The effects of forskolin, cAMP, and cyanoketone on steroid-induced meiotic maturation of yellow perch (Perca flavescens) oocytes in vitro

GENERALANDCOMPARATIVEENDOCRINOLOGY

66,

233-243(1987)

The Effects of Forskolin, CAMP, and Cyanoketone on Steroid-Induced Meiotic Maturation of Yellow Perch (Perca flavescens) Oocytes in Vitro’ DEBORAH

A. DEMANNO*

University of Notre Dame, Department

AND FREDERICK of

Biological

W. GOETZ~

Sciences, Notre Dame, Indiana 46556

Accepted December 10, 1986 Intact yellow perch (Perca flavescens) follicles stimulated by 17u,20P-dihydroxy-4pregnen-3-one (17a,20@PG) to undergo germinal vesicle breakdown (GVBD) in vitro were incubated with several agents which have been shown to increase cellular CAMP levels. Two phosphodiestrase inhibitors, SQ20,006 and isobutyl-methyl-xanthine, blocked GVBD at 1.0 mM. At lower levels (0.5, 0.1 r&) there was a dose-response effect and SQ20,006 was more inhibitory. Forskolin at 1.0-20.0 (LM blocked steroid-induced GVBD, but levels of 0.1 p,M or less were noninhibitory. In time-course experiments, significant inhibition of GVBD was observed when SQ20,006 (1 .O mM) was added within 6 hr after steroid stimulation or forskolin (10.0 CLM) was added within 12 hr. When SQ20,006 was administered in 6-hr pulses and then removed, inhibition was observed only when the steroid was given as a I-hr prepulse which was removed at the start of the incubation period. In this case, GVBD was blocked if the SQ20,006 pulse was given before 18 hr. At 10.0 mM, CAMP completely inhibited GVBD but was noninhibitory at lower levels. However, lower levels of CAMP (1.0, 0.5 mM) and forskolin (0.1 pm were inhibitory if the follicles were also incubated with 1.0 kg/ml of cyanoketone, an inhibitor of steroidogenesis. These results indicate that in vitro, increases in CAMP are inhibitory to steroid-induced meiotic maturation but may stimulate steroidogenesis in the follicle wall as well. Furthermore, in vitro steroid-stimulated maturation can be inhibited by increased CAMP for a relatively long time, following steroid treatment. 0 1987 Academic Press. IIIC

Vitellogenic teleost oocytes are arrested at the first meiotic prophase (Wallace and Selman, 1981). The resumption of meiosis, or final maturation, is controlled in most teleosts by pituitary gonadotropin which stimulates the production of maturation-inducing steroid(s) by the follicle wall (Goetz, 1983). In many teleost species, int This material is based on work supported by the National Science Foundation under Grant DCB-8214055 (to F.G.) and a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society (to D. D.). A preliminary account of this work was presented at the annual meeting of the American Society of Zoologists, Nashville, TN, December 27-30, 1986. z Current address: The Laboratories for Reproductive Biology, Box 4, MacNider Building 202-H, The University of North Carolina School of Medicine, Chapel Hill, NC 27514. 3 To whom reprint requests should be addressed.

eluding yellow perch (Perca J’uvescens), the most effective maturation-inducing steroid (MIS) is 17a,20P-dihydroxy-4pregnen-3-one (17cx,20l%PG) (Goetz and Theofan, 1979; Goetz, 1983; Goetz and Cetta, 1985; Pankhurst, 1985; Greeley et al., 1986). In addition, Nagahama et al. (1985) have identified 17a,20@PG as the major naturally occurring MIS in amago salmon (Oncorhynchus rhodurus), masu salmon (0. masou), chum salmon (0. ketu), and rainbow trout (Salmo guirdneri). Previous in vitro studies in fish have shown that the mechanism of action involved in the steroid stimulation of GVBD has special characteristics not typical of the classic steroid mechanism of action. Steroid-induced GVBD is blocked by inhibitors of translation but not transcription (Detlaff and Skoblina, 1969; Goswami and 233 0016~6480/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AU rights of reproduction in any form reserved.

