Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008219
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/5/1562    most recent
biolreprod.102.008219v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.
Agricola
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.
BIOLOGY OF REPRODUCTION 68, 1562–1568 (2003)
DOI: 10.1095/biolreprod.102.008219
© 2003 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Effects of Luteinizing Hormone and Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I on Aromatase Activity and P450 Aromatase Gene Expression in the Ovarian Follicles of Red Seabream, Pagrus major1

Hirohiko Kagawa2,3, Koichiro Gen4, Koichi Okuzawa4, and Hideki Tanaka3

National Research Institute of Aquaculture,3 Fisheries Research Agency, Nansei, Mie 516-0193, Japan Tamaki Inland Station,4 National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To clarify the mechanism of estradiol-17ß production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17ß) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17ß production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.

estradiol, follicle-stimulating hormone, growth factors, luteinizing hormone, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that, in teleosts, as in other oviparous vertebrates, estrogens play important roles in reproduction, such as induction of the precursor of yolk protein (vitellogenin) in the liver [1] and the positive- and negative-feedback control of gonadotropin synthesis and secretion in the pituitary [2, 3]. Estradiol-17ß, the main estrogen of teleosts, is produced in the ovarian follicle in response to gonadotropins (GTHs). In salmonids, during vitellogenesis, GTH stimulates testosterone production in the thecal cells, which is in turn aromatized to estradiol-17ß in the granulosa cells (two-cell-type model) [4]. Cytochrome P450 aromatase (P450arom) is the key enzyme for conversion of testosterone to estradiol-17ß in the granulosa cells. P450arom cDNAs have been isolated and characterized in several teleost species [5–10], including red seabream [11]. P450arom mRNA levels are increased in association with increases of enzyme activity during vitellogenesis in medaka [7]. Our recent studies demonstrated that P450arom mRNA levels increase in vitellogenic follicles and decrease during the final oocyte maturation of red seabream [11]. However, the mechanisms regulating P450arom gene expression and estradiol-17ß production in the ovarian follicles have not been elucidated precisely.

In teleosts, as in mammals, biochemical analyses [12–14] and molecular cloning studies [15–18] have demonstrated that there are two distinct GTHs: namely, GTH-I, which is homologous to FSH; and GTH-II, which is homologous to LH [19]. In salmonids, FSH is elevated during vitellogenesis, whereas LH appears during final oocyte maturation and ovulation in females [20, 21]. Moreover, in salmonids, P450arom enzyme activity [22] and its mRNA levels [5] also increase during the vitellogenic oocyte growth. These results suggest that FSH regulates estradiol-17ß production through stimulating P450arom gene expression and enzyme activity during the vitellogenic period. In vitro experiments have shown that GTH induces P450arom activity (conversion of T to E2) in ovarian follicles of goldfish [23] and medaka [24] but not in amago salmon [25]. 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) [25] or heterologous GTH, such as human chorionic gonadotropin [23] and pregnant mare serum gonadotropin [24].

Many recent studies in mammals [26, 27] have reported the involvement of growth factors, such as insulin-like growth factor-I (IGF-I), and transforming growth factor-ß (TGFß) in follicular development and oocyte maturation. In teleosts, IGF-I participates in the physiological regulation of the teleost ovary. Our studies have demonstrated that IGF-I is produced in the ovarian follicle of red seabream [28, 29] and induces final oocyte maturation and maturational competence (responsiveness to maturation-inducing steroid) [30]. Maestro et al. [31] showed that recombinant IGF-I stimulated the production of estradiol-17ß in the granulosa cell layers of coho salmon. However, there has been no detailed experiment on the effects of growth factors on aromatase activity in teleost ovarian follicles.

Red seabream provides an interesting and unique model for investigating hormonal regulation of P450arom in fish reproduction. In contrast with salmonids, red seabream have an asynchronous-type ovary and spawn almost every day during the spawning season. Our recent studies have demonstrated that LHß mRNA is maintained at high levels from the beginning of vitellogenesis to spawning period, whereas FSHß mRNA remained at low levels throughout sexual maturation in female red seabream [18]. Moreover, an increase of LHß, but not FSHß, correlated with the increase in P450arom mRNA levels during the course of ovarian development induced by GnRH analogue (GnRHa) implantation [11]. From these results, it is hypothesized that LH, but not FSH, regulates estrogen biosynthesis through an increase in P450arom gene expression. However, the effects of LH and FSH on P450arom gene expression in teleosts have not been elucidated.

