Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an essential step in the biosynthesis of all classes of hormonal steroids. We report the characterization of two types of cDNA clones encoding rat 3 beta-HSD isolated from a rat ovary lambda gt11 cDNA library with a human 3 beta-HSD cDNA probe. Both type I and type II cDNAs encode proteins of 372 amino acids having 94% homology. Transient expression of the type I and the type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that both proteins possess 3 beta-hydroxysteroid dehydrogenase as well as delta 5-delta 4 isomerase activities for both delta 5-pregnene and delta 5-androstene precursors, although the type I 3 beta-HSD protein is more active than the type II. RNA blot analysis using type I 3 beta-HSD cDNA identifies major mRNA transcripts of 1.7 kilobase in rat ovary, testis, and adrenal poly(A)+ RNA. RNase protection assay using type I- and type II-specific cRNA probes revealed the existence of the two corresponding mRNAs in male and female rat adrenals and gonads as well as in female adipose tissue while only type I mRNA is present in male and female kidney. Moreover, in situ hybridization performed using type-specific labeled 24-mer oligonucleotides confirms that type I is the major mRNA species in the ovary and further indicates that both mRNA species have a similar cellular distribution in the ovarian tissue with the highest level of expression found in corpora lutea. Immunoblot analysis using polyclonal antibodies raised against purified human placental 3 beta-HSD identified a single 42-kDa band in rat ovary, testis, and adrenal, which agrees with the calculated molecular masses of 41,911 and 42,150 daltons for the type I and II proteins, respectively. Determination of 3 beta-HSD enzymatic activity using [14C]pregnenolone and [14C]dehydroepiandrosterone as substrates shows that 3 beta-HSD activity is present not only in the gonads and adrenals of animals of both sexes, but also in many peripheral tissues including adipose tissue, mammary gland, kidney, liver, prostate, seminal vesicle, uterus, skin, brain, heart, thymus, pancreas, lung, and spleen. The present data indicate the existence of two mRNAs encoding rat 3 beta-HSD and their differential tissular distribution in both steroidogenic and peripheral tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues. 198 17

In the corpus luteum, prostaglandin F2 alpha (PGF2 alpha) appears to be a physiological agent with both antisteroidogenic and luteolytic actions. It is hypothesized that the antisteroidogenic action of PGF2 alpha acts through altered transport of cholesterol to the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc). However, the effect of PGF2 alpha on the expression of the putative cholesterol transport protein, sterol carrier protein-2 (SCP2; 13.2 kilodaltons), has not been examined. In this study, the decline in serum progesterone after PGF2 alpha injection was examined in parallel with altered ovarian SCP2, P450scc, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) protein and messenger RNA (mRNA) levels. Rats (28 days old) were treated with 8 IU PMSG to induce follicular development and ovulation. Ten days after ovulation, animals were treated with PGF2 alpha (single or multiple injections; 100-250 micrograms each) or left untreated. Ovarian SCP2, P450scc, and 3 beta HSD protein and mRNA levels were examined 0 (time zero), 4, and 8 h post-PGF2 alpha treatment using Western and Northern blot analysis. SCP2 mRNA levels were also examined using a highly sensitive ribonuclease protection assay that detects a 429-base pair SCP2-mRNA specific sequence. The results indicate that serum progesterone was significantly reduced 4 and 8 h after PGF2 alpha injections (P < 0.001; n = 6/time point). The decline in progesterone paralleled a 50-60% reduction in 3 beta HSD protein and mRNA levels by 4 h post-PGF2 alpha. Protein and mRNA levels for 3 beta HSD returned to control values by 8 h post-PGF2 alpha treatment. P450scc expression was also reduced at 4 h (44-54%), but by 8 h, both protein and mRNA levels had increased above the normal control levels (P < 0.02). In contrast, the 0.8-kilobase SCP2-specific mRNA transcript was reduced to 50% and 80% of the pre-PGF2 alpha treatment level at 4 and 8 h, respectively (P < 0.01). SCP2 ribonuclease protection assay analysis also indicated that SCP2 mRNA levels were reduced 65% (P < 0.03) and 85% (P < 0.01) by 4 and 8 h post-PGF2 alpha treatment compared to those in time zero ovarian tissue. Consistent with the loss of SCP2 mRNA expression, Western blot analysis indicated that a 15-kilodalton SCP2-immunoreactive protein (presumably the pro-SCP2 form) was significantly reduced or absent in the PGF2 alpha treated animals (P < 0.04).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Prostaglandin F2 alpha mediates ovarian sterol carrier protein-2 expression during luteolysis. 758 30

