MOLECULAR REPRODUCTION AND DEVELOPMENT 25~147-154 (1990)
Induction and Inhibition of Meiotic Maturation of Amphibian (Rana dybowskii) Follicular Oocytes by Forskolin and cAMP In Vitro HYUK B. KWON,' HYUN J. PARK,' AND ALLEN w. SCHUETZ~ 'Department of Biology, College of Natural Sciences, Chonnam National University, Kwangiu, Republic of Korea; 'Department of Population Dynamics, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland
ABSTRACT Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Ram dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of CAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than CAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous CAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.
meiotic maturation in ovarian follicles of mammalian and non-mammalian vertebrates (Dekel and Beers, 1978; Eppig and Downs, 1984; Schuetz, 1985). In amphibians and fishes, follicle cells are known to produce maturation-inducing steroids that cause oocyte maturation in response to gonadotropin stimulation (Masui and Clarke, 1979; Nagahama and Adachi, 1985; Petrino and Schuetz, 1986; Lin e t al., 1987). Cyclic nucleotides in follicle cells are thought to be a common mediator for many gonadotropin effects in vertebrates. Evidence for the involvement of CAMP in gonadotropin stimulation of steroidogenesis is well established in mammalian ovarian follicles (Lindner et al., 1974; Marsh, 19751, and it was found that gonadotropin increased cAMP levels in ovarian fragments or follicles of amphibians (Gavaud et al., 1979; Jordana e t al., 1982) and fishes (Kuo and Watanabe, 1978; Nagahama and Adachi, 1985; Iwamatsu et al., 1987) during in vitro culture. Recently, we showed that gonadotropin stimulation of progesterone production by amphibian ovarian follicles (Rana pipiens) could be mimicked by manipulation of endogenous cAMP levels using forskolin, a n active adenylate cyclase stimulator (Seamon and Daly, 1981), or 3-isobutyl-l-methylxanthine (IBMX) or exogenously added CAMP. Significantly, oocyte maturation did not occur under these conditions (Kwon and Schuetz, 1985, 1986). A rapid decline in cAMP levels has been observed in oocytes following progesterone exposure and interpreted as being essential for meiosis resumption (Morill et al., 1977; Speaker and Butcher, 1977; Finidori-Lepicard et al., 1981; Baulieu, 1983).The decrease in cAMP is thought to be caused by the suppression of the membrane-bound adenylate cyclase in the oocytes by progesterone (Baulieu, 1983). Steroid-induced oo-
Key Words: Steroidogenesis, Cyclic adenosine monophosphate, Frog, Progesterone Received March 22, 1989; accepted August 7, 1989.
INTRODUCTION It is well known that somatic follicle cells are involved in regulating arrest and resumption of oocyte
0 1990 WILEY-LISS, INC.
Address reprint requests to Dr. Hyuk B. Kwon, Department of Biology, College of Natural Sciences, Chonnam National University, Kwangju 500-757, Republic of Korea.
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cyte maturation is furthermore prevented by stimulators of cAMP production (forskolin, cholera toxin) or inhibitors (IBMX) of cAMP metabolism in amphibians (O’Connor and Smith, 1976; Schorderet-Slatkine and Baulieu, 1982; Kwon and Schuetz, 1985) or fishes (DeManno and Goetz, 1987; Iwamatsu et al., 1987). Thus high levels of cAMP in the oocyte appear to act as a negative regulator of the oocyte maturation process in the presence of maturation-inducing steroid, whereas relatively low levels of cAMP are required for meiosis to be reinitiated. These facts suggest a n explanation of how cAMP levels in follicle cells and oocytes can carry out contradictory functions (stimulate or inhibit oocyte maturation) in follicular oocytes under certain experimental conditions. Experiments were conducted to vary the duration of cAMP stimulation in a n attempt to elucidate how cAMP mediates these dual functions in the ovarian follicles in response to gonadotropin. The data show that induction of oocyte maturation a s well as accumulation of progesterone within the ovarian follicles are induced by transient rather than continuous exposure of the follicles to forskolin or CAMP. Thus the data indicate that temporal changes in cAMP levels in follicle cells and oocytes play a key role in regulating oocyte meiotic arrest and its resumption via its effects on the production and action of progesterone.
