the induction of oocyte maturation: transmembrane signaling … · oocyte maturation has been...

Development 107, 685-699 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

Review Article 685

The induction of oocyte maturation: transmembrane signaling events and

regulation of the cell cycle

L. DENNIS SMITH

Department of Developmental and Cell Biology and Developmental Biology Center, University of California, Irvine, Ca. 92717, USA

Key words: oocyte maturation, induction, transmembrane signaling regulation, cell cycle, MPF, amphibia

Introduction

The induction of oocyte maturation is known to involvean initial action of agonists at the oocyte surface. Thisleads to activation of a cytoplasmic maturation-promot-ing factor (MPF) which induces the observable eventsassociated with maturation (reviews by Smith andEcker, 1970; Smith, 1975; Wasserman and Smith,19786; Baulieu et al. 1978; Masui and Clark, 1979;Mailer and Krebs, 1980; Mailer, 1983; Gerhart et al.1985; Masui and Shibuya, 1987). Recently, major ad-vances have been made in the characterization of MPFand its mode of action in regulating cell cycle events.New evidence also has been obtained concerning thetransmembrane signaling events that lead to MPFactivation. The purpose of this article is to review andintegrate these advances, partly in the context of earlierwork in the field.

Oocyte maturation has been studied in a variety ofvertebrate and invertebrate organisms, but the processhas been investigated most intensively in amphibians.Full-grown amphibian oocytes are arrested in late G2 ofmeiosis I and must progress to the second meioticmetaphase before fertilization is possible. The resump-tion of meiosis in vivo is brought about by the action ofa gonadotropic hormone which acts on ovarian folliclecells, causing them to produce progesterone which actsdirectly on the oocyte to initiate the process of oocytematuration. Similarly, progesterone induces matu-ration in vitro in oocytes dissected from their ovarianfollicles (review by Wasserman and Smith, 1978b).

A few' hours after steroid treatment, the oocytenucleus (germinal vesicle, GV), situated near the centerof the oocyte, starts to migTate towards the animalhemisphere surface and begin the process of dissol-ution. The arrival of the GV at the cortex causespigment to be displaced, producing a whitish circularspot which is later delineated by a dark ring of thedisplaced pigment. This white spot is the first visibleindication that oocyte maturation is proceeding. Afterdissolution of the nuclear membrane (GVBD), thecondensed chromosomes align on the first metaphasespindle, complete meiosis I, and realign on the secondspindle where they remain until the mature egg isfertilized or parthenogenetically activated.

Germinal vesicle breakdown - an assay formaturation

Strictly speaking, an oocyte is not mature until it hasprogressed to the second meiotic metaphase and can beactivated. However, since GVBD is the easiest event toscore, it frequently has been used as the major if notsole criterion that maturation is underway. An exampleof normal GVBD in oocytes induced to mature withprogesterone is shown in Fig. 1A. However, in oocytesfrom some females, and in oocytes treated with certainsubstances, the GV will rise to the surface, displacingpigment, but it does not undergo GVBD. An exampleof this phenomenon has been reported by Coleman etal. (1981) after injection of cytochalasin or colchicineinto oocytes and by Muramatsu et al. (1989) whoinjected oocytes with cloned DNA. The latter authorsrefer to this 'white spot' as indicative that maturationhas been initiated. While it is not clear what process(es)allow GV migration to the oocyte surface (see Lessman,1987), migration per se is not indicative of maturation.At the other extreme, several instances have beenreported in which GVBD actually occurs in response toa stimulus (Deshpande and Koide, 1982) but theoocytes also exhibit obvious degenerative changeswhich precede the onset of GVBD (Fig. IB). Thephysiological significance of GVBD in such oocytes isdifficult to evaluate.

The timing of GVBD can vary considerably inoocytes from different females. This can result in partfrom differing environmental conditions under whichanimals are maintained in different laboratories. Inaddition, a variety of diverse media have been used toculture oocytes and the time at which GVBD occursafter agonist treatment in oocytes in different media canvary as much as 2-fold (Varnold and Smith, unpub-lished data). Finally, the injection of gonadotropins intofemales, either to induce ovulation (human chorionicgonadotropin) or to improve the synchrony of responseto progesterone (pregnant mare serum gonadotropin)can dramatically alter the time of GVBD. Reynhout etal. (1975) reported that oocytes from females notinjected with hCG for at least 4-6 weeks (unstimulated

686 L. D. Smith

females) usually exhibit GVBD beginning about 6hafter progesterone treatment. In animals injected only afew days earlier, GVBD occurred within 2h of pro-gesterone exposure and occasionally oocytes from suchfemales matured spontaneously as a result of dissectionfrom their follicles (no progesterone exposure). At theother extreme, oocytes from animals maintained forsome time in the absence of obvious stimulation mayexhibit GVBD at times greater than 10 h after pro-gesterone exposure. Because of such variability, datafrequently have been normalized such that the timefrom progesterone addition to that at which 50 % of theoocytes exhibit GVBD has been set at 1.0 (Wassermanand Masui, 1975). While this normalization allows acomparison of data from several different laboratories,it obscures a more fundamental point.

If one assumes that oocytes must progress through acertain sequence of events in order for GVBD to occur,then in those cases in which the time of GVBD isrelatively soon after progesterone exposure, it seemsreasonable to suggest that critical early events eitherhave been bypassed or have already occurred prior toprogesterone treatment, i.e. oocytes are downstream inthe pathway that results in maturation. Conversely, incases in which GVBD is very slow in response tostimulation, it would appear that the agonist actsupstream in the pathway. Stated differently, there maybe more than one point in the putative pathway leading

to GVBD and subsequent meiosis at which oocytes canbe arrested. How, then, does one determine at whichpoint oocytes are arrested? Short of defining all of thesteps involved in the initial induction process and thoseinvolved in MPF activation, this question cannot beanswered definitively. However, we have tended toview oocytes taken from unstimulated females as thebenchmark.

Inducers of oocyte maturation

In addition to progesterone which is the physiologicalinducer of maturation (Schuetz and Glad, 1985), sev-eral other steroid hormones as well as a large number ofseemingly diverse drugs and chemicals are reported toinduce oocyte maturation. A partial list of these variousagonists is compiled in Table 1, which also indicateswhich inducers are effective when microinjected intooocytes. Additional lists could be compiled of agentsthat inhibit progesterone-induced maturation as well asthose that both speed up and retard the timing ofGVBD in response to progesterone. It should beemphasized that, in most cases, the only assay formaturation which has been used is GVBD. Further-more, in many cases, the time of GVBD relative toprogesterone controls has not always been monitored.Thus, the various 'agonists' could act at various pointsin the putative pathway leading to GVBD. Neverthe-

Table 1. Inducers of GVBD in amphibian oocytes

Agent Reference

Steroidsprogesterone, deoxycorticosterone, testosterone,cortisol, R5020, pregnenolone

Polypeptidesinsulin, insulin-like growth factor*bee venom melittin

Proteins•MPF*R subunit-PKA*PKI of PKA*ras p21*c-mos•cyclin A and B*calmodulin

IonsCa2 +, Ma2 +, Zn2 + , Co2 + , Ba2+, Mg2*

Amines, weak basesmethylamine, imidazole

Drugs affect Ca fluxA23187 plus Mg2*, Lanthanum verapamil

Local anesthetics, CNS acting drugschloropromazine, fluphenazine, imipramine,tetracaine, trifluoperazine

Organomercurials*p-hydroxymercuriphenylsulfonate, p-hydroxymercuribenzoate,mersayl

Propranolol-like drugspropranolol, alprenolol

Baulieu et al. 1978

Mailer and Koontz, 1981Deshpande and Koide, 1982

see text

Cicirelli and Smith, 1987

Houle and Wasserman, 1983

Baulieu et al. 1978

see Baulieu et al. 1978Hollinger and Alvarez, 1982

Brachet et al. 1975

Baulieu et al. 1978

•Compounds that induce after microinjection.