234

DE MANN0

AND GOETZ

Sundararaj, 1973; Jalabert, 1976; Theofan Xenopus oocytes but it did not stimulate and Goetz, 1981; DeManno and Goetr. GVBD in intact follicles when either in1986). Drugs which presumably increase jected or incorporated into the incubation the level of intracellular cyclic adenosine medium (Sadler and Maller, 1983; Schoro3’S’-monophosphate (CAMP) such as 3- deret-Slatkine and Schoroderet, 1984). isobutyl-I-methylxanthine (IBMX), the- There is also evidence which supports the ophylline, forskolin, and cholera toxin in- involvement of calcium and membrane hibit steroid-stimulated GVBD in \litro in phospholipids in the action of the steroid several salmonid species (Goetz and Hen- (Baulieu ef al., 1978: Morrill et tri., 1981; nessey, 1984; Jalabert and Finet, 1986; De- Schorderet-Slatkine et ui., 1982). Manno and Goetz, 1986). These results inAlthough reported research on rainbow dicate that the steroid mechanism of action trout and book trout (Salvelinus fontinulis) in final maturation is nongenomic and may suggests that steroid-stimulated final matuinvolve CAMP. ration in fish may also involve a nongeSimilar results have been reported for nomic, CAMP-related mechanism of action GVBD in Xenopus laevis and Rana pipiens (Jalabert and Finet, 1986; Goetz and Hennessey, 1984; DeManno and Goetz, 1986), oocytes for which progesterone is considered to be the MIS (for review, Masui and no studies have been performed on fish Clarke, 1979; Maller, 1985). In addition, it species other than salmonids. Regardless is known from amphibian studies that the of the exact mechanism of action, this apsteroid apparently acts via a receptor on pears to be a novel and complex steroid the oocyte plasma membrane and not mechanism of action. Rambo and Szego through cytoplasmic or nuclear receptors (1983) have observed a rapid estrogen ac(Baulieu et al., 1985; Sadler and Maller, tion at the membrane surface of rat endo1982). These results together with more ex- metrial cells, the only other reported extensive research on GVBD in amphibian ample of a steroid-membrane interaction. follicles have led to the proposal of the fol- Given the vast number of fish species and lowing hypothesis: MIS acts at the oocyte the fact that reproduction is evolutionarily plasma membrane to inhibit adenyfyl cy- strongly conserved, it is important to inclase (AC) activity which results in a de- vestigate this phenomenon in other fish crease in the level of intraoocyte CAMP species, especially the higher-order te(Baulieu, 1983; Maller, 1985). The drop in leosts. CAMP leads to an inhibition of cAMPdeThe objectives of this study were to dependent protein kinase catalytic activity termine whether inhibitors of phosphodiesand subsequent dephosphorylation of terase and a stimulator of AC could block target proteins (Maller and Krebs, 1977; 17o,20P-PG-stimulated GVBD in vitro in Maller, 1985; Boyer et al., 1986). While yellow perch, a higher-order teleost. We there have been several reports in am- were further interested in the timing and phibians of an MIS-triggered decrease in characteristics of such inhibition and what role, if any, the follicle wall may play in CAMP, there has been some controversy regarding the magnitude and duration of these types of in vitro studies. the CAMP decrease necessary to trigger MATERIALS AND METHODS GVBD (Cicirelli and Smith, 1985). In addiExperimental anirna/.s. Sexually mature female tion, the decrease in AC activity alone may yellow perch (XI-75 g) were collected in the fieldfrom not be sufficient to stimulate maturation. late January until late April and were held in the laboFor example, the P-site agonist 2’,5’-diratory under natural photoperiods in tanks with nmdeoxyadenosine has been shown to be a ning water at 4”. Since follicles from perch become potent inhibitor of membrane-bound AC in more sensitive to in ~irvo steroid treatment as the re-