MATERIALS AND METHODS Animals Hibernating frogs (Rana dybowslzzz) were collected from streams in the Chonnam area of the Korean Peninsula during the winter. Animals were kept under artificial hibernation conditions in a cold room maintained in darkness at 4°C. Between 10 and 20 animals were kept in glass or plastic boxes containing 10% amphibian Ringer’s solution (AR) that was changed two times per week. These experiments were carried out during midwinter (January 11-February 21, and the data presented here were obtained using ovarian follicles from animals that exhibited no or a low level of spontaneous oocyte maturation in vitro. Hormones and Reagents Progesterone (Sigma) and forskolin (Sigma) were dissolved in vehicle composed of ethanol and propylene glycol (1:1) in a stock of 2 mgiml and 20 mM, respectively. Frog pituitary homogenate (FPH) was prepared from female frogs. Glands were homogenized in AR a t 4°C with a glass homogenizer. The homogenate was centrifuged ( P C , 10,000 rpm, 20 min) to remove debris, and the supernatant was frozen (-20°C) in aliquots until needed (Lin and Schuetz, 1985a). Adenosine 3’: 5’-cyclic monophosphate (CAMP;Sigma) was dissolved in AR solution in a stock solution of 10 mM. Different concentrations of reagents were prepared by diluting the stock solution with AR solution.
In Vitro Follicle Culture Animals were sacrificed by decapitation, and ovaries were removed immediately and placed in Ringer’s solution. Fresh ovaries were used for each experiment. All experimental manipulations were conducted a t room temperature (16-20°C) in amphibian Ringer’s solution (AR solution) (Kwon and Schuetz, 1986). Routine in vitro culture was carried out in a room maintained at 24“C, with 20 follicles cultured in 2 ml of AR per well (24 wellsidish; Costar 3524 culture cluster, Cambridge, MA). Culture dishes were placed in a shaker water bath a t 80 oscillations per minute for 24 hr. Oocyte maturation was assessed 24 h r after hormone addition. The oocytes were heat-fixed by brief boiling and were cracked open individually to check for the germinal vesicle with the aid of a dissecting microscope. Follicle Extraction and Sample Preparation for Radioimmunoassay (RIA) Intrafollicular progesterone concentration was measured by RIA. Extraction of progesterone from follicles employed procedures described by Lessman and Schuetz (1982). The amount of hormone in the medium was not routinely measured. Preliminary studies demonstrated that the relative amount of steroid (pgifollicle) in the medium was about 60% of that present in follicles (62.3% 2 6.276, n = 7). After a specified period of culture, medium was aspirated and follicles were extracted in the culture well using methanol (GR, Merck, 1 ml methanol per 20 follicles per well). Extraction was carried out for 15 min with shaking. Methanol extracts were capped and stored a t -20°C until assayed. Prior to assay, methanol extracts were evaporated in a dry oven a t 30°C and reconstituted using gelatin phosphate-buffered saline (GPBS). An appropriate amount of this extract in GPBS was assayed for progesterone by RIA without further purification. Progesterone RIA General assay procedures were adapted from those described by Fortune et al. (1975) and Lin and Schuetz (1985a), and utilized in previous studies (Kwon and Schuetz, 1986). Labeled progesterone (1,2,6,7-3H-progesterone; 99 Ciimmole) was obtained from Amersham and antiprogesterone serum (progesterone-1la-hemisuccinate-bovine serum albumin) was donated from Dr. Yong-Dal Yoon (Hanyang University, Seoul). Its cross reactivity was determined to be 0.065% with 4-pregnanedione, 0.00016% with 17a-21-dihydroprognen-(4)dion-(3,20), 0.0001% with 17a-OH-progesterone, and less than 0.0001% with A5-pregnen-3p-ol-20-one, cortisol and testosterone (personal communication from Dr. Yoon). Each sample was quantified for tritium using Packard 4530 liquid scintillation counter. Routinely, two sets of progesterone standards (5-2,000 pg) and sample replicates were included in each assay. The
INDUCTION OF OOCYTE MATURATION WITH cAMP
100
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Fig. 1. Effect of CAMP on progesterone-induced oocyte maturation of R. dybowskii in vitro. Dose-response studies. Isolated follicles were cultured in AR in the simultaneous presence of progesterone and cAMP (0.02-2.5 mMi. GVBD was checked after 24 h r culture. Each bar in the figure represents percent GVBD (mean i SEMi of 180 follicles ( n = 9, triplicate wells, three animals).
