Synthesis and aromatization of 19-norandrogens in the stallion testis

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.     

Equine testicular aromatase: substrates specificity and kinetic characteristics.

1. In the stallion, estrogens were synthesized and sulfated in vivo by the testis. 2. The equine testicular enzyme aromatized androgens and 19-norandrogens with similar velocity, but not 16 alpha-hydroxytestosterone or epitestosterone in contrast to the human placental aromatase. 3. One single enzyme was implicated in the aromatization of androstenedione, testosterone, 19-norandrostenedione and 19-nortestosterone by ETMES. 4. During the process of androstenedione aromatization by ETMES, 19-hydroxyandrostenedione and 19-oxoandrostenedione were released and 4-hydroxyandrostenedione was a competitive inhibitor causing an additional irreversible enzyme inactivation which is what occurs with HPMES. 5. Dihydrotestosterone was a potent competitive inhibitor of aromatase activity.     

Aromatase in the human choriocarcinoma JEG-3: inhibition by R 76 713 in cultured cells and in tumors grown in nude mice

The aromatase enzyme and its inhibition by R 76 713 were characterized in the JEG-3 choriocarcinoma cell line in culture and in JEG-3 tumors grown in nude mice. Optimal cell culture parameters and enzyme reaction conditions for the determination of aromatase activity were established. Under these conditions, in vitro JEG-3 aromatase was inhibited by R 76 713 with IC50-values of 7.6 +/- 0.5 nM and 2.7 +/- 1.1 nM using 500 nM of androstenedione and testosterone as substrate respectively. The Km-value of the aromatase enzyme with androstenedione as substrate was 62 +/- 19 nM; with testosterone as substrate, a value of 166 +/- 27 nM was found. In the presence of increasing concentrations of R 76 713, the Km-values increased while the Vmax remained unchanged. Using androstenedione and testosterone as substrate Lineweaver-Burk analysis of the data showed Ki-values for R 76 713 of 0.43 +/- 0.06 nM and 0.47 +/- 0.39 nM respectively. R 76 713 appeared to competitively inhibit the JEG-3 aromatase. Aromatase could easily be measured in homogenates of JEG-3 tumors grown in nude mice and showed Km-values similar to those found for JEG-3 cells in vitro. IC50-values for inhibition of tumor aromatase by R 76 713 were also similar to those found in cultured cells. Tumor aromatase measured ex vivo, 2 h after a single oral administration of R 76 713 was dose-dependently inhibited. An ED50-value of 0.05 mg/kg was calculated. The JEG-3 choriocarcinoma proved to be a useful aromatase model enabling the comparative study of aromatase inhibition in vitro and in vivo.     

Enzyme inactivation by potential metabolites of an aromatase-activated inhibitor (MDL 18,962)

MDL 18,962, 19-acetylenic androstenedione, is an enzyme-activated inhibitor of estrogen biosynthesis which is in Phase I clinical evaluations as a potential therapeutic agent for estrogen-dependent cancers. 19-Acetylenic analogs corresponding to the major metabolites of androstenedione were synthesized as potential metabolites of MDL 18,962. These compounds were 19-acetylenic testosterone, the product of 17 beta-hydroxy steroid oxidoreductase, 6 beta-hydroxy- and 6-oxo-19-acetylenic androstenedione, products of P450 steroid 6 beta-hydroxylase and alcohol dehydrogenase, respectively. All of these analogs showed time-dependent inactivation of human placental aromatase activity. The time-dependent Ki and t1/2 at infinite inhibitor concentration (tau 50) were 4.3 nM, 12.0 min for MDL 18,962; 28 nM, 7.8 min for 17-hydroxy analog; 13 nM, 37 min for 6 beta-hydroxy analog; and 167 nM, 6.1 min for the 6-oxo analog. The 19-acetylenic testosterone, a confirmed metabolite from primate studies, was 25% as efficient as MDL 18,962 for aromatase inactivation, while 6 beta-hydroxy- and 6-oxo analogs were 11% and 5%, respectively as efficient as their parent compound. These data indicate that first-pass metabolism of MDL 18,962 does not cause an obligatory loss of time-dependent inhibition of human aromatase activity.     

Androgen and 19-norandrogen aromatization by equine and human placental microsomes

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Androgen aromatization by luteinized bovine granulosa cells in tissue culture

Luteinized bovine granulosa cells in tissue culture contained an active 19-hydroxylase aromatase enzyme system which converted exogenous androstenedione and testosterone to oestradiol-17beta; no oestrone was detected. In the absence of exogenous androgens, the cells failed to synthesize oestrogens due to a limited capacity to synthesize androgen precursor. Theca-lutein cells, present in those CL which synthesize oestrogens, may provide androgen precursor for aromatization by the granulosa-lutein cells.     

No inhibitory effects of gestrinone and medroxyprogesterone acetate on the estrogen production by ovaries of hypophysectomized rats stimulated by gonadotropins.

The in vivo effects of gestrinone (R2323) and medroxyprogesterone acetate (MPA) on the estrogen production by rat ovaries were investigated. Hypophysectomized immature female rats treated with 2.5 or 5 IU of pregnant mare serum gonadotropin (PMS) were daily given vehicle only, gestrinone (0.5 mg/kg body weight) or MPA (10 mg/kg body weight), and the activities of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 17, 20-lyase, 17 beta-hydroxysteroid dehydrogenase and aromatase in ovaries of these rats were measured. Gestrinone suppressed the 3 beta-hydroxysteroid dehydrogenase activity and increased activities of 17 alpha-hydroxylase, 17, 20-lyase and aromatase in ovaries stimulated by 5 IU of PMS, while MPA suppressed activities of 17 alpha-hydroxylase and aromatase in these ovaries. On the other hand, the aromatase activity in ovaries stimulated by 2.5 IU of PMS was suppressed by gestrinone and increased by MPA, and neither gestrinone nor MPA affected the production of aromatizable androgens from progesterone by these ovaries. Thus, gestrinone and MPA administrated in vivo showed divergent influences on steroidogenic enzyme activities in ovaries, but they did not affect the serum concentration of estradiol-17 beta. The present results suggest that neither gestrinone nor MPA reduced estrogen production by the rat ovary under the gonadotropin stimulation although they influenced some process of its steroidogenesis.     

