FINAL OOCYTE MATURATION AND EGG QUALITY IN EUROPEAN EEL (AGUILLA ANGUILLA) INDUCED IN VIVO
Introduction
Experiments to reproduce European eel (Anguilla anguilla) in captivity involve weekly injections of salmon pituitary extract (PE) to induce oocyte growth. A SPE primer and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP), which has been identified as the follicular maturation-inducing hormone (MIH) in salmonids and Japanese eel, are injected to induce oocyte maturation and ovulation (Ohta et al, 1996). It is possible to obtain fertilizable eggs with this treatment however; fertilization and hatching rates are highly variable, and a main shortcoming for successful offspring production in European eel.
During the course of maturation, oocytes undergo large cytoplasmic changes such as coalescence of lipid yolk droplets and protein yolk globules, migration of the germinal vesicle (GV) towards the animal pole and the breakdown of its nuclear envelope (GVBD) (Lubzens et al., 2010). Furthermore, to undergo maturation, oocytes must develop maturational competence, which includes an increase in MIH receptors on the oocyte cell membrane.
We hypothesize that the level of oocyte maturational competence at the time of SPE primer and DHP injection is an important factor, affecting ovulation and fertilization rates in European eel. Thus, our aim was to assess the expression of relevant hormone receptors and identify oocyte features that can serve as indicators of egg quality.
Materials & Methods
A total of 11 wild-caught female broodstock eels received weekly injections (number of injections varied from 16 to 23) of salmon pituitary extract (SPE) at a constant dosage of 18.75mg/kg body weight (Kagawa et al., 2005; Tomkiewicz, 2012) to induce oocyte growth. To stimulate follicular maturation and ovulation, females received a SPE primer approximately when oocytes reached stage 3 (Palstra et al. 2005) and 24h later a DHP injection at 2mg per kg-1 body weight (Otha et al., 1996).
For each female, samples (ovarian biopsies) were taken before SPE priming, before DHP injection and after stripping from ovulated eggs (OV). Each sample was digitally imaged for cytological analysis and preserved in RNA-later for molecular analysis. Egg dry weight and fertilization success were estimated. Fertilized eggs were incubated (20°C) and hatching success was registered at 55HPF (hours post fertilization).
RNA was extracted from tissue samples using TRIzol reagent and then transcribed into cDNA using High Capacity RNA-to-cDNA kit, following the manufacturer's protocol. qPCR was used to quantify the relative expression of estradiol receptors (ERα, ERβ1, ERβ2, GPER1, GPR2), gonadotropin receptors (FSHR, LHR1, LHR2) and progesterone receptor (PR). 18S was used as reference gene
Results and discussion
Expression level of all estradiol receptors (ERα, ERβ2, GPER1 and GPER2) decreased after ovulation, especially GPER2 where the concentration was lower than the limit for detection in most egg samples. Expression of GPER2 tended to increase after the SPE primer injection. Low gene expression in ovulated egg samples suggests receptors that are mainly expressed by the follicular cells that surround the oocyte. In contrast, the moderate decrease in GPER1 expression in ovulated eggs indicates that this receptor is not only expressed by the follicular cells, but also by the oocyte itself.
PR was expressed in oocytes at the moment of SPE priming and DHP injection, but it was not detectable in ovulated eggs.
These results, showing changes in expression of some hormone receptors in oocytes/eggs, will be related to oocyte stage and may be used as indicator of egg quality in European eel. Together, our preliminary results indicate that a complex of factors may be responsible for the oocyte maturational success in terms of fertilization and hatching rates.
Acknowledgements
The study is part of the project Reproduction of European Eel: Towards a self-sustained Aquaculture (PRO-EEL) funded by EC's FP7 Programme (GA No. 245257). A grant from COST Action FA1205 financed Filipa Silva's research stay at BOREA.
References
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