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Biology of Reproduction 65, 1186-1194 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Correlation Between Messenger RNA Expression of Cytochrome P450 Aromatase and Its Enzyme Activity During Oocyte Development in the Red Seabream (Pagrus major)1

Koichiro Gen2,a, Koichi Okuzawaa,b, Naoki Kumakurab, Sonoko Yamaguchic, and Hirohiko Kagawab,d

a Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan b Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan, Minato-ku, Tokyo 108-8477, Japan c Department of Fisheries, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan d National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 519-0196, Japan

ABSTRACT

In teleosts, estradiol-17ß (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450arom) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450arom during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450arom is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450arom mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450arom mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450arom enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHß, but not FSHß, correlated with increased P450arom mRNA levels during the course of ovarian development. In addition, the levels of P450arom mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450arom mRNA during oocyte development of red seabream.

estradiol, follicle-stimulating hormone, luteinizing hormone, oocyte development, ovary

INTRODUCTION

Estrogen is a pleiotropic hormone that plays a pivotal role in many diverse aspects of the physiological and developmental program. In particular, female reproduction is controlled by the ability of estrogen to regulate proliferation of uterine cells and to regulate various granulosa cell functions [1]. In teleosts, as in other oviparous vertebrates, estradiol-17ß (E2) is the main estrogen that is essential for induction of the precursor of yolk protein (vitellogenin) in the liver and oocyte development (vitellogenesis) [2]. In the ovarian follicle of salmonids, E2 is produced in the granulosa cell layer by conversion of testosterone (T), which is produced in the theca cell layer (two-cell-type model) [3]. Thus, conversion of T to E2 is thought to be the rate-limiting step in E2 biosynthesis.

Cytochrome P450 aromatase (P450arom) is directly involved in E2 production [4]. Recently, isolation and characterization of P450arom cDNAs have been reported for several teleost species [5–10], but little information is available regarding the expression of P450arom in fish reproduction. Studies using rainbow trout [5], medaka [7], and tilapia [8] have shown that mRNA levels of P450arom are increased in association with enzyme activity during vitellogenesis. Even though these findings suggest that the catalytic activity of P450arom is dependent on the levels of mRNA, to our knowledge, precise data and information are not available regarding the relationship between expression of the P450arom mRNA and enzyme activity in these species, except for medaka [7]. Moreover, no data exist concerning concomitant changes in mRNA levels of P450arom, aromatase activity, and serum E2 levels during oocyte development.

Extensive in vivo and in vitro studies of mammals have provided evidence that expression of P450arom is regulated by sequential actions of the gonadotropins (GTHs), FSH and LH, throughout the reproductive cycle [11]. FSH stimulated an increase of P450arom mRNA in rat granulosa cells of the preovulatory follicles [12, 13]. In contrast, P450arom mRNA levels dramatically decreased as a consequence of the LH surge [14, 15]. In teleosts, as in mammals, two distinct GTHs are found: GTH-I, which is homologous to tetrapod FSH; and GTH-II, which is homologous to tetrapod LH [16, 17]. In salmonids, FSH is elevated during vitellogenesis, whereas LH appears during final maturation and ovulation in females [18]. Moreover, previous studies have demonstrated that P450arom enzyme activity [19] and its mRNA levels [5] are also increased during the vitellogenic oocyte growth. These findings tend to suggest that, in salmonids, the expression of P450arom likely is controlled by FSH, similar to the situation in higher vertebrates. However, much less is known about hormonal regulation of P450arom in the reproduction of non-salmonid species.

Red seabream (Pagrus major [order: Perciformes]) provides an interesting and unique model for investigating hormonal regulation of P450arom in fish reproduction. In contrast to salmonids, red seabream have an asynchronous-type ovary and spawn almost every day during the spawning season. Our previous studies indicated that LH, but not FSH, stimulates in vitro E2 production in the vitellogenic follicles of red seabream [20]. Interestingly, a more recent study from our laboratory demonstrated that LHß mRNA is maintained at high levels from the beginning of vitellogenesis to the spawning period, whereas FSHß mRNA remains at low levels throughout sexual maturation in female red seabream [17]. These findings raise the possibility of differences in the regulation of P450arom in perciformes and salmonids during oocyte development.

