6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity

Experimental Cell Research 176 (1988) 68-79

6-Dimethylaminopurine Blocks Starfish Oocyte Maturation Inhibiting a Relevant Protein Kinase Activity

by

I. NEANT and P. GUERRIER’ Developmental

Biology,

Station Biologique,

29211 Roseoff,

France

The puromycin analog N,,N,-dimethyladenine (6-dimethylaminopurine or 6-DMAP) was found to inhibit meiosis reinitiation in starfish oocytes stimulated by the natural hormone I-methyladenine. Increasing concentrations of this agent delayed and eventually blocked germinal vesicle breakdown. They were found to be effective even when applied during the hormone-independent period, after the oocytes had been already committed to reinitiate meiosis. 6-DMAP mimics most of the effects of emetine since it induces protein dephosphorylation, inhibits polar body formation, and promotes the precocious appearance of resting nuclei. However, unlike emetine, 6-DMAP does not affect protein synthesis. The effect of this agent cannot be accounted for by a stimulation of the protease or phosphoprotein phosphatase activities since the rate and extent of protein dephosphorylation do not increase in its presence. Data from in vivo and in vitro endogenous protein phosphorylation experiments suggest rather that 6-DMAP may directly or indirectly affect the activity of a relevant c-AMP and Ca’+-independent protein kinase which is stimulated after hormone addition and seems to support starfish oocyte maturation. 0 1988 Academic RUSS, IX.

Germinal vesicle prophase-arrested oocytes of most animals are triggered to resume synchronous meiotic cleavage in response to various external stimuli [l, 21. This feature proved to be very useful in understanding some of the biochemical processes and cytoplasmic activities which were found to regulate specific events of the cell cycle [3-6]. Thus, it has been established, by cell fusion and heterologous transfers of cytoplasm, that a universal factor, known as M-phase promoting factor (MPF), controls both the release of oocytes from the prophase block and the progression of somatic cells from G2 to mitotis [7-211. It is generally agreed that this factor, which promotes germinal vesicle breakdown (GVBD), nuclear envelope disruption, chromosome condensation, and spindle formation, may be a protein kinase or a phosphoprotein. Indeed, MPF activity appeared always to be associated with a high level of protein phosphorylation, as observed during meiosis reinitiation in amphibians [22], mammals [23, 241, starfishes [25, 261, echiuroids [27], polychaetes [28, 291, and mollusks [21, 301. The same correlation between phosphorylation and MPF activity was also observed after activation of the amphibian oocyte [31], during early synchronous cleavage of the echinoderms [18, 321, or during division of somatic mammalian cells [33]. During starfish oocyte maturation, where two successive phosphorylation bursts are observed, MPF activity and the extent of protein phosphorylation were found to oscillate in parallel with the cell cycle [26]. Thus, MPF activity and phosphorylation dropped transiently during first polar body extrusion, 70 min ’ To whom reprint requests should be addressed. Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827188 $03.00

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after hormone addition, to disappear completely after the second meiotic cleavage which occurs some 50 min later [26, 341. It has been shown that the first increase and decrease in protein phosphorylation and MPF activity are true posttranslational processes that occur normally in the absence of protein synthesis (26, 351. In contrast, the reappearance of MPF after first polar body extrusion [26] or following activation of the ootid [32,34] was found to require the synthesis of new proteins. These newly formed proteins seem to control the cyclic activity of a CAMP and Ca*+-independent protein kinase which is activated following hormone addition [36], cycies during meiosis, and does not reappear after first meiotic cleavage when early protein synthesis is blocked by emetine [26, 321. In this paper, we report about the in uiuo and in vitro effects of 6-DMAP (N6,iV6-dimethyladenine) in inhibiting starfish oocyte maturation. The choice of this structural analog of puromycin has been directed by the fact that it was described to block cell division without affecting protein synthesis [37, 381. Our data confirm this point and show that this inhibitor induces dephosphorylation and loss of MPF activity by affecting the CAMP and Ca*+-independent protein kinase which seems to support starfish oocyte maturation. MATERIALS

