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Biology of Reproduction 64, 382-389 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Polyunsaturated Fatty Acids and Prostaglandins on Oocyte Maturation in a Marine Teleost, the European Sea Bass (Dicentrarchus labrax)1

Lisa Ann Sorberaa, Juan Francisco Asturiano3,a, Manuel Carrilloa, and Silvia Zanuy2,a

a Instituto de Acuicultura (Consejo Superior de Investigaciones Científicas), Torre de la Sal, 12595 Castellón, Spain

ABSTRACT

The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A2 blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE2, or PGF2{alpha}. PGE2 and PGF2{alpha} alone were both effective stimulators of maturation, while PGE1 and PGE3 were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.

follicle, oocyte development, ovary

INTRODUCTION

When examining their involvement in reproduction, lipids are most often considered from an energetic point of view. Thus, their possible role in other physiology processes is often overlooked despite the fact that it is now well established that polyunsaturated fatty acids (PUFAs) and their metabolites produced from cyclooxygenase and lipoxygenase pathways can have different modulatory effects on steroid metabolism or function in a number of other systems [1–3]. Arachidonic acid (AA; 20:4n–6) has been shown to influence gonadal steroidogenesis in mammals and birds [4–7]. In mammals, FSH not only stimulates progesterone release but also PGE2 production in granulosa cells [8–10] via AA metabolism, and it has been shown that PUFAs have inhibitory effects on the production of progesterone and PGE2 in bovine luteal cells in vitro [11]. However, very little information is available on the involvement of eicosanoids in the process of oocyte maturation. In order for oocyte maturation to occur, it is known that gonadotropin (GTH) must stimulate follicular production of a maturation-inducing steroid (MIS) in fish and amphibians, while sterols have been implicated in mammalian maturation [12–15]. In mammals, the mid-cycle LH surge responsible for final oocyte maturation and ovulation is accompanied by increased levels of eicosanoids produced in the follicle prior to ovulation [16] and several studies have suggested that prostaglandins (PGs) are the mediators of ovulation [17–20].

Determination of the role of PUFAs in reproductive physiology and oocyte maturation in particular has been difficult due to the lack of evident changes in these levels during the mammalian reproductive cycle. In contrast, the lipid composition in fish is seasonal and closely related to reproductive status [21, 22] and, as a result, studies have clearly shown that the fatty acid composition of the diet can affect the teleost pituitary and gonadal hormone levels and reproductive performance [23–28]. Polyunsaturated fatty acids can also act at the cellular level to modulate gonadal steroid production in a manner similar to that observed in mammals. Studies using freshwater fish have shown that AA, through conversion to eicosanoids, stimulates testosterone production in goldfish ovaries and testis [29–31], while other unesterified, long-chain PUFAs, including eicosapentaenoic acid (EPA; 20:5n–3) and docosahexaenoic acid (DHA; 22:6n–3), attenuate GTH-stimulated steroid production in both goldfish testis and trout and goldfish ovary [32, 33].

However, even though EPA, DHA, and AA are major lipid components of marine fish tissues, the role of AA, other fatty acids, and eicosanoids in ovarian physiology of the marine teleost is lacking [21]. PGE2 has been shown to be involved in the effects of AA on steroidogenesis in the goldfish [31] and several studies have demonstrated that PGE2 is produced by the ovaries of freshwater species such as trout and goldfish [31, 34, 35]. Other studies in another freshwater species, the yellow perch, suggest that PGs (specifically PGE2 and PGF2{alpha}), although synthesized by follicles in response to AA or MIS, are ineffective or only marginally effective in stimulating ovulation [36]. However, the involvement of these PGs in final maturational processes has been overlooked, especially in marine teleosts.

The present study used the European sea bass (Dicentrarchus labrax), a marine teleost with group-synchronous ovarian development, as a model in an attempt to contribute further to our understanding of the involvement of PUFAs in the process of oocyte maturation. In particular, we examined 1) the effects of AA, EPA, DHA, oleic acid alone and in combination with hCG on oocyte maturation; 2) the involvement of cyclooxygenase metabolites PGE2, PGF2{alpha}, PGE3, and PGE1 on the action of AA; 3) the involvement of lipoxygenase products in the process of maturation; 4) whether phospholipase A2 (PLA2) is involved in AA metabolism; and 5) the hCG-induced maturational behavior and production of PGE2 by oocytes obtained from females fed a natural diet compared with those fed a fatty acid-enriched diet with a lower n-3:n-6 ratio and lower EPA content.

