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Endocrinology Vol. 144, No. 2 491-499
Copyright © 2003 by The Endocrine Society

ARTICLE

Involvement of Cyclic Adenosine 3',5'-Monophosphate in the Differential Regulation of Activin ßA and ßB Expression by Gonadotropin in the Zebrafish Ovarian Follicle Cells

Yajun Wang and Wei Ge

Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

Address all correspondence and requests for reprints to: Wei Ge, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. E-mail: weige{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activin is a dimeric protein consisting of two similar but distinct ß-subunits, ßA and ßB. In our previous studies, both activin A (ßAßA) and activin B (ßBßB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin ßA was cloned and sequenced. The precursor of zebrafish activin ßA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin ßA and ßB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin ßA-subunit; however, it significantly down-regulated the steady-state expression level of activin ßB in a time- and dose-dependent manner. The differential regulation of the two ß-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin ßA expression at 10 µM, but it was unable to reverse the inhibitory effects of hCG and forskolin on ßB expression. This suggests that the hCG-stimulated activin ßA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin ßB expression is likely mediated by PKA-independent pathway(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL ESTABLISHED that pituitary gonadotropins, FSH and LH, play pivotal roles in the regulation of vertebrate ovarian functions including folliculogenesis, steroidogenesis, oocyte maturation, and ovulation (1, 2). Evidence has been increasing that most of gonadotropin actions in the ovary involve local growth factors that serve as intraovarian modulators or mediators. Among these ovarian factors, the family of activin is of particular interest (3, 4, 5).

Activin belongs to the TGF-ß superfamily and is a homo- or heterodimer of two ß-subunits (ßA and ßB) that are covalently linked by a disulfide bond. The dimerization of the ß-subunits generates three biologically active forms of activin, activin A (ßAßA), activin B (ßBßB), and activin AB (ßAßB) (6). Activin was originally identified and purified as an ovarian peptide that specifically stimulates pituitary FSH secretion (7, 8), and it has later been shown to have a wide range of biological activities in a variety of tissues including the ovary (9). In the mammalian ovary, activin stimulates the expression of FSH and LH receptors (10, 11, 12, 13), the production of estradiol, mostly by enhancing aromatase activity (14, 15, 16), and the biosynthesis of inhibin/activin subunits (17, 18); on the other hand, activin suppresses basal and gonadotropin-stimulated progesterone secretion (19, 20, 21, 22) and LH- and IGF-I-stimulated ovarian androgen production (23, 24). In addition, activin also promotes the proliferation of granulosa and thecal-interstitial cells (25, 26), follicle growth (27, 28, 29, 30), and oocyte maturation (22, 31). Both activin ßA- and ßB-subunits are expressed in the ovary; however, they exhibit different patterns of expression during the ovarian cycle (32, 33, 34). The ßA-subunit mRNA is expressed in both the granulosa and thecal cells of human dominant follicles, but its expression is weak in the granulosa cells of small antral follicles. In contrast, activin ßB has a relatively higher expression level in the small antral follicles (34). The differential expression of the two subunits has also been observed in the rat (33), monkey (32), and chicken ovaries (35, 36). These lines of evidence suggest that there exist mechanisms that differentially regulate the expression of the two activin subunits in the vertebrate ovary. Pituitary gonadotropins have been proposed to be the potent regulators of activin ß-subunit genes. In the rat and human, activin ßA expression is up-regulated by gonadotropins in cultured granulosa cells (37, 38) or granulosa-luteal cells (39, 40). As for the regulation of activin ßB expression by gonadotropins, the results are controversial. Although earlier studies showed that gonadotropins also stimulated the expression of activin ßB-subunit in cultured rat granulosa cells (37, 41), recent findings in the human ovary demonstrated that the expression of ßB-subunit was effectively inhibited by FSH and LH in cultured granulosa-luteal cells (42). The differential regulation of the two activin subunits is also supported by the evidence from promoter analysis that reveals significant difference in the promoter region between the two ß-subunit genes (41, 43, 44, 45).

