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a Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and one of the two ß subunits, ßA and ßB, respectively. Activin A and activin B are homodimers of the ßA and ßB subunits, respectively, while activin AB is the heterodimer of the ßA and ßB subunits [1–5]. The in vivo production of these forms of activin has been characterized in mammals [1–6]. All three forms of activin proteins have also been isolated and purified from Xenopus [7, 8]. Molecular cloning and immunological studies in a variety of animals reveal that these forms of activin may be also present in other lower vertebrates. However, cDNA for subunit has been cloned from only several mammals [9–14], and chickens [15], but not from lower vertebrates. Studies in mammals have shown that activins and inhibins are involved in many physiological processes, including reproduction. Within the reproductive system, activins and inhibins have been found to regulate the release of GnRH from the hypothalamus [16] and FSH from the pituitary [1–5]. In addition, activins and inhibins exert many regulatory effects on the testis and ovary, such as steroidogenesis, proliferation of spermatogonia, proliferation of granulosa cells, modulation of FSH receptors, follicle development, and maturation [4, 5, 17]. In most biological systems tested, activin and inhibin have opposite effects [4, 5].
Biological activities of activins are mediated through specific cell surface receptors, known as serine/threonine kinase receptors [18, 19]. A functional receptor is a complex comprising two different proteins, namely the type I and type II receptors. Complementary DNAs encoding two subtypes of the type II receptor (ActR-IIA and -IIB) and two subtypes of the type I receptor (ActR-IA and -IB) have been cloned from a variety of species [18–22]. Activin first binds to the type II receptor, and this complex recruits the type I receptor. The type II receptor subsequently phosphorylates the type I receptor, which in turn phosphorylates downstream signals, such as Smads [23–25]. The structure of the inhibin receptor is unknown, although specific binding sites for inhibin have been characterized in several tissues, such as pituitary [26], ovary [27], and testis [28]. Inhibin also shows affinity for type II activin receptors [29].
Follistatins (FS) are monomeric glycoproteins that bind activins with high affinity and inhibin with lower affinity [30]. Multiple forms of FS have been characterized in mammals. FS-288 and FS-315 are derived from alternative splicing of FS mRNA, whereas FS-300 and FS-303 are proteolytic products of FS-315 [31, 32]. In tissues and biological fluids, FS form complexes with activins and inhibins [33]. FS have been shown to neutralize bioactivities of activin by preventing the interaction of activins with their receptors and by increasing the breakdown of activin through enhancement of activin uptake into cells [30].
Oocyte maturation has been extensively studied in teleosts. Three major factors are known to be important in this process; gonadotropin, maturation-inducing hormone (MIH; 17, 20ß-dihydroxyprogesterone), and maturation promoting factor (MPF) [34]. Gonadotropin stimulates the production of MIH by follicular cells, which in turn acts on the oocyte surface to activate MPF [34]. Recently, we have cloned cDNA encoding ActR-IIB and found that ActR-IIB mRNA is expressed in the oocyte at different stages of development in zebrafish [35]. These results have led us to hypothesize that activins may regulate oocyte development and maturation in fish. To test this hypothesis, we have examined the role of activin A and its related molecules, inhibin A and FS-288, in oocyte maturation. In addition, the presence of these molecules in the zebrafish ovary was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult zebrafish, Danio rerio, were obtained from a local aquarium and maintained in the laboratory according to the procedures of Westerfield [36]. They were kept in filtered and aerated tanks at 28°C under a 14L:10D photoperiod. Experiments were performed according to the Guide to the Care and Use of Experimental Animals published by Canadian Council on Animal Care.