MATERIALS AND METHODS

Ethics

Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Animals

For in vitro studies, 4- to 6-yr-old, mature female sea bass (Dicentrarchus labrax) were held in 2000-liter tanks with flow-through sea water, maintained under natural conditions of photoperiod and temperature, and fed a standard, natural diet consisting of bogue (Boops boops) administered three times a week, supplemented with squid (Loligo vulgaris) one time per week supplied at a ratio of 3:1. For in vivo studies, a different group of females (3- to 4-yr-old) were separated into two groups: one was fed an enriched diet (RD; n = 12), the other was fed a natural diet (ND; n = 10) and were maintained under the same conditions of photoperiod and temperature. Although the ND group received the same natural diet just described, the RD group was first fed with a commercial 7-mm pelleted diet enriched with northern hemisphere fish oil (EWOS Technology Centre, Livingston, UK) for 1 yr (June 1994–June 1995). This diet emulated the most successful diet in previous work in terms of sea bass female reproductive performance [23–28]. From June 1995 until the end of the sampling period, May 1997, the RD group received a pelleted diet that was enriched with tuna orbital oil (Ropufa 30, Roche Product, Heanor, UK), which is relatively low in EPA and contains significantly more AA than standard fish oil. The ND and RD diets had similar energetic values (5.95 and 5.34 kcal/g for the bogue and squid components of ND, respectively, and 5.62 kcal/g for RD). The protein content (% dry weight) of the bogue and squid components of ND were 71% and 79%, respectively. Lipid content (% dry weight) of ND was 20% and 8% in bogue and squid, respectively, and carbohydrates were detected only in the squid component (2%); ash content was 9% and 8% dry weight for bogue and squid, respectively. The protein, lipid, carbohydrate, ash, and fiber contents (% dry weight) of RD were 53%, 21%, 15%, 10%, and 1%, respectively. Differences in n-3 and n-6 PUFA compositions of the diets are shown in Table 1. Fatty acids from the diets were analyzed by capillary gas chromatography as described by Navarro et al. [37]. Individual saturated and monounsaturated fatty acids were similar in each diet, although total saturated fatty acids in the ND diet was approximately 32% (bovine)/40.3% (squid) of total fatty acids compared with 28.3% in the RD diet and total monounsaturated fatty acids was approximately 17.4% (bovine)/13.6% (squid) of the total fatty acids in the ND diet and 30.0% in the RD diet. Fish were hand-fed 4 days a week, once a day at 1.1% body weight (RD) or 2.2% in the ND group to compensate for the higher water content of the ND diet.


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  		TABLE 1. Fatty acid composition of experimental diets (as % of total fatty acids).a

Hormones and Chemicals

Unless otherwise stated, all chemicals and fatty acids were purchased from Sigma Chemical Company (St. Louis, MO). Lyophilized hCG (Laboratorios Serono, S.A., Madrid, Spain) was reconstituted to the appropriate concentrations prior to use in modified sea bass Ringers (SBR) [38, 39]. Media used for follicle isolation and incubation consisted of the following (in mM): 130 NaCl, 5.0 KCl, 1.0 Na2HPO4, 1.0 NaHCO3, 1.0 MgSO4, 2.0 CaCl2, 25 Hepes, and 5.0 glucose titrated to pH 7.4 or pH 7.5 (depending upon the absence or presence of exogenously added PGs) with NaOH. All fatty acids and PGs (Cayman Chemicals, Ann Arbor, MI) were of 98% purity or greater. Indomethacin, nordihydroguaiaretic acid (NDGA), and quinacrine were first dissolved in ethanol and subsequently diluted with SBR. The final concentrations of ethanol in follicle incubations did not exceed 0.1%, which had no influence on basal or stimulated maturation. Control incubations contained ethanol at the appropriate concentrations.

