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Center for Advanced Marine Research, Ocean Research Institute, the University of Tokyo, Tokyo 164-8639, Japan
Address all correspondence and requests for reprints to: Kaoru Kubokawa, Center for Advanced Marine Research, Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639, Japan. E-mail: kubokawa{at}ori.u-tokyo.ac.jp.
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Introduction |
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). Typically, there are three cytochrome P450 enzymes (CYP) such as P450 side chain cleavage (CYP11A), P450c17 (CYP17), and P450 aromatase (CYP19) and two types of hydroxysteroid dehydrogenases (HSDs) such as 3ß-HSD and 17ß-HSD. CYP11A is an enzyme that regulates the conversion from cholesterol to pregnenolone (P5) by its side chain cleavage activity, and it is believed to be a rate-limiting enzyme of steroidogenesis. CYP17 is an important enzyme for the production of androgens, androstenedione, and dehydroepiandrosterone, from progesterone (P4) or P5. CYP19 enzyme is necessary to generate estrogens such as estrone (E1) and estradiol-17ß (E2) from androstenedione and testosterone (T), respectively (1). The 3ß-HSD enzyme catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent two-step reactions, i.e. 3ß-oxidation and isomerization of 
5-steroid to 4-steroid (2). The 17ß-HSD enzyme regulates conversion from 17-ketosteroids (androstenedione, dehydroepiandrosterone, and E1) to 17ß-hydroxysteroids (T, androstenediol, and E2) and vice versa (3). These sex steroid biosynthesis enzymes have been confirmed to be present in vertebrates. Furthermore, in parasite Taenia, the steroid-metabolizing activity to metabolize exogenous androstenedione to testosterone has been observed by thin-layer chromatography (4). In corals and zooxanthellae, 17ß-HSD activities were detected by the assay with E2 and the oxidation of nicotinamide adenine dinucleotide phosphate NADP+ (5).
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The data collected thus far suggest that the existence or function of sex steroids is different between invertebrates and vertebrates. In this point, protochordates are evolutionarily interesting animals because they are positioned on the phylogenetical boundary with vertebrates. Recently the draft genome sequences of urochordata Ciona intestinalis were annotated (9), and no P450 enzymes for biosynthesis of sex steroids have been found in the sequences. Sex steroids also have not been detected in tunicate gonads. The tunicate might have evolved a unique reproductive endocrine system that is different from that of vertebrates and that possibly uses different endocrine mechanisms for the maturation of gonads and the release of gametes. The cephalochordate amphioxus is the closest protochordate to vertebrates and should be investigated for the existence of sex steroids. In amphioxus, sex steroids in gonads such as P4, E2, and T were measured by RIA, although the experimental procedures did not provide definitive evidence of the presence of these sex steroids (10, 11). In addition, Callard et al. (12) demonstrated CYP19 (aromatase) activity by using the radiolabeled 19-hydroxyandrostenedione, which is an intermediate for estrogen generation, as a substrate for in vitro assay. But they did not comment on the ability to produce 19-hydroxyandrostenedione from androstenedione, which is a true substrate for CYP19 (aromatase). Recent molecular techniques confirmed the existence of CYP19 by isolating the partial cDNA of CYP19 from amphioxus Branchiostoma floridae (13). These reports suggest the presence of sex steroidogenesis, at least the pathway for estrogen synthesis, in amphioxus.
Considering the evolutionary importance of the phylogenetic position of amphioxus, an interesting approach is to explore whether it has the same sex steroids and the biosynthesis pathways as vertebrates. To confirm the existence of such steroidogenic pathways in amphioxus, we cloned cDNAs encoding CYP and HSD enzymes from amphioxus B. belcheri. Furthermore, we investigated the presence of sex steroids P4, E2, and T in the amphioxus gonads by RIA using qualitative analysis and evaluated the difference of those steroids between breeding and nonbreeding season by quantitative estimation. In this study, we discuss the evolution from invertebrates to vertebrates as viewed from sex steroid biosynthesis.
