Steroid hormones and many other lipophilic compounds are believed to enter cells solely by free diffusion through the plasma membrane. However, recent work using a megalin-deficient mouse model has identified a new endocytic pathway responsible for the delivery of steroids to renal and gonadal tissues. This review describes these new pathways for uptake of 25-hydroxy-vitamin-D3 and the gonadal sex-steroids (17 beta-estradiol and testosterone) bound to vitamin D-binding protein and sex hormone-binding globulin respectively. Furthermore examples of other lipophilic molecules that enter cells by receptor-mediated pathways will be presented and the receptors responsible for their uptake described.

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an similar to 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.

The mechanisms of action of an estrogenic chemical have been examined in a viviparous fish the eelpout (Zoarces viviparus), by identification of an upregulated estrogenic pathway—the induction of hepatic estrogen receptor mRNA, hepatic estrogen binding activity and plasma vitellogenin. A relative quantitative RT-PCR assay has been established to measure hepatic estrogen receptor α (ER) mRNA levels in eelpout. Assay conditions were optimised using control and induced samples to ascertain its applicability in the actual working range of ER mRNA concentrations. β-Actin was co-amplified and used as an internal standard. Time-course effects of water exposure to 0.5 μg/L 17β-estradiol (E2) and 25 μg/L of the xeno-estrogen 4-tert-octylphenol (4-tert-OP) on ER mRNA levels in the male eelpout was examined. After 48 h of exposure, ER transcripts were induced 15-fold and 6-fold in the E2- and OP-treated fish, respectively. This difference, however, was not apparent after 1 week of exposure, when similar high levels of ER mRNA were present in both groups (20-fold induction). This indicates that the estrogenic capacity of 4-tert-OP increases with exposure time. The effect of treatment was also evaluated by examining the induction of specific E2 binding capacity in hepatic cytosolic extracts and by measuring vitellogenin in plasma. Both parameters were also induced by the treatments, but later in the time course. The measurement of ER mRNA by the RT-PCR assay showed to be the most sensitive method for the detection of estrogenic responses in eelpout.

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.

A cDNA encoding the eelpout (Zoarces viviparus) oestrogen receptor alpha (eERalpha) has been isolated from eelpout liver, cloned and sequenced. The cDNA contains a complete open reading frame encoding 570 amino acid residues (mw: 63.0 kDa). The amino acid sequence of eERalpha showed a high degree of identity to ERalpha of other teleost species. The tissue distribution of eERalpha mRNA was examined using Northern blotting, RT-PCR and in situ hybridisation (ISH). All three methods identified a pronounced expression of eERalpha in liver, pituitary, testis and ovary. In the brain ISH experiments showed that ERalpha mRNA was highly expressed in distinct regions of the preoptic area and the mediobasal hypothalamus. We have provided evidence that the receptor is auto-regulated by 17beta-oestradiol (E(2)) not only in liver but also in the testis, indicating an important role for E(2) during spermatogenesis in male eelpout. RT-PCR analysis showed a broader expression pattern including significant expression in the brain, kidney, heart, and gut of adult eelpout. In eelpout embryos eERalpha expression has also been identified, indicating a possible role for the receptor in early development. This study contributes to the accumulating evidence that in fish E(2) is not only involved in the regulation of liver specific proteins, but has a much broader range of targets.

Exposure to oestrogenic chemicals (xeno-oestrogens) may have severe effects on embryonic development. The present study investigates whether the oestrogenic endocrine disruptor 4-tert-octylphenol (4-tOP) or 17beta-oestradiol (E(2)) is accumulated in the viviparous fish the eelpout (Zoarces viviparus) and transferred to the embryos in ovario and subsequently affects embryonic development, including gonadal differentiation. Pregnant eelpouts were exposed to nominal concentrations of 25 micro gl(-1) or 100 micro gl(-1) 4-tOP (OP25 or OP100, respectively) or 0.5 micro gl(-1) E(2) in water. During 4-tOP exposure, the compound accumulated in both plasma and ovarian fluid in a concentration-dependent manner. In the mother fish, the oestrogenic biomarkers, vitellogenin (Vtg) in plasma, Vtg mRNA in liver and oestrogen-binding activity in liver, were all induced by 4-tOP (and by E(2)) at an actual concentration of 14 micro gl(-1). E(2) and 4-tOP were examined for their potency to disturb the maternal-foetal trophic relationship by disturbing the physiology of the ovary and by changing the distribution of essential nutrients normally transported to embryos during pregnancy. After exposure to E(2) or 4-tOP, calcium was depleted from the ovarian fluid and the level of free amino acids available in maternal plasma was decreased. A marked overall effect on ovarian components, including the ovarian sac, ovarian fluid and embryonic mass, was evident. Embryonic growth was significantly decreased, which might in part be attributed to disturbances of the maternal-foetal trophic relationship. Marked inductions of Vtg mRNA and Vtg protein, determined by RT-PCR and immunohistochemistry, respectively, were found in embryos from the OP100 group - the only group to show considerable accumulation of an oestrogenic compound in the ovarian fluid. A different pattern of gonadal development was found in embryos from the OP100 group compared with embryos from the control, OP25 or E(2) groups, in which approximately 50% had normal ovaries and 50% had normal presumptive male gonads. In the OP100 group, 46% had normal ovaries but, in contrast to controls, only 22% had normal presumptive male gonads, whereas the remaining 32% had abnormal male gonads with structures resembling the endo-ovarian cavity of a female gonad. As oestrogen receptor (ER) expression was detected by in situ hybridisation in early differentiating gonads, these effects could be mediated by direct interaction of the xeno-oestrogens with gonadal ER. In conclusion, this study indicates that the xeno-oestrogen 4-tOP can be transferred from the water via the mother fish to the ovarian fluid and can subsequently disturb the maternal-foetal trophic relationship and cause severe effects on embryonic development, including gonadal differentiation in ovario.