Therefore, the aim of the present study was to elucidate the role of FSH, LH, and IGF-I on aromatase activity and P450arom gene expression in the ovarian follicles of red seabream by means of in vitro incubation of ovarian follicles with these factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Sexually mature 2- or 3-yr-old red seabream, Pagrus major, between 1.5 and 2 kg body weight, which were kept in net pens in Gokasho Bay, Nansei, Mie, Japan, were transferred and kept in a flow-through outdoor tank (2000 L) at the National Research Institute of Aquaculture under natural conditions. In the presence of males, most females spawn daily in the evening (1700–1900 h) during the spawning season (April and May). Fish were deeply anesthetized with 2-phenoxyethanol and killed by decapitation from 1400 to 1700 h. Ovaries were removed and placed in ice-cold Leibovitz culture medium (Flow Laboratories Ltd., Ivine, Scotland), pH 7.4, supplemented with 2.5 g Hepes, 0.1 g streptomycin, 100 000 IU penicillin, and 1 g bovine serum albumin per liter.

Tissue Preparation and Incubation

After ovaries were dissected into small pieces in ice-cold culture medium, oocytes with follicle layers were dispersed by pipetting the ovarian pieces. After full-grown oocytes, which are responsive to maturation-inducing steroid (maturationally competent oocytes), and oocytes at the mature stage were removed by sieving through stainless steel wire mesh (i.d. = 625 µm), the remaining ovarian fragments containing vitellogenic oocytes (approximately 250–450 µm in diameter) and nonvitellogenic oocytes at the perinucleolous stage were collected. Thirty milligrams of ovarian fragments were incubated in 24-well culture plates containing 1 ml of medium. Controls consisted of oocyte incubated in hormone-free medium. Three replicates were made for each treatment and dose. After incubation for 24 h at 20°C in a humidified incubator with atmosphere of 100 % air, incubation media were collected and were frozen at -20°C until RIA for estradiol-17ß [32]. Total RNA extraction from a pool of ovarian fragments and Northern blot analysis were performed as described below.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated using the acid guanidium thiocyanate-phenol-chloroform extraction procedure [33]. Enrichment of poly(A)+ RNA was performed using oligo (dT)18 cellulose column (Amersham Pharmacia Biotech, Uppsala, Sweden). Northern blot analysis was performed as described previously [11]. Briefly, 5 µg of poly(A)+ from ovarian follicles were subjected to electrophoresis in a 1% (wt/vol) agarose/formaldehyde gel, transferred overnight to a Hybond N+ nylon membrane (Amersham Pharmacia Biotech), and fixed to the membrane by baking at 80°C for 2 h. The membrane was then prehybridized for 4 h at 42°C in 5x SSPE (1x SSPE = 150 mM NaCl, 10 mM sodium phosphate buffer, and 1 mM EDTA, pH 7.4) with formamide (50%), BSA (0.1%), Ficoll (0.1%), polyvinylpyrrolidone (0.1%), sodium dodecyl sulfate (SDS; 0.5%), and denatured calf thymus DNA (50 µg/ml). Hybridization with a 32P-labeled 1.5-kilobase (kb) complementary DNA (cDNA) fragment (nucleotides 27–1583) of red seabream P450arom [11] was carried out overnight at 42°C. The probes were labeled with [{alpha}-32P] deoxy-CTP using a Random Primer DNA Labeling Kit (Takara Bio Inc., Shiga, Japan) and diluted to 1 x 106 cpm/ml. The membrane filter was washed at 65°C with several buffer changes of decreasing SSPE (pH 7.4) concentrations from 2x to 0.1x and exposed to x-ray film with an intensifying screen at -70°C. For normalization of data, blot was stripped by boiling in 0.1% SDS before reprobing with a 32P-labeled 0.4-kb cDNA fragment of red seabream ß-actin (kindly provided by Dr. G. Yoshizaki, Tokyo University of Fisheries, Tokyo, Japan). The band intensities were subsequently measured using a BetaScope Model 603 Blot Analyzer (Aloka, Tokyo, Japan). All values for mRNA levels were arbitrary radioactive units after standardizing with the levels of ß-actin.

Hormones and Chemicals

The following human recombinant growth factors (all from Cosmo Bio Co., Ltd., Japan) were used in the present study: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-{alpha} (TGF{alpha}), TGFß1, TGFß2, leukemia inhibitory factor (LIF), stem cell factor (SCF), tumor necrosis factor-{alpha}, and insulin-like growth factor-I (IGF-I). Red seabream FSH and LH were purified from the pituitaries of mature red seabream in the spawning season [14]. Red seabream FSH and LH simulates in vitro production of 11-ketotestosterone, a major androgen in teleost in sliced testis of red seabream in a similar potency [34]. However, LH stimulates in vitro estradiol-17ß production of ovarian fragments, but FSH has much less potency [35] and LH, but not FSH, induces final maturation and the development of maturational competence of oocytes of red seabream in vitro [34].

