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A Novel Type of P450c17 Lacking the Lyase Activity Is Responsible for C21-Steroid Biosynthesis in the Fish Ovary and Head Kidney

Laboratory of Reproductive Biology (L.-Y.Z., D.-S.W., T.K., A.Y., B.P.-P., A.S., F.S., Y.N.), National Institute for Basic Biology, and School of Life Science (L.-Y.Z., Y.N.), Graduate University for Advanced Studies, Okazaki 444-8585, Japan; SORST (D.-S.W., B.P.-P., A.S., F.S., Y.N.), Japan Science and Technology Corp.; School of Life Science (D.-S.W.), Southwest University, 400715 Chongqing, People’s Republic of China; and National Research Institute of Aquaculture (T.K.), Tamaki, Mie 519-0423, Japan

Address all correspondence and requests for reprints to: Professor Yoshitaka Nagahama, Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. E-mail: nagahama{at}nibb.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytochrome P450c17 is the single enzyme that mediates the 17-hydroxylase and 17, 20 lyase activities during the biosynthesis of steroid hormones in the gonads and adrenal gland. However, the mechanism underlying its dual action continues to be a controversy in the field of steroidogenesis in fish. In an attempt to resolve this issue, we identified a novel type of P450c17 (P450c17-II) by an in silico analysis from the genomes of six fish species. We cloned P450c17-II from tilapia and medaka, and comparison with the conventional P450c17-I revealed that they differ in gene structure and enzymatic activity. Enzymatic assays by thin-layer chromatography revealed that P450c17-II possesses only the 17-hydroxylase activity without any 17, 20 lyase activity, unlike P450c17-I, which has both these activities. In testis, both P450c17-I and -II express in the interstitial cells. Remarkable differences, revealed by in situ hybridization, in the expression patterns of the P450c17-I and -II in the ovary and head kidney of tilapia during various stages of development strongly suggest that P450c17-I is responsible for the synthesis of estradiol-17ß in the ovary, whereas P450c17-II is required for the production of C21 steroids such as cortisol in the head kidney. More interestingly, a temporally controlled switching is observable in the expression of these two genes during the steroidogenic shift from estradiol-17ß to the C21 steroid, 17, 20ß-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of fish oocytes) in the fish ovary, revealing a role for P450c17-II in the production of hormones that induce oocyte maturation in fish.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CYTOCHROME P450C17 CATALYZES the 17-hydroxylase activity required for glucocorticoids (cortisol) synthesis in the adrenal gland and the 17, 20 lyase activity required for sex steroid (androgens and estrogens) synthesis in the gonads. The mechanism behind this organ-specific action of P450c17 has been attributed to posttranslational modifications regulated by factors like, the abundance of the electron-donating protein P450 oxidoreductases, the presence of cytochrome b5, and the serine/threonine phosphorylation of P450c17 (1, 2, 3, 4, 5). Alternatively, the existence of P450c17 isozymes has also been proposed in mammals to explain the differential actions of P450c17 in the adrenal and gonad (6).

The vertebrate gonads in general experience a steroidogenic shift from estrogens to progestogens during the transition from the follicular phase to luteal/maturation phase (7). In mammals, the shift is from estradiol-17ß to progesterone. Because the production of progesterone does not need the activity of P450c17, a cessation of the hydroxylase and lyase activities of P450c17 is sufficient to achieve the steroidogenic shift in mammals (8, 9). In fish, however, the situation is different. During the maturation of oocytes, postvitellogenic follicles have to synthesize 17, 20ß-dihydroxy-4-pregnen-3-one (17, 20ß-DP) (10) or 17, 20ß, 21-trihydroxy-4-pregnen-3-one (11) [maturation-inducing hormones (MIHs) in fish] from 17-hydroxyprogesterone. Because 17-hydroxyprogesterone is produced from progesterone by the hydroxylase activity of P450c17, its lyase activity needs to be down-regulated in the fish ovary. Thus, the differential regulation of the activity of P450c17 is critical for the postvitellogenic oocytes to undergo the final maturation step, which is absolutely necessary for their fertilization and subsequent development. Because only a single P450c17 enzyme was found to exist in fish (12, 13, 14, 15, 16, 17, 18), the same mechanisms found in mammalian adrenals were thought to be responsible for the switching in the action of P450c17 during the steroidogenic shift in the gonads of fish (13, 14, 17, 19). However, no studies have proved this claim convincingly.

