Expression of P450(arom) in Malaclemys terrapin and Chelydra serpentina: a tale of two sites.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P(450) enzyme, P(450) aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5 degrees C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5 degrees C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5 degrees C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5 degrees C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5 degrees ), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important.

Embryonic brain-gonadal axis in temperature-dependent sex determination of reptiles: a role for P450 aromatase (CYP19).

Sex determination in egg-laying amniotes may be fundamentally different from that of placental mammals. The mammalian ovary differentiates normally in the absence of estrogen, whereas estrogen seems to be crucial for proper ovarian development in birds, reptiles, and lower vertebrates. Estrogens are produced normally by the biosynthetic conversion of androgens by the enzyme aromatase (CYP19), which is the sole mediator of this reaction. Aromatase inhibitors are capable of reversing females to males in turtles and chickens; therefore, a role for aromatase as the female sex determinant has been postulated for species in which sex determination is temperature-dependent. The entire aromatase coding sequence (1,509 base pairs) from adult terrapin ovaries was cloned, and Northern analysis indicates a single transcript (2.4 kb) for adult ovaries, whereas male and female brains express a 2.4-kb as well as a 9.6-kb transcript. Using a sensitive (attomole sensitivity) competitive RT-PCR technique, aromatase transcript abundance was quantified during embryonic development for embryos treated with and without estrogen. Aromatase is transcribed, well before the temperature-sensitive, (stage 12), at both male and female temperatures in the brain. There is a switch to lower aromatase transcript abundance in the female brain concurrent with an exponential rise of aromatase transcript in the putative ovary. Transcripts remain below the detection limits in the putative testes but exhibit female levels of aromatase transcript when treated with estrogen. Aromatase mRNA levels are generally reduced in the brain by estradiol application. On the basis of these findings, we have postulated a model based on the competition between 5 alpha-reductase and P450 aromatase for androgen substrate in both the brain and the undifferentiated gonad to explain the TSD phenomenon in reptiles.

Temperature-dependent aromatase expression in developing diamondback terrapin (Malaclemys terrapin) embryos.

In the diamondback terrapin, Malaclemys terrapin, males hatch at incubation temperatures below 28 degrees C whereas females hatch at temperatures above 30 degrees C. When estrogen is applied to the eggs at male temperatures early in development, females are produced. These data suggest that the enzyme necessary for estrogen synthesis (CYP19, aromatase) in the developing gonad plays a critical role in sex determination in these vertebrates. Accordingly, we have begun an examination of the role and regulation of the aromatase gene in sex determination in the diamond back terrapin, Malaclemys terrapin. We have obtained full-length cDNAs for terrapin ovarian aromatase. Using reverse transcription-polymerase chain reaction (RT-PCR) on mRNA from various tissues we have determined that aromatase is expressed in the female brain and ovary, whereas it is only expressed in the brain of the male. Brain expression of aromatase occurs before stage 15, the beginning of the temperature-dependent sex determining period. Ovarian expression occurs sometime later. To quantify expression levels, we have developed a competitive RT-PCR technique to study the ontogeny of aromatase transcript levels throughout development. The sensitivity of our assay (0.001-10 atmol of transcript) permits us to analyse individual embryonic adrenal/kidney/gonadal complexes without pooling samples. Female hatchlings (stage 26) brains express higher aromatase mRNA levels than male brains (381 +/- 80 vs 202 +/- 85 atmol/microg RNA, respectively). Similarly, ovaries express significantly higher aromatase mRNA levels than hatchling testes (352 +/- 117 vs <0.001 atmol/microg RNA, respectively).

Role of P-450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin.

Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.