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BOR - Papers in Press, published online ahead of print November 8, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054668
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BIOLOGY OF REPRODUCTION 76, 294–302 (2007)
DOI: 10.1095/biolreprod.106.054668
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Growth Differentiation Factor 9 and Its Spatiotemporal Expression and Regulation in the Zebrafish Ovary1

Lin Liu , and Wei Ge 2 

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

ABSTRACT

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta (TGFB) superfamily. As an oocyte-specific growth factor, GDF9 plays critical roles in controlling folliculogenesis in mammals. In the present study, we cloned a 2.1-kb cDNA of the zebrafish GDF9 homolog (Gdf9, gdf9), which shares ~60% homology with that of mammals in the mature region. RT-PCR analysis showed that zebrafish gdf9 expression was present only in the gonads and Northern blot analysis revealed a single transcript of about 2.0 kb in the ovary. Real-time RT-PCR analysis revealed that gdf9 expression was highest in primary growth (PG, stage I) follicles and gradually decreased during follicular development, with the lowest level being found in fully grown (FG) follicles. The expression of gdf9 was maintained through fertilization and early embryonic development until gastrulation, at which point the expression level dramatically decreased. Expression was barely detectable after the late gastrula stage. Within the follicle, gdf9 mRNA was localized exclusively in the oocytes, as demonstrated by RT-PCR of denuded oocytes and freshly isolated follicle layers as well as by in situ hybridization. Interestingly, when amplified for high numbers of cycles, the expression of gdf9 was detected in cultured zebrafish follicular cells that were free of oocytes. The expression of gdf9 was downregulated by hCG in both ovarian fragments and isolated follicles in dose- and time-dependent manners, and this inhibition appeared to be stage-dependent, with the strongest inhibition observed for the FG follicles and no effect seen for the PG follicles. This correlates well with the expression profile of the LH receptor (lhcgr) in zebrafish follicles. In conclusion, as an oocyte-derived growth factor, GDF9 is highly conserved across vertebrates. With its biological advantages, zebrafish provides an alternative model for studying gene function and regulation.

follicle, GDF9, growth factors, oocyte development, zebrafish

INTRODUCTION

It is well known that in vertebrates the ovary is controlled by gonadotropins, FSH and LH, which are secreted from the pituitary, and by various local intraovarian growth factors secreted from the somatic follicular cells, such as activins [1, 2]. However, in the past few years, evidence has accumulated that the oocyte also plays active roles in ovarian folliculogenesis [3, 4]. By secreting a variety of growth factors, the oocyte can directly regulate gene expression and steroidogenesis in both the granulosa and theca cells and can modulate the effects of gonadotropins on the follicular cells [5, 6].

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta (TGFB) superfamily. Since it was first identified in 1993 [7], GDF9 has attracted increasing attention due to its unique oocyte-specific expression. The expression of GDF9 mRNA and protein has been demonstrated in the oocytes of a variety of mammalian species, including humans, using in situ hybridization or immunocytochemical staining [8–11]. However, there have also been reports on GDF9 expression in other ovarian cell types, such as the granulosa cells [12–14], and in nonovarian tissues, which include the hypothalamus, pituitary, and testis [15, 16].

In the ovary, the temporal expression pattern of GDF9 has been described in a number of species, although the results are somewhat conflicting. In rodents and humans, Gdf9 or GDF9 expression is detectable from the primary follicles to the follicles of all later stages [10, 15], whereas expression starts in the primordial follicles in ovine, bovine, and possum ovaries [16, 17]. In the chicken ovary, Gdf9 expression peaks in the young follicles, which are less than 1 mm in diameter [14], while morphometric analysis in humans has demonstrated that the highest level of GDF9 mRNA is in the oocytes of fully grown secondary follicles [18]. These results suggest that GDF9 has species-specific functions in regulating follicle development, particularly at the early stages. In mice, Gdf9 mRNA is detected continuously in the ovulated oocytes, although it is no longer detectable in Day 1.5 postcoital (pc) embryos [19]. In cows, GDF9 expression is still detectable in 8-cell embryos that are developed in vitro [20, 21].