AND METHODS

Materials. [3SS]methionine (SJ 1515), ‘*P-labeled carrier-free phosphate (PBS 13), and [y-‘*P]ATP (PB 108) were purchased from Amersham. Unlabeled biochemical compounds were obtained from Sigma; sodium dodecyl sulfate (SDS), acrylamide, and N-N’-methylenebisacrylamide from BDH. Molecular-weight-labeled markers were kindly provided by Dr. Nancy Standart (Cambridge). Handling of oocytes. Oocytes from Aster& rubens and Marthasterias glacialis were prepared free of follicle cells in Ca2’-free artificial seawater [39]. As already reported 1353,the presence of lmethyladenine (1-MeAde) in the external medium is required for only a minimum period of time designated as the hormone-dependent period (HDP). The HDP amounts to about 5 min at 24”C, while GVBD occurs from 8 to 13 min later. First and second polar bodies are extruded around 75 and 120 min. Protein synthesis measurements. Protein labeling was performed by adding [‘SS]methionine directly to the medium. For determining the initial rates of incorporation into proteins, l-ml ahquot samples recovered 1, 3, and 5 min after addition of the labeled amino acid (0.5 pCi/ml) were injected into 4 ml ice-cold TCA, washed twice in the same medium, dissolved in 1 ml 0.5 N NaOH, and reprecipitated overnight. The final pellet, washed again with 5 ml TCA, was dissolved in 0.5 N NaOH. Protein concentration was determined [40] and cpm were recorded from 0.5-ml samples suspended in 8 ml ACS liquid scintillant (Amersham). For electrophoresis and autoradiography, the oocyte suspensions (6x 10’ oocytes/ml, 25 or 50 @X~LI) were continuously stirred with a motor-driven glass paddle (60 rpm). At 5-min intervals, 50 ul aliquot samples were withdrawn from the lo- to 12-ml culture and processed as described in [41]. The 15% SDS-polyacrylamide gels were autoradiographed using Amersham hyperfilm B-max. Incorporation of phosphate into proteins. For studying the rate, extent, and kinetics of in uivo endogenous protein phosphorylation, GV-blocked oocytes (lo4 cells/ml) were preloaded with 10 @X/ml 32Pfor 4 h and washed free of external label. Control oocytes and oocytes stimulated with lMeAde in the presence or absence of various concentrations of 6-DMAP were run simultaneously. The cultures were continuously stirred at 60 rpm and maintained at the temperature of 18°C in a water bath. At various intervals, l-ml aliquot samples wee recovered and processed as described for protein synthesis measurements. In vitro protein dephosphorylation was followed according to the same procedure. The extraction medium for preparing the homogenates contained 250 mM sucrose, 1 m&f EDTA, 50 n-&f Tris-HCl, pH 7, and 0.1% j3-mercaptoethanol. Incubations were performed in 0.6 ml (300 pl buffer, 100 ul homogenate, and 100 ~1 of distilled water, dimethylsulfoxide or the drug tested). The reaction was stopped at various time intervals by adding ice-cold TCA (10% final concentration) to different aliquots prepared from the same homogenate.

Fig. 1. Polynuclei and reversibility of the action of 120 @4 6-DMAP applied at GVBD to Asterias rubens oocytes. (A) No polar bodies were formed and polynuclei appeared 90 min after hormone addition. Picture taken 4 h after hormone addition and 2 min after washing out the inhibitor. (S, C, n) The same oocyte observed 4, 8, and 38 mm later. Bar, 50 urn.

Endogenous protein kinase activity was determined in homogenates prepared and assayed as described by Sano [36], using [y-‘*P]ATP as the phosphate donor. Cytological obseruations. GVBD, polar body extrusion, spindles, and resting nuclei were easily observed in uiuo. Semipermanent preparations were also routinely performed after the oocytes were fixed for 10 min in Camoy fluid, stained in some drops of acetic carmine and glycerol, and mounted between the slide and coverslip. The state of chromatin decondensation as well as chromosome individuality were also checked after the oocytes were extracted for 30-60 min in KGE medium containing 1% Triton X-100 [42], fixed in Camoy, and stained with 1 p&I of the fluorochrome bisbenzidine H33342 (Hoechst). Slides were observed through a BH2 Olympus microscope equipped for Nomarski and epifluorescence.

