THE JOURNAL OF EXPERIMENTAL ZOOLOGY 257:115-123 (1991)
Involvement of Protein Kinase C in the Regulation of Oocyte Maturation in Amphibians (Rana dybomskii) HYUK B. KWON AND WON K. LEE Chonnam National University, College of Natural Sciences, Department of Biology, Kwangju 500-757, Republic of Korea ABSTRACT Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed t o mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 pM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50,7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide(1 pg/2 ml), forskolin (9 pM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin (W-7) (100 pM) or a PKC inacantagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide tivator l-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 pM) likewise suppressed TPA- or progesterone-induced maturation. The data suggest that PKC plays a role in the regulation of amphibian oocyte maturation and that the seasonal change in responsiveness (GVBD) of oocytes to steroid occurs after oocytes obtain responsiveness to PKC activation.
Oocytes within fully grown amphibian ovarian follicles undergo germinal vesicle breakdown (GVBD) during in vitro follicle culture following treatment with frog pituitary homogenate (FPH) or meiosis inducing steroid (MIS), progesterone (reviewed by Masui and Clarke, '79; Schuetz, '85). Progesterone is believed to act on the oolemma (Ishikawa et al., '77) and induces a decrease in the intracellular level of cyclic adenosine monophosphate (CAMP)(Maller et al., '79), which triggers a complex chain reaction that eventually induces the oocyte GVBD (Baulieu, '83; Maller, '83; Schuetz, '85). Recently, several investigators reported that protein kinase C activation induced GVBD in Spisula oocytes (Eckberg et al., '87; Eckberg, '88) and amphibians (Stith and Maller, '87; Kleis-San Francisco and Schuetz, '88). In various type of cells, protein kinase C (PKC) is known to be transiently activated by diacylglycerol, which is produced in the membrane during signalinduced turnover of inositol phospholipids (Nishizuka, '84).I t is therefore suggested that 0 1991 WILEY-LISS, INC.
oocytes mediate hormonal signals via the adenylate cyclase system and phosphatidyl inositol turnover system (PI system) for GVBD. Responsiveness of in vitro cultured follicular oocytes of R . dybowskii to progesterone stimulation varies considerably with season. In early hibernation, such oocytes fail to respond to progesterone, whereas during the middle of hibernation, they respond readily to steroid. During the breeding season, oocytes typically matured spontaneously during in vitro culture of follicles without added hormone (Kwon et al., '89). In this study we have assessed whether oocyte responsiveness to protein kinase C activation varies in relationship to the different types of maturation which can be defined (hormone responsive and nonresponsive, spontaneous maturation). The data presented here show that the time course of TPA-induced oocyte GVBD in all Received December 19, 1989; revision accepted May 3, 1990.
116
H.B. KWON AND W.K. LEE
each experiment. Full grown follicles were isolated from ovaries using watchmaker's forceps. For some experiments, oocytes were denuded of somatic components by manually removing the follicular envelope followed by a 2 h treatment in calcium free to remove adherent follicle cells (Lin and Schuetz, '85). All experimental manipulations were conducted at room temperature (1822°C) in amphibian Ringer's solution (Kwon and Schuetz, '86). Routine in vitro culture were carMATERIALS AND METHODS ried out using multiwell culture dish (24 wells,' Animals dish, Nunc) with 20 follicles cultured in 2 ml of Hibernating frogs (Rana dybowskzi) were col- AR per well. Cultures were maintained in a shaklected during late autumn (November) from ing incubator (Kukje Scientific Co., Korea) at streams in the Chonnam area of the southern part 22°C and agitated at 80 oscillations per minute of the Korean peninsula. Animals were kept in a for various periods of time. After culture, the oocold room maintained in darkness at 4°C. Ani- cytes were heat-fixed by brief boiling and cracked mals were contained (10-20) in glass or plastic open individually and checked for the germinal boxes containing 10% amphibian Ringer's solu- vesicle. tion (AR) that was changed two times a week. Follicle extraction and sample preparation Hormones and reagents for radioimmunoassay Progesterone, forskolin, and verapamil were The steroidogenic capacity of cultured follicles dissolved in vehicle composed of ethanol and pro- was monitored by measuring intrafollicular levpylene glycol (1:l)in a stock of 2 mglml, 10 mM, els of progesterone by radioimmunoassay (RIA). and 400 mM, respectively. An active PKC ac- Extraction of progesterone from follicles emtivator, phorbol 12-myristate 13-acetate (TPA) ployed procedures described by Lin and Schuetz (Nishizuka, '84) and a n inactive form of the phor- ('85) and Kwon and Schuetz ('86). After specified (4a-PDD) periods of culture, the medium was aspirated and bol ester, 4au-phorbol-12,13-didecanoate (Clarke et al., '85) were dissolved in dimethyl sul- follicles were extracted in the culture well using foxide (DMSO) in a stock of 8 mM and 12.8 mM, methanol (GR, Merck, 1ml methanol per 20 follirespectively. Calmodulin inhibitor, N-(6-amino- cles per well). Extraction was carried out for 15 hexyl)-5-chloro-l-naphthalenesulfonamide(W-7) min with shaking. Methanol extracts were evapoand PKC antagonist, l-(5-isoquinolinylsulfonyl)- rated in a freeze dryer (Labconco) and stored at 2-methyl-piperazine (H-7) were dissolved in -40°C until assayed. Before assay, methanol exDMSO in a stock of 80 mM and 40 mM, respec- tracts of follicles were reconstituted using gelatin tively. Cycloheximide was dissolved in AR in a phosphate-buffered saline (GPBS). Appropriate stock of 2 mg/ml. All hormones and reagents were amounts of this extract in GPBS was assayed for purchased from Sigma. Frog pituitary homoge- progesterone by RIA without further purification. nate (FPH) was prepared from female frogs. Progesterone radioimmunoassay Glands were homogenized in AR at 4°C using a glass homogenizer. The homogenate was cenGeneral assay procedures were adapted from trifuged (4"C, 10,000 rpm, 20 min) to remove de- those described by Fortune et al. ('751, Lin and bris, and the supernatant was frozen (-40°C) in Schuetz ('85),and used in previous studies (Kwon aliquots until needed (Lin and Schuetz, '85). Dif- et al., '89). Labeled progesterone (1,2,6,7-3H-proferent concentrations of reagents were prepared gesterone; 99 Ci/mmole) was obtained from Amby diluting the stock solution with vehicle and ersham and antiprogesterone serum (progesteradded t o culture wells such that the vehicle con- one-1la-hemisuccinate-bovine serum albumin) centration maintained at 0.25% or 0.5% (v/v). was donated from Dr. Yong-Dal Yoon (Hanyang University, Seoul). Its cross-reactivity was deIn vitro follicle and oocyte culture scribed in a previous report (Kwon et al., '89). Animals were sacrificed by decapitation, and Each sample was quantified for tritium using a ovaries were removed immediately and placed in Packard Tri-Carb 1500 liquid scintillation anaRinger's solution. Fresh ovaries were used for lyzer. Routinely, two sets of progesterone stan-
three types of the oocytes was very similar to that seen in progesterone-treated oocytes. The data suggest that some steps between hormone stimulation and PKC activation i n the oocyte cytoplasm are different among the three types of oocytes. These steps may be closely linked to acquisition of meiotic competence and regulation of the breeding season in amphibians.
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION
dards (10-2,000 pg) and sample replicates were included in each assay. Concentration of progesterone was calculated with SecuRIA program (Packard) by personal computer. The coefficients of variation between and within assays were 11.6% and 7.7% (n = 141,respectively. Statis tics In GVBD data analysis for all experiments, treatments were done in duplicate on individual frogs, and GVBD data of the average of the replications of experiment were transformed using a n arcsin-square root transformation (angular) and analyzed by Student’s t-test. The data of progesterone level were also analyzed by Student’s t-test; 50% GVBD (EDs0) values were estimated for follicles by probit analysis (Statistical Analysis System [SAS], Cary, NC).
