Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. In the red-eared slider turtle (Trachemys scripta), an incubation temperature of 26 degrees C results in all male offspring, whereas an incubation temperature of 31 degrees C results in all female offspring; at temperatures intermediate to these (e.g. 29, 29.2, 29.4 degrees C) a mixed sex ratio is obtained. Administration of exogenous oestrogens will overcome the effects of an all-male producing incubation temperature to cause female sex determination, whereas administration of exogenous dihydrotestosterone (DHT) or testosterone to eggs incubating at an all-female temperature will have no discernible effect. Administration of DHT will cause male sex determination only if administered at intermediate incubation temperatures whereas administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures results in a significant number of female offspring, an effect presumably due to aromatization of testosterone to oestradiol (OE2). Since testosterone serves as the precursor to both DHT and OE2, being metabolized by reductase and aromatase respectively, three experiments were conducted to determine whether various putative reductase and aromatase inhibitors would overcome the effect of incubation temperature. First, while administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures produced females in a dose- and temperature-dependent manner, significant numbers of intersex individuals resulted from high dosage testosterone treatment to eggs incubating at a female-biased intermediate temperature. The reductase inhibitors 4MA and MK906 were capable of producing female offspring if administered at intermediate temperatures, but not in a dose-dependent fashion. Administration of the aromatase inhibitors CGS16949A and CGS20267 resulted in male offspring at both female-biased intermediate and at all female-producing temperatures in a dose-dependent fashion. Second, similar findings were obtained with combined doses of testosterone and reductase or aromatase inhibitors. Combined treatment of eggs at male-biased intermediate incubation temperatures with testosterone and reductase inhibitor resulted in female hatchlings, whereas combined treatment of testosterone and aromatase inhibitor at both female-biased intermediate and at all female-producing temperatures resulted in male hatchlings.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain.

An isoform of the estrogen receptor messenger RNA (ER-mRNA) was identified in RNA from the brain of lizards and rats. Poly(A)+ RNA from brain and uteri was reverse transcribed using gene-specific primer for the ER. The resulting complementary DNA was amplified in a polymerase chain reaction followed by cloning and sequencing of the amplified products. This isoform lacks exon four and is designated delta 4 ER-mRNA. Although several isoforms of the ER have been described from cancerous cells, to our knowledge, none has been identified previously in the brain. Furthermore, the delta 4 isoform is the only isoform detected in normal tissue. The delta 4 isoform appeared most abundant in RNA from brain tissue, whereas uterine RNA contained only trace amounts of the isoform. Apparently, tissue-specific alternative splicing accounts for these differences in abundance. Because exon four encodes a part of the steroid-binding domain, we predict that the corresponding protein encoded by the isoform will not bind estradiol and may therefore belong to a growing subclass of the steroid/thyroid/vitamin superfamily known as orphan receptors. We predict that the putative delta 4 protein may function as a ligand-independent transcription factor that acts on the same DNA response elements as the conventional ER. The abundance of this isoform in the brain relative to the uterus raises fundamental questions regarding the regulation of estrogen-responsive genes in different tissues.