In this study, as a first step in investigating hormonal regulation of the P450arom in female red seabream, we isolated and characterized a red seabream P450arom cDNA derived from ovary. Furthermore, using Northern blot analysis, we examined in detail the expression profiles of P450arom during oocyte development induced by implantation of cholesterol pellets containing gonadotropin-releasing hormone analogue (GnRHa). We provide evidence that expression of P450arom mRNA is associated with increasing levels of P450arom enzyme activity and serum E2, and is detected only in vitellogenic follicles. We also describe, for the first time in teleost species, concomitant changes in the expression of genes encoding P450arom and GTH subunits during oocyte development in female red seabream.

MATERIALS AND METHODS

Animals

Immature, 2-yr-old red seabream, weighing 1.5–2 kg each, were purchased from fishermen in Gokasho Bay, Mie Prefecture, and kept in a flow-through outdoor tank (3000 L) under natural conditions at the National Research Institute of Aquaculture. In November 1998, immature red seabream were implanted with cholesterol pellets containing GnRHa (des-Gly10, [D-Ala6]-LH-RH; Sigma, St. Louis, MO) at a dose of 200 µg/kg body weight. Zero (initial control), 8, and 15 days after implantation, fish were deeply anesthetized with 2-phenoxyehanol (Nacalai Tesque, Kyoto, Japan) before being killed by decapitation. The body weight and length of each fish were recorded before dissection. Blood samples were kept on ice and centrifuged for 15 min at 2500 x g; the serum was separated and kept frozen at -20°C until assayed by RIA [21]. The pituitary glands were collected, frozen individually in liquid nitrogen, and stored at -80°C until used. Ovaries were excised and weighed for calculation of the gonadosomatic index (GSI; gonad weight/body weight x 100%) and then divided into three portions. Small pieces of the ovaries were fixed in Bouin solution for morphological identification of reproductive stage. Other small portions were used to estimate P450arom enzyme activity by in vitro conversion of T to E2, and the remaining portions were frozen immediately in liquid nitrogen and then stored individually at -80°C until isolation of mRNA.

Amplification of P450arom cDNA Fragment and Screening the Ovarian cDNA Library

Degenerate primers based on the nucleotide sequence of other vertebrates were designed to clone red seabream P450arom cDNA (sense: 5'-CCA GCA ACT ACT ACA A(C/T)A (A/G)CA A(A/G)T ATG GAG-3'; antisense: 5'-G(A/G)(C/T) TTC ATC ATC ACC ATG GC-3'). Using the vitellogenic ovarian cDNA as a template, polymerase chain reaction (PCR) was performed using 50 µl of final volume containing 5 µl of 10x reaction buffer, 2 mM MgCl2, 200 µM of each deoxy (d)ATP, dCTP, dGTP, and dTTP, 2 µM of each primer, and 2.5 U of Taq DNA polymerase (Takara Biomedicals, Tokyo, Japan). After an initial 1.5-min denaturing step at 94°C, 30 cycles of amplification were performed using a cycle profile of 94°C for 30 sec, 50°C for 1 min, and 72°C for 1.5 min. After the last cycle, elongation was extended to 10 min at 72°C. The PCR products were T-A cloned into pBluescript II KS- (Stratagene, La Jolla, CA) and sequenced. The ovarian cDNA library was constructed using poly(A)+ RNA from a vitellogenic ovary with a {lambda}ZAP vector system (Stratagene). Screening for red seabream P450arom cDNA clones was carried out by hybridization under high-stringency conditions [22] using a PCR-amplified cDNA fragment encoding the red seabream P450arom as probe. After three rounds of isolation, positive clones were obtained. In vitro excision and rescue of pBluescript phagemids were performed according to the manufacturer's protocol.

DNA Sequencing and Sequence Analysis

Double strand DNA was sequenced on both strands using vector-based and gene-specific primers and an automated sequencing system with fluorescent dye terminator (Perkin-Elmer/Applied Biosystems, Foster City, CA). The nucleotide and amino acid sequences were analyzed using Macvector software (release 7.0; Oxford Molecular Ltd., Madison, WI) and the BLAST network service of the National Center for Biotechnology Information [23]. The accession number of the sequence reported in this paper has been deposited in the GenBank as AB051290.