Harvesting of Follicle-Enclosed Oocytes

For in vitro studies, female fish (n = 4–6) were anesthetized with 3-aminobenzoic acid ethyl ester, methanesulfonate salt (MS-222; 100 mg/L), and small fragments of ovary were obtained via cannulation during the breeding season (December–April). For in vivo studies, different individual females (n = 6) were cannulated once a month (December–April) from the two diet groups. The cannulation procedure involved inserting polyethylene tubing into the oviduct for a distance of approximately 5 cm (depending on the length of the ovary) from the cloaca to the mid portion of the ovary and the oocytes with intact follicles were orally sucked out as the cannula was withdrawn. Oocytes with intact follicles were separated from the ovarian tissue by gentle agitation and, if necessary, with watchmaker's forceps and placed in ice cold SBR. Oocytes were selected manually due to the group-synchronous nature of ovarian development in this species. Only oocytes with an opaque cytoplasm and no indication of yolk clarification or lipid droplet coalescence, indicating a prematurational, postvitellogenic (500–800 µm in diameter) stage [39–44], were chosen for incubation. In these oocytes with intact follicles, the yolk vesicle occupied the entire ooplasm and the appearance was uniform. Oocytes with diameters of <400 µm do not undergo maturation in this system [39, 45, 46].

Follicle Incubation

For each experiment, oocytes with intact follicles were harvested from 4–6 females and pooled. Follicles were placed in a 24-well plastic dish (10–20 follicles per well; Falcon 3047, Becton Dickinson, Lincoln Park, NJ) containing 1 ml/well of SBR with added gentamicin (0.1 mg/ml). Follicles were allowed to stabilize at 18°C for 30 min prior to treatments. Inhibitors (NDGA, indomethacin, quinacrine) were added 15 min before maturation-inducing compounds (hCG, AA, PGs) to facilitate the action of inhibitors. Oocytes were then incubated at 18°C in air for up to 120 h, according to each experimental paradigm. Three replicate incubates were performed for each treatment. Determination was made of percent maturation, which included both mature/preovulatory and ovulated oocytes. Maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown [39]. In the absence of a purified species-specific GTH and because hCG was actively used in vivo in this species [44], this study employed a maximal dose of 100 IU/ml hCG as the maturation-inducing GTH. In addition, previous studies have shown that hCG at this dose in vitro results in similar maturation response time latencies as homologous sea bass pituitary extract [39, 45].

Incubation of oocytes with intact follicles from the RD and ND diet groups was performed as described earlier, however, in addition to assessing maturation, the percent atretic or degenerating follicles [39, 42] was determined from different populations of 100 oocytes at 1, 3, and 6 h after cannulation from each diet group. Media were collected after 72 h of incubation and stored at -80°C for subsequent analysis for PGE by ELISA.

PGE Determination

The protocol for the PGE ELISA was described by Asturiano et al. [47] using reagents and antisera from Cayman Chemicals (Ann Arbor, MI). The sensitivity for the assay was 7.8 pg/ml. Media from each replicate was assayed in duplicate. Cross-reactivities of the PGE2 antisera were = 100%, <43%, <18.7%, and <0.01% for PGE2, PGE3, PGE1, and PGF series, respectively.

Statistical Analysis and Calculations

Percentage data were subjected to an arcsine transformation prior to statistical analysis [48]. PGE values were normalized to PGE production in picograms per oocyte and expressed as the percent increase over basal production. Student's t-test for paired data and ANOVA were used to compare treated groups with their corresponding control groups over time of incubation. Post-ANOVA multiple comparison of means was carried out using Tukey's HSD test. In all cases, differences were accepted as statistically significant when P < 0.05. Within each experiment, all treatments (e.g., dose and times) were repeated in triplicate (i.e., three wells containing 10–20 follicles pooled from 4 to 6 females) and each experiment was repeated a minimum of three times using follicles pooled from different females each time. Data are expressed as the mean ± SEM. All dose-response figures illustrate percent maturation after 72 h of incubation, however, similar results were obtained after 24 and 48 h of incubation. The response time latency (T1/2) was calculated by subtracting the time (in hours) of the minimum percent response (i.e., spontaneous maturation) from the time (in hours) of the maximum response recorded and dividing by two. The ED50 refers to the minimum effective dose for a 50% response and was calculated by subtracting the percent of spontaneous maturation from the maximum response and dividing by two.

