P450arom Gene Research Articles

Testicular gene expression of steroidogenesis-related factors in prepubertal, postpubertal, and aging dogs

Developmental and aging changes in testicular factors related to steroidogenesis are unknown in dogs. Using reverse transcription quantitative real-time PCR, this study examined testicular mRNA levels of CYP11A1 (P450 cholesterol side-chain cleavage enzyme, P450scc), CYP17A1 (P450 17α-hydroxylase/C17–20 lyase, P450c17), HSD3B2 (3β-hydroxysteroid dehydrogenase, 3β-HSD), CYP19A (P450 aromatase, P450arom), STAR (steroidogenic acute regulatory protein, StAR), cyclooxygenase (COX) -1 and COX-2 in prepubertal (4–6 months of age), postpubertal (1 year of age), and aging (2–18 years of age) dogs. Testicular mRNA levels for P450scc, 3β-HSD, StAR, COX-1, and COX-2 did not change from prepubertal to postpubertal stages, whereas that for P450arom markedly and abruptly increased and that for P450c17 gradually decreased. In postpubertal and aging dogs, a negative correlation was found between aging and testicular P450arom mRNA levels. Based on the rapid testicular growth observed during puberty, these results suggested that total testis gene expression for steroidogenesis-related factors, in particular for P450arom, increases during puberty in dogs. In addition, the decline in P450arom gene expression during aging may affect the ability to synthesize steroids in canine testes.

Retrograde and destination transfer of sex steroid hormones in the spermatic cord vessels of the mature boar (Sus scrofa) in short-daylight and long-daylight periods, as well as vernal and autumnal equinox

The aim of this study was to examine the seasonal changes in concentration of steroid hormones in the spermatic cord vessels of the mature boar. Cytochrome P450 aromatase (P450arom) was also localized in the arteries and veins of the spermatic cord. Arterial blood was collected from the common carotid artery and from two branches of the testicular artery supplying the testis and epididymis to determine progesterone (P4), androstenedione (A2), testosterone (T2) and estradiol (E2) plasma concentrations. The greatest concentration of P4 was found in testicular artery during December (P<0.001), when compared with other periods and vessels. In contrast, the greatest A2 concentration was observed in the epididymal artery during the same season (P<0.001). Greater T2 concentrations were found in both testis and epididymal arteries than in common artery in March (P<0.001, P<0.001; respectively) and in September (P<0.01, P<0.001; respectively). The E2 concentration was weakly affected by seasonal periods, but greater E2 concentrations were found within vessels in the testis and epididymis than in the common artery. The P450arom was immunolocalized in all layers of the arteries and veins of the testicular spermatic cord. The intensity of P450arom staining was greater in December than in June (P<0.001). There were greater steroid concentrations in arterial vessels during December in comparison to June and this may explain the summer infertility in boars and may be related to the local retrograde and destination transfer into the spermatic cord area. The P450arom gene expression in this area seems to be involved in the conversion of T2 into E2 to enrich the testes and epididymis.

Effects of phenol on ovarian P450arom gene expression and aromatase activity in vivo and antioxidant metabolism in common carp Cyprinus carpio.

Ovarian cyp19a mRNA expression and P450 aromatase activity were measured in vivo in common carp Cyprinus carpio exposed to phenol for 96 h. Production of reactive oxygen species (ROS) and parameters of antioxidant defense system in serum ovary and liver of this fish after long-term phenol exposure were also studied. In vivo exposure of fish to sublethal dose of phenol for 96 h caused marked attenuation of ovarian cyp19a1a gene expression and P450 aromatase activity. Production of ROS like hydrogen peroxide and hydroxyl radicals in serum, liver and ovary in fish exposed to phenol for 15 days elevated significantly from day 1 to day 7 with no further significant increase thereafter compared to their respective control values. Total superoxide dismutase (SOD) and catalase activities in serum and ovary decreased gradually and significantly from day 1 to day 4, which then increased significantly for the rest of the exposure days. Liver SOD activity seemed to be distinctly responsive to phenol. SOD activity in liver of phenol-exposed fish started to increase gradually from day 1 to 4 with no further increase thereafter. Catalase activities in all the tissues showed significant inhibition up to day 4 which then increased gradually and significantly up to day 15 of phenol exposure compared to their respective control values. From our results, it appears that sublethal dose of phenol has the endocrine disruptive potential and effect is mediated via inhibition of ovarian P450arom gene expression and aromatase activity in vivo. Sublethal dose of phenol also caused oxidative stress, and antioxidant systems are very much effective to prevent the damages caused by the generation of ROS.