Phylogenetic Analysis

Multiple alignments were performed with CLUSTAL W [24]. A phylogenetic tree was constructed based on amino acid sequence alignments by the neighbor-joining method, as implemented in the Neighbor program from PHYLIP version 3.572 [25]. The distance matrices were calculated using the Dayhoff PAM matrix model [26]. The degree of support for internal branches was further assessed by bootstrapping analysis consisting of 1000 replicates [27].

RNA Isolation and Northern Blot Analysis

Total RNA was extracted from each ovary from different stages by the guanidium isothiocyanate method [28]. Poly(A)+ RNA was isolated using an oligo(dT)18 cellulose column (Pharmacia Amersham Biotech, Uppsala, Sweden). Five micrograms of poly(A)+ RNA from each ovary were fractionated on denaturing, 1% (w/v) agarose gel and transferred to Hybond N+ nylon membrane (Pharmacia Amersham Biotech) as previously described for red seabream pituitary [17]. The membrane was air-dried and baked at 80°C for 2 h before hybridization with randomly radiolabeled red seabream P450arom cDNA probe. The membrane filter was washed at 65°C with several buffer changes of decreasing SSPE (pH 7.4) concentrations, from 2x to 0.1x (1x SSPE: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA) and exposed to x-ray film with an intensifying screen at -70°C. The blot was subsequently stripped and rehybridized with a partial ß-actin cDNA probe, which was kindly provided by Dr. G. Yoshizaki (Tokyo University of Fisheries), to control for loading variations. For quantitative analysis of P450arom mRNA levels during oocyte development, the radioactivity of each hybridization signal was counted using a BetaScope 603 blot analyzer (Aloka, Tokyo, Japan), and the amount of P450arom mRNA was normalized to the amount of the ß-actin.

RNase Protection Assay

To generate riboprobes, cDNA was amplified by PCR using primers for red seabream {alpha}-glycoprotein subunit ({alpha}GSU), FSHß, or LHß [17]. The antisense primers were modified to contain a 17-base pair (bp) T7 RNA polymerase recognition sequence (5'-TAA TAC GAC TCA CTA TA-3'), and a 6-bp transcription initiation sequence was appended at the 5' end. The primer sequences and the expected sizes of protected fragments are summarized in Table 1. The amplified PCR products were used as templates for in vitro transcription performed using MAXIscript kit (Ambion, Inc., Austin, TX) in the presence of 32P-deoxyuridine triphosphate (~800 Ci/mmol; Pharmacia Amersham Biotech). Radiolabeled riboprobes were gel-purified before use. The multiprobe RNase protection assay was performed using the Hybspeed RPA kit (Ambion) according to the manufacturer's protocol (with minor modifications). In a single reaction mix, radiolabeled riboprobes (5 x 104 cpm) for {alpha}GSU, FSHß, and LHß were incubated for 30 min at 68°C in 10 µl of Hybspeed hybridization buffer containing 1 µg of total RNA from a single pituitary or synthesized sense RNA standard. After hybridization, unprotected probes were digested with RNase A/RNase T1 for 30 min at 37°C. The specifically protected fragments were then precipitated and separated on a 5% (v/v) Long Ranger gel (Biowhittaker, Rockland, ME) containing 8 M urea. The radioactivity of the mRNA bands was determined with a BAS 1000 image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Each GTH subunit mRNA level in the sample was quantified from the standard curve obtained with known amounts of synthesized sense RNA and expressed as nanograms of mRNA per microgram of total RNA. To control the quality of the assay, we used an external cRNA standard [29], apolipophorin III (apoLp-III), which was obtained from the insect Locusta migratoria [30] and provided by Dr. J. Bogerd (Utrecht University). An equal amount of apoLp-III (100 pg) was added to each sample before hybridization and coanalyzed in each RNase protection assay.


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  		TABLE 1. Primers used in the generation of riboprobes for RNase protection assay

In Vitro Steroid Production

Ovarian fragments were collected at various stages of gonadal development (Day 0; immature, Day 8; vitellogenic and Day 15; mature stages). Thirty milligrams of ovarian fragments were placed in a tissue culture dish (24 well; Corning Inc., Corning, NY) with 1 ml of L-15 medium (Sigma) and then incubated in the presence or absence of T (100 ng/ml) for 24 h at 20°C in a humidified incubator with atmosphere of 100% air. Three replicates were made for each treatment. After completion of the incubation, the media were frozen at -20°C until RIA for E2 [21]. The level of detectability was 30 pg/ml for E2.

