Expression Of Cytochrome P450 Aromatase Research Articles

Estradiol-17beta is produced in bovine corpus luteum.

The aim of this study was to investigate the expression of cytochrome P450 aromatase (aromatase) mRNA, its activity, and estradiol-17beta (estradiol) secretion in bovine corpus luteum (CL) during the estrous cycle. Expression of aromatase mRNA was examined in CL at the early, mid, late, and regressed luteal stages by using a reverse transcription-polymerase chain reaction. Aromatase mRNA was detected in all luteal stages examined, although aromatase expression was significantly lower during the early and regressed luteal phases compared to the mid and late luteal phases. Moreover, cultured midluteal cells clearly converted exogenous [(3)H]androstenedione into estradiol, and an aromatase inhibitor significantly inhibited this conversion. To characterize the local release of estradiol within the CL during the estrous cycle, an in vitro microdialysis system (MDS) of CL was conducted. Estradiol in MDS perfusate was confirmed by a reverse-phase high-performance liquid chromatography in combination with enzyme immunoassays. Basal release of estradiol from microdialyzed CL did not change during the estrous cycle. Additionally, when freshly prepared midluteal cells were exposed to estradiol (10(-14) to 10(-9) M), estradiol stimulated prostaglandin (PG) F(2alpha) secretion (P < 0.05), although it did not affect progesterone and oxytocin secretion. The overall results indicate that estradiol is produced locally in bovine CL throughout the estrous cycle, and they suggest that estradiol plays a role in regulating PGF(2alpha) production in CL as an autocrine/paracrine factor.

Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major).

In teleosts, estradiol-17beta (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450(arom)) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450(arom) during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450(arom) is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450(arom) mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450(arom) mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450(arom) enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHbeta, but not FSHbeta, correlated with increased P450(arom) mRNA levels during the course of ovarian development. In addition, the levels of P450(arom) mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450(arom) mRNA during oocyte development of red seabream.

Interleukin-6 decreases estrogen production and messenger ribonucleic acid expression encoding aromatase during in vitro cytodifferentiation of rat granulosa cell

To elucidate the influence of interleukin-6 (IL-6) on the maturation of rat granulosa cell (GC), we have established a differentiation model of rat GC in vitro and examined the expression of IL-6 and its receptors, and its possible actions during GC maturation. Bioactive IL-6 was detectable in the conditioned media of GC at ∼2.5 ng/ml/24 h per 5×10 5 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that messenger RNAs encoding components of functional IL-6 receptor, namely both ligand-binding subunit (IL-6R) and gp130, were expressed in GC. Treatment of GC with IL-6 for 72 h during the process of in vitro GC maturation dose-dependently inhibited the accumulation of estradiol-17β (E 2) and the expression of cytochrome P450 aromatase (P450arom). IL-6 did not change nitric oxide (NO) production and inducible NO synthase expression, implying that IL-6-induced suppression on E 2 levels is dissociated with NO expression. Further, GC which had been incubated with neutralizing anti-IL-6 antibody showed a distinct increase in the levels of P450arom mRNA. These results suggest that IL-6 may attenuate E 2 production partially by inhibiting the expression of aromatase mRNA as an intraovarian regulator for suppressing GC maturation.

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.

Localized Aberrant Expression of Cytochrome P450 Aromatase in Primary and Metastatic Malignant Tumors of Human Liver

Localized Aberrant Expression of Cytochrome P450 Aromatase in Primary and Metastatic Malignant Tumors of Human Liver

Localized Aberrant Expression of Cytochrome P450 Aromatase in Primary and Metastatic Malignant Tumors of Human Liver1

The present study was designed to demonstrate localized aberrant expression of aromatase in primary and metastatic malignant liver tumors. Immunocytochemistry revealed the presence of locally increased aromatase protein in regions around tumors in all specimens from seven primary and seven metastatic liver tumors. This observation was further confirmed by Western blotting analysis and assay of aromatase activity in tumorous, proximal, and distal regions. Western blots showed most intensely immunoreactive bands at the position corresponding to aromatase in proximal tissues where aromatase activities also were higher (2.75 +/- 1.59 pmol/mg.h) than in tumors (0.137 +/- 0.115 pmol/mg.h) and distal tissues (1.90 +/- 1.47 pmol/mg.h), in spite of a gradient decline of NADPH-cytochrome P-450 reductase activity from the distal regions to the tumors. RT-PCR analysis indicated that the aberrant increase in aromatase protein and enzyme activity in the regions proximal to tumors is caused by locally elevated aromatase messenger RNA.

