The increased awareness that chemicals in the environment can cause endocrine disruption in wildlife and, possibly, humans, has lead international organizations such as OECD to consider developing new test methodologies for detecting EDCs. These methods will eventually be used as standard test procedures in the toxicity testing of new and existing chemicals. Recent work in OECD and the US Environmental Protection Agency has focused on reviewing available methods for detecting endocrine disrupting effects of chemicals in wildlife, including fish. An implementation of Vtg as a core endpoint in a piscine short-term endocrine disrupter screen for chemicals, in combination with e.g. gross morphology and histology, is suggested. The tests should be applicable to different species, in particular zebrafish (Danio rerio), fathead minnow (Pimephales promelas), and medaka (Oryzias latipes) 132. These fish share several attributes that make them ideal test species for reproductive toxicity testing, including small size at maturity, relatively short generation times, asynchronous spawning, and overall ease of culture. Sensitive and quantitative immunoassays for Vtg in these species have recently been developed in our laboratory 133.

Depending on the target organ or tissue, a wide variety of assays have been developed to measure oogenic protein expression in fish. These include radioimmunoassays; enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry using monoclonal and polyclonal antibodies (Abs), RNA protection assay and transcript analysis by Northern blotting or various variants of polymerase chain reaction (PCR). Recently, the use of real-time (quantitative) PCR is increasingly becoming a valuable tool in oogenic protein analysis. In plasma samples, these assays vary in their sensitivity, but some have the ability to detect very low levels of protein expression, i.e. 1 ng/ml or less 134135136137. Vtg assays based on polyclonal antibodies are generally restricted for use with the homologous species, but some antibodies do cross-react with Vtg in other species (e.g. 135138139) (Fig. 5).

The basic principle of a radioimmunoassay (RIA) is the use of radio labeled Abs or antigens (Ags) to detect Ag:Ab reactions. The Abs or Ags are labeled with the ¹²⁵I (iodine-125) isotope, and the presence of Ag:Ab reactions is detected using a gamma counter. RIA techniques are well developed for egg yolk (Vtg) analysis (e.g. 140141), but have not been developed for the zona radiata proteins. Because this technique requires the use of radioactive substances, RIAs are more and more being replaced by other immunologic assays such as ELISAs, that over the last decade have reached similar levels of sensitivity.

The ELISA technique is a sensitive laboratory technique widely used to detect and quantitate Ags or Abs in a variety of biological samples. It can be quantitative (with a standard curve) or semi-quantitative (without a standard curve). The two most widely used principles for quantitative detection of proteins are the competitive ELISA and the sandwich ELISA techniques 142.

In addition to the general issues of antibody specificity and sensitivity, there are some specific challenges related to the development of quantitative immunoassays for the oogenic proteins Vtg and Zrp. For Vtg, although it is relatively easily purified from plasma of estrogenized fish (where it can reach levels of 50–150 mg/ml), it is an inherently unstable protein. The instability of Vtg is due to its role as a precursor for shorter peptide fragments, and it is very sensitive to proteolytic breakdown into these fragments. Care must therefore be taken during sampling to avoid proteolytic breakdown by adding suitable protease inhibitors 96. This instability leads to some problems with immunization, since breakdown products may be more immunogenic than Vtg itself. In addition, it creates an important problem for the use of Vtg as a standard in quantitative assays, since users must ensure that each batch of standard is stored under conditions that prevent breakdown, and is quantitated in a consistent manner (see below). In our own laboratory, we have had success in finding conditions for stabilizing Vtg by lyophilization, although this has not been a straightforward task, and different species behave differently in this process (Goksøyr, Nilsen, Berg et al., unpublished results).

The dynamic range of Vtg concentrations found in fish plasma creates another problem. Plasma Vtg can vary maybe 100 million-fold, from a few ng/ml in unexposed male fish, to the 50–150 or above mg/ml found in estrogenized salmonids (e.g. 136). To be able to quantify this enormous range in blind samples, the working range of the assay should preferentially be as wide as possible. Nevertheless, even with an assay covering several hundredfold variation, all samples need to be serially diluted at least 3–4 times to ensure that at least one dilution falls within the working range of the assay. Many of the recent assays published obtain this range (e.g. 133).

The assay also needs to be robust and reproducible, and current experience in our laboratory demonstrates that the sandwich type ELISA is more robust and reproducible over the working range of the assay compared to the competitive format.