Isolation and Preparation of Ovarian Follicles at Various Developmental Stages

Isolation and preparation of ovarian follicles were performed as described previously [31]. Briefly, ovaries obtained from one mature female red seabream were cut into small pieces in ice-cold L-15 medium. Oocytes with follicular layers were dispersed by pipetting the fragments. After clusters of oocytes were removed by passage of the suspension through a stainless-steel wire mesh (pore diameter, 625 µm), oocytes at the tertiary yolk globule stage (diameter, ~500 µm) were collected by a wire mesh (pore diameter, 425 µm). Clusters of oocytes consist of early vitellogenic oocytes and previtellogenic small oocytes. In the experiment, these clusters of oocytes and isolated oocytes at the tertiary yolk globule stage were used as early vitellogenic oocytes and late vitellogenic oocytes, respectively. Using the same methods, oocytes at the migratory nucleus stage and at the mature stage were isolated from the different female red seabream. Total RNA extraction from a pool of oocytes at various developmental stages and Northern blot analysis were performed as described above.

Data Analysis

All data are present as mean ± SEM. When heterogeneity of variance was present, the data were logtransformed before statistical analysis, since standard deviations were proportional to means. One-way ANOVA followed by the Duncan multiple range test was used to assess statistical differences. For all statistical tests, values were considered significantly different with P values of less than 0.05.

RESULTS

Isolation and Characterization of cDNA Encoding Red Seabream P450arom Derived from Ovary

As a first step in isolating red seabream ovary-derived P450arom cDNA clone, reverse transcription-PCR was performed using degenerate oligonucleotide primers based on nucleotide sequences that were highly conserved among teleosts. The isolated cDNA clone (~1.1 kilobases [kb]) was 88% and 87% identical at the amino acid level to the corresponding region of medaka and flounder, respectively. Using a probe generated by this fragment, approximately 1 x 106 independent plaques from the cDNA library of the red seabream ovary were screened, and 10 positive clones were obtained and sequenced. The sequence revealed that these clones matched completely within the overlapping region. The longest positive clone was comprised of 1779 bp (GenBank accession no. AB051290) and contained an open reading frame of 1557 bp encoding a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. A putative polyadenylation signal, ATTAAA, is located 78 bp upstream of the poly(A)+ tail, which starts at position 1696. Similar to other P450arom derived from the teleost ovary, the red seabream P450arom had a second potential initiation site 30 bp downstream from the first ATG; both of the two ATG codons resulted in a sequence similar to the consensus sequence proposed by Kozak [32]. Figure 1 shows the deduced amino acid sequence of the red seabream P450arom cDNA aligned with counterparts from other species. Red seabream P450arom shared high homology with flounder (85%) and medaka (83%), and it had 62–80% overall sequence identity with other fishes as well as 51% and 49% identity with those of chicken and rat, respectively. The percentage of identity was higher in the regions of high homology in other species, including the I-helix, the aromatic-specific conserved region, and the heme-binding domain region. In contrast, many differences were clustered at the N-terminus and the central part of P450arom molecule, corresponding to putative exons II, V, and VII and the N-terminus of exon IX [33, 34]. Three consensus N-glycosylation sites (Asn29, Asn57, and Asn95) were identified in the N-terminal region of red seabream P450arom protein. The first glycosylation site is conserved in rat, human, and other fish. Further analysis indicated that red seabream ovary-derived P450arom had consensus sequences for phosphorylation by protein kinase C-dependent sites (PKC: S/T-X-R/K) at Thr59, Thr130, Ser212, Thr385, and Ser505 and by casein kinase II-dependent sites (CKII: S/T-X-X-D/E) at Ser5, Thr97, Thr187, Thr207, and Ser270. A phylogenetic tree depicting the relationship of various P450arom amino acids is shown in Figure 2. As expected from homology comparison, red seabream P450arom fell into the cluster with medaka, tilapia, and flounder.



