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Regular Article |
a Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-dihydroxy-4-pregnen-3-one (DHP) serves as the most potent maturation-inducing steroid (MIS) to initiate final oocyte maturation. The maturational competence or the responsiveness of oocytes to DHP increases when the ovarian follicles approach the final stage of growth. In the zebrafish, we demonstrated in the present study that full-grown oocytes (
0.7 mm) exhibited the highest maturational competence, which diminished progressively with decreasing size of the follicles. Using midvitellogenic follicles (0.49–0.56 mm), which had little response to DHP, as the material, the present study aimed at investigating the endocrine and paracrine mechanisms that regulate maturational competence of the oocytes. In agreement with the results of studies in other teleost fish, pretreatment of follicles with gonadotropin (hCG) significantly enhanced the responsiveness of midvitellogenic oocytes to DHP in a clear time- and dose-dependent manner. Interestingly, activin, an ovarian growth factor, also had a potent stimulatory effect on the acquisition of oocyte maturational competence. Pretreatment with either recombinant human activin A or goldfish activin B significantly increased the rate of DHP-induced oocyte maturation from 3% to 70%, also in a clear dose-dependent manner. Similar to the results with hCG, pretreatment with activin alone had no effect in inducing maturation of midvitellogenic oocytes without subsequent DHP treatment, although both exhibited a strong effect in promoting maturation of full-grown oocytes. The effect of activin on maturational competence of oocytes could be reduced by cotreatment with follistatin, a potent activin-binding protein. Interestingly, follistatin treatment also significantly reduced the effect of hCG on maturational competence of oocytes, suggesting a mediating role for endogenous activin or activin-related molecules in the action of gonadotropin. The effects of hCG and activin on maturational competence of oocytes could be significantly inhibited by actinomycin D (1 µg/ml) and completely blocked by cycloheximide (1 µg/ml), suggesting that the hCG and activin-induced acquisition of oocyte maturational competence involves de novo protein synthesis at both the transcriptional and translational levels.
activin, follistatin, gametogenesis, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-dihydroxy-4-pregnen-3-one (DHP) that binds directly to its membrane receptor on the surface of full-grown oocytes to initiate final maturation [4, 5]. In the second stage, gonadotropin stimulates production of MIS by the ovarian follicle cells through the interaction of two ovarian follicle cell layers: the thecal cell layer, and the granulosa cell layer. In most teleosts, the thecal layer produces 17-hydroxyprogesterone, which is converted to DHP in the granulosa cells by 20ß-hydroxysteroid dehydrogenase [6]. The effect of gonadotropin on the acquisition of oocyte maturational competence has been reported in several fish species, including kisu (Sillago japonica) [7, 8], tobinumeri-dragonet (Repomucenus beniteguri) [8, 9], Atlantic croaker (Micropogonias undulatus) [3], killifish (Fundulus heteroclitus) [10], blue gourami (Trichogaster trichopterus) [11], and red seabream (Pagrus major) [12, 13]. In the Atlantic croaker, full-grown oocytes do not respond to MIS, which in this species is 17,20ß,21-trihydroxy-4-pregnen-3-one, and the oocytes develop responsiveness to MIS or maturational competence after pretreatment with gonadotropin [3]. In the red seabream, treatment with LH (formerly called gonadotropin-II or GTH II in fish) largely increased the effect of DHP in inducing germinal vesicle breakdown (GVBD) [13]. Although the two-stage model for gonadotropin induction of oocyte maturation has been demonstrated in a number of teleosts, the signaling mechanism by which gonadotropin works in promoting oocyte maturational competence remains unknown. It is generally accepted that the effects of gonadotropins on vertebrate gonadal steroidogenesis and gametogenesis often involve local factors, particularly protein growth factors, that work through autocrine and paracrine mechanisms. Activin is an important member of the transforming growth factor ß (TGFß) superfamily, and its roles in gonadal functions have been extensively studied in mammals [14]. Using zebrafish (Danio rerio) as a model, we have demonstrated that gonadotropin stimulation of the final maturation of full-grown oocytes in the maturational stage is likely mediated by activation of the local activin system [15]. However, it remains unknown whether activin also regulates the development of oocyte maturational competence in the priming stage and mediates the action of gonadotropin.