Fig. 1. Morphological appearance of oocytes exposed to various agonists. (A) Oocytes incubated in progesterone(10/igmr1) and observed at the time of GVBD; GVBD50 was at 3.5 h. (B) Oocytes incubated in melittin (l^gml"1) andobserved 60min after treatment; GVBD50 in progesterone-exposed oocytes was at 3h. (C) Oocytes incubated in the phorbolester TPA (see text) at 150 nM and observed at 4h after treatment; GVBD50 as in B.

Induction of oocyte maturation 687

less, a variety of the nonhormonal compounds listed areknown to be active at the surface of cells (Baulieu et ah1978). These could function non-specifically to alter theactivity of membrane-bound enzymes, possibly bymodifying hydrophobic lipid-protein interactions. Inthis sense, several of the nonhormonal compoundslisted in the table are reported to decrease membraneadenylate cyclase activity (Schorderet-Slatkine et al.1982) as well as membrane protein kinase C activity(Mori et al. 1980). Thus, they could mimic the effects ofprogesterone on the oocyte surface. The validity of sucha suggestion could be tested more directly if the natureof the progesterone target in the oocyte membrane waswell established.

Steroid receptors in the oocyte membrane

Reports that a specific progesterone receptor existswithin a melanosome fraction derived from oocytes (seeCoffman et al. 1979) are provocative since melanosomesare localized in the oocyte cortex. The equilibriumdissociation constant (Kd) for such receptors was about10~8M, consistent with estimates of the ovarian pro-gesterone concentration in animals stimulated to ovu-late with gonadotropins. Similarly, the minimum con-centration of progesterone that induces maturation invitro is about 10 M. However, oocytes of the albinomutant of Xenopus, which lack melanosomes as well aspremelanosomes, are induced to mature by progester-one. Further, the melanosome component that bindsprogesterone is melanin itself (Coffman et al. 1979).Thus, the melanosome 'receptors' play no physiologicalrole in oocyte maturation.

Sadler and Mailer (1982) reported identification of asteroid receptor on the surface of Xenopus oocyteswhich could be photoaffinity-labeled with the syntheticprogestin R5020, a relatively weak agonist for matu-ration. In this case, a contribution from melanosomeswas eliminated by manually isolating the plasma mem-brane-vitelline envelope complex which does not con-tain pigment. They identified a 110xl03Mr proteinwhich bound the steroid with a Kd of 10~5M to 10~6M,depending on the procedure, and which existed at aconcentration of 4.2X1011 sites/oocyte (6.5xlO4 sitesum~2). While this Kd is close to the effective concen-tration of R5020 that induces maturation, both the /JMKd and the relatively high concentration of putativereceptors are more characteristic of non-specific associ-ation of steroids with a membrane protein. Thus, in theabsence of more compelling evidence, it is difficult toconclude that steroids (or other agonists) induce matu-ration by binding to a specific high-affinity receptor.

Transmembrane signalling events in theinduction of oocyte maturation

Transmembrane signaling events usually involve inter-action of an agonist with membrane receptors which,mediated by GTP-binding proteins (G proteins), leads

to changes in the intracellular concentration of second-messenger molecules. The evidence concerning mem-brane receptors was discussed above and data pertain-ing to G proteins in the oocyte is evaluated in several ofthe subsequent pages.

The two second messenger systems studied mostintensively in the oocyte have been those involvingcAMP and [Ca2+],. Changes in cAMP regulate theactivity of cAMP-dependent protein kinase (PKA)(Krebs, 1972) while alterations in [Ca2+]j lead tomodulation of several enzymes via the calcium-bindingprotein calmodulin (Blackshear et al. 1988). Intracellu-lar calcium levels are regulated by the second messageinositol trisphosphate (IP3), produced by hydrolysis ofmembrane-bound phosphatidylinositol 4, 5-bisphos-phate (PIP2). Phosphodiesterase cleavage of PIP2 alsogenerates yet another second message, 1,2-diacylgly-cerol (DAG), which is involved in regulation of proteinkinase C activity (Berridge, 1986; Nishizuka, 1986). ThePKA, Ca-calmodulin, and PKC pathways all have beenimplicated in the initial response of oocytes to agonistswhich induce maturation. As discussed below, currentevidence suggests that only two of these, PKA andPKC, are actually involved in the induction process,and both appear to be affected by progesterone.

Calcium as a second message in oocytematuration

Several lines of evidence originally led to the hypothesisthat an elevation in [Ca2+], might be necessary andsufficient to induce oocyte maturation (reviews by Corketal. 1987; Cicirelli and Smith, 1987). First, iontophor-esis of calcium into the oocyte cortex or incubation inthe ionophore A23187 have been reported to inducematuration. Second, tracer flux studies showed that therate of 45Ca2+ efflux from preloaded oocytes increasedwithin minutes after progesterone exposure, suggestinga release of bound calcium in response to the hormone.Third, progesterone was observed to induce a transientrise in [CET+]J within minutes after exposure to pro-gesterone in a significant percentage of oocytes injectedwith the calcium photoprotein aequorin. Finally, sev-eral laboratories reported that oocytes injected with thecalcium-binding protein calmodulin were induced toundergo maturation in the absence of steroid treat-ment, although the percentage of oocytes that re-sponded was quite variable.

In contrast to the above experiments, Robinson(1985) was unable to detect any changes in [Ca2+]iduring oocyte maturation using calcium-sensitive elec-trodes. In a subsequent repeat of the aequorin exper-iment (Cork et al. 1987), a small transient increase in[Ca2+], was observed as an early response to progester-one, but in only one oocyte. In all other cases, nochange was observed. Nevertheless, maturation alwaysoccurred in response to progesterone. Thus, while atransient elevation in [Ca ]( could be observed, it wasnot an essential response to progesterone.

