Seasonal changes in plasma levels of gonadal steroids of sea bass, Dicentrarchus labrax L

GENERAL

AND

COhtPARATWE

ENDOCRINOLOGY

78,361-373 (1590)

Seasonal Changes in Plasma Levels of Gonadal Steroids of Sea Bass, Dicentrarchus labrax L. F. PRAT, S. ZANUY, M. CARRILLO,

A. DE MONES,*

AND A. FOPTIER*

Znstituto de Acuicultura de Torre de la Sal, Ribera de Cabanes 12595, Castelk%, Spain, and *Laboratoire Physiologie des Poissons, ZNZU, Campus de Beaulieu, 35042 Rennes, Ckdex, France

de

Accepted July 19, 1989 Levels of plasma testosterone (‘I) and 11-ketotestosterone (1 I-KT) in males and plasma 17P-estradiol (E3, 17a-20l3-dihydroxy4pregnen-3-one (17a,ZOP-diOH-P), and T in females were assayed by radioimmunoassay at monthly intervals throughout the sexual cycle of sea bass (Dicentrarchus labrax L.). 17a,20g-DiOH-P was maintained at low levels (below 1 t&ml) throughout the year, even during the spawning period (January-March). A biiodal seasonal pattern of plasma testosterone was observed. Plasma T and E, levels became significantly increased in December (advanced gametogenesis period) and then showed further increases during January and February (first half of the spawning period) in parallel with the growth of the vitellogenic oocytes. Multiple spawnings of individual females were also observed during the spawning period affecting the relative fecundity of the eggs. A possible role of E, on this behavior is discussed. In males, both plasma T and 1I-KT initially increased in November and then showed further increasings during the rest of the period of gametogenesis (December) to reach their peak levels in the first half of the spawning period (end of January). These increased and sustained higher levels of plasma steroids coincided with the presence of spermiating males. A second peak of plasma testosterone appeared at the end of the postspawning period-beginning of the pregametogenesis period (May-June) both in males and females and their possible role with the preparation of the gonad for the next reproductive cycle is discussed. o 1990Academic RCSS, IN.

In recent years, sea bass, a highly prized fish, has received considerable attention from scientists and aquaculturists with the aim of improving the efficiency of its culture. The control of reproduction is of a particular interest. Knowledge of the role of sex steroids in controlling the maturation cycle in sea bass, especially when spawning time is altered by environmental or hormonal manipulation, is of both theoretical and practical interest. Seasonal changes in serum gonadal steroid hormone levels have been well documented in a number of freshwater teleost species (Fostier et al., 1983). Less information is available for marine species, although seasonal changes in androgens and/ or estrogens have been reported in flatfish and (Campbell et al., 1976; Wingfield Grimm, 1977), black goby (Bonnin, 1979),

bluefish and king mackerel (MacGregor et ul., 1981), striped mullet (Dindo and MacGregor, 1981), Staghom sculpin (de Vlaming et al., 1984), Sparus auruta (Kadmon et al., 1985), Rhabdosargus sarba (Yeung and Chan, 1987), blue cod (Pankhurst and Conroy, 1987), and red seabream (Duchi et al., 1988a, b). Generally, more interest has been focused on the rapid changes which accompany final gonadal maturation in these fish (Goetz et al., 1987; Scott and Canario, 1987; Hirose et al., 1987; Adachi et al., 1988). Various differences have emerged between some marine and freshwater species. In two marine perciform fishes (Tram et al., 1987), et al., 1986; Thomas 17o1,20@dihydroxy-4-pregnen-3-one, the claimed maturation induction steroid (MIS) for salmonids (Fostier and Jalabert, 1982; 361 00166480/90 $1.50 Copyright 0 1990 by Academic Press, Inc. Au lights of reproduction in any form reserved.