Fig. 2. Effect of forskolin on progesterone or FPHinduced oocyte maturation of R. dybowskii i n vitro. Dose-response studies. Follicles were cultured for 24 h r in the presence of progesterone and different doses of forskolin (0.33-9 FMi. Each bar in the figure represents percent GVBD of 240 follicles ( n = 12, triplicate wells, four animals) "P < 0.01 when compared to progesterone induced oocyte GVBD.
coefficients of variation between and within assays were 9.4%and 9.2%(n = 101, respectively.
ure 2 shows that forskolin, a n adenylate cyclase stimulator, significantly suppressed progesterone-induced GVBD of oocytes a t a concentration of 3 pM ( P < 0.001). Forskolin also inhibited FPH (0.01 pituitary equivalentiwell) induced oocyte maturation (Fig. 2) and was more effective in suppressing FPH-induced oocyte GVBD than progesterone-induced oocyte maturation at concentrations of 0.33 pM ( P < 0.01) and 1 pM ( P < 0.001).
Statistics GVBD data analysis: For all experiments, treatments were done in duplicate or triplicate on individual frogs, and the GVBD data for all replications of a n experiment were transformed using a n arcsin-square root transformation (angular) and analyzed by one-way analysis of variance (ANOVA). The data of progesterone level was also analyzed by ANOVA. 50% GVBD (ED501values were estimated for follicles by probit analysis (Statistical Analysis System [SASI, Cary, NC). RESULTS Effect of cAMP or Forskolin on Hormone (Progesterone or FPHbInduced Oocyte Maturation Initially we examined the relationship between cAMP or forskolin dose and inhibition of progesteroneinduced oocyte maturation in cultured R . dybowskii follicles. Follicles were cultured in the simultaneous presence of progesterone (1 pg/well) and various concentrations of cAMP or forskolin for 24 h r before being checked for GVBD. Figure 1 shows that cAMP added to the culture medium strongly suppressed GVBD in oocytes at a concentration of 2.5 mM ( P < 0.001). Fig-
Time Course of Drug Inhibition Effect on Oocyte GVBD An experiment was carried out to determine how long after progesterone stimulation oocytes were sensitive to the inhibitory effects of cAMP and forskolin. Follicles were stimulated with progesterone (1pgiwell) and cAMP (2.5 mM) or forskolin (9 pM) added a t various times (0-8 hr.) during the culture period. Both cAMP and forskolin suppressed oocyte GVBD when added 2 h r after the steroid (Fig. 3). Inhibitory effects of the drugs on oocyte maturation were partially lost when the drugs were added between 4 and 6 hr after progesterone stimulation and completely disappeared when added 8 h r after progesterone stimulation (Fig. 3). The effects of the two drugs were not significantly different from each other at any of the time points except a t 2 h r (Fig. 3). The time required for the progesterone-induced oocyte maturation was 9-12 hr, and
150
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100
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( 9vM)
T
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TIME
DRUG
ADDED ( HOURS)
Fig. 3. Effect of time of CAMP or forskolin addition of progesteroneinduced oocyte maturation of R. dybowshii in vitro. Follicles were cultured in the continuous presence of progesterone and cAMP (2.5 mM) or forskolin (9 JLM)was added to the medium a t designated time point and cultured for up to 24 hr. GVBD was checked after 24 hr
culture. Each bar represents percent GVBD (mean ? SEMI of a t least 300 follicles (triplicate wells, five animals) in CAMP-treated group (open bar) and 480 follicles (triplicate wells, eight animals) in forskolin-treated group (shaded bar).
50% GVBD occurred in these animals after 9.3 h r of culture. The inhibitory influence of forskolin was lost slightly earlier (50% GVBD after 5.2 hr) than that of cAMP (50% GVBD after 6.3 hr).
maturation when cAMP levels declined after removal of forskolin from the medium. The transient exposure experiment was carried out with follicles from nine animals, and maturation was induced in oocytes from five of these animals. Oocytes from four animals did not mature even though their follicles produced considerable amounts of progesterone (data not shown).
