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American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March, 15 June, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggests that aromatase and oestrogen may play a role in re-initiating spermatogenesis.

Granulosa-cumulus and cumulus-corona expansion as well as aromatase localization within ovarian follicles were monitored during the preovulatory period in superovulated cattle that were blood sampled every 2'nd h for LH analyses. Granulosa-cumulus as well as cumulus-corona expansion were studied by means of transmission electron microscopy and computerized image analysis. Localization of aromatase, an enzyme involved in estrogen synthesis, was determined immunocytochemically using anti-human placental aromatase cytochrome P-450 antisera. Nuclear oocyte maturation was determined by aceto:orcein staining. Significant cell dissociation within the granulosa-cumulus stalk occurred before the breakdown of the germinal vesicle, i.e. the oocyte nucleus, during the period up to 5-7 h after the LH peak, i.e. the highest LH concentration during the surge. Significant increase in intercellular spacing between the cumulus-corona cells occurred at 13-15 and 19-21 h after the LH peak. Before the LH peak all layers of granulosa cells were immunocytochemically stained for aromatase. At 5-7 h after the LH peak, however, only the granulosa cell layers located near the basal lamina were stained, and at all later intervals staining was absent. The granulosa cells of primary and secondary follicles, the interstitial gland cells, the theca interna cells and the oocytes in all follicles were immunocytochemically unstained.

Previous anatomical and histochemical studies suggested that interstitial cells were the only steroidogenic cells in the theca layer of small follicles of the chicken ovary. However, the precise cellular site of steroid production in the small follicles is not certain. Therefore, our goal was to identify steroidogenic cells in small follicles (less than 10 mm in diameter) of the chicken ovary which have not entered the follicular hierarchy by localizing steroidogenic enzymes with immunocytochemistry. Polyclonal antisera used were anti-cholesterol side-chain-cleavage cytochrome P450 (P450scc), anti-17 alpha-hydroxylase cytochrome P450 (P450c17), and anti-aromatase cytochrome P450 (P450arom) for pregnenolone-, androgen-, and estrogen-producing cells, respectively. Ovaries were collected 2 hr after oviposition and embedded in Paraplast after fixation with 4% paraformaldehyde, 10% formaldehyde, or Bouin's solution. Tissues were sectioned (4-6 microns) and sections were mounted on poly-L-lysine coated slides. Sections were incubated overnight at room temperature with each specific antiserum raised in rabbits against cytochrome P450 steroidogenic enzymes or normal rabbit serum as a control and were immunostained with an avidin-biotin-peroxidase complex. Immunoreactivity for the P450 enzymes was absent in the granulosa layer but was present in the theca layer of the small follicles (less than 10 mm in diameter). Interstitial cells in the single theca layer of cortical follicles embedded in the ovarian cortex (less than 1 mm in diameter) contained P450scc and P450c17. Cells which contained P450arom, identified as aromatase cells, surrounded the interstitial cells in the theca layer.(ABSTRACT TRUNCATED AT 250 WORDS)

To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-alpha.Seven-week-old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF-alpha (0.5 microg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase.Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF-alpha (GalN/TNF-alpha), but not GalN or TNF-alpha alone. Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-alpha. In a cell-free system, processing of procaspases-9 and -3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF-alpha-treated mice, but not in that from control mice. Processing of procaspase-3 was inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2. In a reconstitution assay system, procaspase-9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-alpha. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation.Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-alpha-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) initiates apoptosis and simultaneously activates the transcription factor, nuclear factor-kappaB (NF-kappaB), which suppresses apoptosis by an unknown mechanism. Pretreatment with TNF-alpha or interleukin-1beta (IL-1beta), which activated NF-kappaB in the liver, dramatically prevented TNF-alpha-induced liver-cell apoptosis in D-galactosamine (GalN)-sensitized mice, but not anti-Fas antibody-induced hepatotoxicity. This protective effect of TNF-alpha continued for 5 hours after TNF-alpha administration, a time course similar to that found in NF-kappaB activation after TNF-alpha administration. In mice treated with adenoviruses expressing a mutant form of IkappaB, the antiapoptotic effect of TNF-alpha was inhibited in part. Prior TNF-alpha administration was not found to block the activation of caspase-8, although caspase-3 was inhibited in mice treated with TNF-alpha plus GalN/TNF-alpha compared with mice treated with GalN/TNF-alpha. These results indicate that TNFR and Fas independently regulate murine apoptotic liver failure, and that a rapid defense mechanism induced by the activation of NF-kappaB blocks death-signaling at the initiation stage of hepatic apoptosis mediated by TNFR, probably downstream of caspase-8, but not by Fas.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.