Plasma androgens during the luteal phase in a case of true hermaphroditism with bilateral ovotestis

A true hermaphrodite with a bilateral ovotestis and a 46 XX karyotype was studied. This 14-year old subject developed ambiguous puberty with bilateral gynecomastia and stage IV public hair. Relatively high level of testosterone (T) (2.80 ng/ml), was found. The 5 alpha-reductase activity for T in the pubic skin was similar to that observed in normal adult males. A hemorrhagic corpus luteum in the left ovotestis was observed at laparotomy. The luteal phase immediately after dexamethasone administration (1 mg/day for 7 days) was characterized by a significant decrease of plasma androgens, T and androstenedione (A). The constantly low level of T (0.30 ng/ml) during the luteal phase in this subject did not appear to be due to the previously administered dexamethasone. This decrease of T production in the luteal phase might be secondary either to the increase of the estradiol-17 beta (E2) secreted by the corpus luteum or to the decrease of LH levels. Both mechanisms might act concomitantly.     

Kinetic studies on androstenedione production in ovarian microsomes from immature rats.

Androstenedione formation from progesterone by P-450(17 alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17 alpha,lyase) were demonstrated by a double-substrate double-label experiment using [14C]progesterone and 17 alpha-[3H]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17 alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17 alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone (Km = 80 nM) to be strongly inhibited by progesterone (Ki = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17 alpha,lyase). 20 alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone with Ki = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20 alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.     

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Aromatase inhibition by R 76 713: a kinetic analysis in rat ovarian homogenates

Reaction kinetics of the aromatase enzyme and of a new nonsteroidal aromatase inhibitor, R 76 713 (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)-methyl]-1-methyl-1H- benzotriazole), were studied in ovarian homogenates obtained from pregnant mare's serum gonadotropin (PMSG)-injected female Wistar rats. The Km (Michaelis constant) of the aromatase enzyme with androstenedione as the substrate was 47 +/- 13 nM; for testosterone as the substrate, a value of 159 +/- 10 nM was found. In the presence of increasing concentrations of R 76 713, the Km increased while the Vmax (maximal velocity of enzyme-catalyzed reaction) remained unchanged. Using androstenedione and testosterone as the substrate, Lineweaver-Burk analysis of the data showed a Ki (dissociation constant of the enzyme-inhibitor complex) for R 76 713 of 0.7 +/- 0.3 nM and 1.6 +/- 0.4 nM, respectively. R 76 713 appeared to competitively inhibit the rat ovarian aromatase.     

A three-dimensional model of CYP19 aromatase for structure-based drug design

Aromatase (CYP450_arom, CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme–substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.     

Androstenedione and testosterone biosynthesis by the adrenal cortex of the horse

An homogenate from cortical tissue of mare adrenals was incubated in the presence of tritiated pregnenolone. The (3H) androstenedione and the (3H) testosterone synthesized during the incubation were extracted, purified, and co-crystallized to constant specific activity in the presence of unlabeled carriers. The rate of conversion of pregnenolone to androstenedione and testosterone was of the order of 5 and 0.15 per cent respectively. The high ratio of (3H) androstenedione to (3H) testosterone observed in this study suggests that androstenedione is the main androgen produced by mare adrenals. It is concluded that adrenals could contribute to the production of blood androgens in normal as well as hyperandrogenic mares.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Serum estradiol-17beta concentrations during spontaneous silent estrus and after prostaglandin treatment in the mare

Serum estradiol-17beta concentrations were determined during silent estrus in the mare. Relationships between serum estradiol-17beta concentration, corpus luteum regression, follicular development, ovulation, prostaglandin treatment and behavioral estrus were investigated. The expression of behavioral estrus was found to be related to the patterns of progesterone and estradiol-17beta secretion during the periovulatory period. When compared to normal estrous cycles, silent estrus was accompanied by a significantly lower maximum serum estradiol-17beta concentration (47.8 vs 34.6 pg/ml), a significantly longer interval from maximum estradiol-17beta concentration to ovulation (1.7 vs 4.0 days), and a significantly shorter interval from corpus luteum regression to ovulation (5.3 vs 2.8 days). Silent estrus following prostaglandin treatment was related to a significantly shorter interval from prostaglandin treatment to ovulation (3.6 +/- 0.4 days) than from normal corpus luteum regression to ovulation (5.3 +/- 0.3 days). Silent estrus appeared to be related to changes in follicular estradiol-17beta secretion and to the pattern of its secretion as related to regression of the corpus luteum. There appeared to be not only less estradiol-17beta present, but also less time available after luteal regression for it to interact with the central nervous system to elicit the changes necessary to cause behavioral estrus. There fore, unusual relationships between luteal function and folliculogenesis can result in one type of silent estrus. Significant correlations (P<0.05) were found between follicle size and serum estradiol-17beta concentration whenever behavioral estrus occurred [follicle diameter in mm = 0.96 (serum estradiol-17beta in pg/ml) + 6.08 and 0.73 (serum estradiol-17beta + 13.32 for control and normal estrus following prostaglandin treatment groups, respectively]. During silent estrus, however, no significant correlations between follicle size and serum estradiol-17beta concentration were observed.