1Fish Physiology and Biochemistry 19: 1–11, 1998.© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

Circulating levels and in vitro production of two maturation-inducinghormones in teleost: 17ααααα,20βββββ-dihydroxy-4-pregnen-3-one and17ααααα,20βββββ,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse,Pseudolabrus japonicus

M. Matsuyama1, K. Ohta1, S. Morita2, M.M. Hoque1, H. Kagawa3 and A. Kambegawa4

1Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan (Phone: 81-092-642-2887;Fax: 81-092-642-2907; E-mail: rinya_m@agr.kyushu-u.ac.jp); 2Hyogo Prefectural Sea Farming Center,Akashi, Hyogo 678, Japan; 3National Research Institute of Aquaculture, Nansei, Watarai, Mie516-01,Japan; and 4The Kambegawa Laboratory, 3-8-5, Motoizumi, Komae, Tokyo 201, Japan

Accepted: October 10, 1997

Key words: oocyte maturation, maturation-inducing hormone, 17α,20β-dihydroxy-4-pregnen-3-one,17α,20β,21-trihydroxy-4-pregnen-3-one, daily spawning, bambooleaf wrasse, teleost

Abstract

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, andexhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1)circulating levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) in females sampled at different times of the day during spawning season incaptivity, and (2) in vitro production of 17,20β-P and 20β-S by follicle-enclosed oocytes at sevendifferent developmental stages. In addition, we developed a microtiter plate enzyme-linkedimmunosorbent assay (ELISA) for 17,20β-P. Serum levels of 17,20β-P and 20β-S showed similar diurnalchanges; substantial increases in these levels occurred around the time of germinal vesicle breakdown(GVBD). In vitro experiments showed that massive production of 17,20β-P and 20β-S occurred infollicles collected just before or during GVBD. Further, acute decreases in 17,20β-P and 20β-S produc-tion were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence ofGVBD in vitro, suggest a role for both 17,20β-P and 20β-S as MIHs in the bambooleaf wrasse.

Introduction

It is generally accepted that final oocyte matura-tion in teleosts is induced by C21 steroid hor-mones (Scott and Canario 1987). 17α,20β-Dihy-droxy-4-pregnen-3-one (17,20β-P) has been iden-tified as the maturation-inducing hormone (MIH)in salmonids (Nagahama and Adachi 1985) and isimplicated as the MIH in most teleosts investi-gated to date (Scott and Canario 1987). Recently,17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S)has been positively identified as the MIH in theperciform family Sciaenidae (Trant et al. 1986;

Thomas and Trant 1989; Trant and Thomas 1989;Patiño and Thomas 1990a). At present, however,available information concerns only a limitednumber of species, and information on marinefishes is scanty.

The bambooleaf wrasse, Pseudolabrus japo-nicus, is abundant, easy to collect and rear, andfemale fish spawn daily in captivity in autumn(Matsuyama et al. 1998). It exhibits diandricprotogyny, with populations consisting of smallinitial-phase (IP) males (primary males), IP fe-males (primary females), and large terminal-phase(TP) males which may be derived from either

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females which have undergone a sex change tobecome males (secondary males), or IP males(TP primary males; Nakazono 1979). The dailycycle of ovarian development in this species hasthe advantage that physiologically differentoocytes can be obtained on the same day. Thesefeatures make the bambooleaf wrasse a goodmodel for studies of the endocrine control ofoogenesis. Recently, we reported the annual re-productive cycle (Morita et al. 1998) and the di-urnal rhythm of oocyte development of bam-booleaf wrasse (Matsuyama et al. 1998). Femalebambooleaf wrasse produce both 17,20β-P and20β-S during the spawning season (Morita et al.1997), and results obtained from a preliminary invitro study show that 17,20β-P and 20β-S areequally potent and highly effective steroids atinducing germinal vesicle breakdown (GVBD) inbambooleaf wrasse oocytes (Matsuyama et al.1997). Thus our prior findings indicate a possiblerole for both hormones as MIHs in this species.