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Materials and Methods |
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Isolation of RNA
The tissues of amphioxus were carefully dissected from sexually mature adults with forceps under a dissecting microscope, immediately frozen in liquid nitrogen, and then stored at –80 C. Total RNA was extracted from ovaries of mature females by using ISOGEN reagent (Nippon Gene, Tokyo, Japan). Homogenization of tissues was performed on a Fastprep FP100A Instrument (Qbiogene, Irvine, CA) with disposable homogenizing tube, Lysing Matrix D (Qbiogene). Approximately 100 mg of frozen tissue collected and pooled from several females were added to the Lysing Matrix D containing 500 µl of ISOGEN reagent, and samples were homogenized on the Fastprep FP100A for 40 sec at the speed setting of 4.5. The homogenate was collected in a new tube, and beads in the Lysing Matrix D were washed by adding 500 µl of ISOGEN reagent. Each 500-µl portion of ISOGEN regent was pooled, and then the total RNA extraction was performed following an ISOGEN protocol. The total RNA eluted in 50 µl of diethylpyrocarbonate water was treated with RNase-free DNase I (TaKaRa Bio, Shiga, Japan) at 37 C for 30 min, and the reaction was stopped by adding 1 ml of new ISOGEN reagent. Total RNA was extracted by the reagent again, immersed in 70% ethanol, and stored at –30 C.
Construction of the first strand of cDNA by reverse transcription (RT)
First-strand cDNAs were constructed from the total RNA extracted from mature ovaries by using RNA PCR kit (AMV) (version 3.0; TaKaRa) with attached dT-adaptor primer. One microgram of the total RNA was used as a template for the RT reaction. The RT reaction was performed at 45 C for 30 min, 55 C for 10 min, 65 C for 10 min, and 70 C for 10 min, and finally denaturation of AMV reverse transcriptase was at 95 C for 5 min. The RT product was stored at –30 C until use. Two-microliter aliquots of the product were used as a template in 20 µl of PCR mixture for cloning of steroid metabolizing enzymes.
Cloning of cDNAs encoding amphioxus CYP11A, CYP17, and 17ß-HSD homolog
The cDNA fragments encoding CYP11A, CYP17, and 17ß-HSD homolog were obtained from the cDNA library of the amphioxus nerve cord. Subsequently a full-length cDNA of an open reading frame (ORF) was amplified from the ovarian first-strand cDNAs by using primers designed according to the sequence of the obtained clones on the ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA). Sequences of the ORF were assembled with those of the 5' and 3' untranslated region obtained from the cDNA library of the software ATGC (Genetyx Co., Ltd., Tokyo, Japan), and then full-length cDNA sequences were constructed. We used the primer pairs of sccCDS5 5'-ATGAAGCGTGCTCTGCACCAG-3' and sccCDS3' 5'-TTAGTTTAACAATTCTGTGAACTTGAATGT-3' for the amplification of CYP11A; c17CDS5 5'-ATGTGGTTGATGACGATAACTGTGGGGGTA-3' and c17CDS3 5'-TCACTTCCGGCAGGTCATCACCACCTTGTA-3' for the amplification of CYP17; and 17b-8CDS5 5'-ATGGCAGAGGGAGGAAAAGGG- CGTCTGACA-3' and 17b-8CDS3 5'-CTAATGCTTTCCTCCAGAAA- TCTCAATGGC-3' for the amplification of 17ß-HSD homolog. The product of the PCR was cloned into pGEM T-easy vector (Promega, Madison, WI) and determined the sequence.
Cloning of amphioxus CYP19 by PCR and rapid amplification of cDNA ends (RACE)
A partial cDNA fragment of CYP19 was obtained by RT-PCR by using degenerate primers of CYP19–1 F 5'-CAGTGCGTNACNGARATG-3' and CYP19–1R 5'-GTGATYAGNACNGCY-3' under the following conditions: initial denaturation at 95 C for 1 min, 40 cycles at 95 C for 5 sec, 45 C for 30 sec, and 72 C for 30 sec and additional extension at 72 C for 2 min. The PCR product was applied to agarose gel electrophoresis, and the gel containing approximately 300- to 600-bp lengths of fragments was excised broadly. DNA fragments in the excised gel were extracted using the QIAEX II gel extraction kit (QIAGEN, Valencia, CA) and used as the second PCR template. The second PCR was performed with the primers of CYP19-F and CYP19-R under the following conditions: initial denaturation at 95 C for 1 min, 40 cycles of denaturation at 95 C for 5 sec, annealing at 55 C for 30 sec, extension at 72 C for 30 sec, and additional extension at 72 C for 2 min. Amplified fragments of approximately 450 bp were subcloned into pCR4 plasmid vector (Invitrogen, Carlsbad, CA) and sequenced.
