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Regular Article |
a Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
b Division of Biological Science, Graduate School of Science, Hokkaido University, Kita-Ku, Sapporo 060-0810, Japan
c Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa-Ku, Tokyo 142-0063, Japan
d Division of Gene Expression and Regulation I, National Institute for Basic Biology, Okazaki 444-8585, Japan
e Sado Marine Biological Station, Niigata University, Niigata 952-2135, Japan
f CREST, Japan Science Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20ß-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20ß-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase.
gametogenesis, granulosa cells, ovary, ovulation, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-dihydroxy-4-pregnen-3-one (17,20ß-DP) has been identified as the MIH, and it can induce maturation of isolated oocytes in vitro [1]. 20ß-Hydroxysteroid dehydrogenase activity in ovarian follicle layers is responsible for the production of MIH from a precursor steroid, 17-hydroxyprogesterone [1, 2].
Although the physiological relevance is not known, high 20ß-hydroxysteroid dehydrogenase activity has been reported in neonatal pig testis [3]. Surprisingly, the purification and cloning of its cDNA revealed that 20ß-hydroxysteroid dehydrogenase shows high similarity to monomeric carbonyl reductase from human and rat [4–7]. Carbonyl reductase is an oxidoreductase with wide substrate specificity for carbonyl compounds, requires NADPH as a cofactor [8–10], and is distributed in many tissues [11–13]. A more recent report [14] indicated that, in rat, liver microsomal carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase has similarity with acetohexamide reductase based on the requirement of NADPH as a cofactor. This further supports the idea that all these enzymes belong to the same family of oxidoreductase or short-chain dehydrogenase superfamily and may be involved in the metabolism of steroids or carbonyl compounds depending on the tissue requirement and localization [14]. Carbonyl reductase has been implicated as serving a housekeeping function to eliminate reactive carbonyl compounds. In addition to metabolism of the xenobiotics, the enzyme also catalyzes the reduction of biologically active compounds, such as prostaglandins and steroid hormones. Recently, two types of carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase cDNAs were cloned from rainbow trout ovary, and one of them was functional in reducing various substrates, including 17-hydroxyprogesterone [15]. In the functional form, isoleucine, at the 15th position, plays a crucial role in coenzyme binding and enzyme activity [16]. However, to our knowledge, the physiological function of carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase in the metabolism of any of these endogenous substrates remains to be elucidated.
Maturation of fully grown oocytes of a teleost fish, the ayu (Plecoglossus altivelis), is initiated by a surge of 17,20ß-DP from ovarian follicle layers [17]. Taking advantage of this temporally discrete and massive production of MIH just before oocyte maturation, we extracted RNA from follicle layers and cloned carbonyl reductase cDNA; we hypothesized that carbonyl reductase is involved in the production of MIH. Here, we describe that ayu carbonyl reductase has 20ß-hydroxysteroid dehydrogenase activity and that the transcripts, which are present in fully grown, immature ovarian follicles, increase substantially in follicles of mature oocytes. These results suggest that one of the physiological functions of carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase is to catalyze the production of MIH in teleost ovary during meiotic maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ayu ovaries containing mature oocytes were obtained from Ueda Station, National Institute of Fisheries Science, Japan. Ovarian follicle layers were prepared as described previously [18]. Total RNA was extracted from the ovarian follicle layers, followed by purification of poly(A)+ RNA using a PolyATract mRNA isolation kit (Promega, Madison, WI). The cDNA was synthesized from the poly(A)+ RNA using a ZAP cDNA synthesis kit (Stratagene, La Jolla, CA) and was packaged into Uni-ZAP XR vector using Gigapack II Gold packaging extract (Stratagene).
