The induction of oocyte maturation and ovulation in the European eel (Anguilla anguilla): in vitro and in vivo comparison of progesterone with 17α,20ß-dihydroxy-4-pregnen-3-one.

Ovulation in European eel is induced by injection of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) as the maturation-inducing hormone (MIH). Female eels need to ovulate within 18 h after injection to release good quality eggs. Progesterone (P), as an upstream precursor of DHP, may promote endogenous DHP production and improve egg quality. The purpose of this study was therefore to compare treatment of P with DHP on batch level, in vitro, to determine dose-response effects, and in vivo, at a single dose. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment: DHP at 1, 10 and 100 ng mL-1, or P at 10, 100 and 1,000 ng mL-1. At the start of incubation, the folliculated oocytes were sampled for histology, microscopy and qPCR. After incubation for 12 and 18 h, the oocytes were sampled for microscopy and qPCR analysis. For the in vivo experiment, females were either injected with DHP or P at a dose of 2 mg kg-1 to assess their effects on ovulation and reproductive success. At the moment of release, eggs were sampled for RNA sequencing to compare effects of DHP and P on the expression of genes involved in egg quality aspects. Remaining eggs were fertilized and larval viability was recorded. Both DHP and P were able to induce GVBD (DHP at 10 and 100 ng mL-1, P at 100 and 1,000 ng mL-1) in vitro. Expression of genes involved in oocyte maturation and ovulation was similar in vitro for both DHP and P treatments. Regarding the in vivo results, RNAseq results reflected similar DHP and P effects on the expression of genes involved in egg quality aspects. Females injected with either DHP or P ovulated, released eggs, and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that DHP and P work equally well in vitro and in vivo. P is more attractive to apply as the price is 3,000 times lower than the price of DHP.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

A mechanistic model for studying the initiation of anguillid vitellogenesis by comparing the European eel (Anguilla anguilla) and the shortfinned eel (A. australis).

An inverse relation exists between the maturation stage at the start of the oceanic reproductive migration and the migration distance to the spawning grounds for the various eel species. The European eel Anguilla anguilla migrates up to 5-6000 km and leaves in a previtellogenic state. The shortfinned eel A. australis migrates 2-4000 km and leaves in an early vitellogenic state. In this study, we compared the early pubertal events in European silver eels with those in silver shortfinned eels to gain insights into the initiation of vitellogenesis. Immediately after being caught, yellow and silver eels of both species were measured and sampled for blood and tissues. Eye index (EI), gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated. Plasma 11-ketotestosterone (11-KT) and 17ß-estradiol (E2) levels were measured by radioimmunoassay. Pituitary, liver and ovaries were dissected for quantitative real-time PCR analyses (pituitary dopamine 2b receptor d2br, gonadotropin-releasing hormone receptors 1 and 2 gnrhr1 and gnrhr2, growth hormone gh and follicle-stimulating hormone-ß fshb; liver estrogen receptor 1 esr1; gonad follicle-stimulating hormone receptor fshr, androgen receptors α and ß ara and arb, vitellogenin receptor vtgr and P450 aromatase cyp19). Silver eels of both species showed a drop in pituitary gh expression, progressing gonadal development (GSI of ∼1.5 in European eels and ∼3.0 in shortfinned eels) and steroid level increases. In shortfinned eels, but not European eels, expression of fshb, gnrhr1 and gnrhr2, and d2br in the pituitary was up-regulated in the silver-stage as compared to yellow-stage females, as was expression of fshr, ara and arb in the ovaries. Expression of esr1 in European eels remained low while esr1 expression was up-regulated over 100-fold in silver shortfinned eels. The mechanistic model for anguillid vitellogenesis that we present suggests a first step that involves a drop in Gh and a second step that involves Fsh increase when switching in the life history trade-off from growth to reproduction. The drop in Gh is associated with gonadal development and plasma steroid increase but precedes brain-pituitary-gonad axis (BPG) activation. The Fsh increase marks BPG activation and increased sensitivity of the liver to estrogenic stimulation, but also an increase in D2br-mediated dopaminergic signaling to the pituitary.