In the present experiment pregnant eelpout Zoarces viviparus females were exposed to 0, 5, 10, 50, 500 ng/l of the synthetic estrogen ethinylestradiol (EE2) and to 500 ng/l estradiol-17beta (E2) under flow-through conditions during 3 weeks and the maternal-fetal trophic response was investigated. The circulating yolk-precursor protein vitellogenin, measured by ELISA, increased from a mean control value of 0.017-36 mg/ml in the plasma of the motherfish exposed to the highest concentration of EE2 This increase in vitellogenin was also depicted by a 288% increase in circulating calcium levels. During pregnancy the ovary represents a new route of calcium loss from the maternal blood for the growth of the embryos. However, a significant decrease (120% in the group exposed to the highest concentration of EE2) in the calcium level in the ambient medium of the embryos, the ovarian fluid, was observed concomitant with the increase in maternal plasma calcium in the EE2-exposed females. In contrast, the level of circulating amino acids decreased in the maternal blood, with a slight concomitant increase in the ovarian fluid of the exposed fish. These and other metabolic observations indicate that exposure to different doses of EE2 and to estradiol-17beta affects the maternal-fetal trophic relationship.

Estrogen binding activity was revealed in the cytosolic fraction of hepatic extracts from adult male and female eelpout (Zoarces viviparus). The binding moiety was characterized by a single class of high affinity binding sites (Kd=0.59±0.05 nM in males and 1.06±0.10 nM in females). The affinity was significantly higher in males. Binding sites were satiable and binding capacity was significantly elevated in vitellogenic females (2.92±0.28 pmol/g) compared to males (1.67±0.11 pmol/g). The binding was specific to known estrogens but not to other tested steroids. The binding moiety was able to bind to DNA–cellulose and was extractable by high salt concentrations. A time-course study of estrogen binding activity in liver cytosol and of vitellogenin (Vtg) in plasma, after intraperitoneal (i.p.) injections of 17β-estradiol (E2) in male eelpout, was carried out. It was shown that both are inducible by E2. Estrogen binding activity was significantly elevated 48 h and Vtg 72 h after E2 treatment. The binding moiety was hereafter designated as a cytosolic estrogen receptor (ER). The estrogenicity of 4-tert-octylphenol (OP) was evaluated by measuring ER and Vtg after i.p. treatment. OP-treatment increased both receptor levels and Vtg concentrations in male fish, indicating that OP acts as an estrogen in male eelpout.

Pregnant females of the eelpout Zoarces viviparus were exposed to different environmental factors to investigate the impact on metabolites which may elucidate physiological interrelations between the embryos and the pregnant motherfish. The maternal organism was exposed to different salinities, elevated levels of nitrogenous endproducts and to different concentrations of the xenobiotic compound octylphenol, respectively. The concentrations of ammonia, urea, low molecular compounds, chloride, and calcium were measured in the maternal plasma and the ovarian fluid, the ambient medium of the embryos. Ammonia increased in the maternal plasma as well as in the ovarian fluid by the highest salinity (28ppt). No change could be observed in the level of urea or chloride by increased salinities. By exposing the embryos to increased levels of urea and ammonia in their ambient medium, the ovarian fluid, it was observed that a steady flow of the nitrogenous compounds occurred from the ovarian fluid to the maternal plasma. In the fish exposed to octylphenol the calcium concentration in the ovarian fluid decreased in a dose-dependent manner by increasing concentrations of the estrogenic compounds. In contrast significant increased levels of total calcium were observed in the plasma of the exposed motherfish.