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Endocrinology Vol. 144, No. 5 1931-1941
Copyright © 2003 by The Endocrine Society

Cloning of Transforming Growth Factor-ß1 (TGF-ß1) and Its Type II Receptor from Zebrafish Ovary and Role of TGF-ß1 in Oocyte Maturation

Gurneet Kohli, Siqin Hu, Eric Clelland, Tamara Di Muccio, Jeffrey Rothenstein and Chun Peng

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

Address all correspondence and requests for reprints to: Dr. Chun Peng, Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. E-mail: cpeng{at}yorku.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGF-ß is a multifunctional factor involved in regulating a variety of cellular activities. In mammals, TGF-ß is known to regulate reproduction, including ovarian functions. The role of TGF-ß in lower vertebrates, such as fish, is poorly understood. To examine the role of TGF-ß in fish reproduction, cDNAs encoding TGF-ß1 and the type II TGF-ß receptor (TßRII) were cloned from the zebrafish ovary using PCR- based strategies. The mature peptide region of the zebrafish TGF-ß1 shows 70–85% identity with TGF-ß1 from other species. The zebrafish TßRII cDNA sequence is the first to be reported from a fish species, and it shows a high level of conservation at the kinase domain. Using RT-PCR, we have detected mRNA expression of TGF-ß1, TßRII, as well as its downstream signaling molecules Smad2, 3, and 4 in ovarian follicles at different stages of development. In addition, we have examined the effect of TGF-ß1 on oocyte maturation. TGF-ß1 significantly inhibited both gonadotropin- and 17{alpha}, 20ß-dihydroxyprogesterone-induced oocyte maturation in a dose- and time-dependent manner. These findings demonstrate, for the first time, that TGF-ß1 plays a role in regulating oocyte maturation in fish and suggest that a TGF-ß/Smad signaling pathway is present in the zebrafish ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TGF-ß SUPERFAMILY comprises a large number of structurally related, secreted, polypeptide growth factors (1). More than 30 members of this family have been identified to date. Based on their structural homologies, TGF-ß superfamily members have been grouped into a number of subfamilies, three of the best characterized being: 1) activins/inhibins; 2) bone morphogenetic proteins; and 3) TGF-ß. Members of the TGF-ß superfamily have been implicated in many diverse developmental and physiological processes, including reproduction (1, 2, 3).

Three different forms of TGF-ß (1, 2, 3) are known to be expressed in the mammalian ovary. They have been detected in follicular cells and oocytes of several species (4, 5, 6, 7). TGF-ß has been implicated in various aspects of ovarian development, including steroidogenesis, maturation, ovulation, and follicular atresia (8, 9, 10, 11, 12, 13). However, inconsistent findings are often reported. TGF-ß has been shown to be a predominant inhibitor of follicle cell growth (10). On the other hand, TGF-ß increased follicle size in a preantral follicle culture system (14) and stimulated (3H)thymidine incorporation into preantral follicle cells (15). Similarly, the data on the role of TGF-ß in oocyte maturation in mammals are also somewhat conflicting. Studies have shown a stimulatory (11), inhibitory (16, 17), and no effect (18) of TGF-ß on oocyte maturation. Thus, the precise role of TGF-ß in the ovary is still largely unclear.

TGF-ß has also been shown to be expressed in fish. TGF-ß1 is present in several teleost fish, such as the rainbow trout, goldfish, carp, and hybrid striped bass (19, 20, 21, 22, 23). TGF-ß2 has been found in the carp (24). TGF-ß3 genes have been identified in the rainbow trout, European eel and the Siberian sturgeon (22). Recently, it was shown that genes for all three different isoforms of TGF-ß are present in the plaice (23), providing the first evidence that three members of the TGF-ß family exist in a single fish species. However, the role of TGF-ß in fish ovary is poorly understood.