Isolation and Sequencing of Ayu Carbonyl Reductase cDNA
The PstI/BstYI fragment of pig 20ß-hydroxysteroid dehydrogenase cDNA, which had been subcloned in M13 vector (M13/42125) [4], was labeled with [-32P]deoxycytidine triphosphate (dCTP) using BcaBEST polymerase (Takara, Tokyo, Japan) and was used to screen the ayu cDNA library under the following conditions. The hybridization was performed in a solution containing 5x SSC (0.75 M NaCl and 0.075 M sodium citrate), 5x Denhardt solution (0.1% [w/v] polyvinylpyrrolidone, 0.1% [w;clv] bovine serum albumin, and 0.1% [w/v] Ficoll), 35% (w/v) formamide, 10% (w/v) dextran sulfate, 100 µg/ml of herring sperm DNA, and 0.2% (w/v) SDS at 47°C. The filters were washed with 1x SSC solution containing 0.1% (w;clv) SDS at 47°C. pBluescript SK vectors harboring cDNA inserts (222b1 and s1111) were generated from isolated positive plaques by in vivo excision according to the protocol of the supplier (Stratagene). Deletion mutants were made from 222b1 and s1111 and subsequently subjected to DNA sequencing as described previously [4]. The sequence was confirmed from both directions. The sequence data discussed in this study have been submitted to GenBank, and the accession number is D82967.
Detection of 20ß-Hydroxysteroid Dehydrogenase Activity in Transformed Escherichia coli, and Identification of Steroid Metabolites
Expression vector, pET15b (Novagen, Inc., Madison, WI) harboring the EcoRI/KpnI s1111 cDNA fragment at the NcoI site (s1111/pET15b) was transformed into BL21 E. coli strain. The transformant (PETS1111) was cultured in 20 ml of Luria-Bertoni (LB) medium for 2 h, and the cDNA product was induced by addition of isopropyl-ß-D(-)-thiogalactopyranoside (IPTG; final concentration, 1 mM). After an additional 2-h incubation, PETS1111 was harvested by centrifugation at 1500 x g; resuspended in 5 ml of the solution containing either 20 mM Tris-HCl (pH 8.0) and 1 mM EDTA or 3 mM potassium phosphate buffer (pH 7.4), 0.2 mM EDTA, 100 µM PMSF, and 1 µM pepstatin; and then disrupted by sonication. Clear lysate was obtained by centrifugation at 105 000 x g for 1 h. Five hundred microliters of the clear lysate were incubated at 28°C with 9 x 106 cpm of [1,2,6,7-3H]17-hydroxyprogesterone (3.44 TBq/mmol; Amersham, Buckinghamshire, England) in the presence of 0.1 mM NADPH. The steroid metabolites were extracted with diethyl ether, separated on a high-performance thin-layer chromatography (TLC) plate (Merck, Whitehouse Station, NJ) using a benzene:acetone (4:1 [w/v]) solvent system followed by cyclohexane:ethyl acetate (1:1 [w/v]), and exposed onto Hyperfilm (Amersham). The spot, which comigrated with cold standard 17,20ß-DP, was extracted with ethanol and chloroform and further identified by recrystallization [19].
Purification of IPTG-Induced cDNA Product
Clear lysate of the PETS1111 clone was prepared from 250 ml of LB medium as described above and dialyzed against 3 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA and 0.1 mM dithiothreitol (DTT). The dialyzed lysate was applied to a DE-52 cellulose column (1.8 x 40 cm). The absorbed proteins were eluted by a linear gradient of 3–100 mM potassium phosphate buffer containing 0.1 mM EDTA and 0.1 mM DTT, and 13-ml fractions were collected. Twenty-five microliters of each fraction were assayed for enzymatic activity. The carbonyl reductase/20ß-hydroxysteroid dehydrogenase activity of each fraction was measured by monitoring the oxidation of NADPH at 340 nm in the presence of 1 mM 4-nitrobenzaldehyde at 25°C. The fraction containing carbonyl reductase/20ß-hydroxysteroid dehydrogenase activity was kept frozen at -70°C.
Substrate Specificity of Ayu Carbonyl Reductase-Like 20ß-Hydroxysteroid Dehydrogenase
Substrate specificity was examined under conditions described previously [4, 8]. Purified ayu carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase (18.9–151 µg) was used to determine substrate specificity using the substrates shown in Table 1. The enzymatic activity was measured by the decrease in absorbance at 340 nm in the presence of 0.08 mM NADPH at 25°C.