RESULTS TPA induction of oocyte maturation of R. dybowskiifollicles isolated during dflerent periods of hibernation In preliminary experiments, we found that oocytes of R. dybowskii isolated in early hibernation period (November) did not mature in response to progesterone stimulation in vitro. Initially, we examined whether PKC activation in such oocytes induced oocyte GVBD by using a strong synthetic activator of the enzyme, TPA. The addition of TPA to the medium (0.01-10 pM) induced GVBD in a dose-dependent fashion during in vitro follicle culture, while an inactive phorbol ester, 4a-PDD (1.6 pM) or progesterone (1 pg/2 ml) were ineffective (Fig. 1).The maturational effects of TPA was also examined using follicular oocytes isolated during the winter (DecemberJanuary). Oocytes collected at these times matured readily to hormone stimulation and did not exhibit spontaneous maturation. Such oocytes underwent GVBD i n the presence of various doses of TPA (Fig. 2B). The dose response for GVBD was essentially the same as that obtained in nonsteroid responsive oocytes (Figs. l,2A). 4a-PDD was likewise ineffective in inducing maturation of these oocytes. However, the maturation rate induced by TPA (-70%) was lower than that produced by progesterone (-90%). Oocytes treated with higher doses (1or 10 pM) of TPA exhibited cytological changes in the cortex different from that seen in progesterone-treated oocytes. The high dose of TPA produced an irregular distribution of pigment on the animal pole and some of
m
> 50
117
1 I
O
00.010.1
1.6
10
I
4a-PDD (pM)
T P A (PM
P4
(pg/2ml)
Fig. 1. TPA induction of oocyte meiotic maturation of R . dybowskii oocytes that did not respond to exogenous progesterone in vitro. Isolated follicles were cultured in the presence of various doses of TPA (0.01-10 KM), 4a-PDD (1.6 FM) or progesterone (1 ~ g / ml) 2 and examined for GVBD after 24 h of culture. Each bar in the figure represents average % GVBD (mean f SEMI of 240 follicles (duplicate wellsianimal, six animals). *P < 0.05, when compared with control (4a-PDD group).
A
z 0
0
0.01 0.1
I
10
-
4D-PDD
Pq
TPA(uM)
100 ~
.--.
Intact Follicle
0-0
Denuded Oocyte
0
0.01 0.1
B
I--
z 0 I
10
4 a - m P,
T PA (rM)
Fig. 2. Effect of follicle cell removal on TPA-induced oocyte maturation of R. dybowskii. Follicles and denuded oocytes obtained from each animal were cultured in medium containing various doses of TPA, 4a-PDD, or progesterone and examined for GVBD after 24 h culture. A Data depicted were obtained using follicular oocytes that did not respond t o progesterone. B: Data depicted were obtained using oocytes that responded to progesterone. Each point or bar in the figure represents average (mean k SEMI % GVBD of 120 follicles (three animals) (A) and 200 follicles (five animals) (B). *P < 0.05, when compared with control (401-PDD group).
118
H.B. KWON AND W.K. LEE
the surface pigment appeared to diffuse into the cytoplasm. Interestingly, in sister oocytes that did not mature with TPA stimulation, no abnormal cytological changes were observed (data not shown). The effect of TPA on follicular oocytes that exhibited spontaneous maturation was also tested. Most oocytes (>go%) underwent spontaneous GVBD after 24 h culture, regardless of the presence of TPA (10 p,M) (data not shown).
1500 *-•
o--0
-0u .-
2
4-4
FPH T PA Control
1000
\ a,
C 0
/
L
0) c v) G)
TPA actions on oocytes and follicles As progesterone produced by follicle cells is known to be a meiosis-inducing hormone, TPA may induce oocyte maturation by stimulating progesterone production by the follicle cells. To test this possibility, experiments were designed t o examine the effect of follicle cell removal on the TPA induction of maturation and to assess the influence of TPA on endogenous intrafollicular levels of progesterone. Removal of the follicle cells did not prevent TPA-induced maturation, regardless of whether oocytes are responsive (Fig. 2B) or nonresponsive to hormone stimulation (Fig. 2A). Furthermore, TPA did not stimulate progesterone accumulation by the follicles (Fig. 3), while FPH (0.1 pituitary equivalent/well) markedly stimulated progesterone accumulation by the follicles (Fig. 3). The data clearly show that the action of TPA on the oocyte is a direct one and is not linked to the follicle cells, which are tightly attached to the oocytes.
0
500
L
a cn P
I’/*** I
*
0
3
, r-0
_g--------*s 6
F
A
9 T I M E(hr)
Fig. 3. Effect of TPA on the progesterone accumulation in follicles of R . dybowskii in vitro. Isolated follicles were cultured in the presence or absence of TPA (10 pM) or FPH (0.1 pituitary equivalenti2 ml) for 524 h. At designated time points, follicles were extracted with methanol for steroid RIA. Each point represents pg progesterone (mean t SEM) per follicles (n = 4, duplicate wells, two animals). **P < 0.01 when compared with control.
divided into two groups and cultured in the presence or absence of TPA for 5 2 4 h. At designated time points, the incidence of oocyte GVBD was compared with that seen in nontreated control oocytes. Experiments with the same design were performed with progesterone treatment using different animals but on the same days. Treatment of TPA remarkably accelerated the time course for the GVBD. Fifty percent of the oocytes matured after 6.8 h of culture in the TPA-treated group, while in nontreated sister follicles a similar incidence of maturation was observed after 19.4 h (Fig. 5A). Progesterone treatment likewise accelerated the time course of spontaneous maturation (GVBD); 50% GVBD occurred by 8.7 h after progesterone treatment, while oocytes in the control nonhormone-treated group did by 18.2 h of culture (Fig. 5B). Thus, the time course for TPAinduced oocyte maturation was essentially the same (ED,,, 7-9 h) for all three types of oocytes.
Time course of the TPA-induced oocyte maturation in vitro The three types of follicles were collected at various times after TPA treatment under standardized culture conditions and examined for GVBD. Follicles were cultured in the presence of 10 pM of TPA for 5 2 4 h and at designated time points oocytes were examined for GVBD. TPAinduced oocyte GVBD, in nonsteroid responsive oocytes, occurred within 12 h of culture (70%). Approximately one-half of the oocytes underwent GVBD after 9 h of culture (Fig. 4A). Progesterone (1 pg/2 ml) treatment induced no oocyte GVBD (Fig. 4A). TPA-treated oocytes, responsive to hormone, exhibited similar time course to that induced by progesterone (Fig. 4B). Fifty percent of Comparison between TPA and the oocytes had undergone maturation by 8 h in progesterone-induced oocyte maturation response t o both types of stimulation (Fig. 4B). in vitro The effects of TPA on the time course of matExperiments were carried out to acertain uration was also examined in follicles exhibiting whether TPA and progesterone induced oocyte spontaneous maturation. Follicles isolated during GVBD by common pathways. A decrease in oothe late hibernation period (early February) were
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION
7
.-.
100
loo
0--0
.-.-.-.
T PA Control
a
0,'
m
> W
119
A
50
z
," ,
I 100-
*-•
Progesterone
O--O
Control
m >50-
r SO%GVBD I
B
(3
ar .o
I
!
0
100
6
0
12
18
24
TIME(hr)
n m 50
> W
ae
0 0
6
12
18 T I M E (hr)
24
Fig. 5. Acceleration of maturation by TPA in R. dybowskii oocytes exhibiting spontaneous maturation in vitro. Follicular oocytes exhibiting spontaneous maturation without added hormone were cultured in the presence or absence of TPA (10 JLM)or progesterone (1 kg12 ml) for 5 2 4 h. Oocytes were examined for GVBD at designated time points. A Acceleration of the onset of oocyte maturation by TPA. B Acceleration of the onset of oocyte maturation by progesterone. Each point represents the average % GVBD of 200 follicles (five animals) (A, B). A: The EDs0 of the TPA-treated and the control group was 6.8 and 19.4 h, respectively. B: The EDSoof progesterone and control groups was 8.7 and 18.2 h, respectively.