Expression of cytochrome P450 aromatase in the channel catfish, Ictalurus punctatus

The cDNA encoding the catfish ovarian aromatase has previously been isolated and described (accession number S75715). As demonstrated previously, the predicted amino acid sequence and enzymatic activity of the encoded protein share a significant degree of similarity to the forms of aromatase found in other vertebrates. Analyses utilizing reverse transcription coupled with the polymerase chain reaction (RT-PCR) demonstrate the expression of aromatase mRNA in catfish brain, testis and ovary. In spite of the evidence provided by Northern blot analysis for a single transcript encoding ovarian aromatase, RT-PCR analysis indicated transcript heterogeneity within the ovary, but not the testis or the brain. Although not characterized, PCR analysis indicated that the transcript complexity of ovarian aromatase was within the encoding region. Until this study, the expression of aromatase and its correlation with the reproductive physiology of fish had not been studied at the molecular level. In the catfish, significant changes in ovarian development were evident following elevation of plasma estradiol titers during October and again in February. Seasonal changes in the expression of ovarian aromatase and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA was reflected in estradiol plasma titers. Ovarian expression of 3β-HSD mRNA commenced a month before the message for aromatase was detected. Both transcripts were present from October to April. As the female approached the time of spawning (in May), the abundance of both aromatase and 3β-HSD transcripts decreased. The aromatase message was not detected in post-spawning females but 3β-HSD transcripts were evident. These data indicate that the timely synthesis of estradiol in catfish is caused by the regulation of both 3β-HSD and aromatase.

Sex differences in androgen-regulated expression of cytochrome P450 aromatase in the rat brain

The basis of functional gender differences in adult responsiveness to testosterone (T) is not yet understood. Conversion of T to estradiol by cytochrome P450 aromatase in the medial preoptic area is required for the full expression of male sexual behavior in rats. High levels of aromatase are found in the medial preoptic nucleus (MPN) and in an interconnected group of sexually dimorphic nuclei which mediate masculine sexual behavior. Within this neural circuit, aromatase is regulated by T, acting through an androgen receptor (AR)-mediated mechanism. This arrangement constitutes a feedforward system because T is both the regulator and the major substrate of aromatase. Preoptic aromatase is thus more active in adult males than in females because of normal sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic because equivalent physiological doses of T stimulate aromatase to a greater extent in males than in females. Dose-response studies indicate that the sex difference is apparent over a range of circulating T concentrations and constitute a gender difference in T efficacy, but not potency. Sex differences in aromatase correlate with sex differences in nuclear AR concentrations in most regions of the sexually dimorphic neural circuit, but not in MPN. These results suggest that males may have larger populations of target cells in which aromatase is regulated by androgen, but the lack of a gender difference in AR levels in the MPN suggests that differences in post-receptor mechanisms could also be involved. Measurements of aromatase mRNA in androgen-treated gonadectomized rats demonstrate that sex difference in regulation is exerted pretranslationally. Taken together these results demonstrate a sexually dimorphic mechanism that could potentially limit the action of T in females, and may relate to the enhanced expression of T-stimulated sexual behaviors in males.

Sex Differences in Androgen-regulated Expression of Cytochrome P450 Aromatase in the Rat Brain

The basis of functional gender differences in adult responsiveness to testosterone (T) is not yet understood. Conversion of T to estradiol by cytochrome P450 aromatase in the medial preoptic area is required for the full expression of male sexual behavior in rats. High levels of aromatase are found in the medial preoptic nucleus (MPN) and in an interconnected group of sexually dimorphic nuclei which mediate masculine sexual behavior. Within this neural circuit, aromatase is regulated by T, acting through an androgen receptor (AR)-mediated mechanism. This arrangement constitutes a feedforward system because T is both the regulator and the major substrate of aromatase. Preoptic aromatase is thus more active in adult males than in females because of normal sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic because equivalent physiological doses of T stimulate aromatase to a greater extent in males than in females. Dose-response studies indicate that the sex difference is apparent over a range of circulating T concentrations and constitute a gender difference in T efficacy, but not potency. Sex differences in aromatase correlate with sex differences in nuclear AR concentrations in most regions of the sexually dimorphic neural circuit, but not in MPN. These results suggest that males may have larger populations of target cells in which aromatase is regulated by androgen, but the lack of a gender difference in AR levels in the MPN suggests that differences in post-receptor mechanisms could also be involved. Measurements of aromatase mRNA in androgen-treated gonadectomized rats demonstrate that sex difference in regulation is exerted pretranslationally. Taken together these results demonstrate a sexually dimorphic mechanism that could potentially limit the action of T in females, and may relate to the enhanced expression of T-stimulated sexual behaviors in males.