Forskolin Induction of Oocyte Maturation An experiment was undertaken to establish whether transient exposure of follicles to forskolin induced oocyte maturation. Isolated follicles from each animal were randomly divided into two or four groups and cultured under various conditions as shown in Table l. In group B, follicles were precultured 6 or 8 h r with forskolin (9 pM) and then transferred to forskolin-free AR medium and incubated for a n additional 18 or 23 hr. Table 1 shows that most of the oocytes exposed to the drug (93%) matured in plain medium without any added hormones. Sister follicles from the same animal did not mature when they were cultured in plain medium (group A) or when they were cultured in the continuous presence of forskolin and progesterone (group C). However, oocytes matured normally (98%) following exposure to progesterone (group D). The data clearly show that transient exposure of the follicles to forskolin triggered oocyte meiotic maturation without any added hormone (Table 1).Our interpretation of the data is that forskolin increased the intracellular levels of CAMPin the follicle cells, which in turn stimulated progesterone production. The steroid subsequently acted on the oocyte t o trigger reinitiation of meiotic
cAMP Induction of Oocyte Maturation In subsequent experiments, we tested the possibility that transient exposure of follicles to exogenous cAMP triggered resumption of meiosis in oocytes. Follicles isolated from each animal were randomly divided into two or four groups and placed in culture under various treatment conditions. Table 2 shows that preincubation of follicles for 6-8 h r in CAMP-containing medium induced oocyte maturation after they were transferred to plain medium and cultured for a n additional 18 or 23 h r (group B). Sister follicles incubated in the presence of cAMP with progesterone (group C ) or no hormone (group A) exhibited low levels of oocyte maturation. This result suggests that sufficient exogenous cAMP entered the follicle cells and stimulated progesterone production, which in t u r n induced oocyte maturation. This experiment was carried out using 13 animals, and follicles from seven of them responded to cAMP with stimulation of GVBD. Essentially all control oocytes treated with progesterone underwent GVBD (group D).
INDUCTION OF OOCYTE MATURATION WITH cAMP
151
TABLE 1. Forskolin Induction and Inhibition of Oocyte Maturation in Rana dybowskii Ovarian Follicles Cultured In Vitro* Culture hours
Group
A
Forskolin (9 pM) 0
R
6-8
C D
Forskolin + progesterone 24 0
No forskolin 24 18-23 Progesterone medium 0 24
No. of animals tested
No. of follicles tested
Percent GVBD (mean ? SEM)
5 5
300 300
15.8 2 6.6 92.8 ? 2.2
3 3
180 180
2.7 ? 2.7 98.3 2 0.9
*Isolated follicles from each animal were randomly divided into two or four groups (A-B or A-D). The follicles in each group were cultured in triplicate wells (20 follicles/well) and checked for GVBD after 24-31 hr culture period. Follicles in group A were cultured in AR without forskolin to establish whether spontaneous maturation occurred. Follicles in group B were precultured for 6-8 hr in forskolin-containing medium and then transferred to plain medium and cultured further for 18-23 hr. Follicles in group C were cultured in the continuous presence of forskolin and progesterone (1 Kgiwell), and those in group D were cultured in progesterone (1 pg/well) containing medium. Percent GVBD (mean ? SEMI of 300 follicles (n = 15, triplicate wells, five animals in groups A and B) or of 180 follicles (n = 9, triplicate wells, three animals in groups C and D).
TABLE 2. cAMP Induction and Inhibition of Oocyte Maturation in Rana dybowskii Follicles Cultured In Vitro*
Group A
B C D
Culture hours cAMP (2.5 mM) Plain medium 0 24 6-8 18-23 Progesterone cAMP + progesterone 24 0 0 24
No. animals tested
No. follicles tested
Perecent GVBD (mean ? SEM)
7 7
420 420
21.4 i 6.5 74.6 2 5.8
2 2
120 120
1.5 2 1.5 96.0 ? 4.0
*Isolated follicles from each animal were divided into two groups (A-B) or 4 (A-D) and were cultured for 24-31 hr in triplicate wells. Follicles in group A were cultured in AR without cAMP or hormone to check the spontaneous maturation. Follicles in group B were precultured for 6-8 h r in the presence of cAMP (2.5 mM) (known to inhibit progesterone-induced GVBD) and then transferred to plain medium and cultured further for 18-23 hr. Follicles in group C were cultured in the continuous presence of cAMP and progesterone (1 kg/well) for 24 hr. Those in group D were cultured in the continuous presence of progesterone (1pgiwell) for 24 hr. Percent GVBD (mean 2 SEM) of 420 follicles (n = 21, triplicate wells, seven animals in groups A and B) or of 120 follicles (n = 6, triplicate wells, two animals in groups C and D).
As was observed previously, cAMP prevented progesterone induced GVBD.
Progesterone Production by R. dybowskii Follicles Stimulated With Forskolin, CAMP,or FPH The ability of forskolin or cAMP to stimulate progesterone production by the follicles during preincubation period (duration of 6 or 8 hr) was investigated. Follicles were cultured in forskolin, CAMP, or FPH containing medium for 6-8 hr and intrafollicular levels of progesterone were measured by radioimmunoassay. Follicles from four animals were used for steroid assays after the exposure to forskolin. Similarly treated sister follicles
were incubated for 24 h r with forskolin and checked for oocyte maturation (GVBD). Two of these animals responded well with GVBD (>go%) and two were relatively nonresponsive to forskolin induction of maturation (
Induction and Inhibition of Meiotic Maturation of Amphibian (Rana dybowskii) Follicular Oocytes by Forskolin and cAMP In Vitro HYUK B. KWON,' HYUN J. PARK,' AND ALLEN w. SCHUETZ~ 'Department of Biology, College of Natural Sciences, Chonnam National University, Kwangiu, Republic of Korea; 'Department of Population Dynamics, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland
ABSTRACT Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Ram dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of CAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than CAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous CAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.