The cDNAs were synthesized by RT, using GeneRacer kit (Invitrogen) with attached GeneRacer oligo dT primer. Reaction conditions of RT by Superscript III transcriptase (Invitrogen) were serially performed at 50 C for 30 min, 55 C for 10 min, 60 C for 10 min, and 65 C for 10 min followed by inactivation of the reaction at 70 C for 15 min. The RACE was carried out with a GeneRacer kit (Invitrogen) to amplify both 5' and 3' ends of CYP19 cDNA. The primer was synthesized according to the CYP19 fragment obtained by the RT-PCR described above and GeneRacer primers contained in the kit. The primer sequences were CYP19GSP5RACE 5'-CGTGGGCCGAGGCCAAACGGCATGAACT-3' and GeneRacer 5' primer for 5'-RACE, and CYP19GSP3RACE 5'-GCTGGCCCGGACACCATGTCGGTCAACA-3' and GeneRacer 3' primer for 3'-RACE. The PCR product was applied for nested PCR as the template with primers CYP19GSP5RACE-Nest 5'-GATCTTCCTCCTCGGCCCGGCGCATGAC-3' and GeneRacer 5' nested primer for 5'-RACE and with CYP19GSP3RACE-Nest 5'-CATGCGCACGAGGCCGGTGGTCACTCT-3' and GeneRacer 3' nested primer for 3'-RACE. Both 5'- and 3'-RACE were performed by touchdown PCR as follows: initial denaturation at 95 C for 1 min, five cycles of denaturation at 95 C for 30 sec, and annealing and extension at 72 C for 2 min; five cycles of denaturation at 95 C for 30 sec and annealing and extension at 70 C for 2 min; 25 cycles of denaturation at 95 C for 30 sec, annealing at 65 C for 5 sec and extension at 72 C for 2 min; and additional extension at 72 C for 10 min. The amplified fragments were subcloned into pCR4 vector and sequenced. The partial sequences obtained from 5'- and 3'-RACE were assembled with ATGC software (Genetyx). The full-length coding region of CYP19 of amphioxus was amplified and confirmed the cDNA sequence derived from a single gene. The identities of amino acid residues and nucleotides of the coding region in CYP19 between B. floridae and B. belcheri were 88 and 87%, respectively. The lengths of base pairs in untranslated regions (UTRs) of CYP19 in B. floridae and B. belcheri were 107 and 240 bp in 5' UTR and 1140 and 1070 bp in 3' UTR, respectively.
Phylogenetic analysis of amphioxus steroidogenic enzyme homologs
Multiple alignments of amino acid sequences from amphioxus were performed using the Clustal X software (17). Phylogenetic trees were constructed by the neighbor-joining method with the evolutionary distance calculated by the Poisson correction (18) and the maximum parsimony method using the MEGA version 3.1 software (19). Selection of the outgroup for the CYP phylogenetic tree was based on the P450 clans proposed by Nelson and colleagues (20, 21). The outgroup used in the HSD phylogenetic tree was determined as described in previous reports (22, 23, 24).