Fig. 4. Time course of TPA-induced oocyte GVBD in R. dybowskii in vitro. Follicles were cultured in the presence or absence of TPA (10 kM) or progesterone (1 kg12 ml) for 524 h. At designated time points, follicles were examined for GVBD. A. Data from follicles that did not respond to progesterone. B: Hormone responsive follicles. Each point represents average % GVBD (mean '-c SEM) of 120 follicles (three animals) (A, B). A ED35(half of maximum % GVBD) was 8.8h (arrow). B: EDs0 was 8 h in TPA and 7.5 h in P4 (progesterone) group.
cyte cAMP levels and protein synthesis are known to be essential for progesterone-induced oocyte maturation in amphibia (Maller, '83). The effect of preventing the decrease of cAMP and protein synthesis on the TPA-induced oocyte maturation was examined using forskolin, a n adenylate cyclase stimulator, and cycloheximide. TPAand progesterone-induced oocyte maturation were both markedly inhibited by both drugs (Figs. 6, 7) in a dose-dependent fashion. Thus, it appears that a decrease in cAMP and protein synthesis are both involved in TPA-induced oocyte GVBD. Experiments were carried out t o test whether
0
I
3
9
F o r s k o l i n (JIM) Fig. 6. Inhibitory effect of forskolin on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of different doses of forskolin (1-9 kM) and TPA (10 pM) or progesterone (1 kgi2 ml) and examined for GVBD after 24 h of culture. Each point represents average (mean & SEM) % GVBD of 200 follicles (5 animals). *P < 0.05, when compared with control. **P< 0.01.
H.B. KWON AND W.K. LEE
120
n m
1
loo
'
(3
50
ap 0
0-
!
-4
+--o \
\
\ \
0--0
P4(1pq/Zml)
.-*
T P A (IOpM)
n m
' a9
0 0.01
0.1
10
I
0
C ycl otlexi m i d e ( u g/2m I)
TPA acts through the calcium-mediated path-. ways for induction of oocyte maturation (Wasserman et al., '80; Morrill et al., '81; Kleis-San Francisco and Schuetz, '86). For these studies, we assessed the effects of a calcium transport blocker (verapamil) and a calmodulin antagonist (W-7)on maturation induction. Both TPA- and progesterone-induced oocyte maturation were inhibited by the two drugs in a dose-dependent fashion (Figs. 8, 9). Verapamil and W-7 in the medium markedly suppressed TPA- and progesterone-induced
1
\ \
I I , I
0
0.01
I
I
PI
0.03 0.09 0.27 0.81
Veropomil ( m M ) Fig. 8. Inhibitory effect of verapamil on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of different doses of verapamil(O.01-0.81 mM) and TPA (10 pM) or progesterone (1 pg/2 ml) and examined for GVBD after 24 h of culture. Each point represents average (mean +- SEM) % GVBD of 200 follicles (five animals). ^P < 0.05, when compared with control.
5c
(3
Fig. 7. Inhibitory effect of cycloheximide on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of various doses of cycloheximide (0.01-10 pg12 ml) and TPA (10 pM) or progesterone (1 pg/2 ml) and examined for GVBD after 24 h of culture. Each point represents average % GVBD (mean ? SEMI of 240 follicles (six animals). **P < 0.01, when compared with control.
0
I00
12.5
25
50
W-7(
VM)
100 200
Fig. 9. Effect of W-7 on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Isolated follicles were cultured in the presence of various doses of W-7 (N46aminohexyl]-5-chloro-1-naphthalenesulfonamide)(12.5-200 pM) and TPA (10 pM) or progesterone (1 pg12 ml). Oocytes were examined for GVBD after 24 h of culture. Each point represents average % GVBD (mean ? SEM) of 240 follicles (six animals). **P < 0.01, when compared with control.
oocyte GVBD at concentrations of 0.27 mM and 100 pM, respectively (Figs. 8, 9). Thus, the data strongly indicate that intracellular Ca2+ plays an important role(s) in TPA-induced oocyte maturation as in hormone-induced maturation. The effect of PKC inactivation on TPA- and progesterone-induced oocyte maturation was examined using H-7, which is known to inhibit purified PKC (Hidaka et al., '84). Inactivation of the enzyme with H-7 suppressed both TPA- and hormone-induced oocyte GVBD in a similar dosedependent manner (Fig. 10). Thus, all the factors associated with progesterone-induced oocyte maturation are also linked t o TPA-induced maturation.
DISCUSSION The present experiments demonstrate that PKC activation induced or altered the maturational response of in vitro cultured follicular oocytes of R. dybowskii at three different stages of physiological responsiveness during the hibernation period, i.e., oocytes that do or do not mature in response to progesterone and those that spontaneously matured during in vitro culture. The time course for TPA-induced oocyte maturation was similar in all three types of the oocytes tested (ED50, 7-9 h) and basically duplicated that seen in progesterone-stimulated maturation (see Fig. 4). TPA appeared t o act directly on the oocyte, since its effects neither required the presence of
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION *-•
I
1 O
i
0
T P A (IOpM)
! l
'
I
I
12.5 25 50 H-7 (NM)
I
100
Fig. 10. Effect of H-7 on TPA- or progesterone-induced oocyte maturation ofR. dybowskii in vitro. Follicles were cultured in the simultaneous presence of various doses of H-7 (l-[5-isoquinolinylsulfonyll-2-methylpiperazine)(12.5-100 pM) and TPA (10 pM) or progesterone (1 kg12 ml) and examined for GVBD after 24 h of culture. Each point represents the average (mean -+ SEM) % GVBD of 160 follicles (four animals) in the TPA-treated group and of 120 follicles (three animals) in the progesterone-treated group. **P < 0.01, when compared with control.
follicle cells (see Fig. 2) nor stimulated follicular progesterone accumulation (see Fig. 3). More recently, other types of protein kinase C activators, 1-oleoyl-2-acetyl-rac-glycerol (OAG) and 1,2-dicaryloyl-rac-glycerol (DAG), were also found to induce oocyte GVBD of R. dybowskii oocytes (unpublished data). Thus, the sum of the evidence suggests t h a t TPA acts via PKC activation t o induce maturation. However, TPA and progesterone exhibit some difference with respect to their maturation-inducing actions. The maturation rate in response to PKC activation between animals varied more and was consistently lower than that produced by progesterone (>go%) in some animals. Furthermore, follicular oocytes isolated from some animals failed to respond to PKC activation, even though they responded readily to progesterone (data not shown). We have insufficient information to explain this phenomenon. Possibly, these facts indicate that PKC activation is not the sole pathway for the induction of GVBD. There might be many other factors associated with PKC activation in determining the ability of the oocyte to mature in response to TPA and progesterone, such as levels of CAMP,cytoplasmic Ca2+, some proteolytic enzymes, and phosphorylation of certain proteins within the oocyte. Thus, PKC activation is not always sufficient for triggering oocyte maturation. Furthermore, cytological changes were also observed in oocytes treated with high doses (1or 10 pM) of TPA. These cy-
121