To confirm this speculation, the following in-vestigations have been made in the present study:(1) circulating levels of 17,20β-P and 20β-S infemales sampled at different times of the dayduring spawning season in captivity, and (2) invitro production of 17,20β-P and 20β-S by folli-cle-enclosed oocytes at seven different develop-mental stages incubated with each steroid precur-sor. In addition, we developed a microtiter plateenzyme-linked immunosorbent assay (ELISA) for17,20β-P and measured the circulating 17,20β-P.

Materials and methods

Fish

Fish were caught in coastal water near the Fish-eries Research Laboratory, Kyushu University,Fukuoka Prefecture, on 14 October, 1995, andwere transferred to the laboratory. The captivepopulation consisted of eight IP fish and three TPmales per tank, established in six round fiberglasstanks of 500 l capacity, supplied with filteredrecirculating sea water, and containing some plas-tic tubes (300 mm in length and 100 mm in diam-eter) as shelters. Fish were kept under conditionsof natural daylength and water temperature, andwere fed with shucked live shellfish or krill oncea day. After confirming daily spawning, for

analysis of circulating levels of steroids, fish infour tanks were sampled at 02:00, 06:00, 10:00,and 16:00 h, respectively from late October toearly November. For investigation of in vitrosteroid production, pairs of fish were sampled at3:00, 6:00, 9:00, 15:00, and 21:00 h.

Histology

After killing the fish by decapitation, the ovarieswere dissected out, and an ovarian tissue samplewas immersed in Bouin’s solution, dehydratedand embedded in Technovit resin (Kulzer,Wehrheim). For light microscopy, 4 µm-thicksections were cut and stained with 1% toluidineblue solution. Developmental stages of theoocytes were classified according to the schemeused for Japanese whiting (kisu) Sillago japonica,a daily-spawning marine teleost (Matsuyama etal. 1990a).

Blood sampling

After anesthetization with 2-phenoxyethanol (300ppm), blood samples were taken from the caudalvessel, centrifuged at 3,000 rpm for 20 min, andthe serum was stored at –30 °C until steroidanalysis. The gonads were then examined histo-logically to determine sexuality and gonadal stage;29 females weighing 25–70 g were used for cir-culating steroid analysis.

In vitro culture

Fish were killed by decapitation, and their ova-ries were removed and placed in ice-coldLeivovitz’s L-15 culture medium (GIBCO,Rockville, MD), buffered with 0.02 M HEPES,pH adjusted to 7.5 with 1 N NaOH. Gentamycinsulfate (200 mg l–1, Sigma, St. Louis, MO) wasadded at the start of incubation. The largest andsecond-largest intact follicles were isolated fromovarian tissue and 20 oocytes at the same devel-opmental stage were placed in each well of aplastic tissue culture plate (24 wells, Falcon)containing 1 ml of medium in the presence orabsence of 100 ng ml–1 of the precursors of ster-

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oids 17α-hydroxy-4-pregnen-3,20-dione (17α-hydroxyprogesterone; 17α-P, Sigma) or 17α,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycorti-sol, Sigma). Steroids were dissolved in ethanol.Ten microliters of ethanol were added as controlto some wells instead of the steroid hormones.The oocytes were incubated for 20 h at 20 °C ina temperature controlled incubator in an atmos-phere of 100% air. All treatments were tested intriplicate. Following the incubation period, me-dia were collected and stored at –20 °C untilexamined by the ELISA for 17,20β-P and 20β-S.

Measurement of steroids

17,20β-P and 20β-S levels in serum and incuba-tion medium were measured by specific ELISAs.The ELISA for 20β-S has been developed byAsahina et al., (1995). The ELISA for 17,20β-Pis described below.

Chemicals: 17,20β-P and horseradish peroxi-dase (Type VI) were purchased from Sigma.Affinity purified goat anti-rabbit IgG was ob-tained from CAPPEL, West Chester, PA. Otherreagents were obtained from Wako ChemicalsCo., Tokyo. 3-Carboxymethyl oxime of 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P-3-CMO),active ester of 17,20β-P-3-CMO, BSA-steroidconjugate, and enzyme-steroid conjugate wereprepared according to Asahina et al. (1995).