The cortisol produced by the hydroxylase activity of P450c17 in the interrenal cells of the head kidney (the piscine counterpart of the mammalian adrenal) is essential for the osmoregulation and energy metabolism in fish (20). No study has confirmed whether the same P450c17 is present in both the gonad and head kidney, except for ambiguous studies in zebrafish and rainbow trout (17, 21). The survival of a natural mutant of medaka [sex characterless (Scl)], in which the P450c17 function was abrogated due to a mutation in the gene, further complicates our understanding of the involvement of P450c17 in the production of cortisol because cortisol is vital to the existence of vertebrates including fish. Moreover, a mutation in the female Scl was shown to have impaired only the advanced stages of oogenesis and secondary sexual characteristics, which are dependent on estrogens (22). This indicates that the mutation in P450c17 had affected the production of the sex steroids in the gonad but not the production of cortisol in the head kidney.

This led us to hypothesize that there could be a second P450c17 gene, responsible for the production of cortisol in the head kidney of fish. Such a gene would also be responsible for the differential action of P450c17 during the steroidogenic shift from estrogen to the MIHs. Thus, we performed an in silico analysis and identified a novel P450c17 gene in the genomes of fugu, medaka, stickleback, tetraodon, and zebrafish. Furthermore, we cloned P450c17-II from tilapia and medaka and studied the enzymatic activity and expression pattern of tilapia P450c17-II, in comparison with the conventional P450c17-I. Our data suggest that P450c17-I is required for the synthesis of estradiol-17ß (E2) in the ovary, whereas P450c17-II is responsible for the production of cortisol in the head kidney and 17, 20ß-DP (the MIH of tilapia) in the ovary.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Tilapias were reared in 1-ton tanks with recirculating aerated fresh water. The fish were maintained at ambient temperature (26 C) under natural light conditions. Mature tilapias (XX) that spawn at an average of 14 d were used in the present study. All animal experiments conformed to the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Institute for Basic Biology).

Identification of novel P450c17 from fully sequenced genomes and expressed sequence tags of vertebrates
Medaka (BAA13252), rainbow trout (CAA46675), Japanese eel (AAR88432), and zebrafish (NP_997971) P450c17-I sequences were used as the query sequences in TBLASTX or BLASTP searches carried out at National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and University of California, Santa Cruz, Genome Bioinformatics (http://genome.ucsc.edu/) portals against the sequenced genomes and, as available, the genome-predicted proteins of the fish Takifugu rubripes (assembly 4 with 5.7x coverage), Tetraodon nigroviridis (assembly 1.1 with 8.3x coverage), Oryzias latipes (assembly 1.0 with 6.7x coverage), Danio rerio (Zv6 with 5–7x coverage), Gasterosteus aculeatus (assembly 1.0 with 11x coverage), the amphibian Xenopus tropicalis (assembly 4.1, 7.65x coverage, searched via DOE Joint Genome Institute), and the bird Gallus gallus (assembly 2.1 with 6.6x coverage). Each matching sequence returned with an expectation value less than e = 0.0001 was used to query the GenBank nonredundant protein database to establish the assignment as a P450c17 and identify which is closer to the mammalian P450c17 sequence. Sequences were also searched by TBLASTX against the database of expressed sequence tags (ESTs) at NCBI for ESTs from the corresponding organism to establish the existence of transcribed sequences corresponding to the open reading frame (ORF) predicted from genomic DNA. In some cases, EST sequences and comparisons with known P450c17 were used to extend or correct the genome-predicted sequences.

The identified genomic contigs/scaffolds corresponding to P450c17-I and -II in fugu, tetraodon, stickleback, medaka, and zebrafish were as follows: fugu, P450c17-I (CAAB01002402) and -II (CAAB01005318.1); tetraodon, P450c17-I (CAAE01021244) and -II (GSTENT00036372001); stickleback, P450c17-I (chrVI:856070–859737, EST sequence DN731810) and -II (chrUn:29090145–29093435, EST sequences DW599621, DN655686, DN721050); medaka, P450c17-I (scaffold2317, golw_scaffold Hd-rR (200506)] and -II (scaffold730); and zebrafish, P450c17-I (NP_997971) and -II (NW_001512620). The deduced ORF of fugu, tetraodon (partial sequence), stickleback, medaka, and zebrafish were used for the following phylogenetic analysis.