Analysis

Data obtained from two or three replicated experiments showed a similar tendency and therefore one of the data of each experiment was used in the present study. All data are expressed as mean ± SEM of three replicate incubations. Group means were compared by ANOVA with Duncan multiple-range test. Comparison between two means was made with Student t-test. For all statistical tests, values were considered significantly different with P values of less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Various Growth Factors and Red Seabream FSH and LH on Aromatase Activity

Aromatase activity, which was estimated by in vitro conversion of testosterone (100 ng/ml) to estradiol-17ß in the ovarian fragments of red seabream, was stimulated by LH at a concentration of 100 ng/ml (P < 0.01) but not by FSH (100 ng/ml) (Fig. 1). Of the growth factors, only IGF-I at a concentration of 100 ng/ml stimulated aromatase activity. The other growth factors, used at a concentration of 100 ng/ml, did not enhance aromatase activity. Aromatase activity was decreased significantly (P < 0.05) by some growth factors (EGF, bFGF, TGF{alpha}, TGFß, LIF, and SCF).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Effects of various growth factors and red seabream gonadotropins on estradiol-17ß production by ovarian fragments of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing 100 ng/ml testosterone for 24 h at 20°C. Concentration of all growth factors was 100 ng/ml. Concentrations of LH and FSH were 100 ng/ml. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series

Effects of Different Doses of IGF-I on Aromatase Activity and Its Time-Course Effects

IGF-I alone at doses of 2, 10, and 50 nM did not affect estradiol-17ß production by ovarian fragments of red seabream (Fig. 2). Addition of testosterone by itself for estimation of aromatase activity increased esradiol-17ß production by ovarian fragments. Moreover, IGF-I stimulated estradiol-17ß production in a dose-dependent manner when testosterone (100 ng/ml) was added into the incubation medium (Fig. 2). IGF-I-stimulated aromatase activities were significantly increased at 12 h (P < 0.01) and attained maximum level at 18 h after incubation (P < 0.01) and maintained high levels thereafter (Fig. 3). IGF-I-stimulated aromatase activities were significantly higher than those of the testosterone-alone group from 12 to 24 h.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Effects of IGF-I alone (IGF-I) or in combination with testosterone (IGF-I+T) on estradiol-17ß production by ovarian follicles of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing various doses of IGF-I alone or in combination with testosterone (100 ng/ml) for 24 h at 20°C. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 3. Time-course experiment of effects of IGF-I on estradiol-17ß production in ovarian follicle of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing testosterone (100 ng/ml) alone (T, closed square) or in combination with IGF-I (10 nM) (T+IGF, closed triangle) for 48 h at 20°C. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series. *, Significant difference between two experiment groups (with and without IGF-I) by Student t-test

Effects of Different Doses of Red Seabream FSH and LH on Aromatase Activities and Its Time-Course Effects

FSH did not stimulate aromatase activity (conversion of testosterone to estradiol-17ß) at any concentrations (11, 33, 100, 300 ng/ml) used in the present experiment (Fig. 4). LH stimulated aromatase activity at concentrations of 100 and 300 ng/ml (P < 0.05). No significant increase of aromatase activity was observed at concentrations of 11 and 33 ng/ml. LH-stimulated aromatase activities were significantly increased and attained maximum levels at 12 h (P < 0.05) and maintained high levels thereafter (Fig. 5). LH-stimulated aromatase activities were significantly higher than those of the group from 12 to 24 h. Aromatase activities slightly increased in FSH and testosterone-alone incubation groups, but no significant difference was observed between these two groups.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4. Effects of various doses of red seabream FSH and LH on aromatase activities in ovarian follicles of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing various concentrations of FSH or LH for 24 h at 20°C. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5. Time course experiment of effects of FSH and LH on estradiol-17ß production in ovarian follicle of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing testosterone (T, 100 ng/ml, open circle) with FSH (100 ng/ml, closed square) or LH (100 ng/ml, closed triangle) for 48 h at 20°C. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series. *, Significant difference between LH- (or FSH) added group and testosterone-alone group by Student t-test

Effects of Red Seabream FSH and LH Alone and in Combination with IGF-I on Estradiol-17ß Production

