Effects of exposure to the xenoestrogen octylphenol and subsequent transfer to clean water on liver and gonad ultrastructure during early development of Zoarces viviparus embryos.

Female eelpouts (Zoarces viviparus L.) are exposed during early pregnancy to nominal concentrations of 100 microg/L of 4-tert-octylphenol (OP) or 0.5 microg/L of 17beta-estradiol (E2). Effects on maternal metabolism and on liver and gonad development in embryos were examined and compared with controls (C) during exposure and after transfer to clean water (depuration). In the mother fish, significantly higher concentrations of plasma vitellogenin (vtg) and calcium were found in the two exposed groups, when compared with the C group after exposure and depuration. When compared, however, with the respective values after exposure, vtg had decreased significantly after depuration. The hepatosomatic index was normalized after depuration. In both exposed groups, the hepatocytes were rounded and not distinctly polygonal as in the controls. The amount of glycogen was considerably less while the number of mitochondria increased, and the rER significantly proliferated after exposure as well as after depuration. The gonads of nine of more than 28 embryos in the group treated with OP exhibited a number of abnormalities as compared with the normal gonad development in both sexes. Feminization of the male gonads in the exposed specimens and a number of histopathological features were observed in all the abnormal gonads, whereas reliable male features, such as formation of seminiferous tubules or spermioduct, were not observed. This study showed that 4t-tert-OP and 17beta-estradiol exert estrogenic effects during very early development of the embryos and that depuration had a positive effect on the motherfish and her embryos.

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study.

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.

Anti-estrogen prevents xenoestrogen-induced testicular pathology of eelpout (Zoarces viviparus).

Estrogenic alkylphenols have been shown to affect the reproductive system of male fish causing induction of vitellogenin synthesis and altered testis structure. However, it is still unknown whether the histopathological effects on the testes is mediated by the estrogen receptor or if it represent general toxicopathological effects. In the present study, the effects of different concentrations of the estrogenic chemical 4-tert-octylphenol on vitellogenin (Vtg) synthesis and testicular structure were investigated in the eelpout Zoarces viviparus during spermatogenesis. Adult male eelpout were exposed to 4-tOP (nominal concentrations: 10, 50 or 100 microg l(-1)) or 17beta-estradiol (E2; 0.5 microg l(-1)) in a continuous flow-through system for 3 weeks. A group of fish were exposed to 4-tOP (50 microg l(-1)) concomitantly with the anti-estrogen ZM 189,154 (20 microg g(-1) week(-1), i.p.). The Vtg concentration in plasma was measured by enzyme-linked immunosorbent assay. The testicular structure was examined by light microscopy and gamma-glutamyl transpeptidase (gamma-GTP) activity was measured in the testes. The testicular localization of gamma-GTP was analysed by enzyme histochemistry. A marked increase in the plasma Vtg concentration was observed after exposure to the actual concentration of 35 microg l(-1) 4-tOP (nominal concentration, 50 microg l(-1)), 63 microg l(-1) 4-tOP (nominal concentration, 100 microg l(-1)) or E2. Co-treatment with ZM 189,154 totally abolished the 4-tOP-dependent induction of Vtg synthesis. Exposure to 4-tOP or E2 caused a marked reduction in the testis mass and severely affected the testicular development and structure including the Sertoli cells (based on histology and gamma-GTP activity), resulting in impairment of spermatogenesis and degeneration of lobular structures. Other cellular abnormalities such as accumulations of yellowish-brown pigmented cells and increased interstitial fibrosis in the testes was also observed in the exposed fish. In the groups exposed to the nominal concentrations of 50 or 100 microg l(-1) all fish had severely affected testes, while both normal, moderately and severely affected testes were found in the group exposed to the nominal concentration of 10 microg l(-1). Co-treatment with ZM 189,154 abolished part of these 4-tOP-induced effects on the testicular growth and histological structure. The study demonstrates that an anti-estrogen can abolish effects on the testis caused by estrogenic chemicals, providing evidence that some of the effects are mediated by the estrogen receptor.

Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus).

Estrogenic chemicals such as alkylphenols (APs) have been shown to disrupt the reproductive system of male fish. In the present study, the effects of the estrogenic chemical octylphenol (100 microg g(-1)) and 17 beta-estradiol on the semen production and the biochemical composition of the seminal fluid of the viviparous eelpout (Zoarces viviparus) were investigated at the time of spawning. After 10 days of octylphenol or estrogen treatment, vitellogenin (Vtg) synthesis was induced as indicated by increased plasma vitellogenin concentration. In accordance with the increased vitellogenin concentration, hepatosomatic index (HSI), total protein concentration, and total calcium concentration were also increased, and free amino acids concentration was decreased in blood plasma. Octylphenol treatment caused a decrease in the gonadosomatic index (GSI) and the milt volume and an increase in the spermatocrit. The histological examination revealed that octylphenol affected the normal lobular structure, including the Sertoli cells. In the majority of the octylphenol-treated fish, trapped sperm cells were observed in parts of the seminiferous lobules and the sperm ducts. The biochemical composition of the seminal fluid was also affected by the octylphenol or estrogen. The seminal plasma concentrations of magnesium, calcium, and total protein were elevated, and the concentration of free amino acids was reduced in the treated fish. This study indicates that octylphenol inhibits the seminal fluid production and changes its biochemical composition in eelpouts.

Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario.

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.