RIA of sex steroids
The mature ovaries and testes used for RIA weighed 790 and 590 mg in July, respectively, and the developing ovaries weighed 400 mg in March. Tissues were homogenized using a Polytron homogenizer (Dremel, Racine, WI) in 1 ml of 10 mM PBS on ice for 1 min. One hundred microliters of 5 M NaCl were added to the homogenate, and it was then centrifuged at 10,000 x g for 15 min at 4 C. The supernatant was transferred to a glass tube. The pellet was suspended with 1 ml of 10 mM PBS and also transferred to a new glass tube. Two milliliters of diethyl ether were added to the supernatant and the suspension of the pellet, respectively. The samples were vortexed for 10 min and centrifuged at 2000 x g for 5 min at 4 C. The ether layer and water layer were separately transferred into new tubes, and the middle layer, which contained mostly undissolved substances and fat, was discarded. The water layer was extracted again with 2 ml of diethyl ether. The first and second ether extracts were combined, dried, and suspended in 500 µl of gelatin-PBS (10 mM PBS containing 0.1% gelatin).
The extraction efficiency was determined by adding 3H-labeled P4 (10,000 cpm), E2 (9,200 cpm), and T (4,200 cpm) to the ovaries and testes before extraction. The average recovery in the mature ovarian extraction was 82.5, 79.2, and 74.9% for P4, E2, and T, respectively. In the extraction of mature testes, the average recovery was 55.6, 66.4, and 69.6% for P, E2, and T, respectively. To examine the E2 concentration in a nonsteroidogenic tissue, we used 25 mg of extracted tissue from the head and tail parts of the body, which did not include gonad tissue. E2 was not detected in the head and tail extract, whereas E2 was detected in an extract of 10 mg of gonads. Furthermore, Callard et al. (12) reported that E2 was not synthesized from radiolabeled precursor steroid in the homogenate of cephalic and caudal body, although sufficient E2 conversion occurred in the central part of the body including gonads.
The RIA procedures were the same as described in previous reports (25, 26). The cross-reactivity of the antibodies used in RIA was the same as described in previous reports. The cross-reactivity of the antibodies used in RIA against the resembled structure steroids was as follows: for the anti-P4 antibody, 5
-pregnanedione (42.6%), 20-hydroxyprogesterone (6.73%), pregnenolone (1.46%), and 17-hydroxyprogesterone (0.89%); for the anti-E2 antibody, E1 (3.20%), estriol (1.77%), and androstenedione (0.44%); for the anti-T antibody, 5-dihydrotestosterone (30.0%), 11-ketotestosterone (1.50%), and androstenedione (1.00%).
The extracted samples were serially diluted by 2- or 4-fold. All samples were measured in duplicate. The detection limits of the steroid hormone concentrations in the ovarian extracts were 65.0, 32.0, and 18.7 pg/ml for P4, E2, and T, respectively. Those in the testicular extract were 15.0 pg/ml for all steroids measured. The concentrations of each steroid hormone were estimated using the commercially available software KyPlot (Keyence, Tokyo, Japan). The final concentration was revised with the extraction efficiency determined by the addition of a tracer.
Statistical analysis
The parallelisms between the standard curve and serially diluted samples were tested using the parallel line test in the commercial software KyPlot (Keyence) in the competitive inhibition analysis. We used a t test to compare estimated concentrations of each ovarian steroid between in March and July following the F test for homogeneity of variance. In addition, we tested the relationship between standard and competition curves by using two-way repeated measures ANOVA in Statview (version 5.0; SAS Institute, Cary, NC).
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Results |
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The cDNA encoding amphioxus CYP11A was 1745 bp long with an ORF of 1590 bp (530 amino acids) (Fig. 2A). The deduced amino acid sequence of the CYP11A cDNA showed 47–50% identity with various vertebrate CYP11A in the comparison of full-length proteins (Table 1). The amphioxus CYP11A protein also showed a similarity of 35–43% with CYP11B of various vertebrates (Table 1). Multiple alignment analysis demonstrated that amino acid residues observed in amphioxus CYP11A were highly conserved and well shared with those in vertebrate CYP11A, especially in the regions involved in the function of enzymatic catalysis (Fig. 2B).
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). The Ono sequence (29), Ozols’ tridecapeptide region (30), and heme ion binding site indicated that the cDNA is a member of CYP17 (Fig. 3B). The BLAST search by predicted amphioxus CYP17 protein showed that amphioxus CYP17 was 51–56% homologous between amphioxus and various vertebrates (Table 1
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). The predicted amphioxus CYP19 protein displayed overall 59–61% homology in comparison with various vertebrates (Table 1). The conserved regions, such as the helix region, Ozols’ tridecapeptide region (30), the aromatic region, and heme ion binding site, indicated that the cDNA is CYP19 (aromatase). The amino acid residues in conserved regions of CYP19 in amphioxus were well shared with those in other vertebrates (Fig. 4B).
