当前位置: 首页 > 期刊 > 《内分泌学杂志》 > 2006年第1期 > 正文
编号:11416166
Bone Morphogenetic Protein-15 in the Zebrafish Ovary: Complementary Deoxyribonucleic Acid Cloning, Genomic Organization, Tissue Di
http://www.100md.com 《内分泌学杂志》
     Department of Biology (E.C., G.K., C.P.), York University, Toronto, Canada M3J 1P3

    Serono Research Institute (R.K.C.), Rockland, Massachusetts 02370

    Marine Biological Laboratory (R.K.C.), Woods Hole, Massachusetts 02543

    Department of Reproductive Medicine (S.Sha., S.Shi.), University of California, San Diego, California 92093-0633

    Abstract

    Bone morphogenetic protein-15 (BMP-15) is a member of the TGF family known to regulate ovarian functions in mammals. The structure and function of BMP-15 in lower vertebrates are less known. In this study, we cloned the zebrafish BMP-15 (zfBMP-15) cDNA and depicted its genomic organization. The zfBMP-15 cDNA encodes a protein of 384 amino acids. The mature protein has 46–51% sequence identities to fugu, chicken, and mammalian BMP-15. It also shares 38–46% homology with growth and differentiation factor-9 in fishes, chicken, and mammals. Phylogenetic analysis further confirms that the zfBMP-15 is most closely related to BMP-15 from other species, whereas the growth and differentiation factor-9 peptides from fish to mammals form a distinct branch. Comparison of zfBMP-15 cDNA with zebrafish genome database revealed that zfBMP-15 is encoded by a gene with two exons and one intron, located on chromosome 6. BMP-15 mRNA is expressed in the ovary and testis and, to a lesser extent, brain, liver, gut, heart, and muscle. Real-time PCR revealed that BMP-15 is expressed in follicles at all stages of development with no significant changes over the course of folliculogenesis. Using in situ hybridization and immunocytochemistry, we detected BMP-15 in both oocytes and follicular cells. Incubation of follicles with antiserum against zfBMP15 increased oocyte maturation, whereas incubation with recombinant human BMP-15 suppressed human chorionic gonadotropin-induced oocyte maturation. These findings suggest that BMP-15 plays a role in regulating gonadal functions in fish, in particular oocyte maturation.

    Introduction

    THE TGF SUPERFAMILY HAS been shown to play important roles in regulating ovarian functions in mammals, such as follicle development and maturation, steroid hormone production, and gonadotropin receptor expression (reviewed in Refs.1, 2, 3, 4, 5, 6). Less is known about the role of TGF superfamily in the ovary of lower vertebrates, such as fish. Several studies have reported that activin and inhibin stimulate oocyte maturation in zebrafish (7, 8). On the other hand, TGF has an inhibitory effect on oocyte maturation in the zebrafish (9) and steroid hormone production in the goldfish (10).

    The discovery of oocyte derived TGF like peptides, growth and differentiation factor (GDF)-9 and bone morphogenetic protein (BMP)-15 (also known as GDF-9B) in mammals has led to significant advances in our understanding of early follicle development. Comprehensive reviews of these factors can be found in earlier publications (3, 5, 6, 11, 12, 13, 14, 15, 16, 17). GDF-9 is the product of a somatic gene and is widely expressed in a variety of tissues such as ovary, testis, brain, bone, etc. (12, 18). BMP-15 is the product of an X-linked gene, and was described independently by two research groups (19, 20). To date, BMP-15 has been reported to be expressed in oocytes (19, 20, 21) and primary pituitary cells (22) and to a much lesser extent in other tissues such as heart and kidney (23). Both GDF-9 and BMP-15 are expressed early in follicle development (primordial follicles and primary follicles, respectively) and their expression persists throughout folliculogenesis (12, 24, 25). GDF-9 and BMP-15 are essential factors regulating the pituitary hormone insensitive phase of mammalian ovarian follicle development, although the relative importance is species dependent. In mouse, knockout of GDF-9 arrests follicle development at the primary stage, whereas BMP-15 knockouts are only subfertile (26). On the other hand, homozygous mutations in BMP-15 genes (FecXG,B,I,H,L) are responsible for sterility in ewes, as is homozygosity for the FecGH in GDF-9 genes (27). Defects in BMP-15 have also been implicated in hypergonadotropic ovarian failure in humans (28). GDF-9 and BMP-15 act as mitogens, stimulating proliferation of granulosa cells (17), and are involved in the regulation of regulatory factors such as kit ligand (29) and Gremlin (30). BMP-15 suppresses FSH receptor mRNA expression and blocks the action of FSH in granulosa cells and is thought to play a role in regulating granulosa cell proliferation and FSH sensitivity in developing follicles (31).

    Whereas the important role of BMP-15 in ovarian function in mammals has become increasingly clear, little is known about the structure and function BMP-15 in nonmammalian species. To further understand the role of TGF superfamily in the ovary, we cloned the cDNA encoding BMP-15 from zebrafish and investigated its tissue distribution and function in oocyte maturation. We report here the sequence and structure of zebrafish BMP (zfBMP)-15 and its chromosomal location. The expression of BMP-15 mRNA in ovarian follicles and other tissues and the effect of recombinant human BMP-15 and antiserum against zfBMP-15 on oocyte maturation are also described. Our observations are discussed with regard to their evolutionary significance and their importance in ovarian follicle development in a lower vertebrate.

