Journal of Fish Biology (2000) 57, 1563–1574doi:10.1006/jfbi.2000.1409, available online at http://www.idealibrary.com on

Effects of polyunsaturated fatty acids and gonadotropin onprostaglandin series E production in a primary testis cell

culture system for the European sea bass

J. F. A*†, L. A. S*, S. Z*‡ M. C**Instituto de Acuicultura de Torre de la Sal (CSIC), Ribera de Cabanes, 12595

Castellon, Spain and †Departamento de Produccion Animal y Ciencia de los Alimentos,Universidad Cardenal Herrera CEU. Av. Seminario, s/n. 46113 Moncada (Valencia),

Spain

(Received 22 January 2000, Accepted 21 July 2000)

Prostaglandin series E (PGE) production by mixed populations of isolated sea bassDicentrarchus labrax testicular cells was measured after culture at 20� C in L-15 for up to 24 hin the absence or presence of arachidonic acid (AA), eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA) and/or gonadotropin (hCG). AA stimulated a significant dose-and time-dependent increase in PGE production. EPA stimulated PGE production only duringthe first 6 h although levels were 26·4% those induced with AA; DHA reduced PGE production.Although hCG alone had no effect, it enhanced AA-induced PGE formation and suppressedEPA-induced PGE production. These results suggest that essential PUFAs modulate signifi-cantly testicular PGE production in vitro and therefore may have important effects onsteroidogenesis and spermiation in the male European sea bass.

� 2000 The Fisheries Society of the British Isles

Key words: fatty acids; gonadotropin; prostaglandin; testis; Dicentrarchus labrax.

‡Author to whom correspondence should be addressed. Tel.: 34 964 319500; fax: 34 964 319509; email:zanuy@iats.csic.es

INTRODUCTION

Ellis & Baptista (1969) demonstrated the relationship between the lipidperoxidation and the synthesis of prostaglandins (PGs) and androgens in the rattestis. Subsequent studies have shown that testicular steroidogenesis is regulatedby PGs from series E and F both in mammals (Abbatiello et al., 1975; Chinoyet al., 1980) and in amphibians (Gobbetti et al., 1992). In fish, several studieshave demonstrated the role of PGs on gonadal steroidogenesis and ovulation(Stacey & Goetz, 1982; Goetz et al., 1989a,b; Goetz & Garczynski, 1997).

The relationship between the composition of fatty acids of the diet andreproduction has been demonstrated in several species of teleosts (Luquet &Watanabe, 1986; Sargent et al., 1989). Polyunsaturated fatty acids (PUFAs) ofthe n-3 series, particularly eicosapentaenoic acid (EPA; 20 : 5n-3) anddocosahexaenoic acid (DHA; 22 : 6n-3) influence gonadal hormone levels,fecundity, egg fatty acid content, egg quality, hatching and survival ratios in theEuropean sea bass (Navas et al., 1997, 1998; Bruce et al., 1999). However, onlya few studies (Sorbera et al., 1998; Asturiano et al., 1999, 2000; Bruce et al.,1999) describe the influence of series n-6 PUFAs (i.e. AA; 20 : 4n-6) on the

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0022–1112/00/121563+12 $35.00/0 � 2000 The Fisheries Society of the British Isles

1564 . . .

reproduction of teleosts. Moreover, little is known about the physiologicalmechanisms involved in regulation within the fish testis.

At the cellular level, PUFAs and their cyclooxygenase and lipoxygenasemetabolites can have different modulatory effects on the gonadal metabolism ofsteroids (Boone et al., 1993; Murdoch et al., 1993; Nunez et al., 1995). Studiesusing freshwater fish have shown that AA, through conversion to PGs, stimu-lates testosterone production in goldfish Carassius auratus L. ovaries and testis(Van Der Kraak & Chang, 1990; Wade & Van Der Kraak, 1993a; Mercure &Van Der Kraak, 1996) while other PUFAs, including EPA and DHA, attenuategonadotropin-stimulated steroid production in both the goldfish testis and troutand goldfish ovary (Wade et al., 1994; Mercure & Van Der Kraak, 1995).However, even though EPA, DHA and AA are the major lipid components ofmarine fish tissues, the role of PUFAs and eicosanoids in testicular and ovarianphysiology of the marine teleosts is still unclear.

In vitro systems allow for a more direct study of testicular steroidogenesis thanin vivo experiments. However, until now, the protocols for in vitro culture ofEuropean sea bass Dicentrarchus labrax (L.) testis were limited to techniquesdeveloped for short-term culture of whole tissue (Colombo et al., 1978). The aimof this study was to use an in vitro system developed for the culture of a mixedpopulation of European sea bass testicular cells to determine the influenceof different PUFAs (AA, EPA and DHA) and/or gonadotropin on PGEproduction in the testis of this marine species.