tological changes occurred only in the oocytes matured by TPA treatment, irrespective of the types of the oocytes used, or the species of Rana tested (R. nigromaculata and R. rugosa). Interestingly, oocytes that did not respond to the TPA treatment (i.e., had a n intact GV) did not show this phenomenon. Similar cytological changes have been described i n the matured oocytes by TPA in Xenopus laeuis (Stith and Maller, '87; Bement and Capco, '89) and in R . pipiens (Kleis-San Francisco and Schuetz, '€48)and may reflect cytotoxic actions of the compound. It is generally accepted that the primary target of progesterone produced by the follicle cells is the oocyte surface, where hormone-receptor binding activates the signal transduction pathway for oocyte GVBD in amphibian ovarian follicles (Masui and Clarke, '79; Maller, '83). The pathway seems to include several complex enzyme systems, such as adenylate cyclase (Sadler and Maller, '83) and the phosphatidyl inositol turnover system (PI system) in the oocyte (Lascal et al., '87). The PI system is known to be linked to Ca2+-dependentprotein kinase whose activation is associated with phosphorylation of some protein kinases in Spisula oocytes. It was suggested that the protein phosphorylation is associated with formation or activation of maturation-promoting factor (MPF) (Eckberg et al., '87; Eckberg, '88). Although this pathway has not been completely elucidated in amphibians, considerable evidence implicates t h a t the same signal transduction pathway also operates in such oocytes. For instance, PKC activation was found to induce the oocyte GVBD in Xenopus laeuis (Stith and Maller, '87) and in R . pipiens (Kleis-San Francisco and Schuetz, '88). Furthermore, PKC, which can be activated with TPA in vitro, was identified and partially purified in Xenopus laeuis oocytes (Laurent et al., '88). Thus, it seems evident that PKC participates in regulating meiotic resumption in amphibia. Except for the cytotoxic changes, oocyte maturation induced by PKC activation resembles in many respects that induced by progesterone. Similarly, the data show that TPA-induced oocyte maturation was inhibited by preventing the drop of CAMPand synthesis of protein, which is known t o be required for the MPF formation (Masui and Clarke, '79). Numerous reports indicate that Ca2+plays crucial role(s) in hormone-induced oocyte maturation in amphibians (Wasserman et al., '80; Morrill et al., '81; Kleis-San Francisco and Schuetz, '86). Interfering with the intracellular calcium action
H.B. KWON AND W.K.LEE
122
with verapamil, a calcium transport blocker, o r W-7, a calmodulin inhibitor also suppressed TPAinduced maturation (Figs. 8, 9). Inasmuch as W-7 also has inhibitory effects on PKC activity (Schatzman et al., '831, it may affect oocyte maturation by interfering with several mechanisms. Furthermore, inactivation of PKC with H-7, a strong PKC antagonist strongly suppressed maturation (Fig. 10).All these treatments also suppressed progesterone-stimulated oocyte GVBD. Thus, the similar effects of the various drug treatments on the response of follicular oocytes t o PKC activation and hormonal stimulation imply that both TPA and progesterone use a common pathway for the GVBD induction in the oocyte. However, we cannot completely exclude the possibility that TPA acts via an alternate pathway than that triggered by progesterone. No information is available to explain how PKC activation is linked t o MPF formation, activation, and eventual GVBD. It is of particular interest that TPA can induce GVBD in oocytes that are relatively nonresponsive t o progesterone during the early hibernation period in R. dybowskii. Furthermore, this phenomenon was not peculiar t o this species. Recently we found that follicular oocytes of R. rugosa, which do not respond t o any hormonal stimulation in vitro during the hibernation period and even in breeding season, readily matured following PKC activation. The time course of GVBD (ED50,-8 h) was the same as that typically seen in cultured follicles of other Rana species (Kwon et al., '89; Snyder and Schuetz, '73). These facts suggest that TPA has a broader range of activity than progesterone in inducing oocyte GVBD. The data also imply that the transduction pathway (cytoplasmic maturation) between progesterone stimulation and PKC activation is not fully developed in the nonhormone-responsive oocytes. Possibly, the cytoplasmic maturation process involves the adenylate cyclase or PI system, which precedes PKC activation in the oocyte may be important in regulating the breeding season in amphibia.
ACKNOWLEDGMENTS This work was supported by grants awarded t o Dr. H.B. Kwon from the Korea Science and Engineering Foundation (KOSEF) and the Ministry of Education of Korea. We wish to thank Professor Allen W. Schuetz for his critical reading of the manuscript.
LITERATURE CITED Baulieu, E.-E. (1983) Steroid-membrane-adenylate cyclase interactions during Xenopus laevis oocytes meiosis reinitiation: A new mechanism of steroid hormone action. Exp. Clin. Endocrinol., 81 :3-16. Bement, W.M., and D.G. Capco (1989) Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs. J . Cell Biol., 108:885-892. Clarke, M.R., Y. Kawai, and J.S. LeMaire (1985) Ovarian protein kinases. In: Proceedings of the Fifth Ovarian Workshop. Champaign, Illinois D.O. Toft and R.J. Ryan, eds. pp. 383. Eckberg, W.R. (1988) Intracellular signal transduction and amplification mechanisms in the regulation of oocyte maturation. Biol. Bull., 174:95-108. Eckberg, W.R., E.Z. Szuts, and A.G. Carroll (1987) Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes. Dev. Biol., 124.5774. Fortune, J.E., P.W. Concannon, and W. Hansel (1975) Ovarian progesterone levels during in vitro oocyte maturation and ovulation in Xenopus laevis. Biol. Reprod., 13.561467. Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki (1984) Isoquinalinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry, 23:5036-5041. Ishikawa, K., Y. Hanaoka, Y. Kondo, and K. Imai (1977) Primary action of steroid hormone at the surface of amphibian oocytes in the induction of germinal vesicle breakdown. Mol. Cell Endocrinol., 9:91-100. Kleis-San Francisco, S., and A.W. Schuetz (1986) Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of R a n a pzppiens. J . Exp. Zool., 240:265-273. Kleis-San Francisco, S., and A.W. Schuetz (1988) Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of R a n a pipiens: Studies with phorbol12-myristate 13-acetate. Gamete Res., 21 :323-334. Kwon, H.B., Y.K. Lim, M.J. Choi, and R.S. Ahn (1989) Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: Seasonal influences, progesterone production and involvement of CAMP.J. Exp. Zool., 252:190-199. Kwon, H.B., and A.W. Schuetz (1986) Role of CAMPin modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Ranapipiens). Dev. Biol., 11 7.354364. Lascal, J.C., P. de la Pena, J. Moscat, P. Garcia-Barreno, P.S. Anderson, and J.A. Aaronson (1987) Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p21. Science, 238533-536. Laurent, A., M. Basset, M. Doree, and C.J. Le Peuch (1988) Involvement of a calcium-phospholipid-dependent protein kinase in the maturation of Xenopus laevis oocytes. FEBS Lett., 226:324-338. Lin, Y.-W.P., and A.W. Schuetz (1985) Intrafollicular action of estrogen in regulating pituitary-induced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: Temporal relationship and locus of action. Gen. Comp. Endocrinol., 58:42 1-435. Maller, J.L. (1983) Interaction of steroids with cyclic nucleotide system in amphibian oocytes. Adv. Cyclic Nucleotide Res., 15:295-336. Maller, J.L., F.R. Butcher, and E.G. Krebs (1979) Early effect
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION of progesterone on levels of cyclic adenosine 3’,5’-monophosphate in Xenopus oocytes. J . Biol. Chem., 254.579582. Masui, Y., and H.J. Clarke (1979) Oocyte maturation. Int. Rev. Cytol., 57~185-281. Morrill, G.A., D. Ziegler, and A.B. Kostellow (1981) The role of Ca2+ and cyclic nucleotides in progesterone initiation of the meiotic divisions in amphibian oocytes. Life Sci., 29: 1821-1835. Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (Lond.), 308:693-698. Sadler, S.E., and J.L. Maller (1983) Inhibition of Xenopus oocyte adenylate cyclase by progesterone and 2’,5’-dideoxyadenosine is associated with slowing of guanine nucleotide exchange. J. Biol. Chem., 258:7935-7941. Schatzman, R.C., R.L. Raynor, and J.F. Kuo (1983) N46aminohexyl)-5-chloro-l-naphthalenesulfonamide(W-71, a
123
calmodulin antagonist, also inhibits phospholipid-sensitive calcium dependent protein kinase. Biochim. Biophys. Acta, 755: 144- 147. Schuetz, A.W. (1985) Local control mechanisms during oogenesis and folliculogenesis. In: Developmental Biology, Vol 1. L. Browder, ed. Plenum Press, New York, pp. 3-83. Stith, B.J., and J.L. Maller (1987) Induction of meiotic maturation in Xenopus oocytes by 12-0-tetradecanoylphorbol 13-acetate. Exp. Cell Res., 169.514323. Snyder, B.W., and A.W. Schuetz (1973) In vitro evidence of steroidogenesis in the amphibian ( R a m pipiens) ovarian follicle and its relationship t o meiotic maturation and ovulation. J . Exp. Zool., 183:333-342. Wasserman, W.J., L.H. Pinto, C.M. OConnor, andL.D. Smith (1980) Progesterone induces a rapid increase in [Ca2+l of Xenopus Zaevis oocytes. Proc. Natl. Acad. Sci. USA, 77: 1534-1536.