RESULTS Biological and Cytological Effects of 6-DMAP Increasing concentrations of 6-DMAP, applied to M. glacialis and A. rubens oocytes 5 min before threshold hormone stimulation, were found to inhibit polar body extrusion. When concentrations ranging from 15 to 60 u&I were applied, first meiotic cleavage might occur in some oocytes, which resulted in the formation of two instead of one female pronucleus. At higher concentrations, however, first and second polar bodies never formed and four or even more resting nuclei were produced (Fig. 1A). These nuclei appeared quite early, 90 min after hormone stimulation, i.e., after the first and before the second polar body were extruded in the control oocytes that never reform a nucleus between these

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Fig. 2. Effect of 6-DMAP in inhibiting GVBD of Asterias rubens oocytes submitted to increasing hormone concentrations. The experiment was run with control oocytes (0) or oocytes which received 120 @4 (O), 150 @I (B), or 300 PM (A.) 6-DMAP simultaneously with the hormone.

events. Protein synthesis inhibitors such as puromycin (1 m&Q, cycloheximide (2 mit4), and emetine (36180 l&0 were found to produce the same cytological modifications as 6-DMAP, except that first polar body extrusion was not suppressed in their presence. Moreover, these modifications did not appear to be reversible by simply washing out the inhibitor as we observed to be the case for 6DMAP-treated oocytes. Under these conditions, the nuclear envelope disappeared, chromosomes recondensed, and spindles reformed within 15-30 min (Fig. 1B, C, and 0). Data presented in Fig. 2 show that GVBD is blocked by concentrations of 6DMAP higher than 100 @4 and that this inhibition may be released by increasing the hormone concentration. Another effect of the drug was to delay GVBD in an exponential manner as its concentration increased (Fig. 3). Since I-MeAde is supposed to bind to its membrane receptors via the N7-N9 region [431, we did check if 6-DMAP could still inhibit meiosis when added after the end of the hormone-dependent period, when oocytes are already committed to resume meiosis. This was found to be the case, as reported in Fig. 3 which shows that the higher the concentration of 6-DMAP was, the later it could be applied to block GVBD. Thus, 2 mM 6-DMAP proved to be effective when added 6 min after the end of HDP, i.e., 3 min only before GVBD occurred in control-stimulated oocytes. Concentrations of 6-DMAP as low as 150 @! were also found to block the meiotic cleavages and to induce polynuclei formation even when added as late as 45 min after hormone stimulation, i.e., 30 min only before first polar body extrusion. Instead, when GVBD occurred in the presence of concentrations equal to or higher than 600 yM, no spindle was formed and the nucleolus persisted within the dispersing nucleoplasm area.

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Fig. 3. Effect of increasing concentrations of 6-DMAP on the kinetics of GVBD in Asterias rubens oocytes. GVBD occurred respectively 13 and 18 min after the addition of 1 pi14I-MeAde to control or 120 @f 6-DMAP-treated oocytes. Ten micromolar I-MeAde was required to release the blocking effect of 150 @4 6-DMAP.

From these data, it appears that 6-DMAP is likely to exert its concentrationdependent effects by affecting a biochemical process which actually overpasses the duration of the HDP. This process seems to control MPF formation since it does regulate nuclear disruption, chromosome condensation, and spindle formation. Effects of 6-DMAP upon Protein Synthesis Initial rates of incorporation were measured in oocytes suspensions stimulated by the hormone (20 @4) in the absence or in the presence of 100 @4 emetine or 150 l.&! 6-DMAP. Determinations were performed immediately or at various times during the meiotic cycle, i.e., after 30, 50, 80, and 110 min. These experiments showed that 6-DMAP, in contrast to emetine, did not block but rather increased the rate of protein synthesis. Moreover, no qualitative or quantitative differences were observed between electrophoretograms obtained from hormonestimulated oocytes incubated in the absence or presence of 6-DMAP. In contrast, a striking difference appeared in the banding patterns observed when emetine or 6-DMAP (up to 600 @4) was added to the oocyte suspensions 10, 30, or 60 min after starting the incubation (Fig. 5). While 6-DMAP did not affect the overall pattern of protein synthesis, emetine addition led to the definitive disappearance of cyclin, a major 54-kDa polypeptide which is continuously synthesized after hormone stimulation but is destroyed at each meiotic cleavage [41]. From these data, it thus appears that the biological effects of 6-DMAP cannot be accounted for by a failure of the protein synthesis machinery.