FSH did not stimulate estradiol-17ß production in the ovarian fragments at concentrations of 33 and 100 ng/ml (Fig. 6). LH stimulated estradiol-17ß production by ovarian fragments at concentrations of 33 (P < 0.05) and 100 ng/ml (P < 0.01). Addition of IGF-I (10 nM) did not alter the effects of either FSH and LH on estradiol-17ß production by ovarian fragments when testosterone was absent from the incubation medium, although IGF-I slightly but significantly decreased LH-stimulated estradiol-17ß production at a concentration of 100 ng/ml of LH.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 6. Effects of red seabream FSH, LH, and IGF-I on estradiol-17ß production in ovarian follicles of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing red seabream FSH or LH alone (33, 100 ng/ml) or in combination with IGF-I (10 nM) for 24 h at 20°C. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series

Effects of Red Seabream FSH and LH and in Combination with IGF-I on Aromatase Activities

LH but not FSH stimulated aromatase activity (conversion of testosterone to estradiol-17ß) at concentrations of 33 and 100 ng/ml (P < 0.05). Aromatase activity was significantly increased (P < 0.05) when ovarian fragments were incubated with LH at a concentration of 100 ng/ml in combination with IGF-I (10 nM) (Fig. 7). However, FSH at concentrations of 33 and 100 ng/ml and the low concentration of LH (33 ng/ml) did not enhance aromatase activity in combination with IGF-I.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7. Effects of red seabream FSH, LH, and IGF-I on aromatase activity in ovarian follicles of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing red seabream FSH or LH alone (33, 100 ng/ml) or in combination with IGF-I (10 nM) for 24 h at 20°C. Testosterone (100 ng/ml) was added to all incubations. Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series

Effects of Red Seabream FSH, LH, and IGF-I on P450arom mRNA Expression in Ovarian Fragments

LH alone (100 ng/ml) but not FSH alone stimulated P450arom mRNA expression in ovarian fragments of red seabream in vitro (P < 0.05). IGF-I alone also stimulated P450arom mRNA expression (P < 0.01), roughly tripling its expression relative to no IGF-I (Fig. 8). P450arom mRNA expression induced by IGF-I was not affected by FSH, but addition of LH-enhanced IGF-I-stimulated P450arom mRNA expression in the ovarian fragments. Addition of LH roughly doubled mRNA expression relative to IGF-I alone, and the combined actions of LH+IGF-I led to a 4-fold increase over the no-treatment control.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 8. Effects of red seabream FSH, LH, and IGF-I on expression of P450arom mRNA in ovarian follicles of red seabream in vitro. Ovarian fragments were incubated in 1 ml incubation medium containing red seabream FSH or LH alone or in combination with IGF-I (10 nM) for 24 h at 20°C. Poly(A)+RNA isolated from ovarian fragments 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). Data were subjected to ANOVA followed by Duncan multiple-range test. Means with different letters differ significantly in each series


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IGF-I was the only growth factor that stimulated the conversion of testosterone to estradiol-17ß (aromatase activity) in the ovarian fragments of red seabream. Other growth factors either did not stimulate or significantly decreased aromatase activity. In mammals, IGF-I [26] potentiates FSH-induced aromatase activity but EGF and TGFß [27] inhibit aromatase activity. FGF [36] exerts a dual inhibitory action on ovarian estrogen production by reducing androgen substrate and by attenuating granulosa cell aromatase activity. In teleosts, Maestro et al. [31] showed that IGF-I stimulated the production of estradiol-17ß by the granulosa cell layers of coho salmon. Behl and Pandey [37] reported that IGF-I stimulated aromatase activity during the 48-h culture of small ovarian follicles of carp. Data from the present study are consistent with these previous data obtained in both mammals and teleost fish. The involvement of IGF-I in the stimulation of aromatase activity in the ovary may have evolved early in vertebrate evolution.

The present study shows for the first time in teleosts that IGF-I stimulated gene expression of P450arom in the ovarian fragments. This is also consistent with the previous results obtained in mammals [38]. Immunocytochemical studies showed that IGF-I immunoreactivity is localized in the granulosa cells of the red seabream and increased in vitellogenic follicles [28]. Moreover, IGF-I receptors are present in both the granulosa and theca-interstitial cell layers of the salmon ovarian follicle [31] and carp ovary [39]. These data indicate that IGF-I produced in the granulosa cells regulates aromatase activity in the granulosa cells through autocrine mechanisms. IGF-I has been shown to induce oocyte maturation and maturational competence (acquisition of responsiveness to maturation-inducing steroids) in the red seabream [30]. The present study in combination with the previous studies indicates that IGF-I may regulate several processes involved in ovarian development in teleost fish.