RESULTS

Effects of PUFAs Administered In Vitro on Oocyte Maturation

Experiments were conducted to investigate the effects of graded doses of PUFAs (3–300 µM) on oocyte maturation. Consistent with earlier studies [39, 45], spontaneous or basal maturation percentages were low (10.1% ± 2.4% after 72 h) while hCG successfully stimulated 89.9% ± 1.7% of the oocytes to mature after 72 h of incubation (Fig. 1). Saturation of the maturational response was achieved at 72 h, although experiments were continued for 120 h. Arachidonic acid administered alone at doses of 3, 30, and 100 µM also stimulated maturation in a time- and dose-dependent manner with maximum stimulation of 63.7% ± 4.1% observed with 100 µM. The high dose of 300 µM was less effective. The response time latency for 100 µM AA (T1/2 = 21.5 h) was more rapid than that of hCG (T1/2 = 25 h).



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  		FIG. 1. Time course of the effects of hCG (100 IU/ml) and graded doses of AA on oocyte maturation. Different letters indicate significant differences between treatments after 72 h of incubation, P < 0.0001

Because mobilization of AA can be achieved through direct hydrolysis of phospholipid by PLA2, we examined whether PLA2 was involved in AA induction of oocyte maturation by administering 10 µM of the a PLA2 inhibitor, quinacrine, to follicle incubations. Figure 2A illustrates that quinacrine significantly suppressed hCG-induced maturation. The quinacrine suppression of hCG-induced maturation could be partially yet significantly reversed by addition of free 100 µM AA, which induced 43.3% ± 2.1% maturation (Fig. 2B).



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  		FIG. 2. A) Effects of graded doses of hCG (IU/ml) in the absence and presence of 10 µM quinacrine (quin) on oocyte maturation after 72 h of incubation. Bar indicates spontaneous maturation in the absence of hCG (control). *Significant differences between treatment groups, P < 0.0001. B) hCG-Induced maturation (100 IU/ml) in the absence and presence of quinacrine (quin) and partial reversal with further addition of 100 µM AA after 72 h of incubation. Different letters indicate significant differences between treatments, P < 0.0001

Specificity of AA Induction of Oocyte Maturation

Next, the specificity of AA induction of maturation was examined. Results demonstrated that the maturation-inducing capability was unique to AA with an ED50 of 5 µM; EPA, DHA, and oleic acid were ineffective at all doses tested (Fig. 3A). To further demonstrate the possibility of differential action of fatty acids, the action of AA and other long chain PUFAs in the presence of hCG was examined. Figure 3B illustrates that AA at doses of 30 and 100 µM significantly enhanced hCG-induced maturation to 96.7% ± 1.7% and 95.8% ± 1.7%, respectively, compared with maturation induced by hCG alone; no significant differences were observed between maturation with hCG alone or in the presence or lower (0.03, 0.3 or 3 µM) AA doses (data not shown). Enhanced maturation was also observed when submaximal doses of hCG were used (data not shown). In contrast, EPA and DHA significantly attenuated hCG-induced maturation with ED50 of 19 and 40 µM, respectively. AA was also inhibitory (75.8% ± 6.0% for AA vs. 89.9% ± 1.7% for hCG), but only at the highest dose tested. Oleic acid had no significant effect (data not shown).



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  		FIG. 3. A) Effects of graded doses of AA, EPA, DHA, and oleic acid on oocyte maturation after 72 h incubation. Bar indicates spontaneous maturation (control). *Significant differences from spontaneous maturation, P < 0.0001. B) Effects of graded doses of AA, EPA, and DHA in the presence of 100 IU/ml hCG on oocyte maturation after 72 h of incubation. Bar indicates maturation in the presence of 100 IU/ml hCG. *Significant differences from hCG-induced maturation (hCG), P < 0.05

Involvement of Cyclooxygenase and Lipoxygenase Products in AA-Induction of Oocyte Maturation