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Fig. 4. Inhibition of GVBD following the addition of increasing concentrations of 6-DMAP after achievement of the hormone-dependent period (HDP). Aliquots from an oocyte suspension of Asterias rubens, stimulated with 0.25 p&f I-MeAde, were either diluted 100 times at the specified times to determine duration of the HDP (@) or injected in vessels containing 100 l&f (0), 300 p&f (W, 600 uJ4 (Cl), or 2225 n&f (A) 6-DMAP to determine the period of sensitivity to these inhibitor concentrations. Control GVBD occurred 13 min after hormone addition.

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Fig. 5. Effects of emetine (100 @4) and 6-DMAP (150 @f) on the patterns of protein synthesis observed after incubating oocyte suspensions (7x10’ cells/ml) of Marthasterias glacialis with 25 @/ml [35S]methionine. Cyclin (54 kDa) was found to persist and to cycle in the presence of 6-DMAP (A), whereas it disappeared at first meiotic cleavage, following emetine treatment (B). The inhibitors were added at 60 min.

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Fig. 6. Endogenous protein phosphorylation as observed during maturation of Asterias rubens oocytes preloaded for 4 h with 10 @i/ml [“PIphosphate. 0, control oocytes stimulated with 1 uM IMeAde; 0, oocytes preincubated for 5 min with 150 fl6-DMAP and further stimulated with 10 uM I-MeAde in presence of the inhibitor. Under these conditions, no polar bodies form, the second phosphorylation burst is lacking, and resting nuclei appear precociously at 90 min.

Effects of 6-DMAP on in Vivo Endogenous Protein Phosphorylation The concentration-dependent effects of 6-DMAP are nicely accounted for by the fact that addition of this inhibitor rapidly and dramatically decreases the extent of endogenous protein phosphorylation as measured from [32P]phosphate preloaded oocytes. While concentrations as low as 150 PM definitively reduce the first phosphorylation burst, which results in the precocious formation of resting nuclei (Fig. 6), higher concentrations were found to block this cycle or to promote a dramatic dephosphorylation which started right after drug addition (Fig. 7). Such a progressive reduction in phosphorylation capacity correlates perfectly with the cytological effects of 6-DMAP which include chromosome decondensation and pronuclear formation, as expected to occur after loss of MPF activity. To check if 6-DMAP-induced dephosphorylation might result from phosphoprotein phosphatase activation, we prepared homogenates from lMeAde-stimulated oocytes and followed the kinetics of their dephosphorylation in vitro. We thus found that this process could be slowed down by the phosphatase inhibitor a-naphtyl phosphate [44] and various protease inhibitors, while emetine or 6-DMAP did not change the rate of protein dephosphorylation (Fig. 8). Moreover, 6-DMAP was found unable to modify the protective effect of these inhibitors.

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Fig. 7. Effect of 6-DMAP on the phosphorylation of endogenous proteins in Asterius rubens oocytes preloaded for 4 h with 10 @i/ml [32P]phosphate.6-DMAP (600 CIM)was added simultaneously with 4 w I-MeAde at 0 time (0) or every 10 min up to 50 min after hormone stimulation (Cl). Phosphorylation of control-stimulated (M) and unstimulated oocytes (0) was also recorded.