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). The predicted amphioxus 17ß-HSD protein possessed the primary structural characteristics shared by other 17ß-HSD8 homologs (Fig. 5B), and the high homology suggested that amphioxus 17ß-HSD protein can be categorized as 17ß-HSD8. The amino acid sequence homologies of 17ß-HSD8 between amphioxus and vertebrates were 57–58% (Table 1).
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). The amphioxus CYP11A was positioned as an outgroup of vertebrate CYP11 clade consisting of CYP11A and CYP11B, which is considered to be evolutionarily the closest enzyme to CYP11A and which regulates adrenocortical steroid biosynthesis (31) (Fig. 6A). Human CYP19A and human CYP4A were used as an outgroup in the CYP11 tree. The amphioxus CYP17 was clustered into the CYP17 clade with vertebrate CYP17 proteins and was clearly distinguished from adrenosteroidogenic enzyme CYP21, which is known to be the closest relative to CYP17 (31) (Fig. 6B). Human CYP11A and human CYP4A were used as the outgroup in the CYP17 tree. The amphioxus CYP19 was located on the ancestral position of vertebrate CYP19 homologs including type B CYP19, which is a brain-specific isoform only confirmed in teleosts (32, 33) (Fig. 6C). Human CYP26A and human CYP11A were the outgroups of the CYP19 tree. The 17ß-HSD8 of amphioxus was positioned between vertebrates and protostome invertebrates (Fig. 6D).
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and were supported with high bootstrap values (data not shown).
Sex steroids in amphioxus gonads detected by RIA
The concentration of P4, E2, and T in the amphioxus gonads was examined by RIA using ovarian and testicular extracts from mature adults in the breeding season (Fig. 7). In July the parallelism of displacement curves drawn from the tissue samples and the standard substance was assessed using two methods. In the result of analysis by Kyplot, the parallelism was not statistically denied in any of the samples of ovarian P4 (P = 0.34), ovarian E2 (0.07), ovarian T (P = 0.20), testicular P4 (P = 0.50), testicular E2 (P = 0.12), and testicular T (P = 0.27), although probability values for E2 were relatively low. In the ANOVA, parallel relationships between standard curves and competitive plots of tissue samples from the breeding season were confirmed (P = 0.28, 0.05, and 0.77 in ovarian P4, E2, and T, respectively, and P = 0.96, 0.22, and 0.77 in testicular P4, E2, and T, respectively), although relatively low probability values were again observed in E2.
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The averages of the concentrations of P4, E2, and T in milligrams of ovary and testis samples in July and those of P4 and E2 in March are shown in Table 2. The immature testes samples taken in March were very small and little tissue could be collected, so we did not show the data of testicular steroid levels in March in this study. The P4 and E2 concentrations in July were significantly higher than those in March (P < 0.01 in P4 and P < 0.05 in E2). In July the P4 concentration in ovary samples was significantly lower than that in testis samples (P < 0.01), and E2 in ovary samples was higher than that in testis samples. The concentration of T was lower than those of P4 or E2 in ovary and also in testis samples taken during the breeding season.
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Discussion |
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It is noteworthy that steroidogenic enzymes (cytochrome P450 enzymes related to sex steroid metabolism) have not been found in invertebrates (34, 35). In the urochordate Ciona intestinalis, a pathway of sex steroid biosynthesis is absent because of the lack of cytochrome P450 enzymes metabolizing sex steroids (34). In view of sex steroidogenesis in animal reproduction, the presence of sex steroid-metabolizing enzymes cloned from amphioxus suggests that the cephalochordate is the closest invertebrate to vertebrates.