meiotic maturation in ovarian follicles of mammalian and non-mammalian vertebrates (Dekel and Beers, 1978; Eppig and Downs, 1984; Schuetz, 1985). In amphibians and fishes, follicle cells are known to produce maturation-inducing steroids that cause oocyte maturation in response to gonadotropin stimulation (Masui and Clarke, 1979; Nagahama and Adachi, 1985; Petrino and Schuetz, 1986; Lin e t al., 1987). Cyclic nucleotides in follicle cells are thought to be a common mediator for many gonadotropin effects in vertebrates. Evidence for the involvement of CAMP in gonadotropin stimulation of steroidogenesis is well established in mammalian ovarian follicles (Lindner et al., 1974; Marsh, 19751, and it was found that gonadotropin increased cAMP levels in ovarian fragments or follicles of amphibians (Gavaud et al., 1979; Jordana e t al., 1982) and fishes (Kuo and Watanabe, 1978; Nagahama and Adachi, 1985; Iwamatsu et al., 1987) during in vitro culture. Recently, we showed that gonadotropin stimulation of progesterone production by amphibian ovarian follicles (Rana pipiens) could be mimicked by manipulation of endogenous cAMP levels using forskolin, a n active adenylate cyclase stimulator (Seamon and Daly, 1981), or 3-isobutyl-l-methylxanthine (IBMX) or exogenously added CAMP. Significantly, oocyte maturation did not occur under these conditions (Kwon and Schuetz, 1985, 1986). A rapid decline in cAMP levels has been observed in oocytes following progesterone exposure and interpreted as being essential for meiosis resumption (Morill et al., 1977; Speaker and Butcher, 1977; Finidori-Lepicard et al., 1981; Baulieu, 1983).The decrease in cAMP is thought to be caused by the suppression of the membrane-bound adenylate cyclase in the oocytes by progesterone (Baulieu, 1983). Steroid-induced oo-
Key Words: Steroidogenesis, Cyclic adenosine monophosphate, Frog, Progesterone Received March 22, 1989; accepted August 7, 1989.
INTRODUCTION It is well known that somatic follicle cells are involved in regulating arrest and resumption of oocyte
0 1990 WILEY-LISS, INC.
Address reprint requests to Dr. Hyuk B. Kwon, Department of Biology, College of Natural Sciences, Chonnam National University, Kwangju 500-757, Republic of Korea.
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cyte maturation is furthermore prevented by stimulators of cAMP production (forskolin, cholera toxin) or inhibitors (IBMX) of cAMP metabolism in amphibians (O’Connor and Smith, 1976; Schorderet-Slatkine and Baulieu, 1982; Kwon and Schuetz, 1985) or fishes (DeManno and Goetz, 1987; Iwamatsu et al., 1987). Thus high levels of cAMP in the oocyte appear to act as a negative regulator of the oocyte maturation process in the presence of maturation-inducing steroid, whereas relatively low levels of cAMP are required for meiosis to be reinitiated. These facts suggest a n explanation of how cAMP levels in follicle cells and oocytes can carry out contradictory functions (stimulate or inhibit oocyte maturation) in follicular oocytes under certain experimental conditions. Experiments were conducted to vary the duration of cAMP stimulation in a n attempt to elucidate how cAMP mediates these dual functions in the ovarian follicles in response to gonadotropin. The data show that induction of oocyte maturation a s well as accumulation of progesterone within the ovarian follicles are induced by transient rather than continuous exposure of the follicles to forskolin or CAMP. Thus the data indicate that temporal changes in cAMP levels in follicle cells and oocytes play a key role in regulating oocyte meiotic arrest and its resumption via its effects on the production and action of progesterone.