Using zebrafish as the model, we and others have demonstrated that both activin ßA and ßB are expressed in the fish ovary (46, 47, 48), and that recombinant human activin A and goldfish activin B significantly promote the final oocyte maturation and the development of oocyte maturational competence (46, 49). Interestingly, follistatin, a specific activin-binding protein, blocks both the activin- and human chorionic gonadotropin (hCG)-induced oocyte maturation (46, 47, 48), suggesting that the ovarian activin system is likely a downstream mediator of gonadotropin actions in the fish ovary (46, 49). This notion is supported by our recent demonstration that hCG stimulated the expression of activin ßA and activin type IIA receptor in the zebrafish ovary (48). Considering that both activin ßA and ßB are expressed in the zebrafish ovary, and that the expression of the two subunits seems to be differentially regulated in mammals despite the inconsistent reports from different models, we have undertaken the present study to investigate the regulation of expression of the two activin subunits by gonadotropin and its mechanisms using a primary culture of zebrafish ovarian follicle cells. Because fish represent the most primitive and diverse group of vertebrates, studies using fish models may serve as useful reference points for those in other vertebrates, and the evidence from the present study not only contributes to the current efforts to understand the differential regulation of the two activin subunits but also provides important insights into the evolution of the mechanisms that control the expression of activin system in the ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
All chemicals were obtained from Sigma (St. Louis, MO) and restriction enzymes from Promega Corp. (Madison, WI) unless otherwise stated. hCG and forskolin were purchased from Sigma, and 3-isobutyl-1-methylxanthine (IBMX), dibutyryl-cAMP (db-cAMP), and H89 from Calbiochem (La Jolla, CA). hCG and db-cAMP were first dissolved in water, forskolin and H89 in dimethylsulfoxide, and IBMX in ethanol. They were diluted to the desired concentrations with the medium before use. The pituitary extract was prepared from the goldfish, Carassius auratus. Briefly, seven goldfish pituitary glands were homogenized in 0.7 ml phosphate buffer followed by centrifugation at 15,000 rpm for 30 min at 4 C. The supernatant was filtered and the protein content determined with Bio-Rad Laboratories, Inc. (Hercules, CA) Protein Assay Kit.

Animals
Zebrafish, Danio rerio, were purchased from local pet stores and maintained in flow-through aquaria (36 liters) at 25 C on a 14-h light, 10-h dark photoperiod. The fish were fed twice a day with the commercial tropical fish food with supplement of live brine shrimp once or twice a week.

Primary follicle culture
The primary culture of zebrafish ovarian follicle cells was performed according to our previous report (48). Briefly, the follicles from about 20 female zebrafish were isolated and washed with Medium 199 (Life Technologies, Inc., Gaithersburg, MD). The follicles were then cultured in medium 199 supplemented with 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT) at 28 C in 5% CO2 for 6 d for the follicle cells to proliferate. The proliferated follicle cells were harvested by trypsinization and plated in 24-well plates for 24 h before treatments.

Total RNA isolation
Three hundred microliters of Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) were added to each well, and the plate was shaken for 10 min at 600 rpm on the Thermomixer Comfort (Eppendorf, Hamburg, Germany). The extract from each well was then transferred to a microtube containing 75 µl chloroform, vortexed, and spun for 30 min at 4 C. The aqueous phase from each sample was transferred to a new tube containing isopropanol, vortexed for 30 sec, left at -20 C for 30 min, and centrifuged for 30 min at 4 C. After washing with 75% ethanol and brief air drying, the RNA pellet was dissolved in 10 µl DEPC-H2O and stored at –80 C.