CYCLIC

AMP AND STEROID-INDUCED

productive season progresses and the germinal vesicle (GV) migrates from the center of the oocyte to the periphery, experiments were standardized by using follicles that were in the same stage. The position of the GV in the oocytes was used as an indicator of maturational stage. Germinal vesicle position was determined by sampling in vivo with a glass catheter according to Goetz and Theofan (1979). Sampled follicles were fixed and cleared in a solution described previously (Goetz and Bergman, 1978) and the position of the GV was determined with a dissecting microscope. Follicles sampled in this manner were representative of the rest of the ovary. Individual fish were chosen for experiments when the oocytes demonstrated nearly complete lipid droplet coalescence and the GV moved to the edge of the lipid droplet cluster. During late winter (January-February), perch were primed intraperitoneally with 50 IU of human chorionic gonadotropin (hCG)/lOO g body wt (Goetz and Theofan, 1979) to obtain follicles at the described stage. Follicles from hCG-injected fish reached this stage l-3 days after priming but did not undergo spontaneous GVBD in in vitro incubations. Later in the season (March-April), priming was unnecessary since by this time the follicles had already progressed to the stage described above. In vitro incubations. Fish were decapitated and the ovaries were removed and placed in ice-cooled Cortland salt solution (Wolf and Quimby, 1969), buffered to pH 7.8 with 0.02 M Tris. This solution also served as the incubation medium. The ovaries were dissected into small groups containing 5-7 follicles. Incubations were performed as described previously (Goetz and Bergman, 1978; Goetz and Theofan, 1979) with the following modifications. Approximately 30 follicles were placed in 25-ml Erlenmeyer flasks containing 3 ml Cortland medium. Follicles were stimulated to undergo GVBD in vifro with 17a,20@PG. This steroid has been determined to be the most potent inducer of meiotic maturation in yellow perch follicles in vitro and stimulatory levels were chosen on the basis of previous dose-response studies (Goetz and Theofan, 1979). The steroid was dissolved initially at high concentrations in 95% ethanol and aliquoted to the individual incubation flasks to obtain the desired final concentrations. The ethanol concentration in the flasks was held constant at 1 PI/ml medium. Treated flasks were incubated at 12” for 48 hr with automatic intermittent shaking. At the end of the incubation period, follicles were assayed for GVBD using the tixative described above. Forskolin and cyanoketone (CK) were also dissolved in 95% ethanol and aliquoted to the individual incubation flasks as described for the steroid above. When these compounds were used, the ethanol concentration in the flasks was 3 PI/ml medium or less, depending on the combinations of steroid, forskolin,

235

GVBD

and CK. The phosphodiesterase inhibitors 3-isobutylI-methyl-xanthine (IBMX) and SQ20,006 (I-ethyl-4hydrazino-lH-pyrazolo[3,4-b]pyridine-5-carboxylic acid, ethyl ester, hydrochloride), CAMP, and the translational inhibitor cycloheximide were dissolved directly in the incubation medium. Effects GVBD

of phosphodiesterase in steroid-stimulated

inhibitors follicles.

or foskolin

Follicles were incubated with stimulatory levels of 17u,20P-PG and different levels of one of the following drugs: SQ20,006, IBMX, or forskolin. Control flasks, containing steroid or ethanol alone, were also run. All flasks were assayed for GVBD at 48 hr. Time course of drug inhibition. Two different types of time-course experiments were performed. In the first protocol, follicles were stimulated with 5.0 rig/ml 17a, 20P-PG at the start of incubation (0 hr). Beginning at the start of incubation and at 6-hr intervals thereafter for 36 hr, separate flasks received 1.0 mM SQ20,006, 1.0 PM cycloheximide, or 10.0 WM forskolin. Incubation was continued and all flasks were assayed for GVBD at 48 hr. Separate flasks of steroidstimulated follicles, without drugs, were assayed at each 6-hr interval to follow the progress of steroid-induced GV migration and GVBD in the absence of drug treatment. In the second protocol, follicles were divided into two groups: one group was incubated with 5.0 rig/ml 17u,20@PG continuously (O-48 hr); the second group was preincubated for t hr in 10.0 rig/ml 17cy,2O@PGbefore the start of incubation. At the end of this preincubation, the steroid was removed and the follicles were washed with and placed in fresh medium. The higher concentration of steroid (10.0 ngiml) was used for the I-hr treatment because results of preliminary experiments indicated that 5.0 rig/ml of 17cx,20@-PGfor I hr was not sufficient to stimulate significant GVBD. Both groups of steroid-stimulated follicles (continuous or I-hr prepulsed) were subjected to the following protocol. At the start of incubation (0 hr), separate flasks were exposed to 6-hr pulses of 1.0 mM SQ20,006. The drug was in the separate flasks during O-6, 6-12, 12-18, 18-24, 24-30, or 30-36 hr. At the end of the pulse period the follicles were removed and then washed with and placed in fresh medium with or without steroid, depending on the steroid-exposure group (continuous or I-hr prepulsed). The incubation was then continued and all follicles were assayed for GVBD at 48 hr. on

Effects of low forskolin roid-stimulated GVBD.