In recent years, the physiological role of GDF9 in folliculogenesis has been a subject of intense study in mammals. Despite this, the function of GDF9 remains elusive and highly controversial [22] and much work remains to be done on this topic. The critical role of GDF9 in primary follicle growth has been clearly demonstrated in GDF9 knockout mice, in which folliculogenesis is blocked at the primary follicle stage [9]. In support of this is the in vivo observation that injection of neonatal rats with recombinant GDF9 promotes the progression of follicle development from the primordial stage to the small preantral stage, but not beyond this stage [23]. When tested in vitro, recombinant GDF9 promotes follicle growth [24, 25], modulates the effect of gonadotropin on follicular cells [24], regulates gene expression [26, 27], enhances FSH-induced cumulus expansion [26, 28], and increases the production of other growth factors [29] and steroids in cultured granulosa [30] and theca cells [24].

In contrast to the attention paid to the biological activities of GDF9, studies on its regulation have been very limited, particularly with respect to the extracellular signals. Recent knockout experiments in mice have demonstrated that Gdf9 expression is inhibited by germ cell nuclear factor (GCNF, Nr6a1), which is an oocyte-specific factor [31], and Nobox, which is an oocyte-specific homeobox gene [32]. As for the endocrine and paracrine regulation of GDF9 expression, little is known. The expression of GDF9 in the porcine oocyte cumulus complex (OCC), but not that of GDF9 in the oocyte, has been shown to be inhibited in vitro by FSH [13].

Although GDF9 and its functions in mammals have captured increased attention in recent years, much remains to be learnt about this molecule. Its existence and function in nonmammalian vertebrates are largely unknown, except for a recent report in the chicken [14]. To examine to what extent the concepts developed in mammals can be applied to other vertebrates and to understand the fundamental roles of GDF9 in vertebrate reproduction, comparative studies in different models are clearly warranted. The present study was undertaken to clone and characterize a GDF9 homolog in the zebrafish, which is a popular vertebrate model of ovarian folliculogenesis [33, 34]. A series of experiments was performed to analyze the temporal and spatial expression patterns of gdf9 in the zebrafish ovary as well as in postfertilization embryos. Considering that information is lacking about GDF9 regulation in the ovaries of mammals and that the zebrafish is an advantageous model in that its ovary contains large numbers of follicles that can be easily isolated and staged, we also carried out experiments to investigate the regulatory effects of gonadotropins on GDF9 expression at both the ovarian and follicle levels.

MATERIALS AND METHODS

Chemicals

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and all enzymes were from Promega (Madison, WI), unless otherwise stated. The hCG was purchased from Sigma-Aldrich, and all culture media were obtained from Gibco Invitrogen (Carlsbad, CA).

Animals

Zebrafish (Danio rerio) were purchased from a local tropical fish market and acclimated in flow-through aquaria at 25–26°C on a 14L:10D cycle. The fish were fed twice a day with commercial tropical fish food. All experiments were performed under license from the Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. The animals were anesthetized with tricaine methanesulfonate (MS-222) before handling.

RNA Isolation, Reverse Transcription (RT) and Polymerase Chain Reaction (PCR)

Total RNA samples were isolated from the tissues, ovarian follicles, cultured follicular cells, and embryos using Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to the protocol of the manufacturer and our previous report [35], and the amount and purity of the RNA were determined by spectrophotometry. The purification of mRNA was performed with the PolyATract mRNA Isolation System III (Promega) according to the manufacturer's protocol. Reverse transcription (RT) was performed at 42°C for 2 h in a total volume of 10 µl that contained 2–3 µg total RNA, 1x MMLV buffer, 0.5 mM of each dNTP, 0.5 µg oligo(dT), and 80 U MMLV reverse transcriptase. PCR amplification was performed in a volume of 25 µl that consisted of 1 µl of RT reaction, 1x PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 0.2 µM of each primer, and 0.5 U Taq polymerase with an annealing temperature of 59°C for gdf9 and 56°C for the housekeeping gene gapdh (for glyceraldehyde-3-phosphate dehydrogenase).

Cloning of Full-length gdf9 cDNA from Zebrafish Ovary

A gene-specific antisense primer for 5'-RACE (rapid amplification of cDNA ends) was designed based on the expressed sequence tag (EST) sequence fi34d02.x1 from the Washington University Zebrafish EST Project. The 5'-RACE was carried out using the SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA), and the amplification products were cloned into pBluescript II KS(+) (Stratagene, La Jolla, CA) and sequenced. Based on the sequence of the 5'-RACE product, new gene-specific sense primers were designed near the 5'-end for nested 3'-RACE, to amplify the full-length cDNA of zebrafish gdf9 (Table 1). The RACE amplification was performed as follows: 1) 5 cycles of 94°C for 5 sec and 72°C for 3 min; 2) 5 cycles of 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min; and 3) 35 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min. The final 3'-RACE product was cloned into pBluescript II KS(+), and both strands were sequenced after exonuclease III and mung bean nuclease deletion. The sequencing reaction was performed with the BigDye Terminator Cycle Sequencing Kit v3.1 and analyzed on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).