Effects of 6-DMAP upon in Vitro Endogenous Protein Phosphoylation It has been shown already that the in uivo addition of I-MeAde stimulated the activity of a Ca*+ and cAMP-independent histone kinase [25, 36, 4.5, 461 which cycled in agreement with MPF activity all along the cell cycle [32]. In this study, we have compared this activity in crude homogenates prepared from oocytes which were stimulated by the hormone in the absence or the presence of 100 PM emetine or various concentrations of 6-DMAP. Alternatively, these drugs were directly added to the incubation medium which was supplemented or not with exogenous histones. In both conditions, we found that emetine inhibited protein kinase activity only after the first phosphorylation burst, which confirmed previous data [32], whereas 6-DMAP was already effective at this early stage. This effect of 6-DMAP is illustrated in Fig. 9 which also shows that a direct addition of this drug to the incubation medium reduces protein kinase activity of both control and stimulated oocytes to the same basal level. The in uiuo and in vitro inhibitions exerted by 6-DMAP were both found to saturate between 300 and 600 fl and to be independent upon the addition of EGTA (Fig. lOA). Even when the addition of exogenous CAMP did not affect endogenous protein kinase activity, it appeared that the residual 6-DMAP resistant activity found in our homogenates

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Fig. 8. Protease and phosphoprotein phosphatase activities as measured in uitro from a homogenate prepared from Asterias rubens oocytes preloaded for 4 h with [“PIphosphate (20 uCi/ml) and stimulated for 50 min with 1 N I-MeAde. X, a-naphtylphosphate (33 mm; l , chymostatin (1.66 mg/ml); 0, chymostatin (0.41 mg/ml); A, 6-DMAP (2.5 mM); 0, DMSO or distilled water.

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Fig. 9. Inhibition of endogenous protein kinase activity by 6-DMAP as observed in homogenates prepared from Asterius rubens oocytes. 0, Control unstimulated oocyte; A, oocytes treated for 20 min with 1 pit4 I-MeAde; 0, oocytes stimulated for 20 mitt in the presence of 150 t&I 6-DMAP. 6DMAP (250 @4) was also added in vitro to aliquots of the homogenates from control (0) and stimulated oocytes (A).

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Fig. 10. In vitro effect of 6-DMAP, EGTA, and protein kinase inhibitor (PKI) upon endogenous protein phosphorylation as observed in crude homogenates prepared from Ask-rim rubens oocytes stimulated for 5 min by 1 p&f I-MeAde. (A) Effect of increasing concentrations of 6-DMAP (initial rate values measured 2 min after adding [Y-~*P]ATP. (E) Effect of PKI upon residual protein kinase activity. 0, I-MeAde-stimulated oocytes + 1 mM EGTA; 0, same without EGTA; A, same +3tM p&! 6-DMAP; Cl, same +300 @4 6-DMAP and 1 mg/ml PKI.

could be further reduced in the presence of PKI, the thermostable inhibitor of CAMP-dependent protein kinase (Fig. 10B). DISCUSSION Although 6-DMAP has already been shown to block cell division both in the bivalve Spisula, the sea urchin [37], the crustacean Artemia salina [38], and the amphibian Xenopus (Charbonneau and Guerrier, unpublished), its target and the mechanisms of its action remained so far poorly understood. Recently, however, we reported that this drug could also block oocyte maturation in the prosobranch mollusk Patella uulgata by directly affecting protein phosphorylation without inhibiting protein synthesis [21]. The aim of this work was thus to investigate the effects of this inhibitor upon both processes of protein synthesis and protein phosphorylation during hormone-induced maturation of M. glacialis and A. rubens starfish oocytes. During early cleavage of the sea urchin [ 181as well as during oocyte maturation and first cleavages of the starfish [26, 321, it has been shown that protein phosphorylation and MPF activity oscillated in parallel with the cell cycle. In both systems [41, 481, as well as in the surf clam Spisula solidissima [49], it has also been reported that a specific protein, known as cyclin, was periodically destroyed before each cleavage and that protein synthesis was required at each cycle for next cleavage to occur [32, 471. Cyclin, protein phosphorylation, and MPF activity must be related in some way since it has been recently shown that