and

cyanoketone

on ste-

All follicles were stimulated with 5.0, 2.5, or 1.3 &ml of 17a,20@-PG. One group of these steroid-stimulated follicles was also incubated with 0.1 PM forskolin alone while a second group was treated with forskolin and 1.0 pg/ml cyanoketone (CK). Follicles treated with CK in this experiment were preincubated in 1.O kg CK/ml for 1 hr before the

236

DE MANNO

start of the incubation. All flasks were assayed for GVBD at 48 hr. Controls of steroid alone or with C‘K were also run. Effects GVBD.

of

CAMP

and

CK

on

steroid-stirnrtiur~‘l

Follicles were treated with stimulatory levels of 17a,20B-PG. Separate flasks then received 10.0. I .O, or 0.5 mM CAMP with and without 1.O *g/ml of CK. As in the previous experiment, those folhcles treated with CK received a I-hr preincubation of 1.0 kg/ml CK. Control flasks of steroid alone or with CK were also incubated. All flasks were assayed at 4X hr for GVBD. GVBD daru unalysis. For all experiments, treatments were done in duplicate on individual fish and experiments were repeated on N number of fish. The GVBD data (GVBD per replicate/total follicles per replicate) for each fish were transformed using an arcsin-square root transformation and the replicate means were statistically analyzed by one- or two-way analysis of variance (ANOVA) and Duncan’s multiple range test or a two-tailed t test where appropriate (Statistical Analysis System @AS). Cary, NC). For the t tests, homogeneity of variances was determined by the Max test (Sokal and Rohlf, 1981). Mureriuh. Cyclic AMP, HCG, 17o,20@-PG, IBMX, cycloheximide, and Tris buffer were obtained from Sigma (St. Louis, MO). Forskolin was purchased from Calbiochem-Behring (San Diego, CA). Cyanoketone (2ol,cyano-4,4,17a-trimethylandrost-5-en-~7~-01-3one) was a gift from the Sterling-Winthrop Research Institute (Rensselaer, NY) and SQ20,006 was a gift from the Squibb Institute for Medical Research (Princeton, NJ).

RESULTS

Effects of Phosphodiesteruse Inhibitors or Forskolin on Steroid-Stimulated Follicles Both IBMX and SQ20,006 significantly inhibited 17a,20p-PG-induced GVBD in yellow perch follicles (Table 1). At 1.0 m44 both inhibitors completely blocked GVBD at all steroid levels tested, but at levels below 1.O rr&!, SQ20,006 was a more effective inhibitor than was IBMX. In addition, a dose-response effect was observed with lower levels of inhibitor and the three different steroid concentrations. The results of a two-way factorial ANOVA (Table 2) demonstrate significant (P < 0.0001) effects due to inhibitor and steroid as well as a steroid-inhibitor interaction. Controls

AND GOETZ TABLE I EFrn?c’rs OP PHOSPHODIESTERASE~NHIBITOR TREATMENTON I~~,ZO~-PG-S~~IMIJLATED PERCH FOLLICLES ~~~~ .~ -._17a,20(j-PCi(ng/ml) ~___~_.._ ~--_~-~.-- ._._.-.~----__-_ Drug 2.2 I .25 0.63 (mM) SQ20.006 1.0 0.5 0.1 IBMX 1.0 0.5 0.1 0.0

0 38 -c 19 98i 2 0 9o_t 982 100

9 2

Note. Values are x% GVBD

0 0 78 ‘- 22 0 8hi 992 100

7 I

0 0 0 0 --7+ 2 83 F 12 932 7

t SEM for three fish.

treated with inhibitor alone or without steroid did not mature. Forskolin (1.0-20.0 PM) also effectively blocked steroid-stimulated GVBD (Table 3). The effect of 0.1 ~.LMforskolin was not significantly different from that of the steroid controls. There was no apparent dose-response effect with forskolin and no significant interaction effect with a twoway ANOVA (results not shown). As above, controls with forskolin alone or without steroid never matured. Time Course of Drug Inhibition Figure 1 shows the response of follicles stimulated with 5.0 rig/ml 17a,20P-PG to 1.O mM SQ20,006 or 1 .O l.& cycloheximide added over time. There were no significant differences between the effects of the two drugs when they were added up to 6 hr TABLE 2 RE~ULTSOFTWO-WAYFA~TORIAL ANOVA~NTHE DATAINTABLE 1 Source Drug Steroid Drug * steroid * P c

0.0001.

