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Biology of Reproduction 61, 987-992 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Activin Stimulation of Zebrafish Oocyte Maturation in Vitro and Its Potential Role in Mediating Gonadotropin-Induced Oocyte Maturation1

Yefei Panga, and Wei Ge2,a

a Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin plays important roles in the regulation of vertebrate reproduction. Using zebrafish, Danio rerio, as a model, the present study aimed at investigating the role of activin in the regulation of final oocyte maturation. Administration of recombinant goldfish activin B significantly increased the rate of oocyte maturation in vitro in a dose- and time-dependent manner. The effect of activin seemed to be additive to the effects of gonadotropin (hCG) and 17{alpha},20ß-dihydroxyprogesterone, a potent maturation-inducing hormone in teleosts. The specificity of the activin action was confirmed by coincubation with recombinant human follistatin, which completely abolished the stimulatory effect of activin B. Interestingly, follistatin also significantly inhibited hCG-induced oocyte maturation, suggesting that endogenous activin may be a downstream mediator of gonadotropin actions. No effect of activin B was observed in the presence of actinomycin D, indicating that the action of activin may involve changes in transcriptional activity. These results, together with the demonstration that activin and its type II receptor are expressed in the zebrafish ovary, strongly suggest a paracrine/autocrine role for activin in the controlling of final oocyte maturation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vertebrate oocytes are arrested at prophase I of meiosis, during which they undergo a lengthy period of growth. Meiosis is resumed during the final oocyte maturation, which is initiated by a surge release of gonadotropins, especially LH, from the pituitary. Although pituitary gonadotropins are critical in inducing oocyte maturation in all vertebrates, increasing evidence also suggests important roles for the local ovarian factors such as steroids and nonsteroidal substances in the event. A major group of nonsteroidal substances that are implicated in the regulation of ovarian functions, including final oocyte maturation, is the activin/inhibin family of growth factors.

Activin (ßAßA, ßAßB, or ßBßB) is a dimeric protein consisting of two similar but distinct subunits, ßA and ßB. Although initially recognized as an ovarian protein that stimulates the secretion of pituitary FSH [1, 2], activin has been shown to have diverse biological activities in a variety of tissues [3]. Activin exerts its actions by binding to its specific membrane receptors. Two types of receptors, namely activin type I and type II receptors, are involved in activin signaling. The type II receptors are responsible for ligand recognition and binding, whereas the type I receptors initiate the process of signal transduction after its activation by the activin type II receptor complex [4]. Inhibin ({alpha}ßA or {alpha}ßB) is also a dimeric protein that shares a ß subunit with activin; however, in many cases, the effects of inhibin are antagonistic to those of activin. Recent evidence supports the hypothesis that inhibin serves as a natural antagonist of activin by binding and blocking activin receptors [5, 6]. Follistatin is a specific binding protein of activin, and its function is probably to fine-tune the activity of activin in local tissues [7]. Although numerous extragonadal activities have been reported for activin including pituitary FSH stimulation [3], the ovary, where activin was initially identified, remains one of the major organs for activin production and function. The expression of activin subunits in the ovary has been localized in the follicular granulosa cells by immunocytochemistry or in situ hybridization in a variety of species including the rat [8], human [9], sheep [10,11], and goldfish [12]. These findings, together with the evidence that activin receptors are also expressed in the ovary [13–15], strongly suggest potential paracrine/autocrine roles for activin in the regulation of vertebrate ovarian functions. Although the possibility still exists that ovarian activin may serve as a hormone to regulate pituitary gonadotropin secretion and expression as initially proposed [16], it is now generally believed that activin mainly acts as a local paracrine/autocrine factor to regulate ovarian functions [17].

The potential role of activin in the regulation of oocyte maturation has been investigated in a number of mammalian species with controversial results. It has been demonstrated that activin A promotes in vitro oocyte maturation in the rat [18, 19], cow [20], rhesus monkey [21], and human [22], and its effect could be blocked by coincubation with follistatin [19, 21]. However, there have also been reports that activin has no effect on the final oocyte maturation in the rat [23] and pig [24]. On the other hand, inhibin has been reported to have either a stimulatory or an inhibitory effect on oocyte maturation in vitro in the cow [20] and rat [23], respectively. Although the discrepancies could be due to different in vitro culture conditions and species used, the difficulty in obtaining sufficient oocytes with consistent quality in mammalian species seems to have limited detailed analysis.