Involvement of Protein Kinase C in the Regulation of Oocyte Maturation in Amphibians (Rana dybomskii) HYUK B. KWON AND WON K. LEE Chonnam National University, College of Natural Sciences, Department of Biology, Kwangju 500-757, Republic of Korea ABSTRACT Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed t o mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 pM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50,7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide(1 pg/2 ml), forskolin (9 pM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin (W-7) (100 pM) or a PKC inacantagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide tivator l-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 pM) likewise suppressed TPA- or progesterone-induced maturation. The data suggest that PKC plays a role in the regulation of amphibian oocyte maturation and that the seasonal change in responsiveness (GVBD) of oocytes to steroid occurs after oocytes obtain responsiveness to PKC activation.
Oocytes within fully grown amphibian ovarian follicles undergo germinal vesicle breakdown (GVBD) during in vitro follicle culture following treatment with frog pituitary homogenate (FPH) or meiosis inducing steroid (MIS), progesterone (reviewed by Masui and Clarke, '79; Schuetz, '85). Progesterone is believed to act on the oolemma (Ishikawa et al., '77) and induces a decrease in the intracellular level of cyclic adenosine monophosphate (CAMP)(Maller et al., '79), which triggers a complex chain reaction that eventually induces the oocyte GVBD (Baulieu, '83; Maller, '83; Schuetz, '85). Recently, several investigators reported that protein kinase C activation induced GVBD in Spisula oocytes (Eckberg et al., '87; Eckberg, '88) and amphibians (Stith and Maller, '87; Kleis-San Francisco and Schuetz, '88). In various type of cells, protein kinase C (PKC) is known to be transiently activated by diacylglycerol, which is produced in the membrane during signalinduced turnover of inositol phospholipids (Nishizuka, '84).I t is therefore suggested that 0 1991 WILEY-LISS, INC.
oocytes mediate hormonal signals via the adenylate cyclase system and phosphatidyl inositol turnover system (PI system) for GVBD. Responsiveness of in vitro cultured follicular oocytes of R . dybowskii to progesterone stimulation varies considerably with season. In early hibernation, such oocytes fail to respond to progesterone, whereas during the middle of hibernation, they respond readily to steroid. During the breeding season, oocytes typically matured spontaneously during in vitro culture of follicles without added hormone (Kwon et al., '89). In this study we have assessed whether oocyte responsiveness to protein kinase C activation varies in relationship to the different types of maturation which can be defined (hormone responsive and nonresponsive, spontaneous maturation). The data presented here show that the time course of TPA-induced oocyte GVBD in all Received December 19, 1989; revision accepted May 3, 1990.
116
H.B. KWON AND W.K. LEE
each experiment. Full grown follicles were isolated from ovaries using watchmaker's forceps. For some experiments, oocytes were denuded of somatic components by manually removing the follicular envelope followed by a 2 h treatment in calcium free to remove adherent follicle cells (Lin and Schuetz, '85). All experimental manipulations were conducted at room temperature (1822°C) in amphibian Ringer's solution (Kwon and Schuetz, '86). Routine in vitro culture were carMATERIALS AND METHODS ried out using multiwell culture dish (24 wells,' Animals dish, Nunc) with 20 follicles cultured in 2 ml of Hibernating frogs (Rana dybowskzi) were col- AR per well. Cultures were maintained in a shaklected during late autumn (November) from ing incubator (Kukje Scientific Co., Korea) at streams in the Chonnam area of the southern part 22°C and agitated at 80 oscillations per minute of the Korean peninsula. Animals were kept in a for various periods of time. After culture, the oocold room maintained in darkness at 4°C. Ani- cytes were heat-fixed by brief boiling and cracked mals were contained (10-20) in glass or plastic open individually and checked for the germinal boxes containing 10% amphibian Ringer's solu- vesicle. tion (AR) that was changed two times a week. Follicle extraction and sample preparation Hormones and reagents for radioimmunoassay Progesterone, forskolin, and verapamil were The steroidogenic capacity of cultured follicles dissolved in vehicle composed of ethanol and pro- was monitored by measuring intrafollicular levpylene glycol (1:l)in a stock of 2 mglml, 10 mM, els of progesterone by radioimmunoassay (RIA). and 400 mM, respectively. An active PKC ac- Extraction of progesterone from follicles emtivator, phorbol 12-myristate 13-acetate (TPA) ployed procedures described by Lin and Schuetz (Nishizuka, '84) and a n inactive form of the phor- ('85) and Kwon and Schuetz ('86). After specified (4a-PDD) periods of culture, the medium was aspirated and bol ester, 4au-phorbol-12,13-didecanoate (Clarke et al., '85) were dissolved in dimethyl sul- follicles were extracted in the culture well using foxide (DMSO) in a stock of 8 mM and 12.8 mM, methanol (GR, Merck, 1ml methanol per 20 follirespectively. Calmodulin inhibitor, N-(6-amino- cles per well). Extraction was carried out for 15 hexyl)-5-chloro-l-naphthalenesulfonamide(W-7) min with shaking. Methanol extracts were evapoand PKC antagonist, l-(5-isoquinolinylsulfonyl)- rated in a freeze dryer (Labconco) and stored at 2-methyl-piperazine (H-7) were dissolved in -40°C until assayed. Before assay, methanol exDMSO in a stock of 80 mM and 40 mM, respec- tracts of follicles were reconstituted using gelatin tively. Cycloheximide was dissolved in AR in a phosphate-buffered saline (GPBS). Appropriate stock of 2 mg/ml. All hormones and reagents were amounts of this extract in GPBS was assayed for purchased from Sigma. Frog pituitary homoge- progesterone by RIA without further purification. nate (FPH) was prepared from female frogs. Progesterone radioimmunoassay Glands were homogenized in AR at 4°C using a glass homogenizer. The homogenate was cenGeneral assay procedures were adapted from trifuged (4"C, 10,000 rpm, 20 min) to remove de- those described by Fortune et al. ('751, Lin and bris, and the supernatant was frozen (-40°C) in Schuetz ('85),and used in previous studies (Kwon aliquots until needed (Lin and Schuetz, '85). Dif- et al., '89). Labeled progesterone (1,2,6,7-3H-proferent concentrations of reagents were prepared gesterone; 99 Ci/mmole) was obtained from Amby diluting the stock solution with vehicle and ersham and antiprogesterone serum (progesteradded t o culture wells such that the vehicle con- one-1la-hemisuccinate-bovine serum albumin) centration maintained at 0.25% or 0.5% (v/v). was donated from Dr. Yong-Dal Yoon (Hanyang University, Seoul). Its cross-reactivity was deIn vitro follicle and oocyte culture scribed in a previous report (Kwon et al., '89). Animals were sacrificed by decapitation, and Each sample was quantified for tritium using a ovaries were removed immediately and placed in Packard Tri-Carb 1500 liquid scintillation anaRinger's solution. Fresh ovaries were used for lyzer. Routinely, two sets of progesterone stan-
three types of the oocytes was very similar to that seen in progesterone-treated oocytes. The data suggest that some steps between hormone stimulation and PKC activation i n the oocyte cytoplasm are different among the three types of oocytes. These steps may be closely linked to acquisition of meiotic competence and regulation of the breeding season in amphibians.
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION
dards (10-2,000 pg) and sample replicates were included in each assay. Concentration of progesterone was calculated with SecuRIA program (Packard) by personal computer. The coefficients of variation between and within assays were 11.6% and 7.7% (n = 141,respectively. Statis tics In GVBD data analysis for all experiments, treatments were done in duplicate on individual frogs, and GVBD data of the average of the replications of experiment were transformed using a n arcsin-square root transformation (angular) and analyzed by Student’s t-test. The data of progesterone level were also analyzed by Student’s t-test; 50% GVBD (EDs0) values were estimated for follicles by probit analysis (Statistical Analysis System [SAS], Cary, NC).