The TGF-ß receptors and Smad signaling molecules are the crucial components of TGF-ß signal transduction. The two TGF-ß receptors, i.e. the type I (TßRI) and type II (TßRII), possess serine-threonine kinase activity. The Smad proteins are important effectors of signal transduction and act downstream of the serine-threonine kinase receptors (1, 2, 3, 25). Smads are of the 3 major types: 1) receptor-regulated Smads (R-Smads); 2) common mediator Smad; and 3) inhibitory Smads. Signaling is initiated by the binding of TGF-ß to the TßRII, which, in turn, recruits and phosphorylates TßRI. Upon activation by TßRII, the TßRI phosphorylates an R-Smad (Smad 2 or 3), allowing it to associate with a common mediator Smad (Smad 4), forming a hetero-oligomeric complex, which translocates to the nucleus. In the nucleus, Smads regulate transcription by interacting with DNA binding cofactors, coactivators, or corepressors (25, 26). Several Smads, such as Smad2, 3, and 4, have been cloned from the zebrafish, and their expression in embryos has been reported (27, 28). However, the type II TGF-ß receptor has not been cloned in any fish species.

Teleost fish have been used extensively to study the mechanisms underlying oocyte maturation. It is now well established that there are three major regulators of oocyte maturation. First, gonadotropin-II (LH) secreted from the pituitary stimulates the release of a maturation-inducing hormone (MIH) from the follicular cells. In a majority of fish species, MIH has been identified as 17{alpha}, 20ß-dihydroxyprogesterone (DHP). MIH then acts on the oocyte to activate a maturation-promoting factor, which is made up of two subunits, cyclin B (a regulatory subunit) and Cdc-2 (a catalytic subunit, reviewed in Refs.29, 30, 31). In addition, several growth factors, such as activin (32, 33, 34), epidermal growth factor (35), insulin (36, 37), and IGF-I/-II (36, 37, 38), have also been shown to stimulate oocyte maturation. The role of TGF-ß in fish oocyte maturation has not been examined.

Zebrafish have been widely used as a model to examine genetic and molecular mechanisms that direct early embryonic development. This model is also very useful for the study of ovarian follicle development and maturation because the zebrafish ovary contains ovarian follicles at different stages of development. We have been using zebrafish to examine the role of TGF-ß superfamily, such as activin in oocyte maturation (32). We report here our recent studies on the structure of zebrafish TGF-ß1 and TßRII, as well as the role of TGF-ß1 in oocyte maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult zebrafish, Danio rerio, were purchased from a local pet store. They were maintained in 9-liter tanks of an AHAB System (Aquatic Habitats, FL) at 26 C, under a 14-h light, 10-h dark photoperiod. The fish were fed twice daily with tropical fish food and supplemented with brine shrimp once or twice a week. Experiments were performed according to the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care.

Cloning of TGF-ß1 cDNA
An overview of the sequencing strategy is provided in Fig. 1AGo. Degenerate primers TGF-ß1–2 and 3 were designed, based on the sequence of the conserved region of TGF-ß1. These primers were used in a PCR with zebrafish ovarian cDNA as template. After sequencing and comparison with GenBank database, an overlapping expressed sequence tag (EST) clone (accession no. AW56657) was identified and additional primers (TGF-ß1–13 and TGF-ß1–15), were designed and used to perform a 3' rapid amplification of cDNA ends (RACE). The 3'RACE products were sequenced directly and yielded the 3' end of the gene. Another overlapping EST clone (WZ3446.1), which had the complete 5' sequence, was identified when searching the zebrafish genome database (Washington University Zebrafish EST project: http://zfish.wustl.edu/). Therefore, forward and reverse primers (TGF-ß1–20 and 21, respectively) flanking the TGF-ß1 coding region were used to do an end-to-end PCR to confirm the coding sequence. The end-to-end PCR product was then ligated into the TOPO PCR-II Vector (Invitrogen Canada Inc., Burlington, Ontario, Canada) and fully sequenced. The cDNA sequence for zebrafish TGF-ß1 has been deposited in GenBank under the accession no. AY178450.