DF

6 2 12

MS

4.89 1.89 0.43

--

F

97.47* 37.65* i3.50*

CYCLIC

AMP AND STEROID-INDUCED

237

GVBD

TABLE 3 EFFECTS OF FORSKOLIN TREATMENT ON 17a,2OB-PG-STIMULATED YELLOW PERCH FOLLICLES 17a,2OB-PG (@ml)

Forskolin (WW

25.0

10.0

20.0

0

0

0

0

0

10.0 1.0 0.1

0 0

0 0

0 0

0 0

99

0.0

100

0 0 100 100

+ 1

98 2 2

100

5.0

2.5

99

1.3

i 1

100

97 -c 2

100

Note. Each figure represents the x% GVBD ? SEM for three fish. Mean responses to forskolin at 1.O pM or greater were significantly (P < 0.0001) different from mean responses for 0.1 pJ4 forskolin or controls.

after the steroid and both were totally inhibitory. When SQ20,006 was added at 12 hr after the steroid, it no longer blocked GVBD. By contrast, cycloheximide was effective when added up to 18 hr after the steroid. The effects of the two drugs were significantly different from each other at 12 hr (P < 0.0001) and at 18 hr (P < 0.05). By 24 hr poststeroid stimulation, neither drug inhibited GVBD and there was no signiticant difference between the two drugs. During final maturation in yellow perch oocytes, the GV first migrates to the edge of the centrally coalescing lipid droplet, and loo

.A”

f. f

Y

then migrates away from the droplet as it continues toward the periphery. SQ20,006 was completely inhibitory if added before the GV had moved away from the lipid droplet, while cycloheximide completely inhibited GVBD if added before the GV had reached the oocyte membrane. GVBD in steroid controls had begun by 30 hr and was complete at 36 hr. By contrast, 10.0 ~.LMforskolin was completely inhibitory when added up to 12 hr following the addition of 5.0 ng 17a,20PPG/ml (Fig. 2). By 18 hr the inhibition due to forskolin was decreased and significantly (Z’ < 0.0001) different from the response for the other time periods. In regard to germinal vesicle migration, forskolin was ef-

+!!!!A,, (96.3%) (100%) La

0

6

TIME

12

DRUG

I8

24

ADDED

30

36

(HRS)

FIG. 1. The effects of time of drug addition on GVBD in yellow perch follicles stimulated with 17a,20B-PG (5.0 rig/ml). Open circles = 1.O mM SQ20,006; open squares = 1.0 ~.LMcycloheximide. Each point represents x% GVBD -C SEM (N = 3). Bar represents time of GVBD in incubates with 5.0 rig/ml steroid alone. x% (N = 3) in steroid controls at 30 and 36 hr is indicated in parentheses. Means significantly different (*P < 0.05 or **P < 0.0001) from other means for a given time period.

2ot

/

l32%)(95%) (100%) --i&F--

FIG. 2. The effects of time of forskolin (10.0 PM) addition on GVBD in perch follicles stimulated with 5.0 &ml of 17a,20B-PG. Each point represents x% GVBD 2 SEM (N = 3). GVBD in steroid controls was assayed at 24, 30, and 36 hr represented as in Fig. 3. *Mean significantly different (P < 0.0001) from means without asterisk at 0, 6 and 12 hrs.

238

DE

MANNO

fective if added before the GV had reached the membrane. In the steroid controls, GVBD had begun by 24 hr (32% GVBD) and was complete by 36 hr. The earlier response to steroid at 24 hr, compared with the above time-course experiment, was due to the response of one of the three fish studied. Six-hour pulses of SQ20,006 in the presence of continuous 5.0 rig/ml steroid treatment did not inhibit GVBD at any time (Fig. 3). However, when the follicles were stimulated with a 1-hr prepulse of steroid (10.0 rig/ml) which was then removed, a 6-hr exposure to SQ20,006 was sufficient to significantly inhibit GVBD if the pulse was given before 18 hr. A gradual decrease in inhibition was observed as the interval between steroid pulse and SQ20,006 pulse increased. In controls of steroid alone, there was 100% GVBD in the 5.0 rig/ml continuous treatment and 86% GVBD in the 10.0 rig/ml 1-hr prepulse.