Stock solutions: Coating buffer, 0.05M carbon-ate buffer pH 8.4, containing 0.05% NaN3; wash-ing solution, 0.85% NaCl; blocking solution,0.05M PBS containing 0.1% BSA, 3% sucrose,and 0.005% thimerosal; assay buffer, 0.05M bo-rate buffer, pH7.8, containing 0.1% BSA and0.01% thimerosal; substrate solution, 0.2M citratebuffer, pH 4.5, containing 0.01% H2O2, addedwith 0.5% o-phenylenediamine immediately be-fore use; stopping solution, 6N H2SO4. All thesolutions except for the stopping solution werestored at 4 °C.

Second antibody-coated microtiter plates:Microtiter plates (MS-3596 F/H plate, SumitomoBakelite Co, Tokyo) were coated with 100 ml perwell of goat anti-rabbit IgG (15 µg µl–1 in coat-ing buffer). The plate was sealed and incubatedat 4 °C for 48 h. After removal of unboundantiserum, the wells were washed three times with

washing solution and dried. Blocking solution(200 µl) was added to each well, which was thensealed and incubated at 4 °C for 24 h. The wellswere emptied and dried by leaving the plates ina refrigerator for 24 h.

Assay procedure: Steroids in serum and incu-bation medium were extracted twice with 10 volof diethyl ether. The ether was evaporated, andthe sample was reconstituted with assay buffer.The wells of a second antibody-coated plate wereloaded with 50 µl of standard or sample, 50 µl ofdiluted steroid-enzyme conjugate solution andanti-17,20β-P solution (all dissolved in assaybuffer), in this sequence. Samples and standardswere applied in duplicate to each plate. Afterincubation at 20 °C for 2 h, the plate was drainedand washed three times with washing solution.150 µl of substrate solution was added to eachwell, and the plate was incubated at 20 °C for 40min. Color development was stopped by adding50 µl of stopping solution to each well. The ab-sorbance of each well was measured at 492 nmwith a microtiter plate analyzer (model 2550,Biorad).

Validation of assay: An antiserum was raisedagainst 17,20β-P-3-CMO-BSA. A standard curvefor 17,20β-P was obtained with the presentELISA (antiserum dilution, 1:100,000; labeledhormone dilution, 1:2,500) and competitioncurves for serial dilutions of serum extract fromtwo female bambooleaf wrasse are shown in Fig-ure 1. These bambooleaf wrasse were sampled at02:00 h when the largest oocyte are at a late stageof germinal vesicle migration. A steep standardcurve covering 1.5–192 pg/well (30–3,840 pg ml-1)was obtained. The intra- and interassay coeffi-cients of variation were 3.1% (n = 4, duplicate)and 6.5% (n = 4, duplicate) respectively, close tothe 50% binding point. Competition curves forserum extract from bambooleaf wrasse were ob-tained (Figure 1). After logarithmic transforma-tion, slope of regressions on each of the curvewas confirmed to be parallel to the standard curve(ANCOVA. p > 0.05). The sensitivity of this as-say was 7.2 pg ml–1. This antiserum cross-reactedwith fewer than 1% of several steroids commonlyobserved in teleost blood (Table 1).

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Statistics

Values below the minimum assay sensitivity (30pg ml–1 serum for steroid assays) were treated aszero. Data were expressed as mean and SEM, andanalyzed by Duncan’s multiple range test.