Using zebrafish, Danio rerio, as a model, the present study aimed to investigate the effect of a recombinant fish activin B (ßBßB) on the rate of oocyte maturation and its relation with other maturation-promoting factors. The zebrafish was chosen because of its small body size, daily spawning behavior, and ability to produce a large number of fully grown oocytes, as well as its increasing popularity as an excellent vertebrate model for developmental studies. The results from the present study not only contribute to our understanding of the mechanisms underlying vertebrate oocyte maturation and its endocrine and paracrine/autocrine regulation; they also pave the way for future investigation into the issue at the molecular level.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), and restriction enzymes were from Promega (Madison, WI) unless otherwise stated. Human CG and 17{alpha},20ß-dihydroxyprogesterone (DHP) were purchased from Sigma. Human CG was dissolved in the medium directly, whereas DHP was first dissolved in ethanol and then diluted to the desired concentrations with the medium before use. Recombinant goldfish activin B was produced by a Chinese hamster ovary (CHO) cell line established in our laboratory and purified according to Schmelzer et al. [25]. One unit (U) of activin B is defined as the amount per milliliter that induces a half-maximal differentiation of F5-5 cells (ED50) in the erythroid differentiation factor assay according to Eto et al. [26] and Schmelzer et al. [25].

Animals

Young zebrafish (Danio rerio) were purchased from local pet stores and maintained without separation of males and females in flow-through aquaria (36 L) at 25°C on a 14L:10D photoperiod. The fish were fed twice a day with commercial tropical fish food with supplement of live brine shrimp once or twice a week.

Extraction of Total RNA and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from the zebrafish ovary with Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the protocol of the manufacturer. Reverse transcription (RT) was performed at 42°C for 3 h in a total volume of 10 µl consisting of 5–8 µg RNA, single-strength first strand buffer (Gibco, Gaithersburg, MD), 10 mM dithiothreitol, 0.5 mM each dNTP, 0.5 µg/µl oligo-dT, and 100 U Superscript II (Gibco). Polymerase chain reaction (PCR) was carried out in a volume of 50 µl consisting of single-strength PCR buffer, 0.2 mM each dNTP, 2.5 mM MgCl2, 0.2 µM each primer, and 1 U of Taq polymerase. The primers used were zActßB-1 (5'-CAACTTAGATGGACACGCTG-3') and zActßB-2 (5'-GTGGATGTCGAGGTCTT GTC-3') for activin ßB [27], and zActRII-1 (5'-TTATACTGAAATGACACGAG-3') and zActRII-2 (5'-AAGAGTGTTGGACCATGAAG-3') for activin type II receptor (GenBank accession no. AA497191). After an initial denaturation for 4 min at 94°C, 30 cycles of reaction were performed on the Thermal Controller PTC-100 (MJ Research, Watertown, MA) with the cycling profile of 30 sec at 94°C, 30 sec at 58°C, and 1 min at 72°C followed by a 5-min extension at 72°C. PCR reaction (5 µl) was electrophoresed on agarose gel to visualize the products. The specificity of the reactions was confirmed by cloning the PCR products into pBluescript II KS(+) (Stratagene, La Jolla, CA) followed by sequencing on ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Isolation and Incubation of Follicles

The ovaries were removed from gravid female zebrafish after decapitation and placed in a 35-mm culture dish containing 60% medium Leibovitz L-15 (Gibco) [28]. The follicles from 4 to 7 females were carefully separated with the aid of fine forceps and blades, and the healthy follicles of 0.58–0.65 mm in diameter were selected, pooled, and randomly distributed in wells of 24-well plates for the experiments.

The incubation of follicles was modified from that reported by Selman et al. [28]. Briefly, the follicles (30–40 per well) were cultured at 28°C for up to 24 h in 1 ml 60% Leibovitz L-15 in 24-well plates (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ). The follicles were scored at different times of incubation for those that had turned translucent due to germinal vesicle breakdown (GVBD), an easily identifiable marker for oocyte maturation. All the experiments were repeated two to three times to confirm the results.

