Frog Pituitary Homogenate Research Articles

Effects of butyltin compounds on follicular steroidogenesis in the bullfrog( Rana catesbeiana)

The effects of butyltin compounds on follicular steroidogenesis in amphibians were examined using ovarian follicles of Rana catesbeiana. Isolated follicles were cultured for 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the steroid levels in the follicles or culture media were measured by radioimmunoassay (RIA). Among the butyltin compounds, tributyltin (TBT) strongly inhibited the FPH-induced synthesis of pregnenolone (P 5), progesterone (P 4) and testosterone (T). It also inhibited the conversion of P 5–P 4 and T to estradiol-17β(E 2) and it partially suppressed the conversion of androstenedione (AD) to T, but not P 4 to 17α-hydroxyprogesterone (17α-OHP 4). A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin (MBT) and tetrabutylin (TeBT) had no effect. These results suggest that the initial step of steroidogenesis (P 5 synthesis) and enzymes such as 3β-HSD, 17β-HSD and aromatase are inhibited by TBT or DBT. However, 17α-hydroxylase was not suppressed by TBT or the other butyltin compounds.

Effect of endocrine disruptors on the oocyte maturation and ovulation in amphibians,rana dybowskii

Abstract Recently, we have shown that some endocrine disruptors, heavy metals, organotins and azoles suppressed steroidogenic enzymes such as P450 side‐chain cleavage enzyme (P450scc) and aromatase in bullfrog ovarian follicles. In the present study, by using an amphibian ovarian follicle culture system, we examined the effects of these endocrine disruptors on maturation and ovulation of oocytes from Rana dybowskii in vitro. Ovarian fragments or isolated follicles were cultured for 24 h in a medium containing frog pituitary homogenate (FPH) or progesterone (P4) with or without endocrine disruptors, and oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation were examined. Among the organotins, tributyltin (TBT) strongly inhibited both FPH‐ and P4‐induced oocyte maturation (ED50: 0.6 and 0.7 μM, respectively); however, tetrabutyltin (TTBT) and dibutyltin (DBT) showed only partial suppression, while monobutyltin (MBT) showed no inhibitory effect. All of the organotins suppressed P4‐induced oocyte...

Effects of heavy metals on thein vitrofollicular steroidogenesis in amphibians

Abstract Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01–100 μM), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH‐induced pregnenolone (P5) production by the follicles (EC50, 4.0 μM, 25.6 μM and 5.7 μM, respectively ), and also suppressed the conversion of testosterone (T) to estradiol 17β (E2) (EC50, 4.2μM, 7.5 μM and 80.0 μM) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as C17–20 lyase and 17β‐hydroxysteroid dehydrogenase (17β‐HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.

Inhibitory action of the gonadopeptide inhibin on amphibian (Rana pipiens) steroidogenesis and oocyte maturation

In view of recent reports on the production of inhibin- and activin-like proteins in lower vertebrates and their important role during development, we have examined the effects of the gonadopeptide inhibin in the process of oocyte maturation using amphibian (Rana pipiens) fully grown preovulatory ovarian follicles cultured in vitro. In the presence of frog pituitary homogenate (FPH), which stimulates progesterone (P4) levels and the subsequent germinal vesicle breakdown (GVBD), purified porcine inhibin (35–50 IU) inhibited both of these responses in a dose-dependent manner. Inhibin also blocked GVBD initiated by exogenously added P4 in intact as well as denuded oocytes. Thus, inhibin seems to act at the follicle (granulosa) cells because it blocked steroidogenesis and at the oocyte because it altered the steroid-induced oocyte maturation. The P4-treated follicles were susceptible to the inhibin action during the first 3 hr of steroid stimulation, which indicates that inhibin affects some early events during the process of GVBD. Maximum inhibitory effect was observed when P4 and inhibin were added simultaneously at the beginning of the incubations. Moreover, the inhibitory effect on GVBD caused by the gonadopeptide was dependent on the length of exposure of the follicles to inhibin. The continuous presence of inhibin in the culture was required to block GVBD efficiently. Data also indicate that the inhibitory effect of inhibin was reversible. Taken together, results from this study present evidence that inhibin may be a relevant paracrine/autocrine regulator of ovarian functions. J. Exp. Zool. 284:232–240, 1999. © 1999 Wiley-Liss, Inc.