Total RNA Extraction, Reverse Transcription (RT), and Polymerase Chain Reaction (PCR)
Zebrafish were anesthetized with MS222 (Sigma-Aldrich Canada, Oakville, ON, Canada) and killed by decapitation. Ovaries were removed, and total RNA was extracted using Trizol reagent (Canadian Life Technologies, Burlington, ON, Canada). Five micrograms of total RNA were reverse-transcribed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Canadian Life Technologies) and oligo-dT12–18 primer (Amersham-Pharmacia Biotech, Oakville, ON, Canada) in a 50-µl reaction. Two microliters from the first-strand cDNA synthesis reaction were used in one PCR. Primers made to detect ßA subunit mRNA were a sense primer based on a conserved region of the known ßA cDNA sequences and an antisense primer specific for the zebrafish activin ßA (according to the partial sequence deposited in the Genbank, accession #AJ238980). Primers for FS were designed on the basis of the zebrafish FS cDNA sequence [37]. The sequences of the primers are were as follows: ßA1, 5' ACT TTG AGA TTT CCA AGG AAG 3'; ßA2, 5' TGT ACC CTC GGA TAC GGT AGT 3'; FS1, 5' GAT CCA TCG GCG TGG CAT ATG 3'; and FS2, 5' GTA GCT TAA CTC TTA GCA GCA 3'. PCR was carried out in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 50 µM dNTPs, 10 pmol primers, and 1 U Taq DNA polymerase (Canadian Life Technologies). Ten cycles of PCR (denaturing at 94°C for 20 sec; annealing for 30 sec at 57–52°C for FS or 55–50°C for activin ßA, with a decrease of 0.5°C for each cycle; and extension at 72°C for 50 sec) were performed, followed by 25 cycles with the same profile except that the annealing temperatures were 52°C for FS and 50°C for activin ßA.
Sequencing Analysis
To confirm the identities of PCR products, DNA fragments were recovered from gels using Gene Clean II kit (Bio 101, Vista, CA) and were sequenced using PCR primers. All sequencing runs were performed using a DNA sequencer (Applied Biosystems, Inc., Foster City, CA) at the York University (Toronto, ON, Canada) Core Facility for Molecular Biology. Sequencing data were analyzed using the Blast program (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD; http:\\www.ncbi.nlm.nih.gov/).
Protein Extraction and Western Blot Analysis
Fish were anesthetized using MS-222 (Sigma) and decapitated. Ovaries were subsequently removed and washed in Cortland's balanced saline. Concentrations of solubilized proteins were determined using a Bio-Rad Protein Assay Kit (Bio-Rad Labs., Richmond, CA). Proteins were separated in a 12.5% SDS-polyacrylamide gel under nonreducing conditions using a Bio-Rad mini-gel apparatus. After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham-Pharmacia). After incubation with the blocking solution (Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk) overnight at 4°C, the membrane was incubated with a monoclonal mouse anti-human activin ßA antibody (1:200 dilution; Cedarlane Laboratories Ltd., Hornby, ON, Canada) or mouse anti-human FS antibody (1:5000 dilution, obtained from Dr. A.F. Parlow, National Hormone and Pituitary Program [NHPP], Rockville, MD), overnight at 4°C. Subsequently, the membranes were washed and then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated (anti-mouse IgG diluted 1:3000 for ßA or anti-rabbit IgG diluted 1:50 000 for FS; Amersham-Pharmacia). Immunoreactive signals were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham-Pharmacia).
In Vivo Injection
Human CG (Sigma) was dissolved in 0.9% NaCl solution in a concentration of 20 IU/ml. Each fish received 50 µl of saline (control) or hCG solution through i.m. injection. At 12 h, 24 h, and 30 h after injections, fish were killed, and ovaries were removed for protein extraction. The ovaries were homogenized in ice-cold 50 mM Tris-HCl (pH 7.2) containing 1 mM EDTA and 1 mM PMSF using a motor-driven polypropylene pestle. The homogenate was centrifuged at 10 000 x g, and the resultant supernatant was used for electrophoresis. Total protein from each of the control or treated samples was mixed with SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 0.05% bromophenol blue, 10% glycerol), and approximately 15-µg protein was loaded in each lane.
In Vitro Culture of Zebrafish Follicles
Ovaries were removed from zebrafish as described above and placed in a Petri dish in modified Cortland's medium [38]. Follicles were isolated through manual dissection and separated into groups according to size. For each experiment, follicles collected from 4–5 fish were pooled, and 15–25 follicles were placed into each well of a 24-well culture plate in 1 ml of medium. They were then incubated with either Cortland's medium alone (control) or hormones at room temperature (25°C). At 18 and 24 h after incubation, the rate of maturation was scored. Germinal vesicle breakdown (GVBD) has been used as an index for maturation, and it can be observed under a dissecting microscope equipped with transmitted light after follicles are placed in a clearing solution (5% acetic acid in Cortland's medium) for 10 min. Follicles that have undergone GVBD can also be identified because they become translucent. Both methods were used to score percentage of GVBD. There was no difference in percentage of GVBD scored between two different methods used. Recombinant human (rh) activin A, rh-inhibin A, and rh-FS-288 were kindly provided by Dr. A.F. Parlow (NHPP).