Cloning of P450c17-I and -II from tilapia and P450c17-II from medaka and zebrafish
A fragment of P450c17-II was obtained from cDNA of tilapia testes using primers designed based on the conserved region of the identified fugu, tetraodon, and medaka P450c17-II. Subsequently four primers were designed to amplify a full-length cDNA sequence by 5'-rapid amplication of cDNA ends (RACE) and 3'-RACE.

The ORF of medaka P450c17-I was amplified from cDNA of the ovary by PCR based on a sequence downloaded from GenBank (BAA13252). The medaka P450c17-II ORF was isolated from testis cDNA by PCR according to the medaka genome sequence [scaffold730, golw_scaffold Hd-rR (200506)]. Zebrafish P450c17-II ORF was isolated from testis cDNA by PCR according to mRNA (XR_029319.1) derived from zebrafish genome sequence.

Analysis of domain architecture of novel P450c17
The domain architecture of the predicted and cloned novel P450c17 proteins was evaluated by searches against the Conserved Domain Database at NCBI to confirm they are members of the P450c17 family.

Phylogenetic analysis
Alignments of P450c17-I and -II amino acid sequences from six species of teleosts were prepared by the progressive, neighborhood-joining alignment method, Clustal X (23). The multiple sequence alignments are presented in Boxshade 3.2. The multiple alignment software Clustal X was also used to analyze homology and calculate phylogenetic trees by the neighborhood-joining method using Japanese eel P450 CYP1C1 (AAR15082) as an outgroup (23). Values on the trees represent bootstrap scores of 1000 trials, indicating the credibility of each branch. The GenBank accession nos. of P450c17 sequences used in this study are as follows: human P450c17-I (AAV38803), mouse P450c17-I (NP_031835), rat P450c17-I (NP_036885), chicken P450c17-I (NP_001001901), Xenopus P450c17-I (AAG42003), Japanese eel P450c17-I (AAR88432), zebrafish P450c17-I (NP_997971), tilapia P450c17-I (AB292401), medaka P450c17-I (BAA13252), rainbow trout P450c17-I (CAA46675), fugu P450c17-I (EF624004), tetraodon P450c17-I (CAAE010212); stickleback P450c17-I (EF624006), fugu P450c17-II (EF624005), medaka P450c17-II (EF423918), tetraodon P450c17-II (GSTENT00036372001), stickleback P450c17-II (EF624007), tilapia P450c17-II (EF423917), and zebrafish P450c17-II (EF624003).

Enzymatic assay by thin-layer chromatography (TLC)
The ORFs of tilapia P450c17-I and -II were subcloned into TOPO pcDNA 3.1 (Invitrogen, Carlsbad, CA) to obtain recombinant constructs. Human embryonic kidney (HEK) 293 cells were transfected with 4 µg pcDNA3.1 vector (Invitrogen) with or without the insert and were cultured in DMEM with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS). These cells were incubated at 37 C and 5.0% CO₂ for 24 h. Then, serum-free medium with ¹⁴C-progesterone (60,000 cpm/well) or ³H-pregnenolone (2000,000 cpm/well) (PerkinElmer, Boston, MA) was used to replace the original medium. After radiolabeled substrates were added, the medium in each well was collected individually at 2, 4, and 8 h. The extraction of steroids, TLC assay, and quantification were carried out according to procedures described elsewhere (24). Lack of lyase activity in P450c17-II was ascertained by performing TLC with the substrate ³H-17-hydoxyprogesterone and in this case, the medium was collected at 24 h.

Three parallel samples were collected at each time point and quantified. The conversion rate was calculated as a percentage of the total radioactivity after extraction. Results were represented as mean ± SE of three independent measurements.