Expression and significance of p63, aromatase P450 and steroidogenic factor-1 in endometrial polyp

To investigate the expression and signification of p63, aromatase P450 (P450 arom) and steroidogenic factor-1 (SF-1) in endometrial polyp, and to explore its role in the pathogenesis of endometrial polyp. Specimen were collected from hysteroscopic resection, pathologically confirmed endometrial polyp specimens of 30 cases of endometrial polyp and the adjoining endometrium around endometrial polyp in 20 cases, endometrial tissue of normal control group of 25 patients. Immunohistochemistry SP method and real- time PCR technology were used to detect the three groups in the expression of p63, P450 arom and SF- 1 protein and gene. P450arom gene (0.274 ± 0.082) and protein (1.2 ± 1.1) expression in endometrial polyp was significantly higher than the adjoining endometrium and normal endometrium (P < 0.05); the expression of SF- 1 protein (1.1 ± 0.8) and p63 protein (0.8 ± 0.5) were also higher in the endometrial polyp than the other two control groups (P < 0.05); while the expression of SF-1 mRNA (0.105 ± 0.049 versus 0.053 ± 0.043) and p63 mRNA (0.261 ± 0.052 versus 0.180 ± 0.018) in endometrial polyp had no significant difference between endometrial polyp and the adjoining endometrial (P > 0.05). p63, P450 arom and SF-1 may play a role in the formation of endometrial polyp.

Effect of Phenol on Ovarian Secretion of 17β-Estradiol in Common Carp Cyprinus carpio

Phenol is a common substance present in many industrial wastewaters and in nonspecific pesticides. Due to its solubility and volatility phenol is often found in marine and freshwater environment. It is lipophilic compound and has a high potential for accumulating along the trophic chain. Phenol thus is not only a threat to natural environment but also to human health. The effects of phenol on the secretion of 17β-estradiol were examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of phenol for 0, 24, 48 and 96h. In the in vitro experiments, vitellogenic follicles were incubated with phenol and dose- and time-course effects on leuteinising hormone (LH) induced steroid production were examined. Exposure of fish with phenol gradually attenuated serum and ovarian 17β-estradiol levels with increasing time and maximum inhibition was noticed after 96h. Administration of phenol significantly inhibited LH-induced secretion of 17β-estradiol by the ovarian follicles in vitro. To clarify the mechanism of attenuated production of 17β-estradiol in phenol-treated follicles, stimulated by LH, in vitro effect phenol and LH on aromatase activity (conversion of testosterone to 17β-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were investigated. Physiological safe dose of phenol significantly inhibited LH-stimulated aromatase activity and P450arom gene expression in ovarian follicles. The present study further demonstrated that LH-induced activation of ovarian steroidogenic factor-1 (SF-1) is strongly inhibited by phenol treatment. These results suggest that physiological safe dose of phenol as endocrine disruption (ED) potential and the effect can be mediated via several cellular pathways including the inhibition of SF-1 activity, aromatase activity and P450arom gene expression.

Effect of cadmium chloride on secretion of 17β-estradiol by the ovarian follicles of common carp, Cyprinus carpio