The present study also shows that IGF-I did not affect LH-induced estradiol-17ß production in the ovarian fragments when testosterone was absent from the incubation medium. These results are in contrast with the stimulatory effects of IGF-I on aromatase activity in the ovarian follicles of red seabream. Maestro et al. [31] showed that IGF-I inhibited the basal and/or LH-stimulated production of testosterone and 17{alpha}-hydoroxyprogesterone, the major steroids produced by isolated theca-interstitial and granulosa layers during the preovulated period. Therefore, in case of the red seabream, IGF-I may inhibit LH-induced precursor production in the theca cell layer, resulting in no significant increase or significant decrease in estradiol-17ß production in the absence of precursor (testosterone) despite IGF-I-stimulated aromatase activity in ovarian fragments. As suggested for the mammalian ovary [26], the opposite effects of IGF-I on the two follicular layers are a reflection of the possible predominant paracrine/autocrine role of IGF-I in the red seabream ovary. Srivastava and Van Der Kraak [40] reported that insulin, but not IGF-I, enhanced the stimulatory effects of human chorionic gonadotropin on steroid production in goldfish vitellogenic follicles. As suggested previously, if the differential regulation of steroidogenesis by IGF-I in theca cell layer and granulosa cell layer are also present in the goldfish ovary, it is conceivable that the net result of the inhibitory and stimulatory steroidogenic effects of IGF-I in theca and granulosa cell layers may be the absence of steroid output from the intact ovarian follicle.

The present study shows that LH, but not FSH, stimulated aromatase activity in the ovarian fragments of red seabream, although it cannot be ruled out that LH itself produces androgen, which in turn metabolizes into estradiol-17ß. Moreover, LH stimulated the expression of P450arom mRNA and IGF-I enhanced LH-induced P450arom mRNA expression. Previous studies have shown that GTH stimulated aromatase activity in ovarian follicles of goldfish [23] and medaka [24]. However, these studies did not use purified LH and FSH of teleosts in their in vitro experiments. They used human chorionic gonadotropin [23] or pregnant mare serum gonadotropin [24]. The present study is consistent with our previous data showing that the biological activity of FSH is much lower than that of LH for inducing in vitro estradiol-17ß production of vitellogenic ovarian fragments [35]. Elevations in the abundance of {alpha}-glycoprotein subunit and LHß transcripts were observed during vitellogenesis induced by GnRH cholesterol pellet implantation, whereas no apparent change was observed in the abundance of FSHß transcripts during the entire sampling period [11]. 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, are high from the beginning of early vitellogenesis to the spawning season in female red seabream [18]. P450arom gene expression in the ovaries increased in association with the increase of LHß and serum estradiol-17ß levels during gonadal development induced by GnRHa implantation [11]. These in vitro results and in vivo data on LH and FSH gene expression strongly suggest that LH, but not FSH, stimulates estradiol-17ß production through regulating aromatase gene expression in the ovarian follicles of red seabream. This is in sharp contrast with the data obtained from salmonid fish. In salmonids, although LH and FSH have almost the same ability to increase estradiol-17ß production by vitellogenic ovarian follicles in vitro [12], FSH is thought to be implicated in estradiol-17ß production during the vitellogenic stage because FSH predominates in plasma during the initial phase of vitellogenesis whereas LH increases during the final stage of oocyte development [20, 21]. In red seabream, LH, but not FSH, induces maturational competence and final oocyte maturation (induction of germinal vesicle breakdown) [34]. Therefore, LH may regulate the entire process of oocyte development in red seabream. It is well known that FSH induces P450arom mRNA expression in rat granulosa cells [41, 42], whereas an LH surge exerts a negative effect on steady-state levels of P450arom mRNA [43]. In salmonids, Maestro et al. [31] reported that LH inhibits estradiol-17ß production in the prematurational ovarian granulosa cell layer of coho salmon and LH, on the other hand, stimulated 17,20ß-dihydroxy-4-pregnen-3-one production. These authors therefore proposed that LH regulates the steroidogenic shift from estradiol-17ß synthesis to DHP synthesis. The shifting secretion of GTHs (from FSH to LH) during final oocyte maturation and ovulation is present in both salmonids and mammals. However, our studies suggest that FSH may not have physiological functions in oogenesis in red seabream. Thus, further studies are needed to address the question of how LH regulates both estradiol-17ß and maturation-inducing steroid production in the ovarian follicle of red seabream. In mammals, FSH stimulates levels of IGF-I mRNA [44] in porcine granulosa cells in vitro, although FSH does not have a major role in regulation of IGF-I mRNA expression in granulosa cells of murine [45]. Moreover, it has been reported that FSH up-regulates expression of the type I IGF-I receptor gene in rat [46]. Therefore, in red seabream, FSH may have roles in these phenomena reported in mammals. Further studies are necessary to clarify the role of FSH in oogenesis.