Tissue distribution analysis by RT-PCR
Total RNA was extracted, cDNA was synthesized, and RT-PCR was carried out to check the expression levels of tilapia P450c17-I and -II in various tissues according to methods described previously (25). Gene-specific primers were used for the RT-PCR analysis. Positive and negative controls were set up with plasmid DNA and water, respectively, as templates to validate the distribution pattern. A fragment of ß-actin was amplified (as internal control) from tilapia to test the quality of the cDNAs used in the PCR. The PCR products were subjected to agarose gel (1.5%) electrophoresis.

In situ hybridization (ISH)
The whole bodies of XX and XY fry at 2, 5, 11, 20, 60 day after hatching (dah; after removal of the yolk and gut) and gonads and head kidneys of 8-month-old tilapia were fixed in 4% paraformaldehyde (Nacalai tesque, Kyoto, Japan) in 0.85x PBS at 4 C to check the expression of P450c17-I and -II in the gonad and head kidney by ISH as described previously (26). Similarly, gonads from regularly spawning fish were sampled and fixed at d 1, 3, 5, 8, 10, 12, and 14 of the spawning cycle. Probes of sense and antisense digoxigenin-labeled RNA strands were transcribed in vitro with an RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) from plasmid DNA containing ORFs of tilapia P450c17-I, P450c17-II, and Cyp19a1 (ovarian type aromatase).

Real-time PCR
Gonads from regularly spawning fish (14 d/spawn) were sampled at d 1, 3, 5, 8, 10, 12, and 14 (2–4 h after spawning) of the spawning cycle. Five fish were used for each time point and three samples (replicates) were collected from each fish. Total RNA was extracted from all the samples, and cDNA was prepared for the real-time PCR. Real-time PCR was carried out according to the protocol of Platinum SYBR Green quantitative PCR SuperMix UDG (Invitrogen). ß-Actin was used as an internal control. The relative expression levels (RNA abundance) were calculated by dividing the number of copies of the target gene by that of ß-actin. Data were expressed as the mean ± SE of the 15 replicates. Furthermore, the results within each type (expressed as a function of days or ovarian stages) were analyzed with a Kruskall-Wallis ANOVA. When the ANOVAs were significantly different, data within each type and between the two types were compared using the Mann-Whitney test.

Primer sequences used for RT-PCR, RACE, and real-time PCR are listed in Table 1.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

View larger version (150K): [in this window] [in a new window]   FIG. 1. Comparison of deduced amino acid sequences of P450c17-I and -II from tilapia, medaka, stickleback, fugu, and zebrafish. The intron positions of P450c17-I and -II are marked with arrows (above the aligned sequences) and arrowheads (below the aligned sequences), respectively. The putative conserved regions are boxed: I, Ono-sequence; II, Ozols’ tridecapeptide region; and III, Heme-binding region. Refer to Materials and Methods for GenBank accession nos.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Schematic representation of the gene structures of medaka P450c17-I (a) and -II (b). Exons are represented as boxes and marked with Roman numerals, whereas introns are indicated by dotted lines. The numbers given under the boxes and above the dotted lines represent the relative base pair sizes of the exons and introns, respectively.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Phylogenetic analysis of P450c17s of vertebrates using Japanese eel cytochrome P4501C1 as an outgroup. Values on the tree represent bootstrap scores of 1000 trials, indicating the credibility of each branch. Branch lengths are proportional to the number of amino acid changes on the branch. Partial sequences (*) may have artificially short branches. Refer to Materials and Methods for GenBank accession nos.

View larger version (48K): [in this window] [in a new window]   FIG. 4. Enzyme assay of P450c17-I and -II recombinant proteins expressed in HEK 293 cells by TLC followed by autoradiography. P450c17-I converts 14C-progesterone and 3H-pregnenolone to androstenedione and DHEA through 17-hydroxyprogesterone and 17-hydroxypregnenolone (a and b), whereas P450c17-II can convert only 14C-progesterone and 3H-pregnenolone to 17-hydroxyprogesterone and 17-hydroxypregnenolone (c and d). Standard sample (S1 and S2) and mock indicate radioisotope-labeled standard substrates and pcDNA3.1 vector without insert, respectively. The right panel indicates the conversion rate of substrates calculated as a percentage of the total radioactivity after extraction. The bars represent the mean ± SE of triplicates measurements.