Cloning and sequencing of the full-length cDNA for zebrafish activin ßA
Based on a cDNA fragment form the GenBank (accession no. CAB43091) (50), a gene-specific primer for activin ßA was designed for 5'-RACE (rapid amplification of cDNA ends) to amplify the 5'-region of the cDNA using SMART-RACE cDNA Amplification Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The amplified 5'-cDNA fragment was cloned into pBluescript II KS (+) (Stratagene, La Jolla, CA) through T/A cloning and sequenced. A new primer was designed near the 5'-end based on the sequence of the 5'-RACE product and used to amplify the full-length cDNA using 3'-RACE. Sequencing of the full-length cDNA was performed on a series of overlapping subclones generated by exonuclease III and mung bean nuclease deletion. Both strands of the cDNA were sequenced using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA) followed by analysis on the ABI PRISM 310 Genetic Analyzer (Perkin-Elmer).

Validation of semiquantitative RT-PCR assays for activin ßA, ßB, and ß-actin
Reverse transcription (RT) was performed at 42 C for 2 h in a total volume of 10 µl consisting of 3 µl total RNA, 1x Single Strand Buffer (Life Technologies, Inc.), 10 mM dithiothreitol, 0.5 mM each deoxynucleotide triphosphate, 0.5 µg oligo-deoxythymidine, and 100 U SuperScript II (Life Technologies, Inc.).

To optimize the cycle number used for semiquantitative PCR analysis, the RT reaction (0.6 µl) from cultured follicle cells was used as template for PCR amplification. The primers used for activin ßA, ßB, and ß-actin were listed in Table 1Go. PCR was carried out in a volume of 30 µl consisting of 1x PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 2.5 mM MgCl2, 0.2 µM each primer, and 0.6 U of Taq polymerase on the Thermal Cycler 9600 (Eppendorf) for various cycles with the profile of 30 sec at 94 C, 30 sec at 56 C, and 70 sec at 72 C. The PCR products from different cycles of amplification were visualized on a UV-transilluminator after electrophoresis on 1.8% agarose gel containing ethidium bromide, and the signal intensity was quantitated with the Gel-Doc 1000 system and Molecular Analyst Software (Bio-Rad Laboratories, Inc.). The cycle numbers that generate half-maximal amplification were used for subsequent semiquantitative analysis of gene expression, and they are 29 cycles for activin ßA and ßB and 19 cycles for ß-actin (see Fig. 2Go). The specificity of PCR amplification was confirmed by cloning the PCR products into pBluescript II KS (+) (Stratagene) followed by sequencing. To validate the feasibility of the semiquantitative RT-PCR assays, PCR amplification (30 µl) was performed on 3 µl of serially diluted plasmids containing activin ßA, ßB, and ß-actin fragments to evaluate the correlation between the input of template and the output of PCR amplification (see Fig. 2Go).


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  		Table 1. Primer sequences

 


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  		Figure 2. Validation of the RT-PCR assays for activin ßA and ßB. Upper panel, Kinetics of PCR amplification for ßA and ßB. The cycle number that generates half-maximal reaction was used to analyze the expression level of each gene. Lower panel, PCR amplification of cloned ßA and ßB cDNA using the cycle number obtained from the upper panel. Each value represents the mean ± SEM of three PCRs.

 
Data analysis
The mRNA level of each gene was first calculated as the ratio to that of ß-actin, which was amplified simultaneously as the internal control, and then expressed as the percentage of the control group. The data were analyzed by one-way ANOVA followed by Dunnett’s test. We performed all the experiments at least twice to confirm the results using different batches of animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of the full-length cDNA encoding zebrafish activin ßA-subunit
Using 5'- and 3'-RACE amplification, we obtained a full-length cDNA of 1834 bp encoding a protein of 395 amino acids (Fig. 1Go). Sequence comparison in the GenBank shows that the cloned cDNA shares the highest homology with goldfish activin ßA-subunit that we previously cloned (51). A hydrophobic signal peptide can be identified at the N-terminal by hydropathy analysis and the method of von Heijne (52). Interestingly, the deduced amino acid sequence of zebrafish activin ßA precursor is shorter than those of other vertebrates. Two deletions have been noticed in the zebrafish precursor when compared with the molecule from the tetrapods. One deletion of 20 amino acids is observed at position 191/192, and the other one of 19 amino acids is located at position 251/252. These two deletions are also present in goldfish activin ßA (51). The mature activin ßA-subunit (116 amino acids), located at the C-terminal and preceded by two basic amino acids (KR), contains nine cysteines that are fully conserved across vertebrates (51, 53) (Fig. 1Go). Zebrafish activin ßA shows 91, 76, and 78% sequence identity over the mature region with that of goldfish (51), chicken (54), and rat (55), respectively; however, it shares only 56% amino acid sequence identity with activin ßB in the same species (56) (Fig. 1Go).