MATERIALS AND METHODS

FISHThree- to four-year-old male European sea bass were maintained under natural

photoperiod and temperature conditions in the Instituto de Acuicultura de Torre de laSal (east coast of Spain, 40� N 0� E) and held in 2000-l tanks supplied with aeratedflow-through sea water (37·8 g l�1 salinity). Fish were hand fed once a day, 3 days aweek with a diet consisting of Boops boops L. at the rate of 2·2% body weight.

TESTIS EXTRACTIONAll experiments were conducted during October and November when the sea bass male

reproductive cycle has been activated already by sex steroids although the quantity ofsperm is still very low allowing for cleaner cell isolation procedures.

In each experiment, testicular tissue from three males (852�23 g) was used. Maleswere killed by immersion in ice prior to decapitation. Testes were extracted under sterileconditions and placed in sterile ice cold calcium- and magnesium-free sea bass Ringers’(SBR), containing (in m): NaCl 130, KCl 5, Na2HPO4 1, HEPES 25, glucose 5, 0·5%BSA fraction V, and 1% (v/v) solution of penicillin (100 IU l�1) and streptomycin(100 mg l�1), titrated to pH 7·4. All subsequent procedures were performed understerile conditions. The following protocol is based on the methods for the isolation andculture of trout testes cells (Loir, 1988a, b; Loir & Sourdaine, 1994), modified for thedevelopment of testes cell cultures of a marine species such as the European sea bass.

ENZYMATIC CELL DISPERSIONTestes were washed three times in SBR to remove blood, and most of the connective

tissue was dissected away. After the final washing, tissue was cut into small pieces(1–3 mm) and transferred to 15 ml tubes (Falcon, Becton Dickinson, Lincoln Park, NJ,

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U.S.A.) (2–4 g tube�1) containing collagenase type IX (Sigma, St Louis, MO, U.S.A.) inSBR (0·8 mg ml�1) and incubated for 30 min at room temperature while shaking gently.Then tissue was filtered through a 40 �m cell strainer (Falcon) and washed with SBR toeliminate the collagenase. All tissue collected in the filter was recovered in 10 ml of SBRand cells were dispersed mechanically by gentle trituration using a plastic pipette. Asecond filtration through a 40 �m cell strainer was performed using 20 ml of SBR andcells were recovered into a 50 ml tube. Cell viability was determined using Trypan blue(germ cells were not counted). Then the cells were centrifuged at room temperature for20 min at 670 g and resuspended in Leibovitz’s L-15 (Sigma) with added HEPES 20 m,0·1% BSA fraction V, 1% (v/v) solution of penicillin (100 IU l�1) and streptomycin(100 mg l�1) solution, and 10% fetal bovine serum (FBS; Sigma) titrated to pH 7·4.

INCUBATIONCells were plated in 24-well culture plates (Falcon) in L-15+FBS at a density of

290 000 cells ml�1 and preincubated overnight at 20� C in a Memmert incubator. Thefollowing morning, L-15+FBS was removed and L-15 without serum was added; cellswere allowed to recover for 10–15 min in the incubator after which fatty acids and/orgonadotropin were added to appropriate wells and cells incubated according to theexperimental paradigm. The initial dilution of fatty acids (AA, EPA or DHA; Sigma)was made with absolute ethanol and subsequent dilutions with L-15 media. Humanchorionic gonadotropin (hCG; Profasi HP, 2500 IU) was diluted to appropriateconcentrations using SBR and L-15. Each treatment and time point was performed intriplicate and media samples were maintained at �80� C until analysis.

PG ANALYSISPGE was measured from media samples using the commercial PGE2 EIA kit

(Prostaglandin E2 Enzyme Immunoassay Kit; Cayman Chemical). The specificity valuesfor the EIA were: PGE2 100%, PGE3 43%, PGE1 18·7%, PGE2� <0·01% and AA <0·01%.Analyses were developed to ensure that there was no interference with the L-15 culturemedium nor with the ethanol used for the initial dilution of fatty acids.