Data Analysis

The data of percentage maturation were analyzed by one-way ANOVA after arcsin transformation followed by Fisher's least-significance difference comparison.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Activin ßB and Activin Type II Receptor in the Zebrafish Ovary

To demonstrate that activin is produced in the zebrafish ovary and that it serves as a potential paracrine/autocrine factor, we examined the expression of activin ßB subunit and activin type II receptor in the zebrafish ovary by RT-PCR. Both activin ßB (382 base pairs [bp]) and activin type II receptor (520 bp) were detected in the zebrafish ovary (Fig. 1). PCR was also performed on zebrafish ovarian RNA without reverse transcription as the negative control, and no visible products were obtained. The identity of the PCR products was confirmed by cloning and sequencing followed by GenBank comparison.



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  		FIG. 1. Detection of activin ßB subunit and activin type II receptor in the zebrafish ovary by RT-PCR. M, Molecular marker; +, with reverse transcription; -, without reverse transcription

Effects of hCG and DHP on Oocyte Maturation

Both hCG and DHP have been demonstrated to promote zebrafish oocyte maturation in vitro [28]. To validate our in vitro follicle isolation and incubation system, we tested the responsiveness of follicles to hCG and DHP. In agreement with the previous study [28], hCG stimulated zebrafish oocyte maturation in a clear dose- and time-dependent manner. Significant effect of hCG was observed as early as 2 h after treatment, and maximal effect was achieved at 1 IU/ml after 12-h incubation (Fig. 2A). As a potent maturation-inducing hormone as demonstrated in many teleost species [29], DHP also significantly promoted zebrafish oocyte maturation in a dose- and time-dependent manner. DHP at 0.2 ng/ml caused maximal response after 12-h incubation, and longer treatment did not seem to further increase the response (Fig. 2B).



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  		FIG. 2. Dose response and time course of zebrafish oocyte GVBD induced by hCG (A), DHP (B), and recombinant goldfish activin B (C). Each value represents the mean ± SEM of four determinations. A and C are each representative of three independent experiments, and B is representative of two independent experiments. *, P < 0.05, compared with the control of the same time point.

Effect of Activin B on Oocyte Maturation

To test whether activin is involved in the regulation of oocyte maturation in the zebrafish, we examined the effect of recombinant goldfish activin B on the maturation rate of zebrafish oocytes in vitro. The results showed that goldfish activin B had a potent stimulatory effect on final oocyte maturation and that the effect was time- and dose-dependent. The effect became significant within 2 h after addition of activin and reached the maximal level after 12 h of treatment, with ED50 of approximately 0.76 U/ml (Fig. 2C). Although the rate of spontaneous maturation varied among experiments, the stimulatory effect of activin was consistent and highly reproducible.

Interactive effects of activin B, hCG, and DHP on oocyte maturation When activin B (2 U/ml) was applied together with hCG (1 IU/ml) or DHP (1 ng/ml), the maturation rate was higher than with either activin B or hCG and DHP alone. The effect of activin seemed to be additive to that of hCG or DHP (Fig. 3, A and B).



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  		FIG. 3. Interactive effects of activin B and hCG (A) or DHP (B) on zebrafish oocyte maturation. Each value represents the mean ± SEM of four determinations. A and B are representative of two and three independent experiments, respectively. *, P < 0.05, compared with the control of the same time point. #, P < 0.05, compared with activin-, hCG-, or DHP-treated groups of the same time point.

Blockade of Activin B and hCG Effects by Recombinant Human Follistatin

To test the specificity of activin's effect on zebrafish oocyte maturation, we examined the effect of activin B in the presence of recombinant human follistatin (350 ng/ml), which was applied 1 h before the addition of activin B. As shown in Figure 4A, the stimulatory effect of activin B was completely abolished by human follistatin, and no effect was observed up to 12 h of treatment. However, follistatin alone did not seem to have significant effect on the rate of spontaneous maturation in the present study. Interestingly, application of follistatin in the same way also significantly suppressed the effect of hCG on oocyte maturation, but not completely (Fig. 4B).



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  		FIG. 4. Blockade of activin (A) and hCG (B) effects on GVBD by recombinant human follistatin. Each value represents the mean ± SEM of four determinations. A and B are each representative of three independent experiments. *, P < 0.05, compared with the control of the same time point. #, P < 0.05, compared with activin- or hCG-treated groups of the same time point.

Blockade of Activin B Effect by Actinomycin D

To understand whether the action of activin in inducing zebrafish oocyte maturation involves changes in the transcriptional activity, we performed experiments to examine the effect of actinomycin D (2 µM), a potent inhibitor of transcription, on the activin-induced oocyte maturation. The results showed that the effect of activin B on oocyte maturation after 12-h treatment was abolished by coincubation with actinomycin D (Fig. 5). However, actinomycin D alone did not cause any significant reduction in the rate of spontaneous maturation.