The sequence similarity of the CYP enzymes between amphioxus and vertebrates is shown in the conserved functional regions such as the substrate binding site and heme ion binding site (27, 28, 29, 30). The cDNAs isolated in our study appear to be the transcripts for CYP11, CYP17, and CYP19 enzymes. Callard et al. (12) reported that amphioxus B. floridae had enzymatic activity for estrogen production from androgen by the method of in vitro conversion using 3H-labeled 19-hydroxyandrostenedione as the substrate. The cloning of CYP19 and CYP17 genes from B. belcheri supports their data by not only the presence of CYP19 but also the existence of CYP17, which generates androgen as a substrate for CYP19.
Vertebrate CYP11A and CYP11B are considered to be derived from an ancestral gene (20, 31). The topology of the CYP11 tree, including the amphioxus CYP11A gene, shows that vertebrate CYP11A and CYP11B form a monophyletic clade, and amphioxus CYP11 is positioned out of the vertebrate enzyme group, although amphioxus CYP11 is more similar to CYP11A than to CYP11B. This topology is caused by the higher similarity between CYP11A and CYP11B in vertebrates. The second isoform of CYP11 in amphioxus was not found in the extracted RNA from ovaries and testes, but a survey of genomic DNA of amphioxus is needed to completely confirm the isoform of CYP11. If there is no CYP11B in amphioxus, our result suggests that the divergence of CYP11A and CYP11B arose in the vertebrate lineage.
In the molecular evolutionary studies of CYP proteins, CYP17 and CYP21 are considered to be monophyletic (20, 31). A homology search with amphioxus CYP17 showed similarity to vertebrate CYP17 and less similarity to CYP21, even in 100 candidates, although the existence of CYP21 in amphioxus should be confirmed in the genomic sequences. In tunicates, no genomic sequences encoding CYP17 and CYP21 were found. The topology of the phylogenetic tree of CYP17 and CYP21 suggests the possibility that these two enzymes diverged at the same time as or previous to the appearance of amphioxus. CYP17 proteins including those of amphioxus have sequence homology to CYP1, which is involved in the metabolism of xenobiotics and is categorized into the CYP2 clan with CYP17 and CYP21 according to previous reports (20, 21). The evolutionary relationships among CYP17, CYP21, and CYP1 might be revealed by a survey of amphioxus genomic sequences in the near future.
CYP19 is well known as an aromatase and a unique gene that is not similar to other members of the CYP superfamily, and it does not share a common clan with other proteins (20, 21). The topology of the phylogenetic tree of CYP19 shows the diversification of CYP19 into CYP19A and CYP19B (which is the brain specific subtype of CYP19, resulting from the duplication of the genomic genes within the teleost lineage). In conclusion, amphioxus CYP19 may be regarded as an ancestral gene of the vertebrate CYP19, which was divided into the two groups of teleosts and other vertebrates.
The other sex steroid-metabolizing enzyme in amphioxus is HSD, which has a similar structure to 17ß-HSD8 of vertebrates. The HSD enzyme is widely represented among invertebrates and vertebrates (3, 22, 35). 17ß-HSD8 is one of the types of 17ß-HSD involved in nicotinamide adenine dinucleotide-dependent conversion from androstenedione to T and interconversion between E1 and E2, and it is present in both invertebrates and vertebrates. The family of 17ß-HSDs is categorized as the short chain alcohol dehydrogenase/reductase superfamily and the isoforms possessing the diverged structures and their 17-oxidoreduction activities that were acquired independently (36). The evolution of the 17ß-HSD family is supported by the presence of 17ß-HSD8 homologs in Drosophila and Caenorhabditis elegans and their absence in C. intestinalis genomes (37). To clarify the evolution of steroidogenesis as inferred from 17ß-HSD, the confirmation needs to reveal which subtype is an ortholog from invertebrates to vertebrates.
From invertebrates to vertebrates, it is known that 3ß-HSD enzyme is necessary for P4 generation from P5. Enzymes other than 3ß-HSD have not been reported to generate P4 from P5 as far as we could determine by searching published papers. In this study, 3ß-HSD was not cloned from amphioxus gonads, but the existence of 3ß-HSD was considered because of the presence of P4 measured by the RIA procedure. This suggests that not only cloned CYP and HSD enzymes but also 3ß-HSD enzyme actually act in sex steroid conversions.