View larger version (81K): [in this window] [in a new window]   FIG. 5. Enzyme assay of P450c17-II recombinant protein expressed in HEK 293 cells by TLC followed by autoradiography. P450c17-II cannot convert 3H-17-hydroxyprogesterone to androstenedione, even after 24 h of incubation, demonstrating the absence of lyase activity of this enzyme. Standard sample (S1, S2) and mock indicate radioisotope-labeled standard substrates and pcDNA3.1 vector without insert, respectively.

View larger version (10K): [in this window] [in a new window]   FIG. 6. RT-PCR analysis of tilapia P450c17-I and -II from various tissues of adult tilapia. B, Brain; P, pituitary; G, gill; H, heart; S, spleen; L, liver; I, intestine; K, kidney; O, ovary; T, testis; A, head kidney (equivalent to adrenal of mammals); M, muscle; –, negative control; +, positive control. Lower panel shows ß-actin (internal control).

View larger version (122K): [in this window] [in a new window]   FIG. 7. Expression of P450c17-I and -II in the gonad and head kidney of tilapia detected by ISH. At 5 dah, P450c17-I was expressed in both XX and XY gonad (a and c), whereas no expression of P450c17-II was detected in the gonad of the same day (b and d). In 8-month-old tilapia, both P450c17-I and -II mRNA were detected in the ovary [granulosa (g) and theca cells (h), respectively] and testis (interstitial cells) (i and j). P450c17-II was strongly expressed in the interrenal cells of the head kidney (f and l), whereas no signal of P450c17-I expression was detected (e and k) in both 5 dah and 8-month-old fish. Arrows indicate P450c17-I and arrowheads indicate P450c17-II.

View larger version (103K): [in this window] [in a new window]   FIG. 8. Expression of P450c17-I, -II, and Cyp19a1 in the granulosa cells and/or theca cells during the spawning cycle of female tilapia. Ovaries collected from regularly spawning female tilapia on d 1, 3, 5, 8, 10, 12, and 14 were subjected to ISH using the amplified RNA probes for P450c17-I, -II, and Cyp19a1. a–g, P450c17-I expression was found in the granulosa cells in the early and midvitellogenic follicles on d 1–8 (a–d), with the expression at its peak on d 3–5 (b and c). Thenceforth, the expression tapered off and had completely ceased by d 14 (e–g). h–n, Expression of P450c17-II persisted throughout the spawning cycle especially in the theca cells. However, a simultaneous increase in the expression of P450c17-II was observed in the granulosa cells from d 8 (k), precisely when P450c17-I started to decrease. The expression reached its peak by d 12 (m) and disappeared on d 14 (n). o–u, Expression of Cyp19a1. Expression pattern of Cyp19a1 in granulosa cells followed a similar pattern of P450c17-I expression during the entire spawning cycle but declined from d 10, whereas the expression of Cyp19a1 remained unchangeable in the theca cells, like the P450c17-II. Arrows and arrowheads indicate the signal in granulosa cells and theca cells, respectively.

View larger version (17K): [in this window] [in a new window]   FIG. 9. Expression pattern of tilapia P450c17-I and -II during the 14-d spawning cycle analyzed by real-time PCR. The solid line represents P450c17-I expression and the dotted line, P450c17-II expression. Each point on the curve represents the relative mRNA expression at each time point in the spawning cycle. Each value represents the mean ± SE of at least 15 samples obtained from five different fish. Mean values with different letters in each type of P450c17 are significantly different (P < 0.05). The asterisks indicate significant differences between the expression levels of P450c17-I and -II for the same day or the same ovarian stage.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is apparent from the sequences and structure of the medaka P450c17-I and -II genes that the two are completely different. The phylogenetic analysis revealed that the teleost P450c17-I is more closely related to the tetrapod P450c17, indicating it to be an archetype of the tetrapod P450c17s. The duplication of P450c17 appears to be a unique incident that occurred only in the course of the evolution of fish. The existence of P450c17-II in the six species of fishes analyzed in this study makes the presence of this gene in other fishes highly probable. A structural analysis of the genes of P450c17-I and -II of fugu, medaka, and zebrafish revealed 8 and 9 exons, respectively. The extra exon appears to be generated by the insertion of an additional intron in P450c17-II, in a position that aligns to the middle of the first exon of P450c17-I. The other introns of P450c17-II are located more or less at the same positions as P450c17-I. These gene structures are quite conserved among all vertebrate P450c17 genes (15, 31). The conservation of gene structures and P450c17 signature domains indicate that in fish, P450c17-II was derived from duplication of P450c17-I.

