Introduction
The European eel is a species of high economic impact for both commercial fisheries and aquaculture in Europe. However, the eel does not reproduce naturally in captivity. Hormonal induction with traditional methods has been applied to address reproduction problems, including weekly administration of human chorionic gonadotropin (hCG) in males (Pérez et al., 2000) and salmon or carp pituitary gland extracts, followed by a final dose of progestogen to induce spawning, in females (Gallego et al., 2012). Despite their effectiveness, these methods are time-consuming and expensive. An alternative approach to induce gametogenesis in captivity can be the use of gene therapy using recombinant gonadotropin plasmids (Mazón et al., 2013). The present study tested the potential of this technique in male and female European eels, by transferring and overexpressing homologous gonadotropin genes.
Materials and methods
Two independent experimental trials were conducted on immature European eel males maintained in seawater at 20 ºC: Experiment 1: Fish with an average weight of 142.64 ± 2.84 g were subjected to three treatments: i) fish injected with empty pCDNA3 plasmid (control), ii) fish injected with eel single-chain follicle-stimulating hormone (scFsh), and iii) fish injected with eel scFsh + eel single-chain luteinizing hormone (scLh). To monitor injection effectiveness, all groups were injected with 15 µg of sea bass (Dicentrarchus labrax) scFsh which can be monitored in plasma and has no functional effect in eel. Injections were performed in weeks 1, 3 and 6 in duplicate (reinjecting after 3 days each time), and total duration of the experiment was 9 weeks.
Experiment 2: Fish with an average weight of 103.54 ± 1.29 g were subjected to three treatments: i) fish injected weekly with hCG (positive control); ii) fish injected every three weeks with empty pCDNA3 plasmid (negative control), and iii) fish injected every three weeks with the combination of eel scFsh at a constant dose and increasing doses of eel scLh plasmids. Total duration of this experiment was 15 weeks.
One experimental trial was conducted on immature European eel females maintained in seawater at 20 ºC. Fish with an average weight of 756.95 ± 12.74 g were subjected to three different treatments similar to experiment 1 for males.
To evaluate maturation progression, blood samples were taken at the times of injections for steroid analysis, and biometric parameters (eye index, fin colour and weight) were recorded. Additionally, at the end of each experiment, the fish were sacrificed, the gonadosomatic index (GSI) was determined and tissue samples were preserved for histological analysis.
Results
In experiment 1, treated male European eels showed significantly higher GSI and plasma steroid levels compared to control groups (Fig. 1). These findings indicate that gene therapy with homologous gonadotropin effectively stimulates spermatogenesis. However, there was some variability among individuals, and only a subset of the treated eels progressed to spermiation.
To improve these results, the treatment protocol was optimized in experiment 2, and a positive control using traditional hCG treatment was included for comparative purposes. Results showed that GSI and steroid levels in plasmid-treated group were similar to the hCG group and significantly different from the control one, indicating enhanced effectiveness of the revised gene therapy protocol. Similarly, biometric parameters such as eye index and fin coloration increased over time in the treated groups, whereas they remained unchanged in the control group (pCDNA3). In addition, nearly all the treated specimens achieved spermiation by the end of the trial.
In the female experiment, treatment with European eel recombinant gonadotropin plasmids led to significant increases in biometric parameters, oogenesis progression, and sex steroid levels compared to the control group, highlighting the potential of gene therapy as an alternative method to induce reproduction of European eel females in captivity.
In conclusion, compared to conventional hormonal treatments, the results obtained in our study show that gonadotropin plasmid-based gene therapy offers a promising, cost-effective, and efficient alternative for overcoming reproductive dysfunctions in European eel aquaculture.
Acknowledgements
This study forms part of the ThinkInAzul programme and was supported by MICIU with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana [GVA-THINKINAZUL/2021/012; Principal investigator: J.F. Asturiano (Universitat Politècnica de València), and GVA-THINKINAZUL/2021/42; Principal investigator: A. Gómez (CSIC)].
References
Pérez L, Asturiano JF, Tomás A, Zegrari S, Barrera R, Espinós FJ, Navarro JC, Jover M (2000) Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. J Fish Biol 57:1488-1504
Gallego V, Mazzeo I, Vílchez MC, Peñaranda DS, Carneiro PCF, Pérez L, Asturiano JF (2012) Study of the effects of thermal regime and alternative hormonal treatments on the reproductive performance of European eel males (Anguilla anguilla) during induced sexual maturation. Aquaculture 354-355: 7-16
Mazón MJ, Zanuy S, Muñoz I, Carrillo M, Gómez A (2013) Luteinizing Hormone Plasmid Therapy Results in Long-Lasting High Circulating Lh and Increased Sperm Production in European Sea Bass (Dicentrarchus labrax), Biol Reprod 88(2):32, 1–7