The method used to quantify the standard must be consistent and reliable. For Vtg, many different methods are presented. In some cases, Vtg is weighed after a lenghty purification procedure. Others have used different protein quantification methods such as Lowry 143, Bradford 144, or the simple A280 absorbance measure. In all these cases, the sample needs to be quantitated towards a known sample. When bovine serum albumin (BSA), ovalbumin, or Immunoglobulin G is used, an assumption is made that Vtg behaves more or less similar to the chosen standard. Generally, this is not the case, and some laboratories develop their own "gold standard" of Vtg, which is used as the standard in quantitation. Again, this gold standard needs to be verified, and this can be done by quantitative amino acid analysis. In this case, one may want to take into account the non-proteinaceous parts of the Vtg, i.e. the lipid, phosphate, and carbohydrate parts. The lipid and phosphate parts have been reported for some species to represent 15–20% and 0.6–0.8%, respectively (e.g. 27), whereas the carbohydrate portion is not well studied. In general, however, the protein part of the molecule is calculated to represent around 65–75% of the weight of the whole molecule, depending on species. The most important aspect of a protein to be used as a standard in an immunoassay is of course that the epitope(s) involved in the immunoassay maintain their stability. This can only be checked by a quality control using the immunoassay itself, so the question becomes a "hen or egg" issue. One way to manufacture a Vtg standard that maintains both proteolytic and epitope stability is to produce a synthetic peptide fragment that contains the epitope(s) of interest.

For Zrp, the challenges are somewhat different. Zrp are found in lower concentrations in plasma compared to Zrp, but recent analyses show that they may reach levels of 1–10 mg/ml in estrogenized rainbow trout 145. The protein is much more stable than Vtg, probably due to the different natures of their fate in the oocyte. Whereas Vtg needs to be broken down to fulfill its role as nutrient for the embryo, the Zrp needs to be incorporated into the eggshell intact. In the eggshell, the Zrps will cross-link by a transglutaminase reaction to form the robust zona radiata structure upon fertilization and hardening 146. The solubilization of Zrp from eggshells requires harsh conditions (ibid.), whereas it is more easily obtained from plasma. Although polyclonal antibodies for Zrp have been developed and used for some time 115119, monoclonal antibodies (MAbs) to Zrp have only recently become available 147. Screening a large panel of MAbs, it has become clear that the α- and β-form of Zrp are closely related to each other, whereas the γ-form is structurally more different (Fig. 6; Berg, Bringsvor, Nilsen, Goksøyr, unpublished results). We have also shown that combining a γ-specific MAb with a polyclonal Zrp-antibody can be used to develop a quantitative sandwich ELISA for γ-Zrp, where the standard γ-form can be purified from plasma using the same MAb in immunoaffinity chromatography 145. Because of the close similarity between the two other isomers, this has proven more difficult for the α- and β-form. However, comparing their relative responses both in ELISA and Western blots, it becomes clear that the α- and β-form are more responsive to estrogens than the γ-form of Zrp (Berg, Bringsvor, Nilsen, Goksøyr, unpublished results).

Oogenic mRNAs can be assayed by reverse transcriptase polymerase chain reaction (RT-PCR, e.g. 25), or quantitative PCR techniques (qPCR, 57). qPCR is a rather new method for the quantification of target mRNA sequences. Unlike conventional PCR, qPCR systems are probe-based PCR product detection. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified. The development of fluorogenic probes made it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET) through space.

Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end. The qPCR has several advantages compared to other hybridization techniques. This includes; fluorogenic probes over DNA binding dyes require specific hybridization between probe and target to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. Another advantage of fluorogenic probes is that they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters, amplification of two distinct sequences can be detected in a single PCR reaction. The disadvantage of fluorogenic probes is that different probes must be synthesized to detect different sequences.

Other mRNA targetting assays for oogenic proteins, such as the RNA protection assay 148, have also been developed.

Cellular localization of hepatic oogenic protein synthesis has also been demonstrated using immunohistochemical analysis of exposed fish with specific antibodies 149150 (Fig. 7). Immunohistochemistry is a valuable tool in the studies of estrogen and estrogen mimicking compound induced hepatic synthesis of Vtg and Zrp in oviparous vertebrates, especially in situations where blood samples are difficult to collect, e.g. when studying small-sized species. Although this technique is time-consuming, localization of Vtg in liver sections may provide insight into responses of different cell types that are important for understanding the role and mechanisms of the estrogens and estrogen mimicking compounds.

Reproductive development is a continuous process throughout ontogeny. Consequently, it is susceptible to the effects of xenoestrogens and/or xenobiotics at all stages of the life-cycle, including fertilization, embryonic development, sex differentiation, oogenesis or spermatogenesis, final maturation, ovulation or spermiation, and spawning. Thus, the sensitivity to a particular compound will vary depending on the stage of reproductive development 157.

