Protein kinase C and progesterone-induced maturation in Xenopus oocytes

The induction of oocyte maturation by progesterone is known to involve an interaction of the steroid with the oocyte surface (review by Smith, 1989). Considering this site of action, considerable attention has been devoted to characterization of the transmembrane signalling events that might mediate progesterone ac tion. There is general consensus that a transient de crease in cAMP levels, resulting at least in part from inhibition of adenylate cyclase activity, is an obligatory first step in the mechanism by which progesterone induces oocyte maturation. This in turn is thought to result in decreased cAMP-dependent protein kinase (PKA) activity which leads to dephosphorylation of a putative maturation-inhibiting phosphoprotein. The large body of evidence supporting this hypothesis has been reviewed extensively (Mailer and Krebs, 1980; Mailer, 1985; Smith 1989).

In addition to progesterone, a large number of other agents also induce oocytes to mature, and it is not known what pathway these agonists might utilize. In some cases, however, it has become clear that a reduction in cAMP is neither sufficient nor necessary for maturation to occur. For example, Birchmeier et al. (1985) reported that injection of the oncogenic protein encoded by H-ras induces maturation when injected into Xenopus oocytes without a corresponding decrease in cAMP levels. More recently, Gelerstein et al. (1988) demonstrated that treatment of oocytes with acetylchol ine rapidly lowered cAMP levels without inducing maturation, while treatment with adenosine, which elevated cAMP levels, nevertheless induced oocyte maturation. These kinds of observations have led to the suggestion that oocytes may contain an alternate path way, bypassing cAMP changes, that is able to induce maturation.

The tumor promoter TPA (12-O-tetradecanoylphor-bol 13-acetate) activates protein kinase C and Stith and Mailer (1987) reported that oocytes treated with TPA underwent GVBD in the absence of hormone treat ment. Recently, Muramatsu et al. (1989) have reported further that a cDNA coding for PKC initiates matu ration when injected into oocytes. These results suggest that activation of PKC might be sufficient to induce oocyte maturation. On the other hand, a number of the diverse agents that induce maturation are phospholipid interacting drugs (Baulieu et al. 1978). Several of these, such as chlorpromazine, tetracaine and verapamil also are reported to inhibit protein kinase C (PKC) (Mori et al. 1980), although their action is by no means specific for the enzyme. In addition, sphingomyelinase is a potent inducer of maturation (see Vamold and Smith, 1989) and this enzyme would be expected to act on membrane sphingolipids leading to the production of sphingosine, a potent inhibitor of PKC (Hannun et al. 1986). These data suggest the possibility that PKC inhibition is sufficient to induce oocyte maturation.

The current study was undertaken to evaluate the potential role of the PKC pathway in the process of oocyte maturation. The intracellular second message diacyglycerol (DAG) was observed to decrease rapidly (within seconds) after progesterone exposure. Prelimi nary experiments aimed at determining the mechanism of progesterone action on DAG levels suggest effects on both DAG synthesis and degradation. However, since DAG regulates protein kinase C activity, these results suggest that a decrease in PKC activity may be an essential event in release of the prophase block. Additional support for this idea comes from obser vations that sphingosine and staurosporine which are potent inhibitors of protein kinase C were able to induce oocyte maturation. Furthermore, DiC₈ and partially purified protein kinase C were found to inhibit or at least delay the onset of GVBD induced by progesterone.

Xenopus laevis were obtained from Nasco (Fort Atkinson, Wl) and maintained in the laboratory. This work was carried out on two separate colonies of Xenopus laevis at Purdue University and the University of California, Irvine, respect ively. Animals at Purdue were maintained at room tempera ture and fed a diet consisting of beef heart mixed with Poly-Vi-Sol with iron (1ml lb⁻¹) twice weekly. At UCI, animals were maintained between 18 and 21 °C and fed a diet of salmon pellets (Rangen, Buhl, Idaho) twice weekly and supplemented with blackworms (Bayou Brine Shrimp Sales, Ontario, CA), once a week. Portions of the ovary were surgically removed and the oocytes manually defolliculated in OR2. Incubations were carried out in OR₂ or with the additions as described in the results.

Diglyceride kinase was from Lipidex (Madison, WI). The [Gamma-³²P]ATP was from NEN or Amersham. The ³H-(5, 6, 8, 9, 11, 12, 14, 15) arachidonic acid (60-100 Ci mmole⁻¹) was from NEN. The triacylglycerol assay kit, diolein and fatty-acid-free bovine serum albumin were from Sigma (St Louis, MO) and the IP₃ radioimmunoassay kit (specific for 1,4,5-inositol trisphosphate) and the ³²P-IP₃ (20-200 Ci mmole⁻¹) used as the recovery marker were from Amersham. The chloroform and methanol were redistilled prior to use.

