Cloning, sequencing and phylogenetic/phenetic classification of an estrogen receptor alpha (alpha) subtype of sheepshead minnow (Cyprinodon variegatus).

The estrogen receptor (ER) is a key component of the reproductive system of both teleosts and tetrapods. In this study, the sequence and evolutionary relationship of sheepshead minnow (Cyprinodon variegatus) ER were examined. Total RNA from livers of adult laboratory-reared gravid female C. variegatus was reverse-transcribed to prepare cDNA. Nested pairs of gene-specific degenerative primers derived from conserved amino acid sequences of the ER DNA binding domain were used to amplify an internal fragment of the ER cDNA of C. variegatus using polymerase chain reaction (PCR) followed by rapid amplification of cDNA ends (RACE). The amplified cDNA products were inserted into pGEM T-Easy Vector for cloning and sequencing. The cloned ER cDNA segments gave a 524-amino-acid ER sequence, which represents approximately 80% of the sequence. The use of PHYLIP for phylogenetic analysis with the maximum parsimony method and for phenetic analysis with the neighbor-joining method, along with bootstrap resampling, using 24 known sequences of alpha and beta ER subtypes (both teleosts and tetrapods) indicated that the ER cDNA sequence of C. variegatus has strong homology to the alpha-subtype (ER alpha) of other teleostean fish, especially the closely related killifish species, Japanese medaka (Oryzias latipes). Because ER alpha was the only subtype found, it appears that the alpha-subtype is predominant in C. variegatus liver.

Reproductive effects of estrogenic and antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegatus).

Environmental estrogens can activate genes of the reproductive system, such as vitellogenin (VTG), a precursor to egg yolk protein, by activating the estrogen receptor (ER), whereas antiestrogens can inhibit ER activation. Adult lab-reared male sheepshead minnows (Cyprinodon variegatus) were exposed to estrogenic 4-tert-octylphenol (OP) and females to antiestrogenic cadmium (Cd), and the effects on four potential indicators of impaired reproductive function were examined: VTG in F0 male blood as sign of feminization, F0 generation fecundity/fertility, embryonic development/egg hatching/survival rate of F1 generation fry, and F0 gonadal histology. Mean VTG in the control, 11.5, 33.6, and 61.1 microg/L OP male fish were 0, 10.7, 38.7, and 65.6 mg/ml postexposure and 0, 2.5, 19.4, and 30.0 mg/ml postreproduction. A significant inverse relationship between increasing VTG in male blood and reproductive success of mating groups involving these males was shown, with higher OP decreasing percent viable eggs (fertility) by approximately 60%. Histology showed increased testis anomalies and decreased spermatozoa with increasing OP exposure. No effects on F1 embryonic development, egg hatching, or fry survival rate were observed. A significant decline in percent viable egg production involving Cd-exposed females occurred only when mated with OP-exposed males, with no eggs produced by fish exposed to the highest OP and Cd concentration. A three-week field exposure near a wastewater treatment plant outfall showed no elevated VTG in male plasma, but significantly higher total egg production per female per collection day (approximately 45%) was observed at the site furthest from the outfall.