Understanding the general principles by which chemical substances or foreign compounds (xenobiotics) interfere with fish reproduction is particularly important for meeting the larger objectives in aquatic reproductive toxicology, as it is impossible to empirically determine the biological specificity or how every compound affect the reproductive life-history strategy of every species. Here we will briefly discuss the specific effects that can be extrapolated from a precocious hepatic synthesis of Vtg and Zrp.

Given the energetic cost of reproduction and the long decision time, it seems most likely that xenobiotically-induced hepatic Zrp and Vtg synthesis may cause an imbalance in the reproductive strategy of a given fish population. The reason is that an organism can only acquire a limited amount of energy for which several processes compete directly; an increase in the energetic allocation to one process must result in a decrease in energy allocation to others 158159160. Thorpe 161 suggested that during maturation, the internal responses that are synchronized by external signals depend upon some genetically determined performance threshold, and that maturation processes will continue if this performance exceeds a set point at this critical time. Furthermore, maturation has developmental priority over somatic growth, and in salmonids survival after spawning implies a chance dependent balance between stored energy and that spent on reproduction 162. Therefore, xenoestrogen-induced Vtg and Zrp synthesis outside the normal maturation period may result in wasteful use of stored energy resources. The ecological implication of this might be failure in the reproduction of affected individual fish, and in the long-term affecting recruitment of the entire population. Another possible deleterious effect is that high Vtg and/or Zrp concentrations might cause kidney failure and increased mortality rates as a result of metabolic stress 163. Furthermore, although not yet demonstrated, there is a possibility that the reduced testicular growth could reduce fertility 121.

Xenoestrogen-induced changes in Zrp synthesis appear to have a higher potential for ecologically adverse effects than Vtg induction, because critical population parameters such as offspring survival and recruitment may be more directly affected. The argument for this, is that whereas subtle changes in Vtg content would not be of great significance to the survival of the offspring, small changes in Zrp synthesis might cause the thickness and mechanical strength of the eggshell to be altered, thus causing a loss in its ability to prevent polyspermy during fertilization and to protect the embryo during development 115.

Intersex is another and a much more common condition caused by early exposure of fish larvae to estrogenic substances. The intersex condition in males usually takes the form of ovotestis. Bortone and Davis 164 have reviewed the subject, particularly with respect to the masculinisation of females caused by pulpmill effluents. Ovotestis is a partial feminization in which oocytes may appear in otherwise normal testes (Fig. 8). Little is known about the implications of this condition for reproductive functionality. Ovotestis can be induced in the laboratory by exposing fish larvae to weak estrogens like NP 110111112, and has also been observed at prevalences ranging from 20 to 100% in wild fish populations exposed to estrogenic effluents 165166.

From an evolutionary point of view, an appreciation of the oogenetic components will not be complete without considering how they evolved and what evolutionary factors have been driving their evolution. In the preceding discussions about oogenic proteins, it is clear that maternal influence in developmental modes that have direct roles in early embryonic patterning and gene regulation is not restricted to informational components such as maternal mRNAs and proteins as developmental biologists will generally consider it 167. Maternal factors also include proteins and lipids that have structural or nutritive roles and that can play a large role in evolution of life histories and embryogenesis. The ability to transport fat, in the form of lipoprotein through the circulatory system by eukaryotes is one of their most significant functions right from the beginning of existence 168. The reason and functional basis for why Vtg transport systems initially evolved provides clues into how energy in the form of water insoluble fat can be distributed from sites of synthesis and absorption to specific tissues and cells. Thus, the evolutionary advancement of storing energy in the form of fat has provided organisms with enormous advantage in adapting to environmental and developmental changes.

During vitellogenesis, oviparous species (i.e. nematodes, decapods, echinoderms, insects, fish, amphibians, birds, reptiles) display over three orders of magnitude increases in the transport of fat, for instance from the liver to oocytes, in order to facilitate egg development 31567168. Thus, Vtgs can be regarded as ancient proteins that are normally encoded by a small variable number of genes 169. One Vtg gene has been demonstrated in sea urchin and silkworm, three in the chicken, four in Xenopus laevis and six in Caenorhabditis elegans (ibid.). However, more recent analysis uncovered 20 Vtg genes and ten pseudogenes in rainbow trout 170. Previously, comparative studies based on amino acid sequences and on gene organization support the hypothesis of a common evolutionary origin of the vertebrate and invertebrate Vtg genes 171172173174175176. More recently, this comparative analysis was extended to include other species groups in order to elucidate the events responsible for the relatively rapid diversification of the Vtg gene family 169.

