In most cases the ligand affinity between subtypes is simila

In most cases, the ligand affinity between subtypes is similar, but their tissue distribution is different. In the case of the estrogen receptors (ER), two subtypes (ERα and ERβ) exist (Kuiper et al., 1996) and ERβ has several isoforms, designated ERβ1–5 (Moore et al., 1998) or ERβcx (Ogawa et al., 1998). Recently, a third subtype (ERγ), derived from ERβ through gene duplication, has been described for a teleost (Atlantic croaker, Micropogonias undulatusHawkins et al., 2000). The ligand binding affinity of rat ERα and ERβ protein for physiological ligands is quite similar, while tissue distribution and/or the relative levels of ERα- and ERβ-mRNA are different (Kuiper et al., 1997). Recently, we isolated two subtypes (α and β) of both AR and PR from the same eel cDNA library. In the present article, we summarize current knowledge about the characteristics of both AR and PR subtypes, such as their structure and function.
Structures and sequence homology with other steroid hormone receptors
Ligand specificity A nuclear receptor is a ligand-dependent transcriptional factor. In this case, the ligand is a transactivator of target genes through the receptor. We aimed to identify the major ligand for each receptor subtype by the reporter assay, rather than by binding assay. It has been reported that the human ER, when expressed in yeast, stimulates initiation of transcription in a strictly hormone-dependent manner, as in mammalian cells, indicating a striking conservation of the underlying regulatory mechanisms (Metzger et al., 1988). Thus, we used mammalian cell lines that do not express endogenous AR and PR, as host cells.
Tissue distribution
Interaction between receptor subtypes ARα and ARβ were detected in the same Cy5.5 maleimide (non-sulfonated) in the testis, and both PR subtypes were expressed in the same tissue. These results raise the question of whether the subtypes influence each other. The molecular interaction of the proteins for both subtypes was demonstrated by a glutathione S-transferase (GST) pull-down assay. In this assay, the GST-fusion protein for each subtype is co-precipitated by a glutathione-affinity column with a radiolabelled subtype that can form a dimer with the fusion protein, and the molecular interaction is autoradiographically detected. Both in vitro translated products of ARα and ARβ were pulled down by GST-fused ARα or ARβ proteins (Fig. 3a). GST protein alone did not pull either ARα or ARβ down. A GST pull-down assay also showed that PRα and PRβ form hetero- as well as homodimers (Fig. 3b).
Conclusions These results indicate that two functional subtypes for both AR and PR exist in eel. It is widely accepted that an ancestral steroid-hormone receptor gene gave rise to four genes (AR, PR, glucocorticoid receptor and mineralcorticoid receptor) through two waves of gene duplication in vertebrates (Laudet, 1997). There is no evidence that eels experienced the ‘third’ gene duplication, unlike salmonids. It is likely that AR and PR diverged before the last gene duplication, because both AR and PR subtypes are encoded by paralogous genes. These findings suggest the existence of previously unrecognized pathways of androgen or progestogen action and will advance our understanding of the mechanisms underlying sex steroid signaling.
Acknowledgements
Introduction The most extensively studied pharmaceutical pollutant is 17α-ethinylestradiol (EE2). This steroidal estrogen compound is used in synthetic oral contraceptives (SOC), and has been shown to impair reproduction in fish even at concentrations below 1ng/L (Caldwell et al., 2008). Recently, another class of contraceptive pharmaceuticals has emerged into focus in ecotoxicology: the progestogens. Progesterone (PRG) is an endogenous steroid hormone involved in the female menstrual cycle, pregnancy and the embryogenesis of humans and other species (Rodriguez et al., 2010). It plays important role in brain function as a neurosteroid (Baulieu and Schumacher, 2000). Progestins are a group of molecules that have effects similar to those exerted by PRG. In fish, the main natural progestin is 17α,20β-dihydroxy-4-pregnen-3-one (DHP). In females, DHP is responsible for final maturation of oocytes (Nagahama and Yamashita, 2008), while in males it is involved in spermiation and sperm motility (Tubbs and Thomas, 2009). Besides natural representatives of PRG analogs, there are several synthetic progestins such as drospirenone (DRO), levonorgestrel (LNG) or norethindrone (NET). The endogenous PRG and its synthetic analog progestins together are generally referred to as progestogens. There are approximately 20 different progestogens used in human and veterinary medicine, which all activate progesterone receptors (Sitruk-Ware and Nath, 2010). Although, progestogens and their metabolites could also interact with other steroid hormone receptors, exerting combinations of progestagenic, (anti)androgenic, (anti)estrogenic, glucocorticoidogenic and anti-mineralocorticoidogenic effects (Africander et al., 2011). Progestogens are widely used as SOCs. More than 100 million women use SOCs yearly (Huezo, 1998). Progestogens or their metabolites are eliminated from the human body mainly through the renal system and a remarkable amount is excreted unchanged or in the form of active metabolites (Besse and Garric, 2009, Liu et al., 2011). These active agents enter waste water treatment plants (WWTP) where the generally applied treatment process is not suitable to eliminate progestogen contaminations of different origins perfectly (Can et al., 2014, Liu et al., 2011).