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the cyclin mRNA from Spisufa could actually reinitiate meiosis when microinjetted in Xenopus GV-arrested oocytes [49]. In this report, we describe that cyclin and other starfish proteins are normally synthesized in the presence of 6-DMAP. Despite this, 6-DMAP was found to block GVBD in a dose-dependent manner, an effect which could be reversed either by washing out this compound or by increasing the hormone concentration. Interestingly, 6-DMAP appeared as the only inhibitor of starfish oocyte maturation known so far which could both delay GVBD and block maturation when applied during the hormone-independent period, after meiosis had been already committed. These concentration-dependent effects are nicely accounted for by the fact that addition of this inhibitor rapidly and dramatically decreases the extent of endogenous protein phosphorylation as measured in ho. This agrees perfectly with the observation that 6-DMAP precociously mimics all the cytological effects of emetine, a drug which has been shown to preclude indirectly the second burst in phosphorylation and MPF activity which occurs after first polar body extrusion [26]. Additional experiments, performed in vitro, showed moreover that 6-DMAP did not stimulate phosphoprotein phosphatases but rather inhibited that CAMP and Ca’+-independent protein kinase which was also inactivated after the first phosphorylation burst, following an early treatment with emetine [32]. Since we found that 6-DMAP had apparently no general or specific effect upon protein synthesis, these two independent observations thus converge to suggest that the newly synthesized proteins required for the second meiotic cycle must undergo some post-translational modification before they can trigger MPF reformation. Our data strongly suggest that such a modification may involve phosphorylation. However, they do not allow us to decide which proteins are affected in that way. These may be the cyclin, which has been shown to be present in two interconvertible forms of 52 and 54 kDA [41], the protein kinase itself, or even other proteins directly involved in MPF function. Work is now in progress to answer these new questions. Thanks are due to Mrs. C. Guerrier for excellent assistance throughout this work and for preparing the illustrations, to Mrs. N. Guyard for typing the text, and to Mrs. N. Standart for introducing us to the tracking of cyclin. This work has been partly supported by ATP 84CO939from the Ministry of Research and Industry and by the ARC (fellowship to 1. Neant).

REFERENCES 1. Masui, Y. (1985) in Biology of Fertilization (Metz, C. B., and Monroy, A., Eds.), Vol. 1, p. 189, Academic Press, New York. 2. Moreau, M., Guerrier, P., and Vilain, J. P. (1985) in Biology of Fertilization (Metz, C. B., and Monroy, A., Eds.), Vol. 1, p. 299, Academic Press, New York. 3. Masui, Y., and Clark, H. J. (1979) Znr. Rev. Cytol. 57, 185. 4. Maher, J. L., and Krebs, E. G. (1980) Curr. Top. Cell. Regul. 16, 271. 5. Meijer, L., and Guerrier, P. (1984) Znr. Rev. Cytol. 86, 129. 6. Masui, Y., and Shibuya, E. K. (1987) Molecular Regulation of Nuclear Events in Mitosis and Meiosis, p. 1, Academic Press, New York. 7. Masui, Y., and Marker& C. L. (1971) J. Exp. Zool. 177, 129. 8. Reynhout, J. K., and Smith, L. D. (1974) Dev. Biol. 38, 394. 9. Balakier, H., and Czolowska, R. (1977) Exp. Cell Res. 110, 466.

Inhibition

of meiosis and protein kinase by 6-DMAP

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10. Balakier, H. (1978) Exp. Cell Res. 112, 137. 11. Wasserman, W. J., and Smith, L. D. (1978) J. Cell Biol. 78, 15. 12. Sunkara, P. S., Wright, D. A., and Rao, P. N. (1979) Proc. Nat/. Acad. Sci. USA 76, 2799. 13. Nelkin, B., Nichols, C., and Vogelstein, B. (1980) FEBS Left. 109, 233. 14. Tarkowski, A. K., and Balakier, H. (1980) J. Embryo/. Exp. Morphol. 55, 319. 15. Weintraub, Buscaglia, Ferrez, Weiller, Boulet, Fabre, and Beaulier, E. E. (1982) Cr. Acad. Sci. Paris 295, 787.