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  		Figure 1. Amino acid sequence of zebrafish activin ßA-subunit (actßA) (GenBank accession no. AF475092) and its comparison with that of zebrafish activin ßB (56 ). The mature region is in bold.

 
Validation of semiquantitative RT-PCR assays for activin ßA and ßB
Using the cycle numbers that generate half-maximal PCR amplification, we performed PCRs on serially diluted plasmid templates that contain target DNA fragments. As shown in Fig. 2Go, there is a clear linear relationship between template input and the output of PCR amplification for both activin ßA and ßB (data for ß-actin not shown), demonstrating the feasibility of the assays for quantitating mRNA levels after RT.

Gonadotropin regulation of activin ßA and ßB expression in cultured zebrafish ovarian follicle cells
Although our previous study demonstrated that gonadotropin stimulated activin ßA expression in the zebrafish ovary, supporting the view that the ovarian activin is likely one of the target molecules of gonadotropin actions in the ovary (46, 48, 49), the role of activin ßB remains unknown in this regard. Considering that the expression patterns of these two activin subunits are different in mammalian ovaries and their regulation by gonadotropins is still controversial, we performed the present experiments to investigate the expression of both activin ßA and ßB in the zebrafish ovary in response to hCG, a human gonadotropin that has been widely used in fish to mimic the endogenous gonadotropin(s). hCG (15 IU/ml) significantly increased the expression level of activin ßA in a clear time-dependent manner. The effect became evident at 40 min of the treatment and reached maximal level at 2 h. In agreement with our previous result (48), longer treatment with hCG (4 h) caused diminishment of the effect. In sharp contrast to the response of activin ßA, the expression of activin ßB was significantly suppressed by hCG (15 IU/ml). The inhibitory effect was also observed at 40 min of hCG treatment and reached the maximal level at 4 h. Longer treatment (8 h) (data not shown) could not further increase the inhibitory effect (Fig. 3AGo). When tested at 2 h of the treatment, hCG stimulated activin ßA, whereas suppressed activin ßB expression in a dose-dependent manner. The maximal stimulation of activin ßA was observed at 5 IU/ml, whereas the inhibitory effect of hCG was evident at 5 IU/ml and reached maximal level at 15 IU/ml (Fig. 3BGo). To further confirm the novel differential regulation of activin ßA and ßB by hCG, we prepared protein extract from the goldfish pituitary glands, which contains both FSH and LH, and examined its effects on the expression of the two activin subunits. In agreement with the effects of hCG, the goldfish pituitary extract also stimulated activin ßA but suppressed ßB expression in a dose-dependent manner (Fig. 4Go).



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  		Figure 3. Time course (A) and dose response (B) of gonadotropin (hCG) effects on the expression of activin ßA and ßB in the cultured zebrafish ovarian follicle cells. In the time course experiment, hCG (15 IU/ml) was applied to the cultured follicle cells for different times (0–4 h) before RNA extraction, whereas in the dose-response experiment, the cells were treated for 2 h with different doses of hCG (0–15 IU/ml). The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates. +, P < 0.05; ++, **, P < 0.01 vs. control.

 


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  		Figure 4. Effects of goldfish pituitary extract on the expression of activin ßA and ßB in the cultured zebrafish ovarian follicle cells. The cells were treated for 2 h with different doses of the extract (0–30 µg/ml). The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates. ++, **, P < 0.01 vs. control.