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Northern Blot Analysis
Ovarian follicle layers were prepared from ayu ovaries containing mature follicles or fully grown, immature follicles. Total RNA was prepared from the ovarian follicle layers, liver, and brain. Twenty micrograms of total RNA were electrophoresed in a formaldehyde denaturing gel [20] and blotted onto Nylon membrane (Hybond N+; Amersham). The KpnI/SacI fragment of s1111 cDNA was labeled with [-32P]dCTP and used as the ayu carbonyl reductase probe. Northern blot hybridization was performed under the following conditions: 5x SSC, 5x Denhardt solution, 0.1% SDS, 100 µg/ml of herring sperm DNA, and 10% dextran sulfate at 60°C. The membrane was washed with 1xSSC containing 0.1% SDS at 60°C. The same membrane was rehybridized with a teleost fish, the medaka (Oryzias latipes), actin probe after removal of the carbonyl reductase probe by boiling the membrane in a solution of 0.1x SSC and 0.1% SDS. The actin DNA template was labeled by polymerase chain reaction (PCR) with [-32P]dCTP using the primers ACACCTTCTACAATGAGCT and CCGTCAGGATCTTCATGAGG.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because we expected carbonyl reductase to be present in ayu ovarian follicle layers surrounding mature oocytes, a cDNA library was constructed from ovarian follicle poly(A)+ RNA. A pig cDNA fragment having high homology with members of the short-chain alcohol dehydrogenase superfamily was used to screen the cDNA library. After three rounds of screening, one phage, designated as 20ßayu222b1, was isolated. However, the 5' portion of the coding region in the 1381-base pair (bp) insert showed no homology with previously reported pig and human carbonyl reductase cDNA sequences. The PCR with primers at the 5' nonhomologous portion and the 3' homologous portion using ayu cDNAs did not amplify any DNA fragment from ayu ovarian cDNA, suggesting that the 5' portion of 20ßayu222b1 was an artifact. Therefore, we rescreened the cDNA library using the conserved region (XhoI/PstI fragment) of 20ßayu222b1 as a probe. Three phages were isolated, and the phage (s1111) with the longest insert (1.3 kilobases [kb]) was chosen for subsequent DNA sequencing. The cDNA sequence is 1279-bp long, includes a poly(A)+ tract, and contains an open reading frame that is expected to code for a 276 amino acid polypeptide (Fig. 1).
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A Blast sequence similarity search was performed against the GenBank database. The sequences identified by the search exhibiting the most significant similarity to the s1111 sequence were pig, trout, rat, rabbit, and human carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase (Fig. 2). Ayu carbonyl reductase shared 60%, 60%, 61%, 63%, and 64% amino acid identity with pig, trout, rabbit, human, and rat carbonyl reductases, respectively, whereas 80–85% homology was found among mammalian carbonyl reductase sequences. The alignment revealed six different domains showing more than 70% local homology in the stretch of 13 or more amino acids among the species (Fig. 2).
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20ß-Hydroxysteroid Dehydrogenase Activity of IPTG-Induced s1111 Product from E. coli
To demonstrate that s1111 cDNA encodes a polypeptide with 20ß-hydroxysteroid dehydrogenase activity, s1111 cDNA was cloned into the prokaryotic expression vector, pET15b. After induction with IPTG, clear lysate was recovered by centrifugation following sonication of E. coli harboring s1111/pET15b (PETS1111). The clear lysate was incubated for 18 h with 3H-labeled 17-hydroxyprogesterone in the presence of NADPH, and metabolites were analyzed by two-dimensional, high-performance TLC with cold standard steroid markers after extraction. The steroids/metabolites were extracted with diethyl ether, evaporated, and dissolved in ethanol. The ethanol was applied onto high-performance TLC plates and analyzed first with a benzene:acetone solvent system (vertical axis) and then with a cyclohexane:ethyl acetate solvent system (horizontal axis). One spot, which was observed only in the IPTG-induced lysate, comigrated with authentic 17,20ß-DP (Fig. 3), indicating that this spot is very likely to be 17,20ß-DP generated by 20ß-hydroxysteroid dehydrogenase activity. To further confirm that the metabolite was 17,20ß-DP, recrystallization was performed by extracting the spot from the plate (before crystallization, 9172 cpm/mg; first crystallization, 7866 cpm/mg; second crystallization, 8416 cpm/mg; third crystallization, 8044 cpm/mg). The extract was crystallized in the three different solvent systems with the same extraction ratio as that for cold 17,20ß-DP. This clearly indicates that 17,20ß-DP was produced from 17-hydroxyprogesterone by 20ß-hydroxysteroid dehydrogenase activity of ayu carbonyl reductase-like cDNA, s1111.