Genomic data indicate that the genomes of most vertebrates (including human) had undergone two whole genome duplications in their evolutionary history, the 2 round hypothesis (32), whereas the genomes of most bony fishes had undergone an additional duplication [the fish-specific genome duplication (FSGD) or the 3 round duplication] at the origin of modern fish (33, 34, 35). FSGD probably occurred after the bichirs (Polypteriformes), sturgeons (Acipenseriformes), gars and bowfins (Semionotiformes) branched off from the fish stem lineage (34). In the present study, two P450c17 types encoded by two different genes were found in six different teleosts species: fugu, tetraodon, stickleback, tilapia (Percomorpha), medaka (Atherinomorpha), and zebrafish (Ostariophysi), which belong to three different clades (36). Phylogenetic analysis of all known P450c17 sequences revealed that the occurrence of two P450c17 types exists only in teleost fish. We did an in silico search on the genomes of Xenopus, chicken, mouse, rat, and human but failed to obtain any P450c17-II-like sequences. Hence, we propose that the P450c17-II sequences probably originated from the FSGD event associated with the emergence of modern teleosts, and therefore most teleosts with the possible exception of bichirs, sturgeons, gars, and bowfins should possess both P450c17 types unless they had experienced a secondary loss after the FSGD event. This claim remains rather speculative as further experimental evidence is yet to be obtained.

Because the nucleotide and amino acid sequences of the two types of P450c17 are clearly different, we further examined the enzymatic activity of P450c17-II by transfection into intact HEK 293 cells and compared it with that of P450c17-I under the same experimental conditions. The inability of P450c17-II to convert 17-hydroxyprogesterone to androstenedione or 17-hydroxypregnenolone to DHEA proves that it lacks the lyase activity, making it different from P450c17-I functionally also. HEK 293 cells were regarded as a cell line rich with cytochrome b5 (4). Even the human P450c17 displayed lyase activity when transfected into this cell line (37), without the addition of any exogenous cytochrome b5. Our data clearly showed that P450c17-II lacks any lyase activity under the above experimental conditions, whereas P450c17-I possesses both the activities. This experiment indicates that P450c17-II does not have any lyase activity even in the presence of cytochrome b5.

Because the novel type of P450c17 was found to have only the hydroxylase activity, we further checked whether this is the gene responsible for the production of cortisol by analyzing its expression pattern in the head kidney of tilapia during different developmental stages. Surprisingly, only P450c17-II was found to be expressed in the interrenal cells of the head kidney from the very early stages (5 dah) to adulthood (8 months old). One of the most important physiological functions of the interrenal cell is to produce cortisol (38). Our data suggest that only P450c17-II is responsible for cortisol production in the interrenal cells, making its presence crucial to the existence of the individual concerned. Although this study has not provided any functional data to support this further due to the unavailability of knockout strategies in fish, inferences on the function of P450c17-II can be drawn from the study of the Scl mutant (22). Despite ablation of the protein, and thus the function of P450c17-I, the Scl embryos survived like normal embryos. This shows that they produced cortisol to regulate the osmoregulation. Cloning and sequencing of P450c17-II from the Scl mutant further confirmed that the fish had an intact P450c17-II gene. This is a staunch proof to the notion that these fish survived successfully due to the existence of another P450c17 gene to orchestrate the synthesis of cortisol in their head kidneys.

Thereafter we checked the expression patterns of both P450c17-I and -II in the gonads during gonadal differentiation and the reproductive cycle. We used the tilapia for this purpose because it is a good model in this context due to the availability of all -XX and -XY genetic populations, making the analysis more robust with respect to the sexuality of the samples being tested. Moreover, the tilapia has a reproductive cycle, spanning 14 d on an average, well suited for the profiling of gene expression during the various stages of oogenesis and maturation.