RESULTS TPA induction of oocyte maturation of R. dybowskiifollicles isolated during dflerent periods of hibernation In preliminary experiments, we found that oocytes of R. dybowskii isolated in early hibernation period (November) did not mature in response to progesterone stimulation in vitro. Initially, we examined whether PKC activation in such oocytes induced oocyte GVBD by using a strong synthetic activator of the enzyme, TPA. The addition of TPA to the medium (0.01-10 pM) induced GVBD in a dose-dependent fashion during in vitro follicle culture, while an inactive phorbol ester, 4a-PDD (1.6 pM) or progesterone (1 pg/2 ml) were ineffective (Fig. 1).The maturational effects of TPA was also examined using follicular oocytes isolated during the winter (DecemberJanuary). Oocytes collected at these times matured readily to hormone stimulation and did not exhibit spontaneous maturation. Such oocytes underwent GVBD i n the presence of various doses of TPA (Fig. 2B). The dose response for GVBD was essentially the same as that obtained in nonsteroid responsive oocytes (Figs. l,2A). 4a-PDD was likewise ineffective in inducing maturation of these oocytes. However, the maturation rate induced by TPA (-70%) was lower than that produced by progesterone (-90%). Oocytes treated with higher doses (1or 10 pM) of TPA exhibited cytological changes in the cortex different from that seen in progesterone-treated oocytes. The high dose of TPA produced an irregular distribution of pigment on the animal pole and some of
m
> 50
117
1 I
O
00.010.1
1.6
10
I
4a-PDD (pM)
T P A (PM
P4
(pg/2ml)
Fig. 1. TPA induction of oocyte meiotic maturation of R . dybowskii oocytes that did not respond to exogenous progesterone in vitro. Isolated follicles were cultured in the presence of various doses of TPA (0.01-10 KM), 4a-PDD (1.6 FM) or progesterone (1 ~ g / ml) 2 and examined for GVBD after 24 h of culture. Each bar in the figure represents average % GVBD (mean f SEMI of 240 follicles (duplicate wellsianimal, six animals). *P < 0.05, when compared with control (4a-PDD group).
A
z 0
0
0.01 0.1
I
10
-
4D-PDD
Pq
TPA(uM)
100 ~
.--.
Intact Follicle
0-0
Denuded Oocyte
0
0.01 0.1
B
I--
z 0 I
10
4 a - m P,
T PA (rM)
Fig. 2. Effect of follicle cell removal on TPA-induced oocyte maturation of R. dybowskii. Follicles and denuded oocytes obtained from each animal were cultured in medium containing various doses of TPA, 4a-PDD, or progesterone and examined for GVBD after 24 h culture. A Data depicted were obtained using follicular oocytes that did not respond t o progesterone. B: Data depicted were obtained using oocytes that responded to progesterone. Each point or bar in the figure represents average (mean k SEMI % GVBD of 120 follicles (three animals) (A) and 200 follicles (five animals) (B). *P < 0.05, when compared with control (401-PDD group).
118
H.B. KWON AND W.K. LEE
the surface pigment appeared to diffuse into the cytoplasm. Interestingly, in sister oocytes that did not mature with TPA stimulation, no abnormal cytological changes were observed (data not shown). The effect of TPA on follicular oocytes that exhibited spontaneous maturation was also tested. Most oocytes (>go%) underwent spontaneous GVBD after 24 h culture, regardless of the presence of TPA (10 p,M) (data not shown).
1500 *-•
o--0
-0u .-
2
4-4
FPH T PA Control
1000
\ a,
C 0
/
L
0) c v) G)
TPA actions on oocytes and follicles As progesterone produced by follicle cells is known to be a meiosis-inducing hormone, TPA may induce oocyte maturation by stimulating progesterone production by the follicle cells. To test this possibility, experiments were designed t o examine the effect of follicle cell removal on the TPA induction of maturation and to assess the influence of TPA on endogenous intrafollicular levels of progesterone. Removal of the follicle cells did not prevent TPA-induced maturation, regardless of whether oocytes are responsive (Fig. 2B) or nonresponsive to hormone stimulation (Fig. 2A). Furthermore, TPA did not stimulate progesterone accumulation by the follicles (Fig. 3), while FPH (0.1 pituitary equivalent/well) markedly stimulated progesterone accumulation by the follicles (Fig. 3). The data clearly show that the action of TPA on the oocyte is a direct one and is not linked to the follicle cells, which are tightly attached to the oocytes.
0
500
L
a cn P
I’/*** I
*
0
3
, r-0
_g--------*s 6
F
A
9 T I M E(hr)
Fig. 3. Effect of TPA on the progesterone accumulation in follicles of R . dybowskii in vitro. Isolated follicles were cultured in the presence or absence of TPA (10 pM) or FPH (0.1 pituitary equivalenti2 ml) for 524 h. At designated time points, follicles were extracted with methanol for steroid RIA. Each point represents pg progesterone (mean t SEM) per follicles (n = 4, duplicate wells, two animals). **P < 0.01 when compared with control.
divided into two groups and cultured in the presence or absence of TPA for 5 2 4 h. At designated time points, the incidence of oocyte GVBD was compared with that seen in nontreated control oocytes. Experiments with the same design were performed with progesterone treatment using different animals but on the same days. Treatment of TPA remarkably accelerated the time course for the GVBD. Fifty percent of the oocytes matured after 6.8 h of culture in the TPA-treated group, while in nontreated sister follicles a similar incidence of maturation was observed after 19.4 h (Fig. 5A). Progesterone treatment likewise accelerated the time course of spontaneous maturation (GVBD); 50% GVBD occurred by 8.7 h after progesterone treatment, while oocytes in the control nonhormone-treated group did by 18.2 h of culture (Fig. 5B). Thus, the time course for TPAinduced oocyte maturation was essentially the same (ED,,, 7-9 h) for all three types of oocytes.
Time course of the TPA-induced oocyte maturation in vitro The three types of follicles were collected at various times after TPA treatment under standardized culture conditions and examined for GVBD. Follicles were cultured in the presence of 10 pM of TPA for 5 2 4 h and at designated time points oocytes were examined for GVBD. TPAinduced oocyte GVBD, in nonsteroid responsive oocytes, occurred within 12 h of culture (70%). Approximately one-half of the oocytes underwent GVBD after 9 h of culture (Fig. 4A). Progesterone (1 pg/2 ml) treatment induced no oocyte GVBD (Fig. 4A). TPA-treated oocytes, responsive to hormone, exhibited similar time course to that induced by progesterone (Fig. 4B). Fifty percent of Comparison between TPA and the oocytes had undergone maturation by 8 h in progesterone-induced oocyte maturation response t o both types of stimulation (Fig. 4B). in vitro The effects of TPA on the time course of matExperiments were carried out to acertain uration was also examined in follicles exhibiting whether TPA and progesterone induced oocyte spontaneous maturation. Follicles isolated during GVBD by common pathways. A decrease in oothe late hibernation period (early February) were
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION
7
.-.
100
loo
0--0
.-.-.-.
T PA Control
a
0,'
m
> W
119
A
50
z
," ,
I 100-
*-•
Progesterone
O--O
Control
m >50-
r SO%GVBD I
B
(3
ar .o
I
!
0
100
6
0
12
18
24
TIME(hr)
n m 50
> W
ae
0 0
6
12
18 T I M E (hr)
24
Fig. 5. Acceleration of maturation by TPA in R. dybowskii oocytes exhibiting spontaneous maturation in vitro. Follicular oocytes exhibiting spontaneous maturation without added hormone were cultured in the presence or absence of TPA (10 JLM)or progesterone (1 kg12 ml) for 5 2 4 h. Oocytes were examined for GVBD at designated time points. A Acceleration of the onset of oocyte maturation by TPA. B Acceleration of the onset of oocyte maturation by progesterone. Each point represents the average % GVBD of 200 follicles (five animals) (A, B). A: The EDs0 of the TPA-treated and the control group was 6.8 and 19.4 h, respectively. B: The EDSoof progesterone and control groups was 8.7 and 18.2 h, respectively.