    Materials and Methods

    Animals

    Zebrafish, Danio rerio, were purchased locally (Fish and Bird Emporium, Brampton, Ontario, Canada). Fish were maintained under 14-h light, 12-h dark, 28 C, in 10-liter tanks in a circulating freshwater AHAB system (Aquatic Habitats, Apopka, FL). Fish were fed twice daily, ad libitum, with Nutrafin staple food (Rolf C. Hagen Inc., Montreal, Quebec, Canada) supplemented two to three times per week with newly hatched brine shrimp (San Francisco Bay Brand, Newark, CA). Zebrafish were killed in accordance with the regulations of the Canada Council for animal care. They were anesthetized in approximately 600 μM Tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich Canada, Oakville, Ontario, Canada) and decapitated.

    Molecular characterization of zfBMP-15

    Sequence similarity searches were performed against zebrafish expressed sequence tag (EST) data using BLAST (www.ncbi.nih.gov/BLAST) (32) and human GDF-9 as the query sequence. Several candidate sequences were identified from the search, and the corresponding EST clones (obtained from Incyte, Willmington, DE) were sequenced using an ABI DNA sequencer (model 310, Applied Biosystems, Foster City, CA). One of the clones, aw170981, was found to have a complete open reading frame. To confirm the sequence obtained from clone aw170981, total RNA was extracted from zebrafish ovary, reverse transcribed, and subjected to PCR using primers that flank the open reading frame. The resulting PCR product was ligated into pCMV5B plasmid (Novagen, Darmstadt, Germany) and sequenced in the York University Molecular Core facility. These sequences were compared with clone aw170981 and found to be identical. The sequence was subsequently BLASTED against the zebrafish genome (http://www.sanger.ac.uk/cgi-bin/blast) to determine its genomic organization. The deduced amino acid sequence was compared against the National Center for Biotechnology Information database to identify homologous proteins. The sequences identified are mostly known BMP-15s and GDF-9s from various species. Because the sequence we obtained consistently showed higher homologies to BMP-15s than to GDF-9s, it was designated as the zfBMP-15. The zfBMP-15 preproprotein sequence was aligned against 18 of the most closely related proteins, including two unnamed protein products (GenBank accession no. CAG01491 and CAF99068.1) [Clustal W, version 1.8, (33)]. Upon comparison, these unnamed proteins showed highest homology to zfBMP-15 and zfGDF-9, respectively, and were therefore considered to be the fugu BMP-15 and GDF-9 in subsequent analyses. A maximum likelihood parsimony phylogram was generated using the protein maximum likelihood program of Phylip 3.6b (34) and Treeview 1.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html), with zfBMP-4 as the outgroup. Signal peptide of the deduced amino acid sequence was predicted using SignalP (35) (www.cbs.dtu.dk/services/SignalP).

    RNA extraction and RT-PCR

    Fish were killed and rinsed in RNase-free Cortland’s medium. Ovary, testis brain, liver, gut, muscle, and heart were excised. Ovarian follicles of differing stages of development were collected as described previously (9). Total RNA was extracted using TRIzol reagent (Invitrogen Canada Inc., Burlington, Ontario, Canada) according to the manufacturer’s instructions. Reverse transcription and PCR were performed as previously described (8, 9). All primers used in this study are listed in Table 1. Primer BMP15–4 and -5 were used to determine tissue distribution of BMP-15 mRNA, and BMP15–6 and -7, incorporated with restriction sites, were used for the cloning of the coding region. Annealing temperature for PCR ranges from 55 to 65 C, depending on the primer set used. Quantitative PCR of ovarian follicles was carried out using an ABI Prism 7700 (Applied Biosystems) with Quantitect SYBR Green (Qiagen Canada Inc., Mississauga, Ontario, Canada). The reaction mix contained 7 pmol primers, 10 μl SYBR green mix, template cDNA, diluted to 20 ml in RNase-free water. The comparative cycle threshold method (outlined in ABI Prism 7700 sequence detection system bulletin 2) was used to determine the relative quantity of BMP-15 mRNA in follicles of developmental stages I and II (previtellogenic), III-1 (small vitellogenic), III-2 (large vitellogenic), and IV (maturing) (9, 36), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous reporter. Primers used in the real-time PCR were BMP15–1 and -3 and GAPDH-1 and -2 (Table 1). A validation curve using serial dilutions of mixed oocyte cDNA was produced to ensure replication efficiencies of BMP-15 and GAPDH were similar.

    In situ hybridization

    To obtain riboprobes for in situ hybridization, PCR was performed using clone aw170981 as the template and BMP15–3 and BMP15–4 as the primers. The resulting 502-bp fragment was subcloned into the pCR II TOPO vector (Invitrogen), and orientation of the cloned BMP-15 fragments was confirmed by DNA sequencing. Both sense and antisense RNA probes were generated using a digoxigenin (DIG) RNA labeling kit (Roche Diagnostics, Laval, Quebec, Canada) following the manufacturer’s instructions. The probes were used to detect zfBMP-15 mRNA in sectioned and whole-mount ovarian tissues.

