AIMTo study the effects of L-tyrosine on 3beta-HSD activity of rat luteal cells in vitro.

METHODSLuteal cells were isolated from ovary tissues of female rats pretreated with PMSG and hCG. Luteal cells were cultured with 95% oxygen and 5% carbon dioxide in 37 degrees C. 3beta-HSD activity was measured by radioimmunoassay (RIA).

RESULTS(1) 0.2 mmol x L(-1) and 2.0 mmol x L(-1) L-tyrosine significantly inhibited 3beta-HSD activity. (2) 0.2 mmol x L(-1) L-tyrosine exerted different effects on 3beta-HSD activity at different concentrations of pregnenolone (Ph). It increased 3beta-HSD activity at 0.1 micromol x L(-1) and 1 micromol x L(-1) of Pn concentration. With further increase in the concentration of Pn to 100 micromol x L(-1), the stimulating effect of L-tyrosine was switched to suppression effect. (3) L-tyrosine and L-tyrosine hydrazide both inhibited 3beta-HSD activity induced by hCG.

CONCLUSIONL-tyrosine affects 3beta-HSD activity of rat luteal cells in vitro. L-tyrosine and tyrosine hydrazide inhibits hCG induced 3beta-HSD activity.

3-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cells, Cultured ; Female ; Luteal Cells ; drug effects ; enzymology ; Rats ; Rats, Wistar ; Tyrosine ; pharmacology

The aim of the present study was to investigate the expression changes of three steroidogenic enzymes in the polycystic ovary syndrome (PCOS). Thirty Sprague-Dawley (SD) rats were randomly divided into normal control (NC) group and PCOS group. PCOS rat model was established by DHEA injection. The serum levels of progesterone, estrogen and testosterone were measured by immunoradioassay or enzyme immunoassay. The cellular distributions of 3β-hydroxy steroid dehydrogenase (3β-HSD), 17β-hydroxy steroid dehydrogenase (17β-HSD) and cytochrome P450 aromatase (P450arom) in ovaries were detected by immunohistochemistry. The expression levels of 3β-HSD, 17β-HSD and P450arom were detected by RT-PCR and Western blot. The results showed that the serum levels of estrogen and testosterone of PCOS group were significantly higher than those of the NC group. There was no significant difference of serum progesterone level between the PCOS and NC groups. Compared with the NC group, the PCOS group showed increased mRNA and protein expressions of both 3β-HSD and 17β-HSD, as well as reduced P450arom mRNA and protein expressions. These results suggest that 3β-HSD and 17β-HSD, but not P450arom, may participate in the ovarian hormonal regulation in the present rat model of PCOS.
17-Hydroxysteroid Dehydrogenases ; metabolism ; 3-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Aromatase ; metabolism ; Disease Models, Animal ; Estrogens ; blood ; Female ; Polycystic Ovary Syndrome ; enzymology ; Progesterone ; blood ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood

OBJECTIVETo set up a stable primary culture system of Leydig cells with higher purity.

METHODSWe separated Leydig cells from other testicular cells, such as Sertoli and germ cells, by enzymatic digestion in combination with Percoll density gradient centrifugation and identified Leydig cells by 3beta-HSD staining.

RESULTSThe purity achieved by this method was above 95% and the total number of Leydig cells obtained from one testicle was about 1 x 10(6). The cytoplasm of Leydig cells was stained in deep blue by 3beta-HSD staining, and these cells possessed testosterone-secreting capability.

CONCLUSIONLeydig cells can be separated by enzymatic digestion combined with Percoll density gradient centrifugation, and 3beta-HSD staining to identify Leydig cells is simple and feasible with high purity.

3-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Centrifugation, Density Gradient ; methods ; Histocytochemistry ; Leydig Cells ; cytology ; enzymology ; physiology ; Male ; Rats ; Rats, Sprague-Dawley

3-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cadmium ; toxicity ; Cells, Cultured ; DNA ; drug effects ; DNA Damage ; Leydig Cells ; drug effects ; enzymology ; secretion ; Male ; Rats ; Testosterone ; biosynthesis ; secretion

OBJECTIVETo establish a primary culture method of rat testis Leydig cell.

METHODSThe primary rat Leydig cells were treated with or without 4 U/ml human chorionic gonadotropin (hCG), and testosterone in culture medium was detected by radioimmunoassay. The morphology and biological characteristics of Leydig cell were observed.