Cadmium (Cd2+) is a common environmental pollutant present in wastes associated with mining, smelting and electroplating. It is a major constituent of the tobacco smoke. Exposure of this heavy metal has been linked to wide range of detrimental effects on mammalian reproduction particularly on ovarian steroidogenesis. Low doses of Cd2+ are reported to stimulate ovarian luteal progesterone synthesis whereas high doses inhibited it. Cd2+ exposure is also reported to inhibit gonadal function in fish. In the present study the effects of cadmium chloride (CdCl2) on the secretion of gonadotropin-induced 17β-estradiol was examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of CdCl2 for 0, 24, 48 and 96h and serum and ovarian 17β-estradiol levels were estimated. In the in vitro experiments, vitellogenic follicles were incubated with CdCl2 and a dose- and time-dependent effects on steroid production were estimated induced by LH. Exposure of fish with CdCl2 gradually attenuated serum and ovarian 17β-estradiol levels with increasing time and maximum inhibition was noticed after 96h. Administration of CdCl2 to the incubations significantly inhibited LH-induced release of 17β-estradiol in vitro. To clarify the mechanism of attenuated production of 17β-estradiol, in vitro effects of CdCl2 on LH induced P450 aromatase activity (conversion of testosterone to 17β-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were evaluated. Results show that LH-stimulated P450 aromatase activity and P450arom gene expression in ovarian follicles were significantly inhibited by CdCl2. The present study further demonstrated that LH-induced stimulation of ovarian steroidogenic factor-1 (SF-1) which activates aromatase enzyme, is strongly inhibited by cadmium chloride treatment.

Regulation of ovarian steroidogenesis in vitro by IGF-I and insulin in common carp, Cyprinus carpio: stimulation of aromatase activity and P450arom gene expression

Regulation of ovarian steroidogenesis in vitro by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) was investigated in intact follicles and isolated follicular cells of carp, Cyprinus carpio at vitellogenic stage of oocyte maturation. In intact follicles, IGF-I and b-insulin stimulated testosterone and 17beta-estradiol production in vitro. In isolated theca cells, IGF-I and b-insulin stimulated testosterone production, whereas in granulosa cells, they stimulated 17beta-estradiol production when testosterone was added in the incubation medium as precursor substrate. In intact follicles and in theca cells, IGF-I and b-insulin had no effect on HCG-stimulated testosterone production. HCG-stimulated 17beta-estradiol production, however, was significantly increased by IGF-I and b-insulin. To clarify the mechanism of 17beta-estradiol production by the ovarian follicles during vitellogenic stage of carp, effects of IGF-I and b-insulin either alone or in combination with HCG on aromatase activity (conversion of testosterone to 17beta-estradiol) and cytochrome P450 aromatase (P450arom) gene expression were investigated in vitro. IGF-I and b-insulin alone stimulated aromatase activity and P450arom gene expression and significantly enhanced HCG-induced enzyme activity and P450arom gene expression. Our results thus indicate that IGF-I and b-insulin alone can stimulate testosterone and 17beta-estradiol production in vitellogenic follicles of C. carpio by stimulating aromatase activity and P450arom gene expression. Evidence also provided for the modulation of HCG-induced aromatase activity and P450arom gene expression by IGF-I and b-insulin in such follicles.

Androgens and estradiol-17β production by porcine uterine cells: In vitro study

Porcine ( Sus scrofa domestica) uterine slices harvested during both early pregnancy and luteolysis produce steroid hormones. The aim of the present study was to determine (1) which porcine separated uterine cells secrete androgens: androstenedione (A 4) and testosterone (T), and estradiol-17β (E 2) in culture; (2) if the production of A 4, T and E 2 in the uterine cells is regulated by P4 and OT; (3) if uterine tissues expressed cytochrome P450arom gene ( CYP19). Uteri were collected on Days 14 to 16 of early pregnancy and the estrous cycle. Enzymatically separated epithelial cells, stromal cells, and myocytes were cultured in vitro for 2, 6, and 12 h with control medium, progesterone (P 4; 10 –5 M), oxytocin (OT; 10 –7 M), and both hormones (P 4 + OT). The studied cells secreted A 4, T, and E 2 in vitro. Progesterone served as a substrate for steroid synthesis in the uterine cells. Isolated uterine cells, cultured separately, contributed in equal portion to the basal production of androgens (A 4 and T) during both early pregnancy and luteolysis. In pregnant pigs, the epithelial and stromal cells were rich sources of E 2 compared with myocytes. Myocytes produced E 2 mainly during luteolysis. Pregnant porcine endometrium and myometrium expressed the gene CYP19, which encodes for P450 aromatase, a steroidogenic enzyme. The results indicate an active steroidogenic pathway in porcine uterine cells. The epithelial cells, stromal cells, and myocytes participate in steroid production as an alternative source for their action in pigs.