GOETZ

Effects of’ Low Forskolin and c’k on Steroid-Stimulated GVBD As shown in Fig. 4, significant inhibition of GVBD due to 0.1 PM forskolin alone occurred only when the follicles were stimulated with 1.3 ngiml of steroid. However, the combination of 1.O ygiml of CK and forskolin resulted in significant (P < 0.01) decreases in GVBD at all three steroid concentrations tested. In controls of steroid and 1.O pgiml CK, there was 94% GVBD. Effects oj’cAMP and CK on Steroid-Stimulated GVBD This experiment was replicated on three different fish which had oocytes that were in slightly different initial stages. As a result, the follicles from different fish did not demonstrate the same response to each level of steroid used and cAMP/CK treatments were effective at different steroid concentrations depending on the fish tested. Representative results for follicles from one fish stimulated with 0.63 rig/ml 17d,20P-PG are presented in Fig. 5. At 10.0 mM, CAMP always inhibited GVBD. At lower levels (0.5. 1.0 m&4),

** .* loo? 80

ii

AND

60

:-

40

E

20

2

0

0 6

12 18 24 30 36

SQ 20,006 PULSES (HR.%

FIG. 3 The effects of 6-hr pulses of 1.0 mM SQ20,006 on steroid-induced GVBD. Pulse of drug given for time periods indicated on x axis. Open bars: follicles stimulated with 5.0 rig/ml 17a,208-PG continuously, beginning at the start of incubation (0 hr). Hatch bars: follicles preincubated in 10.0 rig/ml 17a,20p-PC 1 hr before the start of incubation after which the steroid was removed. Values are x% GVBD with SEM indicated by bars (N = 4). Steroid controls: 5.0 rig/ml continuous = 100% GVBD; 10.0 ng/rnJ for 1 hr = 86% GVBD. Significant differences between means for a given time period: **P < O.ooO1; *P < 0.01.

, I

1701,ZOp

25 -PG

50 (q/ml)

Frc. 4. The effects of low farskohn (0.1 n&f) combined with 1.0 pg/ml CK on GVBD induced by three levels of 17a,208-PG. Open bars = steroid alone; hatched bars = steroid and forskolin; solid bars = steroid, forskolin, and CK. Responses are 2% GVBD rfr SEM for (AI) fish. Different numbers of asterisks indicate significantly different means for a given steroid level. P < 0.0001 for 1.3 and 2.5 rig/ml steroid; P < 0.01 for 5.0 rig/ml steroid.

CYCLIC AMP AND STEROID-INDUCED

loo-

lxr

o-

0 CLJOKETONE

0 (:;/mll

FIG. 5. The effects of CAMP with and without 1.0 *g/ml CK on GVBD stimulated by 0.63 rig/ml 17a,20B-PG. Points are x% GVBD r SEM (N = 2) for one fish. Closed circles = no CAMP; open circles = 10.0 mM CAMP; triangles = 1.0 mM CAMP; squares = 0.5 mM CAMP. Mean responses to 0 or 1.0 (*g/ml CK for each level of CAMP were analyzed by two-tailed t tests: *P < 0.05, **P < 0.01.

CAMP by itself generally did not significantly inhibit GVBD. However, significant (P < 0.01) inhibition was observed when follicles were treated with 1.0 or 0.5 mM CAMP together with 1.0 kg/ml CK. In several cases, 1.0 *g/ml CK significantly (P < 0.01) inhibited steroid-induced GVBD by itself, but the level of inhibition was always much less than when CK was combined with CAMP. DISCUSSION

Both SQ20,006 and IBMX at 1.0 mM completely blocked steroid-induced GVBD. At drug concentrations below 1.0 mM there was a dose-response effect with 17o,20@PG stimulation as demonstrated by the significant drug-steroid interaction (Table 2). This indicates that the action of the steroid on the oocyte was countered by the presumed effect of SQ20,006 and IBMX, that of increasing intracellular CAMP levels. Similar inhibition has been observed for 17a,20@PG stimulated GVBD in rainbow and brook trout (Jalabert and Finet, 1986; Goetz and Hennessey, 1984; DeManno and Goetz, 1986) as well as