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  		FIG. 1. Comparison of the deduced amino acid sequence of red seabream P450arom with those of rainbow trout [5], catfish [6], medaka [7], tilapia [8], goldfish [9], chicken [54], rat [14], and human [55]. Roman numerals I, II, and III indicate the I-helix region, aromatic-specific conserved region, and heme-binding region, respectively. Triangles indicate the location of the exon-intron boundary as reported in human and medaka P450arom [33, 34]. Consensus glycosylation sites are underlined, and PKC and CKII are the putative sequences for phosphorylation by protein kinase C and casein kinase II, respectively



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  		FIG. 2. Phylogenetic tree of vertebrate P450arom proteins. The unrooted phylogenetic tree was constructed by neighbor-joining method after alignment of deduced amino acid sequences of P450arom protein. Values at the interior nodes are bootstrap percentages derived from 1000 replications. The scale bar indicates an evolutionary distance of 0.1 amino acid substitution per position in the sequence. The sequences for P450arom were extracted from the GenBank database and the published literature as follows: red seabream (AB051290), rainbow trout [5], catfish (S75715), medaka (D82968), tilapia (U72071), goldfish (AF020704), flounder (AB017182), zebrafish (AF004521), Xenopus (AB031278), chicken (J04047), zebra finch (S75898), rabbit (Z70301), rat (M33986), mouse (D00659), and human (J04127)

Serum E2 Levels, P450arom mRNA, and In Vitro Synthesis of E2 During Ovarian Development

Changes in the GSI of female red seabream during oocyte development induced by implantation of cholesterol pellets containing GnRHa are shown in Figure 3. The GSI increased from Day 8 (1.38% ± 0.1%, P < 0.05) and reached a high value on Day 15 (4.36% ± 0.87%, P < 0.05) after GnRHa implantation, when spawning started. Histological examination revealed that previtellogenic oocytes at the perinucleolus stage were present in the ovaries of fish collected before implantation of GnRHa, when the GSI was 0.88% ± 0.11% (Fig. 4A). Vitellogenic oocytes at the secondary yolk globule stage were observed in the ovaries of fish collected on Day 8 (Fig. 4B). On Day 15, oocytes at various developmental stages, from the perinucleolus to the tertiary yolk globule stage, were observed (Fig. 4C). Figure 5 shows changes in serum E2 levels during oocyte development of female red seabream. Although serum E2 levels before GnRHa implantation were very low (58.86 ± 9.43 pg/ml), E2 levels increased during ovarian development, with the greatest increase occurring between Day 8 (667 ± 216 pg/ml, P < 0.05) and Day 15 (1958 ± 286 pg/ml, P < 0.05).



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  		FIG. 3. Changes in the GSI during oocyte development induced by GnRHa implantation. Each point represents the mean ± SEM of the indicated number of samples. Lack of error bars on some points is due to the errors being too small to show graphically. Significant (P < 0.05) differences between samples are denoted by different letters



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  		FIG. 4. Representative light micrographs of red seabream ovaries during oocyte development induced by GnRHa implantation. A) Initial control. B) Day 8 after GnRHa implantation. C) Day 15 after GnRHa implantation. O, Oocyte at the oil stage; PN, oocyte at the perinucleolus stage; PYG, oocyte at the primary yolk globule stage; SYG, oocyte at the secondary yolk globule stage; TYG, oocyte at the tertiary yolk globule stage. Bars = 100 µm. Photographs were taken at x90 magnification



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  		FIG. 5. Changes in serum E2 levels during oocyte development induced by GnRHa implantation. Each value represents the mean ± SEM of the indicated number of samples. Lack of error bars on some points is due to the errors being too small to show graphically. Means with different letters differ significantly (P < 0.05) in each series

To evaluate P450arom mRNA levels during oocyte development, we performed Northern blot analysis. Poly(A)+ RNA (5 µg) isolated from each individual red seabream ovary was visualized using a radiolabeled P450arom cDNA capable of detecting a single transcript of approximately 2.0 kb (Fig. 6A). The blot was rehybridized with a cDNA probe for ß-actin to control for loading variations. The amount of P450arom mRNA in each sample was then normalized to the amount of ß-actin; this arbitrary ratio was used to express relative P450arom mRNA levels. Quantitation of the blot with the BetaScope 603 Blot Analyzer is presented in Figure 6B. In GnRHa-implanted female red seabream, P450arom mRNA levels were increased at Days 8 and 15 to 2.5- and 18.3-fold the initial levels, respectively. One-way ANOVA followed by the Duncan multiple range test revealed significant differences (P < 0.05) in P450arom mRNA levels between Days 0 and 8, Days 0 and 15, and Days 8 and 15.