Results

Ovarian histology

All the 29 IP fish used for circulating steroidanalysis were females. The ovarian stage repre-sented by the developmental stage of the largestoocytes in the ovary, is shown in Table 2. Fishsampled at 10:00 h were in the post-spawningcondition and the largest oocytes were at the ter-tiary yolk stage (TY) with vitellogenesis com-pleted (Figure 2A). At 16:00 h, oocytes were atthe early stage of germinal vesicle migration(EMN), when the germinal vesicle is situatedbetween the center of the oocyte and the half wayto the animal pole (Figure 2B). At 02:00 h, fiveof nine fish had oocytes at the late stage of ger-minal vesicle migration (LMN), when the germi-nal vesicle is located near the zona radiata wherethe micropyle is situated (Figure 2C). Two of the

remaining fish had oocytes at EMN and two atthe early mature stage (EM). The oocytes at EMseemed to be just after GVBD, with a translucentappearance (Figure 2D) likely due to yolk prote-olysis in progress (Matsubara et al. 1995). At06:00 h, all of seven fish had transparent oocytes

Figure 1. A standard curve for 17,20β-S obtained from thepresent ELISA system (antiserum dilution, 1: 100,000;labeled hormone dilution, 1:2,500) and competition curvesfor serial two-fold dilution of plasma extract from maturingfemale bambooleaf wrasse.

Table 1. Cross reaction of various steroids measured in thespecific enzyme immunoassay for 17,20β-P

Steroids* Cross (%)

17,20β-P 10020β-S 0.9Androstendione 0.4Testosterone 0.411-Deoxycortisol 0.18Pregnenolone 0.15Progesterone < 0.120β-P > 0.117α-P 0.07Corticosterone 0.02Estradiol-17β 0.02Cortisol 0.002

*Abbreviations:17,20β-P, 17α,20β-dihydroxy-4-pregnen-3one; 20β-S, 17α,-20β,21-trihydroxy-4-pregnen-3-one; Androstendione, 4-androstene-3,17-dione; Testosterone, 17β-hydroxy-4-andros-ten-3-one; 11-Deoxycortisol, 17α,21,dihydroxy-4-pregnene-3,20-dione; Pregnenolone, 5-pregnen-3β-hydroxy-20-one;Progesterone, 4-pregnene-3,20-dione; 20β-P, 20β-hydroxy-4-pregnen-3-one; 17αp-P, 17α-hydroxy-4-pregnene-3,20 dione;Corticosterone, 11β,21-dihydroxy-4-pregnene-3,20-dione;Estradiol-17β, 3,17β-dihydroxy-estra-1,3,5(10)-triene; Corti-sol, 11β,17α,21-trihydroxy-4-pregnene-3,20-dione.

Table 2. Developmental stages of the largest oocytes in thebambooleaf wrasse ovary sampled at different times of theday during the spawning season

10:00 h 16:00 h 02:00 h 06:00 h

TY EMN EMN LMTY EMN EMN LMTY EMN LMN LMTY EMN LMN LMEMN EMN LMN LM

EMN LMN LMEMN LMN LMEMN EM

EM

A total of 29 females was examined for oocyte stage. TY,tertiary yolk stage; EMN, early migratory nucleus stage; LMN,late migratory nucleus stage; EM, early mature stage; LM,late mature stage. See Figure 2 and text for histologicalcharacteristics of each stage of oocyte.

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at the late mature stage (LM) in which yolk pro-teolysis had been completed (Figure 2E).

Six IP fish used for in vitro steroid productionanalysis were all females. The developmentalstages of the largest oocytes and the second-larg-

est oocytes of fish sampled at six different timesof the day are shown in Table 3, in which thetime before ovulation for each oocyte group sam-pled at different times was estimated on the as-sumption that ovulation occurred at 06:00 h every

Figure 2. Photomicrographs of the bambooleaf wrasse oocytes. A, completion of vitellogenesis (tertiary yolk stage, TY); B, onsetof germinal vesicle migration (early migratory nucleus stage, EMN); C, Late stage of germinal vesicle migration (LMN); D, matureoocyte just after GVBD (early mature stage, EM); and E, mature oocyte just prior ovulation (late mature stage, LM). See text forhistological characteristics of each stage oocyte. GV, germinal vesicle; O, oil droplet. Scale bars indicate 100 µm.