MATERIALS AND METHODS Animals Hibernating frogs (Rana dybowslzzz) were collected from streams in the Chonnam area of the Korean Peninsula during the winter. Animals were kept under artificial hibernation conditions in a cold room maintained in darkness at 4°C. Between 10 and 20 animals were kept in glass or plastic boxes containing 10% amphibian Ringer’s solution (AR) that was changed two times per week. These experiments were carried out during midwinter (January 11-February 21, and the data presented here were obtained using ovarian follicles from animals that exhibited no or a low level of spontaneous oocyte maturation in vitro. Hormones and Reagents Progesterone (Sigma) and forskolin (Sigma) were dissolved in vehicle composed of ethanol and propylene glycol (1:1) in a stock of 2 mgiml and 20 mM, respectively. Frog pituitary homogenate (FPH) was prepared from female frogs. Glands were homogenized in AR a t 4°C with a glass homogenizer. The homogenate was centrifuged ( P C , 10,000 rpm, 20 min) to remove debris, and the supernatant was frozen (-20°C) in aliquots until needed (Lin and Schuetz, 1985a). Adenosine 3’: 5’-cyclic monophosphate (CAMP;Sigma) was dissolved in AR solution in a stock solution of 10 mM. Different concentrations of reagents were prepared by diluting the stock solution with AR solution.
In Vitro Follicle Culture Animals were sacrificed by decapitation, and ovaries were removed immediately and placed in Ringer’s solution. Fresh ovaries were used for each experiment. All experimental manipulations were conducted a t room temperature (16-20°C) in amphibian Ringer’s solution (AR solution) (Kwon and Schuetz, 1986). Routine in vitro culture was carried out in a room maintained at 24“C, with 20 follicles cultured in 2 ml of AR per well (24 wellsidish; Costar 3524 culture cluster, Cambridge, MA). Culture dishes were placed in a shaker water bath a t 80 oscillations per minute for 24 hr. Oocyte maturation was assessed 24 h r after hormone addition. The oocytes were heat-fixed by brief boiling and were cracked open individually to check for the germinal vesicle with the aid of a dissecting microscope. Follicle Extraction and Sample Preparation for Radioimmunoassay (RIA) Intrafollicular progesterone concentration was measured by RIA. Extraction of progesterone from follicles employed procedures described by Lessman and Schuetz (1982). The amount of hormone in the medium was not routinely measured. Preliminary studies demonstrated that the relative amount of steroid (pgifollicle) in the medium was about 60% of that present in follicles (62.3% 2 6.276, n = 7). After a specified period of culture, medium was aspirated and follicles were extracted in the culture well using methanol (GR, Merck, 1 ml methanol per 20 follicles per well). Extraction was carried out for 15 min with shaking. Methanol extracts were capped and stored a t -20°C until assayed. Prior to assay, methanol extracts were evaporated in a dry oven a t 30°C and reconstituted using gelatin phosphate-buffered saline (GPBS). An appropriate amount of this extract in GPBS was assayed for progesterone by RIA without further purification. Progesterone RIA General assay procedures were adapted from those described by Fortune et al. (1975) and Lin and Schuetz (1985a), and utilized in previous studies (Kwon and Schuetz, 1986). Labeled progesterone (1,2,6,7-3H-progesterone; 99 Ciimmole) was obtained from Amersham and antiprogesterone serum (progesterone-1la-hemisuccinate-bovine serum albumin) was donated from Dr. Yong-Dal Yoon (Hanyang University, Seoul). Its cross reactivity was determined to be 0.065% with 4-pregnanedione, 0.00016% with 17a-21-dihydroprognen-(4)dion-(3,20), 0.0001% with 17a-OH-progesterone, and less than 0.0001% with A5-pregnen-3p-ol-20-one, cortisol and testosterone (personal communication from Dr. Yoon). Each sample was quantified for tritium using Packard 4530 liquid scintillation counter. Routinely, two sets of progesterone standards (5-2,000 pg) and sample replicates were included in each assay. The
INDUCTION OF OOCYTE MATURATION WITH cAMP
100
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0
4/
0
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0.3 3
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Fig. 1. Effect of CAMP on progesterone-induced oocyte maturation of R. dybowskii in vitro. Dose-response studies. Isolated follicles were cultured in AR in the simultaneous presence of progesterone and cAMP (0.02-2.5 mMi. GVBD was checked after 24 h r culture. Each bar in the figure represents percent GVBD (mean i SEMi of 180 follicles ( n = 9, triplicate wells, three animals).