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  		TABLE 1. Primers used in RT-PCR analysis.

Northern Blot Analysis

Northern blot hybridization was performed based on our previous study [36]. Briefly, total RNA (20 µg) or mRNA (3 µg) from the whole ovary was resolved on a 1% denaturing agarose gel that contained 2.2 M formaldehyde, transferred to a positively charged nylon membrane (Roche, Mannheim, Germany), and UV cross-linked with the GS Gene Linker (Bio-Rad, Hercules, CA). The membrane was then hybridized with DIG-labeled cRNA probe prepared from the cloned zebrafish gdf9 cDNA by in vitro transcription, detected with the Chemiluminescent Detection Kit according to the manufacturer's instruction (Roche), and analyzed on the Lumi-Imager F1 workstation (Roche).

In Situ Hybridization

The ovaries were freshly fixed in Bouin solution and processed for paraffin sectioning. The sections were mounted on slides that were coated with poly-(L)-lysine, and hybridized with sense (control) or antisense gdf9 cRNA probes according to the reported protocol [37], with some modifications. Briefly, the sections were deparaffinized, washed with PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, [pH 7.4]), and treated with proteinase K (10 µg/ml in PBS) for 20 min at 37°C. After rinsing with cold glycine-PBS buffer (2 mg/ml), the sections were postfixed for 10 min with 4% paraformaldehyde in PBS before acetylation for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine (TEA) buffer (pH 8.0). After a 10-min wash with 4x SSC, the sections were prehybridized at 58°C for 1 h in 50% formamide with 2x SSC, and hybridized at 58°C overnight with DIG-labeled antisense or sense probes in the hybridization buffer (50% formamide, 10% dextran sulfate, 1x Denhardt solution, 10 mM DTT, 1 mg/ml tRNA, 1 mg/ml salmon sperm DNA). After a series of washes with 2x SSC and 50% formamide/50% 2x SSC at 58°C, the sections were incubated at 37°C in NTE buffer (0.5 M NaCl, 10 mM Tris [pH 8.0], 1 mM EDTA) for 5 min, followed by a 30-min treatment at 37°C with RNase A in NTE buffer (20 µg/ml). The sections were further washed with NTE buffer and 0.1x SSC (58°C), equilibrated in TBS buffer (100 mM Tris [pH 7.5], 150 mM NaCl) for 10 min, and blocked for more than 30 min with blocking solution that contained 10% sheep serum and 1% Blocking Reagent (Roche). The probes were then detected with anti-DIG-alkaline phosphatase according to the manufacturer's protocol (Roche). Color development was stopped by rinsing the slides with distilled water. Finally, the sections were mounted in gelatin-glycerol and viewed under the Microphot-FX microscope (Nikon, Tokyo, Japan). All images were captured with the DXM 1200 digital camera (Nikon) and analyzed with the ACT-1 version 2.12 software (Nikon).

Real-Time and Semiquantitative RT-PCR

Most quantitative measurements of gdf9 expression were carried out using real-time RT-PCR. The standards for gdf9 and gapdh were prepared by RT-PCR amplification of cDNA fragments with specific primers (Table 1). The amplicons were resolved by agarose gel electrophoresis, purified, and quantitated by electrophoresis together with the Mass Ruler DNA marker (MBI Fermentas, Hanover, MD). These amplified amplicons were used to construct the standard curves in the real-time PCR assays.

Total RNA (3 µg) from each sample was reverse-transcribed in a 10-µl reaction volume, as described above. After reverse transcription, each reaction was diluted to 200 µl with water. Real-time PCR was carried out on the iCycler iQ Real-Time PCR Detection System (Bio-Rad) in a volume of 30 µl that contained 10 µl diluted RT reaction mix, 1x PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 0.2 µM of each primer, 0.75 U Taq polymerase, SYBR Green (1:35 000 dilution; Molecular Probes, Leiden, The Netherlands), and 20 nM fluorescein (Bio-Rad). The reaction profile consisted of 38 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, and 80°C for 7 sec for signal detection. A melt curve analysis, which consisted of 180 cycles of 7 sec with temperature increase of 0.2°C/cycle, was performed at the end of the reaction to demonstrate the specificity of the reaction. A single peak was revealed by this analysis, which was confirmed by agarose gel electrophoresis and sequencing.