Guanabenz (Wytensin) was shown to inactivate nitric oxide synthase (NOS) activity in vitro and in vivo. In in vitro studies with the use of a cytosolic fraction from penile tissue, the inactivation was found to depend on NADPH, time, and the concentration of guanabenz. The L-, but not the D-, isomer of arginine could protect from the inactivation, suggesting an active site-directed event. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (Ki) and a pseudo first-order inactivation constant (kinact) of 38.5 microM and 0.179 min-1, respectively. In in vivo studies, guanabenz was shown to inhibit penile cytosolic NOS activity in a dose- and time-dependent manner. Treatment of rats with guanabenz (5 mg/kg/day) for 4 days caused a decrease of approximately one-half in the NOS activity of the penile cytosolic fraction with a concomitant loss in the amount of immunodetectable NOS protein. Treatment for 4 days at a dose of 0. 5 mg/kg/day showed a similar decrease in activity, whereas a dose of 0.05 mg/kg/day showed no effects. Due to the multitude of processes that are regulated by NO, the inactivation of NOS is a potential mechanism to be considered in a variety of biological effects associated with drugs.

Although testosterone is the principal sex steroid produced by the testis, estrogen is known to be produced by both Leydig and Sertoli cells during different developmental periods. Additionally, evidence is unfolding to suggest that germ cells might also participate in the synthesis of estrogen within the male reproductive tract. We have recently reported that the messenger ribonucleic acid (mRNA) for P450 aromatase (P450arom), the enzyme that converts androgen to estrogen, is synthesized by rat germ cells. Therefore, the present study was conducted to determine which germ cell types synthesize active P450arom and to measure the activity of this enzyme in germ cells throughout spermatogenesis and in maturing sperm during epididymal transit. First, P450arom activity was measured in pachytene spermatocytes, round spermatids, and a mixture of round spermatids, elongating spermatids, and residual bodies using the tritiated water (3H2O) assay. Second, sperm isolated from different regions of the epididymis were assayed for P450arom activity. Sperm isolated from the caput epididymis with attached efferent ductules had the higher P450arom activity, whereas sperm isolated from the corpus and cauda epididymides had lower P450arom activity. The decrease in P450arom activity in cauda sperm was further confirmed by immunocytochemistry. On the basis of these observations, we conclude that rat testicular germ cells from pachytene spermatocytes through elongating spermatids and epididymal sperm contain active P450arom and that sperm lose aromatase activity as they mature during epididymal transit. Therefore, both post-pachytene rat germ cells and epididymal sperm are capable of estrogen synthesis and are an additional, potentially significant, source of estrogen in the male reproductive tract.

We recently found that cytochrome P450 aromatase (P450arom) is present in germ cells of the mammalian testis and is capable of converting androgens to estrogens in the male reproductive tract. The objective of the present study was to determine whether testicular germ cells and epididymal sperm of an avian species are also capable of synthesizing estrogen. P450arom was localized in the rooster testis and epididymal region by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. Immunostaining was present in pachytene spermatocytes, round spermatids, elongated spermatids, flagella of late spermatids, and sperm in the epididymal region. A positive reaction was also found in nonciliated cells of the epididymal region. However, the absence of mRNA for P450arom in the epididymal region indicated that the immunoreactive protein present in the epididymal region is not synthesized in this region. The immunoreactive P450arom found in epididymal sperm was shown to be active through use of a 3H2O assay. On the basis of these data, we conclude that rooster testicular germ cells and epididymal sperm are sites for the synthesis of estrogen, a potential regulator or modulator of germinal epithelium in the testis and the epithelium of the epididymal region of the avian species.