Stimulation of salmon calcitonin on secretion of 17β-estradiol by the ovarian follicles of common carp, Cyprinus carpio

The effects of salmon calcitonin (sCT) on the secretion of 17beta-estradiol (E(2)) were examined in female common carp, Cyprinus carpio. Vitellogenic stage fish adapted to high-Ca water were i.p. injected with vehicle, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. To determine whether ovarian follicles are equipped with CT receptors, a CT binding assay was conducted. In the in vitro experiments, vitellogenic follicles were incubated with stimulators and inhibitors. Administration of sCT increased the basal and hCG-stimulated E(2) release in vivo and in vitro. Binding characteristics of [(125)I]sCT to plasma membrane preparation of carp ovarian follicles showed saturability with high-affinity (K(d)=48.48 pmol/l and B(max)=1.2 pmol/mg protein). To clarify the mechanism of E(2) production by sCT, in vitro effect of sCT and hCG on aromatase activity (conversion of testosterone to E(2)) and cytochrome P450 aromatase (P450arom) gene expression in carp ovarian follicles were investigated. Salmon CT-stimulated both aromatase activity and P450arom gene expression in ovarian follicles of carp. sCT-stimulated E(2) release by the ovarian follicles in vitro was augmented in the presence of dibutyryl cAMP. Inhibitor of protein kinase A (PKA), SQ 22536 inhibited sCT-stimulated steroid production in a dose-dependent manner. Specific inhibitor of protein kinase C (PKC), NPC-15437 dihydrochloride had no inhibitory effects on sCT-induced E(2) release. The present study indicates that sCT binds specifically to carp ovary and stimulates E(2) production by increasing the activity of cytochrome P450 aromatase and P450arom gene expression. The results further suggest that stimulatory action of sCT on E(2) production is mediated through cAMP pathway.

Up-regulation of P450arom and down-regulation of Dmrt-1 genes in the temperature-dependent sex reversal from genetic males to phenotypic females in a salamander

When larvae of the salamander Hynobius retardatus were reared at a high temperature (28 degrees C) during their thermosensitive period (TSP=15-30 days after hatching), all larvae developed to phenotypic females irrespective of their genetic sexes. Hynobius P450 aromatase (P450arom) and Dmrt-1 complementary DNAs were isolated and their expression patterns were analyzed by competitive and conventional reverse transcriptase-polymerase chain reaction. While the P450arom gene was expressed predominantly in the ovary, Dmrt-1 was expressed exclusively in the testis. When larvae were reared at the female-producing temperature (28 degrees C) during the TSP, a strong expression of the P450arom gene and a complete suppression of the Dmrt-1 gene were induced in all experimental larvae. Up-regulation of the P450arom gene and down-regulation of the Dmrt-1 gene even in genetic males constitute a part of the molecular biological cascade for the temperature-dependent sex reversal from genetic males to phenotypic females in this salamander.

Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole

In the present study, we implanted 2-year-old female red-spotted grouper, Epinephelus akaara, with a non-steroidal aromatase inhibitor (AI), fadrozole, in the breeding season and examined changes in gonadal histology, serum sex steroids, aromatase activities and P450 aromatase (P450arom) gene expression in gonads after AI implantation. Aromatase inhibitor at doses from 0.1 to 10.0 mg kg−1 BW induced a sex inversion and completion of spermatogenesis up to the functional male phase, but doses of 1.0 and 10.0 mg kg−1 BW AI produced more males than 0.1 mg kg−1 BW AI. Serum estradiol-17β (E2) levels decreased, but 11-ketotestosterone (11-KT) levels increased significantly in all the AI-implanted groups, whereas testosterone (T) levels increased significantly only in the 1.0 mg kg−1 BW AI-implanted group. Aromatase activities and P450arom gene expression in gonads were inhibited significantly in the AI-implanted groups, which was in accordance with the decrease in serum E2 levels. These results suggested the optimal dose of AI to induce sex inversion to be 1.0 mg kg−1 BW. Furthermore, the sex inversion induced by AI may be attributed to the inhibition of P450arom gene expression and aromatase activity and the resultant decrease in the biosynthesis of endogenous E2. Meanwhile, the elevated 11-KT levels were also associated closely with the occurrence of sex inversion in protogynous red-spotted grouper.

