• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1750-1759
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• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.464-474
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• 2021

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37℃. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.12
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• pp.1566-1577
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• 2010

Nandrolone, 19-nortestosterone, is a synthetic androgenic-anabolic steroid promoting muscle growth. Nandrolone is also present in pig meat and sera at non-negligible levels. A number of scientific reports have suggested a positive relationship between incidence of infertility and increased meat consumption in humans. The present study was designed to determine out the effect of feeding nandrolone on the testis of the male reproductive tract. Mixtures of food and nandrolone at different concentrations (0.005 ppm and 0.5 ppm) were supplied to pubertal male rats for 6 weeks. Body weight was recorded every week during the entire experimental period. At the end of the treatment, the testis, epididymis, and epididymal fat were collected and weighted. Sperm numbers in the caudal epididymis were counted. Differential gene or protein expression of steroidogenic enzymes in the testes among experimental groups was determined by semi-quantitative real-time PCR or western blotting analysis, respectively. Histological changes of the testis induced by nandrolone treatment were examined by hematoxylin and eosin staining. Immunohistochemical analysis was employed to detect changes in the localization of steroidogenic enzymes in the testes among experimental animals. There were no significant changes on body, testis, epididymis, and epididymal fat weights among experimental groups. A significant increase of caudal sperm number was found in the 0.5 ppm nandrolone-treated group. Histological examination of the testes noted a high frequency of germ cell sloughing in seminiferous tubules of 0.5 ppm nandrolone-treated rats. Even though transcript levels of $3{\beta}$-hydroxysteroid dehydrogenase (HSD) I, $17{\beta}$-HSD4, and $17{\alpha}$-hydroxylase were influenced by nandrolone treatments, protein levels of all molecules examined in the present study were not significantly affected. Immunohistochemical analysis showed no visible changes in the localization of steroidogenic enzymes in the testes among experimental groups. The current study showed that oral intake of nandrolone in male rats for 6 weeks did not cause significant damage to the testis. It is considered that a feeding effect of nandrolone on male fertility would not be remarkable.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.5
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• pp.658-671
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• 2018

Objective: Nandrolone decanoate (ND) is an anabolic-androgenic steroid frequently used for clinical treatment. However, the inappropriate use of ND results in the reduction of serum testosterone level and sperm production. The suppressive effect of ND on testosterone production has not been investigated in detail. The present study was designed to examine the effect of ND on the expression of steroidogenic enzymes in the rat testis. Methods: Male Sprague Dawley rats at 50 days of age were subcutaneously administrated with either 2 or 10 mg of ND/kg body weight/week for 2 or 12 weeks. The changes of transcript and protein levels of steroidogenic enzymes in the testis were determined by real-time polymerase chain reaction and western blotting analyses, respectively. Moreover, immunohistochemical analysis was employed to determine the changes of immunostaining intensity of these enzymes. The steroidogenic enzymes investigated were steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage enzyme, $17{\alpha}-hydroxylase$, $3{\beta}-hydroxysteroid$ dehydrogenase, and cytochrome P450 aromatase. Results: The treatment of ND resulted in depletion of Leydig cells and sloughing of germ cells in the testis. The ND treatment caused significant expressional decreases of steroidogenic enzymes at transcript and protein levels, and the destructive effects of ND on the testis were more apparent with a higher dose and a longer period of the treatment. Evident reduction of immunostaining intensity present in Leydig cells was clearly detected by the ND treatment. Conclusion: The exposure to ND in young male results not only in histological changes of the testis but also in aberrant gene expression of testicular steroidogenic enzymes, consequently leading into the reduction of testosterone production in the testis and thus likely disruption of spermatogenesis.

• Development and Reproduction
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• v.13 no.3
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• pp.163-172
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• 2009

Some organotin compounds such as butyltins and phenyltins are known to induce impo-sex in various marine animals and are considered to be endocrine disruptors. In this study, the effect of organotins on follicular steroidogenesis in amphibians was examined using ovarian follicles of Rana dybowskii and Rana catesbeiana. Isolated follicles were cultured for 6 or 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the levels of product steroids in the culture media oassay. Among the butyltin compounds, tributyltin (TBT) strongly and dose-dependently inhibited the FPH-induced synthesis of pregnenolone ($P_5$) and progesterone ($P_4$) by the follicles. TBT also strongly suppressed the conversion of cholesterol to $P_5$ and partially suppressed the conversion of $P_5$ to $P_4$. A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin and tetrabutyltin had negligible effects. The toxic effect of TBT or DBT was irreversible and a short time of exposure (30 min) was enough to suppress steroidogenesis. All the phenyltin compounds significantly inhibited FPH-induced $P_5$ synthesis by the follicles. The effective dose of 50% inhibition by diphenyltin was $0.04\;{\mu}M$ and those of monophenyltin and triphenyltin were $0.24\;{\mu}M$ and $0.3\;{\mu}M$, respectively. However, none of the phenyltin compounds significantly suppressed the conversion of $P_4$ to $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) (by $17{\alpha}$-hydroxylase), $17{\alpha}$-OHP to androstenedione (AD) (by $C_{17-20}$ lyase), or AD to testosterone by the follicles. Taken together, the data show that among the steroidogenic enzymes, P450scc in the follicles is the most sensitive to organotin compounds and that an amphibian follicle culture system can be a useful screening model for endocrine disruptors.