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  		FIG. 6. Changes in P450arom mRNA during oocyte development induced by GnRHa implantation. A) Poly(A)+ RNA (5 µg) isolated from ovary of individual red seabream at various stages of sexual maturation was subjected to Northern blot analysis. The blot was hybridized first with the P450arom cDNA probe (upper panel) and then stripped and hybridized with the ß-actin cDNA probe (lower panel). B) The amount of P450arom mRNA was calculated relative to an arbitrary unit of ß-actin. Error bars represent the mean ± SEM of the indicated number of samples. Lack of error bars on some points is due to the errors being too small to show graphically. Means with different letters differ significantly (P < 0.05) in each series

To determine the profile of P450arom enzyme activity during gonadal development, in vitro production of E2 was assayed in ovarian fragments obtained from ovary at various stages of oocyte development. Figure 7 shows the levels of E2 in media with or without T. Nondetectable or low levels of E2 were present in media at Day 0 (<30 pg/ml), Day 8 (180 ± 3 pg/ml), and Day 15 (268 ± 13 pg/ml) when ovarian fragments were incubated without T. In contrast, E2 production by ovarian fragments incubated with T (100 ng/ml) increased significantly from Day 8 (533 ± 88 pg/ml, P < 0.01) to Day 15 (945 ± 45 pg/ml, P < 0.01).



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  		FIG. 7. In vitro production of E2 by ovarian follicles of red seabream at different stages of oocyte development induced by GnRHa implantation. Follicular preparations were incubated in Ringer alone (R) or in 100 ng/ml of T. Each column represents the mean of triplicate determinations, and vertical bars represent the mean ± SEM. Lack of error bars on some columns is due to the errors being too small to show graphically. ND, Not detected

Changes in mRNA Levels of GTH Subunits During Oocyte Development

Expression of GTH subunit genes during oocyte development was assessed by RNase protection assay. Figure 8 shows changes in mRNA levels of {alpha}GSU, FSHß, and LHß. The {alpha}GSU levels significantly (P < 0.05) increased at Days 8 and 15 to 2.5- and 3.4-fold the initial levels, respectively. Similarly, the levels of LHß mRNA elevated to 3.1-fold the initial levels at Day 8 (P < 0.05) and to 4.6-fold at Day 15 (P < 0.05). No significant changes in the levels of FSHß mRNA were observed during oocyte development. In addition, the levels of FSHß mRNA were approximately two orders of magnitude lower than those of {alpha}GSU and LHß mRNA throughout oocyte development.



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  		FIG. 8. Changes in mRNA levels of {alpha}GSU, FSHß, and LHß during oocyte development induced by GnRHa implantation. One microgram of total pituitary RNA from a single pituitary was assayed for GTH subunit mRNA levels using a multiprobe RNase protection assay. The amount of radioactivity in each sample was determined by comparison with the amount of reference RNA calculated by regression analysis. Values represent the mean ± SEM of the indicated number of samples. Lack of error bars on some points is due to the errors being too small to show graphically. Data were subjected to ANOVA followed by a Duncan multiple range test. Significant differences (P < 0.05) in each series are denoted by different letters

Expression of P450arom mRNA at Different Stages of Follicular Development in Red Seabream Ovary

Because red seabream is a multiple spawner, they have ovaries that contain large numbers of ovarian follicles at different developmental stages throughout the spawning period. We examined the P450arom mRNA levels at various stages of oocyte development by Northern blot analysis. The results demonstrated that the levels of P450arom mRNA in isolated ovarian follicles increased during vitellogenesis and became undetectable in follicles at the migratory nucleus stage and the mature stage (Fig. 9).