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  		FIG. 5. Blockade of activin stimulation of zebrafish oocyte maturation by 12-h coincubation with actinomycin D (2 µM). Each value is the mean ± SEM of four determinations, and data shown are typical of three independent experiments. *, P < 0.05, compared with control treatment and treatment with activin plus actinomycin D.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vertebrate oocyte maturation is a dramatic yet complex physiological process whose regulation involves multiple endocrine and paracrine/autocrine factors [30]. The present study demonstrated that recombinant goldfish activin B has a potent stimulatory effect on final oocyte maturation in the zebrafish. This is in agreement with results reported in the rat [18, 19], cow [20], rhesus monkey [21], and human [22], indicating that promotion of oocyte maturation by activin is a well-conserved function across vertebrates. In fishes, Garg and Peng [31] also observed a stimulatory effect of human activin A on zebrafish oocyte maturation; however, there has been no detailed report of that study. On the other hand, activin A has been reported to have no effect on oocyte maturation in vitro in the red seabream, Pagrus major [32]. The discrepancy could be due to differences in the sources of activin and species used. Instead of using mammalian activin A, we used recombinant goldfish activin B in the present study. Our previous work demonstrated that activin ßB is structurally much more conserved in vertebrate evolution than ßA subunit [33], and goldfish activin ßB shares 98% amino acid sequence identity with that of zebrafish activin ßB [27, 34]. The specificity of activin action observed in the present study was confirmed by the demonstration that the effect of recombinant goldfish activin B could be completely abolished by cotreatment with recombinant human follistatin, a specific binding protein for activin [7]. The stimulatory effect of activin on final oocyte maturation, together with our evidence that activin ßB subunit and activin type II receptor are both expressed in the zebrafish ovary, strongly suggests potential paracrine/autocrine roles for endogenous activin in the regulation of the event. This may also be true of other teleost species, as the expression of activin and its receptor has also been demonstrated in the ovaries of goldfish, Carassius auratus [14, 34], and medaka, Oryzias latipes [27].

Similar to those in mammalian species, fully grown zebrafish oocytes undergo spontaneous maturation in vitro with the percentage varying between experiments. It should be noted that although follistatin could abolish the effect of exogenous activin, it did not seem to affect the rate of spontaneous zebrafish oocyte maturation, implying that the occurrence of spontaneous oocyte maturation in vitro may not involve endogenous activin. Interestingly, follistatin treatment also significantly inhibited the stimulation of oocyte maturation by hCG. This leaves open the possibility that activation of the activin system in the follicles may be a downstream event of gonadotropin action in the induction of final oocyte maturation. Studies are now under way in our laboratory to investigate whether gonadotropin-induced oocyte maturation in the zebrafish involves increased expression of activin subunits or activin receptors. In mammals, it is well documented that pituitary FSH stimulates the expression of activin subunits in the granulosa cells [35–38]. In the Japanese eel, Anguilla japonica, it has been demonstrated that gonadotropin (hCG) stimulates activin ßB expression in the testis; and, as a downstream mediator of gonadotropin action, activin B seems to play a critical role in promoting spermatogenesis both in vivo and in vitro [39, 40]. By analogy with the function of activin B in eel spermatogenesis, the regulation of zebrafish oocyte maturation by activin B as demonstrated in the present study raises the possibility that similar endocrine and paracrine/autocrine mechanisms may be involved in the regulation of gametogenesis in both male and female teleosts.

It is generally accepted that most of activin's actions in various biological systems involve alteration of transcriptional activity. Our experiment using actinomycin D clearly demonstrated that the effect of activin in inducing final oocyte maturation in the zebrafish may be mediated at the transcriptional level. Further characterization of the genes regulated by activin during oocyte maturation will be necessary for a better understanding of the regulatory mechanism of activin in the event.

In summary, the present study demonstrated that activin and its type II receptor are expressed in the zebrafish ovary, and that exogenous goldfish activin B stimulates zebrafish oocyte maturation in vitro. The effect could be blocked by recombinant human follistatin, therefore confirming the specificity of the action. Evidence from the present study also suggests that pituitary gonadotropin(s) may, at least partially, promote oocyte maturation by stimulating the activity of the endogenous activin system, which modulates the activities of other genes at the transcriptional level.