GVBD

239

in progesterone-stimulated GVBD in Xenopus and R. pipiens (O’Connor and Smith, 1976; Morrill et al., 1977; Bravo et al., 1978; Huchon and Ozon, 1979). In the present study, SQ20,006 was more inhibitory at lower levels than was IBMX and thus was chosen for use in the remaining experiments. The reverse was observed in in vitro brook trout experiments where IBMX was more inhibitory than was SQ20,006 (DeManno and Goetz, 1986). SQ20,006 is a synthetic, nonxanthine-derived phosphodiesterase inhibitor (Morrill et al., 1977) which may account for the differences in potency between the two drugs. There is also the possibility of phosphodiesterase isozymes (Wells and Kramer, 1981) in the follicle which may be differentially inhibited by SQ20,006 and IBMX. Results of experiments using phosphodiesterase inhibitors to raise CAMP levels in intact cell systems should be interpreted carefully due to possible side effects resulting from their use. The alkylated xanthines, including IBMX, have reportedly inhibited protein synthesis (Bravo et al., 1978)) decreased intracellular pH (Wasserman and Houle, 1984), altered calcium levels, and inhibited adenosine-receptor interactions (Wells and Kramer, 1981). Although side effects have not been reported for SQ20,006, the same precautions should hold. Forskolin, a potent stimulator of adenylyl cyclase (Seamon et al., 1981), also blocked steroid-stimulated GVBD in perch follicles. Levels of forskolin at 1.0 ~.LM or greater completely inhibited GVBD stimulated by a range of 1.3 to 25.0 rig/ml of 17au,20@PG. There was no difference between the response to steroid and forskolin at 0.1 pA4 and to steroid controls. No dose-response effect was found between forskolin and steroid but this could be related to the dilution series of forskolin concentrations used. forskolin also inhibited GVBD in brook trout and rainbow trout (DeManno and Goetz, 1986; Jalabert and

240

DE

MANN0

Finet, 1986) and progesterone-induced maturation in Xenopus and R. pipiens oocytes (Schorderet-Slatkine and Baulieu, 1982; Kwon and Schuetz, 1985). Forskolin, SQ20,006, and cycloheximide demonstrated three different time courses of inhibition when they were added separately to steroid-treated follicles. When SQ20,006 was added to the incubation flasks up to 6 hr after the steroid, it effectively blocked GVBD. Given that GVBD was complete in steroid controls by 36 hr, SQ20,006 was inhibitory for one-sixth of in vitro meiotic maturation or the time to undergo GVBD. By contrast, forskolin effectively inhibited GVBD if it was added up to 12 hr after the steroid or during one-third of maturation. Steroid-stimulated maturation was sensitive to cycloheximide treatment up to 18 hr poststeroid treatment or for the first half of maturation. However, the time course of inhibition depended on the experimental condition used. For example, when SQ20,006 was added as a 6-hr pulse and then removed, there was no inhibition of GVBD at any time provided the steroid (5.0 rig/ml) treatment was continued. But, when the steroid (10.0 rig/ml) was administered as a 1-hr prepulse and removed, the processes triggered by the steroid were blocked with a 6-hr pulse of SQ20,006 and this inhibition occurred if the SQ20,006 pulse was given as late as 12 hr after the steroid was removed. When these pulse experiments were performed using 1 .O $14 cycloheximide instead of SQ20,006 (results not shown), the time course of inhibition was similar to that presented in Fig. 1. In similar studies on brook trout (DeManno and Goetz, 1986), the time course of inhibition for forskolin (one-third of maturation) and cycloheximide (one-half of maturation) was the same as for perch. However, in brook trout the time course of inhibition for 1.0 m&f IBMX was one-half, the same as for cycloheximide. Cycloheximide and theophylline (1.0 m&I) also dem-

AND

COET%

onstrated the same time pattern of inhibition of GVBD in progesterone-stimulated R. pipiens (Morrill et ai., 1977) and Xenopus (Bravo cf ul., 1978) oocytes. Forskolin inhibited progesterone-induced GVBD in Xenopus for one-third of the time to GVBD (Schorderet-Slatkine and Baulieu, 1982). In both Xenopus and Rana, the steroid-stimulated decrease in oocyte CAMP has been described as a rapid event occurring anywhere from 20 set to 3 hr after progesterone exposure with a return to basal levels within l-3 hr (Speaker and Butcher, 1977; Bravo et ul., 1978; Maller et al., 1979; Cicirelli and Smith, 1985). However, the results presented here demonstrate that phosphodiesterase inhibitors and forskolin block steroid-stimulated GVBD in fish and amphibian follicles for a relatively long period, even when added well after the steroid. Thus it appears that the proposed steroid-induced drop in CAMP levels must be maintained for longer than the measured CAMP decreases would suggest.