Largest oocytes Second-largest oocytes

Time Individual Time before Time beforeNo. Stage ovulation(h)*1 Stage ovulation (h)

06:00 h 1 LM 0 TY -*2

05:00 h 2 EM 1 SY 2503:00 h 3 LMN 3 SY 2721:00 h 4 EMN 9 SY 3315:00 h 5 EMN 15 PY 3909:00 h 6 TY 21 PY -

*1 It is assumed that the ovulation occurs at 06:00 h; PY, primary yolk stage; SY, secondary yolk stage; TY tertiary yolk stage;EMN, early migratory nucleus stage; LMN, late migratory nucleus stage; EM, early mature stage; LM, late mature stage. See Figure2 and text for histological characteristics of each stage of oocyte; *2 Oocytes were not used for experiment of in vitro steroidproduction.

Table 3. Developmental stages of the largest and the second-largest oocytes in the bambooleaf wrasse ovaries sampled at differentsix times of the day for in vitro steroid production analysis

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day. Stages PY, SY, and TY represent the begin-ning of vitellogenesis, active vitellogenesis, andthe completion of vitellogenesis, respectively.

Serum steroid levels

As shown in Table 2, during most of the matu-ration cycle the ovarian developmental stage rep-resented by the stage of the largest oocyte, wasconsistent within groups of fish sampled at thesame time. However, there was less consistencywith respect to the developmental stage of theovary, which is reflected by the serum hormonelevels. Therefore, data for establishing serum17,20β-P and 20β-S levels were obtained fromfish grouped according to gonadal stage (Figure3). Additionally, the number of fish at stage EMwas so small that data from fish at EM and LMstages were combined as the mature stage (M).

Serum 17,20β-P showed a peak level (946 ± 91pg ml–1) at the mature stage (vs. EMN, p < 0.05;vs. LMN, p < 0.01). After spawning (TY), theabsolute level of serum 17,20β-P was relativelyhigh (871 ± 285 pg ml–1), but there was no statis-tically significant difference (TY vs. EMN andLMN) (p > 0.05).

Serum 20β-S levels showed a statistically sig-nificant difference (p < 0.05) between the maturestage (M, 735 ± 240 pg ml–1) and the early mi-gratory nucleus stage (EMN, 202 ± 122 pg ml–

1). The absolute level of serum 20β-S was high-

Figure 3. Changes in serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) in female bambooleaf wrasse sampledat different times of the day in captivity during spawningseason. Each value is mean ± SEM. Numbers indicate samplesizes. TY, tertiary yolk stage; EMN, early migratory nucleusstage; LMN, late migratory nucleus stage; M, mature stage;data from fish at early and late mature stages were combinedas the mature stage.

Figure 4. Diagrammatic representation of diurnal changes of serum levels of 17,20β-P and 20β-S. TY, tertiary yolk stage; EMN,early migratory nucleus stage; LMN, late migratory nucleus stage; GVBD, germinal vesicle breakdown.

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est post-spawning (TY, 765 ± 329 pg ml–1), butthere was no statistically significant differencebetween the stages (p > 0.05).

Thus, both serum 17,20β-P and 20β-S levelsshowed significant increases from MN stage tomature stage, and were maintained at relativelyhigh levels post spawning.

The diurnal changes in developmental stage ofthe largest oocytes (first clutch of oocytes) andthe serum levels of the two steroids in thebambooleaf wrasse are diagrammatically repre-sented in Figure 4.

In vitro steroid production

Figure 5 shows the levels of 17,20β-P and 20β-S production in vitro from their radioinert precur-sors (100 ng ml–1) by 20 intact follicles at 10different times before ovulation.