Fig. 2. Effect of forskolin on progesterone or FPHinduced oocyte maturation of R. dybowskii i n vitro. Dose-response studies. Follicles were cultured for 24 h r in the presence of progesterone and different doses of forskolin (0.33-9 FMi. Each bar in the figure represents percent GVBD of 240 follicles ( n = 12, triplicate wells, four animals) "P < 0.01 when compared to progesterone induced oocyte GVBD.
coefficients of variation between and within assays were 9.4%and 9.2%(n = 101, respectively.
ure 2 shows that forskolin, a n adenylate cyclase stimulator, significantly suppressed progesterone-induced GVBD of oocytes a t a concentration of 3 pM ( P < 0.001). Forskolin also inhibited FPH (0.01 pituitary equivalentiwell) induced oocyte maturation (Fig. 2) and was more effective in suppressing FPH-induced oocyte GVBD than progesterone-induced oocyte maturation at concentrations of 0.33 pM ( P < 0.01) and 1 pM ( P < 0.001).
Statistics GVBD data analysis: For all experiments, treatments were done in duplicate or triplicate on individual frogs, and the GVBD data for all replications of a n experiment were transformed using a n arcsin-square root transformation (angular) and analyzed by one-way analysis of variance (ANOVA). The data of progesterone level was also analyzed by ANOVA. 50% GVBD (ED501values were estimated for follicles by probit analysis (Statistical Analysis System [SASI, Cary, NC). RESULTS Effect of cAMP or Forskolin on Hormone (Progesterone or FPHbInduced Oocyte Maturation Initially we examined the relationship between cAMP or forskolin dose and inhibition of progesteroneinduced oocyte maturation in cultured R . dybowskii follicles. Follicles were cultured in the simultaneous presence of progesterone (1 pg/well) and various concentrations of cAMP or forskolin for 24 h r before being checked for GVBD. Figure 1 shows that cAMP added to the culture medium strongly suppressed GVBD in oocytes at a concentration of 2.5 mM ( P < 0.001). Fig-
Time Course of Drug Inhibition Effect on Oocyte GVBD An experiment was carried out to determine how long after progesterone stimulation oocytes were sensitive to the inhibitory effects of cAMP and forskolin. Follicles were stimulated with progesterone (1pgiwell) and cAMP (2.5 mM) or forskolin (9 pM) added a t various times (0-8 hr.) during the culture period. Both cAMP and forskolin suppressed oocyte GVBD when added 2 h r after the steroid (Fig. 3). Inhibitory effects of the drugs on oocyte maturation were partially lost when the drugs were added between 4 and 6 hr after progesterone stimulation and completely disappeared when added 8 h r after progesterone stimulation (Fig. 3). The effects of the two drugs were not significantly different from each other at any of the time points except a t 2 h r (Fig. 3). The time required for the progesterone-induced oocyte maturation was 9-12 hr, and
150
H.B. KWON ET AL.
100
-I
L
a FORSKOLIN
( 9vM)
T
n m
60-
>
8
TIME
DRUG
ADDED ( HOURS)
Fig. 3. Effect of time of CAMP or forskolin addition of progesteroneinduced oocyte maturation of R. dybowshii in vitro. Follicles were cultured in the continuous presence of progesterone and cAMP (2.5 mM) or forskolin (9 JLM)was added to the medium a t designated time point and cultured for up to 24 hr. GVBD was checked after 24 hr
culture. Each bar represents percent GVBD (mean ? SEMI of a t least 300 follicles (triplicate wells, five animals) in CAMP-treated group (open bar) and 480 follicles (triplicate wells, eight animals) in forskolin-treated group (shaded bar).
50% GVBD occurred in these animals after 9.3 h r of culture. The inhibitory influence of forskolin was lost slightly earlier (50% GVBD after 5.2 hr) than that of cAMP (50% GVBD after 6.3 hr).
maturation when cAMP levels declined after removal of forskolin from the medium. The transient exposure experiment was carried out with follicles from nine animals, and maturation was induced in oocytes from five of these animals. Oocytes from four animals did not mature even though their follicles produced considerable amounts of progesterone (data not shown).