For semiquantitative RT-PCR analysis of gdf9 expression during embryonic development, the cycle numbers used were optimized according to our previous report [38]. Briefly, PCR was carried out in a volume of 30 µl, which consisted of 1x PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 0.3 µM of each primer, and 0.6 U Taq polymerase, using the Thermal Cycler 9600 (Eppendorf, Hamburg, Germany) for various number of cycles with the following profile: 94°C for 30 sec, 60°C for 30 sec for gdf9 or 56°C for gapdh, and 72°C for 60 sec. The numbers of cycles used were: 29 for gdf9; 35 for lhcgr (luteinizing hormone/choriogonadotropin receptor); and 27 for gapdh and bactin1. The specificity of PCR amplification was confirmed by cloning and sequencing.

Preparation of Ovarian Fragments

After anesthetization and decapitation, the two ovaries from each individual (4–6 fish used in each experiment) were carefully removed and placed in a dish that contained 60% Leibovitz L-15 medium. Each ovary was halved in the middle, and each half was transferred to a well of a 12-well plate, where it was briefly dispersed into small fragments before drug treatment. Each treatment group consisted of the ovarian fragments from all individuals incubated in separate wells. In some experiments, one fourth of the ovary was used for each treatment group.

Isolation of Ovarian Follicles

The ovaries were removed from 15–20 female zebrafish at noon after anesthetization and decapitation, and placed in a 100-mm culture dish that contained 60% Leibovitz L-15 medium. The follicles of different stages were manually isolated and grouped according to the following stages: fully grown but immature (FG; ~0.65 mm), midvitellogenic (MV; ~0.50 mm), early vitellogenic (EV; ~0.40 mm), previtellogenic (PV, stage II or cortical alveolus stage; ~0.30 mm), and primary growth follicles (PG, stage I; ~0.1 mm). The process of isolation normally lasted for 4–6 h at room temperature before incubation and drug treatment at 28°C for different periods of time.

Separation of Oocytes and Follicle Layers

As in many teleosts, the follicle of the zebrafish contains a thin follicle layer that consists of only one monolayer of granulosa and theca cells, which makes it extremely difficult to separate the follicle layer from the oocyte. It has recently been reported that cold-shock treatment of the follicles makes it easier to separate mechanically the two follicle compartments [39]. In the present study, we pretreated the FG follicles with low temperature (4°C) for 30 min. The follicle layer was then carefully peeled off with fine forceps without damaging the oocyte. The isolated follicle layers and denuded but intact oocytes from 5–10 follicles were pooled and subject to RNA extraction with Tri-Reagent, respectively. Some denuded oocytes were fixed with Bouin solution for histological examination, to demonstrate complete removal of the follicle layer.

Embryo Collection

One mature female and two males were placed in a tank at 26°C for breeding before the day of embryo collection. The postfertilization embryos (5–7) of different developmental stages were collected at different incubation times for RNA extraction and real-time semiquantitative RT-PCR analysis. The embryos were staged according to the method published previously [40].

Primary Follicular Cell Culture

The primary culture of zebrafish ovarian follicular cells was performed according to our previous report [35]. Briefly, the EV and MV follicles from about 20 female zebrafish were isolated, washed with medium M199, and incubated in M199 that was supplemented with 10% fetal calf serum at 28°C in 5% CO2 for 6 days, in order for the follicular cells to proliferate. The proliferated follicular cells were then harvested by trypsinization, and plated in 6-well plates for more than 24 h. After washing twice with the medium, the follicular cells, which were free of any oocytes, were extracted with Tri-Reagent for total RNA.

Data Analysis

The ratio of the zebrafish gdf9 expression level to the level of the internal control gapdh was calculated for each sample, and then expressed as the percentage of the reference group for statistical analysis. All the experiments were repeated at least three times using different batches of fish. All values were expressed as the mean ± SEM, and the data were analyzed by one-way ANOVA followed by the Dunnett test (for comparisons with the control group only) or the Newman-Keuls test (for comparisons of all pairs of groups) using Prism 4.0b for Macintosh OS X (GraphPad Software, San Diego, CA). P < 0.05 was considered to be statistically significant.