16. Kishimoto, T., Kuriyama, R., Kondo, H., and Kanatani, H. (1982) Exp. Cell Res. 137, 126. 17. Kishimoto, T., Yamazaki, K., Kato, Y., Koide, S., and Kanatani, H. (1984) J. Exp. Zoo!. 231, 293.

18. Schatt, P., Moreau, M., and Guenier, P. (1983) Cr. Acad. Sci. Paris 296, 551. 19. Gerhart, J., Wu, M., and Kirschner, M. (1984) J. Cell Rio/. 98, 1247. 20. Sorensen, R. A., Cyert, M. S., and Pedersen, R. A. (1985) J. Ce// Biol. 100, 1637. 21. Neant, I., and Guerrier, P. Development, in press. 22. Maller, J. L., Wu, M., and Gerhart, J. C. (1977) Deu. Biol. 58, 295. 23. Wassarman, P. M., Schultz, R. M., and Letourneau, G. E. (1979) Deu. Biol. 69, 94. 24. Crosby, I. M., Osbom, J. C., and Moor, R. M. (1984) J. Exp. Zool. 229, 459. 25. Guerrier, P., Moreau, M., and Doree, M. (1977) Mol. Cell. Endocrinol. 7, 137. 26. Doree, M., Peaucellier, G., and Picard, A. (1983) Deu. Biol. 99, 489. 27. Meijer, L., Paul, M., and Epel, D. (1982) Deu. Biol. 94, 62. 28. Peaucellier, G., Do&e, M., and Picard, A. (1984) Deu. Biol. 106, 267. 29. Peaucellier, G., Doree, M., and Demaille, J. G. (1982) Gamete Res. 1, 115. 30. Dube, F., and Golsteyn, P. (1986) Biol. Bull. 171, 485. 31. Capony, J. P., Picard, A., Peaucellier, G., Labbe, J. C., and Doree, M. (1986) Deu. Rio/. 117, 1. 32. Picard, A., Labbe, J. C., Peaucellier, G., Le Bouffant, F., Le Peuch, C., and Doree, M. (1987) Deu. Growth Differ.

29, 93.

33. Westwood, J. T., Church, R. B., and Wagenaar, E. B. (1985) J. Biol. Chem. 260, 10308. 34. Guerrier, P., and Neant, I. (1986) Proc. Natl. Acad. Sci. USA 83, 4814. 35. Guerrier, P., and Doree, M. (1975) Deu. Biol. 47, 341. 36. Sano, K. (1985) Deu. Growth Differ. 27, 263. 37. Rebhun, L. I., White, D., Sander, G., and Ivy, N. (1973) Exp. Cell Res. 77, 312. 38. Fautrez, J., and Fautrez-Firlefyn, N. (1975) Arch. Eio/. 86, 467. 39. Dorte, M., and Guerrier, P. (1975) Exp. Cell Res. 96, 296. 40. Lowry, 0. M., Rosebrough, N. J., Farr, A. L., and Randall, R. G. (1951)J. Biol. Chem. 193,265. 41. Standart, N., Minshull, J., Pines, J., and Hunt, T. Cell, in press. 42. Paweletz, N., Mazia, D., and Finze, E. M. (1984) Exp. Cell Res. 152, 47. 43. Doree, M., Guerrier, P., and Leonard, N. J. (1976) Proc. Natl. Acad. Sci. USA 75, 1669. 44. Pondaven, P., and Meijer, L. (1986) Exp. Cell Res. 163, 477. 45. Guerrier, P., Doree, M., and Freyssinet, G. (1975) Cr. Acad. Sci. Paris 281, 1475. 46. Mazzei, G., and Guerrier, P. (1982) Deu. Rio/. 91, 246. 47. Wagenaar E. B. (1983) Exp. Cell Res. 144, 393. 48. Evans, T., Rosenthal, E. T., Youngblom, J. Distel, D., and Hunt, T. (1983) Cell 33, 389. 49. Swenson, K. I., Farrell, K. M., and Ruderman, J. V. (1986) Ce// 47, 861. Received August 28, 1987

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