For example, Vtg genes of the fruit fly, Drosophila melanogaster, are not obviously related to the Vtg genes of chicken, X. laevis and C. elegans 67. The chicken and Xenopus genes both have 34 introns and have the same exon-intron organization. These genes are distantly related to the C. elegans gene with only four introns, whose positions are apparently conserved in vertebrates, suggesting their presence at the same positions in the ancestral Vtg gene 177178. It has been suggested that prokaryotes and lower eukaryotes have streamlined their genome by intron sequence elimination. Given that this is true, the structural organization of the comtemporary vertebrate Vtg gene may be more representative of the earliest gene than those of the invertebrates 67177178. This assumption is supported by the study of Mouchel et al 169, which suggests that almost half of the splicing junctions identified in invertebrates are related neither to each other nor to vertebrate genes. The observed differences between vertebrate and invertebrate Vtg gene structure may also be explained by the "intron-late" theory 179180, which hypothesizes that introns became inserted more recently, therefore assuming that insertions may be specific to each lineage. Irrespective of what the explanation may be, it has been suggested that Vtg genes have been re-organized through multiple insertions and deletions of intervening sequences during the evolution of the various lineages. Characterization of the Vtg region in the genome of the rainbow trout, e.g., revealed that this locus contains twenty complete genes and ten pseudogenes per haploid genome 170. The Vtg genes differed from each other by insertion, deletion and rearrangement events, although, at the sequence level, they showed a high degree of similarity. Fluorescent in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE) and Southern analysis indicated that all gene copies are contained in a single 1,500-kb region, and that most of the genes form tandem arrays separated by a conserved 4.5-kb intergenic region. The presence of large reiterated fragments indicates that this region has been subjected to several amplification events. The presence of a retroposon element in Vtg intron 9 appeared to be responsible for the silencing of at least nine of the ten pseudogenes (ibid.).

It has become increasingly clear that the proteins of the zona pellucida are conserved among eutherian mammals and that the proteins of the zona radiata are conserved among teleostean fish. In most fish, sperm lack an acrosome and penetrate the zona radiata surrounding fish eggs via a discrete micropyle 40. Most commonly, the micropylar channel is sufficiently narrow to permit the passage of a single sperm, and subsequent fusion with the plasma membrane induces the cortical granule reaction, resulting in a block to polyspermy 52. In contrast, a prerequisite to successful fertilization in all vertebrates is penetration of sperm through an acellular envelope surrounding ovulated eggs. In mammals, sperm bind to the zona pellucida, the mammalian equivalent of fish zona radiata. Following the induction of the acrosome reaction and release of lytic enzymes, sperm penetrate the zona and fuse with the egg's plasma membrane, triggering the post fertilization block to polyspermy 181. More recently, it has become apparent that, although critical for speciation, the proteins from the mammalian egg envelope are distinctly related to those of the teleostean envelope 48. Recently, the mouse zona proteins was successfully incorporated into the extracellular envelope surrounding Xenopus eggs, showing that they have been sufficiently conserved through 350 million years of evolution 182. In general, the exon conservation at the same region in mammalian zona pellucida and fish zona radiata protein suggests that not only has this protein domain been duplicated in mammals, but that it has been conserved and used as an egg envelope protein in species that diverged 650 million years ago.

How ancient are these important components of eukaryotic reproduction? Recently, Walther 183 summarized his hypothesis that the oocyte and the sperm represent cellular lineages dating back to the two prokaryotic cell domains (eubacteria and archea, respectively), which gradually evolved ever more complete but reversible coalescence or syngamy instead of permanent fusion to form an equilibrium between the two moneric prokarya and the prototypic zygote under photoseasonal polar conditions two billion years ago. The now commonly accepted theory of endosymbiosis as the origin of eukaryotic cells was presented independently by Jostein Goksøyr 184 and Lynn Margulis (Sagan) 185 to account for a eukaryotic cell with organelle. Walther's theory instead inserts a primordial syngamy to a dimeric prokaryotic cell (termed A-KARYON), and proposes that such sexual syngamy was the origin of symbiosis leading to organelles. According to this theory further endosymbiosis 184185 created in one event the eukaryotic nucleolemma and the outer membrance of the mitochondrion, as the second step in cell evolution from moneric prokarya to dimeric eukarya. This theory depicts cell evolution by a dynamic interaction between only two moneric species in a unique event in cell evolution, which established sexuality as the dynamic fusion of these two cells or species. This dynamic model contrasts markedly from the view of an evolutionary past where cell evolution occurred by fusing a multitude of prokaryotic cell types to yield the static eukaryote, among which some later acquired sexuality (see also Margulis and Bermudes, 186).

The evolution of the eggshell and egg yolk protein genes would appear to have been driven by different factors (protection vs. nutrition), but still in modern oviparous vertebrates they are being synthesized in close concert by the hepatic machinery under a common endocrine regulation. There is still a lot to be learnt about when these genes appeared and how they evolved in the interplay between hormones, environmental cues, speciation, reproductive strategies, and the hepatic organ as their major site of synthesis today.