It now seems clear that an elevation in [Ca2+]; per se

688 L. D. Smith

is not sufficient to induce maturation. For example, inthe experiments described above with A23187, matu-ration was obtained only when the concentration ofCa2+ (and Mg2+) in the medium was relatively high(10-20ITIM). Treatment of oocytes incubated in lmM-Ca2+ with A23187 does not induce maturation althoughit does cause an increase in [Ca2+]( (Cicirelli and Smith,1987). Also, injection of oocytes with LP3 which releasesbound Ca2+ in Xenopus oocytes (Berridge, 1988), doesnot induce maturation (Picard et al. 1985). While thecalcium efflux data referred to above were interpretedas indicating an increased calcium ion concentration inthe cytoplasm, an alternative interpretation is thatincreased efflux results from an increased efficiency ofthe calcium extrusion pump induced by progesterone(O'Connor et al. 1977). This would imply an actualdecrease in [Ca2+]j in response to the steroid.

cAMP as a second message in oocyte maturation

There is general consensus that a transient decrease incAMP levels, resulting from an inhibition of adenylatecyclase activity, is an obligatory first step in the mechan-ism by which progesterone induces oocyte maturation.This in turn is thought to lead to a decrease in cAMP-dependent protein kinase activity (PKA) which wouldresult in the dephosphorylation of a putative matu-ration-inhibiting phosphoprotein. As with the calciumstory, the evidence supporting this sequence of eventsfalls into four categories (review by Mailer, 1983). First,activators of adenylate cyclase, such as cholera toxin,which elevate cAMP levels in Xenopus oocytes, inhibitprogesterone-induced maturation. Second, oocytecAMP levels are reported to decrease within minutesafter progesterone exposure. Third, progesterone isreported to partially inhibit adenylate cyclase activity.Finally, injection of the regulatory subunit of PKA,expected to reduce activity of the catalytic subunit inoocytes, induces maturation, while injection of thecatalytic subunit inhibits progesterone-induced matu-ration.

A scheme depicting the events described above isshown in Fig. 2. Additional data supporting the modelcomes from the observation that injection of the heat-stable inhibitor protein (PKI) of the catalytic subunitinto oocytes also induces maturation. Finally, injectionof phosphatase inhibitor proteins 1 and 2, which mightbe expected to prevent dephosphorylation of the puta-tive inhibitory phosphoprotein, delays GVBD in pro-gesterone-stimulated oocytes (see Mailer, 1983). In thiscontext, inhibitor-1 is activated by PKA, suggestingthat a decrease in cAMP could also lead to increasedphosphatase-1 activity. This would enhance dephos-phorylation of the putative meiosis-inhibitory protein(see Cyert and Kirschner, 1988). In contrast to theabove, several recent studies have indicated that adecrease in cAMP content is not always an obligatory oreven sufficient step in the induction of maturation (seelater section). This has led to the suggestion thatoocytes may contain a pathway independent of that

Progesterone

1-oAMP

PKA PKI

Meiosla

Inhibitoryphoaphoprotaln

I

JMPF

- phosphatase

THphosphatase

Inhibitor

Fig. 2. Proposed sequence of events involving the cAMPpathway in oocyte maturation. Progesterone leads to adecrease in membrane-bound adenylate cyclase (AC)activity which results in lower cAMP levels. This in turndecreases cAMP-dependent protein kinase (PKA) activitywhich results in dephosphorylation of a putative meiosis-inhibitory phosphoprotein. PKA activity also can bereduced via a heat-stable inhibitor (PKI) of PKA, whiledephosphorylation of the putative phosphoprotein also canbe accomplished by phosphatase action on the protein.MPF refers to maturation-promoting factor.

involving cAMP which can result in maturation. In viewof this, it seems desirable to discuss in more detailcertain aspects of the data supporting the involvementof the cAMP pathway in oocyte maturation.

Reports of cAMP changes in oocytes as a response toprogesterone have been quite variable, but most haveshown a modest decrease of about 20 % within minutesafter progesterone exposure (Mailer, 1983; Cicirelli andSmith, 1985; Gelerstein etal. 1988). Since the endogen-ous cAMP pool in stage 6 oocytes averages between1.5-2.5 pmole oocyte" , these data suggest a progester-one-induced decrease of 0.3-0.5 pmole within minutesafter agonist stimulation.

The mechanism by which progesterone could causesuch decreases in unclear. An increase in the phospho-diesterase activity, which hydrolyzes cAMP, is reportednot to occur in response to progesterone (Sadler andMailer, 1987). On the other hand, several laboratorieshave observed that adenylate cyclase activity in isolatedoocyte membranes is altered by progesterone (seeMailer, 1983) in that the steroid can prevent stimulationof activity by cholera toxin. However, the effects ofprogesterone on basal activity were quite variable. This


is due in part to the very low level of basal activity(0.05 pmol oocyte"1 h-1) observed in control oocytes(no cholera toxin or progesterone exposure) (Sadlerand Mailer, 1981). Thus, even if progesterone resultedin complete inhibition of this activity, several hourswould be required to generate the reported decreases incAMP content.

The most thoroughly characterized GTP-bindingproteins (G proteins) involved in transmembrane sig-naling events are those associated with regulation ofadenylate cyclase activity (Freissmuth et al. 1989).Xenopus oocytes contain the two guanine nucleotide-binding proteins, Gs and Gj, normally associated withregulation of adenylate cyclase. However, progesteronedoes not appear to regulate cAMP levels through theseproteins in a conventional manner. For example, per-tussis toxin, which inhibits G,, elevates oocyte adeny-late cyclase activity but does not prevent inhibition ofadenylate cyclase by progesterone (Sadler et al. 1984).Curiously, the toxin is reported not to affect oocytecAMP levels (Mulner et al. 1985), but is reported toboth delay (Sadler et al. 1984) and accelerate (Mulner etal. 1985) the time course of progesterone-inducedGVBD. One solution to these apparent discrepancies isthe suggestion by Sadler and Mailer (1983) that themechanism by which progesterone acts on oocyteadenylate cyclase is novel, with properties in commonboth with the P site described for adenosine agonists aswell as the more conventional receptor-mediated sys-tems. They suggest that progesterone decreases the'turn-on' reaction of Gs, i.e. decreases the rate ofguanine nucleotide exchange on the Gs protein (alsoJordana et al. 1984). However, even if correct, thismechanism again would not account for the magnitudeof the decrease in cAMP content observed after pro-gesterone treatment.

The 'glue' that holds the cAMP hypothesis together isthe study of Mailer and Krebs (1977) on injection ofPKA subunits into oocytes. The holoenzyme of proteinkinase A exists as an inactive complex consisting ofregulatory and catalytic subunits which, in the presenceof cAMP, dissociates into free catalytic subunit andcAMP-regulatory subunit complex as follows (Krebs,1972):

R2C2 + R(cAMP)n + 2C (1)

Several lines of evidence suggest that PKA is fullydissociated in the oocyte (Masaracchia et al. 1979;Huchon et al. 1981). Thus, elevating cAMP levelsshould have little effect on catalytic activity of thekinase. Accordingly, it is not clear why agents such ascholera toxin, that increase oocyte cAMP should alsoinhibit maturation, unless inhibition is unrelated toeffects on cAMP levels.

Based on the equilibrium reaction in equation (1),injecting R subunits into oocytes would be expected todrive the reaction to the left, decreasing catalyticactivity and releasing cAMP; cAMP levels would in-crease temporarily. In the experiments reported byMailer and Krebs, R subunit of the type II isozyme wasinjected into oocytes as the subunit-cAMP complex.