RESULTSThe culture cells were highly homogeneous, proliferative and had a high differentiation rate. The high purified Leydig cells were verified by their dynamic morphological changes and 3beta-hydroxysteroid dehydrogenase delta4-delta5 isomerase (3beta-HSD) histochemical staining. The testosterone secretion induced by hCG significantly increased (P < 0.05) 24 hours after inoculation than that induced without hCG in the control.

CONCLUSIONIt suggests that the Leydig cell cultured in vitro may secrete high concentration of testosterone, and this study laid the basis of androgen replacement therapy for partial androgen deficiency in aging male.

3-Hydroxysteroid Dehydrogenases ; Animals ; Cell Culture Techniques ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Humans ; Leydig Cells ; cytology ; drug effects ; secretion ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; cytology ; drug effects ; Testosterone ; metabolism

OBJECTIVETo explore the effects of di(2-ethylhexyl)phthalate (DEHP) on neonatal mice's testes and Leydig cells in vivo.

METHODSPregnant mice were exposed to DEHP at the dose of 100 mg/kg, 200 mg/kg or 500 mg/kg (body weight) per day by gavage from gestation day 12 (GD 12) through postnatal day 3 (PND 3), respectively. The testis and body weights, testicular histopathology and the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the neonatal mice were investigated.

RESULTSThe body and testis weights of the male mice's offspring were significantly reduced following DEHP exposure. Leydig cell morphology was affected significantly by DEHP as compared with the controls. Leydig cells obviously increased in the neonatal mice's testes on PND 15 and PND 30 when exposed to DEHP (500 mg/[kg x d]). Activities and positive area of the steroidogenic enzymes 3beta-HSD immunoexpression decreased markedly when exposed to DEHP (100 mg/[kg x d] or 200 mg/[kg x d]). Image analysis showed a decrease in the activities of 3beta-HSD in the animals exposed to DEHP (500 mg/[kg x d]), but an increase in the positive area of 3beta-HSD immunoexpression as compared with the control animals on PND 15 (P < 0.01).

CONCLUSIONDEHP affects the Leydig cell morphology, the activity of 3beta-HSD, the testis and body weights and the testicular histopathology of neonatal mice, and it may function as an antiandrogenic agent.

3-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Animals, Newborn ; Diethylhexyl Phthalate ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Leydig Cells ; cytology ; drug effects ; Male ; Mice ; Mice, Inbred Strains ; Pregnancy ; Prenatal Exposure Delayed Effects ; Testis ; drug effects

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.
3-Hydroxysteroid Dehydrogenases/metabolism* ; Animals ; Animals, Newborn ; Anti-Mullerian Hormone/metabolism* ; Aromatase/metabolism* ; Cell Culture Techniques ; Enzyme-Linked Immunosorbent Assay ; Follicle Stimulating Hormone/pharmacology* ; Hormones/pharmacology* ; In Vitro Techniques ; Inhibins/metabolism* ; Leydig Cells/metabolism* ; Luteinizing Hormone/pharmacology* ; Male ; Models, Biological ; Real-Time Polymerase Chain Reaction ; Receptors, FSH/metabolism* ; Receptors, LH/metabolism* ; Sertoli Cells/metabolism* ; Swine ; Testis/metabolism* ; Testosterone/metabolism*

OBJECTIVEThis study was designed to examine the in vitro effects of fenvalerate on steroid production and steroidogenic enzymes mRNA expression level in rat granulosa cells.

METHODSUsing primary cultured rat granulosa cells (rGCs) as model, fenvalerate of various concentrations (0, 1, 5, 25, 125, 625 micromol/L) was added to the medium for 24 h. In some cases, optimal concentrations of 22(R)-hydroxycholesterol (25 micromol/L), Follicle stimulating hormone (FSH, 2 mg/L), or 8-Bromo-cAMP (1 mmol/L) were provided. Concentrations of 17 beta-estradiol(E2) and progesterone (P4) in the medium from the same culture wells were measured by RIA and the steroidogenic enzyme mRNA level was quantified by semi-quantitative RT-PCR.

