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

Abstract

Ovulation in European eel is induced by injection of 17α,20β-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.

Keywords: RNA-seq; aquaculture; eel reproduction; maturation-inducing hormone; spawning induction.

FIGURE 1

Histological sections of maturing oocytes in European eel that are folliculated (A) and de-folliculated (B). Insets show parts of sections at higher magnification. OC, oocyte; OM, oocyte membrane; TC, theca cells and GC; granulosa cells. Contrary to the de-folliculated oocytes, folliculated oocytes still show some theca and granulosa cells (Tosaka et al., 2010).

FIGURE 2

Maturing oocytes and germinal vesicle breakdown (GVBD). (A) Maturing oocytes fixed in Serra’s solution to stain the germinal vesicle GV (yellow arrow) and (B) percentage of oocytes where GVBD had occurred shown as percentage (GVBD%). Oocytes were treated with various doses of DHP (0, 1, 10, and 100 ng mL⁻¹) and P (0, 10, 100, and 1,000 ng mL⁻¹) in vitro at the start of incubation (dark blue; hardly visible as GVBD% was 0), and after 12 (mild blue) and 18 h (light blue) of incubation. Oocytes incubated without hormone have a visible GV at the start and after 12 and 18 h of incubation. Similarly, oocytes incubated at low doses of DHP (1 ng) and P (10 ng) still have a visible GV after 12 and 18 h of incubation. Some oocytes incubated with doses of 10 ng DHP after 12 and 18 h of incubation did not show presence of a GV since GVBD was induced in ∼27% of oocytes. Most oocytes incubated with doses of 100 ng DHP, 100 ng P and 1,000 ng P after 12 and 18 h of incubation did not show presence of a GV since GVBD was induced in ∼70% of oocytes. GVBD% was compared between timepoints and doses for DHP and P. Bars with no overlap in letters are significantly different (p < 0.05). Data are displayed as barplots with averages ± standard deviation and individual datapoints as circles. Data are based on oocytes originating from N = 11 eels.

FIGURE 3

Lipid diameter when treated with various doses of (A) DHP (0, 1, 10, and 100 ng mL⁻¹) and (B) P (0, 10, 100, and 1,000 ng mL⁻¹) at the start of incubation (dark blue), and after 12 (mild blue) and 18 (light blue) hours of incubation in vitro. Measurements were compared between timepoints and doses for DHP and P. Lipid diameter increased over time as a result of fusion of lipid droplets. Treatment with a dose of 100 ng DHP after 18 h of incubation tended to increase lipid diameter but results were not statistically different from the 18 h control; probably due to the high variations in the data. Treatment with a dose of 1,000 ng P after 18 h of incubation induced lipid fusion. Bars with no overlap in letters are significantly different (p < 0.05). Data are displayed as barplots with averages ± standard deviation and individual datapoints as circles. Data are based on oocytes originating from N = 10 eels.

FIGURE 4

Expression of the (A) nuclear progestin receptors pgr1, (B) nuclear progestin receptors pgr2, (C) luteinizing hormone receptor lhcgr1, (D) follicle-stimulating hormone receptor fshr, (E) prostaglandin receptor ptgr4b and (F) androgen receptor ara when treated with various doses of (1) DHP (0, 1, 10, and 100 ng mL⁻¹) and (2) P (0, 10, 100, and 1,000 ng mL⁻¹) at the start of incubation (dark blue) and after 12 (mild blue) and 18 (light blue) hours of incubation in vitro. Receptor expression was normalized to the start of incubation and expressed as fold change. Expression was compared between timepoints and doses for DHP and P. Expression of pgr1 was decreased in oocytes incubated with doses of 100 ng DHP, 100 ng P and 1,000 ng P after 18 h of incubation. Expression of pgr2 was decreased in oocytes incubated with doses of 10 ng DHP, 100 ng DHP, 100 ng P and 1,000 ng P after 12 and 18 h of incubation. Expression of lhcgr1 decreased with time and treatment. The lowest doses of DHP (1 and 10 ng) increased the expression of lhcgr1 after 12 and 18 h of incubation when compared to the corresponding controls. Oocytes incubated with the lowest dose of P (10 ng) followed a similar expression pattern than oocytes incubated with DHP. Expression of fshr was increased in oocytes incubated with doses of 100 ng DHP, 100 ng P and 1,000 ng P after 12 h of incubation. Similarly, doses of 100 ng DHP, 100 ng P and 1,000 ng P increased the expression of ptger4b after 12 h of incubation. Expression of ara was decreased with doses of 100 ng DHP and 1,000 ng P after 18 h of incubation. Bars with no overlap in letters are significantly different (p < 0.05). Data are displayed as barplots with averages ± standard deviation and individual datapoints as circles. Data are based on oocytes originating from N = 10 eels.