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  		Figure 1. Schematic structure and cloning strategies of TGF-ß1 (A) and TGF-ß type II receptor (B). A, TGF-ß1 precursor contains a signal peptide, an N-terminal peptide and a C-terminal mature peptide. Partial zebrafish TGF-ß1 cDNA sequence was initially obtained by using a pair of degenerate primers (TGF-ß1–2 and 3). The resulting sequence was compared and assembled with an EST clone (AW566567). 3' RACE was then performed using primers TGF-ß 15 and 13 to obtain the 3'-end sequence. The sequence was assembled with another EST clone (WZ3446.1), which has a complete 5'-end sequence. Finally, an end-to-end PCR was performed using primers TGF-ß1–20 and 21, which flank the entire coding region. B, TGF-ß type II receptor precursor contains a signal peptide (SP) followed by the mature receptor peptide. The receptor has an extracellular domain (ECD), a transmembrane domain (TM), and an intracellular kinase domain. The cDNA sequence for zebrafish TßRII was obtained from two sequences (WZ6900.1 and WZ6900.2) of an EST clone using primers TßRII-1 and 2.

 
Cloning of TßRII cDNA
An EST clone (wz6900.1) that shows high homologies to other TßRII was identified from the Washington University Zebrafish EST project database. Another sequence (wz6900.2) derived from the same EST clone showed no homology to TßRII and was believed to be the 3'-end sequence of the zebrafish TßRII. As shown in Fig. 1BGo, sense and antisense primers, TßRII-1 and TßRII-2, derived from sequences of wz6900.1 and wz6900.2, respectively, were designed and PCR was performed. The PCR product was ligated again into the PCR-II Vector and cloned. Several positive clones were sequenced using the M13F and M13R primers as well as internal primers. The zebrafish TßRII cDNA sequence has been deposited in GenBank (accession no. AY178449).

Total RNA extraction
Gravid female zebrafish were anesthetized using 3-aminobenzoic acid ethyl ester (Sigma-Aldrich Canada Inc., Oakville, Ontario, Canada) and decapitated. The ovaries were removed and total RNA was extracted using an RNAeasy mini kit (QIAGEN Inc., Mississauga, Ontario, Canada) following the manufacturer’s suggested protocol. Ribonuclease-free deoxyribonuclease Set (QIAGEN) was also used during RNA isolation to remove any potential genomic DNA contamination.

RT-PCR
Five micrograms of total RNA were reverse transcribed to cDNA at 37 C for 1.5 h in a total volume of 50 µl as previously described (32). All primers used in this study are listed in Table 1Go. PCR was carried out in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 50 µM deoxynucleotide triphosphate, 1 U Platinum High Fidelity Taq DNA polymerase (Invitrogen, for cloning experiments) or Hotstart Taq (QIAGEN, for expression studies), and 5 pmol primers for 20–40 cycles, depending on the cDNA target to be amplified. Annealing temperature for PCR ranges from 55 to 65 C, depending on the primer sets used.


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  		Table 1. List of primers and their sequences

 
3'RACE
Five micrograms of total RNA from zebrafish ovary were reverse transcribed to 3'RACE cDNA at 42 C, using SuperScript II Reverse Transcriptase (Invitrogen) in a 20 µl reaction, containing 20 mM Tris.HCl, 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, 500 nM oligo deoxythymidine adapter primer, 500 µM deoxynucleotide triphosphate, and 2 U of ribonuclease H. The first round of PCR was carried out using the manufacturer’s supplied primer, Universal Adapter Primer, and TGF-ß1–13, a primer specific for TGF-ß1 (Table 1Go). Ten cycles of PCR (denaturing at 94 C for 20 sec, annealing at 62–57 C for 30 sec, with a decrease of 0.5 C for each cycle and extension at 72 C for 50 sec) were performed, followed by 30 cycles with the same conditions, except that the annealing temperature was 57 C. The first round PCR product was diluted 1:30 and used as template for the second round of PCR, which was performed using Universal Adapter Primer and a nested primer, TGF-ß1–15 (Table 1Go) under the same conditions as the first round.