RESULTS

Cloning and Characterization of Zebrafish gdf9

The full-length cDNA for gdf9 (AY833104) was cloned from the zebrafish ovary by 3'-RACE. This clone is 2.1 kb in length and encodes 418 amino acids. The overall amino acid sequence of the zebrafish Gdf9 precursor shares only about 40% homology with those of mammals and chickens. However, the mature region (125 amino acids) of the zebrafish Gdf9 precursor shows much higher homology (60% to 70%), with the C-terminus being more conserved. The six characteristic cysteine residues are fully conserved in the mature region (Fig. 1). Unlike the corresponding molecule in mammals, which has four potential N-glycosylation sites, the zebrafish Gdf9 has only three such sites, none of which is located in the mature region.


Figure 76021601
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  		FIG. 1. Upper panel: Comparison of the amino acid sequences of the mature region of zebrafish Gdf9 and those of other vertebrates. The mature sequence is preceded by a putative tetrabasic cleavage site (RRRR). The light-gray areas indicate identical amino acid sequences. The six fully conserved cysteine residues are marked by asterisks. Lower panel: Phylogenetic relationship of zebrafish Gdf9 with its counterparts and related proteins of other species, as demonstrated by bootstrap analysis using the UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) method. Zebrafish Tgfb1 was used as the outlier because it is the prototype of the TGFB superfamily that is distantly related to gdf9. The GenBank accession number for each protein is indicated in parentheses. The numbers at the forks are the bootstrap proportions.

RT-PCR analysis using specific primers designed from the cloned cDNA was performed to demonstrate the tissue distribution of zebrafish gdf9 expression. Several tissues were tested, including the gill, liver, kidney, muscle, brain, pituitary, testis, and ovary. The expression of gdf9 was restricted exclusively to the testis and ovary, and no signal was detected in nongonadal tissues (Fig. 2A). Northern blot analysis of total RNA or mRNA from the ovary revealed a single transcript of about 2 kb in size (Fig. 2B). Interestingly, when amplified for high numbers of cycles (35 and 40), a weak signal was detected in cultured somatic follicular cells (Fig. 2C). Although we cannot completely rule out the possibility that the weak signal detected in the cultured follicular cells was due to oocyte contamination, especially of the early PG stage, this seems unlikely, as we thoroughly washed the cultured cells (PG follicles do not attach well during the incubation) and carefully examined each well before RNA extraction.


Figure 76021602
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  		FIG. 2. A) Tissue distribution of gdf9 in the zebrafish body. RT-PCR was performed for 35 cycles for gdf9 and 25 cycles for gapdh. +, RT with reverse transcriptase; -, RT without reverse transcriptase. B) Northern blot analysis for gdf9 expression in the ovary for both total RNA (20 µg) and mRNA (3 µg). A single transcript of about 2 kb is detected in both RNA preparations. The arrows indicate the locations of the 28S and 18S rRNAs. C) Detection of gdf9 expression in cultured zebrafish ovarian follicular cells. Total RNA (3 µg) from a freshly isolated ovary and cultured follicular cells was reversed-transcribed followed by PCR amplification for different cycles (30 to 40 for gdf9 and 23 for gapdh). The expression level of gapdh was consistently higher in the cultured follicular cells than in the ovary. +, RT with reverse transcriptase; -, RT without reverse transcriptase.

Localization of gdf9 Expression in the Ovarian Follicle

GDF9 is regarded as an oocyte-specific factor in mammals. To confirm that this also holds true in the zebrafish, we separated the somatic follicle layer from the fully grown oocyte (Fig. 3A) and analyzed the expression of GDF9 in the two compartments. Clean separation was confirmed by histological examination of denuded oocytes, which revealed the absence of the follicle layers (Fig. 3B). Furthermore, as a follicle cell-specific marker, the expression of lhcgr was demonstrated in both the intact follicles and isolated follicle layers, but not in the denuded oocytes (Fig. 3, C and D). In contrast to lhcgr, gdf9 was expressed exclusively in the oocytes, and each oocyte contained about 25 000 copies of reverse-transcribable mRNA, which was comparable to the level in the intact follicle. However, the level of gapdh expression in either the denuded oocyte or follicle layer was nearly half that in the intact follicle, and there was no significant difference between the two compartments (Fig. 3C). A 32-cycle PCR amplification revealed no expression of gdf9 in the freshly isolated follicle layer (Fig. 3D).