The present study shows that IGF-I stimulated the conversion of testosterone to estradiol-17ß (aromatase activity) and P450arom gene expression in the ovarian fragments of red seabream. The present study also shows that LH, but not FSH, stimulated both aromatase activity and the expression of P450arom mRNA. IGF-I amplified the LH-induced aromatase activity in the ovarian fragments. These results and our previous data indicate that LH, but not FSH, stimulates aromatase activity through its effects on P450arom gene expression in the ovarian follicle of red seabream during the vitellogenic stage. IGF-I, which is produced in the granulosa cells, enhance the LH-induced P450arom gene expression and aromatase activity. In IGF-I knockout mice [45], FSH receptor mRNA was significantly reduced in ovaries, suggesting that IGF-I serves to enhance granulosa cell FSH responsiveness by augmenting FSH receptor expression. Therefore, in teleosts, we hypothesize that, during estradiol-17ß production, IGF-I increases the responsiveness to LH in estradiol-17ß production through an increase in LH receptor gene expression. Further studies are necessary to clarify the effects of IGF-I on LH or FSH receptor gene expression. These studies are now in progress in our laboratory.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Graham Young (University of Otago) for his critical reading of the manuscript and valuable comments.


   FOOTNOTES
 
1 Supported in part by a grant-in-aid from the Ministry of Agriculture, Forestry, and Fisheries, Japan (Bio-Design Program). Back

2 Correspondence. FAX: 81 599 66 1962; hkagawa{at}fra.affrc.go.jp Back

Received: 5 June 2002.

First decision: 2 July 2002.