Assuming no degradation of the protein after injection,sufficient subunit was injected to result in a decrease incatalytic activity of 20-50 %. This point was not testeddirectly, although Masaracchia et al. (1979) reported adecreased level of free catalytic subunit in extracts fromprogesterone-treated oocytes. In contrast, Cicirelli etal. (1988) reported no change in PKA activity duringthe entire course of oocyte maturation. Masaracchia etal. (1979) also reported that the regulator)' subunit fromtype I PKA does not induce maturation when injectedinto oocytes. This would be somewhat surprising sinceRI also binds to C. However, we have observed that thetype I regulatory subunit does induce oocyte matu-ration, albeit less efficiently than that from type II,when injected into oocytes (Varnold and Smith, unpub-lished data).

One might expect that injection of excess catalyticsubunit also would drive the reaction in equation (1) tothe left, resulting in a decrease in catalytic activity. Thisappears not to be the case. There are suggestions thatinjection of C into oocytes alters the phosphorylation ofnumerous proteins, both quantitatively (see Mailer,1983) and qualitatively (Boyer et al. 1987). However,identification of these substrates and their role inmaturation has not been accomplished. Mailer andKrebs (1980) state that injected catalytic subunitseverely depresses protein synthesis; this alone wouldinhibit maturation. They suggest that the putativeinhibitory phosphoprotein might function to preventthe synthesis of essential proteins early in the matu-ration response. In summary, while the data presentedby Mailer and Krebs (1977) provides strong support forthe sequence of events depicted in Fig. 2, many ques-tions remain unanswered. Clearly, identification andcharacterization of the putative maturation-inhibitoryphosphoprotein would go a long way towards answeringthese questions.

Alternative pathways in the Induction of oocytematuration

There exist several examples in which oocyte matu-ration has been obtained without obvious involvementof the cAMP pathway. For example, Birchmeier et al.(1985) reported that the oncogenic protein encoded byH-rasv£U induced maturation when injected into Xeno-pus oocytes without any corresponding change incAMP levels. It should be pointed out that the onco-genic ras protein did not induce GVBD in choleratoxin-treated oocytes indicating that ras protein doesnot overcome the effects of elevating cAMP levels.Nevertheless, Birchmeier et al. (1985) suggested thatoocytes contain an alternate pathway able to triggermeiosis, which bypasses changes in intracellular cAMPlevels. In support of this, the injection of a monoclonalantibody against the oncogenic ras protein is reportedto inhibit insulin-induced but not progesterone-inducedmaturation (Korn et al. 1987; Deshpande et al. 1987).

Allende et al. (1988) reported that activated rasprotein (H-rasval 12) induces GVBD in Xenopus oocytes

690 L. D. Smith

incubated with cycloheximide or puromycin, as doesMPF, while both inhibitors block maturation inducedby progesterone. This suggests the possibility that rasprotein acts downstream of the protein synthesis re-quirement in oocyte maturation, possibly at the level ofMPF, rather than at a point in the initial inductionprocess. This observation has not been confirmed and issuspect on other grounds. If the oncogenic protein wasacting downstream of the protein synthesis require-ment, it seems reasonable to anticipate that maturation(GVBD) would be faster in ras-injected oocytes than inthose exposed to progesterone. That is not the case(Birchmeier et al. 1985). In addition, while both theprotein and rasvaU2 transcript induce maturation instage 6 oocytes, neither activates MPF (inducesGVBD) in smaller stage 4 oocytes (Johnson and Smith,unpublished data) which contain preMPF (Taylor andSmith, 1987). This implies an action by ras protein earlyin the sequence of events induced by progesterone, i.e.prior to MPF activation.

Gelerstein et al. (1988) demonstrated that treatmentof oocytes with acetylcholine (ACh) shortly after pro-gesterone exposure caused GVBD sooner than inoocytes exposed to progesterone alone, and also rapidlylowered intracellular cAMP levels. However, AChalone did not induce maturation suggesting that adecrease in cAMP per se is not sufficient to triggermaturation. In contrast, addition of adenosine tooocytes elevated endogenous cAMP levels and abol-ished the progesterone-induced decrease in cAMP.Nevertheless, adenosine alone induced GVBDalthough the time of GVBD was slower than thatobserved in response to progesterone. Gelerstein et al.(1988) suggested the coexistence in oocytes of differentand parallel mechanisms for the induction of matu-ration.

The protein kinase C pathway In oocytematuration

The tumor-promoting phorbol ester TPA (12-o-tetra-decanoylphorbol 13-acetate) can substitute for DAG inactivating protein kinase C and Stith and Mailer (1987)reported that oocytes treated with TPA undergoGVBD in the absence of hormone treatment. Thisresult suggests that activation of the PKC pathway caninduce oocyte maturation. However, when PKC iso-lated from bovine tissue was injected into oocytes itneither induced maturation nor changed the time ofGVBD in oocytes treated at the time of injection withprogesterone. It did result in GVBD within a shortertime after treatment of oocytes also with insulin com-pared to oocytes induced to mature with insulin alone.Based on this observation and other data, Stith andMailer suggested that while the PKC pathway is notinvolved in progesterone-induced maturation, insulinmight work by this route.

With respect to PKC injections, Muramatsu et al.(1989) have reported that a mutant construct of PKCwhich expresses activity in the absence of phorbol ester

activation initiates maturation when injected into Xeno-pus oocytes. However, that effect involved only mi-gration of the oocyte GV to the animal hemispheresurface and not GVBD. We have confirmed the obser-vation of Stith and Mailer (1987) that injection of amixture of PKC isozymes (rat brain) at the time ofprogesterone treatment has no effect on maturation.However, when PKC is injected into oocytes 30-60 minprior to progesterone exposure, the time of GVBD issignificantly delayed (Varnold and Smith, unpublisheddata). It should be pointed out that injected proteinsmust diffuse through the cytoplasm to potential sites ofaction before exerting an effect; injecting prior toprogesterone exposure may provide sufficient time fordiffusion. Thus, one interpretation of these exper-iments is that elevated PKC activity is inhibitory duringearly times after progesterone treatment.

The observation that TPA induces GVBD has notbeen confirmed. In fact, Bement and Capco (1989) haveobserved that treatment of oocytes containing folliclecells with TPA results in cytolysis. We also haveobserved that such treatment leads to extreme mottlingof the pigmented animal hemisphere (Fig. 1C) but thatsuch oocytes usually contain an intact GV (Varnold andSmith, unpublished data; see also Stith and Mailer,1987). Bement and Capco further observed that, inoocytes with no follicle cells, the phorbol ester inducedcortical granule breakdown and elevation of the vitel-line envelope, a typical response to activation stimuli.In no case was GVBD obtained.

Maturation in starfish oocytes is induced by theaction of 1-methyladenine acting on the oocyte surface,resulting in the activation of MPF (Kishimoto andKanatani, 1976). In essentially all aspects, the processparallels that observed with amphibian oocytes. In thiscase, phorbol esters inhibit the induction of maturationby 1-MA (Kishimoto et al. 1985). In contrast, surf clamoocytes are shed with an intact GV and GVBD isinduced by fertilization; TPA is reported to induceGVBD is these oocytes (Bloom et al. 1988).