The purpose of the present study was to further test the two-stage hypothesis for gonadotropin induction of oocyte maturation in the zebrafish and whether the activin system is involved in both stages. Considering that the zebrafish has a short life cycle and that each individual produces a large number of oocytes each day, we believe that this animal model offers advantages for detailed molecular analysis of the two-stage regulation of oocyte maturation. Different from those of Atlantic croaker but similar to those of mammals, the full-grown oocytes of zebrafish undergo spontaneous maturation after isolation from the ovary, and they respond well to stimulation by DHP [15, 16]. To study the development and regulation of oocyte maturational competence in this species, we used midvitellogenic follicles that do not mature spontaneously and that have little response to DHP treatment. The effects of both gonadotropin and activin on the acquisition of maturational competence were examined, and the role of the activin system in the action of gonadotropin was investigated by cotreatment with follistatin, a specific activin-binding protein. To demonstrate if the actions of gonadotropin and activin on the development of oocyte maturational competence involve de novo synthesis of proteins, we also examined the effects of these factors in the presence of actinomycin D or cycloheximide.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually mature zebrafish 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 using commercial tropical fish food (Momizi Tropical; Yeaster Co., Tokyo, Japan) with a supplement of live brine shrimp once or twice a week. The fish were maintained for 4 wk under these conditions before use.
Chemicals and Hormones
All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise stated. Human chorionic gonadotropin and DHP were dissolved in water and ethanol, respectively, and diluted to the desired concentrations with the medium before use. Recombinant human activin A and follistatin were supplied by Dr. A.F. Parlow (National Hormone & Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA). Recombinant goldfish activin B was produced by a Chinese hamster ovary (CHO) cell line established in our laboratory using a cloned cDNA [17] and was partially purified according to the method of Schmelzer et al. [18]. One unit 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 [18, 19]. One unit of goldfish activin B is equivalent to 8 ng of human activin A in the assay.
Isolation and Incubation of Ovarian Follicles
The ovarian follicles were isolated and incubated according to the protocol described earlier [15]. Briefly, the ovaries were removed from four to eight gravid female zebrafish after decapitation and placed in a 50-mm culture dish containing 60% (v/v) medium Leibovitz L-15 (Gibco BRL, Grand Island, NY) [16]. The follicles were carefully separated under a dissecting microscope with the aid of fine forceps and blades, and healthy midvitellogenic follicles of 0.49–0.56 mm in diameter were selected, pooled, and randomly distributed in wells of a 24-well Falcon plate (Becton Dickinson Labware, Franklin Lakes, NJ) for the experiments (30–40 follicles/1 ml/well). The follicles were first incubated at 28°C for 1–6 h in the presence or absence of gonadotropin (hCG) or activin (pretreatment). After changing the medium, the follicles were further incubated for 10 h in the presence or absence of DHP (treatment). The follicles were scored at the end of incubation for those that turned translucent due to GVBD, an easily identifiable marker for oocyte maturation (Fig. 1A). All experiments were repeated three times to confirm the results.
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Data Analysis
All treatments were carried out in quadruplicate. Data regarding the percentage of maturation were analyzed by one-way ANOVA after arc-sine transformation followed by the Fisher least-significance-difference comparison [20].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most of the full-grown zebrafish oocytes gained maturational competence after isolation and responded to DHP in vitro. To investigate the mechanism that regulates development of oocyte maturational competence, it is necessary to use follicles that either lack or have a low level of maturational competence. In this experiment, we examined the maturational competence of the oocytes at different developmental stages, and the stage that exhibited poor responsiveness to DHP was used in all subsequent experiments. The maturational competence of the oocytes increased when the ovarian follicles approached the final stage of growth (vitellogenesis), and midvitellogenic follicles had no spontaneous maturation and exhibited poor response to DHP treatment (5–10%, P < 0.05) (Fig. 1B). The midvitellogenic follicles of 0.49–0.56 mm in diameter were therefore chosen for all subsequent experiments.
Effect of Gonadotropin on Development of Oocyte Maturational Competence
Gonadotropin (hCG) has a potent stimulatory effect on final oocyte maturation in the zebrafish [15, 16, 21]. However, it remains unknown whether hCG also acts in the priming stage to promote development of oocyte maturational competence in this species, as it does in several other species of teleost fish. To answer this question and to validate our assay system, the time course and the dose response of the hCG effect on maturational competence of midvitellogenic oocytes were examined. We found that hCG significantly enhanced the maturational competence of oocytes in a time-dependent manner. Pretreatment of follicles with hCG (20 IU/ml) could significantly promote the responsiveness of the oocytes to subsequent treatment with DHP (5 ng/ml), and the promoting effect of hCG reached the maximal level after 4 h of pretreatment (P < 0.01) (Fig. 2A). Therefore, a pretreatment of 6 h was used in all subsequent experiments. The effect of hCG on the development of maturational competence also exhibited strong dose dependence after the follicles were pretreated with hCG for 6 h, with an ED50 of 2.8 ± 0.3 IU/ml (mean ± SEM) (P < 0.01) (Fig. 2B). The maximal maturational response of the oocytes to DHP treatment (10 h, 5 ng/ml) was achieved by pretreating the follicles with hCG at 20 IU/ml for 6 h (P < 0.01) (Fig. 2C). Pretreatment with hCG alone and without subsequent DHP treatment had no effect on oocyte maturation, and treatment with DHP alone and without hCG pretreatment had poor or little effect in inducing oocyte maturation (Fig. 2).
