatory oocytes was not apparently affected by heat shock. DISCUSSION

The purpose of the present work was to understand whether mouse preovulatory oocytes displayed an hs response similar to the typical hs response of mammalian somatic cells, and this was performed by taking granulosa cells as a reference tissue for normal thermotolerance and hsp inducibility. In fact we show here that heat-shocked mouse granulosa cells maintained a normal rate of protein synthesis and synthesized a number of typical hsps (see Burdon, 1986) such as a “small” hsp (33 kDa apparent MW), three hsps belonging to the socalled hsp70 family (68 and 70 kDa apparent MW), and two high MW hsps (89 and 110 kDa apparent MW). Additional hsp synthesis in heat-shocked granulosa cells may have been undetected by protein labeling and separating procedures used in the present experiments. An inducible synthesis of small MW hsps is a common feature in mammals, and a variety of these proteins have been described in different mammalian systems studied. Synthesis of hsp16 has been shown in rat lens (de Jong et ak, 1986), of hsp25 in rat myoblasts (Kim et ah, 1983), of hsp26 and hsp31 in Chinese hamster ovary fibroblasts (Li and Werb, 1982), of hsp28 in rat embryo fibroblasts (Welch and Feraminisco, 1985), and of hsp37 in HeLa cells (Slater et ah, 1981). It remains to be elucidated whether hsp33 of mouse granulosa cells is structurally and/or functionally related to the above mentioned small hsps. Beside hsp33, heat-shocked granulosa cells newly synthesized two 68-kDa polypeptides, resembling isoelectric point variants of hsp68 already described in mouse L cells (Lowe and Moran, 1984). Three heat-shock inducible hsp68 genes have been recently cloned in the mouse (Lowe and Moran, 1986). In addition, heatstressed granulosa cells displayed an enhanced synthesis of a 70-kDa polypeptide, resembling 70 kDa heat shock cognate protein (hsc70) of mouse L cells (Lowe and Moran, 1984,1986). The present results therefore further indicate that in mouse the hsp70 family consists of (i) strictly heat-inducible members such as 68-kDa polypeptides described in the present work and corresponding to heat-inducible mammalian hsp70 (Voellmy et ah,

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FIG. 5. Autoradiograms of radiolabeled proteins synthesized by unstressed and heat-shocked mouse granulosa cells and preovulatory oocytes, analyzed by two-dimensional electrophoresis. Granulosa cells and oocytes were prepared, incubated at 37°C or heat-shocked at 43°C for 30 min, and then labeled at 37°C for 3 hr as described under Materials and Methods. Equivalent amounts of TCA-precipitable radioactivity were loaded for each sample. Arrowheads indicate the position of polypeptides typically induced by hyperthermia in mouse granulosa cells. Numbers indicate the molecular mass of standards (daltons X lo-‘). (A) Unstressed granulosa cells; (B) heat-shocked granulosa cells; (C) unstressed oocytes; (D) heat-shocked ooeytes.

1985; reviewed by Pehlam, 1986), Xenopus hsp68-70 (Heikkila et al, 1985; King and Davis, 1987), Lkosophyla hsp70 (Ashburner and Bonner, 1979), and Saccharomices hsp70 (see Kurtz et al, 1986); and (ii) slightly heat-inducible members such as 70-kDa polypeptide described in this work, corresponding to constitutively-expressed and/or cell-cycle-regulated hsc/hsp70 of a number of mammalian systems (Kao et aZ., 1985; O’Malley et cd., 1985; Lowe and Moran, 1986; reviewed by Pehlam, 1986), to developmentally regulated Xenop hsp70 (Bienz, 1984), and to Drosophila and yeast hscs70 (Ingolia and Craig, 1982; Lindquist, 1984). In addition to the previously mentioned hsp/hsc, heatstressed mouse granulosa cells also displayed an enhanced synthesis of 89-kDa polypeptide, similar to hsp89 of mouse L cells (Lowe and Moran, 1984), as well as to hsp83-90 of a number of vertebrate systems (see Burdon, 1986). In mouse L cells hsp89 can be resolved from one of similar size synthesized in unstressed cells (Lowe and Moran, 1984). It remains to be elucidated whether this is also true for 89-kDa polypeptides of unstressed and heat-shocked mouse granulosa cells. Finally, hs response

of mouse granulosa cells was also represented by enhanced synthesis of a llO-kDa polypeptide, resembling the nucleolus-associated, major mammalian hspll0 (Subjeck et al, 1983). Although a direct cause-effect relationship between synthesis of individual hsps and granulosa cell resistance to heat was not demonstrated in the present experiments, it is reasonable to conclude that maintenance of a normal rate of protein synthesis after a heat shock was dependent on heat-elicited synthesis of hsps in these cells. In contrast to granulosa cells, preovulatory mouse oocytes displayed a high degree of sensitivity to hyperthermia. In fact, when the heat effect on oocyte viability was measured in terms of both spontaneous in vitro resumption of meiosis and amino acid incorporation into total protein, it was apparent that these cells were severely affected by heat treatments that were well tolerated by granulosa cells. Therefore it may be concluded that in mammals hyperthermia directly damages the ovulating oocytes and is not likely to affect somatic components of Graafian follicles. It is interesting to note

