The effect of progestin on rabbit endometrial aromatase activity

The ability of rabbit endometrium to synthesize estrogen was investigated. Aromatase activity was measured by incubating the viable tissue fragments with [3H] testosterone at 37 degrees C and quantitated the [3H]estrogens at the end of 6 h incubation. The activity was not detectable in endometrium and myometrium of non-pregnant rabbits. In pregnant rabbit, the activity increased from 2 to 12 fmol/h/g of tissue in endometrium obtained from both gravid and nongravid uterine horns 4-8 days after mating. Aromatase activity in endometrium at implanted sites on the 8th day after mating reached up to 550 fmol/h/g of tissue. Hormonal regulation of the aromatase activity was studied directly in cultured rabbit endometrial stromal cells. Both progesterone and medroxyprogesterone acetate stimulated the activity with 2- to 25-fold increase over the control values (0.2-1 fmol/h/mg protein). The stimulation of aromatase activity by progestin was found to be both time- and dose-dependent. Estrogen, Bu2cAMP, forskolin, HCG or relaxin had no effect on the activity. Aromatase activity in glandular epithelial cells was neither detectable nor affected by progestin. These results indicate that aromatase activity in rabbit endometrium is concentrated in stromal cells and progestin stimulates the activity. However, estrogen, forskolin and relaxin which enhanced the stimulation of the activity by progestin in human endometrium had no effect on aromatase in rabbit endometrium. The increase of aromatase activity observed after the onset of conception may be physiologically important to increase the local estrogen content for decidualization of the endometrium.     

Effects of gonadectomy and androgen treatment on aromatase activity in the fetal monkey brain

We studied the ability of neural tissues from fetal rhesus macaques to aromatize androgens to estrogens and assessed whether androgens are involved in the regulation of aromatase activity during development. Fetuses of both sexes, obtained on approximately Day 100 of gestation, were gonadectomized and immediately given intraabdominal Silastic capsules containing dihydrotestosterone. Sham-gonadectomized (intact) and gonadectomized, sham-implanted groups were also studied. Three weeks after the initial operation, the fetuses were delivered by cesarean section. To examine the developmental pattern of aromatase activity in the brain, fetuses were also delivered at two earlier times in gestation (at approximately 50 days and approximately 80 days). Whole-homogenates of preoptic area plus anterior hypothalamus (POA), hypothalamus (HT), amygdala (AMYG), and cerebral cortex (CTX) were incubated for 1 h in a phosphate buffer with saturating concentrations of [1 beta-3H] androstenedione. The amount of 3H2O formed was used as an index to estimate aromatase activity. The aromatase reaction exhibited Michaelis-Menten kinetics with an apparent Km of approximately 0.03 microM in all tissues from 120-day-old fetuses. Activity measured with the 3H2O assay closely corresponded with levels determined by product isolation. The highest levels of aromatase activity were found in the POA. Neither gonadectomy nor treatment with androgen affected aromatase in fetal tissue; activities in males were significantly greater than in females for the AMYG and CTX, but not for the POA or HT. The levels of aromatase activity detected in the HT and CTX of both sexes on Days 50 and 80 of gestation were at least ten times greater than the levels measured in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of Sertoli cell aromatase activity and plasminogen activator production by hormones, retinoids and the testicular paracrine factor, PModS.

Testicular peritubular cells produce a paracrine factor termed PModS that has dramatic effects on Sertoli cell function in vitro. The current study was designed to examine the actions of PModS and hormones on Sertoli cell aromatase activity and plasminogen activator production at various stages of pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats (ages correspond to prepubertal, midpubertal, and late-pubertal stages of development). Aromatase activity was found to be high and hormone-responsive in prepubertal Sertoli cells and to decline and be nonresponsive to hormones in late-pubertal Sertoli cells. FSH was the only hormone found to influence aromatase activity and estrogen production. PModS alone was not found to affect aromatase activity at any of the developmental stages examined. Interestingly, PModS was found to suppress the ability of FSH to stimulate aromatase activity and estrogen production in midpubertal Sertoli cells. Results imply that PModS may promote Sertoli cell differentiation to a more adult stage of development that is less responsive to FSH in stimulating aromatase activity. In contrast to aromatase activity, plasminogen activator production was found to increase during pubertal development. Production of Sertoli cell tissue-type plasminogen activator (tPa) was stimulated by FSH at each of the developmental stages examined, whereas production of urokinase-type plasminogen activator (uPa) was influenced by FSH only in prepubertal Sertoli cells. Insulin also stimulated uPa and tPa production by prepubertal Sertoli cells, and retinol significantly suppressed uPa production and the ability of FSH to stimulate tPa production by midpubertal Sertoli cells.     

