The Measurement Of Vitellogenin Biology Essay

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Other than these two studies, using ELISA method as well, Segner and his coworkers assessed the reproduction fitness on zebrafish, a freshwater vertebrate, whereby the plasma Vtg levels were significantly increased in both males and females upon exposure to xenoestrogenic compounds under full life cycle test. Since estrogen regulates reproductive development and coordinates sexual differentiation at different specific times and dosage responses, Segner and colleagues (2003) evaluated the effects caused by estrogenic chemicals in zebrafish under partial life cycle test at various developmental stages. This allowed them to find out the sensitivity of the freshwater fish towards the estrogenic effect at certain critical period. Not surprisingly, the Vtg synthesis in the partial life cycle test was induced and Vtg levels were elevated, as similar to what had been observed in the full life cycle test.

Basically, Vtg induction and production effects are dependent on the potency of each and every estrogenic chemicals present in the aquatic environments. For instance, the lowest observed-effect concentration value for the induction of vitellogenin for EE2 is typically lower than BPA and OP, indicating the higher potency of EE2 in exerting threatening effects to environment (Segner et al., 2003). The effects of different estrogenic chemicals in relation to their respective potencies will be discussed in Section 2.2.1. Another phenomenon which can be seen from every reproductive response curve is that the Vtg induction effect is dose-dependent, in which a sigmoid pattern can be observed as pointed out by Jobling et al. (2003). As mentioned before, Vtg is usually synthesized only in the liver of sexually mature females in response to circulating estrogens, thus control females have approximately 60-fold higher Vtg amount as compared to control males (Johnson et al., 2008; Jobling et al., 2003). Notable augmentation in plasma vitellogenesis happened in both male and female aquatic organisms but Jobling and colleagues (2003) showed that the increase in this plasma egg protein in the exposed males was comparatively higher than in exposed females, as compared to the control males and females.

The study of Calò et al. (2010) explained that the increased Vtg gene expression and protein production are related to the estrogenicity of EDCs, such as dioxin and polychlorobiphenyl. These chemicals exert their toxicity by agonistically stimulating a ligand activating transcription factor, the aryl hydrocarbon receptor (AhR) that acts as mediator and regulator in sex steroid hormone-related actions. This receptor performs its regulatory functions by directly activating other transcription factors such as sex hormone receptors as for example estrogen receptors, through cross-talk activation pathway. This group observed the increase in synthesis of Vtg protein in liver of fish exposed to the chemicals by immunohistochemical staining method. In another study by Zha's and colleagues (2008), the substantial rise in plasma Vtg production was associated with the accumulation of material stained by eosin dye (eosinophilic substance) in the liver of adult males. They have stated that the increase in Vtg synthesis requires the expenditure of energy derived from hepatic energy storage, proving the reduction of glycogen and lipid in liver following exposure to xenoestrogens.

Besides vertebrate, Segner et al. (2003) have also assessed on invertebrates such as molluscs and insects whose reproductive control can be dependent on their steroid neurosecretory hormones, or commonly called ecdysteroids. These steroids are involved in vitellogenesis, most probably they have similar structural components that can interact with the estrogen receptors and interfere with the activity of endogenous estrogens (Segner et al., 2003; deFur, 2004). Exposure of mollucs to xenoestrogens such as EE2 can result in an alteration in protein metabolism including vitellogenin-like protein, called vitellin, by inducing its local production for uptake into the gonad rather than entering the 'blood' haemolymph (deFur, 2004; Jobling et al., 2003).

Eggs and Embryo Productions

Besides egg-protein production, xenoestrogens can also stimulate eggs and embryo production in invertebrates, particularly in the prosobranch snails. The endpoints can be seen in dose-response curves which showed an inverted U-shape manner (often known as hormesis) (Calabrese and Baldwin, 2001). In other words, there is pronounced low-dose stimulatory effect followed by higher-dose inhibitory consequence in response to increasing exposure concentrations of xenoestrogenic compounds (Jobling et al., 2003). Jobling and coworkers (2003) stated that many xenoestrogens are being reported to stimulate egg production and the mechanism underlying the egg-laying response is related to the neural hormones such as ovulation-inducing hormone or caudo-dorsal cell hormone, and gonadotropin-releasing hormone-like peptide. Furthermore, for embryo production, they ruled out that the invertebrates are more sensitive to some kinds of estrogenic chemicals which are weak in potency at environmentally relevant concentrations, at lower levels than lowest observed-effect concentrations which are ineffective in vertebrates. Thus, invertebrates such as snails are sensitive to weak estrogens but give a slower dose-response as compared to vertebrates, with reduced critical threshold for the embryo production response as the exposure time increased (Jobling et al., 2003).