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Purification of IPTG-Induced s1111 Product from Clear Lysate
Because E. coli has endogenous enzymatic activities that may interfere with the measurement of carbonyl reductase activity, we purified the s1111 cDNA product from IPTG-treated E. coli. The clear lysate was applied to a DE-52 column and fractionated. Carbonyl reductase activity in each fraction was monitored by the decrease in absorbance of NADPH at 340 nm in the presence of 4-nitrobenzaldehyde (Fig. 4A). Three peaks of enzymatic activity were identified. Fractions 23–26, 29–32, and 66–70 were pooled and designated as fractions I, II, and III, respectively. The results of SDS-PAGE showed that fractions I and II were purified to homogeneity with molecular masses of 32 and 30.5 kDa, respectively (Fig. 4B), whereas several bands were observed in fraction III. Because the first two peaks of activity (fractions I and II) were not detected in clear lysate from noninduced E. coli, and because the last peak (fraction III) was consistently observed in both preparations of the clear lysate, the latter is very likely to be derived from endogenous activity of E. coli. Amino acid sequencing revealed that the 30.5-kDa band consists of an amino acid sequence starting with a serine residue just one amino acid downstream from the putative initiation codon of s1111 cDNA. The partial sequence perfectly matches the deduced amino acid sequence from s1111 cDNA. The 32-kDa band (fraction I) was found to be a protein fused with a seven-amino-acid vector sequence (data not shown). Therefore, we used fraction II for subsequent measurement of carbonyl reductase/20ß-hydroxysteroid dehydrogenase activity.
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Carbonyl Reductase-Like 20ß-Hydroxysteroid Dehydrogenase Activity of s1111 cDNA Product
Carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase activity was examined by measuring the decrease in absorbance at 340 nm in the presence of NADPH. Table 1 shows the specific activity of ayu carbonyl reductase. 9,10-Phenanthrenequinone was the best substrate for ayu carbonyl reductase, followed by cyclohexanone and 4-nitrobenzaldehyde. A very high concentration (1 mM) of prostaglandin (PG) E1 and PGE2 could also be utilized as substrates. Ayu carbonyl reductase more preferentially reduced 5- and 5ß-dihydroxytestosterone compared to pig and human carbonyl reductase, whereas the enzyme did not reduce testosterone. The specific activity of ayu carbonyl reductase for 17-hydroxyprogesterone, the immediate precursor of 17,20ß-DP, was as high as that of pig, indicating that 17-hydroxyprogesterone may serve as a good substrate for ayu carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase. To confirm that the s1111 product catalyzes the reduction of the C20-oxo-position in 17-hydroxyprogesterone, the metabolites of 17-hydroxyprogesterone were analyzed by TLC. The resultant metabolite comigrated with authentic 17,20ß-DP (data not shown). All these results demonstrate that the s1111 product functions not only as 20ß-hydroxysteroid dehydrogenase but also as carbonyl reductase.