The gonadotropin-stimulated steroidogenesis which occurs in the oocyte follicle wall (Goetz, 1983) is mediated by AC and CAMP (Nagahama et al., 1985). It is believed that both the follicle wall components and the oocyte have AC systems which respond to forskolin stimulation with increased CAMP production (Kwon and Schuetz, 1985; DeManno and Goetz, 1986). This increased endogenous CAMP could then stimulate steroidogenesis in the follicle wall cells or it could counteract the effects of steroid on the oocyte. In either case, the presence of the follicle wall in these types of in vitro experiments should be considered. Previous attempts in our lab to remove the follicle wall from perch oocytes were unsuccessful and oocytes induced to ovulate before undergoing GVBD are not viable (Goetz and Theofan, 1979). Therefore, CK was used to chemically denude the oocytes by blocking steroidogenesis. Cyanoketone is a pregnenolone analog

CYCLIC AMP AND STEROID-INDUCED

which binds specifically and irreversibly to 3p-hydroxy-A5-dehydrogenase and inhibits the conversion of pregnenolone to progesterone (Young et al., 1982). Low levels of forskolin (0.1 pLM) and CAMP (1 .O and 0.5 mM) were generally not inhibitory to 17ol,20P-PG-stimulated GVBD. When follicles were incubated with 1.0 pg/rnl CK in addition to steroid and forskolin or CAMP, it became evident that at these lower concentrations, forskolin and CAMP-stimulated steroidogenesis in the follicle wall was overriding their inhibitory effect on oocyte maturation. Similarly, Kwon and Schuetz (1985) reported that in Rana, forskolin stimulated steroidogenesis in intact follicles and inhibited GVBD in denuded oocytes. At times in the present study, the CK did have an antagonistic effect on the action of the steroid in the CAMP experiments, as shown by the slight decrease in GVBD in steroid controls incubated with CK. Since a number of different CZ, steroids can stimulate teleost oocyte maturation in vitro (Goetz, 1983), the receptors’ physical specificity is not limited to 17a,20@PG alone. The CK may have occupied some of the membrane steroid receptors and thereby decreased the receptors available to 17a,20@PG. The results presented here demonstrate that compounds which raise the intraoocyte level of CAMP can block 17a,20@PGinduced GVBD, are effective for a relatively long time, and may also stimulate steroidogenesis in the follicle wall. These results led us to measure CAMP levels over time in control and steroid-stimulated perch follicles. Although there were a few instances when the CAMP levels were significantly decreased in steroid-stimulated follicles as compared with those of controls, the time and magnitude of the decreases were not reproducible (results not shown). Similar measurements have also been made in our lab on brook trout follicles and partially denuded oocytes but significant decreases have not been de-

GVBD

241

tected thus far. In rainbow trout, CAMP levels in 17a,20p-PG-incubated follicles were significantly lower than control levels at various times within the first 10 hr of incubation, but again the time and magnitude of the decreases were not reproducible between fish (Jalabert and Finet, 1986). Unlike the reports of steroid-induced decreases in oocyte CAMP cited above, Thibier et al. (1982) reported that it was not possible to detect a reproducible decrease in steroid-stimulated Xenopus denuded oocytes unless they were pretreated with cholera toxin. The toxin boosted the basal CAMP levels and served to magnify the effect of the steroid. The requisite drop in CAMP has been calculated, on the basis of protein kinase inhibitor studies, to be IO-20% below basal (Thibier et al., 1982; Cicirelli and Smith, 1985), and the inconsistencies in reported values are often attributed to lack of sensitivity in the assay used. Furthermore, the discrepancy between the inhibitor and forskolin time courses and the duration of the measured CAMP decrease has rarely been addressed. One explanation is that CAMP is linked to the appearance of maturation-promoting factor which occurs shortly before GVBD in amphibians (Schorderet-Slatkine et al., 1982; Kwon and Schuetz, 1985). Another possibility which should be considered is an interaction between CAMP and calcium and/or membrane phospholipids (Baulier et al., 1978; Baulieu, 1983). It may be that the steroid effect at the oocyte plasma membrane indirectly inhibits AC and that the resulting drop in CAMP serves to modulate a longer-lasting response triggered by the steroid. ACKNOWLEDGMENTS We thank the Squibb Institute for Medical Research for their gift of SQ20,006 and the Sterling-Winthrop Research Institute for donating the cyanoketone.

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