Accepted: 14 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wallace RA. Vitellogenesis and oocyte growth in nonmammalian vertebrate. In: Browder LW (ed.), Developmental Biology. New York: Plenum Press; 1985: 127–177
  2. Trudeau VL, Peter RE. Functional interactions of neuroendocrine systems regulating GTH-II release. In: Proceedings of the Fifth International Symposium of the Reproductive Physiology of Fish; 1995: Austin, TX: 44–48
  3. Linard B, Anglade I, Bennani S, Salbert G, Navas JM, Bailhache T, Pakdel F, Jego P, Valotarie Y, Salgaut C, Kah O. Some insights into sex steroid feedback mechanisms in the trout. In: Proceedings of the Fifth International Symposium of the Reproductive Physiology of Fish; 1995: Austin, TX: 49–51
  4. Kagawa H, Young G, Adachi S, Nagahama Y. Estradiol-17ß production in amago salmon (Oncorhynchus rhodurus) ovarian follicles: role of the thecal and granulosa cells. Gen Comp Endocrinol 1982 47:440-448[CrossRef][Medline]
  5. Tanaka M, Telecky TM, Fukada S, Adachi S, Chen S, Nagahama Y. Cloning and sequence analysis of the cDNA encoding P-450 aromatase (P-450arom) form a rainbow trout (Oncorhynchus mykiss) ovary: relationship between the amount of P450arom mRNA and the production of oestradiol-17ß in the ovary. J Mol Endocrinol 1992 8:53-61[Abstract/Free Full Text]
  6. Trant JM. Isolation and characterization of the cDNA encoding the channel catfish (Ictallurus punctatus) form of cytochrome P450arom. Gen Comp Endocrinol 1994 95:155-168[CrossRef][Medline]
  7. Fukada S, Tanaka M, Matsuyama M, Kobayashi D, Nagahama Y. Isolation, characterization, and expression of cDNA encoding the medaka (Oryzias latipes) ovarian follicle cytochrome P-450 aromatase. Mol Reprod Dev 1996 45:285-290[CrossRef][Medline]
  8. Chan XT, Kobayashi T, Kajiura H, Nakamura M, Nagahama Y. Isolation and characterization of the cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis. J Mol Endocrinol 1997 18:57-66[Abstract/Free Full Text]
  9. Tchoudakova A, Callard GV. Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary. Endocrinology 1998 139:2179-2189[Abstract/Free Full Text]
  10. Kitano T, Takamune K, Kobayashi T, Nagahama Y, Abe S-I. Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese founder (Paralichthys olivaceus). J Mol Endocrinol 2000 23:167-176
  11. Gen K, Okuzawa K, Kumakura N, Yamaguchi Y, Kagawa H. Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major). Biol Reprod 2001 65:1186-1194[Abstract/Free Full Text]
  12. Suzuki K, Kawauchi H, Nagahama Y. Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands. Gen Comp Endocrinol 1988 71:292-301[CrossRef][Medline]
  13. Swanson P, Suzuki K, Kawauchi H, Dickhoff WW. Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II. Biol Reprod 1991 44:29-38[Abstract]
  14. Tanaka H, Kagawa H, Okuzawa K, Hirose K. Purification of gonadotropins (PmGTH I and II) from red seabream (Pagrus major) and development of a homologous radioimmunoassay for PmGTH II. Fish Physiol Biochem 1993 10:409-418[CrossRef]
  15. Sekine S, Saito A, Itoh H, Kawauchi H, Itoh S. Molecular cloning and sequence analysis of chum salmon gonadotropin cDNAs. Proc Natl Acad Sci U S A 1989 86:8645-8649[Abstract/Free Full Text]
  16. Gen K, Maruyama O, Kato T, Tomizawa K, Wakabayashi K, Kato Y. Molecular cloning of cDNAs encoding two types of gonadotropin a subunit from the masu salmon, Oncorhynchus masou: construction of specific oligonucleotides for the a 1 and a 2 subunits. J Mol Endocrinol 1993 11:265-273[Abstract/Free Full Text]
  17. Kato Y, Gen K, Maruyama O, Tomizawa K, Kato T. Molecular cloning of cDNAs encoding two gonadotrophin ß subunits (GTH-Iß and -IIß) from the masu salmon, Oncorhynchus masou: rapid divergence of the GTH-Iß. J Mol Endocrinol 1993 11:275-282[Abstract/Free Full Text]
  18. Gen K, Okuzawa K, Senthilkumaran B, Tanaka H, Moriyama S, Kagawa H. Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation. Biol Reprod 2000 63:308-319[Abstract/Free Full Text]
  19. Melamed P, Rosenfeld H, Elizur A, Yaron Z. Endocrine regulation of gonadotropin and growth hormone gene transcription. Com Physiol Biochem 1998 119C:325-338
  20. Gomez JM, Weil C, Ollitrault M, Le Bail P-Y, Breton B, Le Gac F. Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 1999 113:413-428[CrossRef][Medline]
  21. Prat F, Sumpter JP, Tyler CR. Validation of radioimmunoassays for two salmon gonadotropins throughout the reproductive cycle in male and female rainbow trout (Oncorhynchus mykiss). Biol Reprod 1996 54:1375-1382[Abstract]
  22. Kagawa H, Young G, Nagahama Y. Relationship between seasonal plasma estradiol-17ß and testosterone levels and in vitro production by ovarian follicles of amago salmon (Oncorhynchus rhodurus). Biol Reprod 1983 29:301-309[Abstract]
  23. Kagawa H, Young G, Nagahama Y. In vitro estradiol-17ß and testosterone production by ovarian follicles of the goldfish, Carassius auratus. Gen Comp Endocrinol 1984 54:139-143[CrossRef][Medline]
  24. Nagahama Y, Matsuhisa A, Iwamatsu T, Sakai N, Fukada S. A mechanism for the action of pregnant mare serum gonadotropin on aromatase activity in the ovarian follicle of the medaka, Oryzias latipes. J Exp Zool 1991 259:53-58[CrossRef]
  25. Young G, Kagawa H, Nagahama Y. Evidence for a decrease in aromatase activity in the ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus) associated with final oocyte maturation. Biol Reprod 1983 29:310-315[Abstract]
  26. Adashi EY, Resnick CE, Brodie AMH, Scoboda ME, Van Wyk JJ. Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells. Endocrinology 1985 117:2313-2320[Abstract/Free Full Text]