In mature hamster ovaries, many follicles undergo atresia and this atretic change occurs in any estrous day. In this study, the localization of enzymes involved in estrogen biosynthesis, Bromodeoxyuridine (BrdU) incorporation, and Proliferating Cell Nuclear Antigen (PCNA) were immunohistochemically examined in the atretic follicles always provided with an atrum variable in size. Moreover, the granulosa cells from the atretic follicle in various but parallel stages of development were examined by scanning and transmission electron microscopes. Very early morphological signs of atresia in these follicles was the pyknotic change of a few granulosa cells lining the antral cavity. In this kind of follicles, the number of BrdU incorporating granulosa cell was decreased and the immunoreactivity of PCNA and aromatase was gradually decreased. Even in the early stage of atresia, some granulosa cells showed a remarkable morphological change characteristic of apoptosis as revealed by scanning and transmission electron microscopy.

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17α-hydroxylase/C17–C20 lyase (17α-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17α-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles. Even in the early stage of atresia, the granulosa cells showed remarkable morphological change characteristic of apoptosis, as visualized by scanning and transmission electron microscopy. Cessation of granulosa cell proliferation may occur earlier than apoptotic change and the degeneration of the granulosa cells becomes rapid once atresia starts.

Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Sertoli or germ cells. Heterogeneity in the level of immunostaining among individual Leydig cells was observed. The results of this study indicate that in postpubertal, adult, and aged stallions, testicular aromatase is located in Leydig cells.

The 7-substituted 6H-pyrazolo[4,5,1-de]acridin-6-ones with (aminoalkyl)amino and/or (hydroxy-alkyl)amino groups in the side chains were synthesized by bromination using N-bromosuccinimide and the subsequent reaction with amines from the 7-substituted 5-bromo-2-methyl-6H-pyrazolo-[4,5,1-de]acridin-6-one. The substitution reaction of the amines with alkyl bromide (the C2 position) and aryl bromide (the CS position) was accomplished by choosing the proper reaction conditions. These compounds show DNA intercalating ability in ethidium fluorescence assay and antiproliferative activity against Hela S 3 cells. Impressive antitumor activity in vivo against murine P388 leukemia and murine sarcoma 180 solid tumor in mice was demonstrated for the 7-hydroxy analogs. In addition, some of these showed excellent antitumor activity against adriamycin-resistant murine P388 leukemia (P388/ADM) in mice

Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.

The stoichiometric reductive debromination of BrCCl3 to a trichloromethyl radical by myoglobin caused the prosthetic heme to become covalently cross-linked to the protein moiety and transformed myoglobin from an oxygen storage protein to an oxidase. This was shown in experiments in which oxygen consumption was measured during redox cycling of the altered myoglobin in the presence of ascorbate or an enzymatic reducing system containing diaphorase and NADH. Redox cycling eventually led to loss of the protein-bound heme adduct and oxidase activity of myoglobin. We have used molecular modeling and the known structure of the protein-bound heme adduct to identify probable mechanisms for transformation of myoglobin to an oxidase. Based on these modeling studies, the most likely structure of the experimentally observed adduct involves ligation to the heme iron of the epsilon-nitrogen atom of histidine 97 and/or that of histidine 64. The model structures revealed access of solvent to the heme active site, which could facilitate oxygen reduction. The transformation of myoglobins and perhaps other hemoproteins to oxidases may have toxicological importance in causing the tissue damage resulting from exposure to various xenobiotics and endogenous chemicals as well as explaining how hemoproteins are inactivated during catalysis.

In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00 am) of Day 1 to the evening (5:00 pm) of Day 4; in the night (9:00 pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction. As for 17 alpha-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00 am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00 pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00 pm) of Day 4 or morning (10:00 am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17 alpha-lyase in the theca interna cells of the ovary. On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17 alpha-lyase antibody. Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17 alpha-lyase show ultrastructural features typical of steroid secretory cells.