Polyclonal goat anti mouse IgG (Fc specific; absorbed with bovine, horse and humanserum proteins; Sigma) at 10 �g ml�1 in 0·05 phosphate buffer was used to coat themicrotitration plates (Nunc Maxisorp). After adding 200 �l well�1 of the antibodysolution, plates were incubated overnight at room temperature. The next day, theantibody solution was recovered and 300 �l well�1 of EIA buffer (potassium phosphatebuffer 0·1 , pH 7·4, containing 0·01% sodium azide, 0·4 NaCl, 0·001 EDTA and0·1% BSA, fraction V; Sigma) was added and plates were incubated for at least one nightat 4� C. The antibody solution recovered previously was used for coating a second groupof plates adding 200 �l well�1, maintaining the plates overnight at room temperature,and adding 100 �l well�1 of saturation buffer (potassium phosphate buffer 0·1 , pH 7·4,containing 0·03% sodium azide, 0·4 NaCl, 0·001 EDTA and 0·3% BSA, fraction V;Sigma). Comparative analyses were performed using plates from the two batches withoutfinding differences in the results.

The remainder of the assay was carried out according to the instructions of theCayman Chemical EIA kit, described by Pradelles et al. (1985, 1990). PGE2 recovery wasmeasured by preparing a wide range (from 1000 to 15 pg ml�1) of PGE2 standards inL-15 medium. Samples were assayed as described above and the mean percentage ofrecovery was 93·5%. The resulting displacement curve for diluted samples was parallel tothat of the PGE2 standard (Fig. 1). The interassay coefficient of variation was 10·59%(n=23).

STATISTICSAll the treatments were assayed in triplicate and experiments were repeated a minimum

of twice. Results are expressed as mean�standard error (..). A one-way ANOVA wasapplied to data followed by a Student Newman–Keuls test for multiple comparisons.

1566 . . .

When normality or equal variance failed, a one way ANOVA on ranks was used followedby a Dunn’s test. The effect of different fatty acids was compared using a t-test.Differences were considered significant at P<0·05.

RESULTS

CELL YIELD AND VIABILITYThe method of enzymatic dispersion described yielded a mean of

107 000�28 000 cells mg�1 testicular tissue. The percentage of viable cellsafter dispersion was 94·1�1·7%. Cells survived for up to 100 h of incubationat 20� C maintaining good morphological aspect and physiological activity,actively producing both prostaglandins and testosterone (data not shown). Thedispersed population included several cell types, including Leydig, Sertoli andgerm cells as assessed according to the criteria of Loir (1988a,b, 1989) andby immunocytochemistry using antibodies against testosterone following celldispersion and plating.

01000

100

PGE2 (pg ml–1)

B:B

o (%

)

0 800600400200

80

60

40

20

F. 1. Binding curves for the PGE2 standard (obtained as a serial dilution of PGE2 in EIA buffer; �) anddifferent dilutions of PGE2 in L-15 medium (�).

EFFECTS AA, EPA AND DHA ON THE PGEs PRODUCTIONIn the first experiments, testis cells were incubated in the presence of different

doses (3–300 �) of AA, EPA or DHA and samples were collected from 0·75 to24 h. The mean levels of PGE basal secretion observed from all experiments was55·03�14·16 and 81·79�21·53 pg ml�1 at 12 and 24 h, respectively. AAstimulated a significant dose- and time-dependent increase in PGE production[Fig. 2(a)].

EPA (1–100 �) also stimulated significant dose-dependent increases in PGEproduction during the first 6 h which declined gradually to control levels by 24 h[Fig. 2(b)]. EPA at a dose of 300 � also increased PGEs although levels weresubmaximal to the 100 � dose.

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Time of incubation (h)

1.50.75

3 6 12 24

}0

1200

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(c)

c

b

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b

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F. 2. Levels of PGE obtained after treatment of primary sea bass testis cultures with differentdoses (3, 30, 100 and 300 �) of (a) AA, (b) EPA, or (c) DHA and the respective controls.Results are expressed as mean�.. Different letters indicate significant differences between treat-ments within one time point of incubation. �, Controls; �, 3 �; �, 30 �; �, 100 �; , 300 �.

Only the highest dose of DHA (300 �) stimulated PGE production signifi-cantly above control levels at all the times of incubation [Fig. 2(c)]. AlthoughDHA at a dose of 100 � increased levels slightly but significantly during the first3 h, 3 and 30 � had no effect.

Given that the effect caused by 300 � DHA or EPA did not follow adose-dependent pattern, and because 100 � was the dose stimulating maximumPGE production in most cases, the degree of secretion of PGE caused by 100 �

1568 . . .

AA, EPA or DHA at 12 and 24 h was compared also. Addition of AA (100 �)increased PGE levels significantly, that reached over 23-fold with respect to thecontrol at 12 and 24 h [Fig. 3(a)]. AA-stimulated PGE levels were significantlyhigher than those induced by EPA or DHA both at 12 and 24 h.

