Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption

Abstract

The oocyte is the starting point for a new generation. Most of the machinery for DNA and protein synthesis needed for the developing embryo is made autonomously by the fertilized oocyte. However, in fish and in many other oviparous vertebrates, the major constituents of the egg, i.e. yolk and eggshell proteins, are synthesized in the liver and transported to the oocyte for uptake. Vitellogenesis, the process of yolk protein (vitellogenin) synthesis, transport, and uptake into the oocyte, and zonagenesis, the synthesis of eggshell zona radiata proteins, their transport and deposition by the maturing oocyte, are important aspects of oogenesis. The many molecular events involved in these processes require tight, coordinated regulation that is under strict endocrine control, with the female sex steroid hormone estradiol-17beta in a central role. The ability of many synthetic chemical compounds to mimic this estrogen can lead to unscheduled hepatic synthesis of vitellogenin and zona radiata proteins, with potentially detrimental effects to the adult, the egg, the developing embryo and, hence, to the recruitment to the fish population. This has led to the development of specific and sensitive assays for these proteins in fish, and the application of vitellogenin and zona radiata proteins as informative biomarkers for endocrine disrupting effects of chemicals and effluents using fish as test organisms. The genes encoding these important reproductive proteins are conserved in the animal kingdom and are products of several hundred million years of evolution.

Figure 1

Schematic representation of the hypothalamus-pituitary-gonadal-liver (HPGL) axis during oogenic protein synthesis in female teleosts. The HPGL is regulated through the negative feedback mechanism by estradiol-17β. The hypothamalus, pituitary, gonad and liver are all potential targets for endocrine disruptors, as discussed in the text. GtH = gonadotropin I & II.

Figure 2

Immunohistochemical staining of a cod (Gadus morhua) ovarian follicle with oocyte, probed with rabbit antiserum to cod zona radiata proteins. The zona radiata proteins (Zr) and the yolk (Y) protein vitellogenin are both synthesized in the liver of most fish species and transported to the ovary. (A) Section of whole oocyte, demonstrating specific immunohistochemical staining of the zona radiata, with no cross-reaction to yolk material (Y). (B) Higher magnification of the cod follicle. Zr denotes the zona radiata (positively stained). The follicle cells (theca, T, and granulosa, G) are indicated with arrowheads. Spherical bodies represent unstained yolk granules. Reproduced from Oppen-Berntsen et al. [19], with permission from University of the Basque Country Press (UBC Press) and the author.

Figure 3

Simplified diagram of estradiol-17β (E₂) or E₂-mimic stimulated oogenic protein synthesis. Eggshell zona radiata proteins and the egg yolk protein precursor, vitellogenin are synthesized and secreted by the hepatocyte. They are transported in blood to the ovary and incorporated into maturing oocytes in female teleosts.

Figure 4

Immunochemical analysis using indirect ELISA of oogenic proteins in plasma of juvenile Atlantic salmon (Salmo salar) exposed to different concentrations of oil refinery treatment plant (ORTP) effluent. Proteins were detected with homologous antisera against Atlantic salmon zona radiata proteins (Zr-protein) and vitellogenin (Vtg). Data are given as mean ELISA absorbance values (492 nm) ± SD (n = 6 per treatment group). Data were analyzed using Dunnett's tests for comparison with control group. *Significantly different from control (p < 0.001). Reproduced with permission from Arukwe et al. [113].

Figure 5

Cross-reactivity of a monoclonal zebrafish (Danio rerio) vitellogenin antibody to different cyprinid fish species. Monoclonal mouse anti-zebrafish vitellogenin IgG JE-10D4 (Biosense Laboratories AS, Bergen, Norway) was used to probe a Western blot with samples of: (1) Pre-stained molecular weight standard (Bio-Rad), (2) purified zebrafish Vtg, (3) whole-body homogenate sample of estradiol-17β (E₂) treated zebrafish, (4) whole-body homogenate sample of control zebrafish, (5) plasma sample of E₂ treated carp (Cyprinus carpio), (6) plasma sample of control carp, (7) plasma sample of E₂ treated fathead minnow (Pimephales promelas), (8) plasma sample of control fathead minnow, (9) plasma sample of E₂ treated roach (Rutilus rutilus), (10) plasma sample of control roach. Reproduced with permission from Biosense Laboratories AS.

Figure 6

Specificty of Atlantic salmon (Salmo salar) zona radiata protein antibodies. A plasma sample from estradiol-17β treated salmon was probed with different monoclonal Zrp antibody supernatants and the polyclonal mouse antiserum. (1) Clone 2C4, showing equal specificty for the α- and β-isomer, (2) clone 3D7, showing highest specificty for the α-isomer, (3) clone 7F2, a γ-specific clone, (4) clone 8C4, a predominantly α-specific clone, and (5) polyclonal mouse antiserum, showing reactivity with all three isomers. (Berg, Nilsen, Goksøyr, unpublished results).

Figure 7

Immunohistochemical localization of vitellogenin (Vtg) in liver sections of control (a), nonylphenol- (b) and estradiol-17β-treated (c) juvenile Atlantic salmon (Salmon salar). Cellular Vtg levels were detected with mouse monoclonal antibody (BN-5) against salmon Vtg. Yellow colors show strong Vtg-specific staining and as demonstrated primarily in endothelial cells, hepatic sinusoids, and cytoplasm of hepatocytes (labeled C). Blue stains show nuclei of hepatocytes. Goat anti-mouse horseradish peroxidase (GAM-HRP) was used as secondary antibody. Reproduced from Arukwe et al. [143] with permission from Taylor and Francis .

Figure 8

Histological section of a grossly intersex gonad of the gudgeon, Gobio gobio, containing testicular tissues and both primary and secondary (vitellogenic) oocytes. Picture is a kind donation from Ronny van Aerle and Charles Tyler.