Results obtained from this study also suggest that the metabolites of AA from cyclooxygenase, lipoxygenase, or both may also be involved in oocyte maturation. Figure 4 illustrates that indomethacin (10 µM), a specific cyclooxygenase inhibitor, completely suppressed hCG-induced maturation in addition to inhibiting basal spontaneous maturation to 3.3% ± 1.7%; addition of free AA (100 µM) partially reversed the indomethacin block of hCG-induced oocyte maturation and, moreover, indomethacin was also shown to significantly block AA-induced maturation (data not shown). On the other hand, Figure 5 shows that PGE2 and PGF2{alpha}, AA cyclooxygenase metabolites, administered alone at doses of 1, 100, and 1000 ng/ml significantly induced maturation in a time- and dose-dependent manner. The maximal maturation response was achieved with 1000 ng/ml PGE2 (84.4% ± 1.8%), which was comparable to hCG-induced maturation. PGF2{alpha} was slightly less potent, inducing only 71.1% ± 4.8% maturation. However, PGE3 and PGE1, which are cyclooxygenase metabolites of EPA and dihomo-{gamma}-linolenic acid, respectively, were ineffective. Further support for eicosanoid involvement in oocyte maturation was achieved when submaximal doses of 100 ng/ml PGE2 were tested and found capable of reversing the indomethacin block of hCG-induced oocyte maturation (73.3% ± 6.7% recovery; Fig. 6). PGF2{alpha} also reversed the indomethacin block only less potently with 33.3% ± 3.3% hCG-induced maturation observed in the presence of indomethacin (data not shown). It is interesting to note from Figure 6 that spontaneous maturation is not completely inhibited by indomethacin, which suggests that other factors may be also involved. Therefore, in the next experiment, maturation of oocytes was observed in the presence and absence of the lipoxygenase inhibitor, NDGA, which nonselectively inhibits various lipoxygenase pathways. Figure 7 shows that hCG-induced maturation in the presence of 10 µg/ml NDGA is significantly suppressed to a maximum maturation rate of only 40.2% ± 9.6%.



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  		FIG. 4. Effects of graded doses of hCG (IU/ml) in the absence and presence of 10 µM indomethacin (indo) on oocyte maturation after 72 h of incubation. Bar indicates spontaneous maturation in the absence of hCG (control). *Significant differences between treatment groups, P < 0.0001



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  		FIG. 5. Effects of graded doses of PGE2, PGF2{alpha}, PGE1, and PGE3 on oocyte maturation after 72 h of incubation. Bar indicates spontaneous maturation in the absence of hCG (control). *Significant differences from spontaneous maturation within a treatment, P < 0.0001



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  		FIG. 6. Effects of PGE2 (100 ng/ml) in the absence and presence of 10 µM indomethacin (Indo) in oocyte maturation after 72 h of incubation in the absence (control) and presence of hCG (100 IU/ml). Different letters indicate significant differences within each treatment group, P < 0.0001



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  		FIG. 7. Effect of NDGA (10 µg/ml) on spontaneous (control) and hCG-induced (100 IU/ml) maturation after 72 h of incubation. Different letters indicate significant differences between all treatments, P < 0.0001

Comparison of hCG-Induced Maturation in Follicles from Females Fed PUFA-Enriched and Natural Diets

Figure 8A illustrates the time course of the maturational response of oocytes from females in the RD and ND groups in the absence and presence of 100 IU/ml hCG. There was no significant difference in spontaneous maturation between the two groups. However, the RD group exhibited a significantly higher rate of hCG-induced maturation compared with the ND group (96.8% ± 1.1% vs. 79.6% ± 4.3%, RD and ND, respectively) at 72 h.



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  		FIG. 8. A) Percent maturation in the absence (basal maturation) and presence of hCG (100 IU/ml) after 72 h of incubation from oocytes obtained from fish fed an enriched diet (RD) and a natural diet (ND). Different letters indicate significant differences between all treatments, P < 0.001. B) Percent atresia observed 1, 3, and 6 h after cannulation in different populations of oocytes (n = 100) obtained from fish fed RD or ND. *Significant differences within each sampling time, P < 0.05

In addition, the percentage of atresia observed in RD and ND follicle populations was noted and is shown in Figure 8B. At 1, 3, and 6 h after cannulation, the ND group had a significantly higher percentage of atresia, ranging from 66.0% ± 17.0% to 77.3% ± 13.0% compared with the RD group (5.3% ± 0.3% to 9.7% ± 0.3%).

It should be noted that the maturational responses observed in the present in vivo study were not unique to this particular population of fish. Several preliminary experiments have shown similar responses using other fish fed similar diets or other high-lipid commercial diets (data not shown).