PGE secretion was significantly higher in EPA (100 �)-treated wells at 12 h incomparison with basal level [Fig. 3(b)]. However, EPA-stimulated PGE levelswere just 29·45% that of those stimulated by AA. Levels were similar to controls24 h after the addition of EPA to the cells [Fig. 3(b)], being just 3·55% that ofthose stimulated by AA.

DHA-stimulated PGE levels were lower than those induced by AA or EPA at12 and 24 h. DHA (100 �)-treated cultures showed PGE levels significantlylower than controls (53·8 and 50·18% at 12 and 24 h, respectively) [Fig. 3(c)].

0

250

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**

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500

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6000

4000

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(a)

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12 24

Time of incubation (h)

F. 3. Levels of PGE of primary sea bass testis cultures in the absence and presence of 100 �(a) AA, (b) EPA, or (c) DHA at 12 and 24 h. Results are expressed as mean�.. Asterisksindicate significant differences as compared with controls ().

EFFECTS OF GONADOTROPIN ON PGE PRODUCTIONIn the first experiments, sea bass testis cells were incubated in the presence of

hCG at different concentrations (0–1000 IU ml�1) and the levels of PGE weremeasured from media samples collected from 0 to 24 h of incubation.

1569

At all time points, levels of PGE in the presence of hCG were similar to thosefound in untreated controls with the exception of levels found at 6 h with 100 and1000 IU ml�1 hCG which were significantly higher than controls (Fig. 4). Asignificant increase in PGE concentration was observed over time of incubationalthough this increase was independent of treatment.

Two other experiments revealed no significant differences in PGEproduction between hCG (100 IU ml�1)-treated and control cultures (datanot shown).

024

300

Time of incubation (h)

PG

E (

pg m

l–1)

0 1.5 3 6 12

250

200

150

100

50

Control0.11101001000

* *

F. 4. Levels of PGE obtained from primary sea bass testis cultures in the absence and presence ofdifferent doses (0·1, 1, 10, 100 and 1000 IU ml�1) of hCG. Results are expressed as mean�..Asterisks indicate significant differences as compared with controls within one time point ofincubation.

COMBINED EFFECTS OF PUFAs AND GONADOTROPIN ON PGEsPRODUCTION

After 12 and 24 h, AA stimulated a significant increase in PGE with respect tothe control and the addition of hCG together with the AA resulted in asignificantly enhanced effect on PGE secretion, while hCG alone caused nosignificant differences in PGE production [Fig. 5(a)].

EPA increased PGE production significantly with respect to the control at12 h. However, the presence of hCG produced no significant differences with thecontrol, but suppressed EPA-stimulated PGE production. At 24 h, EPA-treatedcultures showed PGE levels similar to controls and hCG-treated cultures, whileEPA+hCG-treated cells displayed significantly lower PGE concentrations[Fig. 5(b)].

Cultures treated with DHA or DHA+hCG at 12 and 24 h both producedsignificantly lower concentrations of PGE as compared with controls andhCG-treated cultures [Fig. 5(c)].

1570 . . .

ControlhCGPUFAPUFA + hCG

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F. 5. Levels of PGE obtained at (a) 12 and (b) 24 h after treatment of primary sea bass testis cultureswith PUFAs (AA, EPA or DHA 100 �) and/or hCG (100 IU ml�1) and the respective controls.Results are expressed as mean�.. Different letters indicate significant differences betweentreatments.

DISCUSSION

The present study reports for the first time a method for dispersing andculturing a mixed population of European sea bass testicular cells. The methodyielded a high efficiency (c. 105 cells mg�1), a high percentage of viable cells(c. 94%) and cells survived up to 100 h at 20� C. Results obtained using thismethod suggest that essential PUFAs modulate testicular PG series E productionin vitro significantly and therefore, may have important effects on steroidogenesisand spermiation in the male European sea bass.

PGE2 is derived from AA via cyclooxygenase and therefore the availability ofAA is the limiting factor for PGE2 and also PGF2� production. This has beendemonstrated extensively in the ovary of teleosts (Tan et al., 1987; Goetz et al.,

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1989b; Van Der Kraak & Chang, 1990; Mercure & Van Der Kraak, 1996).Moreover, in mammals, cyclooxygenase is synthesized constitutively (Morris &Richards, 1993; Creminon et al., 1995) and its activity is limited by the substrateavailability (Mercure & Van Der Kraak, 1996). The present study demonstratedthis in the European sea bass testis. Addition of exogenous AA stimulated asignificant dose- and time-dependent increase of PGE production.