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Effects of Activins A and B on Development of Oocyte Maturational Competence
To test if activin is involved in the regulation of maturational competence of zebrafish oocytes, the effects of recombinant human activin A and recombinant goldfish activin B on the maturational competence of these oocytes were examined. Both activin A and activin B had a strong stimulatory effect on maturational competence. The responsiveness of the oocytes to subsequent stimulation by DHP was dramatically increased by a 6-h pretreatment of follicles with activin A or B, and the effect was clearly dose dependent. Activin A and activin B started to exhibit a significant effect on the maturational competence at 10 ng/ml and 1 U/ml, respectively, and the maximal response to DHP was induced by 40 ng/ml of activin A and by 4 U/ml of activin B, with an ED50 of 18.8 ± 1.9 ng/ml and 1.8 ± 0.2 U/ml, respectively (P < 0.01) (Fig. 3). As with hCG pretreatment, pretreatment with activin alone for 6 h and without subsequent DHP treatment had no effect on the maturation of oocytes, and treatment with DHP alone and without activin pretreatment had only a limited effect (Fig. 3).
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Blockade of hCG and Activin B Effects by Actinomycin D and Cycloheximide
To investigate the involvement of RNA transcription and protein synthesis in the stimulation of zebrafish oocyte maturational competence by gonadotropin and activin, we examined the effects of actinomycin D, an inhibitor of transcription, and of cycloheximide, an inhibitor of protein synthesis, on hCG- and activin-induced maturational competence. Both drugs were applied to the cultured follicles 2 h before pretreatment with hCG or activin. Due to the limited supply of activin A, we only used recombinant goldfish activin B in this and the subsequent experiment. Actinomycin D significantly suppressed the hCG effect in a dose-dependent manner, with the maximal effect being reached at 1 µg/ml (P < 0.01) (Fig. 4A), and it also significantly attenuated the effect of activin B on the development of oocyte maturational competence (P < 0.01) (Fig. 4B). Interestingly, actinomycin D could not completely block the effects of hCG or activin on the maturational competence of the oocytes, even at a high dosage. By contrast, the stimulatory effects of hCG and activin could be dramatically reduced by cycloheximide in a dose-dependent manner and abolished at 1 µg/ml (P < 0.01) (Fig. 5).
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Blockade of hCG and Activin Effects by Recombinant Human Follistatin
To confirm the specificity of the activin effect on maturational competence and to test the involvement of activin in the action of hCG on maturational competence, we examined the effects of activin B and hCG in the presence of recombinant human follistatin (350 ng/ml), which was applied 2 h before the addition of activin B or hCG. After 6 h of pretreatment with activin B (4 U/ml) or hCG (20 IU/ml), the follicles were challenged with DHP (5 ng/ml) for 10 h. As shown in Figure 6, neither activin nor hCG alone had any effect on oocyte maturation, and DHP alone and without pretreatment had only a limited effect on oocyte maturation (P < 0.01) (Fig. 6). Pretreatment with hCG or activin B could dramatically increase the maturational competence of oocytes. As expected, cotreatment with follistatin significantly suppressed the stimulatory effect of activin B on the acquisition of maturational competence by the oocytes. Interestingly, the effect of hCG was also significantly inhibited by follistatin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we demonstrated that full-grown oocytes (0.7 mm) exhibited the highest maturational competence or responsiveness to DHP, which diminished progressively with decreasing size of the follicles. Using incompetent midvitellogenic follicles as the material, we demonstrated that the two-stage model for oocyte maturation may also be applied to the zebrafish. Unlike the full-grown follicles, the incompetent zebrafish vitellogenic follicles exhibited no spontaneous maturation when incubated in vitro and poor response to DHP stimulation. However, pretreatment of the follicles with gonadotropin (hCG) dramatically increased the responsiveness of vitellogenic follicles to DHP in a clear time- and dose-dependent manner, with the maximal effect being achieved at 4 h of treatment. The promoting effect of hCG could be significantly suppressed by actinomycin D, a potent transcription inhibitor, and completely abolished by cycloheximide, a protein synthesis inhibitor, suggesting the involvement of de novo gene transcription and protein synthesis in the action of gonadotropin. This is in agreement with results reported for other teleosts [22].