Figure 8 indicates the tentative pathway of androgen and estrogen biosynthesis in amphioxus. The pathway matches part of that of vertebrates. Furthermore, phylogenetic analysis suggests that the sex steroidogenic enzymes of amphioxus might represent ancestral molecules of vertebrates. To confirm the metabolizing activity of the sex steroidogenesis in amphioxus, we are currently conducting an experiment using radiolabeled substrate and gonadal extraction.
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P4 and E2 elevation in the breeding season
The ovarian P4 and E2 levels of amphioxus in the breeding season were significantly higher than those in the nonbreeding season. The level of ovarian P4 is 2.4-fold lower than that in the testis, and the level of E2 in the ovary is 2.3-fold higher than that in the testis. However, E2 was not detectable by RIA in the nongonadal tissues. In the mature ovary and testis in breeding season, gene expressions for CYP11A, CYP17, CYP19, and 17ß-HSD were detected by RT-PCR (data not shown), although a similar study in the nonbreeding season remains to be done. The different concentrations of sex steroids and the expression of sex steroid-metabolizing enzymes in mature gonads support that the sex steroids in amphioxus might be involved with reproductive phenomena during the breeding period.
Interestingly, we demonstrated that E2 was present in the testis of amphioxus, suggesting that CYP19 exists for E2 generation. In almost all the vertebrate species, it was well understood that androgen synthesis is a major part of steroidogenesis in the testis. However, a recent report described that CYP19 was expressed in the testis and that estrogen is important for testicular development and spermatogenesis in mammals (41, 42, 43). The biosynthesis of E2 and its role in the amphioxus testis should be confirmed by the CYP19 gene expression and the conversion activity from T to E2 in a future study.
Evolution of reproduction in amphioxus
Considering the possibility of sex steroid biosynthesis in the urochordate C. intestinalis as viewed from the annotation of several steroidogenic enzyme homologs, it is unable to generate androgens and estrogens by vertebrate-like metabolizing pathways because CYP17- and CYP19-like genes are absent (34). In the consideration of urochordate reproduction, at least C. intestinalis controls reproductive phenomena without sex steroids. This is strongly supported by the absence of steroid receptors in the C. intestinalis genome (9). In addition, the sex steroidogenic CYP enzymes are absent in other invertebrates such as echinoderms and protostome species (35). Therefore, sex steroidogenesis from cholesterol by a serial reaction of CYP and HSD enzymes might have been evolutionarily acquired at least in the ancestor of the cephalochordate amphioxus and vertebrates as discussed previously (44, 45). Recently it has been reported that urochordates are considered to be the evolutionarily closest invertebrates to vertebrates by molecular phylogenetic analysis (46). However, in this study, the reproductive system regulated by sex steroidogenesis is specialized in both amphioxus and vertebrates but not in urochordates. In summary, amphioxus is the key animal in the study of evolution from invertebrates to vertebrates. The analysis of genomic and cDNA sequences of amphioxus would be expected to reveal a comprehensive comparison among urochordates, cephalochordates, and vertebrates.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online April 19, 2007
Abbreviations: CYP, Cytochrome P450 enzyme; E1, estrone; E2, estradiol-17ß; HSD, hydroxysteroid dehydrogenase; ORF, open reading frame; P4, progesterone; P5, pregnenolone; RACE, rapid amplification of cDNA ends; RT, reverse transcription; T, testosterone; UTR, untranslated region.
Received January 24, 2007.
Accepted for publication April 11, 2007.
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5-4 isomerase gene family. Endocr Rev 26:525–5825-4 isomerase: molecular cloning and regulation of their messenger ribonucleic acids in equine follicles during the ovulatory process. Biol Reprod 64:206–215This article has been cited by other articles:
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  M. E. Baker Amphioxus, a Primitive Chordate, Is on Steroids: Evidence for Sex Steroids and Steroidogenic Enzymes Endocrinology, August 1, 2007; 148(8): 3551 - 3553. [Full Text] [PDF]
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