17,20β-P production from 17α-P (Figure 5,upper row): The level of in vitro 17,20β-P pro-duction from 17α-P, which directly reflects theactivity of 20β-hydroxysteroid dehydrogenase(20β-HSD) in the intact follicles, was very low(under 1.0 ng ml–1) during vitellogenesis (PY,SY, and TY). Thirty nine to 21 h before ovula-tion, levels showed a small increase at EMN (1.59ng ml–1, 15 and 9 h) (p < 0.05, vs. 21 h) and,following a drastic increase at LMN (18.97 ngml–1, 3 h) (p < 0.01, vs. 15 h), remained high atEM (15.94 ng ml–1, 1 h) and thereafter decreasedrapidly at LM (2.44 ng ml–1, 0 h) (p < 0.01, vs.1 h).

20β-S production from S (Figure 5, middlerow): In vitro 20β-P production from 11-deoxy-cortisol (S), which also reflects the activity of20β-hydroxysteroid dehydrogenase (20β-HSD) inthe intact follicles, exhibited a pattern similar tothat of 17,20β-P production from 17α-P, withlower levels (1.28–2.75 ng ml–1) during vitello-genesis and a small increase at EMN (15 and 9h), peaking (25.07 ng ml–1) at LMN, remaining ata high level (22.56 ng ml–1) at EM, and decreas-ing (1.34 ng ml–1) at LM.

20β-S production from 17α-P (Figure 5, lowerrow): In vitro 20β-S production from 17α-P,which reflects the combined activities of 20β-HSD and 21-hydroxylase in the intact follicles,

Figure 5. In vitro 17,20β-P and 20β-S productions by 20intact follicles of bambooleaf wrasse sampled at ten differenttimes before ovulation. Each value is mean ± SEM. PY,primary yolk stage; SY, secondary yolk stage; TY, tertiaryyolk stage; EMN, early migratory nucleus stage; LMN, latemigratory nucleus stage; EM, early mature stage; and LM,late mature stage. Stages PY and SY mean beginning ofvitellogenesis and active vitellogenesis, respectively.

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also showed a pattern similar to those of 17,20β-P production from 17α-P and 20β-S from S,although its absolute levels were relatively low.20β-S production was at low levels (0.60–1.66ng ml–1) during vitellogenesis with a small in-crease at EMN, peaks at LMN (12.51 ng ml–1)and EM (12.60 ng ml–1), and a rapid decrease atLM (0.61 ng ml–1). The levels of 20β-S produc-tion from the two precursors (S and 17α-P) dif-fered significantly at LMN (p < 0.05) and at EM(p < 0.01).

Discussion

The sensitivity and precision of the 17,20β-PELISA were almost the same as those of ordinarysteroid radioimmunoassays (Young et al. 1983).The antiserum cross reacted relatively little withsteroids other than 17,20β-P, and competitioncurves for serum collected from bambooleafwrasse were almost parallel to the standard curve.These results indicate that the present ELISA isvery specific for 17,20β-P, and serum 17,20β-Plevels can be measured after simple collection.

As stress can have profound (generally inhibi-tory) effects upon the endocrine profiles ofteleosts, it is desirable that sampling programmes

be designed so as to minimize exposure to stress.In the studies based on hatchery or laboratorystocks of fish, like the present study, stress ef-fects are less clear (Pankhurst and Carragher1991). Many species do not undergo normal de-velopment and spawning in captivity (Donaldsonand Hunter 1983). The bambooleaf wrasse, how-ever, in captivity under conditions of naturaldaylength and water temperature spawns daily fora long period like the wild fish (Matsuyama et al.1998). Furthermore, the developmental stages ofthe oocytes at different times of the day duringthe spawning season have been clarified (Mat-suyama et al. 1998). Based on this previousstudy, in the present study sampling programmeswere designed to take advantage of the plentifulinformation available on spawning periodicities.The changes in the levels of the two steroid hor-mones in serum observed in the present studylikely reflect real events in the reproductive cycleof the bambooleaf wrasse, not artifacts of thesampling regime. The present study showed thatfish underwent GVBD at 06:00 h, ovulation oc-curred between 06:00 and 09:00 h, and germinalvesicle migration lasted for 12 h or more over,quite similar to the observations in our previousstudy (Matsuyama et al. 1998). Thus, femalebambooleaf wrasse have a constant, unchanging

Figure 6. Diagrammatic representation of in vitro 17,20β-P and 20β-S productions by 20 intact follicles of bambooleaf wrassesampled at ten different times before ovulation. Time of GTH surge was cited from Matsuyama et al. 1998. PY, primary yolkstage; SY, secondary yolk stage; TY, tertiary yolk stage; EMN, early migratory nucleus stage; LMN, late migratory nucleus stage;EM, early mature stage; and LM, late mature stage.