 
Response of activin ßA and ßB expression in the follicle cells to IBMX, forskolin, and db-cAMP
Because cAMP has been viewed as the principal second messenger involved in gonadotropin signaling, we further studied the effects of increased intracellular cAMP level on the expression of activin ßA and ßB to demonstrate whether cAMP is involved in the gonadotropin regulation of both activin subunits. Three drugs, IBMX (an inhibitor of phosphodiesterase), forskolin (an activator of adenylate cyclase), and db-cAMP (a cAMP analog), all of which increase the intracellular cAMP level, were used in the experiments. Treatment of the cultured zebrafish follicle cells with IBMX for 2 h significantly increased the expression of activin ßA-subunit in a dose-dependent manner with the peak level at 1 mM. Higher concentration (4 mM) of IBMX had desensitizing effect on the cells and the expression of activin ßA returned to the basal levels at this and higher dosage. In agreement with the effect of hCG, IBMX (1 mM) had strong inhibitory effect on the express of activin ßB-subunit (Fig. 5AGo). Consistent with IBMX, forskolin and db-cAMP also significantly increased activin ßA expression while suppressing the expression of activin ßB (Fig. 5Go, B and C). However, no significant desensitization was noticed for forskolin and db-cAMP over the concentration ranges tested.



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  		Figure 5. Effects of IBMX, db-cAMP, and forskolin on the expression of activin ßA and ßB in the cultured zebrafish ovarian follicle cells. The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates. ++, **, P < 0.01 vs. control.

 
Evaluation of protein kinase A (PKA) involvement in the differential regulation of activin ßA and ßB expression by gonadotropin and cAMP
To examine whether the inverse effects of gonadotropin and cAMP on the expression of the two activin ß-subunits are mediated by cAMP-dependent PKA, we examined the effects of hCG and forskolin on the expression of activin ßA and ßB in the zebrafish follicle cells in the presence or absence of H89, an inhibitor commonly used to suppress PKA. H89 (10 µM) effectively suppressed both basal and hCG (15 IU/ml)- or forskolin (10 µM)-stimulated expression of activin ßA (Fig. 6AGo). As for the expression of activin ßB, hCG (15 IU/ml) and forskolin (10 µM) consistently suppressed its expression level as described above; however, in sharp contrast to the stimulation of activin ßA, the inhibition of activin ßB expression by hCG and forskolin could not be reversed by H89 and the suppression was even enhanced by H89 cotreatment. Interestingly, treatment of the follicle cells with H89 alone also caused a significant decrease in the basal level of activin ßB expression (Fig. 6BGo).



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  		Figure 6. Effects of hCG and forskolin (FK) on the expression of activin ßA and ßB in the absence or presence of H89. The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates. +, *, P < 0.05; ++, **, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have cloned a full-length cDNA of activin ßA-subunit from the zebrafish ovary, which shares 76–78% sequence homology with that of mammals, considerably lower than that of ßB (95%). The discrepancy of sequence conservation between activin ßA and ßB suggests that the two activin subunits have been under different selective pressure in evolution and therefore may play different roles in vertebrate life. Studies on the temporal and spatial patterns of expression of the two activin subunits and their regulation will shed light on their developmental and physiological roles in vertebrates.

The vertebrate ovary undergoes dynamic cyclic changes in the reproductive cycle. Because both activin ßA and ßB are expressed in the ovary of vertebrates including the zebrafish, and they seem to exhibit distinct patterns of expression during the ovarian cycle (32, 33, 34, 46, 48), the ovary therefore serves as an excellent model to investigate the differential function and regulation of the two activin subunits.