362

PRAT

Goetz, 1983), is low and does not change significantly during final oocyte maturation. Furthermore, data from S. aura& a multispawner, show high levels of plasma 17B-estradiol (E,) during the spawning season (Kadman et al., 1985). The sea bass, Dicentrurchus lubrax L., is a marine teleost that reproduces annually upon reaching sexual maturity. Although histological aspects of the gonads and spawning are well documented for this species (Caporiccio, 1976; Barnabe, 1980; Brusle and Roblin, 1984; Zanuy et al., 1986; Mayer et al., 1988; Carriho et al., 1989a), apart from Prat et al. (1985) very little is known about seasonal levels of circulating gonadal steroids. It is also not clear whether this species undergoes multiple spawnings in captivity. The purpose of the present study was to determine how serum estrogen, progestagen, and androgen concentrations change during seasonal reproduction and to determine if correlations exist among these changing levels, environmental factors, oocyte growth (based on oocyte diameters), number of spawnings, and fecundity. METHODS Facilities Adult female [1.07 2 0.085 kg (mean rt SEM); 46.8 + 1.30 cm] and male (0.89 f 0.054 kg; 44.7 f 0.80 cm) sea bass, bred within the facilities of the Instituto de Acuicultura de Terre de la Sal, were maintained in 12000-liter tanks, supplied with aerated running seawater, under natural conditions of photoperiod and temperature (Fig. 1).

ET AL.

der to avoid unnecessary damage to the developing gonadal ducts. Oocyte diameter was measured and the stages of maturation were assessed according to Zanuy et a/., (1986). Eggs were collected from the outflow water of the tank in special line gauge nets and the number of natural spawnings was recorded each day. Unfortunately, it was not possible to ascertain the exact number of spawnings which had been carried out by the individual female fish as the eggs from each tank were pooled in the oufflow collection nets. The total number of eggs produced and the relative fecundity (i.e., the number of eggs/kg of postspawned female body weight) were recorded, as were the proportion of floating (good quality) to sinking (poor quality) eggs in a measuring cylinder of seawater. Sinking eggs included both infertile and damaged ova, whereas the floating group always had fertilization rates in excess of 90%.

Steroid Assays Steroids. Nonradioactive steroids to be used as standards were purchased from Sigma Chemical Co. (St. Louis, MO). Radiolabeled steroids ([1,2,6,7-‘HItestosterone (93 Ci/mmol), [ 1,2-3H]1 l-ketotestosterone (93 Ci/mmol), and [2,4,6,7-3H]estradio1 (93 Ci/ mmol) were purchased from Amersham International (Amersham, England). Radiolabeled lla-hydroxy 20B-dihydro-progesterone was prepared from [1,2,6,7-3H]-17a-hydroxyprogesterone (56 Wmmol) (Amersham International) by reduction with NaBH, according to Fostier er al. (1973). Antisera. Anti-testosterone-3-carboxymethyl BSA; anti-17p-hydroxyandrost-4-ene-3,1 I-dione-3-carboxymethyl BSA; anti-17P-estradiol-6carboximethyl BSA; and anti-l7a-20@dihydroxy-4-pregnen-3-one-3carboxymethyl BSA were used as antigens to raise antisera in rabbits. Antisera dilutions for the assays was 113500 for anti-testosterone, l/7500 for anti1I-ketotestosterone, 112500 for anti-17l3-estradiol, and l/5700 for anti-17a-hydroxy-20$-dihydroxyprogesterone. The specilicity of the antisera is shown in Table 1.