Fig. 4. Time course of TPA-induced oocyte GVBD in R. dybowskii in vitro. Follicles were cultured in the presence or absence of TPA (10 kM) or progesterone (1 kg12 ml) for 524 h. At designated time points, follicles were examined for GVBD. A. Data from follicles that did not respond to progesterone. B: Hormone responsive follicles. Each point represents average % GVBD (mean '-c SEM) of 120 follicles (three animals) (A, B). A ED35(half of maximum % GVBD) was 8.8h (arrow). B: EDs0 was 8 h in TPA and 7.5 h in P4 (progesterone) group.
cyte cAMP levels and protein synthesis are known to be essential for progesterone-induced oocyte maturation in amphibia (Maller, '83). The effect of preventing the decrease of cAMP and protein synthesis on the TPA-induced oocyte maturation was examined using forskolin, a n adenylate cyclase stimulator, and cycloheximide. TPAand progesterone-induced oocyte maturation were both markedly inhibited by both drugs (Figs. 6, 7) in a dose-dependent fashion. Thus, it appears that a decrease in cAMP and protein synthesis are both involved in TPA-induced oocyte GVBD. Experiments were carried out t o test whether
0
I
3
9
F o r s k o l i n (JIM) Fig. 6. Inhibitory effect of forskolin on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of different doses of forskolin (1-9 kM) and TPA (10 pM) or progesterone (1 kgi2 ml) and examined for GVBD after 24 h of culture. Each point represents average (mean & SEM) % GVBD of 200 follicles (5 animals). *P < 0.05, when compared with control. **P< 0.01.
H.B. KWON AND W.K. LEE
120
n m
1
loo
'
(3
50
ap 0
0-
!
-4
+--o \
\
\ \
0--0
P4(1pq/Zml)
.-*
T P A (IOpM)
n m
' a9
0 0.01
0.1
10
I
0
C ycl otlexi m i d e ( u g/2m I)
TPA acts through the calcium-mediated path-. ways for induction of oocyte maturation (Wasserman et al., '80; Morrill et al., '81; Kleis-San Francisco and Schuetz, '86). For these studies, we assessed the effects of a calcium transport blocker (verapamil) and a calmodulin antagonist (W-7)on maturation induction. Both TPA- and progesterone-induced oocyte maturation were inhibited by the two drugs in a dose-dependent fashion (Figs. 8, 9). Verapamil and W-7 in the medium markedly suppressed TPA- and progesterone-induced
1
\ \
I I , I
0
0.01
I
I
PI
0.03 0.09 0.27 0.81
Veropomil ( m M ) Fig. 8. Inhibitory effect of verapamil on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of different doses of verapamil(O.01-0.81 mM) and TPA (10 pM) or progesterone (1 pg/2 ml) and examined for GVBD after 24 h of culture. Each point represents average (mean +- SEM) % GVBD of 200 follicles (five animals). ^P < 0.05, when compared with control.
5c
(3
Fig. 7. Inhibitory effect of cycloheximide on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Follicles were cultured in the presence of various doses of cycloheximide (0.01-10 pg12 ml) and TPA (10 pM) or progesterone (1 pg/2 ml) and examined for GVBD after 24 h of culture. Each point represents average % GVBD (mean ? SEMI of 240 follicles (six animals). **P < 0.01, when compared with control.
0
I00
12.5
25
50
W-7(
VM)
100 200
Fig. 9. Effect of W-7 on TPA- or progesterone-induced oocyte maturation of R. dybowskii in vitro. Isolated follicles were cultured in the presence of various doses of W-7 (N46aminohexyl]-5-chloro-1-naphthalenesulfonamide)(12.5-200 pM) and TPA (10 pM) or progesterone (1 pg12 ml). Oocytes were examined for GVBD after 24 h of culture. Each point represents average % GVBD (mean ? SEM) of 240 follicles (six animals). **P < 0.01, when compared with control.
oocyte GVBD at concentrations of 0.27 mM and 100 pM, respectively (Figs. 8, 9). Thus, the data strongly indicate that intracellular Ca2+ plays an important role(s) in TPA-induced oocyte maturation as in hormone-induced maturation. The effect of PKC inactivation on TPA- and progesterone-induced oocyte maturation was examined using H-7, which is known to inhibit purified PKC (Hidaka et al., '84). Inactivation of the enzyme with H-7 suppressed both TPA- and hormone-induced oocyte GVBD in a similar dosedependent manner (Fig. 10). Thus, all the factors associated with progesterone-induced oocyte maturation are also linked t o TPA-induced maturation.
DISCUSSION The present experiments demonstrate that PKC activation induced or altered the maturational response of in vitro cultured follicular oocytes of R. dybowskii at three different stages of physiological responsiveness during the hibernation period, i.e., oocytes that do or do not mature in response to progesterone and those that spontaneously matured during in vitro culture. The time course for TPA-induced oocyte maturation was similar in all three types of the oocytes tested (ED50, 7-9 h) and basically duplicated that seen in progesterone-stimulated maturation (see Fig. 4). TPA appeared t o act directly on the oocyte, since its effects neither required the presence of
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION *-•
I
1 O
i
0
T P A (IOpM)
! l
'
I
I
12.5 25 50 H-7 (NM)
I
100
Fig. 10. Effect of H-7 on TPA- or progesterone-induced oocyte maturation ofR. dybowskii in vitro. Follicles were cultured in the simultaneous presence of various doses of H-7 (l-[5-isoquinolinylsulfonyll-2-methylpiperazine)(12.5-100 pM) and TPA (10 pM) or progesterone (1 kg12 ml) and examined for GVBD after 24 h of culture. Each point represents the average (mean -+ SEM) % GVBD of 160 follicles (four animals) in the TPA-treated group and of 120 follicles (three animals) in the progesterone-treated group. **P < 0.01, when compared with control.
follicle cells (see Fig. 2) nor stimulated follicular progesterone accumulation (see Fig. 3). More recently, other types of protein kinase C activators, 1-oleoyl-2-acetyl-rac-glycerol (OAG) and 1,2-dicaryloyl-rac-glycerol (DAG), were also found to induce oocyte GVBD of R. dybowskii oocytes (unpublished data). Thus, the sum of the evidence suggests t h a t TPA acts via PKC activation t o induce maturation. However, TPA and progesterone exhibit some difference with respect to their maturation-inducing actions. The maturation rate in response to PKC activation between animals varied more and was consistently lower than that produced by progesterone (>go%) in some animals. Furthermore, follicular oocytes isolated from some animals failed to respond to PKC activation, even though they responded readily to progesterone (data not shown). We have insufficient information to explain this phenomenon. Possibly, these facts indicate that PKC activation is not the sole pathway for the induction of GVBD. There might be many other factors associated with PKC activation in determining the ability of the oocyte to mature in response to TPA and progesterone, such as levels of CAMP,cytoplasmic Ca2+, some proteolytic enzymes, and phosphorylation of certain proteins within the oocyte. Thus, PKC activation is not always sufficient for triggering oocyte maturation. Furthermore, cytological changes were also observed in oocytes treated with high doses (1or 10 pM) of TPA. These cy-
121