Statistical Analysis
All values are expressed as the mean ± SEM of pooled data from 2–4 experiments. Multiple group comparisons were performed by one-way ANOVA, followed by Scheffe's multiple-comparison procedure, using the Statistical Analysis System (SAS) program (Cary, NC). Unpaired Student's t-tests were used if only two groups were to be compared. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the expression of activin A, inhibin A and FS in the zebrafish ovary, RT-PCR was first performed using cDNA prepared from zebrafish ovary. A sense primer from a conserved region of ßA cDNA and an antisense primer specific for zebrafish ßA subunit were used to detect ßA mRNA expression. As shown in Figure 1A, a 645-base pair (bp) DNA fragment of the expected size was generated upon amplification of ovary cDNA, but not of the control (omitted cDNA sample) or of the corresponding total RNA sample. Sequencing of the PCR product revealed that it was highly similar to the ßA cDNA sequence of other species (data not shown). Similarly, using primers specific for zebrafish FS cDNA, a DNA fragment with the expected size of 384 bp was obtained from the ovary cDNA sample, but not from the corresponding total RNA sample or the water control (Fig. 1A). The sequence of the PCR product was identical to the published zebrafish FS cDNA sequence [37].
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To further confirm the expression of activin and FS, Western blot analyses were carried out using antibodies against human activin ßA subunit and FS, respectively. Three bands, with molecular sizes of approximately 75, 32, and 26 kDa, were observed when ßA antibody was used (Fig. 1B). The large band may represent a precursor for ßA subunit, while the respective smaller bands are similar in size to mammalian inhibin and activin. The FS antibody also detected several bands, ranging from 30 to 50 kDa (Fig. 1B). These molecular sizes are consistent with the known structure of FS [30].
Activin A and Inhibin A Stimulated Zebrafish Oocyte Maturation
Previous studies in the zebrafish have shown that only follicles that reach certain sizes can undergo maturation in vitro in response to exogenous hormones [38]. To determine which sizes of follicles are responsive to activin A, follicles were isolated from ovaries and grouped according to their sizes. Different groups of follicles were then incubated with control medium or activin A (100 ng/ml) for 18 h. As shown in Figure 2A, follicles within the category of 0.34–0.51 mm did not show any response to activin A while follicles of 0.52 mm or greater had an enhanced rate of GVBD after activin A treatment. Similarly, the rate of spontaneous maturation was increased with the increasing sizes of follicles, and spontaneous maturation did not occur in follicles smaller than 0.52 mm. Subsequent experiments were conducted using follicles greater than 0.52 mm.
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When follicles were incubated with different concentrations of activin A for 18 or 24 h, a dose-dependent stimulation of GVBD was observed. Activin A, at 10–100 ng/ml, significantly increased the rate of GVBD compared to that of the control group (Fig. 2B). Similarly, inhibin A also induced oocyte maturation in a dose-dependent manner (Fig. 3). Follicles treated with 10–100 ng/ml of inhibin A had a significantly higher percentage of GVBD. Similar patterns were observed when follicles were incubated with inhibin A for 18 h and 24 h, although the rates of GVBD were slightly higher in both control and treated groups after 24-h incubation (Fig. 3B) than those observed at 18 h (Fig. 3A).
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To determine the specificity of activin A and inhibin A action, the interaction between activin A and FS or inhibin A and FS was examined. Furthermore, the combined effect of activin A and inhibin A on oocyte maturation was also tested. As shown in Figure 4, activin A (25 ng/ml) or inhibin A (25 ng/ml) alone significantly increased the maturation rate after 24-h incubation. The stimulatory effects of activin A and inhibin A were completely neutralized by FS; in the presence of FS-288 (100 ng/ml), neither activin A nor inhibin A induced oocyte maturation. FS-288 alone did not significantly alter basal level of maturation. When activin A and inhibin A were added together, there was no further increase in maturation rate compared to that of either activin A- or inhibin A-treated groups, indicating that activin A and inhibin A do not have additive effects. These results were consistently seen in follicles incubated with these agents for either 18 h or 24 h.