Steroid Extraction Sampling

Procedure

Fish were sampled monthly for 1 year. They were anesthetized in (100 ppm) MS-222 (Sigma), weighed, and measured. A l-ml blood sample was taken from each fish by caudal puncture and the plasma was separated and stored at -30” until analysis. Males were checked for milt production by gentile abdominal massage, and females were sampled by cannulation from November to April in order to determine gonadal development (Zanuy et al., 1986; Carrillo et al., 1989a). In earlier months, females were not cannulated in or-

and Chromatography

Radiolabeled steroid (0.1 ml) (2000 dpm) was added to 0.1 ml of plasma for recovery. Samples were extracted twice with a cyclohexane:ethylacetate mixture (1: 1, v/v). After evaporation of the solvent, the dry residue was redissolved in dichloromethane:methanol (95:5, v/v), and in order to purify the steroids these were transferred to a Sephadex LH-20 column (12 x 0.5 cm) (Pharmacia Fine Chemicals). Elution was performed in the same solvent system. After purification, a further evaporation of the solvent was performed and the dry residues were dissolved in 0.35 or 0.5 ml of

SEX STEROIDS IN SEA BASS hrr

363

of light

B’r

Jun

I

1

Jul

Aug

I

6ep

1

I

I

I

1

Oot

Nov

Deo

Jan

I Jan

Fob

I

Mar

I

I

Apr

May

I

Jun

tUIIPU8tUn(OO) 2'1 2421 1616129,

I Jun Jul teatortorono 6(rig/ml)

I, Au9

6ep

I1 Oot

Nov

I Dw

1 Aug

1 SOP

1 Oot

1 Nou

1 Deo

I Feb

b Mar

I Apr

I May (14)

I Jun

64a2lo-,

6-

Jun

, Jul

1 Jan

I Feb

I Mar

I Apr

I May

I Jun

(6)

4-

ll-kMOt@OtO8tWOn~

(6)

3lb rpormiatlng

2l-

0’

I Jun I

I I Jul Aug p~metogsnda

I 6ep

1 I 1 Oot Nov Dee grnW0gene~~ I

I Jan I

I I Fob Mu Ipawning

1 Apr

I 1 May Jun P-t I atmwnho I

FIG. 1. Seasonal changes of plasma levels of testosterone and 1I-ketotestosterone and percentage of spermiating males (white bars at the bottom) of male sea bass, kept under natural conditions of photoperiod and temperature. The number of fish per group is indicated in parentheses. Vertical bars represent means f SEM. Significant differences are reported in the text.

364

PRAT

SPECIFICITY

Antiserum Anti-testosterone”

Anti-l I-ketotestosterone’

Anti-17B-estradiol”

Anti-17a-hydroxy-20B-dihydroprogesteroneb

OF THE

ET

AL.

TABLE

1

ANTISERUM

USED

IN THE ASSAYS

Steroid Testosterone 1I-Ketotestosterone Sa-Dihydrotestosterone Androstendione 5a-Androstan-3B,17f3-diol Sa-Androstan-3a,17B-diol 11B-Hydroxyandrostendione 1IB-Hydroxytestosterone De.hydroepiandrosterone 17B-Estradiol 17-Hydroxy-20B-dihydroprogesterone 1l-Ketotestosterone Testosterone 1lB-Hydroxytestosterone Sa-Androstan-17B-ol-3-one Dehydroepiandrosterone Androstendione Androsterone Sa-Androstan-3B,17$-diol Sa-Androstan-3a,l7B-diol 17B-Estradiol 17a-Hydroxy-20Bdihydroprogesterone 17B-Estradiol Estrone 6-Ketoestradiol-17B 16Ketoestradiol-17@ IbEpiestriol Estriol 17aEstradiol Testosterone 1 I-Ketotestosterone 17a-Hydroxy-20Bdihydroprogesterone 17a-Hydroxy-20Bdihydroprogesterone 17a-Hydroxy-2Oadihydroprogesterone 20g-Dihydroprogesterone Sa-Pregnan-3a-ol-2&one Progesterone 1 la-Hydroxyprogesterone 1I-Ketoprogesterone 17a-Hydroxyprogesterone Testosterone 1l-Ketotestosterone 17B-Estradiol Estrone

Cross-reaction (94 100.0 31.0 40.7 14.0 2.2 6.2 0.2 co.1