    For tissue sections, ovaries were excised, blotted dry, and flash frozen in supercooled isopentane. Cryostatic sections (8 μm) were fixed to poly-L-lysine-coated slides and allowed to dry. In situ hybridizations were completed following the protocol of Braissant and Wahli (37), with the addition of a RNase A digestion step. Briefly, after fixation in 4% paraformaldehyde, tissue sections were washed and prehybridized for 2 h at 58 C in 5x saline sodium citrate (SSC)/formamide (1:1) containing 40 μg/ml salmon sperm DNA. The sections were then incubated overnight at 58 C in SSC/formamide containing 1 μg/ml DIG-labeled zfBMP-15 sense or antisense RNA probe. After hybridization, sections were washed in 2x SSC at room temperature (RT) and then 2x and 0.1x SSC at 65 C. After RNase A digestion (1 μg/ml) for 30 min at 37 C, sections were washed again in 0.1x SSC and probed with an anti-DIG antibody (Roche; 1:5000 dilution) for 2 h at RT. Color was developed using 5-bromo-4-chloro-3-indoyl-phosphate/4-nitro blue tetrazolium chloride (NBT) for 3 h at RT. Photographs were taken under light microscope using Ektrachrome 160T film (Kodak, Rochester, NY).

    For whole mounts, each ovary was excised, washed, and split approximately into quarters. Hybridization was conducted following the protocol of Shinomiya et al. (38). In brief, tissues were digested for 2 min in 10 mg/ml proteinase K in PBS and Tween 20 (0.1%), rinsed in 1 mg/ml glycine, and refixed in 4% paraformaldehyde. They were prehybridized at 65 C for 1 h in hybridization buffer containing 50% formamide/5x SSC, 0.1% Tween 20, 0.5 mg/ml Torula yeast RNA, and 50 μg/ml heparin (Sigma-Aldrich). The solution was then replaced with hybridization buffer containing 1 μg/ml sense or antisense RNA probe and tissues incubated overnight at 65 C. Subsequently tissues were washed several times and probed with anti-DIG (1:5000) for 2 h at RT. Color was developed overnight using 5-bromo-4-chloro-3-indoyl-phosphate/NBT, nonspecific staining removed in 95% ethanol, and ovaries stored in 70% glycerol. Photos were taken under a dissecting microscope using Ektachrome 160T.

    Antibody production and immunohistochemistry

    A peptide (MQTFISELGVADIPL) located at the C-terminal mature zfBMP-15 region was selected as the antigen for production of polyclonal antiserum. The sequence was submitted to Sigma-Genosys (Oakville, Ontario, Canada), in which the peptide was synthesized and used to inoculate white rabbits for antibody production. For immunocytochemistry, the following procedures were conducted at RT, unless otherwise stated. Sections were postfixed for 10 min in 4% paraformaldehyde in PBS, washed twice in PBS, blocked for 60 min in 5% normal goat serum in PBS, and incubated with either the zfBMP-15 antiserum or preimmune serum overnight at 4 C. The sections were washed and probed with donkey antirabbit IgG horseradish peroxidase-conjugated secondary antibody (Amersham Canada, Baie d’Urfe, Quebec) applied at 1:100 dilution. After a 2-h incubation period, the sections were washed in PBS, color developed with DAB-H202 (Sigma-Aldrich), dehydrated through graded ethanol, mounted via xylene, and photographed.

    Follicle incubations

    Zebrafish were killed and ovaries excised as described above. Follicles ranging from 0.52 to 0.68 mm were selected and incubated (20 follicles /well) in 24-well culture plates for 24 h at 28 C. For incubations with exogenous BMP-15, follicles were incubated with 400 ng/ml recombinant human (rh) BMP-15 produced as previously described (21), 2 μg/ml recombinant human chorionic gonadotropin (hCG) (obtained from Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA), or a combination of rhBMP-15 and hCG, and scored for maturation. Oocytes that underwent germinal vesicle breakdown were identified by their ooplasmic clearing (due to proteolytic cleavage of vitellogenin) (9, 36). To determine the effect of BMP-15 antiserum on oocyte maturation, follicles were incubated with either preimmune serum or zfBMP-15 antiserum. The sera were diluted in Cortland’s medium to final concentrations of 1:25, 1:50, 1:100, or 1:200. Each group had four replicate wells and each experiment was repeated two to three times. Data were analyzed by one-way ANOVA with Student-Newman-Keuls post testing, using GraphPad Instat (GraphPad Software, San Diego, CA).