P450c17-I was expressed in the gonads of both XX and XY tilapia from 5 dah, the time when gonadal sex differentiation becomes apparent in this species (39). It continued to be expressed strongly in the gonads of both the adult male and female, reemphasizing its requirement in the production of androgen and E2. In contrast, P450c17-II was not found in the gonads of either XX or XY fish during the early stages of gonadal differentiation (5 dah). Because it lacks the lyase activity, its expression may not be required in the gonads during the initiation of sexual differentiation. The difference in the temporal expression patterns of P450c17-I and -II suggests that they are required for apparently different functions in the gonads.

The data from the ISH and quantitative analysis during the spawning cycle of tilapia suggest that P450c17-I is needed mainly for oocyte growth, whereas P450c17-II is required for oocyte maturation. The expression of P450c17-I in the granulosa cells reached its peak around d 3–5, signifying its importance during early and midvitellogenesis for the production of estrogen. P450c17-I started to decline in the granulosa cells from around d 8, probably to reduce the production of the precursors of E2. A simultaneous initiation of P450c17-II expression was observed in the granulosa cells may be to orchestrate the synthesis of 17, 20ß-DP. Before d 8, P450c17-II expression was confined only to the theca cells. Furthermore, P450c17-I was reduced by d 12 significantly, and there was a synchronous culmination in the expression of P450c17-II in the granulosa cells. The significant decline in the expression of P450c17-I between d 8 and 12 and the sharp increase in the expression of P450c17-II at the same time indicate that the steroidogenic shift from estrogen to 17, 20ß-DP does occur somewhere between d 8 and 12. A previous study on the steroid hormone profiles of tilapia during a spawning cycle of 14–15 d has shown peaks in estrogen and 17, 20ß-DP, respectively, at 9 and 12 d after spawning (40), providing support to our expression data.

The present study using tilapia has resolved a longstanding issue in the field of steroidogenesis with respect to the differential regulation of the two enzymatic activities displayed by the fish P450c17 during oocyte maturation (13, 14, 17, 19). Recently 17, 20ß-DP has been suggested to be an essential factor for the initiation of the meiosis in spermatogenic cells of the Japanese eel (41). Very intriguingly, we could notice in tilapia gonads that the initiation of P450c17-II expression is sexually dimorphic. In XX gonad its expression starts at 11 dah, whereas in XY gonad the expression is initiated from 60 dah only. This sex-specific stage-dependency in the initiation of P450c17-II expression matches with the pattern of meiosis, which starts in the XX and XY tilapia gonad at around 35 and 75 dah, respectively. We assume that P450c17-II expression may have a role in the production of 17, 20ß-DP which in turn initiates the meiosis in tilapia, as suggested in Japanese eel. In this context, our finding might open another research area for P450c17-II in the field of fish reproductive physiology.

	Acknowledgments

 
The sequences reported in this paper have been deposited in the GenBank database [accession no. of P450c17-I: AB292401 (tilapia-I), EF624004 (fugu-I), EF624006 (stickleback-I); P450c17-II: EF423917 (tilapia-II), EF423918 (medaka-II), EF624003 (zebrafish-II), EF624005 (fugu-II), EF624007 (stickleback-II)].

	Footnotes

 
This work was supported in part by a Grant-in-Aid for Scientific Research from the SORST Research Project of Japan Science and Technology Corp.; the Ministry of Education, Science, Sports, and Culture of Japan; and Environmental Endocrine Disruptor Studies from the Ministry of the Environment.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 14, 2007

Abbreviations: dah, Day after hatching; DHEA, dehydroepiandrosterone; 17, 20ß-DP, 17, 20ß-dihydroxy-4-pregnen-3-one; E2, estradiol-17ß; EST, expressed sequence tag; FSGD, fish-specific genome duplication; HEK, human embryonic kidney; ISH, in situ hybridization; MIH, maturation-inducing hormone; NCBI, National Center for Biotechnology Information; ORF, open reading frame; RACE, rapid amplification of cDNA ends; Scl, sex characterless; TLC, thin-layer chromatography.

Received April 16, 2007.

Accepted for publication June 4, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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