In cattle, a dramatic increase in plasma estradiol occurs during the short 2- to 3-day follicular phase. The objective of this study was to investigate the molecular mechanisms that mediate this critical change, specifically whether increases in the steroidogenic ability of granulosa and thecal cells of the preovulatory follicle are associated with increases in the levels of messenger RNA (mRNA) for steroidogenic enzymes. Luteolysis and a follicular phase were induced cycling Holstein heifers (n=15) by injection of a luteolytic dose of prostaglandin F2 alpha (PGF 2 alpha) on day 6 or 7 of the estrous cycle (day 0 = estrus), and preovulatory follicles were obtained at three stages of differentiation (0, 12, or 24 h post-PGF2 alpha treatment). To assess developmental changes in steroidogenesis in vivo, estradiol and androstenedione were measured in follicular fluid and in culture medium after a 3-h incubation of granulosa and thecal cells in defined medium with or without gonadotropins. To determine whether changes in mRNA for steroidogenic enzymes are associated with changes in follicular steroidogenesis, levels of mRNA for cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase, and cytochrome P450 aromatase (P450arom) were measured in thecal and granulosa cells using ribonuclease protection assays. Concentrations of estradiol in follicular fluid were relatively high at time zero, increased significantly by 12 h, and increased further by 24 h post-PGF2 alpha treatment. However, the aromatizing activity of granulosa cells was high at the time of PGF2 alpha injection and did not increase significantly during the first 24 h after the initiation of luteolysis. The aromatizing activity of granulosa cells was reflected in levels of mRNA for P450arom, which was relatively abundant in granulosa cells obtained before luteolysis and did not increase further during the first 24 h of the follicular phase. Concentrations of androstenedione were virtually undetectable in follicular fluid at time zero and had increased dramatically by 12 and 24 h post-PGF2 alpha treatment. Similarly, thecal cells isolated at 24 h secreted 3-fold more androstenedione than cells isolated at the time of PGF2 alpha injection. Androstenedione production by thecal cells in response to LH was also markedly higher at 12 and 24 h than at the time of PGF2 alpha injection. Likewise; levels of mRNA for P450 17 alpha-hydroxylase increased significantly by 12 h post-PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Differentiation of bovine preovulatory follicles during the follicular phase is associated with increases in messenger ribonucleic acid for cytochrome P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, and P450 17 alpha-hydroxylase, but not P450 aromatase. 758 47

We have isolated and characterized two molecular types of guinea pig (GP) apolipoprotein D (apoD) cDNA. The sequences of cDNA clones GP APO D-20 and -38 are 100 % homologous in their putative exons 2-5, as determined by analogy within human apoD gene, but they differ totally in their putative exon 1. RNase protection assays showed the presence of both apoD RNA types 20 and 38 in cauda epididymis. Northern blot analysis revealed four polyadenylated apoD bands at 3.2, 2.7, 1.7, and 1.0 kb. Types 20 and 38 specific probes hybridized with the major 1-kb mRNA and two of the three other minor RNA transcripts, respectively. Southern blot analysis revealed that the guinea pig genome probably contains one apoD gene. Our data also demonstrated that the cauda epididymis and fallopian tubes had an apoD mRNA concentration 100-fold higher than the liver, suggesting that the apoD gene expression could be associated with the presence of steroids. The levels of the 1-kb mRNA increased in the fallopian tubes and ovaries during gestation and were lower in fetal reproductive tissues and liver than in mature animals. No positive correlation was found between apoD and 3 beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3 beta-HSD) mRNA levels in these tissues, thus suggesting that high amounts of apoD mRNA are not necessarily associated with in situ progesterone synthesis. Taken together, our results indicate that both the guinea pig epididymis and fallopian tubes are excellent models to study the local role of apoD in steroid target tissues.
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PMID:Guinea pig apolipoprotein D RNA diversity, and developmental and gestational modulation of mRNA levels. 766 86

In classical target tissues, progesterone (P) down-regulates its own receptor, yet in the primate corpus luteum, progesterone receptors (PRs) exist within a very high local P milieu. The percentage of luteal cells staining PR-positive by immunocytochemistry is highest at the midluteal phase of the menstrual cycle during the period of peak serum P. To investigate the regulation of luteal PRs, we developed a solution hybridization/ribonuclease protection assay for the analysis of PR messenger RNA (mRNA) in macaque corpora lutea (n = 3-4/group). A 332-basepair fragment of the macaque PR complementary DNA corresponding to the hormone-binding region was used as a template for riboprobe production; the specific hybridization of this riboprobe with PR mRNA was confirmed with Northern analysis. P regulation of luteal PR mRNA was investigated by administering trilostane, a 3 beta-hydroxysteroid dehydrogenase inhibitor, to female rhesus macaques beginning on day 6 or 7 of the luteal phase, which reduced serum P until the time of lutectomy. By 18 h after trilostane treatment, luteal PR mRNA levels were significantly elevated compared to untreated control values (mean +/- SEM, 2.0 +/- 0.4 vs. 0.7 +/- 0.3; P < 0.05). Reduction in P levels for 4 days after trilostane administration decreased luteal PR mRNA levels compared with control values (0.50 +/- 0.02 vs. 1.1 +/- 0.2; P < 0.05). To characterize changes in PR mRNA during the lifespan of the corpus luteum, mRNA levels in luteal tissues from the early, mid-, mid-late, and late luteal phases were determined. PR mRNA levels were lowest during the early luteal phase and increased (P < 0.05) 3-fold by the mid-late luteal phase; this higher PR mRNA level was maintained throughout the remainder of the luteal phase. These data indicate that P or a metabolite may acutely regulate primate luteal PR mRNA in a manner consistent with PR regulation in classical P target tissues. In contrast, PR mRNA levels parallel increases in P and PR-positive luteal cells during the early, mid-, and mid-late portions of the luteal phase. High PR mRNA levels are maintained during luteal regression as P and the percentage of PR-positive cells decline, suggesting that PR and PR mRNA are regulated in an asynchronous manner during the lifespan of the corpus luteum in the menstrual cycle.
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PMID:Progesterone receptor messenger ribonucleic acid in the primate corpus luteum during the menstrual cycle: possible regulation by progesterone. 772 Jun 32

Regression of the CL causes a dramatic decrease in plasma progesterone levels. To test the hypothesis that the decrease in progesterone involves the down-regulation of mRNA encoding the steroidogenic enzymes, cytochrome P450 side-chain cleavage (P450scc) and/or 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), levels of plasma progesterone and luteal mRNA for P450scc and 3 beta-HSD were measured and correlated during induced luteolysis. Holstein heifers (n = 25) were injected with 25 mg prostaglandin F2 alpha (PGF2 alpha) on Day 6 or 7 of the estrous cycle (Day 0 = estrus) to induce luteal regression. To determine acute changes in plasma progesterone during luteolysis, jugular blood samples were obtained from 5 heifers hourly for 12 h, beginning immediately before injection of PGF2 alpha, and assayed for progesterone by RIA. A significant decrease in plasma progesterone levels was observed as early as 1 h after the PGF2 alpha injection (3.62 vs. 2.72 ng/ml, p < 0.05). Progesterone levels continued to decline with time through 12 h after administration of PGF2 alpha. The other 20 animals were ovariectomized at 0 (n = 6), 2 (n = 4), 12 (n = 4), or 24 (n = 6) h after PGF2 alpha. Levels of P450scc and 3 beta-HSD mRNA were determined in extracts of total luteal RNA by ribonuclease (RNase) protection assay. By 2 h after PGF2 alpha, 3 beta-HSD mRNA levels had decreased by about 40% as compared with levels at time 0 (p < 0.05), and a further significant decrease occurred between 2 and 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Changes in levels of messenger ribonucleic acid for cytochrome P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase during prostaglandin F2 alpha-induced luteolysis in cattle. 814 50

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.
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PMID:Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells. 847 48

Reproductive dysfunction in the diabetic female rat is associated with impaired folliculogenesis, reduced corpus luteum progesterone output, and spontaneous abortion. The underlying mechanism for reduced steroid production remains unresolved. In this study we examined whether or not diabetes alters levels of P450 side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), or the cholesterol transport proteins, steroidogenic acute regulatory (StAR) protein and sterol carrier protein-2 (SCP2), leading to lower progesterone levels and pregnancy loss. Rats (Day 3 pregnant) received an injection of streptozotocin (STZ, 60 mg/kg; i.v.) to induce a diabetic state; P450scc, 3 beta-HSD, and SCP2 were examined by Western and Northern blot analysis in ovarian tissue 12 days after injection of STZ (diabetic rats, n = 12) or vehicle (nondiabetic rats, n = 12). Serum progesterone, triglyceride, and beta-hydroxybutyrate (beta-HBA) levels were also examined. Results indicate that diabetic rats that aborted (diabetic-fetus [Ft], n = 6) had significantly lower progesterone levels (7.04 +/- 2.6 ng/ml; p < 0.004) than nondiabetic animals (108.6 +/- 5.15 ng/ml) and diabetic +Ft animals (74.3 +/- 8.9 ng/ml, n = 6). Western blot analysis of ovarian P450scc and 3 beta-HSD in the nondiabetic rats and the diabetic rats with fetuses indicated no significant difference. In contrast, ovaries from diabetic animals without fetuses had significantly lower SCP2 levels (p < 0.017) compared to controls. Concomitant with the reduction in SCP2, a 58-kDa SCP2-immunoreactive protein, referred to as sterol carrier protein-X (SCPx), increased significantly (p < 0.001). The C-terminal sequence of SCPx is identical to SCP2, while its N-terminal region is homologous with 3-oxoacyl coenzyme A thiolase, an enzyme involved in fatty acid metabolism. Increased SCPx expression coincided with increased serum triglyceride and beta-HBA levels, suggesting that the enhanced SCPx level may coincide with an ovarian shift to fatty acid metabolism. When SCPx steady-state mRNA levels were measured using an SCPx-specific riboprobe (280-bp protected fragment) in a ribonuclease protection assay, ovarian SCPx mRNA levels in the diabetic animals were increased 4.2-fold compared to control SCPx mRNA levels. Ovarian StAR mRNA levels were increased slightly in the diabetic animals, and ovarian P450scc and 3 beta-HSD mRNA levels were increased 3-fold in the diabetic animals that aborted relative to the nondiabetic animals and the +Ft diabetic animals. Results of this study confirm that SCPx mRNA levels are elevated following diabetes onset and that StAR, P450scc, and 3 beta-HSD mRNA levels do not correspond with the reduced steroid hormone profile associated with diabetes. These results are concordant with the possibility that reduced steroid levels in the diabetic animals reflect a loss of SCP2-mediated cholesterol transport capacity as SCPx/3-oxoacyl coenzyme A thiolase expression is enhanced.
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PMID:Altered ovarian sterol carrier protein expression in the pregnant streptozotocin-treated diabetic rat. 879 56

Previous studies in our laboratory indicated that the midcycle gonadotropin surge stimulates progesterone receptor (PR) expression in granulosa cells of the macaque preovulatory follicle. The current experiments were designed to determine whether gonadotropin or steroids continue to regulate PR in luteinized granulosa cells that contain these receptors after the LH surge. Luteinizing granulosa cells obtained from gonadotropin-treated rhesus macaques were cultured in chemically defined medium in the presence of low density lipoprotein (LDL; 100 micrograms/ml) with or without hCG (100 ng/ml) for up to 4 days. Cells were also cultured with various concentrations (0.25-250 ng/ml) of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) inhibitor trilostane to reduce progesterone (P) production in vitro. P and estradiol (E) in the media were assayed by RIA; PR mRNA was assessed by RNase protection assay, and cells expressing PR were identified by immunocytochemistry. Whereas hCG stimulated cellular P production through 4 days of culture, trilostane reduced hCG-stimulated P production in a dose-dependent fashion, with P levels decreasing more than 90% during incubation with 250 mg/ml trilostane (p < 0.05). When trilostane was removed from the media, P production returned to hCG-stimulated levels, indicating that trilostane (250 ng/ml) alone did not alter levels compared to those in controls. Before culture, 68 +/- 11% of luteinizing granulosa cells expressed PR; intense nuclear staining was typically observed. After 2 days of culture, 78 +/- 3% of cells remained PR-positive, but nuclear staining was more heterogeneous. Incubation with hCG did not alter the percentage of luteinized granulosa cells staining positive for PR but increased the intensity of PR staining. Trilostane treatment (25 ng/ml) in combination with hCG significantly reduced the percentage of PR-positive cells (54 +/- 9%) when compared with hCG treatment (83 +/- 2%, p < 0.05). These in vitro data suggest that macaque luteinized granulosa cells retain some PR expression in the absence of luteotropic hormones, but that gonadotropin stimulates PR mRNA levels and enhances PR expression as assessed by intensity of nuclear PR staining. In the presence of gonadotropin, trilostane effectively inhibited P production ad reduced the number of PR-positive cells, suggesting that P or a metabolite modulates PR expression in primate luteinized granulosa cells.
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PMID:Progesterone receptor messenger ribonucleic acid and protein in luteinized granulosa cells of rhesus monkeys are regulated in vitro by gonadotropins and steroids. 892 10

In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha-hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E(2)) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E(2) and T titers. Alternatively 3 beta-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
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PMID:Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle. 1109 Apr 35


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