    Results

    Cloning of zfBMP-15 cDNA

    We obtained the full-length cDNA sequence for zfBMP-15 (Fig. 1, GenBank accession no. AY954923) by sequencing an EST clone (aw 170981) and confirmed by RT-PCR of RNA samples extracted from zebrafish ovaries and subsequent cloning. The nucleotide sequence encodes a protein of 384 amino acids. The signal peptide is predicted to be 26 residues. A cleavage site (RXXR), known to be recognized by proteases involved in processing members of the TGF superfamily (6), is found in position 254–257. Therefore, the mature protein of zfBMP-15 is predicted to have 127 residues, from the position 258 to 384 (Fig. 1). Most members of the TGF family have been reported to possess seven conserved cysteine residues in the mature protein, six contributing to the formation of a cysteine knot and the remaining cysteine forming an intersubunit disulfide bond between two subunits. BMP-15 and GDF-9 are notable exceptions in that they lack the residue responsible for dimerization (17). zfBMP-15 resembles tetrapod BMP-15 and GDF-9, possessing only six cysteines in the mature region (Fig. 1).

    Comparison of zfBMP-15 cDNA sequence with zebrafish genomic sequences revealed that the bmp-15 gene is located in chromosome 6 and has two exons and one intron (Fig. 2). Exon 1 encodes for the 5' untranslated region, the coding sequence of the signal peptide and the first 90 amino acids of the proregion. Exon II encodes for the remaining proregion, the C-terminal mature protein, and the 3' untranslated region.

    Phylogenetic analysis

    The phylogenic relationship between the full-length sequences of zfBMP-15, GDF-9, and BMP-4, fugu BMP-15 and GDF-9, mammalian GDF-9 and BMP-15, frog Vg1, and Drosophila DPP predicted by the maximum likelihood protein parsimony analysis program in PHYLIP, is shown in Fig. 3A. This phylogram was predicted by both the Jones-Taylor-Thornton and the Henikoff/Tillier models (described in PHYLIP). Because BMP-15 and GDF-9 have previously been shown to form a distinct branch in a phylogram of TGF family members (11), with the exception of zfBMP-4, DPP, and Vg1, other family members were not considered. Alignment with Clustal W revealed that the location of all cysteine knot residues has been conserved in all the peptides examined. BMP-15s and GDF-9s were seen to form separate branches on the phylogram as expected. zfBMP-15 most closely aligned with fugu BMP-15, whereas zfGDF-9 was most similar to the fugu homolog. zfBMP-4, the outlier for this analysis, grouped with the amphibian Vg1 and Drosophila DPP and served to illustrate the distinctive nature of BMP-15 and GDF-9 relative to other members of the TGF superfamily.

    When the mature zfBMP-15 sequence was compared with related proteins in the National Center for Biotechnology Information database, it was found that zfBMP-15 has about 46–55% sequence identity to BMP-15 of fish, bird, and mammals (Fig. 3B). The homology of zfBMP-15 to GDF-9 from other species is slightly lower at 38–46% (Fig. 3B).

    Expression of BMP-15

    To determine the mRNA expression of BMP-15, total RNA was extracted from ovary, testis, heart, gut, liver, brain, and muscle. RT-PCR revealed strong expression of BMP-15 mRNA in ovary and testis. BMP-15 mRNA was also detected in the brain, liver, gut, heart, and muscle, although the expression level in these tissues was much lower than that in the gonads (Fig. 4). In this experiment, the possibility of amplifying genomic DNA was ruled out by selecting intron-spanning primers.

    Because BMP-15 is known to play important roles in the ovary, we further examined the expression pattern of BMP-15 in the ovary by in situ hybridization and immunocytochemistry. As shown in Fig. 5A, when ovarian tissues were incubated with DIG-labeled antisense BMP-15 probe, small follicles were positively stained after 2 h of color development. Positive staining was observed in all follicles after 24 h of color development (data not shown). In tissues incubated with the sense probe, no specific signals were observed. In ovarian sections probed with antisense zfBMP-15 RNA, it was found that BMP-15 mRNA is expressed in not only oocytes but also granulosa and theca cells but was generally absent from ovarian stroma (Fig. 5B). To confirm the findings from in situ hybridization, we also carried out immunocytochemistry studies using an antiserum against a synthetic peptide of zfBMP-15. When the antiserum was used to incubate ovarian sections, positive signals were observed in oocytes and follicular cells (Fig. 6A). No signal was detected in control sections incubated with preimmune serum (Fig. 6B).

    To measure BMP-15 mRNA levels in different stages of follicles, a real-time PCR assay was developed. To validate the use of GAPDH as the endogenous control, dilution curves, representing threshold cycle vs. log of cDNA dilutions were performed for BMP15 and GAPDH (Fig. 7A). Both curves had similar slopes, thus indicating similar amplification efficiencies of BMP-15 and GAPDH. Subsequently total RNA was extracted from: 1) previtellogenic follicles (stages I and II, smaller than 0.38 mm); 2) small vitellogenic follicles (stage III-1, 0.39–0.51 mm); 3) large vitellogenic follicles (stage III-2, 0.52–0.69 mm); and 4) maturing follicles (stage IV, greater than 0.69 mm). The RNA samples were treated with DNase to remove any genomic DNA and then reverse transcribed. Real-time PCR revealed that there is no significant difference in BMP-15 mRNA levels among follicles of different sizes (Fig. 7B). Real-time PCR was also conducted using TaqMan probes (Applied Biosystems), and similar results were obtained (data not shown).