Inhibitory action of the gonadopeptide inhibin on amphibian (Rana pipiens) steroidogenesis and oocyte maturation.

In view of recent reports on the production of inhibin- and activin-like proteins in lower vertebrates and their important role during development, we have examined the effects of the gonadopeptide inhibin in the process of oocyte maturation using amphibian (Rana pipiens) fully grown preovulatory ovarian follicles cultured in vitro. In the presence of frog pituitary homogenate (FPH), which stimulates progesterone (P4) levels and the subsequent germinal vesicle breakdown (GVBD), purified porcine inhibin (35-50 IU) inhibited both of these responses in a dose-dependent manner. Inhibin also blocked GVBD initiated by exogenously added P4 in intact as well as denuded oocytes. Thus, inhibin seems to act at the follicle (granulosa) cells because it blocked steroidogenesis and at the oocyte because it altered the steroid-induced oocyte maturation. The P4-treated follicles were susceptible to the inhibin action during the first 3 hr of steroid stimulation, which indicates that inhibin affects some early events during the process of GVBD. Maximum inhibitory effect was observed when P4 and inhibin were added simultaneously at the beginning of the incubations. Moreover, the inhibitory effect on GVBD caused by the gonadopeptide was dependent on the length of exposure of the follicles to inhibin. The continuous presence of inhibin in the culture was required to block GVBD efficiently. Data also indicate that the inhibitory effect of inhibin was reversible. Taken together, results from this study present evidence that inhibin may be a relevant paracrine/autocrine regulator of ovarian functions.

Evidence for two-cell model of steroidogenesis in four species of amphibian

Previously, based on studies conducted using Rana nigromaculata, a two-cell model involving the theca and granulosa cells was proposed to account for the steroidogenic activity of amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was applicable to four other frog species with different reproductive cycles (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii, these treatments stimulated secretion of progesterone (P4) and 17α-hydroxyprogesterone (17α-OHP4) into the culture medium. Intact follicles obtained from all species readily converted pregnenolone (P5), P4, and 17α-OHP4 to AD, T, and E2. In contrast GCEO converted P5, P4, and 17α-OHP4 to AD and E2, but not to T. However, AD, but not P5, P4, or 17α-OHP4, was converted to T when cultured in the presence of isolated THEP. The microdissection procedure was also modified to isolate THEP without contaminating granulosa cells. The steroidogenic capacities of “impure” THEP and “pure” theca-epithelium (P-THEP) were then compared. Basal amounts of P4 were produced when P5 was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P5 or P4 to 17α-OHP4 occurred following culture with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD → T is localized in the THEP, whereas the metabolic capacities to convert P5 → AD and T → E2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is applicable to other frog species. J. Exp. Zool. 284:91–99, 1999. © 1999 Wiley-Liss, Inc.

Evidence for two‐cell model of steroidogenesis in four species of amphibian

Previously, based on studies conducted using Rana nigromaculata, a two-cell model involving the theca and granulosa cells was proposed to account for the steroidogenic activity of amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was applicable to four other frog species with different reproductive cycles (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii, these treatments stimulated secretion of progesterone (P4) and 17α-hydroxyprogesterone (17α-OHP4) into the culture medium. Intact follicles obtained from all species readily converted pregnenolone (P5), P4, and 17α-OHP4 to AD, T, and E2. In contrast GCEO converted P5, P4, and 17α-OHP4 to AD and E2, but not to T. However, AD, but not P5, P4, or 17α-OHP4, was converted to T when cultured in the presence of isolated THEP. The microdissection procedure was also modified to isolate THEP without contaminating granulosa cells. The steroidogenic capacities of “impure” THEP and “pure” theca-epithelium (P-THEP) were then compared. Basal amounts of P4 were produced when P5 was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P5 or P4 to 17α-OHP4 occurred following culture with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD → T is localized in the THEP, whereas the metabolic capacities to convert P5 → AD and T → E2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is applicable to other frog species. J. Exp. Zool. 284:91–99, 1999. © 1999 Wiley-Liss, Inc.