Comparison of PGE Production of Follicles from Females Fed PUFA-Enriched and Natural Diets

Follicle-enclosed oocytes obtained from the RD group also produced more PGE compared with the ND group after 72 h of incubation in the presence of hCG (Fig. 9). No significant differences were observed in basal levels of PGE production in the absence of hCG between oocytes from the different diet groups (16.1 ± 2.4 pg/oocyte). However, 72 h after addition of 100 IU/ml hCG, media collected from RD oocytes had a significantly higher percent increase in PGE production compared with oocytes from the ND group (158.0 ± 29.3 vs. 69.1 ± 18.6 pg/oocyte, RD and ND groups, respectively). Similar differences were also observed after 12, 24, 36, and 60 h of incubation in the presence of hCG (data not shown).



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  		FIG. 9. PGE production of follicle-enclosed oocytes from fish fed an enriched (RD) and natural (ND) diet after 72 h of incubation. PGE production expressed as percent increase of basal production. *Significant difference, P < 0.05

DISCUSSION

The results of the present study demonstrate a role for dietary essential fatty acids and their cyclooxygenase and lipoxygenase metabolites in oocyte maturation. AA was shown to stimulate oocyte maturation in a dose- and time-dependent manner. Results using quinacrine, a PLA2 blocker, suggest that GTH can mobilize AA possibly through PLA2, making free AA available as a potential stimulator of oocyte maturation in the sea bass. In addition, data obtained in this study indicate that GTH-induced maturation is indomethacin-sensitive, suggesting product mediation by cyclo oxygenase, lipoxygenase, or both. This action was indeed found to be in part mediated by cyclooxygenase products PGE2 and possibly PGF2{alpha}, and lipoxygenase products may also be involved because NDGA partially suppressed GTH-induced maturation. More experiments are needed in order to conclusively determine which fatty acids (hydroxy, hydroperoxy, or both) are involved. In contrast, EPA, which is known to compete with AA for cyclooxygenase action, and its cyclooxygenase metabolite, PGE3 were ineffective. In fact, when EPA was administered, hCG-induced maturation was attenuated, suggesting that when high levels of free EPA are present, AA induction of maturation is depressed. Similarly, an increased production of PGE3 would have no effect on maturation. Moreover, PGE1, a metabolite of linolenic acid, had no significant effect on oocyte maturation. The results obtained concerning the effects of PGs are consistent with other studies using goldfish and trout that showed that PUFAs actively effect testosterone production in the ovary and testis [29–33]. Thus, results from this study suggest that not only do unesterified long-chain PUFAs and their cyclooxygenase metabolites participate in the regulation of ovarian steroidogenesis, but also that they may be directly involved in oocyte maturation processes in marine teleosts.

The doses of PUFAs used in the follicle incubations described in this study may appear to be exceptionally high. However, these levels can be considered close to physiological in fish and are comparable to those doses used in other studies [21, 31–33]. In fact, the levels of unesterified PUFAs measured in fish [21] are actually higher than the doses tested in this study. The decreased maturation response induced by 300 µM AA could be attributed to the hydrophobicity and lability of high doses of fatty acids due to metabolism and esterification into complex lipids, resulting in a poor availability of the fatty acid to converting enzymes [34, 49]. A similar effect on steroidogenesis was observed with administration of high AA doses in freshwater teleosts [29, 31, 33].

Although the ND and RD diets had differences in the percentage of proteins, previous studies have demonstrated that while protein deficiencies in the diet have detrimental effects on sea bass reproduction, crude protein levels provided at a minimum of 40% or more do not influence reproductive performance [23, 25, 26]. Differences in carbohydrate content have also been shown to have no effect on reproductive performance [23, 24, 26–28] in this species. Although the percentage of total lipids were similar in the two diets, both components of the ND diet had a higher percentage of EPA and DHA and a higher n-3:n-6 ratio, while the RD diet contained a higher percentage of linoleic acid (18:2n–6), the precursor to AA (Table 1). The results obtained from this study demonstrate that oocytes obtained from fish fed for approximately 2 yr with the commercial RD diet enriched with fatty acids yet lower in EPA content, had a significantly higher GTH-induced maturation rate compared with oocytes taken from fish fed a natural diet, which is higher in EPA. Furthermore, a significantly higher rate of atresia was observed in populations of oocytes from fish in the ND group, suggesting a lower quality of available oocytes for maturation and fertilization. Although basal levels of PGE secretion from oocytes obtained from fish fed the two diets were the same, RD oocytes produced significantly higher amounts of PGE compared with ND oocytes in the presence of GTH. It is possible that because the ND diet contained more EPA and DHA compared with the RD diet (Table 1), potential PGE production by oocytes was reduced due to the higher levels of available EPA and DHA and, instead, there was an increase in production of PGE3, a PG that is ineffective in stimulating maturation in this system. Further studies in vivo with more selective diets such as a PUFA-deficient diet and diets with clear differences in AA and EPA content are necessary to further elucidate a direct effect of these diets on oocyte maturation in vitro. The results obtained from this study can only suggest that the fish fed the enriched diet exhibit higher quality oocytes that attain higher maturation rates in response to GTH.