    Effect of BMP-15 on oocyte maturation

    To determine the effect of BMP-15 on oocyte maturation, two approaches were used. First, follicles were incubated with rhBMP-15, either alone or in combination with hCG. It was observed that rhBMP-15 had no apparent effect on basal oocyte maturation, but when added in combination with hCG, it suppressed oocyte maturation induced by hCG (Fig. 8A). When follicles were incubated with zfBMP-15 antiserum, there were significant increases in oocyte maturation rates, compared with follicles in the control Cortland’s medium or Cortland’s supplemented with preimmune serum (Fig. 8B). The effect was observed for all serum concentrations tested. Finally, follicles were incubated with hCG and either the preimmune serum or antiserum against zfBMP-15. As shown in Fig. 8C, preimmune serum had no significant effect on basal oocyte maturation but slightly reduced the effect of hCG. Antiserum against zfBMP-15 again induced a significant increase in oocyte maturation; however, in the presence of hCG and anti-BMP-15, there was no further increase in oocyte maturation (Fig. 8C).

    Discussion

    BMP-15 has been shown to be an important factor involved in the regulation of early folliculogenesis in mammals. However, no studies on BMP-15 have been reported in lower vertebrates. In this study, we obtained the cDNA sequence and depicted the genomic organization of zfBMP-15. We also examined its expression in various tissues, especially in the ovary and demonstrated that blocking the effects of endogenously secreted BMP-15 (using antiserum) leads to an increase in oocyte maturation rate, whereas addition of exogenous BMP-15 suppresses hCG-induced oocyte maturation.

    The zfBMP-15 has six cysteine residues in its mature protein region, a characteristic of mammalian BMP-15 and GDF-9 (17), and Clustal W alignment of BMP-15, GDF-9, and the related proteins before PHYLIP analysis revealed that the location of the cysteine knot residues is 100% conserved. Phylogenetic analysis demonstrates that the zfBMP-15 is more closely related to BMP-15 from other species than to GDF-9. These findings support our assertion that the molecule we cloned is in fact BMP-15. However, compared with many other members of the TGF family, BMP-15 is less conserved. For example, the zebrafish activin-A and -B sequences are over 90% identical with the mammalian counterparts. Similarly, the zebrafish TGF1 has over 70% identities with mammalian TGF1 (9). However, we found only 46–51% homology between the zebrafish and tetrapod BMP-15. The low degree of conservation of zfBMP-15 is in agreement with mammalian studies in which the mouse and human BMP-15 amino acid sequences in the mature domain exhibit only 70% identity, whereas other BMP proteins have a greater than 96% homology between mouse and human (19). Despite the apparent low homology between zebrafish and human BMP-15, the rhBMP-15 has activities in zebrafish as determined in oocyte maturation experiments.

    Surprisingly, whereas zfBMP-15 mature protein has 42–46% homologies with mammalian GDF-9, it shares only 39% identity with zfGDF-9 and 38% with fugu GDF-9. Comparison between fugu BMP-15 and GDF-9 also reveals less than 40% similarities. The identities between fish BMP-15 and GDF-9 within the same species are lower than those in mammals, which have approximately 50% homologies (26). In mammals, it is well documented that BMP-15 and GDF-9 have overlapping and synergistic roles in regulating ovarian functions (6, 26, 39, 40, 41). The reason for such low degree of identity between fish BMP-15 and GDF-9 is unclear, and description of additional piscine homologs is required for any further interpretation. Similarly, whether BMP-15 and GDF-9 have synergistic functions in fish ovary remains to be determined.

    Comparison of the zfBMP-15 cDNA sequence with zebrafish genome database reveals that the zfBMP-15 mRNA is contained within two exons separated by an intron. This is consistent with the human and mouse bmp15 genes (19). BMP-15 is known as an X-linked protein in mammals (19), whereas the zebrafish bmp-15 gene is mapped to chromosome 6. This is not surprising because zebrafish do not appear to have sex chromosomes (42).

    Using RT-PCR, we detected a high level of BMP-15 mRNA in ovary and testis. This is consistent with results from mammalian studies. In mouse, strong expression of BMP-15 was found in the oocytes (19). In human and bovine, BMP-15 has been reported to be expressed in both testis and ovary (24, 43). We also detected low level of mRNA in other tissues such as the heart, liver, gut, brain, and muscle, which is similar to the expression of BMP-15 in sheep (23). Recently Otsuka and Shimasaki (22) demonstrated that gene expression and protein synthesis also occur in primary pituitary cells of mice and in the gonadotrope cell line LT2. In the pituitary, BMP-15 stimulated synthesis and release of FSH but had no effect on LH secretion, suggesting that BMP-15 acts in an autocrine manner to regulate the monotropic rise of FSH during the estrous and menstrual cycles. GDF-9 is expressed in oocytes (44, 45, 46) but has also been shown to be expressed in a number of other tissues, including brain, bone, testes, and uterus (47, 48). Thus, the tissue distribution pattern of BMP-15 in zebrafish is similar to that of GDF-9 and BMP-15 in mammals.