Sequencing analysis
To confirm the identity of the PCR products, DNA fragments separated in agarose gels were recovered using a Gel Extraction Kit (QIAGEN). DNA fragments or plasmid DNAs were sequenced using an automatic DNA sequencer (Applied Biosystems Inc., Foster City, CA) at the York University Core Facility for Molecular Biology. Sequencing data were analyzed using the BLAST program. The open reading frames and the predicted amino acid sequences were determined using Genscan (http://bioweb.pasteur.fr/seqanal-/interfaces/genscan.html). Structural domains were predicted using the Simple Modular Architecture Research Tool (SMART) program (http://smart.embl-heidelberg.de/). A multiple sequence alignment of different species was produced using the Baylor College of Medicine Search Launcher (http://dot.imgen. bc-m.tmc.edu). Sequences of TGF-ß1 and TßRII from other species were obtained from GenBank database.

In vitro culture of zebrafish follicles
Ovaries were removed from zebrafish and follicles isolated as previously described (32). For oocyte maturation experiments, follicles greater than 0.52 mm in diameter were isolated from a pool of four to five fish. Previous studies in the zebrafish have shown that only follicles greater than 0.52 mm can undergo maturation in vitro in response to hormones (32). Approximately 20 follicles were placed into each well of a 24-well culture plate and incubated at 26 C in either 1 ml of modified Cortland’s medium (MCM) alone (Control) or MCM + hormones, such as human chorionic gonadotropin (hCG; kindly provided by Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA), 17{alpha}, 20ß-DHP (Sigma-Aldrich Canada) and human recombinant TGF-ß1 (R&D Systems, Minneapolis, MN). The rate of maturation was scored at various times of incubation. Follicles that underwent germinal vesicle breakdown (GVBD) could be identified by their acquired translucency. Each treatment was conducted in four wells and each experiment was repeated two to three times.

Statistical analysis
All values are expressed as mean ± SEM of three or four experiments. To determine the statistical difference among different groups at the same time point, multiple group comparisons were performed by one-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons test, using InStat Software (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of zebrafish TGF-ß1
Using PCR, 3' RACE and database searching, we have obtained the cDNA sequence for zebrafish TGF-ß1 (Fig. 2Go). The nucleotide sequence encodes a protein of 377 amino acids. Residues 1–17 make up the signal peptide, whereas the mature peptide has 112 residues, from positions 280–377 (Fig. 2Go). The zTGF-ß1 amino acid sequence is highly homologous to those of other fish, with 80–85% sequence identities. Also, a 70% identity was found upon comparison with the TGF-ß1 amino acid sequence of higher vertebrates, such as Xenopus, chicken, and mammals (Fig. 3Go). Thus, TGF-ß1 is reasonably well conserved at the amino acid level.



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  		Figure 2. Full-length cDNA and deduced amino acid sequences of zebrafish TGF-ß1. The coding region is predicted using Genscan. The asterisk indicates the stop codon.

 


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  		Figure 3. Comparison of the TGF-ß mature peptide sequence across different species. Deduced amino acid sequence from sheep, human, pig, rat, mouse, horse, chicken, Xenopus, rainbow trout, striped bass, carp, and zebrafish were compared using the Baylor College of Medicine Search Launcher program. Conserved amino acid residues are indicated by dots. Numbers on the top indicate the position of nine conserved cysteine residues, characteristic of the TGF-ß family.

 
Members of the TGF-ß family have been reported to have nine conserved cysteine residues. Cysteines 2, 4, 5, 7, 8, and 9 contribute to the formation of the "cysteine-knot" structure, characteristic of this family (39). Nine cysteine residues were found in the region of the zTGF-ß1 mature peptide. Cysteines 2–9 have identical positions as those in higher vertebrates. Interestingly, in all fish species studied, the first cysteine has a different position from those of all other species (Fig. 3Go). The mature peptide of zTGF-ß1 shows the presence of a stop codon at the second position after the sixth knot-forming cysteine, the characteristic cysteine knot signature (CXCX-stop; Refs.40 and41). Thus, zTGF-ß1 bears all expected structural resemblances to the TGF-ß1 family.