In making comparisons among diverse organisms, itshould be emphasized that, while all of the oocytesmentioned contain an intact GV at the time maturationis induced, it is less clear that they are arrested inmeiosis at the same point relative to the G2/M tran-sition. For example, full-grown Xenopus oocytes con-tain active lampbrush chromosomes while, in the surfclam, oocytes contain chromosomes already partiallycondensed, i.e. they are downstream in the sequence ofevents leading to meiosis I compared with Xenopus.Thus, if the TPA data overall is taken at face value, itmight suggest that both a decrease and increase in PKCactivity are involved in maturation, depending on thepoint in the pathway at which oocytes are arrested.

Is the PKC pathway involved In progesterone-induced maturation?

Recently, Cicirelli and Krebs (unpublished data) ob-served that sphingomyelinase is a potent inducer of


maturation in Xenopus oocytes and we have confirmedthis observation (Varnold and Smith, 1989, unpublisheddata). Sphingomyelinase acts on membrane sphingo-lipids to produce ceramide, which in turn is hydrolyzedto sphingosine, a potent inhibitor of PKC (Hannun etal. 1986) as well as Ca-calmodulin-dependent enzymes(Jefferson and Schulman, 1988). We have observedfurther that sphingosine induces oocyte maturation, asdoes staurosporine, also an inhibitor of PKC (Varnoldand Smith, 1989, unpublished data). Taken together,these observations again suggest the possibility thatinactivation of PKC, similar to the situation proposedfor PKA, could represent a mechanism for the induc-tion of oocyte maturation. The question then becomes,is the PKC pathway affected by progesterone or is it aseparate pathway?

In response to extracellular signals, the increase inDAG which activates PKC is brought about byincreased phosphodiesterase activity (phospholipase C)which hydrolyzes PIP2. That enzyme is coupled to a Gprotein (Cockcroft and Stutchfield, 1989), which pre-sumably acts like the Gs protein coupled to adenylatecyclase. By analogy, we have made the assumption thatextracellular signals could act to decrease DAG levels,possibly via an inhibitory G protein that reduces phos-pholipase C activity. This would result in a decrease in

PKC activity. On that basis, Varnold and Smith (1989;unpublished data) measured the mass of DAG inXenopus oocytes as a function of time after progester-one exposure. The results are shown in Fig. 3. Within15 s after exposure of full-grown (stage 6) oocytes toprogesterone, DAG levels decreased by about 30%compared to control oocytes. The DAG levelsremained low for at least the first 2min, returned tocontrol levels by about 15min, and then begin tocontinuously increase up to the time of GVBD. Alsoshown in the figure are the effects of progesterone onDAG levels in stage 4 oocytes (800/xm in diameter),which are not responsive to progesterone. In this case,there is no change in DAG mass, arguing against atotally nonspecific effect of steroids on the oocytemembrane. A similar decrease in IP3 mass was ob-served in response to progesterone (data not shown).Thus, the data imply that progesterone acts rapidly toreduce hydrolysis of PIP2, resulting in a decrease in thelevel of both second messengers.

The data described above were obtained by extract-ing lipids from whole oocytes and thus would havemeasured DAG changes in both the cytoplasm andoocyte membrane. In additional experiments, wemeasured changes in DAG mass in oocyte plasmamembranes isolated manually (Sadler and Mailer,

1.8-

1.6-

1.4-

1.2-

oQ

T T "o

o

Jl.5

oc

-—-_

1.0*1/ /

0.8-

0.6-

0.4-

o 0.2-Z

o

V

1 2 3 4 5

~———ji

200 300 400 500Min after progesterone

600

Fig. 3. Changes in DAG levels in responseto progesterone. Groups of 5 stage 6 or 15stage 4 control and progesterone-treatedoocytes were homogenized inchloroform/methanol (1:2) and washedonce with lM-NaCl. The organic phase wasdried under N2 and assayed for DAGaccording to Preiss et al. (1987). (A) EarlyDAG changes in stage 4 oocytes (triangles)and stage 6 oocytes (circles). Each point isthe mean±SEM from 11 experiments.Insert represents DAG levels in isolatedmembranes in response to progesterone.Each point is the mean for 2 experiments.(B) Long term changes in DAG levels afterprogesterone exposure. The arrowindicates the time of GVBD.

692 L. D. Smith

Progesterone

PI—PIP,

CaM/CaENZYMES

Fig. 4. Model depicting the roles of both PKA and proteinkinase C (PKC) in the induction of oocyte maturation.Progesterone acts at the oocyte membrane to reduce bothadenylate cyclase (AC) activity and that of thephosphodiesterase (PLC) which cleaves PIP2.Reduction in AC activity leads to decreased PKA activity asshown on Fig. 2. Decreased PLC activity lowers the level ofinositol trisphosphate (IP3) and diacylglycerol (DAG).Decreased DAG is postulated to inactivate PKC, resultingin dephosphorylation of the putative inhibitoryphosphoprotein and/or reducing cAMP levels via action onAC. Dashed lines refer to reactions which are not yetdocumented in the oocyte.

1981). The membrane DAG averaged about 6% oftotal DAG mass in the oocyte. However, when thesemembranes were exposed to progesterone, DAG levelsalso began to decrease with 15 s and continued todecrease, reaching a level 41% of controls by 5min(Fig. 3). The continued decrease would be expectedsince replacement of membrane inositol phospholipidsfrom the cytosol would not occur in isolated mem-branes. This early decrease in DAG in oocyte mem-branes supports the hypothesis that a decrease in PKCactivity, while not yet measured directly, is a very earlyresponse of oocytes to progesterone.

A summary of data discussed on the several preced-ing pages is as follows. Progesterone induces an earlytransient decrease in the content of a known secondmessenger molecule, cAMP, which then is presumed toresult in decreased PKA activity. The same agonist alsoinduces an early transient decrease in another knownsecond messenger molecule, DAG, which is presumedto result in decreased activity of a second proteinkinase, PKC. Unlike cAMP, DAG levels then increaseas oocytes approach GVBD, implying that increasedPKC activity might be involved in later events ofmaturation.

At least two models can be considered in which boththe PKA and PKC pathways would be involved in theinduction of oocyte maturation by a single agonist.These are shown schematically in Fig. 4. The firstsuggests that the putative maturation-inhibitory protein

is phosphorylated at multiple sites by both kinases. Thisidea is not without precedent as several examples existin the literature in which a single substrate is phos-phorylated by both protein kinases (Nishizuka, 1986).Thus, meiotic arrest could be released by decreasing thetotal phosphate content of the protein via inhibition ofeither (or both) of the kinase pathways. The secondmodel is based on many observations indicating exten-sive cross-talk between the PKC and PKA pathways(Nishizuka, 1986). For example, changes in PKC ac-tivity have been linked to modulation in adenylatecyclase activity (Yoshimasa et al. 1987; Rozengurt et al.1987). Reciprocally, examples exist in which changes incAMP alter phospholipid hydrolysis and PKC activity(Supattapone et al. 1988; Kato et al. 1989). In postulat-ing interactions within the oocyte, one is influenced bythe observation that the progesterone-induced decreasein DAG mass appears to precede the decrease in cAMPlevels. This suggests the possibility that interactionbetween the two pathways is sequential, i.e. a decreasein PKC activity would lead to a decrease in PKAactivity. This could be mediated by PKC effects oncAMP levels due in part to inactivation of adenylatecyclase.

Both models clearly are speculative at this point.However, the essential features of each model appearto be testable. Furthermore, the involvement of twopathways in the induction of maturation, especially one(PKC) that can be regulated by several products ofmembrane lipid catabolism (Blackshear et al. 1988)would help explain how a relatively large number ofdiverse 'agonists' can induce oocytes to mature.

Nuclear membrane breakdown - maturationpromoting factor

Early experiments on Rana pipiens and Xenopus laevisoocytes established that MPF activity appears in thecytoplasm prior to GVBD, appears at the same time inmanually enucleated oocytes and can be continuallyamplified through serial cytoplasmic transfers into re-cipients not exposed to hormone (review by Wassermanand Smith, 19786; Masui and Clark, 1979). The latterobservation in particular suggested that oocytes containa store of inactive MPF which can be activated andamplified by small amounts of active MPF. Reynhoutand Smith (1974) and Wasserman and Masui (1975)further demonstrated that injection of cytoplasm con-taining MPF activity always induced precocious GVBDin recipient oocytes. Since the initial appearance ofMPF activity, but not amplification, requires proteinsynthesis (Wasserman and Masui, 1975; Gerhart et al.1984), this suggests that much of the time lag betweenprogesterone treatment and GVBD is involved inproduction of active MPF, i.e. injected MPF can bypassearly progesterone-induced steps. The ability of MPF toinduce maturation in the absence of protein synthesis isunique and has been used as a diagnostic assay for MPFactivity (Gerhart et al. 1984).

Wasserman and Smith (1978) first reported that MPF


activity cycles during the mitotic divisions of earlycleavage in both Rana pipiens and Xenopus laevisembryos. The peak of MPF activity coincided approxi-mately with M-phase of the cell cycle, and activitydisappeared when cells completed mitosis. Theyshowed further that protein synthesis was necessary forreappearance (but not disappearance) of MPF activityat each division, suggesting either that an activator ofMPF, or MPF itself, must be synthesized during eachcell cycle. Shortly thereafter, Sunkara et al. (1979)showed that synchronized HeLa cells contain MPFactivity, assayed using Xenopus oocytes, which ap-peared in G2, peaked at metaphase, and disappeared inG! of the cell cycle. These key experiments and severaladditional studies on MPF in diverse cells (Nelkin et al.1980; Weintraub et al. 1982; Doree et al. 1983; Kishi-moto et al. 1982, 1984; Gerhart et al. 1984; Sorensen etal. 1985) established the generality of MPF as an M-phase promoting factor in mitotic as well as meiotic cells(Gerhart et al. 1984, 1985).

Purification of MPF

Attempts to purify MPF began about 15 years ago, butin spite of efforts in several laboratories (Wassermanand Masui, 1976; Drury, 1978; Wu and Gerhart, 1980;Adlakha et al. 1985; Nguyen et al. 1986), progress untilvery recently has been very slow. The two majordifficulties have been the extreme lability of MPF andthe fact that the only assay involved microinjection intoXenopus oocytes. The development of systems tomonitor nuclear membrane breakdown in vitro greatlyfacilitated assays of MPF activity (Lohka and Mailer,1985; Miake-Lye and Kirschner, 1985). Concerninglability, Wu and Gerhart (1980) developed proceduresin which MPF activity could be obtained by gentlehomogenization and in which MPF activity survivedlimited dilution. They purified MPF about 100-fold andsuggested it might be a phosphoprotein with an appar-ent relative molecular mass of about lOOxlO3. Thebreakthrough in purification was achieved by Lohka etal. (1988). By using essentially the conditions developedby Wu and Gerhart (1980) and a very large amount ofmaterial, they obtained small amounts (1 % yield) of afraction purified approximately 3000-fold. That prep-aration contained two predominant proteins, with rela-tive molecular masses of 34 and 45X103, respectively.

The 34K protein has been identified as the Xenopushomolog of a fission yeast protein encoded by a gene(cdc2+), which is required for the G2/M transition inthe mitotic cell cycle (Gautier et al. 1988; Dunphy et al.1988). It has now been implicated as a component ofMPF in starfish (Labbe et al. 1988, 1989; Arion et al.1988) and clam oocytes (Draetta et al. 1989) as well as inmouse (Morla et al. 1989) and human (Draetta andBeach, 1988; Brizuela et al. 1989) tissue culture cells.The cdc2 protein (p34cdc2) is a serine/threonine proteinkinase, which can phosphorylate a number of substratesunder varying conditions but exhibits a strong prefer-ence for histone HI as cells progress from G2 to M. The

45K protein, which is also a substrate for p34cdc2, hasnot been identified definitively, but is thought to beanalogous to a protein called cyclin.

Cyclins and MPF activity

Cyclins were first identified in sea urchin and clamembryos as products of maternal mRNA which aresynthesized and accumulated during interphase andthen rapidly degraded near the end of each mitosis(Evans et al. 1983). They have now been identified inseveral eukaryotic cells including yeast (Goebl andByers, 1988; Solomon etal. 1988), Xenopus (Minshull etal. 1989; Murray and Kirschner, 1989), Drosophila(Lehner and O'Farrell, 1989; Whitfield et al. 1989),starfish (Standart et al. 1987) and probably in mam-malian tissue culture cells (Draetta and Beach, 1988).Based on the nucleotide sequence of cDNAs fromclams, they fall into two classes, A and B, withpredicted relative molecular masses of 42K and 48K,respectively (Swenson et al. 1986; Westendorf et al.1989). However, they usually display apparent relativemolecular masses on SDS gels of about 55K; theputative cyclin in mammalian cells is a protein of 62K.

Swenson et al. (1986) originally reported that themRNA for clam cyclin A induces the resumption ofmeiosis when injected into Xenopus oocytes. Similarresults now have been obtained with the sea urchincyclin mRNA (Pines and Hunt, 1987) and the mRNAfor clam cyclin B (Westendorf et al. 1989). These resultssuggested that cyclin either is a component of activeMPF or that it functions as an activator of MPF.Additional observations suggest that interaction of thecyclins with p34cdc2 is necessary for the histone HIkinase associated with MPF to be active (Draetta et al.1989; Meijer et al. 1989; Brizuela et al. 1989). Forexample, both cyclins A and B in clam oocytes arefound in association with p34cdc2 as assayed by immuno-precipitation with either anti-cyclin A, anti-cyclin B, oranti-cdc2 sera (Draetta etal. 1989). Meijer etal. (1989)also have demonstrated that anti-cyclin antibodies pre-cipitate p34cdc2 in sea urchin eggs. Finally, anti-cdc2sera coprecipitates p34cdc2 and a protein (p62) thoughtto be cyclin in mitotic HeLa cell extracts (Draetta andBeach, 1988).

Activation of MPF in oocytes

Protein synthesis is required for the appearance ofactive MPF in mitotically dividing cells (see Swenson etal. 1989). Since p34cdd2 is constitutively present individing cells, this would imply that synthesis anddegradation of cyclin regulates the activity of MPF.Recently, this view has been confirmed with the demon-stration that cyclin is the only newly synthesized proteinnecessary to induce MPF activity and drive the cell cyclein a Xenopus egg extract (Minshull et al. 1989; Murrayand Kirschner, 1989; Murray et al. 1989). Severaladditional lines of evidence show that the rapid degra-

694 L. D. Smith

Metaphase Metaphase

kAct. Act.

Inact.

'cydindestroyed

Fig. 5. Model depicting the interaction between cdc2 andcyclin protein in regulating MPF activity. cdc2 protein ispresent continuously but cyclin is periodically synthesizedand destroyed. Active MPF at the G2/M transition resultsfrom cdc2/cyclin association while cyclin proteolysisinactivates MPF at the metaphase/anaphase transition.Adapted from Draetta et al. (1989) and presented in'Research News', Science 245, 252-255 (1989).

dation of cyclin at the end of each M phase is requiredfor loss of MPF activity (Draetta et al. 1989; Murray etal. 1989; Luca and Ruderman, 1989). This view of cyclinregulation of the cell cycle is shown in Fig. 5.

Since the induction of maturation in Xenopus oocytesalso requires protein synthesis, one might suggest thatprogesterone stimulation leads to the synthesis of cyc-lin, which then results in active MPF. This possibilityremains uncertain. Frog, starfish, clam and mouseoocytes all are arrested at the G2/M border of meiosis I.In response to the appropriate stimulus, MPF activityappears, induces meiosis I, and then cycles betweenmeiosis I and II (Gerhart et al. 1984; Hashimoto andKishimoto, 1988; Arion etal. 1988; Draetta etal. 1989).Protein synthesis is required in all cases for entry intomeiosis II but, with the exception of frog oocytes, notfor meiosis I. Nevertheless, at least in clam oocytes,both cyclins A and B are normally synthesized duringthe transition from G2 to meiosis I (Westendorf et al.1989). A resolution to this discrepancy is provided bythe observation that clam oocytes contain a store ofcyclin B protein which is present as large aggregates.Shortly after the induction of meiosis, cyclin B proteinappears in a more soluble disperse form, which allows itto interact with stored cdc2 protein, generating activeMPF (Westendorf et al. 1989).

Westendorf et al. (1989) speculate that Xenopusoocytes also might contain a pool of cyclin protein,which is released in response to progesterone andassociates with stored cdc2 protein. To explain theprotein synthesis requirement, they suggest that pro-gesterone stimulation leads to synthesis of a non-cyclinprotein which somehow unmasks the stored cyclin. Onecandidate for such a protein is the product of the c-mosproto-oncogene (Freeman et al. 1989; Sagata et al.1989). Thus, the rate-limiting step in MPF activationwould be release of preexisting cyclin rather than

synthesis and accumulation of new cyclin. While Xeno-pus oocytes contain maternal mRNA for cyclin B (andprobably cyclin A), cyclin synthesis has not beenobserved in the oocyte in response to progesterone(Minshull et al. 1989); it has been observed afteractivation of mature eggs (Murray and Kirschner,1989). On the other hand, the existence of stored cyclinprotein in Xenopus oocytes has not been documented.These possibilities remain open questions.

Resolution of the sequence of steps involved inactivation of MPF in oocytes would be gTeatly facili-tated by a clearer understanding of the interactionbetween p34cdc2 and cyclins, i.e. what is MPF? On thispoint, two schools of thought have developed. On theone hand, active MPF is viewed as a complex of p34cdc2

and cyclin (Draetta et al. 1989). Thus, neither com-ponent would exhibit MPF activity independently. Theevidence for this comes largely from studies referred toearlier in which antisera against one or the other of theproteins precipitates both, and the complex exhibitshistone HI kinase activity. It is not clear from suchstudies whether the antisera quantitatively precipitatedall of the respective proteins as the complex. At least insea urchin eggs, the content of p34cdc2 is in large excessrelative to cyclin, and the majority of the p34 is notcomplexed with cyclin (Meijer etal. 1989). On the otherhand, Gautier et al. (1989) have observed that onlyabout 10% of the p34cdc2 actually functions as MPFduring Xenopus oocyte maturation.

The second view is that cyclin activates p34cdc2 but isnot necessarily a stably bound component of MPF;p34cdc2 alone could exhibit MPF activity once activated(Murray et al. 1989). This is based in part on the reportthat histone HI kinase (MPF) purified from starfishoocytes contains only p34cdc2 (Labbe et al. 1988). Inaddition Murray and Kirschner (personal communi-cation) have observed that MPF activity induced bycyclin activation of pre-MPF in a Xenopus egg extract ismaintained after removal of the cyclin with anti-seaurchin cyclin B antibody (Murray et al. 1989). Crucial tointerpretation of this latter experiment is the completeremoval of cyclin from activated MPF by immuno-precipitation. Murray and Kirschner (personal com-munication) found that the antibody precipitationremoved only 80% of the cyclin, but with no loss ofMPF activity in the supernatant. Since the assay forMPF involved microinjection into cycloheximide-treated oocytes, the results imply that p34cdc2 alone canfunction as MPF in the oocyte. Further, since amplifi-cation in response to MPF can occur in cycloheximide-treated oocytes the results imply that p34cdc2 alone canactivate preMPF.

Post-translational modifications of cyclins andp34cdc2

Draetta and Beach (1988) reported that the phosphoryl-ation of p34cdc2 and cyclin (p62) both are subject to cellcycle regulation in HeLa cells and that such regulationaffects the activity of cdc2 kinase. In Gj cells, cdc2 was


unphosphorylated, not associated with p62, and inac-tive as a kinase. In G2 cells, p34cdc2 became phosphoryl-ated on both tyrosine and threonine/serine residuesand formed a complex with p62 which itself becamephosphorylated, presumably by the cdc2 kinase; thecomplex exhibited kinase activity. By the time celldivision was completed, p34cdc2 was dephosphorylated,dissociated from p62, and protein kinase activity was nolonger observed.

Since only the hyperphosphorylated form of cdc2 wasobserved in association with p62, Draetta and Beach(1988) suggested that complete phosphorylation ofp34cdc2 is a necessary precondition for complex forma-tion. These observations have been extended with thereport that p34cdc2 is inactive as a histone HI kinaseuntil it associates with p62, although the p34cdc2 alonedoes exhibit casein kinase activity (Brizuela etal. 1989).They suggest that one role of cyclin might be to confer'M-phase specificity' on the cdc2 kinase.

Draetta et al. (1988) have shown that cdc2 is a majorphosphotyrosine-containing protein in HeLa cells andthe level of tyrosine phosphorylation is subject to cellcycle regulation. Morla et al. (1989) have shown furtherthat while increasing tyrosine phosphorylation of cdc2correlates with formation of the cdc2/p62 complex inmouse 3T3 fibroblasts, the complex is inactive as ahistone HI kinase. Quantitative tyrosine dephosphoryl-ation occurs during entry into mitosis and this correlateswith maximal HI kinase activity. Further, in vivoinhibition of tyrosine dephosphorylation correlates withG2 arrest. These observations are not restricted tomammalian tissue culture cells.

Recently, Gautier et al. (1989) have shown thatmaximal histone HI kinase activity of p34cdc2 in matur-ing Xenopus oocytes correlates with dephosphorylationof the protein. Conversely, phosphorylation of p34cdc2

led to inactivation of HI kinase activity in egg extractsthat contained active MPF. Dunphy and Newport(1989) have extended these observations in showingthat tyrosine phosphorylation of Xenopus cdc2 is highin interphase (oocytes) but absent during M phase(unfertilized eggs). Activation of preMPF in oocyteextracts resulted in dephosphorylation of tyrosine oncdc2 which correlated with activation of HI kinaseactivity. Furthermore, the product of the yeast genesucl (pl3), which inhibits entry into mitosis in theextracts, blocks tyrosine phosphorylation and kinaseactivation. While these findings support the view thattyrosine dephosphorylation of cdc2 is an important stepin MPF activation, this step alone may not be sufficient.At least in HeLa cells, entry into mitosis is associatedwith both tyrosine and threonine dephosphorylation ofp34cdc2

The possibility considered earlier that active p34cdc2

alone can activate preMPF appears to pose a dilemmawhen one considers the data discussed above. Thus, ifp34cdc2 is active as MPF only when dephosphorylatedand if only phosphorylated p34cdc2 is able to bind cyclin,what role does cyclin play in the activation of preMPFin the oocyte? One solution to this apparent dilemmacould be obtained by minor modification in the se-

quence of events depicted above. For example, if onespeculates that phosphorylation of cyclin facilitatesbinding to cdc2 and that cyclin binding in turn facilitatescdc2 dephosphorylation, then injection of active cdc2would amplify MPF activity via cyclin phosphorylation,which in turn would generate more active cdc2. Thissuggestion necessitates that some cyclin preexists in theoocyte and that it be available as substrate for the cdc2kinase. Obviously, this idea could be tested for byinjection of purified p34cdc2 into oocytes.

The induction of oocyte maturation - anintegrated model

The initial interaction of steroids at the oocyte surfaceresults in a transient decrease in two intracellularsecond messages, one (cAMP) involved in regulatingthe protein kinase A pathway and a second (DAG)which regulates a membrane-bound protein kinase(PKC). Both are serine/threonine protein kinases.Thus, the initiation of maturation, like the activationand functioning of MPF, appears to be controlled bynegative regulation of protein kinases and/or activationof the appropriate phosphatases. The molecular detailsof this initial event remain to be fully elucidated.However, based on the discussion in the several preced-ing pages, a model that integrates the transmembranesignaling events with activation of MPF activity can besuggested.

Crucial to understanding the mechanism by whichprogesterone releases oocytes from G2 arrest is thehypothesis, proposed by Mailer and Krebs (1977), thatoocytes contain a putative phosphoprotein which main-tains meiotic arrest. Progesterone-induced dephos-phorylation of this putative protein would then lead toactivation of MPF and subsequent meiotic events. Thesimplest model that integrates these events is one inwhich the putative phosphoprotein is p34cdc2. Thus,within minutes after progesterone treatment, transientinactivation of one or more protein kinases wouldinitiate the process of preMPF activation by dephos-phorylating the cdc2 kinase.

Both serine/threonine and tyrosine kinases appear tobe involved in cdc2 kinase activation, and both could beinactivated concurrently in response to progesterone.However, to be consistent with available data, acti-vation of MPF is viewed as occurring by two dephos-phorylation steps. First, progesterone would result inpartial dephosphorylation of cdc2 by inactivation of aserine/threonine protein kinase(s). Concurrently,either synthesis of cyclin or a protein that releases cyclinwould occur. The cdc2 protein at this point, stillphosphorylated on tyrosine residues, would not exhibithistone HI kinase activity but would be able to phos-phorylate other substrates including cyclin (Brizuela etal. 1989; Morla etal. 1989; Meijer etal. 1989). Thus, themajor function of the first step would be to phos-phorylate cyclin which would facilitate binding to cdc2kinase. The role of cyclin phosphorylation relative to itsinteraction with cdc2 is not clear. Murray et al. (1989)

696 L. D. Smith

Sertne/Threonlne Melosls p34 Active Tyrosine P 3 4 f u " yklnases Inhibitory as casein dephosphorylatlon active as

inactivated phosphoproteln kinase of p34 occurs histone Hip34 inactive kinase -

()Progesterone —|

Protein Synthesis — (cyclin

Fig. 6. Model indicating the sequence of events from initial progesterone action to the activation of MPF activity.Progesterone results in partial dephosphorylation of the putative meiosis-inhibitory protein, p34, by inactivation of proteinkinase A (PKA) and/or protein kinase c (PKC), and in the synthesis of a protein(s) which produces cyclin. Partiallydephosphorylated p34 then phosphorylates cyclin, which facilitates p34/cyclin association. This prevents continuedphosphorylation of p34 by a tyrosine kinase, leading the fully dephosphorylated p34 and active MPF.

suggest that phosphorylation of cyclin is a prerequisitefor cyclin degradation. However, cyclin breakdowndoes not appear to require phosphorylation, at leastduring cleavage in sea urchin embryos (Neant et al.1989). On the other hand, Meijer et al. (1989) havedemonstrated that phosphorylation of cyclin correlateswell with maximal histone HI kinase activity, sugges-ting that cyclin phosphorylation might be a crucial stepin MPF activation. At any rate, the model suggests thatonce bound, phosphorylated cyclin prevents additionalphosphorylation of the cdc2 by a tyrosine kinase,resulting in dephosphorylated and fully active p34cd

with histone HI kinase activity (Fig. 6).The model clearly is speculative at this point, and

appears to be at odds with the observations of Cyert andKirschner (1988) that activation of preMPF in a stage 6Xenopus oocyte extract involves two components,preMPF and an inhibitor of activation (INH), separablein different ammonium sulfate fractions. They suggestthat preMPF is activated by phosphorylation and thatINH inhibits the activation step, but only when it also isphosphorylated; INH could be the putative phospho-protein. This certainly remains a possibility. On theother hand, their studies did not involve highly purifiedpreparations and there is no direct evidence that INH isfunctional in vivo. More relevant, the original premisethat MPF is active only when phosphorylated appearsto be incorrect.

Because of the intense interest in MPF as a regulatorof mitosis in actively dividing cells, less attention hasbeen paid in the past few years to molecular details ofthe progesterone-dependent release from G2 arrest inthe oocyte. This also traces in part to the view that themechanism of release might be specific to only those celltypes that arrest in G2, and that the progesterone-dependent release might be specific to amphibianoocytes (Gerhart et al. 1984). However, the proteinproducts of two oncogenes, c-mos (Freeman et al. 1989;Sagata et al. 1989) and ras (Birchmeier et al. 1986), bothinduce maturation when injected into oocytes, and bothappear to act very early in the sequence of eventsinduced by progesterone. These observations suggest

that studies on the initial response(s) of oocytes toprogesterone may have wider applicability to under-standing cell-cycle regulation than previously sus-pected.

I thank my colleagues Peter Bryant, Hans Bode and JohnScott for helpful suggestions in preparing this manuscript, andRobert Varnold for technical assistance. The original researchpresented in this paper was supported by NIH grant HD04229.

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{Accepted 21 September 1989)

the induction of oocyte maturation: transmembrane signaling … · oocyte maturation has been...

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