    Using in situ hybridization and immunocytochemistry, we detected BMP-15 mRNA and protein expression in all stages of ovarian development. Quantification of mRNA levels by real-time PCR revealed that there was no significant difference in BMP-15 mRNA levels among follicles at different stages of development. However, we cannot exclude the possibility that BMP-15 protein expression and/or secretion may vary with follicles development and oocyte maturation, and this will be tested in the future. Nevertheless, our observation that BMP-15 is expressed in follicles at all stages of development is consistent with mammalian studies in which BMP-15 has been reported to be expressed from early to late stages of follicle development (49). The important role of BMP-15 in folliculogenesis has been well established. Naturally occurring mutations in the bmp-15 genes of Inverdale, Hanna, F700 Belclare, Cambridge, and Booroola sheep produce infertility in ewes homozygous for the mutation, and hyperfertility in heterozygotes (27). The follicles of sterile ewes do not proceed beyond the primary stage and possess an enlarged oocyte and an incomplete complement of granulosa cells owing to a failure of granulosa cell proliferation. Rams do not appear to be affected by these mutations (23, 49, 50, 51). The role of BMP-15 in early follicle development of fish is unknown.

    In the present study, we demonstrated that BMP-15 has an inhibitory effect on oocyte maturation in zebrafish. Similar to previous studies, treatment of hCG significantly induced oocyte maturation (8, 9, 36). The basal and hCG-induced maturation rates observed in this study were consistent with previous reports from our (8, 9) and other (36) laboratories. Although efforts to synthesize recombinant zfBMP-15 have been unsuccessful, we have observed that hCG failed to induce oocyte maturation in zebrafish follicles incubated with rhBMP-15, suggesting that BMP-15 has an inhibitory effect on oocyte maturation. This notion is further supported by the finding that incubation of zebrafish follicles with antiserum against zfBMP-15 significantly induced oocyte maturation. When zfBMP-15 antiserum was added with hCG, we observed no further increase in hCG-induced oocyte maturation. It may be possible that hCG down-regulates BMP-15 expression, and thus, neutralization of endogenous BMP-15 has little effect over hCG-induced maturation. In addition, because the effect of hCG on oocyte maturation is dependent on follicle sizes (larger follicles have greater response to hCG) (36) as well as the dose and incubation time (36), it is also possible that the lack of facilitation of hCG effects by BMP-15 antiserum is due to the presence of unresponsive follicles and/or the hCG concentration used. Interestingly, preimmune serum slightly reduced the effect of hCG on oocyte maturation, suggesting the presence of inhibitory growth factors/hormones in the serum. The observation that antiserum against zfBMP-15 induced oocyte maturation indicates that the antiserum we produced has neutralizing activity. Several studies in mammals have reported that antibodies raised against peptides located in the C-terminal region of BMP-15 or GDF-9 have neutralizing effects (40, 41). The peptide selected for the production of zfBMP-15 antibody has little homology with other BMPs and GDFs, including the zebrafish GDF-9. Therefore, the antiserum is expected to have high specificity.

    Although the mechanisms underlying the inhibitory effect of BMP-15 on oocyte maturation remain to be investigated, several possibilities exist. First, BMP-15 may inhibit inhibin and activin expression and therefore decrease oocyte maturation. In zebrafish, it has been demonstrated that activin and inhibin enhance hCG-induced oocyte maturation (7, 8) and hCG up-regulates activin/inhibin-A subunit expression (8, 52, 53). In mammals, BMP-15 has been shown to inhibit FSH-induced inhibin expression (31). Second, BMP-15 may inhibit the production of 17, 20, dihydroxyprogesterone, which is known to be the maturation-inducing hormone in zebrafish (54). Again, BMP-15 is known to inhibit gonadotropin-induced progesterone production in mammals (21, 40, 41). Finally, BMP-15 may down-regulate LH receptor, thereby reducing the effect of hCG on oocyte maturation. It has been reported that BMP-15 suppresses FSH-induced LH receptor mRNA levels (31). Our preliminary data also suggest that in zebrafish follicles, hCG-induced LH receptor mRNA expression is inhibited by BMP-15 (Clelland, E., and C. Peng, unpublished results).

    Several studies in mammals have suggested that in addition to promoting early follicle development, BMP-15 and GDF-9 are also involved in the regulation of late stages of follicle development. However, unlike our findings in zebrafish follicles, which indicate an inhibitory role of BMP-15 in oocyte maturation, BMP-15 and GDF-9 appear to have a positive role in later stages of follicle development, such as cumulus expansion and oocyte maturation (55). In double-mutant mice lacking both gdf9 and bmp15 genes, FSH failed to induce cumulus cell expansion (55). In addition, there was a delay in LH-induced oocyte maturation in vivo but no significant change in spontaneous in vitro oocyte maturation (55). The effects of BMP-15 and GDF-9 are thought to be mediated by their actions on cumulus cells because no BMP-15 receptors are expressed on oocytes (55). In isolated murine cumulus-oocyte complexes, which develop normally and enlarge in culture, knockdown of GDF-9 but not BMP-15 inhibits cumulus expansion and decreased expression of prostaglandin synthase-2 and hyaluron synthase-2, which are necessary for antral expansion (56), supporting the notion that GDF-9 is the cumulus expansion-enabling factor. However, BMP-15 and GDF-9 may also have an inhibitory role in ovulation in the sheep because short-term immunization of ewes against either BMP-15 or GDF-9 has been shown to increase ovulation rate without apparent detrimental effect on ovulated follicles (57), although long-term immunization results in anovulation due to the cessation of follicular growth (58).