EPA competes with AA as a substrate for cyclooxygenase which results in areduction in PGE2 and PGF2� levels and an increase in synthesis of PGE3

(Zhang et al., 1992). In the present study, the addition of EPA stimulatedsignificantly the production of PGE. At 12 h, EPA (100 �) stimulated PGElevels that were 29·45% of those observed in the presence of AA. Since EPA isthe precursor of trienoic eicosanoids and the EIA used in the present study forthe PGE2 analysis has a 43% cross reactivity with PGE3, it is probable that theincreased PGE levels observed with EPA were in fact PGE3.

On the other hand, although a slight but significant increase in PGE levels wasobserved with 100 � DHA during the first 3 h of incubation, doses of �100 �,reduced PGE production with respect to the controls at 12 and 24 h of incubation.DHA at a dose of 300 � stimulated the synthesis of PGE at all incubation timepoints. In spite of the high percentage of this PUFA in fish cell membranes, noDHA metabolites from the cyclooxygenase pathway are known (German et al.,1983). The high 300 � dose of DHA could be considered a pharmacologic effectattributed to the hydrophobicity and lability of fatty acids at high doses (Goetzet al., 1989b; Delton et al., 1995; Mercure & Van Der Kraak, 1995). This mustalso be considered with the results obtained with 300 � AA and EPA.

In the present study, basal production of PGEs was not altered by hCG(Figs 4 and 5). In rat Leydig cells, PGE2 metabolism from AA can be stimulatedby LH (Molcho et al., 1984). However, studies in goldfish preovulatory ovarianfollicles (Kellner & Van Der Kraak, 1992; Mercure & Van Der Kraak, 1996) ormature testis (Wade & Van Der Kraak, 1993b) did not find hCG stimulation ofPGE2 production. Wade & Van Der Kraak (1993b) proposed that regulation oftestis steroidogenesis differs in fish and mammals. Wade & Van Der Kraak(1993a) observed a stimulatory effect of hCG on cAMP production in thegoldfish testis supporting previous studies in teleost gonads which suggested aninfluence of gonadotropin on steroidogenesis via activation of adenylate cyclasesimilar to that observed in mammals (Salmon et al., 1984; Tan et al., 1986;Kanamori & Nagahama, 1988; Van Der Kraak, 1992). Moreover, studies haveshown that an increase in cAMP accumulation using agents such as forskolin orIBMX inhibited PG production (Tan et al., 1987; Kellner & Van Der Kraak,1992). In the present experiments, it is possible that the testes used for cellisolation were in an early reproductive condition and intracellular cAMPaccumulation was relatively low due to an activated state reflected in thebasal levels of secretion obtained. Further stimulation of the adenylate/cyclasesignalling pathway with hCG may have been impossible or undetectable.

Treatment of sea bass testis cultures with hCG+AA resulted in enhancedPGE production. In the mammalian ovary, in addition to constitutivecyclcooxygenase, a second form of cyclooxygenase is induced by gonadotropin(Morris & Richards, 1993; Creminon et al., 1995). If the fish testis presents asimilar mechanism, it is possible that hCG is increasing the inducible form of

1572 . . .

cyclo-oxygenase. Moreover, as in some cases with the inducible form, it ispossible that both the substrate and enzyme are the limiting factors. This couldexplain the finding that combined treatment with AA and hCG had greater effecton the synthesis of PGE as compared with AA or hCG alone (Fig. 5). WhenhCG is added alone, it could still induce inducible cyclooxygenase, but then onlya slight effect on the PGE2 production would be observed, which confirms theidea that AA availability is the limiting factor.

In contrast, cultures treated with EPA+hCG or DHA+hCG exhibited lowerPGE levels than cultures treated with these PUFAs alone. In the case of EPA, anyfurther stimulation of AA by hCG may have depleted the endogenous substratetherefore leaving only EPA as an available cyclooxygenase substrate. This wasshown by the lower PGE levels obtained possibly reflecting predominately PGE3

production. Accepting the existence of a gonadotropin-inducible cyclooxygenasein the fish testis, results suggest that the activation of this second form of theenzyme could coincide with a reduction in activity of the first constitutive form,limiting the synthesis of PGE3 from EPA and having specific action over AA,increasing the synthesis of PGE2. This could explain the observed differentialeffects of hCG on EPA and DHA stimulated PGE production.

In summary, the dispersion and culture system for primary sea bass testis cellsdescribed was functional and provided results indicating that PUFAs are capableof regulating PGE production. Additional studies are underway examining theeffects of these PUFAs on steroidogenesis to elucidate further their role inthe regulation of male sea bass reproduction.

This study was supported by EEC (AIR-CT93-1005) and CICYT (MAR96-1859) toM.C. J.F.A. was supported by the Generalitat Valenciana.

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