Transcriptional modulation of brain and hepatic estrogen receptor and P450arom isotypes in juvenile Atlantic salmon ( Salmo salar) after waterborne exposure to the xenoestrogen, 4-nonylphenol

The molecular basis for vitellogenin (Vtg) and Zr-protein gene (and protein) expression shows that the Vtg and Zr-protein gene activations are receptor-mediated responses that are ligand structure-dependent interactions with ER, probably involving all isoforms, in addition to other co-activators. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of the male and female reproductive tracts. In vertebrates, the cytochrome P450 aromatase (P450arom) is a crucial steroidogenic enzyme catalyzing the final step in the conversion of androgens to estrogens. The present study was undertaken to investigate the effects of nonylphenol on brain and liver ER and P450arom isotypes gene expressions as sensitive biomarker of effect. Fish were exposed to static and continuous waterborne nonylphenol at 5, 15 and 50 μg/L. Blood, brain and liver samples were collected after 0 (control), 3 and 7 days post-exposure. Plasma levels of Vtg and Zr-proteins were analyzed using immunoblotting and enzyme linked immunosorbent assay (ELISA) methods. A quantitative RT-PCR method was used to analyse gene expression patterns using cloned plasmid containing amplicons of interest as standards. Our data demonstrate that both ER and P450arom gene isotypes showed differential organ-specific, nonylphenol concentration- and time-dependent expression patterns after exposure to environmental relevant concentrations of nonylphenol. Liver and plasma Vtg and Zr-protein gene and protein expression showed a concentration- and time-dependent induction at day 3 and 7 post-exposure. This is the first study evaluating, in parallel, the ERs, P450aroms and protein xenoestrogen biomarkers of effect in any fish species or lower vertebrate. Therefore, the present study shows that the brain and hepatic ER and P450arom isotypes evaluated using state-of-the-art molecular approaches are sensitive xenoestrogen biomarkers of effect. These responses should be evaluated in parallel with plasma protein biomarkers such as Vtg and Zr-proteins.

Organ-specific patterns of P450arom gene isoforms are modulated by p,p′-DDE in adult male European common frog, Rana temporaria

The organ-specific gene expression patterns of P450arom isoforms have been studied in European common frog, Rana temporaria after exposure to DDE, using real-time PCR. Four groups of frogs were subcutaneously injected with DDE at 0.01, 0.1, 1 and 10 mg/kg body weight and one group, serving as the control was injected with pure corn oil. Brain, kidney, testis and liver P450aromA and P450aromB gene expressions were evaluated at day 14 after exposure. P450aromB data show that 0.1 and 10 mg DDE/kg doses caused 76% and 63% (testis) and 79% and 80% reductions, respectively, of mRNA levels. Brain P450aromB mRNA decreased 10% and 34%, respectively, after exposure to 0.01 and 0.1 mg DDE/kg. Thereafter, a 185% and 177% induction response of brain P450aromB was observed in the groups treated with 1 and 10 mg DDE/kg, respectively. In the kidney, 0.01, 0.1, 1 and 10 mg DDE/kg induced a 516%, 178%, 466% and 247% induction of P450aronB mRNA, respectively. P450aromA expression was not quantified in any of the organs. The relative abundance of P450aromB gene expression in different organs is in the order: kidney > brain > liver > testis. The present data suggest potential detrimental effect of organochlorine pesticides (OCs) and their metabolites on the European frog steroidogenic pathways. Given the high persistency in the environment and continued use in developing countries coupled with the tendency for global atmospheric transport, OCs and their metabolites such as DDE will remain a focus of concern both for scientific and societal reasons.