Cloning of zebrafish TßRII
A nucleotide sequence of approximately 2 kb was obtained for the zebrafish TßRII. The cDNA encodes a protein of 556 amino acids, which displays characteristics of the TGF-ß receptor family, including a cysteine-rich extracellular domain, a transmembrane domain, and a kinase domain with the predicted serine/threonine specificity. The first 19 amino acids represent the signal peptide. The extracellular domain (amino acids 20–148) has 12 cysteine residues, a characteristic of the TßRII. Amino acids between positions 149 and 171 make up the transmembrane domain, whereas the kinase domain is located between amino acids 232 and 525 (Fig. 4Go).



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  		Figure 4. Full-length cDNA and deduced amino acid sequences of zebrafish TßRII. The signal peptide and the transmembrane domain are underlined. Arrowheads indicate the beginning and end of kinase domain. The coding region is predicted by Genscan, and the structural domains are determined by SMART program. The asterisk indicates the stop codon.

 
Comparison of the zebrafish TßRII sequence with that of other species, such as human, American mink, rat, mouse, chicken, and Xenopus revealed that the kinase domain is the most conserved region (Fig. 5Go). The kinase domain of the zebrafish TßRII has over 87% identity to chicken and mammalian TßRIIs. Surprisingly, the homology between zebrafish and Xenopus TßRIIs appeared to be lower. Although the extracellular domain of zebrafish TßRII has less than 50% of sequence identity with the corresponding region of TßRII from other species, the number and position of the cysteine residues are identical, with the exception of the Xenopus sequence, which has one insertion between cysteines 11 and 12 (Fig. 5Go).



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  		Figure 5. Comparison of zebrafish TßRII sequence with that of other species, including human, American mink, rat, mouse, chicken, and Xenopus. Conserved residues are indicated by dots. *, Location of cysteine residues.

 
Expression of TGF-ß1, TßRII, and Smads in the zebrafish ovary
As an initial step to understand the role and signaling mechanisms of TGF-ß1 in the zebrafish ovary, the presence of TGF-ß1, TßRII, and the Smads 2, 3, and 4, which are believed to be important effectors of the TGF-ß signal transduction (1) was examined. Antisense and sense primers specific to each molecule were used in PCR using cDNAs prepared from ovarian follicles at different stages of development: 1) previtellogenic follicles (stage 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). To rule out the possibility of cross-contamination and genomic DNA contamination, two negative controls were also performed; one with water as the template and the other with the total RNA (without RT) as the template. As shown in Fig. 6Go, DNA fragments of the expected sizes were detected for all molecules in follicles at all stages of development. No amplification was found in negative controls. All PCR products have been sequenced to verify their identities.



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  		Figure 6. Expression of mRNAs for TGF-ß1, TßRII, Smad2, Smad3, Smad4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in zebrafish ovarian follicles at different stages of development. Total RNA were extracted from follicles at different stages and reverse transcribed. PCRs were performed using primers specific for TGF-ß1 (40 cycles), TßRII (40 cycles), Smad2 (30 cycles), Smad3 (30 cycles), Smad4 (40 cycles), and GAPDH (20 cycles). C, No template control; RT-, no RT control; RT+, PCR using cDNA as the template. MW, Molecular weight marker.

 
Role of TGF-ß1 in oocyte maturation
The detection of mRNAs for TGF-ß1 and its signaling components in the zebrafish ovary suggests that TGF-ß1 may be involved in regulating ovarian function. We therefore determined the effect of TGF-ß1 on spontaneous and hormone-induced oocyte maturation. hCG was used in this study as an LH analog because it is known that hCG can interact with fish LH receptor (42) and has similar bioactivity as fish LH, including regulation of steroid production and oocyte maturation (32, 33, 34, 43). Follicles were treated with TGF-ß1 (10 ng/ml), hCG (100 IU/ml), DHP (100 ng/ml), either alone or in combination, for 2, 6, 18, and 24 h. Similar to previous studies (32), hCG and DHP significantly induced oocyte maturation (Fig. 7Go). TGF-ß1, by itself, did not significantly alter the rate of maturation relative to control levels. However, the rate of hCG-induced maturation was significantly reduced by coincubation with TGF-ß1 at 6 h after treatment (Fig. 7AGo). Similarly, TGF-ß1 inhibited DHP- induced oocyte maturation; a significant inhibitory effect of TGF-ß1 on DHP-induced oocyte maturation was observed at 2, 6, 18, and 24 h after treatment (Fig. 7BGo).