Figure 76021603
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  		FIG. 3. Spatial distribution of gdf9 expression within the fully grown follicle. (A) A mechanically separated oocyte (arrow head) and the somatic follicle layer (arrow) are shown. B) Histological confirmation of the separation of the follicle layer from the oocyte. The denuded oocyte (upper) is shown to be free of the follicle layer, which is present in the intact follicle (lower, arrow). C) The expression levels of gdf9, gapdh, and lhcgr in the intact follicles, denuded oocytes, and follicle layers. Real-time RT-PCR was used to analyze gdf9 and gapdh, whereas semiquantitative RT-PCR was used for lhcgr. The expression levels of gdf9 and gapdh are expressed as the numbers of reverse-transcribed mRNA transcripts, since the standards used in the assays consisted of DNA. The values are the mean ± SEM (n = 4) from a representative experiment. Different letters indicate statistical significance (P < 0.05). D) Agarose gel electrophoresis for the expression of gdf9, lhcgr, gapdh, and bactin1 in the intact follicles, denuded oocytes, and isolated somatic follicle layers.

The spatial expression of gdf9 in the zebrafish follicle was also confirmed by in situ hybridization, which showed strong staining for gdf9 mRNA in the oocytes (Fig. 4).


Figure 76021604
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  		FIG. 4. Stage-dependent expression pattern of gdf9 in developing follicles. Upper panel: Real-time RT-PCR quantitation of gdf9 expression levels in the follicles of different stages. The values are the mean ± SEM (n = 3) from a representative experiment. Different letters indicate statistical significance (P < 0.05). PG, primary growth (stage I); PV, previtellogenic stage (stage II); EV, early vitellogenic stage (early stage III); MV, midvitellogenic stage (midstage III); FG, fully grown (late stage III). Lower panel: Detection of gdf9 mRNA in the zebrafish ovary by in situ hybridization with antisense (left) and sense (right) probes prepared by in vitro transcription. Arrow head, PG follicles; arrow, PV follicles.

Stage-Dependent Expression of gdf9 in the Ovarian Follicles and Postfertilization Embryos

Real-time RT-PCR was performed to assess the relative levels of gdf9 expression in the ovarian follicles of different developmental stages from PG to FG. A significant change in gdf9 expression was observed during follicle development. Although it could be easily detected in all the stages, the level of gdf9 mRNA was highest in the PG follicles and gradually decreased with follicle development (Fig. 4). The decreasing trend of gdf9 remained after normalization with gapdh. Consistent with this result, in situ hybridization also showed that the oocytes at the PG stage exhibited the strongest staining and the signal seemed to decline progressively with the growth of the follicles (Fig. 4).

Zebrafish gdf9 mRNA was still detectable after fertilization, as demonstrated by both real-time and semiquantitative RT-PCR assays. However, the gdf9 mRNA level declined dramatically at the stage of gastrulation (7–10 h postfertilization) and had disappeared by the later stages (Fig. 5).


Figure 76021605
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  		FIG. 5. Temporal expression pattern of gdf9 in developing zebrafish embryos. Embryos were collected at different times postfertilization for RT-PCR analysis. Upper panel: Relative levels of zebrafish mRNA at different developmental stages after fertilization. The values are the mean ± SEM (n = 3–4) from a representative experiment using real-time RT-PCR. Different letters indicate statistical significance (P < 0.05). Lower panel: Agarose gel electrophoresis for the expression of gdf9 and gapdh using semiquantitative RT-PCR.

Effects of Gonadotropin on the Expression of Zebrafish gdf9

As demonstrated above, zebrafish gdf9 expression showed a significant trend of decline during follicle development, with the highest level detected in the PG stage. This suggests potential roles for endocrine hormones, particularly gonadotropins from the pituitary, in regulating gdf9 expression, especially since our recent work has demonstrated that both FSH and LH receptors show sequential and progressive increases in expression during zebrafish follicle growth [41]. Two in vitro approaches using ovarian fragments and isolated follicles were adopted to address this issue. The isolated follicles allowed us to analyze stage-dependent responses to gonadotropin treatment, whereas in the ovarian fragments, the spatial relationship of follicles of different stages reflected that in the intact ovary.

When tested on the isolated FG follicles, hCG inhibited gdf9 expression in a time-dependent manner, with the maximal effect reached after 2 h of treatment (Fig. 6A). In the dose-response experiments, treatment of freshly isolated zebrafish ovarian fragments or FG follicles with hCG for 2 h consistently reduced the expression of gdf9 in a dose-dependent manner (Fig. 6, B and C). As a control, the ovarian aromatase exhibited increased expression in response to hCG (data not shown). Interestingly, the effect of hCG seemed to be stage-dependent. When tested on the follicles of different stages, hCG caused significant inhibition of gdf9 expression in the FG follicles, but it had low or negligible effects on the follicles of earlier stages (PV and EV) (Fig. 6D).