    In summary, this study is the first in lower vertebrates to examine the structure and expression of BMP-15 and provides preliminary evidence, suggesting BMP-15 plays a role in regulating gonadal function in zebrafish. Future studies on the function of BMP-15 in fish, such as early follicle development, will provide further insight into the structural and functional evolution of this protein.

    Acknowledgments

    We thank Ms. Lee Wong for performing DNA sequencing and Dr. A. F. Parlow and National Hormone and Peptide Program for providing recombinant human hCG.

    Footnotes

    This study was supported in part by an Natural Sciences and Engineering Research Council grant (to C.P.) and National Institutes of Health Grant HD41494 (to S.S.). C.P. is a recipient of an Ontario Premier’s Research Excellent Award and a Mid-Career Award from Ontario Women’s Health Council and Canadian Institutes for Health Research.

    E.C, G.K., R.C., S.S., S.S., and C.P. have nothing to declare.

    First Published Online October 6, 2005

    Abbreviations: BMP, Bone morphogenetic protein; DIG, digoxigenin; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDF, growth and differentiation factor; hCG, human chorionic gonadotropin; NBT, 4-nitro blue tetrazolium chloride; rh, recombinant human; RT, room temperature; SSC, saline sodium citrate; zfBMP, zebrafish BMP.

    Accepted for publication September 28, 2005.

    References

    Peng C, Mukai ST 2000 Activins and their receptors in female reproduction. Biochem Cell Biol 78:261–279

    Peng C 2003 The TGF superfamily and its roles in the human ovary and placenta. J Obstet Gynaecol Can 25:834–844

    Shimasaki S, Moore RK, Erickson GF, Otsuka F 2003 The role of bone morphogenetic proteins in ovarian function. Reprod Suppl 61:323–337

    Ingman WV, Robertson SA 2002 Defining the actions of transforming growth factor in reproduction. Bioessays 24:904–914

    Knight PG, Glister C 2003 Local roles of TGF superfamily members in the control of ovarian follicle development. Anim Reprod Sci 78:165–183

    Juengel JL, McNatty KP 2005 The role of proteins of the transforming growth factor- superfamily in the intraovarian regulation of follicular development. Hum Reprod Update 11:143–160

    Pang Y, Ge W 1999 Activin stimulation of zebrafish oocyte maturation in vitro and its potential role in mediating gonadotropin-induced oocyte maturation. Biol Reprod 61:987–992

    Wu T, Patel H, Mukai S, Melino C, Garg G, Ni X, Peng C 2000 Activin, inhibin, and follistatin in zebrafish ovary: expression and role in oocyte maturation. Biol Reprod 62:1585–1592

    Kohli G, Hu S, Clelland E, Di Muccio T, Rothenstein J, Peng C 2003 Cloning of transforming growth factor-1 (TGF1) and its type II receptor from zebrafish ovary and role of TGF1 in oocyte maturation. Endocrinology 144:1931–1941

    Calp MK, Matsumoto JA, Van Der Kraak G 2003 Activin and transforming growth factor- as local regulators of ovarian steroidogenesis in the goldfish. Gen Comp Endocrinol 132:142–150

    Chang H, Brown CW, Matzuk MM 2002 Genetic analysis of the mammalian transforming growth factor- superfamily. Endocr Rev 23:787–823

    Wu X, Matzuk MM 2002 GDF-9 and BMP-15: oocyte organizers. Rev Endocr Metab Disord 3:27–32

    Fortune JE 2003 The early stages of follicular development: activation of primordial follicles and growth of preantral follicles. Anim Reprod Sci 78:135–163

    McNatty KP, Juengel JL, Wilson T, Galloway SM, Davis GH, Hudson NL, Moeller CL, Cranfield M, Reader KL, Laitinen MP, Groome NP, Sawyer HR, Ritvos O 2003 Oocyte-derived growth factors and ovulation rate in sheep. Reprod Suppl 61:339–351

    Gilchrist RB, Ritter LJ, Armstrong DT 2004 Oocyte-somatic cell interactions during follicle development in mammals. Anim Reprod Sci 82–83:431–446

    Juengel JL, Bodensteiner KJ, Heath DA, Hudson NL, Moeller CL, Smith P, Galloway SM, Davis GH, Sawyer HR, McNatty KP 2004 Physiology of GDF9 and BMP15 signalling molecules. Anim Reprod Sci 82–83:447–460

    Shimasaki S, Moore RK, Otsuka F, Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocr Rev 25:72–101

    Fitzpatrick SL, Sindoni DM, Shughrue PJ, Lane MV, Merchenthaler IJ, Frail DE 1998 Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues. Endocrinology 139:2571–2578

    Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM 1998 The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12:1809–1817

    Laitinen M, Vuojolainen K, Jaatinen R, Ketola I, Aaltonen J, Lehtonen E, Heikinheimo M, Ritvos O 1998 A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis. Mech Dev 78:135–140

    Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF, Shimasaki S 2000 Bone morphogenetic protein-15. Identification of target cells and biological functions. J Biol Chem 275:39523–39528