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  		Figure 7. Effects of TGF-ß1 on spontaneous and hormone-induced oocyte maturation. A, Ovarian follicles were incubated with medium only (control), TGF-ß1 (10 ng/ml), hCG (100 IU/ml), or a combination of hCG and TGF-ß1 for 2, 6, 18, and 24 h and the rate of maturation scored as percentage of follicles that underwent GVBD. B, Ovarian follicles were incubated with medium only (control), TGF-ß1 (10 ng/ml), DHP (100 ng/ml) or a combination of DHP and TGF-ß1 for 2, 6, 18, and 24 h and then scored for % GVBD. Data represent mean ± SEM of four experiments. Different letters denote statistical significance (P < 0.05) within the same time point.

 
The dose-dependent effect of TGF-ß1 on hormone- induced oocyte maturation was also determined. Follicles were incubated with control medium, hCG (100 IU/ml) or DHP (100 ng/ml) alone or in the presence of different concentrations of TGF-ß1 (0.1, 1, and 10 ng/ml) for 18 h. Again, hCG significantly increased the rate of oocyte maturation. All doses of TGF-ß1 significantly reduced the effect of hCG; however, a stronger inhibition was seen for higher doses of TGF-ß1 (Fig. 8AGo). Similarly, TGF-ß1 dose dependently inhibited DHP-induced oocyte maturation (Fig. 8BGo).



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  		Figure 8. Dose-dependent effects of TGF-ß1 on (A) hCG- and (B) DHP-induced oocyte maturation. Ovarian follicles were treated with medium (control), TGF-ß1 (10 ng/ml), hCG (100 IU/ml), or DHP (100 ng/ml) alone or in combination with 0.1, 1, or 10 ng/ml TGF-ß1 for 18 h. Data represent mean ± SEM of three experiments. Different letters denote statistical significance (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have cloned cDNAs encoding zebrafish TGF-ß1 and TßRII. Notably, the zebrafish TßRII cDNA is the first piscine TßRII sequence to be reported. We have also detected mRNA expression of TGF-ß1, TßRII, Smad2, Smad3, and Smad4 in the zebrafish ovary. Most interestingly, we demonstrated, for the first time, that TGF-ß1 inhibits in vitro oocyte maturation induced by a maturation-inducing steroid. These findings strongly suggest that TGF-ß1 is a physiological regulator of fish ovarian function.

TGF-ß1 has been cloned from other species of fish, such as the rainbow trout (19), carp (20), and hybrid striped bass (21). The present study reports the cloning of the zebrafish TGF-ß1 full-length cDNA. The structure of the zebrafish TGF-ß1 is very similar to TGF-ß1 cloned from other piscine species at the level of amino acids. Although the mature peptide region of zTGF-ß1 only shows 67% amino acid sequence identity with mammalian TGF-ß1, the zTGF-ß1 bears all the features conducive to the formation of a cysteine knot, which facilitates protein-protein interactions (40). Recently, the crystal structure of the human TßRII ectodomain-TGF-ß3 complex has been determined and ten residues in TGF-ß3 are found to be directly involved in binding to the type II receptor (44). These residues are R25, K31, W32, H34, K37, Y90, Y91, V92, G93, and R94 (44). Consistent with the finding that TGF-ß1 and TGF-ß3 bind to TßRII with similar affinities, these residues are identical between human TGF-ß1 and TGF-ß3 (44). Interestingly, all of the ten residues are conserved in zTGF-ß1. These indicate that all structural domains critical for ligand/receptor interaction are conserved in the zebrafish TGF-ß1 molecule. The high degree of conservation of TGF-ß1 functional domains between human and zebrafish also suggests that the human TGF-ß1 used in the present study can interact with zebrafish TGF-ß receptors and has similar activities as the native zebrafish TGF-ß1. Cloning of the zebrafish TGF-ß1 provides an effective tool for studying the in vivo function of TGF-ß1 in the future. For example, the zTGF-ß1 cDNA sequence can be used to design antisense oligonucleotides for gene knockdown studies to investigate the physiological role of TGFß1 on follicle development and maturation.