In the zebrafish, both activin A and activin B promote the development of oocyte maturational competence and stimulate final oocyte maturation. Follistatin, a specific activin-binding protein that can neutralize biological activities of activin, blocks the activin-stimulated oocyte maturational competence and oocyte maturation in the zebrafish (46, 47, 49). Interestingly, follistatin also suppresses the hCGinduced oocyte maturational competence and oocyte maturation (46, 49), suggesting a role of ovarian activin in the action of gonadotropins. Our previous study showed that the expression of activin ßA-subunit was stimulated by hCG in the cultured zebrafish ovarian follicle cells (48); however, the regulation of activin ßB remains unknown. The present study demonstrated that hCG (15 IU/ml) rapidly increased activin ßA expression in a time- and dose-dependent manner with a strong desensitization after elongated treatment (4 h), consistent with our previous result (48) and the result of Western blot analysis (47). In contrast to its stimulation of activin ßA, hCG significantly suppressed the basal expression of activin ßB under the same conditions. Similarly, the extract prepared from the goldfish pituitary glands also exhibited the same effects on the levels of activin ßA and ßB mRNA, therefore confirming the actions of hCG on the two activin subunits. Because the goldfish pituitary extract contains both FSH and LH, it remains unknown whether these two gonadotropins play any differential roles in controlling activin ßA and ßB biosynthesis, and it would be interesting to investigate this issue when recombinant fish FSH and LH are available. The rapid up-regulation of zebrafish activin ßA by gonadotropin and its response kinetics agree well with the reports in humans (39, 40). Compared with the consistent evidence for the up-regulation of mammalian activin ßA by gonadotropins, there has been limited information on the regulation of activin ßB expression in the ovary, and the results from different models have been somehow inconsistent probably due to the different animals or cell types used (37, 39, 41, 42). In humans, both FSH and LH stimulate activin ßA while suppressing ßB expression in cultured human granulosa-luteal cells within 24 h (39, 42), and hCG also suppresses activin A-stimulated activin ßB expression in the same system (17). The inhibition of ovarian activin ßB expression by gonadotropins has also been noticed in the chicken (Gallus domesticus). FSH treatment for 4 h slightly but significantly suppresses the expression of activin ßB in cultured chicken granulosa cells from large F4 and F5 follicles; however, the effect appeared to be dependent on the stage of follicles and the duration of treatment (57). The stimulation of activin ßA and suppression of activin ßB expression by gonadotropins in the ovary of human and chicken agree well with our findings in the zebrafish ovary, suggesting that the regulatory mechanisms for the biosynthesis of different forms of activin in the ovary by gonadotropins may be well conserved during vertebrate evolution. The physiological relevance of the differential regulation of activin ßA and ßB by gonadotropins in the zebrafish ovary remains unknown, and studies on their expression profiles in vivo during the cycle of follicle development and maturation will provide useful clues to the roles played by the two peptides in ovarian function. In the human ovary, activin ßB is predominantly expressed in the small antral follicle, whereas ßA has a low expression level at this stage. The expression of ßA increases significantly in the preovulatory follicles, whereas ßB expression decreases (34). Interestingly, similar expression patterns of activin ßA and ßB during folliculogenesis have also been demonstrated in the chicken with ßA being abundantly expressed in the large follicles and ßB mainly expressed in the small follicles of early developmental stages (35, 54). Although the mechanisms by which these two subunits are differentially controlled in the human and chicken ovaries in vivo are not very clear yet, the inverse effects of FSH and LH on their expression in vitro in the human suggest that pituitary gonadotropins are likely one of the factors involved.