Northern Blot Analysis
Northern blot hybridization was performed to determine whether the amount of carbonyl reductase transcripts changes during the course of final maturation of ayu oocytes. A single band, which was estimated to be 1.4 kb long (Fig. 5A), was detected. Transcripts were also found in brain, as reported previously [8]. Although barely detectable in liver, expression was confirmed by reverse transcription-PCR (data not shown). When the level of transcripts were normalized against the level of actin transcripts, the increase of the transcripts during maturation becomes more apparent. Mature follicles expressed 1.6-fold more transcript compared with fully grown, immature follicles (Fig. 5B). Northern blot analysis was repeated with tissues from four different individuals and found 1.6-, 1.8-, 3.2-, and 3.5-fold increases.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-DP, have been identified as steroid hormones that induce final maturation of oocytes in ovarian follicles [1, 4]. Plasma levels of 17,20ß-DP and the capacity of ovarian follicles to produce 17,20ß-DP are elevated during the transition from vitellogenesis to oocyte maturation [1, 22], concomitant with an increase in production of the endogenous precursor, 17-hydroxyprogesterone [1]. Furthermore, addition of 17-hydroxyprogesterone and forskolin to isolated granulosa cells results in enhanced 17,20ß-DP production, which is associated with a dramatic increase in 20ß-hydroxysteroid dehydrogenase activity [1, 22]. These physiological observations correlate very well with the results of Northern blot analysis that show a marked increase in carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase transcripts during final maturation of ayu oocytes. In accordance with our findings, a recent report by Espey et al. [24] demonstrated a temporal pattern of expression of ovarian carbonyl reductase in rat after the induction of ovulation, suggesting that expression of ovarian carbonyl reductase activity is related to the ovulatory process [24, 25]. More recently, Espey et al. [26] have also reported the expression of another carbonyl reductase-like enzyme, 3-hydroxysteroid dehydrogenase, in immature rat ovary and have indicated its role in ovulation. These reports, together with the present study, strengthen the role of carbonyl reductase-like, 20ß-hydroxysteroid dehydrogenases in the preparation of final oocyte maturation and ovulation processes in vertebrates. In addition to human carbonyl reductase, the nucleotide sequences of carbonyl reductase cDNA from rat testis [27] and rabbit liver [28] have been reported. Rat carbonyl reductase shows only 64% positional identity with ayu carbonyl reductase at the amino acid level. However, a putative cofactor-binding site around region I (Fig. 2) is well conserved between species at 75% amino acid homology [29], and 77% homology is found in an extraloop of 13 amino acid residues (region V), which may affect the substrate pocket [30]. Moreover, a long stretch of conserved region VI (79% homology) and of regions IV and V (71% and 77% homology, respectively) characterize the carbonyl reductase family, whereas characteristics of the short-chain alcohol dehydrogenase superfamily are also found in region II (87% homology). The motif Tyr-X-X-X-Lys, which is regarded as being the active site for a nucleophilic attack on the substrate, is found from positions 193 to 197 [31]. These local, high homologies suggest that ayu carbonyl reductase simply represents a species-specific form of rat and human carbonyl reductases.
However, ayu carbonyl reductase shows more preference for steroid hormones, such as 17-hydroxyprogesterone and 5/ß-dihydroxytestosterone, than for prostaglandins. Ayu carbonyl reductase cannot utilize 0.1 mM prostaglandins (PGE1 and PGE2), whereas, in contrast, the Km of rat carbonyl reductase for prostaglandins is 0.05–0.1 mM [32, 33]. The substrate preference of ayu carbonyl reductase for 17-hydroxyprogesterone is as high as that previously reported for the neonatal pig enzyme (Km = 9.42 µM) [34], which suggests that ayu carbonyl reductase is involved more in the metabolism of steroids rather than of prostaglandins. As Wermuth et al. [27] pointed out, two types of carbonyl reductases may exist on the basis of substrate specificity. One isozyme, present in pig neonatal testis, ayu ovary, and probably rat ovary, exhibits a high selectivity for steroids, whereas the other has low selectivity for steroids and predominates in rat testis and human brain. Nevertheless, all these oxidoreductases require NADPH as a cofactor [14].