Aromatase in testis: Expression and role in male reproduction

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids, among them estrogens are the end products obtained from the irreversible transformation of androgens by aromatase (P450arom). In the rat the pattern of P450arom expression differs among the testicular somatic cell types according to age; in addition, we have shown that gonocytes, spermatogonia, spermatocytes (preleptotene, pachytene), spermatids and spermatozoa, represent an important source of estrogens; the expression of aromatase is three-fold higher in pachytene spermatocyte (PS) compared to gonocytes. In man both Leydig cells and immature germ cells (PS and round spermatids, RS) as well as ejaculated spermatozoa expressed a biologically active aromatase revealed as a single band of 49 kDa on western blots. Up today P450arom has been demonstrated in male germ cells of all mammals so far studied (mice, bank vole, bear and monkey). The aromatase gene is highly conserved and is unique in humans; its expression is regulated in a cell-specific manner via the alternative use of various promoters located in the first exon. Nevertheless, data concerning the regulation of P450arom especially in germ cells are scarce. We have demonstrated that TGFβ inhibits the expression of Cyp19 in PS and RS via the SMAD pathway although TNFα exerts a stimulatory role in PS, which is amplified in presence of dexamethasone. It is noteworthy that dexamethasone alone exerts a positive effect on Cyp19 expression in PS and a negative one in RS. Cyclic AMP is also a positive regulator of P450arom gene expression in germ cells. In addition, we have shown that androgens and estrogens modulate Cyp19 gene expression, whatever the testicular cell type studied, which favored the presence of androgens and estrogens responsive elements on the Cyp19 gene promoter(s). Moreover, in presence of seminiferous tubules conditioned media, the amount of aromatase transcripts is increased in Leydig cells, therefore, suggesting that other locally produced modulators are involved in the regulation of the aromatase gene expression and among them the liver receptor homolog-1 (LRH-1) from germ cells origin is concerned. Using RACE-PCR we have confirmed that promoter II directs the expression of aromatase gene, whatever the testicular cell type studied in the rat but the involvement of another promoter, such as PI.4 is suggested. Finally, the aromatase gene is constitutively expressed both in somatic and germ cells of the testis and the identification of the promoter(s) concerned as well as their detailed regions which direct(s) the expression of Cyp19 gene is obviously very important but largely unknown especially according to the ontogeny of the male gonad.

Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed.The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5'-UTR of 18 bp and at least 407 bp in 3'-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.

No evidence of mutations in the P450 aromatase gene in patients with polycystic ovary syndrome

Etiology and inheritance pattern in polycystic ovary syndrome (PCOS) remain uncertain. Granulosa cells from follicles of women with PCOS have little, if any, aromatase (encoded by the CYP19 gene) activity; follicles contain low levels of estradiol, P450arom mRNA and aromatase stimulating bioactivity. Mice with targeted disruption of the CYP19 gene present cystic follicles. It has been proposed that chronic exposure to high levels of LH, because of aromatase deficiency, determines the development of ovarian cysts. Herein, we investigated if mutations in the CYP19 gene and/or its ovary promoter are causal in patients with PCOS. Twenty-five patients with PCOS and 50 control women were studied. PCR analysis of genomic DNA and complete sequence of all exons of the aromatase gene and its ovary promoter were performed. No heterozygous or homozygous mutant alleles were present in any of the patients studied. In the population studied, mutations of the P450arom gene or its promoter are not the cause of PCOS. However, these findings do not preclude the possible importance of an aromatase disorder in PCOS etiology. Variations in aromatase complex function could play a role in PCOS etiology, but the determinants of such variations might be located in other genes.

P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads

Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.