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FS-288 Blocked Gonadotropin- and MIH-Induced Maturation
Gonadotropin and MIH are known to play major roles in oocyte maturation. To test the possibility that activins and/or inhibins are mediators of gonadotropin and MIH action, the interaction between FS-288 and hCG or MIH was examined. Human CG, previously shown to function as a gonadotropin in fish [39], stimulated oocyte maturation. Combined treatment of hCG and FS-288 resulted in a significant reduction in the rate of maturation compared to that of the hCG-treated group. Furthermore, in the presence of FS-288, hCG no longer had a significant stimulation effect on oocyte maturation (Fig. 5A). MIH alone also increased the rate of maturation significantly. When FS-288 was added together with MIH, the rate of maturation was significantly lower than that found in the MIH-treated group but was still significantly higher than the control value (Fig. 5B). These results indicate that FS completely blocks gonadotropin-induced maturation but only partially blocks MIH-stimulated oocyte maturation.
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Gonadotropin Up-Regulated the Expression of Activin A- and Inhibin A-Like Molecules
The finding that FS-288 neutralized the effect of hCG on oocyte maturation suggests that activins and/or inhibins are local regulators mediating gonadotropin-induced oocyte maturation. To further test this hypothesis, fish were given injections of hCG, and ovaries were removed at 12, 24, and 30 h after injection. Proteins were then extracted from the ovaries and subjected to Western blot analysis using the antibody against the ßA subunit. At 12 and 24 h after hCG injection, there were significantly higher levels of both activin A- and inhibin A-like molecules in the ovaries of hCG-treated fish than in those of the control fish (Fig. 6). However, the expression levels of activin A and inhibin A-like molecules were similar between the control group and the hCG-treated group at 30 h after injection (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit has not been examined, the true identity of the 32-kDa inhibin A-like molecule requires further investigation. The detection of activin A and an inhibin A-like molecule in the zebrafish ovary is in agreement with previous studies in the goldfish. Using immunocytochemistry, Ge et al. [40] showed the expression of , ßA, and ßB subunits in goldfish follicular cells and oocytes, suggesting that activin- and inhibin-like molecules may be produced by the ovary. In mammals, several molecular forms of FS have been characterized, which are derived from alternative splicing of the FS gene [31], proteolytic cleavage, and variable glycosylations [41–43]. In the present study, we observed several FS-immunoreactive molecules with molecular sizes ranging from 30 to 50 kDa. Although only the truncated form of FS has been cloned, Northern blot analysis revealed two transcripts [37]. These results, together with the detection of multiple bands from proteins extracted from zebrafish ovary, suggest that, similar to the case in mammals, multiple forms of FS are present in fish.
The present study demonstrates that activin A and inhibin A stimulate oocyte maturation in the zebrafish. Both activin A and inhibin A increased GVBD in a dose-dependent manner, with significant stimulation at 10–100 ng/ml. Although the physiological concentrations of activin A and inhibin A have not been determined in fish, the doses used in this study are within the concentrations of activins and inhibins found in mammalian follicular fluid [44] and have been widely used to study the role of activins and inhibins in mammals. The observation that activin A induces final oocyte maturation is consistent with several other studies. Activin A was found to increase GVBD in the rat [45], rhesus monkey [46], human [47], and cow [48]. Recently, Pang and Ge [49] also reported that recombinant goldfish activin B induced zebrafish oocyte maturation. Thus, both activin A and activin B have a similar function in inducing final oocyte maturation. In contrast, earlier studies in the rat [50, 51], pig [52], and seabream [53] showed that activin A was ineffective in inducing GVBD. Similarly, previous studies on inhibin in oocyte maturation have also yielded somewhat conflicting results. For example, O et al. [50] reported that inhibin A inhibited spontaneous maturation of rat oocytes while Tsafriri et al. [51] found no effects of inhibin A during in vitro maturation of oocytes taken from eCG-treated rats. In seabream, inhibin A was also found to be ineffective in inducing GVBD [53]. However, inhibin A was shown recently to stimulate rhesus monkey oocyte maturation [46]. Although the exact causes of such discrepancies are not known, species variation, developmental stages of the oocytes used, and culture conditions (whether or not serum is present), as well as forms of activin and inhibin used, may contribute to the different results reported.