TßRII cDNA has been cloned from several mammalian species and chicken. Recently, Partial sequence of Xenopus TßRII has also been reported (45). Our present study provides the first piscine TßRII cDNA sequence. The zTßRII exhibits all structural features characteristics of the type II TGF-ß receptor family, including cysteine-rich extracellular domain, a transmembrane domain and a predicted serine/threonine kinase domain (1, 2, 3). Interestingly, alignment of zTßRII sequences from various species reveals that the extracellular domain of chicken, Xenopus and zebrafish TßRIIs is quite divergent when compared with their mammalian counterparts. However, despite the low degree of conservation in this region, studies using both cloned chicken (46) and Xenopus TßRII (45) cDNAs have shown that they are functional TGF-ß receptors. Notably, the positions of cysteine residues are highly conserved. The crystal structure of the human TßRII ectodomain has revealed that the 12 cysteine residues form six intrastrand disulfide bonds to maintain a proper folding of the receptor (44, 47). Two of the cysteine residues, C67 and C71, have also been examined by site-directed mutagenesis and found to be critical for receptor binding and signaling (48). Furthermore, eleven residues found to be involved in ligand binding, L50, F53, D55, S72, I73, T74, S75, I76, E78, D142, and E143 (44), are highly conserved; ten of these residues are identical between human and zebrafish TßRII. The same degree of conservation is also found in the mammalian TßRIIs (44). On the other hand, some of the nonconserved residues, such as V85, V87, L97, V100, F133, and M135 of the human TßRII, have been found to have no effect on receptor binding when individually mutated (48). Again, the conservation of functional domains of the TßRII in zebrafish further supports the notion that human TGF-ß1 can interact with zebrafish TßRII.

The role of TGF-ß in oocyte maturation has been previously examined in several mammalian species and yielded contradictory findings. Coskun and Lin (16) reported that TGF-ß inhibited spontaneous porcine oocyte maturation in cultured cumulus-oocyte complexes. However, in a similar culture model, TGF-ß was found to have no effect on oocyte maturation in pigs (18). Similarly, in pregnant mare’s serum gonadotropin-treated rats, TGF-ß has been reported to stimulate spontaneous oocyte maturation of both follicle- enclosed oocytes and cumulus-oocyte complexes (11) or to inhibit LH-induced oocyte maturation in follicle-enclosed oocytes (17). In the present study, we demonstrated that TGF-ß1 inhibited both gonadotropin- and MIH-induced oocyte maturation. To our knowledge, this study is the first to report that the action of MIH can be inhibited by a growth factor. We found that gonadotropin-induced oocyte maturation can be completely blocked by TGF-ß1, whereas MIH-induced maturation is only partially blocked. It is well documented in fish that hCG-induced oocyte maturation involves transcription and translation, such as MIH production and activin-A expression (29, 30, 31, 32, 33, 34, 35), whereas MIH- induced oocyte maturation does not involve gene transcription (29, 30, 31). It is possible, therefore, that the relative ability of TGF-ß1 to affect hCG- vs. MIH-induced maturation is due to its action at multiple sites. TGF-ß1 may act at levels both upstream and downstream of MIH. It may inhibit gonadotropin-induced MIH production and down-regulate MIH receptors, leading to a decreased response to MIH. Recently, it has been shown that TßR-II can physically associate with cyclin B and inactivate cdc-2, thus preventing the cells from entering the G2/M phase (49). Thus, it is also possible that TGF-ß blocks MIH-induced oocyte maturation by inhibiting the activation of maturation-promoting factor. The mechanisms underlying the inhibitory role of TGF-ß1 on oocyte maturation will be investigated in the future.