Aromatase cytochrome P450: cloning, intron variation, and ontogeny of gene expression in southern flounder (Paralichthys lethostigma)

Aromatase cytochrome P450 (P450arom) is the enzyme complex responsible for conversion of androgens to estrogens in vertebrates. Consequently, in some fishes its activity appears critical to ovarian differentiation. Southern flounder (Paralichthys lethostigma) is a commercially important flatfish in which females grow larger than males and sex determination is temperature sensitive. Through cloning of the P450arom gene in ovary and quantitative reverse transcription-polymerase chain reaction, we developed a biomarker for early female differentiation in southern flounder. The deduced amino acid sequence for southern flounder P450arom is similar to other teleosts. Comparison of P450arom intron sequences from fish of different populations revealed substantial inter-individual variation. Adult ovary and spleen exhibited high levels of P450arom mRNA, while P450arom mRNA was only weakly detected in testes. Brain, liver, intestine, kidney, gill, muscle, and heart showed little or no P450arom mRNA expression. Gonads of wild and hatchery-produced juvenile flounder of sizes spanning the period of sex differentiation initially exhibited low levels of P450arom mRNA followed by increases in some individuals and bifurcation into two clearly segregated groups (i.e., putative males and females) beginning at approximately 65 mm in total length. Gonadal histology confirmed predictions of sex based on P450arom expression in juvenile flounder, demonstrating that the patterns of P450arom expression observed relate to sex-specific differentiation. This research represents a unique approach to assessing sex differentiation in a natural population, and a powerful technique for better understanding mechanisms of flounder sex determination and rapidly defining conditions for controlling sex for aquaculture.

Sequence and expression of cytochrome P450 aromatase and FTZ-F1 genes in the protandrous black porgy ( Acanthopagrus schlegeli)

In this study, a cDNA encoding cytochrome P450 aromatase ( P450arom) was cloned from black porgy Acanthopagrus schlegeli ovary. The deduced amino acid sequence had high homology with ovarian P450arom of other teleost fish. Moreover, we partially cloned two FTZ-F1 homologues ( asff1a and asff1b) from black porgy. Comparative sequence analysis grouped asff1a and asff1b in NR5A2 and NR5A4 clades, respectively. Among the various tissues tested, P450arom mRNA was highly expressed in the ovary and weakly in the brain and testis, asff1a was expressed in brain, liver, intestine, kidney, testis, and ovary, asff1b was expressed in brain, kidney, testis, and ovary. The transcript levels of P450arom, asff1a, and asff1b were measured in the ovary and testis of 1 +-year-old, 2 +-year-old, and 5 +-year-old black porgy. The transcript level of P450arom in the ovary of 2 +-year-old fish was significantly higher than those of 1 +-year-old and 5 +-year-old fish. The results suggest that P450arom gene may be involved in the mechanism of natural sex change of protandrous black porgy. No change in ovarian expression of asff1a or asff1b was observed among different ages. These results suggest that up-regulation of the transcript levels of P450arom during the course of natural sex change of black porgy was not regulated via FTZ-F1.

In Vivo and in Vitro Inhibition of cyp19 Gene Expression by Prostaglandin F2α in Murine Luteal Cells: Implication of GATA-4

A major function of the corpus luteum (CL) is to secrete progesterone. In rats, this gland also produces significant amounts of 17beta-estradiol. Progesterone and 17beta-estradiol are important regulators of rat luteal cell function. Estrogen biosynthesis is catalyzed by P450aromatase (P450arom), which is encoded by the cyp19 gene. In the rat CL, P450arom is expressed throughout pregnancy until the day before parturition, when it rapidly decreases. The mechanisms that control P450arom expression in luteal cells, particularly, the one or more factors that cause its rapid fall before parturition, are not known. Inasmuch as prostaglandin (PG) F(2alpha) plays a key role in the regulation of luteal function at the end of pregnancy, the purpose of this investigation was to determine whether PGF(2alpha) affect the expression of P450arom in the CL before parturition. PGF(2alpha) decreased luteal P450arom mRNA and protein levels in vivo and in vitro. A decrease in P450arom mRNA was also observed in mice CL just before parturition, but this change did not take place in PGF(2alpha) receptor knockout mice. The time course of the decrease in P450arom mRNA by PGF(2alpha) reflected the P450arom mRNA half-life determined by actinomycin D. Moreover, nuclear run-on assay showed that PGF(2alpha) attenuates P450arom gene transcription. Gel shift assays revealed that GATA-4 binds to the P450aromatase promoter, and that such binding is increased by PGF(2alpha). It is concluded that PGF(2alpha) decreases luteal P450arom mRNA levels at the end of pregnancy in rodents by inhibiting cyp19 expression.