The conversion of androgen to estrogen is mediated by aromatase activity, and 19-hydroxylase and C10-19lyase are seen to be involved in C19-demethylation. The present study is to demonstrate C10-19lyase activity in adrenal tissue. Fetal bovine and pig adrenal tissue homogenates were incubated with 14C-androstenedione(A) and NADPH under air. The products 14C-19-OHA, 14C-19-norA, with added carrier standard 3H-19-OHA, 3H-19-norA were separated and purified by T.L.C.. The identification of 19-norA was established by recrystallization and that of 19-OHA by C.C.D.. The 3H/14C ratios of the products 19-norA were found to be constant in repeated crystallization. The amount of 19-OHA from A in fetal adrenal tissue was 30.0nmol/h/g protein, and that of 19-norA from A was 12.59pmol/h/g protein. C10-19lyase activity from 19-OHA, calculated by the amounts of 19-norA in fetal, bovine and pig adrenal tissue, was 49.7, 3.50, 10.3pmol/h/g protein, respectively. This is the first report to demonstrate androgen C10-19lyase in human fetal adrenal tissue, and it is assumed that fetal adrenal tissue forms 19-norA via 19-OHA.

Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-450 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-450 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-450 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for androstenedione on reconstitution with NADPH-cyt P-450 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-450 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-450 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, 4-14C]androstenedione led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.

NADPH-cytochrome c (P-450) reductases from human placental aromatase II and from horse placental microsomes were solubilized and purified to show a single band of 83,000 daltons in SDS-polyacrylamide gel electrophoresis. Rabbits were immunized with purified human placental aromatase II NADPHcytochrome c (P-450) reductase. The resulting antibodies (Reduc-Ab) were used to examine the species specificity of estrogen biosynthesis and the reductase activity in humans, baboons, horses and rats. Rcduc-Ab suppressed androstenedione aromatase activity in human, baboon and horse placental microsomes with sensitivities in the order of human ⪢ baboon ⪢ horse. Reduc-Ab was much less effective in inhibiting baboon and horse placental NADPH-cytochrome c reductase activity. On the other hand, rat liver microsomal NADPH-cytochrome c reductase activity and its purified preparation were effectively suppressed by Reduc-Ab and the sensitivity was much closer to that of human than to horse. The results suggest that there are common antigenic structure(s) in human, baboon and horse placental and rat liver NADPH-cytochrome c (P-450) reductases and also that there are distinct structural portions or multiple forms of NADPH-cytochrome c reductase among different species.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.

The metabolic pathway leading to equilin and equilenin biosynthesis in the pregnant mare is different from that of estrone and estradiol and it is apparently cholesterol-independent. The precise precursors and intermediates and the stereomechanism of equine placental aromatization have not been established. [1,2-3H, 4-14C]3-Hydroxy-3,5,7-androstatrien-17-one was synthesized as a potential substrate and the 3H-distribution was analyzed by biochemical and chemical derivatization methods. The substrate was converted to equilin, equilenin and Heard's ketone by horse placental microsomes with a sp. act. of 74, 18 and 2.8 pmol/h/mg, respectively, and only to equilin by human placental microsomes with a rate of 26 pmol/h/mg. Analysis of the loss of 3H-labeling during aromatization showed the stereospecific 17 beta,2 beta-cis hydrogen elimination for equine estrogen biosynthesis both by horse and human placental microsomes. This is the same as for estrone and estradiol biosynthesis by both placentas. The biosynthesis of Heard's ketone, a non-phenolic ring-B aromatic C18 steroid, by horse placental microsomes was found to involve none of the four hydrogens at C-1 and C-2. This refutes the previous postulate that Heard's ketone arises from equilenin by reduction of the ring-A.

Phencyclidine, 1-(1-phenylcyclohexyl)piperidine, was found in this study to be a mechanism-based inactivating agent for cytochrome P-450 form 2, the major phenobarbital-inducible cytochrome of rabbit liver microsomes. This process is highly selective for P-450 form 2, both in microsomes and in the reconstituted enzyme system, in that forms 3a, 3b, 4, and 6 are not affected. However, phencyclidine iminium ion, an oxidative metabolite, inactivates both P-450 form 2 and form 3b in a metabolism-dependent manner. Both phencyclidine and its iminium ion give biphasic kinetics of inactivation with similar rate constants, which supports the hypothesis that the iminium ion is an intermediate in the inactivation of P-450 form 2 by the parent compound. The absorption of the oxidized cytochrome and of the ferrous-carbonyl complex in the visible spectrum are both decreased upon inactivation by phencyclidine, indicating modification of the heme moiety. Several modified hemes produced by the action of phencyclidine were isolated by HPLC.

The immunocytochemical localization of aromatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets, these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.