RESULTSFenvalerate decreased both P4 and E2 production in a dose-dependent manner while it could significantly stimulate rGCs proliferation. This inhibition was stronger in the presence of FSH. Furthermore, it could not be reversed by 22(R)-hydroxycholesterol or 8-Bromo-cAMP. RT-PCR revealed that fenvalerate had no significant effect on 3 beta-HSD, but could increase the P450scc mRNA level. In addition, 17 beta-HSD mRNA level was dramatically reduced with the increase of fenvalerate dose after 24 h treatment.

CONCLUSIONFenvalerate inhibits both P4 and E2 production in rGCs. These results support the view that fenvalerate is considered as a kind of endocrine-disrupting chemicals. The mechanism of its disruption may involve the effects on steroidogenesis signaling cascades and/or steroidogenic enzyme's activity.

3-Hydroxysteroid Dehydrogenases ; analysis ; metabolism ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Animals ; Base Sequence ; Cells, Cultured ; Dose-Response Relationship, Drug ; Estradiol ; analysis ; metabolism ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Hydroxycholesterols ; pharmacology ; Nitriles ; pharmacology ; Progesterone ; analysis ; metabolism ; Pyrethrins ; pharmacology ; RNA, Messenger ; analysis ; metabolism ; Rats ; Steroids ; metabolism

OBJECTIVETo study the clinical features of children with 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency and review the literature.

METHODClinical features and treatment of one Chinese infant with 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency confirmed by HSD3B7 gene mutation analysis were retrospectively reviewed, and 51 cases of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency who were internationally reported since 2000 were also reviewed in this paper.

RESULT(1) A 3-month-old infant with neonatal cholestasis was admitted to our hospital because of hyperbilirubinemia and abnormal liver dysfunction (total bilirubin 110.7 µmol/L, direct bilirubin 74.5 µmol/L, γ-glutamyltransferase 24.4 IU/L, total bile acid 0.1 µmol/L).His jaundice disappeared within a few weeks, serum liver biochemistries improved and his growth in weight and height was excellent after oral cholic acid therapy.HSD3B7 gene analysis using peripheral lymphocyte genomic DNA from the patient identified compound heterozygous mutations. This child was confirmed as the most common inborn error of bile acid metabolism-3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency by molecular analysis.(2) Retrospective review of the literature showed that the clinical features of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency included neonatal cholestasis, some patients progressed to severe liver disease and needed liver transplantation without effective therapy; however, serum biochemical characteristics of normal γ-glutamyltransferase activity, normal or low total bile acid concentrations were not consistent with cholestasis, the replacement treatment with cholic acid produced a dramatic improvements in symptoms, biochemical markers of liver injury; 31 cases were diagnosed by HSD3B7 gene mutation analysis.

CONCLUSIONThe clinical characteristics of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency include neonatal cholestasis, normal serum γ-glutamyltransferase activity, and normal or low serum total bile acid concentration.Oral cholic acid replacement is an effective therapy; definitive diagnosis of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency can be identified by molecular genetic testing technology.

3-Hydroxysteroid Dehydrogenases ; deficiency ; genetics ; Administration, Oral ; Bile Acids and Salts ; biosynthesis ; blood ; Bilirubin ; blood ; Chenodeoxycholic Acid ; administration & dosage ; therapeutic use ; Cholestasis, Intrahepatic ; diagnosis ; drug therapy ; enzymology ; DNA Mutational Analysis ; Humans ; Infant ; Liver ; drug effects ; metabolism ; physiopathology ; Liver Function Tests ; Male ; Metabolic Diseases ; drug therapy ; physiopathology ; Molecular Sequence Data ; Mutation ; genetics ; Retrospective Studies

AIMTo study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis.

METHODSPurified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined.

RESULTSGnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05).

CONCLUSIONGnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.

3-Hydroxysteroid Dehydrogenases ; biosynthesis ; genetics ; Animals ; Blotting, Western ; Cell Separation ; Cells, Cultured ; Cholesterol Side-Chain Cleavage Enzyme ; biosynthesis ; Dose-Response Relationship, Drug ; Gonadotropin-Releasing Hormone ; agonists ; pharmacology ; Leydig Cells ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Phosphoproteins ; biosynthesis ; genetics ; RNA ; biosynthesis ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sexual Maturation ; physiology ; Steroids ; biosynthesis ; Testosterone ; biosynthesis