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  		FIG. 9. Expression of P450arom mRNA at different stages of follicular development in red seabream ovary. Ovarian follicles at four stages of development were isolated: immature and early vitellogenic stage, late vitellogenic stage, migratory nucleus stage, and mature stage. Poly(A)+ RNA (5 µg/lane) samples isolated from ovarian follicles at various developmental stages were hybridized to the 32P-labeled P450arom cDNA probe (upper panel). The blot was then stripped and rehybridized with the ß-actin cDNA probe to confirm equal loading of RNA (lower panel)

DISCUSSION

We have isolated and characterized a red seabream P450arom cDNA derived from ovary. The predicted amino acid sequences of the cloned red seabream ovary-derived P450arom display a high degree of overall homology with those reported for other teleosts and tetrapods. In particular, the functional amino acids of P450arom enzyme are highly conserved. These include the aromatic-specific conserved region, which is predicted to contribute to the active aromatase site, and the I-helix, which is believed to form the substrate-binding pocket. Moreover, the heme-binding domain region containing the cysteine residue that serves as the fifth coordinating ligand for the heme group iron atom is well conserved. Based on mutational analysis and molecular modeling, amino acids known to be essential for catalytic functions in human P450arom (i.e., Ile133 [35], Glu302 and Pro308 [36], Asp309 and Thr310 [37], and Arg435 and Cys437 [38]) were identical in red seabream P450arom. Also, His128 of human P450arom, which may be involved with orientation of substrate in the active site, was present in an analogous position in the red seabream P450arom [39]. Therefore, these amino acids in red seabream P450arom may be involved in catalytic functions. Similar to previous reports regarding other teleosts, the red seabream P450arom has an extra 18 amino acid extension at the N-terminus upstream of the sequence, which is common to P450arom of higher vertebrates. According to Kyte-Doolittle hydropathic analysis, this extended N-terminus region is enriched in hydrophobic residues (data not shown). In most microsomal cytochrome P450 isoforms, a region of hydrophobic amino acids characterizes the N-terminus, which is believed to comprise a membrane-spanning domain. Interestingly, the N-terminus region (first 20 amino acids) has no obvious hydrophobic stretches in the case of tetrapod P450arom. Rather, the region that can be described as hydrophobic lies between amino acids 20 and 40, and it seems to serve as the membrane-spanning region [40]. Therefore, the ancestral P450arom gene likely had an N-terminus hydrophobic region comprising a membrane-spanning domain that, subsequently, might have been lost with the substitution of the second membrane-spanning region lying between amino acids 20 and 40 during the course of vertebrate evolution. It remains unclear whether the extension at the N-terminus is functionally important for teleost ovary-derived P450arom at this time. Efforts to analyze the function of the N-terminal extension of teleosts may contribute to understanding of the molecular evolution of P450arom. In addition, we noted three potential N-glycosylation sites in the N-terminus of red seabream P450arom. The first of these is present in an analogous position (Asn12) to a glycosylation position in human P450arom [41], suggesting a possible functional site. A previous study indicated that aromatase enzyme activity may be regulated by a protein kinase A-dependent phosphorylation mechanism [42]. Although the red seabream P450arom lacks the protein kinase A-dependent site, it has several protein kinase C- and casein kinase II-dependent sites. The biological ramifications of these differences in phosphorylation sites remain to be determined. Northern blot analysis revealed that one transcript of approximately 2.0 kb is present in red seabream ovary, similar to the situation previously found in chicken, rainbow trout, catfish, and medaka. Despite the similarities in coding regions, the numbers and sizes of P450arom transcripts in mammals differed markedly. For example, two sizes of transcripts (2.9 and 3.4 kb) are found in human P450arom [43], and three sizes of transcripts (1.7, 2.2, and 2.7 kb) are found in rat P450arom [14, 44]. These differences in numbers and sizes are due to an alternative RNA processing method using different polyadenylation signals.

This study also described in detail changes in P450arom mRNA levels in relation to serum E2 levels and enzyme activity during oocyte development. Northern blot analysis showed that P450arom mRNA levels in ovaries increased in association with the increase in GSI and serum E2 levels during gonadal development induced by GnRHa implantation. This is consistent with a recent physiological study showing that E2 biosynthesis and P450arom mRNA levels rise in parallel during sexual maturation in catfish [45]. Furthermore, the increase of aromatase activity of ovarian fragments assessed by in vitro conversion of T to E2 was associated with the ascending pattern of P450arom mRNA levels. Therefore, the increase in E2 production during oocyte development may be due primarily to the increased levels of P450arom transcripts. However, the levels of P450arom mRNA were increased even more sharply between Day 8 and Day 15 (7.4-fold increase), whereas in vitro E2 production showed only a 2-fold increase or less, suggesting that the correlation between mRNA expression and enzyme activity does not seem to be linear during this period. The abundance of enzyme may not always be directly correlated with its activity, because enzyme activity can sometimes be enhanced without any changes in abundance.