tological changes occurred only in the oocytes matured by TPA treatment, irrespective of the types of the oocytes used, or the species of Rana tested (R. nigromaculata and R. rugosa). Interestingly, oocytes that did not respond to the TPA treatment (i.e., had a n intact GV) did not show this phenomenon. Similar cytological changes have been described i n the matured oocytes by TPA in Xenopus laeuis (Stith and Maller, '87; Bement and Capco, '89) and in R . pipiens (Kleis-San Francisco and Schuetz, '€48)and may reflect cytotoxic actions of the compound. It is generally accepted that the primary target of progesterone produced by the follicle cells is the oocyte surface, where hormone-receptor binding activates the signal transduction pathway for oocyte GVBD in amphibian ovarian follicles (Masui and Clarke, '79; Maller, '83). The pathway seems to include several complex enzyme systems, such as adenylate cyclase (Sadler and Maller, '83) and the phosphatidyl inositol turnover system (PI system) in the oocyte (Lascal et al., '87). The PI system is known to be linked to Ca2+-dependentprotein kinase whose activation is associated with phosphorylation of some protein kinases in Spisula oocytes. It was suggested that the protein phosphorylation is associated with formation or activation of maturation-promoting factor (MPF) (Eckberg et al., '87; Eckberg, '88). Although this pathway has not been completely elucidated in amphibians, considerable evidence implicates t h a t the same signal transduction pathway also operates in such oocytes. For instance, PKC activation was found to induce the oocyte GVBD in Xenopus laeuis (Stith and Maller, '87) and in R . pipiens (Kleis-San Francisco and Schuetz, '88). Furthermore, PKC, which can be activated with TPA in vitro, was identified and partially purified in Xenopus laeuis oocytes (Laurent et al., '88). Thus, it seems evident that PKC participates in regulating meiotic resumption in amphibia. Except for the cytotoxic changes, oocyte maturation induced by PKC activation resembles in many respects that induced by progesterone. Similarly, the data show that TPA-induced oocyte maturation was inhibited by preventing the drop of CAMPand synthesis of protein, which is known t o be required for the MPF formation (Masui and Clarke, '79). Numerous reports indicate that Ca2+plays crucial role(s) in hormone-induced oocyte maturation in amphibians (Wasserman et al., '80; Morrill et al., '81; Kleis-San Francisco and Schuetz, '86). Interfering with the intracellular calcium action
H.B. KWON AND W.K.LEE
122
with verapamil, a calcium transport blocker, o r W-7, a calmodulin inhibitor also suppressed TPAinduced maturation (Figs. 8, 9). Inasmuch as W-7 also has inhibitory effects on PKC activity (Schatzman et al., '831, it may affect oocyte maturation by interfering with several mechanisms. Furthermore, inactivation of PKC with H-7, a strong PKC antagonist strongly suppressed maturation (Fig. 10).All these treatments also suppressed progesterone-stimulated oocyte GVBD. Thus, the similar effects of the various drug treatments on the response of follicular oocytes t o PKC activation and hormonal stimulation imply that both TPA and progesterone use a common pathway for the GVBD induction in the oocyte. However, we cannot completely exclude the possibility that TPA acts via an alternate pathway than that triggered by progesterone. No information is available to explain how PKC activation is linked t o MPF formation, activation, and eventual GVBD. It is of particular interest that TPA can induce GVBD in oocytes that are relatively nonresponsive t o progesterone during the early hibernation period in R. dybowskii. Furthermore, this phenomenon was not peculiar t o this species. Recently we found that follicular oocytes of R. rugosa, which do not respond t o any hormonal stimulation in vitro during the hibernation period and even in breeding season, readily matured following PKC activation. The time course of GVBD (ED50,-8 h) was the same as that typically seen in cultured follicles of other Rana species (Kwon et al., '89; Snyder and Schuetz, '73). These facts suggest that TPA has a broader range of activity than progesterone in inducing oocyte GVBD. The data also imply that the transduction pathway (cytoplasmic maturation) between progesterone stimulation and PKC activation is not fully developed in the nonhormone-responsive oocytes. Possibly, the cytoplasmic maturation process involves the adenylate cyclase or PI system, which precedes PKC activation in the oocyte may be important in regulating the breeding season in amphibia.
ACKNOWLEDGMENTS This work was supported by grants awarded t o Dr. H.B. Kwon from the Korea Science and Engineering Foundation (KOSEF) and the Ministry of Education of Korea. We wish to thank Professor Allen W. Schuetz for his critical reading of the manuscript.
LITERATURE CITED Baulieu, E.-E. (1983) Steroid-membrane-adenylate cyclase interactions during Xenopus laevis oocytes meiosis reinitiation: A new mechanism of steroid hormone action. Exp. Clin. Endocrinol., 81 :3-16. Bement, W.M., and D.G. Capco (1989) Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs. J . Cell Biol., 108:885-892. Clarke, M.R., Y. Kawai, and J.S. LeMaire (1985) Ovarian protein kinases. In: Proceedings of the Fifth Ovarian Workshop. Champaign, Illinois D.O. Toft and R.J. Ryan, eds. pp. 383. Eckberg, W.R. (1988) Intracellular signal transduction and amplification mechanisms in the regulation of oocyte maturation. Biol. Bull., 174:95-108. Eckberg, W.R., E.Z. Szuts, and A.G. Carroll (1987) Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes. Dev. Biol., 124.5774. Fortune, J.E., P.W. Concannon, and W. Hansel (1975) Ovarian progesterone levels during in vitro oocyte maturation and ovulation in Xenopus laevis. Biol. Reprod., 13.561467. Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki (1984) Isoquinalinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry, 23:5036-5041. Ishikawa, K., Y. Hanaoka, Y. Kondo, and K. Imai (1977) Primary action of steroid hormone at the surface of amphibian oocytes in the induction of germinal vesicle breakdown. Mol. Cell Endocrinol., 9:91-100. Kleis-San Francisco, S., and A.W. Schuetz (1986) Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of R a n a pzppiens. J . Exp. Zool., 240:265-273. Kleis-San Francisco, S., and A.W. Schuetz (1988) Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of R a n a pipiens: Studies with phorbol12-myristate 13-acetate. Gamete Res., 21 :323-334. Kwon, H.B., Y.K. Lim, M.J. Choi, and R.S. Ahn (1989) Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: Seasonal influences, progesterone production and involvement of CAMP.J. Exp. Zool., 252:190-199. Kwon, H.B., and A.W. Schuetz (1986) Role of CAMPin modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Ranapipiens). Dev. Biol., 11 7.354364. Lascal, J.C., P. de la Pena, J. Moscat, P. Garcia-Barreno, P.S. Anderson, and J.A. Aaronson (1987) Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p21. Science, 238533-536. Laurent, A., M. Basset, M. Doree, and C.J. Le Peuch (1988) Involvement of a calcium-phospholipid-dependent protein kinase in the maturation of Xenopus laevis oocytes. FEBS Lett., 226:324-338. Lin, Y.-W.P., and A.W. Schuetz (1985) Intrafollicular action of estrogen in regulating pituitary-induced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: Temporal relationship and locus of action. Gen. Comp. Endocrinol., 58:42 1-435. Maller, J.L. (1983) Interaction of steroids with cyclic nucleotide system in amphibian oocytes. Adv. Cyclic Nucleotide Res., 15:295-336. Maller, J.L., F.R. Butcher, and E.G. Krebs (1979) Early effect
INDUCTION OF OOCYTE MATURATION BY PKC ACTIVATION of progesterone on levels of cyclic adenosine 3’,5’-monophosphate in Xenopus oocytes. J . Biol. Chem., 254.579582. Masui, Y., and H.J. Clarke (1979) Oocyte maturation. Int. Rev. Cytol., 57~185-281. Morrill, G.A., D. Ziegler, and A.B. Kostellow (1981) The role of Ca2+ and cyclic nucleotides in progesterone initiation of the meiotic divisions in amphibian oocytes. Life Sci., 29: 1821-1835. Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (Lond.), 308:693-698. Sadler, S.E., and J.L. Maller (1983) Inhibition of Xenopus oocyte adenylate cyclase by progesterone and 2’,5’-dideoxyadenosine is associated with slowing of guanine nucleotide exchange. J. Biol. Chem., 258:7935-7941. Schatzman, R.C., R.L. Raynor, and J.F. Kuo (1983) N46aminohexyl)-5-chloro-l-naphthalenesulfonamide(W-71, a
123
calmodulin antagonist, also inhibits phospholipid-sensitive calcium dependent protein kinase. Biochim. Biophys. Acta, 755: 144- 147. Schuetz, A.W. (1985) Local control mechanisms during oogenesis and folliculogenesis. In: Developmental Biology, Vol 1. L. Browder, ed. Plenum Press, New York, pp. 3-83. Stith, B.J., and J.L. Maller (1987) Induction of meiotic maturation in Xenopus oocytes by 12-0-tetradecanoylphorbol 13-acetate. Exp. Cell Res., 169.514323. Snyder, B.W., and A.W. Schuetz (1973) In vitro evidence of steroidogenesis in the amphibian ( R a m pipiens) ovarian follicle and its relationship t o meiotic maturation and ovulation. J . Exp. Zool., 183:333-342. Wasserman, W.J., L.H. Pinto, C.M. OConnor, andL.D. Smith (1980) Progesterone induces a rapid increase in [Ca2+l of Xenopus Zaevis oocytes. Proc. Natl. Acad. Sci. USA, 77: 1534-1536.