It is generally accepted that gonadotropins regulate the target gene expression primarily via activation of cAMP-PKA pathway (58). To confirm the inverse effects of hCG on the expression of the two activin subunits, we carried out experiments to examine the response of activin ßA and ßB in the zebrafish follicle cells to pharmacological agents IBMX, forskolin, and db-cAMP, which increase the intracellular cAMP levels by different mechanisms. All the three drugs could mimic the differential regulation of activin ßA and ßB by hCG, suggesting that cAMP is likely the major second messenger involved in the inverse effects of gonadotropin on the two activin subunits. These results are similar to the studies in humans in which cAMP stimulates activin ßA but inhibits ßB expression in cultured granulosa-luteal cells (40, 42). To further evaluate the role of PKA in the differential regulation of the two activin subunits, we examined the effects of hCG and forskolin in combination with H89, a potent PKA inhibitor. Application of H89 not only suppressed the hCG- and forskolin-stimulated ßA expression, but also significantly reduced the basal level of activin ßA expression, suggesting that the cAMP-dependent PKA pathway is most likely a major mechanism for the basal and gonadotropin-stimulated expression of activin ßA, although we cannot rule out the involvement of other kinases that may be influenced by H89. This is well consistent with the fact that a cAMP response element or an element like it exists in the promoters of human and rat activin ßA genes (44, 45). In contrast to its blockade of basal and hCG- or forskolin-stimulated activin ßA expression, H89 could not reverse the inhibitory effects of hCG and forskolin on activin ßB, and its presence seemed to further enhance the inhibition of ßB expression by hCG and forskolin. Furthermore, treatment of the follicle cells with H89 alone significantly reduced the basal expression of activin ßB. These results suggest that although cAMP is likely the second messenger, the inhibition of zebrafish activin ßB expression by hCG and forskolin in the follicle cells may involve an alternative cAMP-dependent but PKA-independent pathway. Based on the lines of evidence presented herein, we hypothesize that although cAMP is the major second messenger in the zebrafish ovary that mediates gonadotropin actions, two distinct pathways, namely PKA-dependent and -independent pathways, are involved in the stimulation of activin ßA and inhibition of activin ßB expression, respectively. However, although the PKA-dependent pathway does not seem to be directly involved in the ßB inhibition, it may have a negative impact on the PKA-independent pathway. The inhibition of PKA by H89 may enhance the inhibitory tone of the latter, leading to a decline in the basal expression of activin ßB and a further suppression in the presence of either hCG or forskolin (Fig. 7Go). The components of the PKA-independent pathway that inhibit activin ßB remain unknown. Recently, Richards and her colleagues (59, 60) demonstrated that in addition to activating PKA in the rat granulosa cells, cAMP also activates several other pathways in response to FSH, including protein kinase B/Akt, serum and glucocorticoid-induced kinase, and MAPK. The roles of these kinases in the zebrafish ovary and their involvement in the regulation of activin expression, particularly the regulation of activin ßB, remain to be elucidated.



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  		Figure 7. Hypothetical model for the mechanisms by which gonadotropin(s) and cAMP stimulate activin ßA and suppress ßB expression in the zebrafish follicle cells. Gonadotropin(s) stimulates activin ßA through the typical cAMP-PKA pathway; however, although cAMP is also the second messenger in the gonadotropin suppression of activin ßB expression, its effect seems to be mediated by a PKA-independent pathway. PKA is likely to influence the expression of activin ßB by exerting a negative impact on this PKA-independent pathway.

 
In summary, a full-length cDNA coding for activin ßA-subunit has been cloned from zebrafish ovary in the present study. Using a primary culture of zebrafish ovarian follicle cells and semiquantitative RT-PCR assays, the present study not only demonstrated that hCG-stimulated activin ßA but suppressed the activin ßB expression, in agreement with the studies in humans, but provided evidence for the first time that different cAMP-dependent signal transduction pathways are involved. The stimulatory effect of hCG on the expression of activin ßA was dependent on the activation of PKA pathway, whereas its inhibition of activin ßB did not seem to involve the activation of PKA. The search for the putative cAMP-dependent but PKA-independent pathway involved in the inhibition of activin ßB expression is now underway in our laboratory, which will contribute to our understanding of the molecular mechanisms of gonadotropin actions in the vertebrate ovary.


   Footnotes
 
The work was substantially supported by Grants CUHK200/96M and CUHK4176/99M from the Research Grants Council of the Hong Kong Special Administrative Region (to W.G.).

Abbreviations: ßAßA, Activin A; ßBßB, activin B; db-cAMP, dibutyryl cAMP; hCG, human chorionic gonadotropin; IBMX, 3-isobutyl-1-methylxanthine; PKA, protein kinase A; RACE, rapid amplification of cDNA ends; RT, reverse transcription.

Received July 22, 2002.

Accepted for publication November 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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