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FIG. 6. Autoradiogram of 70- and 89-kDa polypeptides synthesized by heat-shocked mouse granulosa cells and by unstressed preovulatory mouse oocytes. (A) Radiolabeled proteins synthesized by granulosa cells after a heat shock at 43°C for 30 min were mixed with radiolabeled proteins synthesized by unstressed oocytes, and the mixture was then analyzed by two-dimensional electrophoresis. Note concomitant presence of granulosa cell- and oocyte-specific gene products in the autoradiogram. Arrowheads indicate polypeptides typically induced by heat in mouse granulosa cells. Bracket indicates oocyte LDH-1. Numbers indicate molecular mass of standards (daltons X lo-‘). (B) Seventy-kilodalton polypeptides synthesized by mouse granulosa cells after a heat shock at 43°C for 30 min (lane G) and by unstressed preovulatory mouse oocytes (lane 0) were subjected to limited proteolysis by V8 protease, and peptide digestion patterns were analyzed by SDS-PAGE, as described under Materials and Methods. Numbers indicate molecular mass of standards (daltons X 10e3).

that in rabbit the temperature value of antral Graafian follicles is significantly lower than that of surrounding preantral follicles and of the body core (Grinsted et al., 1980). In this respect mammalian Graafian follicles may therefore be equated to mammalian seminiferous tubules, where germ cells are sensitive to heat (Nakamura and Hall, 1980), while Sertoli cells survive well at an elevated temperature (Hall et aZ., 1985). It has been shown in the present experiments that preovulatory mouse oocytes constitutively synthesize 70and 89-kDa polypeptides, similar to corresponding heatenhanced polypeptides of granulosa cells (here demonstrated by comigration on two-dimensional gel electrophoresis and, at least for 70-kDa polypeptides, by similar V8 protease digestion patterns), and do not display heatinducible or heat-enhanced synthesis of hsps. If, as it is likely, oocyte thermosensitivity is dependent on the lack of hs response in these cells, it can be concluded that in mammalian oocytes constitutively made hsc70 and hsp89 do not confer thermotolerance per se. A developmentally induced synthesis of hsp-related proteins and the absence of strictly heat-inducible synthesis of hsps is a feature common to oogenesis in different vertebrate and invertebrate species, as well as to yeast ascospore development. For example, Xenoms oocytes constitutively express an hsp70 and do not display heat-inducible hsp synthesis, while follicle cells do (King and Davis, 1987). In Drosophila oogenesis ovarian nurse cells normally synthesize and pass to developing oocytes hsp26, hsp28, and hsp83 mRNAs and respond to heat shock by synthesis of hsp70, but in late egg chambers hsp70 is not induced even after heat shock, nor is it transferred from

nurse cells (Zimmerman et al., 1983; Ambrosio and Schedl, 1984). During ascospore development in Saccharomices hsp26 and hsp84 are strongly developmentally induced, but hsp70 is neither developmentally induced nor inducible by heat shock (Kurtz et ah, 1986). The significance of the widely spread absence of hsp68/ 70 inducibility in meiotic cells is a matter of speculation, and it has been proposed to represent a very ancient developmental pathway in eukaryotes (Kurt.2 et aL, 1986). Interestingly, both hsp68 and hsc70 represent the first developmentally induced major products of zygotic gene activity in mouse embryos and have been proposed to play an important role in controlling diploid genome expression after fertilization (Bensaude et ah, 1983). Taken together, the present observations on oocytes and previous findings on early embryos (Bensaude et aL, 1983) show that in the mouse (i) hsc70 is a major product of both preovulatory oocytes and early embryos, and (ii) hsp68 expression is completely blocked in preovulatory oocytes, but is thereafter developmentally induced following fertilization. Therefore the regulatory mechanisms of hsp gene expression are deeply rearranged throughout ovulation and fertilization in mammals. We thank Drs. Carla Boitani, Rita Canipari, Rosella Colonna, Fioretta Palombi, and Giulio Cossu for critical reading of the manuscript. This work was supported by Grants MPI 40% (Gruppo Nazionale sul Differenziamento), and CNR Progetto Finalizzato Oncologia, Contract No. 86.0046'7.44. REFERENCES AMBROSIO, L., and SCHEDL, P. (1934). melanogaster oogenesis: Analysis sections. Deu. Biol. 105, 80-92.

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BURDON,R. H. (1986). Heat shock and the heat shock proteins. Biochxm J. 240,313-324. CHO, W. K., STERN, S., and BIGGER% J. D. (1974). Inhibitory effect of dibutyryl CAMP on mouse oocyte maturation in vitro. J. Exp. ZooL 187,383-386. CLEVELAND, D. W., FISHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. BioL Chem. 252, 1102-1106. COLONNA,R., and MANGIA, F. (1983). Mechanisms of amino acid uptake in cumulus-enclosed mouse oocytes. BioL Reprod. 28,797-803. DE JONG,W. W., HOEKMAN, W. A., MULDERS,J. W. M., and BLOEMENDAL, H. (1986). Heat shock response of the rat lens. J. Cell BioL 102,104111. GRINSTED, J., BLENDSTRUP, K., ANDREASEN, M. P., and BYSKOV, A. G. (1980). Temperature measurements of rabbit antral follicles. J. Re-

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