  27. Mulheron GW, Schomberg DW. The intraovarian transforming growth factor system. In: Adashi EY, Leung PCK (eds.), The Ovary. New Yori: Raven Press; 1993: 337–362
  28. Kagawa H, Moriyama S, Kawauchi H. Immunocytochemical localization of IGF-I in the ovary of red seabream, Pagrus major. Gen Comp Endocrinol 1995 95:293-300
  29. Kagawa H, Moriyama S, Okuzawa K. In vitro production of IGF-I by ovarian follicles of the red seabream, Pagrus major. In: Kwon HB, Joss JMP, Ishii S (eds.), Recent Progress in Molecular and Comparative Endocrinology. Korea: Kwangju; 1999: 144–148
  30. Kagawa H, Kobayashi M, Hasegawa Y, Aida K. Insulin and insulin-like growth factors I and II induce final maturation of oocytes of red seabream, Pagrus major, in vitro. Gen Comp Endocrinol 1994 95:293-300[CrossRef][Medline]
  31. Maestro MA, Planas JV, Moriyama S, Gutierrez J, Planas J, Swanson P. Ovarian receptors for insulin and insulin-like growth factor I (IGF-I) and effects of IGF-I on steroid production by isolated follicular layers of the preovulatory coho salmon ovarian follicle. Gen Comp Endocrinol 1997 106:189-201[CrossRef][Medline]
  32. Kagawa H, Takano K, Nagahama Y. Correlation of plasma estradiol-17ß and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char, Salvelinus leucomaenis. Cell Tissue Res 1981 218:315-329[Medline]
  33. Chomczynski P, Sacchi N. Single-step methods of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162:156-159[Medline]
  34. Kagawa H, Tanaka H, Okuzawa K, Kobayashi M. GTH II but not GTH I induces final maturation and the development of maturational competence of oocytes of red seabream in vitro. Gen Comp Endocrinol 1998 112:80-88[CrossRef][Medline]
  35. Tanaka H, Kagawa H, Hirose K. Steroidogenic activities of two distinct gonadotropins in red seabream, Pagrus major. In: Goetz FW, Thomas P (eds.), Proceedings of the Fifth Symposium on Reproductive Physiology of Fish, 1995; Austin, TX; 1995: 10–12
  36. Hurwitz A, Hernandez ER, Resnick CE, Packman JN, Payne DW, Adashi EY. Basic fibroblast growth factor inhibits gonadotropin-supported ovarian androgen biosynthesis: mechanism(s) and site(s) of action. Endocrinology 1990 126:3089-3095[Abstract/Free Full Text]
  37. Behl R, Pandey RS. Effects of insulin-like growth factor-I on steroidogenesis in cultured carp ovarian follicles: interactions with estradiol. Indian J Exp Biol 1999 37:138-142[Medline]
  38. Steinkampf MP, Mendelson CR, Simpson ER. Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human ovarian granulosa cells. Mol Cell Endocrinol 1998 59:93-99
  39. Gutie'rrez J, Pa'rrizas M, Carneiro N, Maestro JL, Maestro MA, Planas J. Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycles. Fish Physiol Biochem 1993 11:247-254[CrossRef]
  40. Srivastava RK, Van Der Kraak G. Insulin as an amplifier of gonadotropin action on steroid production: mechanism and sites of action in goldfish prematurational full-grown ovarian follicles. Gen Comp Endocrinol 1994 95:60-70[CrossRef][Medline]
  41. Hickey GJ, Chen S, Besman MJ, Shively JE, Hall PF, Gaddy-Kurten D, Richards JS. Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 1988 122:1426-1436[Abstract/Free Full Text]
  42. Fitzpatrick SL, Richards JS. Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 1991 129:1452-1462[Abstract/Free Full Text]
  43. Fitzpatrick SL, Carlone DL, Robker RL, Richards JS. Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge. Steroids 1997 62:197-206[CrossRef][Medline]
  44. Hsu C-J, Hammond JM. Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro. Endocrinology 1987 120:198-207[Abstract/Free Full Text]
  45. Zhou J, Kumar TR, Matzuk MM, Bondy C. Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol Endocrinol 1997 11:1924-1933[Abstract/Free Full Text]
  46. Hernandes ER. Regulation of the genes for insulin-like growth factor (IGF-I) I and II and their receptors by steroids and gonadotropins in the ovary. J Steroid Biochem Mol Biol 1995 53:219-221[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
S. Paul, D. Mukherjee, K. Pramanick, S. Kundu, S P Bhattacharyya, P. De, and D. Mukherjee
Stimulation of salmon calcitonin on secretion of 17{beta}-estradiol by the ovarian follicles of common carp, Cyprinus carpio
J. Endocrinol., February 1, 2008; 196(2): 413 - 424.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
H. Ko, W. Park, D.-J. Kim, M. Kobayashi, and Y. C. Sohn
Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies
J. Mol. Endocrinol., January 1, 2007; 38(1): 99 - 111.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
B. Campbell, J. Dickey, B. Beckman, G. Young, A. Pierce, H. Fukada, and P. Swanson
Previtellogenic Oocyte Growth in Salmon: Relationships among Body Growth, Plasma Insulin-Like Growth Factor-1, Estradiol-17beta, Follicle-Stimulating Hormone and Expression of Ovarian Genes for Insulin-Like Growth Factors, Steroidogenic-Acute Regulatory Protein and Receptors for Gonadotropins, Growth Hormone, and Somatolactin
Biol Reprod, July 1, 2006; 75(1): 34 - 44.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
T.-T. Wong, S. Ijiri, and Y. Zohar
Molecular Biology of Ovarian Aromatase in Sex Reversal: Complementary DNA and 5'-Flanking Region Isolation and Differential Expression of Ovarian Aromatase in the Gilthead Seabream (Sparus aurata)
Biol Reprod, May 1, 2006; 74(5): 857 - 864.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/5/1562    most recent
biolreprod.102.008219v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.
Agricola
Right arrow Articles by Kagawa, H.
Right arrow Articles by Tanaka, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS