Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG,and in pregnant rat ovaries

Summary Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 17 alpha-hydroxylase/C17-C20 lyase by bifluranol and its analogues

A simple assay for the measurement of the activities of both 17 alpha-hydroxylase and C17-C20 lyase is described. No extraction procedures are required. The separation of substrate and products is achieved using HPLC which allows the collection of the components of interest and the monitoring of the recovery of various steroids. Using this assay, bifluranol (known to show anti-prostatic activity in vivo) and some analogues were tested for inhibitory activity towards these enzyme activities. Each compound was active, although less potent than ketoconazole, and this activity may contribute towards the in vivo action.     

Immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase in the ovary of pregnant pigs and fetal gonads

The study was designed to localize P450 17alpha-hydroxylase/c17-20 lyase (P450c17) in the ovaries of pregnant pigs and fetal gonads. Immunoexpression of P450c17 was investigated in the porcine ovaries (follicles and corpora lutea; CL) collected on days 10, 18, 32, 50, 70 and 90 post coitum (p.c.), and fetal gonads (testes and ovaries) on days 50, 70 and 90 p.c. The presence of P450c17 in ovarian follicles was demonstrated on all examined days of pregnancy but was restricted to theca interna cells. In CL, P450c17 was detected on all examined days of pregnancy but only in small luteal cells. In the female porcine fetuses, P450c17 immunostaining was found in oocyte nests and granulosa cells of primary ovarian follicles, while in the male fetuses in fetal Leydig cells. In conclusion, the immunolocalization of P450c17, detected in the ovaries of pregnant pigs and fetal porcine gonads, indicates the potential sites of androgen synthesis. We suggest that androgens may play a role in the maintenance of pregnancy and in the development of prenatal gonads in pigs.     

Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co

Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Histochemical and biochemical studies on esterase activity in the rat ovary

A correlative histochemical and biochemical study has been made of the changes in esterase positive sites in immature (10-, 20- and 30-days old), mature normal cycling (3-, 5- and 8-months-old), pregnant and lactating rat ovaries. The typical perivascular esterase-positive sites localized in the hilar portion, branch along the blood vessels and traverse into medullary and cortical portions of the ovary. The stromal vascularity surrounding the normal developing follicles, corpora lutea, atretic follicles and interstitial gland tissue showed rich activity of this enzyme system. On semiquantitative basis the number, intensity and quantity of esterase-positive sites vary with the maturation and reproductive states of the rat. The administration of estradiol-17 beta increased the fine perifollicular and theca externa perivascular esterase-positive sites, whereas atropine and reserpine affected severely both the large and fine meshwork of esterase-positive sites. Biochemical estimates of acetylcholine esterase activity endorse these histochemical observations. The possible roles of AChE activity in varied ovarian functions are discussed.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.     

Human chorionic gonadotropin treatment prevents depressed 17 alpha-hydroxylase/C17-20 lyase activities and serum testosterone concentrations in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

It is known that administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes decreased serum testosterone concentrations in the rat. Previous studies in this laboratory have shown that in rats TCDD exposure results in decreased 17 alpha-hydroxylase and C17-20 lyase activities. The decreases in these activities paralleled decreases in testicular microsomal heme and cytochrome P450 contents. As reported herein, neither testicular mitochondrial cytochrome P450 content nor the activity of cholesterol side-chain cleavage was altered in rats exposed to TCDD. Since the production of testosterone in the testis is dependent on LH, it is important to determine the early effects of TCDD on serum LH concentrations in the rat. Male Sprague-Dawley rats were given a single, oral dose of TCDD (50 micrograms/kg). Serum LH concentrations were determined by RIA on Days 1, 2, 3, 5, and 7 following TCDD treatment. Rat serum LH concentrations were decreased to 60% of controls as early as Day 1 and continued to be depressed on Days 2 and 3 at 53% and 59% of control values, respectively. Rat serum LH returned to control values by Day 5 in spite of continued depression of serum testosterone concentrations. The early depression in serum LH levels caused by TCDD may be related to the subsequent androgenic deficiency in the rat. Treatment of rats with hCG was found to be able to prevent the depression of the activities of testicular microsomal 17 alpha-hydroxylase and C17-20 lyase and serum testosterone concentrations caused by TCDD. These data indicate that TCDD decreases serum testosterone by decreasing P450(17 alpha) and C17-20 but not P450sec activities and that hCG treatment prevents the TCDD-induced decrease.     

Acute effect of human chorionic gonadotropin (hCG) on 17 alpha-hydroxylase and C17,20-lyase activity in neonatal or prepubertal rat testis

In contrast to the situation in adults, desensitization of androgen production, secondary to loss of enzyme activity, was not found in testes of neonatal rats exposed to human Chorionic Gonadotropin (hCG). In the present study attention was given to the acute effects of a single injection of hCG upon the activity of testicular 17 alpha-hydroxylase, C17,20-lyase and the concentration of testosterone in the serum of 5, 10 or 28-30 day old rats was investigated. Tritiated H2O from 17 alpha-[3H]progesterone and 14CH3COOH from 21-[14C]progesterone were the products measured to evaluate hydroxylase and lyase activities respectively. Large increases in hCG in the serum were detected within 2 h of a subcutaneous injection. Testosterone, which was highest in 5 day animals, increased quickly in all animals given hCG. In 28-30-day old animals, the concentration of this steroid began to fall 24 h after injection of hCG. 17 alpha-Hydroxylase activity decreased in the testes of all animals given hCG, but only after a brief increase. Activity returned to the starting level, or above, within 24 h in 5 or 10-day old animals. In 28-30-day old rats the activity of both enzymes decreased dramatically to a nadir at 24 h, but increased thereafter. The results indicate that desensitization of testicular androgen synthesizing enzymes occurs in neonatal as well as older testes stimulated with hCG, but the desensitization was very brief in neonatal animals and no desensitization of lyase was found in 5-day old rat testes.     

Production of 17-hydroxypregnenolone and 17-hydroxyprogesterone by the immature rat ovary

The concentration of 17-hydroxyprogesterone (17-OH-prog), but not 17-hydroxypregnenolone (17-OH-preg), was detectable in the sera of 5 day old female rats. The level of 17-OH-preg increased dramatically between day 5 and 10, remained high for 5 days and then decreased to low levels by day 25; a second increase was found on day 35. 17-OH-prog began to increase in the serum after day 10, reached a peak by day 20 and then decreased by day 25 and remained the same through day 35. Stimulation of the ovaries of intact females with 20 IU of pregnant mare's serum gonadotropin resulted in a prompt increase in both progestins, but a much larger increase in 17-OH-preg than in 17-OH-prog. Increases were similar, but quantitatively less, in hypophysectomized females. The results demonstrate that the ovaries of immature rats contain an active 17-hydroxylase system.     

Relationship between in vivo and in vitro 17 alpha-hydroxylase and C17,20-lyase activity in ovaries of immature hypophysectomized rats treated chronically with human chorionic gonadotropin

The production of 3H2O from 17 alpha-3H-progesterone and 14CH3COOH from [21-14C]progesterone were used to measure the 17 alpha-hydroxylase and C17,20-lyase activities respectively in the microsomal + mitochondrial fraction of homogenates of ovaries from immature hypophysectomized rats chronically treated with human chorionic gonadotropin (hCG). The highly stimulated thecal and interstitial tissues were considered the only source of enzyme. hCG produced an increase in 17-hydroxyprogesterone, and androstenedione, but a drastic decrease in enzyme activity within 6 h; this could be largely prevented by pretreatment of the rats with cycloheximide or aminoglutethimide but actinomycin D was ineffective. After a nadir at 24 h, enzyme activities increased to more than double those of the starting level; this could be prevented by cycloheximide. Maximal activity levels were greatly decreased by cycloheximide and modestly increased by aminoglutethimide. Cessation of treatment at 60 h followed by a single injection of hCG 24 h later did not cause a loss, but delays of 36 or more hours produced a dramatic decrease in enzyme activity, which could be prevented by aminoglutethimide. The results indicate that the level of activity of these enzymes attained in the ovary following exposure to hCG is determined by a balance between the amount of substrate provided and production of enzyme and/or stimulating factors. Therefore, maintenance of increased enzyme activity induced by gonadotropin appears to be under genomic control.     

Studies of the human testis. XX--Effect of NADH on 17 alpha-hydroxypregnene C17-20 lyase activity

A study of the cofactor requirements of C17-20 lyase was carried out using human testis tissue obtained at the time of orchiectomy for treatment of prostatic carcinoma. Washed microsomal fractions were prepared from frozen human testes using a KCl containing buffer. The preparation revealed dose-dependent activity of C17-20 lyase in the presence of either NADPH or commercial or purified NADH. The Km of NADH for the enzyme was of the order of 10(-3) M and the Km of NADPH was determined as 1.6 X 10(-5) M. NADH also provided synergistic enhancement of NADPH-mediated lyase activity, and decreased the Km of NADPH for the lyase but did not change the Vmax of NADPH-mediated lyase activity. Carbon monoxide inhibited both NADH and NADPH-mediated lyase activities indicating that both activities are catalyzed by cytochrome P-450. Cations including Ca2+, Mg2+ and Mn2+ were found to inhibit the NADPH-mediated lyase activity but enhanced the lyase activity in the presence of NADH. The results indicate both the presence of NADH-mediated C17-20 lyase activity and the synergistic effect of NADH on NADPH-mediated lyase activity in the human testis.     

Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

Background  

17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS) was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary.     

Modulation of 17 alpha-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17 alpha-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 x 10(6) cells/6 ml/well) were cultured in Medium 199 (M199) containing only insulin (10 micrograms/ml) and transferrin (5 micrograms/ml). At 24 h, theca cells were treated with M199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27-72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1 microCi [3H]progesterone +0.5 mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.