• Journal of Animal Science and Technology
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• v.63 no.6
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• pp.1247-1264
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• 2021

Anabolic steroids are frequently used to increase the growth rate of meat-producing animals. Exposure to an anabolic-androgenic steroid, nandrolone decanoate (ND), is associated with expressional reduction of testicular steroidogenic enzymes. However, the effect of withdrawal of ND exposure on the expression of these testicular molecules has not been thoroughly explored. The current research investigated expression changes of testicular steroidogenic enzymes in rats at several recovery periods (2, 6, and 12 weeks) after the stop of ND treatment with different doses (2 and 10 mg/kg body weight) for 12 weeks. Body and testis weights were recorded, and transcript levels of molecules were determined by quantitative real-time polymerase chain reaction (PCR). The immunohistochemistry was used to examine the changes of immuno-intensities of molecules. At 6 and 12 weeks of the recovery period, the 10 mg/kg ND-treated rats were lighter than other experimental groups. The interstitial compartment vanished by ND treatment filled up as the recovery period became longer. The expression of steroidogenic acute regulatory protein was returned to the control level at 12 weeks of the recovery period. Expression levels of cytochrome P450 side-chain cleavage and 17a-hydroxylase were increased in 2 mg/kg ND-treated group at 6 weeks of the recovery period, and transcript levels of these molecules in 2 and 10 mg/kg ND-treated groups at 12 weeks of the recovery period were significantly lower than the control. Expression levels of 3β-hydroxysteroid dehydrogenase (HSD) type I and 17β-HSD type 3 in 2 mg/kg ND-treated group were comparable with those of control at 12 weeks of the recovery period, but not in 10 mg/kg ND-treated group. Expression of cytochrome P450 aromatase (Cyp19) was reverted to the control level at 2 weeks of the recovery period. Except for Cyp19, there was a visible increase of immuno-staining intensity of other testicular steroidogenic enzymes in the Leydig cells as the recovery period progressed. This research has demonstrated that the cease of ND administration could restore the expression of testicular steroidogenic enzymes close to the normal level. Nevertheless, a relatively long recovery period, compared to the ND-exposure period would be required to retrieve normal expression levels of testicular steroidogenic enzymes.

• Development and Reproduction
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• v.6 no.2
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• pp.105-110
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• 2002

Numerous hormones are involved in the regulation of reproduction. Among them, estrogen and progesterone are the most important ovarian steroid hormones regulating female fertility. On the other hand, diverse stressors impede female receptivity and fertility. Since norepinephrine(NE) and epinephrine(E) are released from the adrenal during stress, it might play a role in stress-induced disruptions of fEmale reproductive parameters. The present study was performed to analyze the changes in adrenal catecholaminergic activities in cycling rats. The tissue content and secretion level of catecholamines were determined by high performance liquid chromatography coupled with electrochemical detector(HPLC-ECD). Adrenomedullary content of norepinephrine(NE) was increased on proestrus stage (59.47 $\pm$ 6.86 ug/gland), peaked on diestrus I stage(65.22 $\pm$ 5.99 ug/gland), and was nadir on diestrus II stage(41.63 $\pm$ 1.33 ug/gland). The highest E content was observed on proestrus stage(361.86 $\pm$ 15.58 ug/gland) while the lowest level was on diestrus II stage(285.58 $\pm$ 12.25 ug/gland). In addition to these observations, a significant reduction of the NE : E ratio was observed (1 : 4.81 on diestrus I vs 1 : 6.13～7.02 on other stages). In vitro secretion of adrenal NE and E was increased on proestrus stage, peaked on estrus stage, and decreased on diestrus II stage. Interestingly, the NE : E ratio in conditioned media was significantly increased on estrus stage (1 : 3.32 vs 1 : 2.34～2.65 on other stages. The biosynthesis of NE and E is mediated by tyrosine hydroxylase(TH) and phenylethanolamine-N-methyltransferase(PNMT) which acts conversion of tyrosine into DOPA and NE into E, respectively. These finding demonstrated that sex steroids, during setrous cycle, seem to be able to modify the adrenal catecholamines biosynthesis and secretion with stage-specific manner by modulation of the enzyme activities.