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diurnal rhythm of oocyte development, and pro-vide reproducible material for experiments.

The profiles of circulating levels of 17,20β-Pand 20β-S that we observed in naturally maturingfish, showing a diurnal rhythm in the reproduc-tion of these two steroids, peaked at around thetime of GVBD. Our previous experiment showedthat 17,20β-P and 20β-S are equally potent andhighly effective steroids at inducing GVBD invitro in bambooleaf wrasse (Matsuyama et al.1998). These findings suggest that 17,20β-P and20β-S are implicated in the final oocyte matura-tion of bambooleaf wrasse, and further supportthe concept that final oocyte maturation may becontrolled by more than one MIH in someteleosts, such as the tobinumeri-dragonet, Repo-mucenus beniteguri (Asahina et al. 1991), andstriped bass, Morone saxatilis (King et al. 1994).The equipotence of 17,20β-P and 20β-S could bedue to conversion of 17,20β-P to 20β-S, as re-ported in Atlantic croaker (Patiño and Thomas1990b), or could be based on a relative non-specificity of 17,20β-P and 20β-S for the MIHreceptor, as seen in the rainbow trout, Oncorhyn-chus mykiss (Yoshikuni et al. 1993). Further stud-ies on the characteristics of the MIH receptor inbambooleaf wrasse have to be conducted in orderto clarify this phenomenon.

A diurnal rhythm of circulating levels of pro-gestin, regarded as an MIH of the species, hasbeen reported in two daily spawning marine fish,Japanese whiting (Matsuyama et al. 1990a) andred seabream, Pagrus major (Kagawa et al.1991). In both species, 17,20β-P is one of themost potent steroids for inducing GVBD in vitro(Adachi et al. 1988; Matsuyama et al. 1990b) andblood 17,20β-P levels increased during theGVBD period (Matsuyama et al. 1990a; Kagawaet al. 1991), although there is no direct evidencethat 17,20β-P is a true MIH in either species. Thepeak blood level of 17,20β-P in bambooleafwrasse (946 pg ml–1) is higher than that in Japa-nese whiting (100 pg ml–1; Matsuyama et al.1990a) and red seabream (389 pg ml–1; Kagawaet al. 1991). However, circulating 20β-S levels inthese two species have not been measured.

The results of the present in vitro investigationshow that there is a marked change in ovariansteroidogenesis in bambooleaf wrasse in the pro-duction of MIH(s), coincident with final oocyte

maturation. Marked increases in the rates of con-version of 17α-P to 17,20β-P and S to 20β-S wereobserved in follicles collected prior to or duringGVBD (between 3 and 1 h before ovulation).These results suggest that the activity of 20β-HSD is high during this period. There is nowample evidence that, in salmonids, gonadotropin(GTH) stimulates follicular (granulosa cell) 20β-HSD activity via the gonadotropin receptor-ade-nylate cyclase-cAMP-dependent step (Nagahamaand Yamashita 1989). Our previous in vitro studyon the diurnal rhythm of oocyte development inbambooleaf wrasse suggests that an endogenousGTH surge occurs at some point between 09:00and 12:00 h, 21–18 h prior to spawning (Mat-suyama et al. 1998), exactly when the most ad-vanced oocytes completed their vitellogenesis.Thus, it is likely that this pre-maturational GTHsurge is responsible for the increase of 17,20β-Pand 20β-S production via 20β-HSD activation inthe bambooleaf wrasse ovarian follicles. Thisdaily GTH surge probably controls the diurnalmaturation cycle of oocytes in bambooleafwrasse.