Sphingosine was from Calbiochem or Sigma. Staurosporine and the DiC_s were from Calbiochem. A stock solution of staurosporine was made by dissolving it in DMSO. The concentrations used in the Results section were made by diluting the staurosporine stock solution with the appropriate amount of OR₂. A sphingosine stock solution was made by mixing Imgml⁻¹ sphingosine or sphingosine sulfate in equi molar quantities of fatty-acid-free BSA in OR₂ and sonicating in a Branson model 1200 sonicator bath for 1 h on ice. The resulting stock solution was a suspension that was diluted to the appropriate concentrations in OR₂ or injected directly into the oocyte.

The protein kinase C was the kind gift of Curtis Ashendel (Purdue University) and consisted of mixed isomers of protein kinase C isolated from rat brain.

Stage 4 or stage 6 control oocytes, progesterone (10 μg ml⁻ ‘)-treated oocytes or β-estradiol (10μml^−l)-treated oocytes (stage 6 only) were homogenized in 3 ml chloroform/metha-nol (1:2) at the times specified in the Results using a Brinkman Polytron homogenizer. 1 ml of chloroform and 1.8ml IM NaCl were added and the tubes vortexed vigor ously. The organic phase was transferred to a 13×100 mm screw cap tube, dried under N₂ and stored at -20°Cover night. The mass of diglyceride was quantitated by using the DG kinase assay of Preiss et al. (1987). The [³²P]phospholipids obtained were resuspended in chloroform/methanol (2:1) and an aliquot spotted on Whatman K6 TLC plates and developed in chloroform/methanol/acetic acid (130:30:10). After autoradiography, the spot corresponding to DAG was scraped and counted by LSC in PPO-toluene or Redi-Safe (Beckman Instruments). The mass of the DAG was deter mined by comparing the radioactivity incorporated into the [³²P]phosphatidic acid band in the oocyte sample with the same band in a diolein standard curve run at the same time. In our hands, the standard curve was linear from 0.035 to 0.52 nmoles with a slope typically greater than 150,000ctsmin⁻¹ nmole⁻¹ DAG. All measurements were within the linear portion of the curve.

When the mass of DAG in the oocyte membrane was measured, the plasma membrane-vitelline envelope complex was removed according to Sadler and Mailer (1981). Briefly, groups of 15 oocytes were incubated in ice-cold buffer (20 mM NaCl, 25 mM Hepes, pH 7.5) 5–10 min, the oocytes were broken open with a pair of forceps, and then allowed to incubate on ice for at least an additional 5 min. The membra ne-vitelline envelope complex was removed by manually pulling it away from the cytoplasm. The membrane complex was transferred to fresh buffer, washed and then transferred to a Eppendorf microcentrifuge tube containing 1 ml of ice cold buffer. After centrifugation at 14,000 revs min⁻¹ for 20min, the supernatant was carefully removed, and the membranes were incubated in OR₂ or in 1 ; μml⁻¹ progester one in OR₂ for the times indicated in the Results. The reaction was terminated with 3 ml chloroform/methanol (1:2) and the extraction of DAG and the DG kinase assay carried out as for whole oocytes. To correct for variable amounts of membrane material, the data obtained with individual samples were normalized to triacylglyceride mass. In this case, an aliquot of the resuspended [³²P]phospholipids (used for spotting the TLC plates) was dried under N₂ and assayed for triacylglycerides using the GPO-trinder kit from Sigma.

IP₃ levels were measured using a radioimmunoassay kit obtained from Amersham which is highly specific for inositol 1,4,5-trisphosphate; cross-reactivity with inositol 1,3,4-tris-phosphate is claimed to be less than 0.3 %. Groups of 5–15 stage 6 oocytes were homogenized in 10 % trichloroacetic acid as per the protocol that came with the RIA kit. Approxi mately 10,000cts min⁻¹ of ³²P-IP₃ were added to each sample prior to extraction to correct for recovery. After centrifu gation, the supernatant was washed 3 times with 10 volumes of water saturated diethyl ether. Duplicate aliquots from each sample were assayed and IP₃ levels were calculated by comparison with a standard curve based on assay of different concentrations of authentic IP₃; the curve was linear from 0.19–25 pmoles, the range of values estimated for the oocytes.

Cicirelli and Krebs have observed that sphingomyeli nase is a highly potent inducer of Xenopus oocyte maturation (personal communication). We have con firmed these results (Varnold and Smith, 1989). The metabolism of sphingolipids might be expected to result in an increase in the production of sphingosine, a potent inhibitor of protein kinase C (reviews by Merrill and Stevens, 1989; Hannun and Bell, 1989) with an IC50 of 5 μM in vitro. Sphingosine is not specific for PKC since it also inhibits Ca²⁺-calmodulin-dependent enzymes (Jef ferson and Schulman, 1988), but it is reported to have no effect on cAMP-dependent protein kinase (PKA). Incubation of oocytes with sphingosine induced GVBD as shown in Table 1, but at a relatively high external concentration (350 μM), and the time of GVBD was considerably slower than in oocytes exposed to pro gesterone. This time differential appears to result in part from poor diffusion of the sphingosine-BSA com plex across the oocyte’s vitelline envelope, since re moval of the envelope prior to incubation with the inhibitor results in GVBD at about the same time as did progesterone and at a lower concentration (170 M). When sphingosine was injected directly into oocytes as a BSA-sphingosine suspension, internal concentrations of 0.3– 0.9μM all induced GVBD (Table 1). However, again the time at which GVBD occurred after injection was delayed compared to controls exposed to pro gesterone.

Staurosporine is considered to be the most potent inhibitor of PKC, with an IC50 of less than 5 nM measured in vitro (Tamaoki et al. 1986). It also is not specific for PKC but can inhibit PKA and myosin kinase activities at higher concentrations (Tamaoki et al. 1986; Watson et al. 1988). In two separate experiments, 120 oocytes were incubated with staurosporine at concen trations ranging from 6.5 to 210nw; GVBD was ob tained in one group (10 oocytes) at the highest concen tration and at approximately the same time as progesterone-treated controls. In two additional exper iments, staurosporine was injected into oocytes in amounts that produced internal concentrations ranging from 0.5 HM to 44 μ M (8 different groups). We observed GVBD in one group of oocytes from each female at internal concentrations of 1 and 9HM, respectively. However, GVBD again was considerably slower than in oocytes exposed solely to progesterone, and was evi dent only after overnight incubation.

While the results described above suggest that inhi bition of PKC can induce oocytes to undergo matu ration (GVBD), they are by no means unequivocal. We have no simple explanation for the variability obtained with the different inhibitors. It is becoming increasingly clear, however, that the effectiveness of protein kinase inhibitors varies considerably with different cell types (Sako et al. 1988) and with the type of agonist used to elicit a response in the same cell type (Schachtele et al. 1988). Moreover, depending on the intracellular half life of the inhibitors (hours for sphingosine, unknown for staurosporine), long-term inhibition of protein kinase C might prevent maturation even if the induction phase has taken place (see later).

If, as suggested above, inhibition of PKC activity can induce oocyte maturation, then elevation of PKC ac tivity prior to progesterone exposure might be expected to prevent or delay oocyte maturation. In order to test this hypothesis, we treated oocytes with DiC₈ (dioctyl glyceride), a synthetic water-soluble analog of diglycer ide, which can activate protein kinase C (May et al. 1986). The results of these experiments are shown in Table 2. Continuous incubation of stage 6 oocytes with 0.1 mg ml⁻¹ (0.29 μ M) DiC₈ beginning 15 min prior to progesterone treatment completely inhibited progester one-induced maturation. While lower concentrations of DiC₈ did not completely inhibit progesterone-induced maturation, they did in some cases delay it. Since DiC₈ is rapidly metabolized by cells (May et al. 1986), the inhibitory effect of DiC₈ should be readily reversible. This was the case. When oocytes were incubated with the dioctyl glyceride for 3h, transferred to OR2 for an additional 3 hours, and then exposed to progesterone, GVBD occurred at the same time as in control oocytes treated with progesterone alone (data not shown).

In related experiments, we attempted to elevate endogenous PKC levels by injecting oocytes with pro tein kinase C isolated from rat brain. As reported by Stith and Mailer (1987), injection of the mixture of PKC isozymes did not induce maturation nor did it have any obvious effect on progesterone-induced maturation provided the steroid was added shortly after injection of the kinase. However, as summarized in Table 2, when PKC was injected 15– 60 minutes prior to addition of progesterone, GVBD was significantly delayed com pared to progesterone-induced controls. The lowest amount of PKC that delayed maturation represents an internal concentration of 0.14μ M.

The results obtained with the PKC inhibitors, sphin gosine and staurosporine, the PKC activator, DiC₈, and the partially purified PKC all indirectly support the hypothesis that in order to obtain re-entry into the cell cycle, a transient decrease in PKC activity is required. In order to more directly assess the activity of protein kinase C, we measured the mass of DAG in control and progesterone-treated oocytes. The measurement of DAG mass was carried out using two separate colonies of Xenopus laevis (one at Purdue University and one at the University of California, Irvine). The mass of DAG measured from these two colonies was significantly different, averaging 168±9pmoles oocyte⁻’ at the University of California (8 females) and 48±6pmoles oocyte⁻¹ at Purdue (3 females). Nevertheless, in re sponse to progesterone, DAG mass decreased in both cases an average of 29 % with 15 sec and levels remained reduced for at least the first 2 min (Fig. 1A). By 5 min after progesterone addition, DAG levels had increased to 32 % above control levels, followed by a reduction to control levels by about 15 minutes. Stage 4 oocytes which are not responsive to progesterone did not show a decrease in DAG levels during the same time period and, in fact, may have slightly increased DAG levels. The steroid β -estradiol, which does not induce maturation, also did not change DAG levels in stage 6 oocytes (data not shown). Thus, the decrease in DAG levels in stage 6 oocytes correlates with the physiological response to progesterone. After the initial decrease, DAG levels increase from 30 min until GVBD (at 540 min) where it is 2.2-fold higher than controls (Fig. 1B).

The data described above were obtained by extract ing lipids from the whole oocyte and would have measured DAG changes in both the oocyte plasma membrane and intracellular sources. Since PKC is a membrane-bound kinase in its active state, it was of interest to see if the changes in DAG could also be measured in the plasma membrane. The mass of DAG in stage 6 oocyte membranes average 10±lpmole oocyte⁻¹ (2 females, measured at UCI) or about 6% of the total DAG mass in the oocyte. Removal of follicle cells prior to isolation of the plasma membrane did not alter the mass of DAG found in the membrane (data not shown).

When the isolated membranes were exposed to progesterone, DAG levels were also decreased although the time course was different from that found in whole oocytes. This is not necessarily surprising since membrane phospholipids normally are continually re plenished from the cytoplasm. This would not occur in the isolated material. Thus, by 15sec, progesterone induced only a 10 % decrease in DAG levels compared to 29 % for the whole oocyte. It required between 1 and 2 min for the membrane DAG levels to be reduced by 30%. In contrast to the intact oocyte, DAG levels in the membranes continued to decrease, reaching a level of 41% of controls by 5 min, the latest time point measured.

Within recent years, considerable attention has been devoted to studies on the hydrolysis of membrane bound PIP₂ by phospholipase C. The lipase is regulated by a GTP-binding protein coupled to plasma membrane receptors in several cell types (Cockcroft and Stutch-field, 1988), providing a mechanism by which extra cellular factors can modulate intracellular levels of the second messengers DAG and IP₃. To approach the possibility that progesterone acts in this manner, we have initiated experiments to measure progesterone-induced changes in IP₃ levels.

The results of two experiments involving oocytes from different females are shown in Fig. 2, which plots changes in IP₃ mass after progesterone treatment of intact oocytes relative to control values at each time point. The actual IP₃ values varied widely in the two experiments, a result also reported by Tigyi et al. (1990) for Xenopus oocytes. Nevertheless, the relative change in both cases was identical. In response to progester one, IP₃ decreased by about one-third during the first 15 seconds, followed by a later return to control levels. These changes mimic the time course for DAG changes and suggest that one effect of progesterone is to reduce the hydrolysis of membrane-bound PIP₂.

Clearly, the total decrease in DAG levels in response to progesterone (as much as 50pmoles/oocyte) greatly exceeds that which could result from the mechanism postulated above. However, diacylglycerol is an im portant intermediate in phospholipid synthesis and could be regulated at several levels (Tijburg et al. 1989). For example, hydrolysis of phosphatidylcholine pro duces DAG and decreased PC turnover could result in lowered DAG mass. Alternatively, an increase in the activity of enzymes that degrade DAG would also result in lowered DAG. In this case, if DAG labeled with arachidonic acid is metabolized to phosphatidic acid by DAG kinase, one would expect to find radioactive PA. Degradation of DAG by lipase action would result in a loss of radioactivity in DAG. The results of three separate experiments of this type are shown in Fig. 3. In response to progesterone, there was a loss of radioac tivity ([³H]arachidonic acid) in DAG of more than 30 % within the first minute. We have not followed exten sively the fate of the released arachidonate. Neverthe less, this result suggests a progesterone-stimulated in crease in DAG lipase activity.