Indeed, the change in rat ovarian carbonyl reductase during the estrous cycle shares some characteristics with the change in 20ß-hydroxysteroid dehydrogenase activity in the teleost ovary. The content of rat carbonyl reductase in the proestrous ovary increases approximately twofold over that in the diestrous ovary [35], and hCG causes an estrogen-mediated induction of rat carbonyl reductase [36]. Two carbonyl reductase cDNAs were isolated from rat ovary, but only one was rapidly and strongly induced by eCG [37]. Interestingly, two types of carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase have been isolated from rainbow trout ovary, one of which possesses the enzymatic activity [15]. Results of subsequent studies warranted that the presence of isoleucine at the 15th position in one of the enzymes is responsible for cofactor binding and enzymatic activity [16]. In fact, ayu carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase showed high homology to its inducible (active form) counterpart in rainbow trout. In most teleost fish, intracellular cAMP levels in ovarian follicle layers correlate with the stimulation of steroidogenesis and elevation of intracellular cAMP by hCG, and forskolin or salmon gonadotropin (GTH) results in increased 20ß-hydroxysteroid dehydrogenase activity [1, 2, 22]. The dramatic increase of 20ß-hydroxysteroid dehydrogenase enzyme activity induced by GTH/hCG in isolated ovarian follicles of teleosts [1, 2] is inhibited by actinomycin D, suggesting that 20ß-hydroxysteroid dehydrogenase is transcriptionally regulated [1]. These results tend to indicate that the carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase in the ayu ovary is probably under the transcriptional control of the pituitary-gonadal axis via GTH. Further studies are required to confirm this phenomenon. The involvement and regulation of carbonyl reductase in the estrous/ovarian cycle may have been developed phylogenetically from the origin of lower vertebrates.
Several lines of evidence (localization in ovary, enzymatic characteristics, and increase in transcripts with oocyte maturation) suggest that ayu carbonyl reductase functions as 20ß-hydroxysteroid dehydrogenase in the production of MIH, although the involvement of other types of 20ß-hydroxysteroid dehydrogenase in oocyte maturation cannot be ruled out. Nevertheless, existing knowledge confirms that ayu ovary carbonyl reductase is the carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase of the species. The temporal expression and regulation patterns of 20ß-hydroxysteroid dehydrogenase during the entire process of sexual maturation are worthy of further analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS-RFTF 96L00401 to Y.N.); Scientific Research (07283103, 08454266, and 10440247 to Y.N. and 08740642 to M.T.) from the Ministry of Education, Science, Culture, and Sports, Japan, and CREST of Japan Science Technology Corporation; and a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry, and Fisheries. 
2 Correspondence. FAX: 81 564 55 7556; nagahama{at}nibb.ac.jp
Accepted: December 11, 2001.
Received: November 14, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-Dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 1997; 62:190-196[CrossRef][Medline]-hydroxyprogesterone, and 17,20ß-dihydroxy-4-pregnen-3-one during synthetic LH-RH-induced final oocyte maturation and ovulation in the ayu Plecoglossus altivelis. Bull Jpn Soc Sci Fish 1983; 49:1165-1169-Hydroxysteroid dehydrogenase messenger RNA transcription in the immature rat ovary in response to an ovulatory dose of gonadotropin. Biol Reprod 2001; 65:72-78This article has been cited by other articles:
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  C Mittelholzer, E Andersson, D Consten, T Hirai, Y Nagahama, and B Norberg 20{beta}-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua) J. Mol. Endocrinol., October 1, 2007; 39(4): 319 - 328. [Abstract] [Full Text] [PDF]
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 M Kusakabe, I Nakamura, J Evans, P Swanson, and G Young Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes. J. Endocrinol., June 1, 2006; 189(3): 541 - 554. [Abstract] [Full Text] [PDF]
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 B. Senthilkumaran, C.-C. Sudhakumari, X.-T. Chang, T. Kobayashi, Y. Oba, G. Guan, Y. Yoshiura, M. Yoshikuni, and Y. Nagahama Ovarian Carbonyl Reductase-Like 20{beta}-Hydroxysteroid Dehydrogenase Shows Distinct Surge in Messenger RNA Expression During Natural and Gonadotropin-Induced Meiotic Maturation in Nile Tilapia Biol Reprod, October 1, 2002; 67(4): 1080 - 1086. [Abstract] [Full Text] [PDF]
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). Closed diamonds (
) show the absorbance of each fraction at 280 nm. B) Fractions I (no. 23–26), II (no. 29–32), and III (no. 66–70) were separated by 10% SDS-PAGE gel and stained with Coomassie brilliant blue R-250 to indicate the presence of purified product. 
, Conductivity