   ACKNOWLEDGMENTS
 
We thank NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases, USA) for providing recombinant human follistatin.


   FOOTNOTES
 
1 This research was supported by the Earmarked Research Grant CUHK200/96M from the Research Grants Council of Hong Kong to W.G. Back

2 Correspondence: Wei Ge, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. FAX: 852 2609 5646; weige{at}cuhk.edu.hk Back

Accepted: May 14, 1999.

Received: February 22, 1999.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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W.-K. So, H.-F. Kwok, and W. Ge
Zebrafish Gonadotropins and Their Receptors: II. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Subunits--Their Spatial-Temporal Expression Patterns and Receptor Specificity
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H.-F. Kwok, W.-K. So, Y. Wang, and W. Ge
Zebrafish Gonadotropins and Their Receptors: I. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Receptors-- Evidence for Their Distinct Functions in Follicle Development
Biol Reprod, June 1, 2005; 72(6): 1370 - 1381.
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Y. Wang and W. Ge
Developmental Profiles of Activin {beta}A, {beta}B, and Follistatin Expression in the Zebrafish Ovary: Evidence for Their Differential Roles During Sexual Maturation and Ovulatory Cycle
Biol Reprod, December 1, 2004; 71(6): 2056 - 2064.
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 EndocrinologyHome page
T.-T. Wong and Y. Zohar
Novel Expression of Gonadotropin Subunit Genes in Oocytes of the Gilthead Seabream (Sparus aurata)
Endocrinology, November 1, 2004; 145(11): 5210 - 5220.
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Y. Wang and W. Ge
Cloning of Epidermal Growth Factor (EGF) and EGF Receptor from the Zebrafish Ovary: Evidence for EGF as a Potential Paracrine Factor from the Oocyte to Regulate Activin/Follistatin System in the Follicle Cells
Biol Reprod, September 1, 2004; 71(3): 749 - 760.
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Y. Wang and W. Ge
Spatial Expression Patterns of Activin and Its Signaling System in the Zebrafish Ovarian Follicle: Evidence for Paracrine Action of Activin on the Oocytes
Biol Reprod, December 1, 2003; 69(6): 1998 - 2006.
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 EndocrinologyHome page
Y. Wang, A. O. L. Wong, and W. Ge
Cloning, Regulation of Messenger Ribonucleic Acid Expression, and Function of a New Isoform of Pituitary Adenylate Cyclase-Activating Polypeptide in the Zebrafish Ovary
Endocrinology, November 1, 2003; 144(11): 4799 - 4810.
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 EndocrinologyHome page
Y. Wang and W. Ge
Involvement of Cyclic Adenosine 3',5'-Monophosphate in the Differential Regulation of Activin {beta}A and {beta}B Expression by Gonadotropin in the Zebrafish Ovarian Follicle Cells
Endocrinology, February 1, 2003; 144(2): 491 - 499.
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 Toxicol PatholHome page
J. M. Spitsbergen and M. L. Kent
The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research--Advantages and Current Limitations
Toxicol Pathol, January 1, 2003; 31(1_suppl): 62 - 87.
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C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer
Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium
Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752.
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A. E. Drummond, M. T. Le, J.-F. Ethier, M. Dyson, and J. K. Findlay
Expression and Localization of Activin Receptors, Smads, and {beta}glycan to the Postnatal Rat Ovary
Endocrinology, April 1, 2002; 143(4): 1423 - 1433.
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Y. Pang and W. Ge
Gonadotropin and Activin Enhance Maturational Competence of Oocytes in the Zebrafish (Danio rerio)
Biol Reprod, February 1, 2002; 66(2): 259 - 265.
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Y. Pang and W. Ge
Epidermal Growth Factor and TGF{alpha} Promote Zebrafish Oocyte Maturation in Vitro: Potential Role of the Ovarian Activin Regulatory System
Endocrinology, January 1, 2002; 143(1): 47 - 54.
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T. Wu, H. Patel, S. Mukai, C. Melino, R. Garg, X. Ni, J. Chang, and C. Peng
Activin, Inhibin, and Follistatin in Zebrafish Ovary: Expression and Role in Oocyte Maturation
Biol Reprod, June 1, 2000; 62(6): 1585 - 1592.
[Abstract] [Full Text]

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