In vitroovulation and prostaglandin synthesis by ovarian follicles ofRana dybowskii

Changes in the levels of prostaglandin F2α (PGF2α) and E2 (PGE2) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12‐O‐tetradecanoylphorbol‐13‐acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF2α to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE2 increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF2α induced ovulation but PGE2 suppressed FPH‐ or PGF2α‐induced oocyte ovulation. Basal levels of PGs increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell‐enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOs. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF2α plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) theca/ epithelium layer is responsible for the PGs production in Rana.

Relative Roles of Theca and Granulosa Cells in Ovarian Follicular Steroidogenesis in the Amphibian, Rana nigromaculata

Using frog ovarian follicles in vitro we assessed the role of the theca/epithlium (THEP) layer and granulosa cells in the production of steroids. The THEP layer and granulosa cell-enclosed oocytes (GCEOs) were separated from ovarian follicles of the frog, Rana migromaculata, by microdisxection. Intact follicles (IFs), THEP layer, or GCEOs were cultured for 6 hr in amphibian Ringer's in the presence or absence of various steroid precursors (100 ng/ml) or of frog pituitary homogenate (FPH, 0.05 gland/ml). The amounts of progesterone (P 4), 17α-hydroxyprogesterone (17α-OHP 4), androstenedione (AD), testosterone (T), and estradiol (E 2) secreted into the medium by the different types of follicular components during culture were measured by radioimmunoassay. Addition of pregnelone resulted in a marked increase in P 4 concentrations produced by GCEOs (1858 pg/follicle) and IFs (1549 pg/follicle), but a much smaller increase in P 4 occurred in the THEP layer (427 pg/follicle). Likewise, addition of P 4 stimulated greater production of 17α-OHP 4 by GCEOs (659 pg/follicle) and by IFs (877 pg/follicle) than by the THEP layer (330 pg/follicle). Exogenous 17α-OHP 4 markedly increased AD levels secreted by GCEOs (1139 pg/follicle) and by IFs (841 pg/follicle), but not by the THEP layer (<15 pg/follicle) and by IFs (841 pg/follicle), but not by the THEP layer (<15 pg/follicle). In contrast, AD addition resulted in the secretion of markedly elevated levels of T by the THEP layer (2326 pg/follicle) and IFs (3652 pg/follicle) as compared to the GCEOs (408 pg/follicle). Addition of T markedly increased E 2 concentrations produced by GCEOs (378 pg/follicle) and by IFs (673 pg/follicle), but not by the THEP layer (<12 pg/follicle). Without addition of FPH or steroid precursors, none of the follicular components examined produced significant amounts of the steroids measured (<20 pg/follicle). Denuded oocytes incubated in the presence of exogenous substrates or FPH secreted nearly nondetectable amounts of steroids. Theses data suggests that the main sites for synthesis of P 4, 17α-OHP 4, AD, and E 2 are granulosa cells and those of T are the theca/epithelium layer in amphibian ovarian follicles.

Steroidogenic shift by cultured ovarian follicles of Rana dybowskii at breeding season.

The steroid secretory activity of cultured ovarian follicles of Rana dybowskii and the activities of relevant steroidogenic enzymes were examined during the natural hibernation period (October-February). Enzyme activities were measured indirectly by monitoring the conversion of exogenous substrates to products by isolated follicles. Follicles were incubated for 6 h in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors. Progesterone (P4), 17 alpha-hydroxyprogesterone (17 alpha-OHP) or testosterone (T) secreted by the follicles into the medium were measured by RIA. In the presence of FPH, high levels of P4 were produced by follicles at the early and mid-hibernation period (695 and 898 pg/follicle, respectively) whereas markedly elevated levels of P4 were produced during late hibernation (1,393 pg/follicle) (just prior to or breeding season, February). In contrast, high levels of T were produced by the follicles early in hibernation (1,206 pg/follicle) while negligible levels were produced in late hibernation (69 pg/follicle). Higher levels of 17 alpha-OHP were produced by follicles at early and mid hibernation (594 and 705 pg/follicle, respectively) than in later hibernation (221 pg/follicle). Addition of exogenous pregnenolone markedly increased P4 levels in a dose-dependent manner when added to follicles at early and late stages of hibernation: FPH addition further enhanced conversion of pregnenolone. Similarly, addition of androstenedione (AD) increased T levels in a dose-dependent manner by these follicles. However, following addition of exogenous P4, less 17 alpha-OHP was produced by follicles collected in late hibernation as compared to those collected earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the Activities of Steroidogenic Enzymes during the Development of Ovarian Follicles in Rana nigromaculata