Forskolin Induction of Oocyte Maturation An experiment was undertaken to establish whether transient exposure of follicles to forskolin induced oocyte maturation. Isolated follicles from each animal were randomly divided into two or four groups and cultured under various conditions as shown in Table l. In group B, follicles were precultured 6 or 8 h r with forskolin (9 pM) and then transferred to forskolin-free AR medium and incubated for a n additional 18 or 23 hr. Table 1 shows that most of the oocytes exposed to the drug (93%) matured in plain medium without any added hormones. Sister follicles from the same animal did not mature when they were cultured in plain medium (group A) or when they were cultured in the continuous presence of forskolin and progesterone (group C). However, oocytes matured normally (98%) following exposure to progesterone (group D). The data clearly show that transient exposure of the follicles to forskolin triggered oocyte meiotic maturation without any added hormone (Table 1).Our interpretation of the data is that forskolin increased the intracellular levels of CAMPin the follicle cells, which in turn stimulated progesterone production. The steroid subsequently acted on the oocyte t o trigger reinitiation of meiotic
cAMP Induction of Oocyte Maturation In subsequent experiments, we tested the possibility that transient exposure of follicles to exogenous cAMP triggered resumption of meiosis in oocytes. Follicles isolated from each animal were randomly divided into two or four groups and placed in culture under various treatment conditions. Table 2 shows that preincubation of follicles for 6-8 h r in CAMP-containing medium induced oocyte maturation after they were transferred to plain medium and cultured for a n additional 18 or 23 h r (group B). Sister follicles incubated in the presence of cAMP with progesterone (group C ) or no hormone (group A) exhibited low levels of oocyte maturation. This result suggests that sufficient exogenous cAMP entered the follicle cells and stimulated progesterone production, which in t u r n induced oocyte maturation. This experiment was carried out using 13 animals, and follicles from seven of them responded to cAMP with stimulation of GVBD. Essentially all control oocytes treated with progesterone underwent GVBD (group D).
INDUCTION OF OOCYTE MATURATION WITH cAMP
151
TABLE 1. Forskolin Induction and Inhibition of Oocyte Maturation in Rana dybowskii Ovarian Follicles Cultured In Vitro* Culture hours
Group
A
Forskolin (9 pM) 0
R
6-8
C D
Forskolin + progesterone 24 0
No forskolin 24 18-23 Progesterone medium 0 24
No. of animals tested
No. of follicles tested
Percent GVBD (mean ? SEM)
5 5
300 300
15.8 2 6.6 92.8 ? 2.2
3 3
180 180
2.7 ? 2.7 98.3 2 0.9
*Isolated follicles from each animal were randomly divided into two or four groups (A-B or A-D). The follicles in each group were cultured in triplicate wells (20 follicles/well) and checked for GVBD after 24-31 hr culture period. Follicles in group A were cultured in AR without forskolin to establish whether spontaneous maturation occurred. Follicles in group B were precultured for 6-8 hr in forskolin-containing medium and then transferred to plain medium and cultured further for 18-23 hr. Follicles in group C were cultured in the continuous presence of forskolin and progesterone (1 Kgiwell), and those in group D were cultured in progesterone (1 pg/well) containing medium. Percent GVBD (mean ? SEMI of 300 follicles (n = 15, triplicate wells, five animals in groups A and B) or of 180 follicles (n = 9, triplicate wells, three animals in groups C and D).
TABLE 2. cAMP Induction and Inhibition of Oocyte Maturation in Rana dybowskii Follicles Cultured In Vitro*
Group A
B C D
Culture hours cAMP (2.5 mM) Plain medium 0 24 6-8 18-23 Progesterone cAMP + progesterone 24 0 0 24
No. animals tested
No. follicles tested
Perecent GVBD (mean ? SEM)
7 7
420 420
21.4 i 6.5 74.6 2 5.8
2 2
120 120
1.5 2 1.5 96.0 ? 4.0
*Isolated follicles from each animal were divided into two groups (A-B) or 4 (A-D) and were cultured for 24-31 hr in triplicate wells. Follicles in group A were cultured in AR without cAMP or hormone to check the spontaneous maturation. Follicles in group B were precultured for 6-8 h r in the presence of cAMP (2.5 mM) (known to inhibit progesterone-induced GVBD) and then transferred to plain medium and cultured further for 18-23 hr. Follicles in group C were cultured in the continuous presence of cAMP and progesterone (1 kg/well) for 24 hr. Those in group D were cultured in the continuous presence of progesterone (1pgiwell) for 24 hr. Percent GVBD (mean 2 SEM) of 420 follicles (n = 21, triplicate wells, seven animals in groups A and B) or of 120 follicles (n = 6, triplicate wells, two animals in groups C and D).
As was observed previously, cAMP prevented progesterone induced GVBD.
Progesterone Production by R. dybowskii Follicles Stimulated With Forskolin, CAMP,or FPH The ability of forskolin or cAMP to stimulate progesterone production by the follicles during preincubation period (duration of 6 or 8 hr) was investigated. Follicles were cultured in forskolin, CAMP, or FPH containing medium for 6-8 hr and intrafollicular levels of progesterone were measured by radioimmunoassay. Follicles from four animals were used for steroid assays after the exposure to forskolin. Similarly treated sister follicles
were incubated for 24 h r with forskolin and checked for oocyte maturation (GVBD). Two of these animals responded well with GVBD (>go%) and two were relatively nonresponsive to forskolin induction of maturation (