Sex Changes

Apart from the Vtg induction, the second observable fact that has been alarming treat in the ecosystem and receiving considerable attention recently in scientific community is the sex change in many species of organisms. These sex changes include the occurrence of imposex or intersex and sex reversal in many species. Imposex or intersex can be considered as partial sex changes as it involved feminization of male organisms or masculinisation of female organisms in response to estrogenic compounds, which is the presence of one sex germ cells within another predominantly gonad (Blazer et al., 2007). For example, according to deFur (2004), one of the chemical that seriously affects invertebrates, the tributyltin (TBT) can cause imposex by imposing growth of penis, the male reproductive organ, in female snails, shifting the activity of testosterone and eventually averts reproduction. On the other hand, sex reversal can be thought as an "all-or-none" process (Stoker et al., 2003) where fully sex change is observed and this is able to alter the sex ratio of a species among a population. Kuhl's group (2005) revealed the role of brain aromatase in male-female sex changes in response to estrogenic EDCs. This will be further discussed in Section on how the EDCs can give effects on hormonal responses by affecting this crucial enzyme that has been mentioned earlier.

To further discuss on the intersexuality, one of the indicator used by many study groups is the transformation from male reproductive tissues (testis) to the female one (ovaries) or the presence of oocytes, the female reproductive tissues, in the testis of male aquatic organisms as they are exposed to the environment xenoestrogenic compounds during early gonadal development in sexual differentiation (Zha et al., 2008; Blazer et al., 2007; Aravindakshan et al. 2004). These are known as testicular oocytes. The testes of suffered male organisms are predominantly consisted of testicular tissue, however, with one or more oocytes randomly situated within the tissue (Aravindakshan et al. 2004). Aravindakshan's group's experiment (2004) had also depicted that the presence of intersexuality can be associated to the Vtg induction by proportional relationship, in the sense that both can be the biomarkers for exposure to endocrine disruptors.

The occurrence of testicular oocytes can be observed either macroscopically or microscopically as gonadal abnormalities and severity can be ranked according to the association between the oocytes (Blazer et al., 2007). Observations on the gonadal sections showed an altered histoarchitecture or shape as abnormal seminiferous tubules were found in male organisms exposed to low-dose xenoestrogen chemicals, distinguished by the appearance of large empty lumens and significantly increased perimeters of the ducts, together with the presence of Müllerian duct, ovarian cortex and medulla with lacunae that only occur in female gonads (Stoker et al., 2003). This has been related to the presence of testicular oocytes in male gonads where the oocytes are found in the large lumens of seminiferous tubules; they can be shed from the epithelial layer and released together with the sperm or else they are degenerated (Blazer et al., 2007). Occasionally, the occurrence of intersexuality was accompanied by one or more skin and gonadal lesions including tumor formations and degenerative changes; this was more common in urban sites where industrial contaminants which are estrogenic can be found in the aquatic systems (Johnson et al., 2008; Sayed et al. 2012).

The influence of EDCs on sex reversal can be clearly seen in the reptile species, particularly Crocodilians, with temperature-dependent sex determination (TSD) and is predominantly in male organisms. TSD is a characteristic illustrates the capability to induce one sex or the opposite one even though for a little incubation temperature changes (Parachú Marcó et al., 2010). For most cases, the animals have their respective sex-determined incubation temperatures for male and female for which male- and female-producing temperatures are fixed and must be monitored correctly to yield 100% progenies of one of the two sexes. TSD can become one of the sensitive biomarkers for endocrine disruptors' effects as for the fact that exposure to estrogenic chemicals can override the effects of temperatures on sexual determination and induce sex reversal in species with TSD; for instance, administration of xenoestrogens prior to temperature-sensitive period can induce the production of female animals from the eggs incubated at appropriate male incubation temperatures (Stoker et al., 2003; Parachú Marcó et al., 2010).

The occurrence of sex reversal in organisms with TSD establishes the role of aromatase and estrogens in sexual differentiation. Many EDCs can act through estrogen receptors to alter the expression and activity of the aromatase through the binding affinity of these xenoestrogenic chemicals to the estrogen receptors, whereby the chemicals exert estrogenic responses onto the enzyme to inhibit its normal functioning in reproductive system and alter the ovarian differentiation upon embryonic exposure to these chemicals (Kuhl et al., 2005; Guillette Junior et al., 2000). Kuhl's team's (2005) study had also suggested the role of up-regulated aromatase to produce more estrogens from aromatization in sex reversal in a male to female direction in response to environmental estrogenic pesticide o,p-DDT, proven by a significant female-skewed adult sex ratio in medaka fishes when the exposure concentration is at the maximum level. Even at low concentrations of xenoestrogens exposure, they can act in an addition manner with other estrogenic chemicals to cause some altered reproduction effects, particularly for those chemicals that are weak estrogen such as BPA.