Earlier studies showed that the predominant steroid (estradiol [E 2], testosterone [T], progesterone [P 4]) secreted by in vitro cultured amphibian ( Rana nigromaculata) ovarian follicles varied with their size. E 2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P 4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E 2, T, androstenedione (AD), 17α-hydroxyprogesterone (17α-OHP 4), or P 4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P 5) was converted to P 4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P 4 in all sized follicles. Highest levels of P 4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P 4 to AD or T. EPH suppressed conversion of exogenous 17α-OHP 4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E 2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles. Taken together, the data suggest that (1) the transition from E 2 to T secretion (Class III to IV) is associated with a loss of aromatase activity, (2) the transition from T to P 4 production (Class IV to V) is correlated with a decrease in the activity of enzymes which metabolize P 4, and (3) activities of steroidogenic enzymes varied in their responsiveness to EPH stimulation.

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.

Steroid production by amphibian (Rana nigromaculata) ovarian follicles at different developmental stages

AbstractRana ovarian follicles at different developmental stages were obtained from frogs collected at different seasons. Isolated follicles were sorted into five different classes (Class I–V) by size and cytoplasmic pigmentation. They were incubated for 6 hours in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.05 pituitary/ml) and changes in steroid production by follicles were assessed by measuring estradiol (E2), testosterone (T), and progesterone (P4) in media and follicles by radioimmunoassay. Small follicles (size range 0.22–0.7 mm) secreted very low or non‐detectable levels of the three steroids in the presence or absence of FPH. Medium‐sized vitellogenic follicles (0.70–1.11 mm) actively secreted all the three types of steroids tested (E2, P4, and T). Larger follicles (1.15–1.58 mm), isolated during September–February, secreted much more T than E2 and P4. In contrast, fully grown follicles (&gt; 1.50 mm) collected late in the hibernation period secreted more P4 and less T than the large follicles and very low levels of E2; however, follicles (Class V) collected in breeding season (May) secreted solely P4 and negligible amounts of T and E2. All types of follicles produced very low levels of the steroids without FPH stimulation. Intrafollicular levels of steroids essentially reflected those which accumulated in the medium. Oocyte responsiveness to exogenous hormone, as evidenced by germinal vesicle breakdown (GVBD), increased as the breeding season approached. The data indicate that only the medium‐sized follicles produced considerable amounts of E2 while larger follicles produced mainly T and full‐grown follicles collected during breeding season produced only P4.

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

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 Rana 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.

Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: Seasonal influences, progesterone production, and involvement of cAMP

Seasonal and hormonal influences regulating oocyte maturation (germinal vesicle breakdown, GVBD) in ovarian follicles of Rana dybowskii were investigated. During the early winter (Dec.-Jan.) GVBD occurred at a low incidence following in vitro culture of intact follicles. Addition of progesterone of frog pituitary homogenate (FPH) to such follicles induced oocyte maturation, whereas IBMX or forskolin inhibited hormone-induced oocyte maturation. The time course of spontaneous in vitro maturation varied markedly with the seasons and between animals. Follicles isolated from the ovaries in early February required 21-24 hours of culture to mature spontaneously, and addition of FPH or progesterone to the culture medium markedly accelerated the time course of GVBD. In contrast, follicles isolated in late February matured very rapidly (less than 6 hours), and FPH or progesterone were ineffective in accelerating the time course of GVBD. IBMX and forskolin separately or in combination stimulated follicular progesterone production, which resembled that seen following FPH stimulation. FPH addition to such follicles shifted the steroid peak to the left (accelerated) and increased the absolute amount of hormone detected in late-maturing follicles (50% GVBD, about 18 hours) but not in rapidly maturing follicles (50% GVBD, 3 hours). In contrast to other amphibians, a high incidence of spontaneous oocyte maturation occurred during in vitro culture. Essentially all animals exhibited spontaneous maturation during the normal breeding season, even those animals collected in the early winter and kept in artificial hibernation at 4 degrees C for extended periods.

Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β-Hydroxysteroid Dehydrogenase: (ovarian steroidogenesis/cAMP, 3β-hydroxysteroid dehydrogenase/progesterone/estradiol regulation/Rana pipiens).

Previous studies demonstrated that estradiol interferes with pituitary-induced progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP-induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX-phosphodiesterase inhibitor) and exogenously added cAMP. Progesterone production induced by forskolin alone or forskolin in combination with frog pituitary homogenate (FPH) was inhibited by estrogen. Addition of estradiol to culture medium markedly inhibited follicular progesterone accumulation following treatment of follicles with cAMP and IBMX. In the presence of exogenous pregnenolone, non-FPH stimulated ovarian follicles effectively converted the 3β-HSD substrate to progesterone. Treatment of follicles with estradiol inhibited conversion of pregnenolone to progesterone. The results indicate that estradiol acts, following FPH stimulation, at one or more steps subsequent to elevation of cAMP levels to regulate intrafollicular progesterone accumulation and oocyte maturation. Estrogen appears to directly influence the enzymatic (3β-HSD) conversion of pregnenolone to progesterone.

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.

Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles.

Treatment of isolated amphibian ovarian follicles with frog pituitary homogenate (FPH) increases follicular progesterone levels, which, in turn, initiate oocyte maturation. Recent studies have demonstrated that follicular progesterone production requires concomitant protein synthesis at some stage preceding pregnenolone formation. Experiments were carried out to determine whether cholesterol metabolism plays a role in mediating these biochemical and physiological processes. Aminoglutethimide (AGI, and inhibitor of P450 side-chain cleavage enzyme) inhibited FPH-induced intrafollicular progesterone accumulation and oocyte maturation (or germinal vesicle breakdown, GVBD) in a dose-dependent manner. Follicular progesterone accumulation and GVBD were both stimulated, in the absence of FPH, after addition of 25-OH-cholesterol, but not cholesterol, to the culture medium. Higher levels of progesterone were present in defolliculated oocytes as compared to intact ovarian follicles after incubation with 25-OH-cholesterol. The results indicate that the surface epithelium and theca layer in the follicle wall retard 25-OH-cholesterol access to steroid-producing follicle cells. AGI blocked 25-OH-cholesterol-induced accumulation of progesterone and GVBD in defolliculated oocytes, suggesting that 25-OH-cholesterol does not directly induce GVBD and is metabolized by the follicle cells. The capacity of follicles to accumulate progesterone following preincubation with FPH or 25-OH-cholesterol along with AGI was compared. Intrafollicular levels of progesterone increased after AGI- and 25-OH-cholesterol-treated follicles were washed. In contrast, progesterone levels decreased in follicles pretreated with AGI and FPH after washing. The results indicate that considerable 25-OH-cholesterol, but not endogenous cholesterol (FPH stimulation), remains available for steroidogenesis after removal of AGI. A significant, but incomplete, inhibition of progesterone accumulation occurred when follicles were incubated in the presence of 25-OH-cholesterol and cycloheximide. This partial blockage produced by the protein synthesis inhibitor indicates that some basal protein synthesis is required for progesterone accumulation from exogenous 25-OH-cholesterol. We conclude that intracellular cholesterol stores in the follicle wall are utilized to mediate FPH induction of progesterone accumulation and oocyte maturation in amphibian follicles.

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians ( Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian ( Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0–3 μ M), cAMP (0–3 m M), or dibutyryl cAMP (dbcAMP, 0–2.5 m M) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 m M) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 m M) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0–3 μ M) and IBMX (0-0.09 m M) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 m M) and IBMX (0.09 m M). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.

Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin

AbstractProstaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro‐cultured ovarian follicles. Exposure of follicles to PGF2α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF2α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF2α to FPH‐treated follicles. PGE, in contrast to PGF2α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF2α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF2α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH‐induced ovulation and maturation. PGF2α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH‐induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF2α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.