Figure 76021606
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  		FIG. 6. Regulation of gdf9 expression in the zebrafish ovary by gonadotropin (hCG) in vitro. The values are the mean ± SEM (n = 3) from a representative experiment. A) Time course of hCG (10 and 100 IU/ml) effect on gdf9 expression in FG follicles. *, P < 0.05; **, P < 0.01 vs. control. B) Dose-response of hCG effect on gdf9 expression in ovarian fragments. C) Dose-response of hCG effect on gdf9 expression in FG follicles. Different letters indicate statistical significance (P < 0.05). D) Stage dependence of hCG effect (2 h) on gdf9 expression; hCG shows the strongest effect on FG follicles, but not at the earlier stages (PV and EV). *, P < 0.05 vs. the control.

DISCUSSION

In the present study, we cloned and sequenced a full-length cDNA for growth differentiation factor 9 (gdf9) in the zebrafish. The overall amino acid sequence homology of the GDF9 precursor between zebrafish and other tetrapods is not high, although the predicted mature region at the C-terminus shows much higher conservation. In contrast to the protein in mammals [8], zebrafish Gdf9 does not have consensus glycosylation sites in the mature region.

GDF9 was first demonstrated as an oocyte-specific factor [19]. However, some later studies have shown its expression in several other tissues, such as the hypothalamus, testis, and pituitary [15, 16]. Using RT-PCR with sequence-specific primers, we examined a variety of zebrafish tissues for the expression of gdf9, and the results clearly show that this molecule is expressed only in the testis and ovary. This supports the conclusion from studies in mammals that GDF9 functions as an important growth factor in the gonads, particularly the ovary [5, 15, 42, 43]. The gonad-specific expression of zebrafish gdf9 contrasts sharply with that of the gene for the closely related bone morphogenetic protein 15 (bmp15). Although both GDF9 and BMP15 have been demonstrated to be oocyte-specific factors in mammals [11, 44], a recent report has shown that the expression of zebrafish bmp15 is not restricted to the ovary but has widespread tissue distribution in this species [45].

To localize the expression of gdf9 in the zebrafish ovary, we mechanically separated the somatic follicle layer and oocyte of the FG follicle and analyzed the mRNA levels in the two compartments by real-time RT-PCR. In agreement with the findings in mammals, zebrafish gdf9 shows exclusive expression in the oocyte, with its transcript being barely detectable in the freshly isolated follicle layer. Interestingly, after a high number of amplification cycles, the mRNA of gdf9 was detected in cultured zebrafish follicular cells, which are free of oocytes. This observation is consistent with the studies in some other organisms, including the human [12], pig [13], and chicken [14], which have also reported the expression of GDF9 in follicular cells at levels lower than those in oocytes. Although the functions of GDF9 as an oocyte-specific factor have been well documented and are generally accepted, the physiological relevance of this molecule from the somatic follicular cells and its regulation remain largely unknown. This would be an interesting issue to address in the future.

To provide clues as to the roles of zebrafish Gdf9 in controlling folliculogenesis, we analyzed its temporal expression pattern during follicle growth using quantitative real-time RT-PCR. Interestingly, the expression of gdf9 at the follicle level was found to be highest in the PG stage (stage I), which contained no cortical alveoli (a marker of the PV stage or stage II) in the oocytes. The expression of gdf9 declined progressively with the growth of the follicles, with the lowest level being reached at the FG stage. This result was confirmed by in situ hybridization, which revealed the strongest staining in the oocytes of the PG stage. This pattern of expression differs from that observed in mammals, in which GDF9 is usually expressed when the follicles enter the primary stage [15, 19], but is similar to the finding recently reported for the chicken, the first oviparous vertebrate from which GDF9 was cloned [14]. Chicken Gdf9 also showed the highest expression level in the smallest follicles [14]. Although the functions of Gdf9 in the zebrafish are currently unknown, the progressive decrease in its expression during follicle growth suggests potential roles for this molecule in the recruitment of follicles from stage I (PG stage) to stage II (cortical alveolus or PV stage) and thereafter during vitellogenic growth.