One of the most interesting findings from this study is that activin A and inhibin A had the same effect and did not antagonize each other in oocyte maturation. In agreement with these findings, an earlier study on goldfish pituitary also found that both activin and inhibin stimulated gonadotropin-II release [54]. Interestingly, while most studies in mammals have shown that activin and inhibin have opposite functions and that inhibin is a functional antagonist of activin [16], Alak et al. [46] reported that activin A and inhibin A both enhanced oocyte maturation in rhesus monkeys. Since activin A and inhibin A had additive effects on monkey oocyte maturation, it was suggested that they might act at different levels and/or by different mechanisms [46]. However, no additive effects of activin A and inhibin A on oocyte maturation were observed in our study, suggesting a similar mechanism of action for both activin A and inhibin A. In mammals, inhibin has an affinity for activin receptors [55] and has been shown to block activin action by binding to activin type II receptors and preventing the association between type I and type II [29]. Whether or not inhibin can bind to activin receptors in fish is not known.
FS are known to be binding proteins for activins and inhibins. FS-288 has been shown to neutralize activin functions in many tissues and cells [30]. In the present study, the effect of activin A and inhibin A was completely neutralized by FS-288, supporting the specificity of activin A and inhibin A action. Furthermore, FS-288 also suppressed gonadotropin- and MIH-induced final oocyte maturation. These findings suggest that activins and/or inhibins are autocrine/paracrine regulators that mediate, at least in part, the action of gonadotropin and MIH in the induction of final oocyte maturation. These results are consistent with the findings of Pang and Ge [49].
The notion that activins and/or inhibins are local mediators of gonadotropin within the ovary is further supported by the finding that gonadotropin increased the expression of activin A- and inhibin A-like molecules. In vivo administration of hCG increased intensities of the 26- and 32-kDa bands in a time-dependent manner. In addition, hCG appears to differentially regulate the expression of inhibin A- and activin A-like molecules. The expression of inhibin A-like molecules reached maximal level at 12 h after hCG injection and remained elevated at 24 and 30 h post-injection. However, a significantly higher level of expression of the activin A-like molecule was observed only at 24 h after hCG injection. In mammals, the regulation of activin and inhibin production are also under the control of gonadotropins. FSH and hCG stimulated both activin and inhibin production in a dose-dependent fashion in cultured porcine granulosa cells [56]. Similarly, in human granulosa-luteal cells, FSH and LH also enhanced the secretion of inhibin A and activin A [57]. However, in rat granulosa cells, FSH or activators of the cAMP signalling pathway stimulated inhibin, but not activin, production [58].
The site(s) of activin A and inhibin A actions in the zebrafish ovary remains unclear at present. Both inhibin and activin have been reported to modulate steroidogenesis, including progesterone production, in mammals [5, 59, 60]. It is possible that activin A and inhibin A may stimulate MIH production, which in turn, induces oocyte maturation. On the other hand, our finding that FS-288 partially blocked MIH-induced maturation suggests that activin A and/or inhibin A may act downstream of MIH, possibly at the level of the oocyte. A direct action on oocytes for activin is also supported by the expression of activin type II receptors in zebrafish oocytes [35, 61].
In summary, this study demonstrates that activin A, inhibin A-like molecule, and FS are present in the zebrafish ovary. In addition, activin A and inhibin A stimulate final oocyte maturation. Furthermore, FS neutralizes the effect of activin A and inhibin A on oocyte maturation and blocks gonadotropin- and MIH-induced final oocyte maturation. These findings, together with our previous study demonstrating the presence of activin receptor mRNA in the zebrafish ovary, suggest that activin A and inhibin A are physiological regulators of final oocyte maturation. The sites of activin A and inhibin A actions are currently under investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by a grant from the Natural Science and Engineering Research Council (NSERC) of Canada to C.P. C.P. is a recipient of NSERC Women's Faculty Awards. T.W. and J.C. were supported by visiting professorships from the Chinese Academy of Sciences. 
2 Correspondence: Chun Peng, Department of Biology, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3. FAX: 416 736 5698;cpeng{at}yorku.ca
Accepted: January 5, 2000.
Received: November 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1987; 1:561–568.[Abstract], 20ß-Dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 1997; 62:190–196.[CrossRef][Medline]This article has been cited by other articles:
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E. Clelland, G. Kohli, R. K. Campbell, S. Sharma, S. Shimasaki, and C. Peng Bone Morphogenetic Protein-15 in the Zebrafish Ovary: Complementary Deoxyribonucleic Acid Cloning, Genomic Organization, Tissue Distribution, and Role in Oocyte Maturation Endocrinology, January 1, 2006; 147(1): 201 - 209. [Abstract] [Full Text] [PDF]
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