In the present study, Northern blot analysis showed that the P450arom mRNA levels increased in isolated vitellogenic follicles during oocyte growth, whereas these levels decreased drastically in the migratory nucleus and the mature stages. These data agree with the observed reduction in aromatase activity of granulosa cells after the completion of vitellogenesis in amago salmon [46]. Moreover, P450arom mRNA in rainbow trout [5] and tilapia [8] was present in ovarian follicles during vitellogenesis but not during the subsequent stages of oocyte development, such as the postvitellogenic stage and the oocyte maturation stage. Therefore, the present results, together with those of the previous studies, suggest that the expression of P450arom is restricted to the period of vitellogenesis in red seabream ovary. However, we cannot rule out the possibility that red seabream P450arom is expressed in previtellogenic oocytes, because we detected a small amount of conversion of T to E2 by ovarian fragments containing previtellogenic oocytes. Further studies are required to determine both when and what factors affect P450arom gene expression in red seabream.

It is well known that P450arom mRNA is induced by FSH in rat granulosa cells both in vivo [12] and in vitro [13], whereas the LH surge exerts a dramatic negative effect on steady-state levels of P450arom mRNA [14, 15]. Based on recent studies, induction of P450arom gene expression in granulosa cells by FSH is mediated by a cAMP-signaling cascade and phosphorylation of key substances, including transcription factors such as CREB [47] and SF-1 [48, 49]. In teleosts, GTH has been reported to induce P450arom activity (conversion of T to E2) in ovarian follicles of goldfish [50] and medaka [51], but not in amago salmon [46]. However, it remains unclear whether FSH or LH can stimulate the expression and activation of P450arom in teleost fish, because most in vitro studies have been performed with either partially purified chinook salmon GTH (SG-G100) [46] or heterogeneous GTH, such as hCG [50] and eCG [51]. In this study, to elucidate the roles of FSH and LH in promoting P450arom gene expression, we observed changes in the mRNA levels of GTH subunits during oocyte development. Interestingly, elevations in the abundance of {alpha}GSU and LHß transcripts were observed during oocyte development, whereas no apparent change was observed in the abundance of FSHß transcripts during the entire sampling period. This is consistent with our recent observations that FSHß mRNA of female red seabream is maintained at low levels during sexual maturation, whereas LHß mRNA levels, which are correlated with serum LH levels [52], are high from the beginning of early vitellogenesis to the spawning season in female red seabream [17]. Furthermore, from our in vitro studies using purified red seabream GTHs [53], the biological activity of FSH is much lower than that of LH for inducing in vitro E2 production of vitellogenic follicles [20]. Taken together, these results support the hypothesis that, unlike salmonids and mammals, FSH may have no significant role in the regulation of P450arom gene expression in female red seabream. Further studies are needed to address the question of how LH controls the expression of the P450arom gene in red seabream follicles in vivo and in vitro.

ACKNOWLEDGMENTS

The authors thank Dr. J. Bogerd and Mr. M. Bruysters (Utrecht University) for significant advice in setting up the multiprobe RNase protection assay, Dr. Y. Kazeto (University of Maryland Biotechnology Institute) for thoughtful discussions, and Dr. T. Kitagawa (Mie University) for advice concerning phylogenetic analysis. We also thank Drs. G. Young (University of Otago) and B. Senthilkumaran for critical reading of this manuscript.

FOOTNOTES

First decision: 2 February 2001.

1 Supported in part by a grant-in-aid from the Ministry of Agriculture, Forestry and Fisheries (BDP-01-IV-2-2). Back

2 Correspondence. FAX: 81 596 58 6413; kgen{at}affrc.go.jp Back

Accepted: May 25, 2001.

Received: December 14, 2000.

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