芳香化酶在文昌鱼神经系统、哈氏窝和性腺特异性定位:原位杂交和免疫细胞化学研究

芳香化酶活性发现在脊椎动物脑、脑垂体和性腺中,但在文昌鱼脑和哈氏窝的组织特异性定位尚无可利用资料。本文用免疫细胞化学和原位杂交技术,首次发现芳香化酶活性组织特异性定位在幼年和性腺发育不同时期雌、雄文昌鱼神经系统(脑和脊髓)、轮器、哈氏窝和性腺中。芳香化酶蛋白和转录物在前脑、中脑、脊髓、轮器和哈氏窝十分丰富,而后脑、早期卵巢和精巢不够丰富;没有芳香化酶表达的部位是哈氏窝另两种细胞(不规则形细胞和带纤毛粘液细胞)以及成熟卵巢和精巢;芳香化酶免疫活性物质分布在胞质,核为阴性。芳香化酶在文昌鱼神经系统、哈氏窝和性腺的分布模式与低等脊椎动物中的分布模式极为类似,尤其是芳香化酶在脑内调节哈氏窝分泌活动的神经内分泌中枢表达,并形成类似脊椎动物的文昌鱼原始的脑-芳香化酶调节系统。这些结果有力地证明,文昌鱼脑和哈氏窝高水平的芳香化酶活性像在其它脊椎动物中一样,对局部介导睾酮芳香化起着关键作用,同时还可能影响脑-芳香化酶系统参与调节哈氏窝的分泌活动[动物学报49(6)：800～806,2003]。     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and thesexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

Developmental Changes in Calmodulin-Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). This kinase is comprised of a major 50-kilodalton (kDa) and a minor 60-kDa subunit. Experiments examined the developmental properties of CaM-kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM-kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that forebrain CaM-kinase II undergoes a striking age-dependent change in subunit composition. In early postnatal forebrain the 60-kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM-kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM-kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

Aromatase activity of human adipose tissue stromal cells: effects of thyroid hormones and progestogens

In order to determine the direct effects of thyroid hormones and progestogens on extraglandular aromatization, human adipose stromal cells in monolayer culture were used as a model system for this study on the regulation of aromatase enzyme activity. It was found that 1-thyroxine at 2- and 4-fold normal concentrations (220 and 440 nM, respectively) and triiodothyronine at 4-fold normal concentration (7.4 nM) had no effect on basal, dibutyryl cyclic AMP ((Bu)2 cAMP)-induced, or dexamethasone-induced aromatization. Medroxyprogesterone acetate at a concentration of 25.9 nM, but not progesterone, 47.7 nM, stimulated basal aromatization slightly but not significantly. In contrast, both medroxyprogesterone acetate and progesterone potentiated the effect of (Bu)2 cAMP on aromatase activity (P less than 0.05 and P less than 0.01, respectively) but had no effect on dexamethasone-stimulated aromatase activity. We concluded that (i) the increased peripheral aromatization associated with hyperthyroidism is not due to the direct effect of thyroid hormones on aromatase activity, and (ii) neither progesterone nor medroxyprogesterone acetate inhibit aromatase activity of adipose tissue stromal cells, but may stimulate this activity under certain conditions.     

The potential role of estrogen in aromatase regulation in the breast

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3–4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.     

Aromatase and other inhibitors in breast and prostatic cancer

Estrogens have an important role in the growth of breast and other hormone-sensitive cancers. We have shown that 4-hydroxyandrostenedione (4-OHA) selectively blocks estrogen synthesis by inhibiting aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. In postmenopausal men and women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either by daily oral or parenteral weekly treatment with 4-OHA, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. We recently studied the effects of 4-OHA and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione (PED) and imidazo[1,5-]3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile) (CGS 16949A) as well as 5-reductase inhibitors, N,N-diethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxyamide (4-MA) and 17β-hydroxy-4-aza-4-methyl-19norandrost-5-en-3-one (L651190) in prostatic tissue from 11 patients with prostatic cancer and six patients with benign prostatic hypertrophy (BPH), and from normal men at autopsy. We attempted to measure aromatase activity in tissue incubation by quantitating ³H₂O released during aromatization of androstenedione or testosterone labeled at the C-1 position. The amount of ³H₂O released from all samples was at least twice that of the heat inactivated tissue samples. The ³H₂O release was significantly inhibited by 4-OHA and 4-MA, but not by the other aromatase inhibitors. However, when HPLC and TLC were used to isolate steroid products, no estrone or estradiol was detected in the incubates. Furthermore, no aromatase mRNA was detected following amplification by PCR. The 4-OHA was found to inhibit 5-reductase in both BPH and cancer tissue, although to a lesser extent than 4-MA. The other aromatase inhibitors were without effect. Although a mechanism involving intraprostatic aromatase is not likely, inhibitors may act to reduce peripherally-formed estrogens. In postmenopausal breast cancer, the results indicate that 4-OHA is of significant benefit.     