An interest finding in the present in vitro inves-tigation was that the production of 17,20β-P from17α-P and 20β-S from S drastically decreased infollicles isolated just prior to ovulation. Theseresults suggest that the activity of 20β-HSD sud-denly became low, or that 17,20β-P and 20β-Swere largely converted to any other metabolitesat this time by an unknown enzyme(s) while 20β-HSD activity remained high. In another dailyspawner, the medaka, Olyzias latipes, in which17,20β-P is a major MIHs, 17α,20β-dihydroxy-5β-pregnan-3-one appeared immediately after theappearance of 17,20β-P, suggesting that 5β-re-ductase may be activated in medaka folliclesimmediately after the activation of 20β-HSD (Fu-kada et al. 1994). 17α,20β-Dihydroxy-5β-preg-nan-3-one was found to be much less effective ininducing oocyte maturation in medaka than17,20β-P, and it has been suggested that the con-version of 17,20β-P represents an inactivationprocess. It is possible, therefore, that the rapidinactivation of MIH(s) may also occur in bam-booleaf wrasse ovarian follicles and be importantin maintaining the spawning cycle in a dailyspawning fish, as suggested for medaka (Fukadaet al. 1994).

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In vitro 20β-S production from 17α-P, whichreflects the combined activities of 20β-HSD and21-hydroxylase in the ovarian follicles, alsoshowed a pattern similar to those of 17,20β-Pproduction from 17α-P and 20β-S from S. It ispossible that profiles of 20β-S production from17α-P may be regulated by the stage-specificchange in 20β-HSD activity previously men-tioned. However, there are two possible pathwaysfrom 17α-P to 20β-S; one is a pathway via S andthe other via 17,20β-P. In the present study, theactivity of 21-hydroxylase, the enzyme whichconverts 17α-P to S and 17,20β-P to 20β-S, wasnot investigated. Future studies, some in vitro,including the establishment of an ELISA for S,are needed to elucidate the pathways of 17,20β-P and 20β-S production.

There was one problem with the study in thatthe recorded in vitro fall in 17,20β-P and 20β-Sjust before ovulation is inconsistent with the cir-culating changes which showed that serum levelsof both steroids remain high either side of spawn-ing. The high levels of circulating 17,20β-P and20β-S in the mature fish are likely due to themarkedly increased production of both steroids infollicles at LMN and EM stages occurred between3 and 1 h before ovulation. The remaining17,20β-P and 20β-S in plasma may result in therelatively high levels of both steroids after ovu-lation, in spite of the drastical decreases in bothsteroids in the follicles at LM stage just beforeovulation.

In summary, we established an ELISA for17,20β-P in the present study, and demonstratedthe circulating levels of two MIHs of teleosts,17,20β-P and 20β-S, during the diurnal reproduc-tive cycle of bambooleaf wrasse. Serum levels of17,20β-P and 20β-S showed similar diurnalchanges; substantial increases in these serum lev-els occurred around the time of GVBD. Moreo-ver, in vitro experiments clearly showed that dra-matic production of 17,20β-P and 20β-S occurredin follicles collected just before or during GVBD.The results of the present study, taken togetherwith our previous data on in vitro occurrence ofGVBD (Matsuyama et al. 1998), suggest a rolefor both 17,20β-P and 20β-S as MIHs in bam-booleaf wrasse. Further, an acute decrease in17,20β-P and 20β-S production in the ovarianfollicles occurred just prior to ovulation, suggest-

ing inactivation of MIH. However, the enzymaticmechanisms involved in this phenomenon areunknown. Further studies of the kinetics and path-ways of 17,20β-P and 20β-S production relativeto GVBD should elucidate details of their in-volvement in final oocyte maturation in this spe-cies.

Acknowledgements

This study was supported in part by grants-in-aidfor scientific research from the Ministry of Edu-cation, Science and Culture of Japan.

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