Members of the TGF-ß family exert their functions by interacting with type I and type II membrane serine/threonine kinase receptors and subsequently activating the Smad signaling pathway. TGF-ß actions have been shown to be mediated by TßRI, TßRII, Smad2, Smad3, and Smad4. Although TßRI and TßRII are specific to TGF-ß, Smad2, 3, 4 are common mediators of several members of the TGF-ß family, including activin and Nodal (1, 2, 3). As an initial step to examine TGF-ß signal transduction events in the zebrafish ovary, we have detected mRNA expression of TGF-ß1, TßRII, Smad2, Smad3, and Smad4. The detection of TßRII, Smad2, Smad3, and Smad4 mRNAs in the zebrafish ovary is consistent with several studies in mammals, such as rats, hamsters, and human (50, 51, 52, 53). The role of Smad2 on inhibin production from human granulosa cells has been recently reported (54). The role of Smad3 in follicle development has also been suggested from studies using Smad3-deficient mice (55). In the present study, we found that mRNAs of TGF-ß1, TßRII, and Smads are expressed in follicles at all stages of development, suggesting that they may play a role in regulating follicle development and maturation. Interestingly, there appeared to be a decrease in TGF-ß1 mRNA levels in maturing follicles. Our preliminary real-time RT-PCR and Western blot analysis have confirmed that TGF-ß1 expression levels decrease during maturation of follicles (Kohli, G., T. Wu, and C. Peng, unpublished observation). The decreased level of TGF-ß1 expression is consistent with the finding that TGF-ß1 inhibits oocyte maturation. If and how Smads mediate the function of TGF-ß1 in the zebrafish ovary will be determined in future studies.

Although activin and TGF-ß act through a similar Smad signaling pathway, they have differential effects on zebrafish oocyte maturation. Activin-A and -B enhance spontaneous and MIH-induced oocyte maturation (32, 33, 34), whereas TGF-ß1 inhibits MIH-induced oocyte maturation (present study). Opposing effects of activin-A and TGF-ß1 on rat follicle development (14) and progesterone production (56) have also been reported. The mechanisms underlying the opposing actions of activins and TGF-ß1 are not clear at present. However, it is unlikely that this is due to competition at the receptor level because activins and TGF-ßs interact with distinct receptors and there is no cross-reactivity between their receptors (1, 25, 26). Recent structural studies have also shown that the ligand/receptor interaction between the activin and TGF-ß systems is quite different (44, 48). It is possible that activins and TGF-ß1 may regulate different target genes involved in oocyte maturation. Alternatively, activin may stimulate oocyte maturation by suppressing the expression of TGF-ß1 expression or vise versa. Finally, activin and TGF-ß actions may be mediated by different R-Smads (Smad2 or Smad3).

In summary, we have cloned the first fish TßRII cDNA. We have also obtained the cDNA sequence for zebrafish TGF-ß1 and established a potential role for TGF-ß1 in the zebrafish ovary. Cellular and molecular mechanisms underlying TGF-ß action on oocyte maturation will be investigated in the future.


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


   Footnotes
 
This study was supported by a grant from Natural Science and Engineering Research Council of Canada and an Ontario Premier’s Research Excellent Award (to C.P.).

Abbreviations: DHP, 17{alpha}, 20ß-Dihydroxyprogesterone; EST, expressed sequence tag; GVBD, germinal vesicle breakdown; hCG, human chorionic gonadotropin; MIH, maturation-inducing hormone; RACE, rapid amplification of cDNA ends; R-Smad, receptor-regulated Smad; TßRI and II, types I and II TGF-ß receptor.

Received December 11, 2002.

Accepted for publication January 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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