To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT.

Quantitative real-time PCR (qPCR) has been the method of choice for the quantification of mRNA. Due to the various artifactual factors that may affect the accuracy of qPCR, internal reference genes are most often used to normalize qPCR data. Recently, many studies have employed computer programs such as GeNorm, BestKeeper and NormFinder in selecting reference genes, but very few statistically validate the outcomes of these programs. Thus, in this study, we selected reference genes for qPCR of liver and ovary samples of yellow (juvenile), migratory (silver) and 11-KT treated juveniles of New Zealand shortfinned eels (Anguilla australis) using the three computer programs and validate the selected genes statistically using REST 2009 software and the Mann-Whitney test. We also tested for the repeatability of use for the best reference genes by applying them to a data set obtained in a similar experiment conducted the previous year.

Estradiol-17β (E₂) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesised that in combination, 11KT and E₂ would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E₂ (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver and ovarian tissues by histological examination, qPCR, immunoblotting or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analysed to determine mechanisms by which steroid effects may be exerted. When administered alone, E₂ increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E₂ could induce uptake of Vtg into oocytes, although E₂ treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E₂, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

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 5-6,000 km and leaves in a previtellogenic state. The shortfinned eel A. australis migrates 2-4,000 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 17β-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-β fshb; liver estrogen receptor 1 esr1; gonad follicle-stimulating hormone receptor fshr, androgen receptors α and β 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.

Lipoprotein receptors, including low-density lipoprotein receptor (LDLr) relatives (Lrs) and LDLr-related proteins (Lrps), belong to the LDLr supergene family and participate in diverse physiological functions. In this study, novel sequences of lr and lrp genes expressed in the ovary of the short-finned eel, Anguilla australis, during early gonadal development are presented. The genes encoding the LDLr-like, Lrp1-like, Lrp1b-like, Lrp3, Lrp4-like, Lrp5-like, Lrp6, Lrp10, Lrp11, Lrp12-like, and Lr11-like proteins were found and identified by sequence and structure analysis, in addition to phylogenetic analysis. Genes encoding proteins previously implicated in follicle development and vitellogenin (Vtg) uptake in oviparous vertebrates were also identified, i.e. lr8 (including lr8 + and lr8- variants) and lrp13; their identification was reinforced by conserved synteny with orthologues in other teleost fish. Compared to other lr/lrp genes, the genes encoding Lr8 + , Lr8-, and Lrp13 were highly expressed in ovary during early development, decreasing as oocyte development advanced when induced by hypophysation. Furthermore, lr8 + , lr8-, and lrp13 were dominantly expressed in the ovary when compared with 17 other tissues. Finally, this study successfully detected the expression of both lr8 variants, which showed different expression patterns to those reported in other oviparous vertebrates and provided the first characterisation of Lrp13 in Anguilla sp. We propose that lr8 + , lr8-, and lrp13 encode putative Vtg receptors in anguillid eels.