Although the promotion of oocyte maturational competence by gonadotropin has been well reported in teleosts [2, 9, 12, 13], the signaling mechanisms by which gonadotropin works remain unknown. Local ovarian growth factors, but not steroids, have been implicated in the gonadotropin stimulation of oocyte maturational competence [22]. Insulin-like growth factor-I (IGF-I) is one of the growth factors with a potential role in the acquisition of oocyte maturational competence [23, 24]; IGF-I stimulates the appearance of gap junctions, which is considered to be a marker for maturational competence, in follicles of the red seabream [2]. In the present study, we demonstrated, to our knowledge for the first time, that activin, an important member of the TGFß superfamily, had a powerful effect in stimulating the development of oocyte maturational competence. Both recombinant human activin A and recombinant goldfish activin B significantly increased the responsiveness of incompetent follicles to the subsequent stimulation by DHP. The effect of activin B could be suppressed by actinomycin D and abolished by cycloheximide, suggesting that activin may act through regulating gene activity or protein synthesis. That actinomycin D could only partially suppress the effects of hCG and activin B, whereas cycloheximide could completely abolish them, suggests that the mechanisms of their actions may involve translation of proteins from the existing messengers. In addition, cotreatment with follistatin, a specific activin-binding protein, could significantly suppress the stimulatory effect of activin B, therefore confirming the specificity of activin action. Interestingly, when applied together with hCG, follistatin also partially inhibited the stimulatory effect of hCG on the development of oocyte maturational competence. This strongly suggests that the endogenous ovarian activin is likely a downstream mediator of gonadotropin action during the developmental course of oocyte maturational competence, and this result further strengthens the hypothesis we formulated in our previous study, that pituitary gonadotropin regulates fish ovarian functions by activating, at least partially, the ovarian activin system [15]. To provide direct evidence for the role of activin in gonadotropin-regulated ovarian functions, studies are now underway in our laboratory to examine the regulation of expression of activin subunits and activin receptors by gonadotropin.
Although the effect of activin during the development of oocyte maturational competence involves new protein synthesis, as suggested by the results of the present study, the identities of these proteins that contribute to the acquisition of oocyte maturational competence remain unknown. One possibility is that activin may directly act on the surface of the oocytes to stimulate biosynthesis of the receptor for MIS (DHP in the zebrafish), which has been localized on the surface of oocytes in several teleost species and increases in abundance during in vivo oocyte maturation [22, 25–27]. The expression of activin type II and/or IIB receptors in oocytes has been demonstrated in a variety of vertebrates, including the zebrafish [28], Xenopus [29], and mammals [30, 31], suggesting direct actions of activin on the oocytes in these species. It has also been documented in the spotted seatrout (Cynoscion nebulosus) that, during the development of oocyte maturational competence, the concentration of membrane receptor for MIS increases significantly [32], suggesting that this could be a potential target molecule regulated by various endocrine or paracrine factors, including activin, in inducing oocyte maturational competence. This notion is supported by recent evidence from the spotted seatrout that the abundance of MIS receptor on the oocyte plasma membrane is closely correlated with the development of oocyte maturational competence, and that gonadotropins (FSH, LH, and hCG) and IGF-I, both of which stimulate the development of oocyte maturational competence and final maturation in teleosts, could significantly increase the level of membrane MIS receptor in the oocytes of spotted seatrout, although IGF-I seemed to have a lower potency than gonadotropins [32]. Further studies regarding the effect of activin on the concentration of MIS receptor in oocytes will shed light on the mechanism of activin action in this regard.
In summary, the present results not only support the two-stage hypothesis for oocyte maturation and its regulation by gonadotropin but also provide important information regarding the paracrine role played by the ovarian activin system in oocyte maturation. Both gonadotropin and activin had a significant promoting effect in the priming stage to stimulate the acquisition of oocyte maturational competence, and the effect of gonadotropin could be inhibited by follistatin, suggesting involvement of the ovarian activin system in the action of gonadotropin. More studies on the regulation of zebrafish ovarian activin subunits and receptors by gonadotropin are necessary to shed light on the functional role of activin in the ovary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Earmarked Research Grant CUHK200/96M from the Research Grants Council of Hong Kong to W.G. 
2 Correspondence. FAX: 852 2603 5745; weige{at}cuhk.edu.hk
Accepted: September 5, 2001.
Received: May 8, 2001.
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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. [Abstract] [Full Text] [PDF]
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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. [Abstract] [Full Text] [PDF]
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R. Patino, G. Yoshizaki, D. Bolamba, and P. Thomas Role of Arachidonic Acid and Protein Kinase C During Maturation-Inducing Hormone-Dependent Meiotic Resumption and Ovulation in Ovarian Follicles of Atlantic Croaker Biol Reprod, February 1, 2003; 68(2): 516 - 523. [Abstract] [Full Text] [PDF]
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