    Otsuka F, Shimasaki S 2002 A novel function of bone morphogenetic protein-15 in the pituitary: selective synthesis and secretion of FSH by gonadotropes. Endocrinology 143:4938–4941

    Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O 2000 Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nat Genet 25:279–283

    Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, Hovata O, Dale L, Ritvos O 1999 Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 84:2744–2750

    Duffy DM 2003 Growth differentiation factor-9 is expressed by the primate follicle throughout the periovulatory interval. Biol Reprod 69:725–732

    Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 15:854–866

    McNatty KP, Smith P, Moore LG, Reader K, Lun S, Hanrahan JP, Groome NP, Laitinen M, Ritvos O, Juengel JL 2005 Oocyte-expressed genes affecting ovulation rate. Mol Cell Endocrinol 234:57–66

    Di Pasquale E, Beck-Peccoz P, Persani L 2004 Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene. Am J Hum Genet 75:106–111

    Otsuka F, Shimasaki S 2002 A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis. Proc Natl Acad Sci USA 99:8060–8065

    Pangas SA, Jorgez CJ, Matzuk MM 2004 Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin. J Biol Chem 279:32281–32286

    Otsuka F, Yamamoto S, Erickson GF, Shimasaki S 2001 Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression. J Biol Chem 276:11387–11392

    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Higgins DG, Bleasby AJ, Fuchs R 1992 CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8:189–191

    Felsenstein J 2004 PHYLIP (Phylogeny Inference Package) version 3.6b. Seattle: Department of Genome Sciences, University of Washington

    Nielsen H, Engelbrecht J, Brunak S, von Heijne G 1997 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Selman K, Petrino TR, Wallace RA 1994 Experimental conditions for oocyte maturation in the zebrafish Brachydanio rerio. J Exp Biol 269:538–550

    Braissant O, Wahli W 1998 A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs in tissue sections. Bichemica 1:10–16

    Shinomiya A, Tanaka M, Kobayashi T, Nagahama Y, Hamaguchi S 2000 The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes. Dev Growth Differ 42:317–326

    Moore RK, Otsuka F, Shimasaki S 2003 Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells. J Biol Chem 278:304–310

    McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP 2005 Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction 129:481–487

    McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP 2005 Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduction 129:473–480

    Traut W, Winking H 2001 Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy. Chromosome Res 9:659–672

    Pennetier S, Uzbekova S, Perreau C, Papillier P, Mermillod P, Dalbies-Tran R 2004 Spatio-temporal expression of the germ cell marker genes MATER, ZAR1, GDF9, BMP15, and VASA in adult bovine tissues, oocytes, and preimplantation embryos. Biol Reprod 71:1359–1366

    Bodensteiner KJ, Clay CM, Moeller CL, Sawyer HR 1999 Molecular cloning of the ovine Growth/differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries. Biol Reprod 60:381–386

    Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM 1999 Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary. Mol Endocrinol 13:1018–1034

    Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM 1999 Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 13:1035–1048

    Elvin JA, Yan C, Matzuk MM 2000 Oocyte-expressed TGF superfamily members in female fertility. Mol Cell Endocrinol 159:1–5

    Erickson GF, Shimasaki S 2000 The role of the oocyte in folliculogenesis. Trends Endocrinol Metab 11:193–198

    Mazerbourg S, Hsueh AJ 2003 Growth differentiation factor-9 signaling in the ovary. Mol Cell Endocrinol 202:31–36

    Bodensteiner KJ, McNatty KP, Clay CM, Moeller CL, Sawyer HR 2000 Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the inverdale prolificacy gene [FecX(I)]. Biol Reprod 62:1479–1485

    Mandon-Pepin B, Oustry-Vaiman A, Vigier B, Piumi F, Cribiu E, Cotinot C 2003 Expression profiles and chromosomal localization of genes controlling meiosis and follicular development in the sheep ovary. Biol Reprod 68:985–995

    Di Muccio T, Mukai ST, Clelland E, Kohli G, Cuartero M, Wu T, Peng C 2005 Cloning of a second form of activin A cDNA and regulation of activin A subunits and activin type II receptor mRNA expression by gonadotropin in zebrafish ovary. Gen Comp Endocrinol 143:287–299

    Pang Y, Ge W 2002 Gonadotropin regulation of activin A and activin type IIA receptor expression in the ovarian follicle cells of the zebrafish, Danio rerio. Mol Cell Endocrinol 188:195–205

    Rahman M, Ohta K, Yoshikuni M, Nagahama Y, Chuda H, Matsuyama M 2002 Characterization of ovarian membrane receptor for 17,20-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in yellowtail, Seriola quinqueradiata. Gen Comp Endocrinol 127:71–79

    Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ 2004 Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276:64–73

    Gui LM, Joyce IM 2005 RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice. Biol Reprod 72:195–199

    Juengel JL, Hudson NL, Whiting L, McNatty KP 2004 Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes. Biol Reprod 70:557–561

    Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O’Connell AR, Laitinen MP, Cranfield M, Groome NP, Ritvos O, McNatty KP 2002 Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biol Reprod 67:1777–1789(Eric Clelland, Gurneet Kohli, Robert K. )