The results obtained in vitro suggesting that oocytes from females fed an enriched diet attain maturation more readily have been substantiated in vivo in this species because alterations in essential dietary fatty acids have been shown to impair reproductive performance, including spawning rates, fertilized egg viability, hatching rates, and survival rates [23–25, 26–28]. Studies have shown that variation in AA and EPA/DHA ratios in the diet effectively alter plasma GTH and sex steroid levels, resulting in decreased spawning quality and activity. In addition, other studies have shown that PGE2 and PGF2{alpha} are produced by the teleost freshwater fish ovary [34–36, 50] and it has been previously suggested that PGE2 may be important for the prematurational period [31, 51]. However, the results from this study suggest that these eicosanoids may also be the mediators of AA induction of maturation and therefore important during a later period of oocyte development in a marine teleost.

It is interesting to note that in the present study, hCG had a stimulatory effect on oocyte PGE production. This appears to be unique to the sea bass. Other in vitro studies in goldfish, a fresh water fish with a synchronous ovary, have not found any effects of gonadotropin on PG production [31, 52].

The physiological process of oocyte maturation is a complex phenomenon involving GTH induction of MIS production. Studies in rats have demonstrated that dietary essential fatty acids can affect the onset of puberty and maturation of the reproductive tract, although a direct role of fatty acids on oocyte maturation has not been demonstrated [53, 54]. Results from this study demonstrate that AA and its metabolites, PGE2 and PGF2{alpha}, stimulate oocyte maturation and enhance GTH-induced maturation in a marine teleost in vitro. Mobilization of AA may be achieved through PLA2 and AA may subsequently affect MIS production. However, this or another MIS of the sea bass must first be identified before the effects of AA on this hormone or others can be explored. Differential action of long-chain PUFAs has also been shown in this system in which EPA and DHA have no stimulatory effects on maturation, yet attenuate GTH-induced maturation. Moreover, oocytes obtained from fish fed a PUFA-enriched diet exhibited higher GTH-induced maturation rates, less atresia, and higher hCG-induced production of PGE compared with fish that were fed a natural diet with a higher EPA content and n-3:n-6 ratio. Further work is necessary to identify the effective ratios of AA and EPA/DHA, which are crucial for optimal oocyte maturation and, more importantly, to determine the interactions of PUFAs with the as-yet-unidentified MIS in the sea bass [39]. In summary, the present study provides further evidence for the importance of essential dietary fatty acids in the physiological function of the reproductive system in a marine teleost.

ACKNOWLEDGMENTS

_Fatty acid analysis of the natural squid portion of the natural diet was provided by Drs. M. Redón and J.M. San Feliu (Instituto de Acuicultura de Torre de la Sal, Castellón, Spain). The authors would like to thank Dr. F. Piferrer for his careful review of the manuscript.

FOOTNOTES

First decision: 4 April 2000.

1 These studies were supported by a C.I.C.Y.T. grant (Mar 96-1859) to M.C., a grant from the European Union (AIR2-CT93-1005), a postdoctoral fellowship to L.A.S. from the Spanish Ministry of Science and Education, and a predoctoral fellowship from the regional government of Valencia, Spain (Generalitat Valenciana) to J.F.A. Back

2 Correspondence: Silvia Zanuy, Instituto de Acuicultura de Torre de la Sal, Torre de la Sal, 12595 Ribera de Cabanes (Castellón), Spain. FAX: 34 964 319509; zanuy{at}iats.csic.es Back

3 Current address: Laboratorio de Acuicultura, Departamento de Ciencia Animal, Universidad Politécnica de Valencia, Camino de Vera, 14. 46071 Valencia, Spain. Back

Accepted: September 5, 2000.

Received: February 17, 2000.

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