Obviously, the effects of progesterone on lipid metabolism in the oocyte are complex, and appear to involve different action at the plasma membrane versus intracellular sites. The manual isolation of membranes provides one approach to discrimination between these different effects, but that procedure is time consuming and only limited quantities of material can be obtained. Thus, sorting out the various progesterone effects is not a simple task. Regardless, the results show clearly that the earliest response yet detected of oocytes exposed to progesterone is a significant decrease in the level of an intracellular second message (DAG) known to regulate the activity of protein kinase C.

The current study shows that within seconds of ex posure to progesterone, DAG levels decrease in both isolated oocyte plasma membranes and in intact oocytes. Thus, this change precedes those mentioned above and represents the earliest documented response to progesterone stimulation. Based on the known role of DAG in regulating protein kinase C activity (reviews by Bell, 1986; Nishizuka, 1986), the data on DAG changes suggest that the induction of oocyte maturation by progesterone involves a transient decrease in PKC activity. Two kinds of additional evidence support this view. First, treatment of oocytes prior to progesterone exposure with agents expected to elevate PKC activity (DiC₈, injected PKC) prevents or delays GVBD. Second, treatment of oocytes with known inhibitors of PKC can induce GVBD in the absence of hormone treatment.

In contrast to the above suggestion, Muramatsu et al. (1989) reported that injection of a cDNA encoding PKC into the oocyte germinal vesicle initiates maturation, since the germinal vesicle was observed to migrate to the animal hemisphere surface. Since GVBD did not actually occur, it seems inappropriate to refer to nuclear migration alone as a manifestation of oocyte matu ration. On the other hand, several other studies involv ing treatment of oocytes with known or suspected activators of PKC have led to the suggestion that activation of PKC can induce GVBD by a pathway that does not involve progesterone.

Stith and Mailer (1987) reported that continuous incu bation of oocytes with the phorbol ester 12-0-tetradeca-noylphorbol 13-acetate (TPA), an activator of PKC, induced GVBD in the absence of hormone treatment. Pan and Cooper (1990) recently have reported the same result. Notably, in both cases, the oocytes exposed to the phorbol ester were taken from females recently primed with a gonadotropin (pregnant mare serum gonadotropin, PMSG). We also have observed that TPA induces GVBD in oocytes taken from females recently injected with gonadotrotin (human chorionic gonadotropin, hcG), although only about 10% of the oocytes exhibited this response. In oocytes from un stimulated females, no GVBD was observed after treatment (unpublished data). Bement and Capeo (1989) also observed that neither continuous nor short term exposure of oocytes to phorbol ester resulted in GVBD.

Protein kinase C has been localized to the nuclei of NIH 3T3 cells (Leach et al. 1989) and other cell types (Cambier et al. 1987), and has been implicated in the phosphorylation of nuclear envelope lamins (Fields et al. 1988; Hornbeck et al. 1988). Since the only assay for phorbol ester effects on Xenopus oocytes has been presence or absence of an intact GV, conceivably agents that activate PKC could result in nuclear mem brane dissolution independent of other events. How ever, this does not adequately explain the differential effect of phorbol esters on oocytes from stimulated versus unstimulated females.

In Xenopus oocytes from stimulated females, GVBD in response to progesterone occurs much earlier than in oocytes from unstimulated females. Starfish oocytes are arrested in meiosis I with an intact nucleus (GV) and maturation is induced by the action of 1-methyadenine acting on the oocyte surface. In this case, treatment with phorbol esters inhibits the induction of maturation (Kishimoto et al. 1985). This is not different in principle from the inhibitory effect of DiC₈ on progesterone-induced maturation in Xenopus oocytes. Surf clam oocytes also are arrested in meiosis I with an intact GV and fertilization initiates GVBD. In this case, TPA is reported to induce GVBD (Bloom et al. 1988).

