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.     

Role of steroidogenic factor 1 and aromatase in temperature-dependent sex determination in the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider.     

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.     

Estrogen-Independent Ovary Formation in the Medaka Fish, Oryzias latipes

To investigate whether a female sex steroid, estrogen, acts as a natural inducer of female gonadal sex determination (or ovary formation) in the medaka fish, Oryzias latipes, the effects of an aromatase inhibitor and anti-estrogens on sexual differentiation of gonads were examined. We found that both drugs did not show any discernible effects on the genetically determined sex differentiation in both sexes. However, the aromatase inhibitor impaired the paradoxical effects of androgen (a male sex steroid), and the anti-estrogens inhibited the male-to-female sex reversal caused by estrogen. Treatments of the fertilized eggs with androgen disturbed the gonadal sex developments in both sexes, suggesting that sex steroid synthesis is detrimental to the gonadal sex developments in the medaka embryos. These results are consistent with the previous observation that sex steroids are not synthesized before the onset of gonadal sex differentiation, and suggest that ovary formation in the genetic females of the medaka fish is not dependent on estrogen.     

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.     

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.