Expression of Cytochrome P450 Aromatase in the Channel Catfish, Ictalurus punctatus

Expression of Cytochrome P450 Aromatase in the Channel Catfish, Ictalurus punctatus

Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor.

Ovarian estradiol (E2) production is regulated by complex interaction of different hormones, such as gonadotropins, steroids, and growth factors. Despite the key role of 17 beta-hydroxysteroid dehydrogenase (17HSD) in E2 biosynthesis, little is known about its regulation in the ovary. Recently, we have characterized the structure of rat 17HSD type 1 and demonstrated that its expression is regulated by gonadotropins and diethylstilbestrol (DES) in rat ovary in vivo. In the present study, the hormonal regulation of 17HSD type 1, and the expression of cytochrome P450 aromatase were examined in parallel in cultured granulosa cells obtained from DES-primed immature rats. Under these conditions, the cells show high expression of 17HSD type 1. Both the enzyme activity and 17HSD type 1 messenger RNA expression in these cells decreased over 2 days of culture in serum-free medium. However, recombinant FSH (recFSH) partially prevented the decreases in enzyme activity and messenger RNA expression in a dose-dependent manner. This effect appears to be mediated by a cAMP-dependent pathway. In contrast to recFSH, neither estrogens (DES or E2) nor androgens (testosterone or dihydrotestosterone) alone affected expression of the enzyme in the cultured cells. However, both estrogens and androgens clearly enhanced the effect of recFSH on 17HSD type 1 expression and 17HSD activity in a dose-dependent manner. Among the growth factors, epidermal growth factor (EGF) has previously been shown to decrease the expression of cytochrome P450 aromatase and E2 biosynthesis in granulosa cells. In the present study, we found that treatment with EGF caused a marked decrease in the effect of recFSH on 17HSD type 1 expression and 17HSD activity. The fact that 17HSD type 1 expression and 17HSD activity always behaved in parallel suggests that 17HSD type 1 is the major 17HSD enzyme involved in estradiol biosynthesis in rat granulosa cells. In conclusion, these data indicate that expression of 17HSD type 1 in rat granulosa cells is under multihormonal regulation. The enzyme is regulated by FSH, via cAMP, and the effect of FSH is modulated by estrogens, androgens, and EGF.

Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle- stimulating hormone, estrogens, androgens, and epidermal growth factor

Ovarian estradiol (E2) production is regulated by complex interaction of different hormones, such as gonadotropins, steroids, and growth factors. Despite the key role of 17 beta-hydroxysteroid dehydrogenase (17HSD) in E2 biosynthesis, little is known about its regulation in the ovary. Recently, we have characterized the structure of rat 17HSD type 1 and demonstrated that its expression is regulated by gonadotropins and diethylstilbestrol (DES) in rat ovary in vivo. In the present study, the hormonal regulation of 17HSD type 1, and the expression of cytochrome P450 aromatase were examined in parallel in cultured granulosa cells obtained from DES-primed immature rats. Under these conditions, the cells show high expression of 17HSD type 1. Both the enzyme activity and 17HSD type 1 messenger RNA expression in these cells decreased over 2 days of culture in serum-free medium. However, recombinant FSH (recFSH) partially prevented the decreases in enzyme activity and messenger RNA expression in a dose-dependent manner. This effect appears to be mediated by a cAMP-dependent pathway. In contrast to recFSH, neither estrogens (DES or E2) nor androgens (testosterone or dihydrotestosterone) alone affected expression of the enzyme in the cultured cells. However, both estrogens and androgens clearly enhanced the effect of recFSH on 17HSD type 1 expression and 17HSD activity in a dose-dependent manner. Among the growth factors, epidermal growth factor (EGF) has previously been shown to decrease the expression of cytochrome P450 aromatase and E2 biosynthesis in granulosa cells. In the present study, we found that treatment with EGF caused a marked decrease in the effect of recFSH on 17HSD type 1 expression and 17HSD activity. The fact that 17HSD type 1 expression and 17HSD activity always behaved in parallel suggests that 17HSD type 1 is the major 17HSD enzyme involved in estradiol biosynthesis in rat granulosa cells. In conclusion, these data indicate that expression of 17HSD type 1 in rat granulosa cells is under multihormonal regulation. The enzyme is regulated by FSH, via cAMP, and the effect of FSH is modulated by estrogens, androgens, and EGF.