Zebrafish gdf9 expression continued after fertilization until gastrulation, at which point there was a dramatic decrease in expression. In the mouse, Gdf9 expression decreases dramatically by 6 h postfertilization and reaches a marginal level at the 2-cell stage (9 h postfertilization) [46]. Similarly, bovine GDF9 expression could only be detected in the embryos of the 8-cell stage [20]. The persistent expression of gdf9 in the early zebrafish embryos implicates this molecule in early embryogenesis, although there is no evidence to support such roles. Some members of the GDF family, such as GDF1 and GDF5, have been demonstrated to be essential for left-right patterning [47] and formation of joints [48], respectively. The abundance of zebrafish gdf9 mRNA quickly dropped to undetectable level at the stage of gastrulation. It is of interest to know when the gene starts to be expressed again during development. A recent study on rainbow trout has shown that gdf9 expression starts in the late phase of ovarian differentiation [49].

Although GDF9 was identified more than a decade ago and this molecule has since attracted increasing attention, little is known about its regulation by extracellular endocrine or paracrine factors. In the pig, the expression of GDF9 in the oocytes remains relatively stable during the preovulatory period or after FSH stimulation [13]. Recently, it has been reported for the mouse that FSH regulates the expression of Bmp15 but not Gdf9 [50]. The decreasing expression of gdf9 during zebrafish follicle development prompts us to speculate that this oocyte-derived factor is subject to endocrine regulation, particularly by gonadotropins from the pituitary, since these hormones play vital roles in driving ovarian development. A recent study from our laboratory has shown that with the PG follicles entering the cortical alveolus or PV stage, the expression of FSH receptor (fshr) significantly increases, and continues to rise thereafter. This increase in fshr expression is followed by a progressive increase in lhcgr expression [41].

To determine whether gonadotropins play any role in regulating gdf9 expression in the zebrafish ovary, we performed a series of experiments to study the effects of hCG, which specifically signals through the zebrafish Lhcgr [41], on gdf9 expression using both ovarian fragments and isolated follicles of different stages. The zebrafish is an excellent model for this type of study because large numbers of ovarian follicles of different stages are readily available year round. Interestingly, treatment with hCG in vitro significantly decreased the expression of gdf9, and the effect appeared to be stage-dependent, with the greatest suppression observed in the FG follicles. This is probably due to the increasing expression of lhcgr, the mRNA level of which started to rise at the EV stage and peaked at the FG stage [41]. The inverse correlation between gdf9 and lhcgr expression during zebrafish follicle development and the inhibitory effect of hCG on gdf9 expression in vitro suggest that pituitary gonadotropin is one of the factors responsible for the decreasing expression of gdf9 during follicle growth. Since hCG signals specifically via Lhcgr, the role of Fshr activation and signaling in regulating gdf9 expression remains largely unknown. The elucidation of this issue will depend on the availability of recombinant zebrafish FSH and LH. Although our laboratory has established stable cell lines that express these hormones [51], the extremely low yields hindered their application in the present study. Since zebrafish fshr and lhcgr are expressed exclusively in the follicular cells, another interesting issue to address in the future is how gonadotropins signal the oocytes through the follicular cells. Stage-dependent regulation of GDF9 expression in oocytes has also been reported in mammals. In Nr6a1 knockout mice, it was only at the diestrus stage that Gdf9 expression was significantly different from that of the control [31].

In summary, a full-length cDNA for gdf9 has been cloned and characterized in the zebrafish. Its expression is gonad-specific, with the transcript being detectable only in the ovary and testis. In the ovary, gdf9 is predominantly expressed in the oocyte, although its expression is also detected in cultured follicular cells albeit at a much lower level. The expression of gdf9 appears to be stage-dependent and the highest levels were observed in the follicles at early stages. Pituitary gonadotropins are probably involved in regulating gdf9 expression in the zebrafish ovary, since treatment of the ovarian fragments or follicles in vitro with hCG decreased its expression level in time- and dose-dependent manners.

FOOTNOTES

1Supported by grants (CUHK4258/02M, CUHK4422/04M and CUHK4578/05M) from the Research Grants Council of the Hong Kong Special Administrative Region to W.G. Back

Correspondence: 2FAX: 852 2603 5646; e-mail: weige{at}cuhk.edu.hk

Received: 14 June 2006.

First decision: 21 July 2006.

Accepted: 23 October 2006.

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E. S. Clelland, Q. Tan, A. Balofsky, R. Lacivita, and C. Peng
Inhibition of Premature Oocyte Maturation: A Role for Bone Morphogenetic Protein 15 in Zebrafish Ovarian Follicles
Endocrinology, November 1, 2007; 148(11): 5451 - 5458.
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