One explanation for these diverse results is that oocytes from diverse organisms, or even from the same animal under different physiological conditions, can be arrested at various points relative to the G2/M tran sition even though they all contain intact GVs. Thus, oocytes from stimulated Xenopus females are ‘down stream’ in the sequence of events leading to meiosis I relative to those from unstimulated females (see Smith, 1989). In this context, preliminary experiments indicate that DAG levels in stage 6 oocytes taken from stimu lated females are transiently increased after progester one treatment, in contrast to the data shown in Fig. 1A (unpublished data). Since the data in Fig. IB show further that DAG levels increase substantially in oocytes from unstimulated females as they approach GVBD, increased PKC activity may be a relatively late requirement in the events leading to GVBD. In short, dependent on the actual point in the cell cycle at which oocytes are arrested, one faces the apparent paradox that both activation and inactivation of PKC could induce oocyte maturation.

Perhaps the most perplexing set of data on PKC involves the several studies which have shown that injection of oncogenic ras protein into stage 6 oocytes induces maturation (Birchmeier et al. 1985; Laçai et al. 1987; Barrett et al. 1990; Pan and Cooper, 1990). Since antibodies to ras protein inhibit insulin-induced but not progesterone-induced maturation (Deshpande and Kung, 1987; Korn et al. 1987), it has been suggested that ras activates a different pathway than that induced by progesterone. Oncogenic ras protein is reported to activate protein kinase C in several cell types (Price et al. 1989), and ras protein is reported to cause a rapid (within 20 min) increase in DAG levels after injection into stage 6 oocytes (Laçai et al. 1987; Lacal, 1990). However, these latter experiments did not monitor changes in DAG mass, and the reported increases in DAG, based on incorporation of [³H]glycerol, required relatively large amounts of the oncogenic protein. Furthermore, posttranslational modification of the injected protein and transport to the oocyte membrane require about 3 hours (Pan and Cooper, 1990), sugges ting that early ras effects on DAG levels occur in the cytoplasm with unmodified protein. On the other hand, Pan and Cooper (1990) reported no change in DAG levels aftrer injection of p21 into oocytes, but did observe a relatively late increase in phosphatidylinosit ide metabolism. In this sense, Allende et al. (1988) reported that oncogenic ras protein induces GVBD in cycloheximide-treated oocytes, suggesting the protein acts downstream of the protein synthesis requirement seen when maturation is induced by progesterone. Finally, Sadler and Mailer (1989) observed that injec tion of oncogenic ras protein into Xenopus oocytes stimulates cAMP phosphodiesterase. Based on this discussion, we suggest that elucidating the mechanism by which ras protein induces oocyte maturation re quires further investigation; studies with ras are not necessarily inconsistent with our current hypothesis.

It has been known for some time the oocytes enucleated prior to progesterone exposure still exhibit most of the events associated with maturation. These include ap pearance of MPF activity, increased protein synthesis and cytoplasmic changes that allow mature oocytes to activate in response to appropriate stimuli (reviewed by Smith, 1989). However, since MPF alone can induce all of the events associated with maturation, it has been assumed that all agonists that induce maturation, regardless of mode of action, converge at the level of MPF activation.

A series of recent studies on characterization of MPF have shown that the active component is a protein kinase (p34^cdc2) whose activity is regulated by phos-phorylation/dephosphorylation by another protein, cyclin, which also is phosphorylated (review by Smith, 1989). When the p34^cdc2 kinase is phosphorylated by both a serineμhreonine and a tyrosine protein kinase(s), MPF is inactive; dephosphorylation corre sponds to activation. These data all reinforce the view that multiple protein kinases are involved in the pro cesses of oocyte maturation leading to GVBD (Cicirelli et al. 1988).

The initial action of progesterone at the oocyte surface now is known to result in a decrease in the intracellular content of two second messages, cAMP and DAG, which regulate protein kinase A and protein kinase C activity, respectively. At least one additional protein kinase, the product of the c-mos proto-onco gene also has been implicated in the initiation of oocyte maturation (Sagata et al. 1989). Thus, the induction of maturation as well as MPF activation appears to involve multiple protein kinases. The molecular details of the initial agonist-induced events, the nature of potential interactions between various protein kinases, and the link between induction and MPF activation remain to be elucidated. However, the involvement of more than one pathway in the initiation of maturation, especially one (PKC) that can be regulated at several levels by the products of lipid catabolism, would certainly help explain how a large number of seemingly diverse ‘agonists’ can induce oocyte maturation.

We thank Christa Florea, Charissa Castro and Denise Martella for their technical assistance during the preliminary phases of this study. We thank Dr Curtis Ashendel for supplying the protein kinase C and Dr Charles Glabe and Dr Ricardo Miledi for their comments and helpful suggestions in the preparation of this manuscript. This work was supported by NIH grant HD04229 awarded to L.D.S.