Thyroid hormones modulate ornithine decarboxylase in the immature rat cerebellum

In this study, we measured ornithine decarboxylase (ODC) activity as a potential parameter to evaluate the response of the developing rat brain to thyroid hormones. In cerebellum, neonatal hyperthyroidism (40 micrograms thyroxine/100 g body weight daily from birth) increased ODC activity at 2 and 5 days of age and then accelerated its developmental decline. Conversely, ODC activity was decreased in 2- and 5-day-old hypothyroid rats (propylthiouracil to the mother), but it was not significantly different from normal thereafter. No significant differences were observed in the forebrain following either treatment. In hypothyroid rat cerebellum, a single injection of triiodothyronine (T3, 100 micrograms/100 g 18 h before sacrifice) increased significantly ODC activity at all ages. A dose-response study showed that 0.5 micrograms T3/100 g is sufficient to obtain maximal stimulation. Finally, administration of antiserum against rat growth hormone had no significant effect on ODC response to T3. These results show that ODC is a useful marker of thyroid state and tissue response in the neonatal rat cerebellum.     

Rat testis 17 beta-estradiol: identification by gas chromatography-mass spectrometry and age related cellular distribution

The aromatization of testosterone into 17 beta-estradiol (E2) was assessed in purified Leydig and Sertoli cells from rats aged 10-80 days. E2 was identified by gas chromatography-mass spectrometry (GC-MS) and measured both by radioimmunoassay (RIA) and GC-MS associated with stable isotope dilution. A potent competitive inhibitor of the aromatase activity, 4-hydroxyandrostenedione (4-OH-A) was used to test the enzymatic specificity. The basal aromatase activity was present in both cell types whatever the age of the animals. The basal E2 levels did not vary in Sertoli cells while a gradual increase was noted in Leydig cells until day 40, followed by a slight decrease in mature rats. In 10-day old animals, the aromatase activity was localized in Sertoli cells and highly stimulated by FSH; on day 20, both Sertoli and Leydig cells synthesized E2 although E2 from Sertoli cell origin was still predominant. Starting on day 20 until adulthood, the aromatase activity was under LH control in Leydig cells with a maximum around 40 days. The FSH and LH effects were mediated by cyclic AMP.     

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.     

Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

芳香化酶抑制剂letrozole对赤点石斑鱼(Epinephelus akaara)性逆转的作用

在繁殖季节,采用腹部埋植方式,用非类固醇型芳香化酶抑制剂（aromatase inhibitor,AI）letrozole以5mg／kg体重的剂量处理2龄雌性赤点石斑鱼（每4周埋植1次,共埋植2次）,检查埋植后性腺组织结构、血清性类固醇激素以及脑和性腺芳香化酶活性的变化。结果显示：一次埋植AI即可有效诱导雌性赤点石斑鱼发生不同程度的性逆转;性腺成熟指数明显下降;性腺中卵细胞退化,精原细胞增殖,出现大量精母细胞和精子细胞;性逆转雄鱼的精巢在组织结构上与正常雄鱼精巢没有明显差异,部分鱼成为功能性雄鱼。第一次埋植AI后4周轻微挤压腹部有14．3％的鱼可排精,精子活力与正常雄鱼相同。第二次埋植后明显提高性逆转效果,排精率在第6、8周分别达到35．3％和48．4％。此外,埋植AI后性腺芳香化酶活性显著降低,化脑郝芳香化酶活性的变化不明显;血清11-酮基睾酮（11-ketotestosterone,11-KT）浓度显著增加,雌二醇（estradiol-17β,E2）水平显著降低,而睾酮（testosterone,T）含量无明显变化。这些结果表明,AI主要通过抑制内源性E2的产生并提高11-KT水平,从而诱导赤点石斑鱼由雌性转变为雄性。