OBJECTIVE: This study aimed to compare 9 perfluoroalkyl sulfonic acids (PFSA) with carbon chain lengths (C4-C12) to inhibit human placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), aromatase, and rat 3β-HSD4 activities. METHODS: Human and rat placental 3β-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS-MS, and human aromatase activity was determined by radioimmunoassay. RESULTS: PFSA inhibited human 3β-HSD1 structure-dependently in the order: perfluorooctanesulfonic acid (PFOS, half-maximum inhibitory concentration, IC ₅₀: 9.03 ± 4.83 μmol/L) > perfluorodecanesulfonic acid (PFDS, 42.52 ± 8.99 μmol/L) > perfluoroheptanesulfonic acid (PFHpS, 112.6 ± 29.39 μmol/L) > perfluorobutanesulfonic acid (PFBS) = perfluoropentanesulfonic acid (PFPS) = perfluorohexanesulfonic acid (PFHxS) = perfluorododecanesulfonic acid (PFDoS) (ineffective at 100 μmol/L). 6:2FTS (1H, 1H, 2H, 2H-perfluorooctanesulfonic acid) and 8:2FTS (1H, 1H, 2H, 2H-perfluorodecanesulfonic acid) did not inhibit human 3β-HSD1. PFOS and PFHpS are mixed inhibitors, whereas PFDS is a competitive inhibitor. Moreover, 1-10 μmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells. Docking analysis revealed that PFSA binds to the steroid-binding site of human 3β-HSD1 in a carbon chain length-dependent manner. All 100 μmol/L PFSA solutions did not affect rat 3β-HSD4 and human placental aromatase activity. CONCLUSION Carbon chain length determines inhibitory potency of PFSA on human placental 3β-HSD1 in a V-shaped transition at PFOS (C8), with inhibitory potency of PFOS > PFDS > PFHpS > PFBS = PFPS = PFHxS = PFDoS = 6:2FTS = 8:2FTS.
Humans ; Pregnancy ; Female ; Rats ; Animals ; Placenta ; Progesterone/pharmacology* ; Aromatase/pharmacology* ; Cell Line, Tumor ; Fluorocarbons ; Alkanesulfonic Acids ; Structure-Activity Relationship ; Hydroxysteroid Dehydrogenases/pharmacology*

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*

OBJECTIVETo investigate the expression of 17 beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) in the kidney of rats and explore the capacity of the kidney for synthesizing sex hormones.

METHODSThe expressions of 17-HSD1 and sex hormones were detected by Western blotting and radioimmunoassay in rat renal cells in primary cultured for 24 and 48 h in the presence or absence of follicle-stimulating hormone (FSH) and luteinizing hormone (LH).

RESULTSAfter cell culture for 24 h, the primary rat renal cells expressed a low level of 17β-HSD1 (0.1843±0.076), which increased to 1.6651±0.044 (P<0.01) in response to co-stimulation by FSH and LH. Low levels of estradiol, progesterone and testosterone were also detected in rat renal cells (3.30±3.78, 62.60±12.33, and 22.12±3.36, respectively), and co-stimulation of FSH and LH significantly increased their levels to 8.50±2.64, 117.80±9.79, and 45.04±4.39, respectively (P<0.05). The levels of these hormones showed no significant differences between cells cultured for 24 h and 48 h (P>0.05).

CONCLUSIONThe rat renal cells express 17β-HSD1 and are capable of stably secreting sex hormones in response to co-stimulation with FSH and LH, suggesting the capacity of the rat kidneys for synthesizing sex hormones. These findings enrich the understanding of the endocrine function of the kidney.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cells, Cultured ; Estradiol ; biosynthesis ; Follicle Stimulating Hormone ; pharmacology ; Kidney ; enzymology ; Luteinizing Hormone ; pharmacology ; Progesterone ; biosynthesis ; Rats ; Testosterone ; biosynthesis

OBJECTIVETo evaluate the inhibitory effect of genistin combined with anastrozole on the growth and apoptosis of breast tumor tissue, and to study their anti-cancer mechanism by using the model of 7,12-dimethylbenz [alpha] anthracene (DMBA)-induced mammary tumors following ovariectomy in Sprague-Dawley (SD) rats.

METHODSThe DMBA induced postmenopausal SD rats were randomly divided into the control group, the genistein group, the anastrozole group, and the genistein combined with anastrozole group. The growth of tumors was observed in each group. The proliferation index and apoptosis index of tumor cells were determined. Moreover, estradiol (E2) and 17beta-HSD1 mRNA levels were determined by ELISA and RT-PCR respectively.

RESULTSThe tumor growth was inhibited in the genistein group and the anastrozole group. The inhibitory ratio was significantly higher in the genistein combined with anastrozole group (P < 0.05). Compared with the control group, levels of E2 and 17beta-HSD1 mRNA decreased more significantly in the genistein combined with anastrozole group (P < 0.05).

CONCLUSIONSGenistein could suppress the growth of mammary tumors in postmenopausal rats. It showed synergistic effect when combined with anastrozole, which resulted in reduced levels of E2 and 17beta-HSD1 mRNA. It had inhibitory effect on the growth of breast tumors.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; metabolism ; Female ; Genistein ; administration & dosage ; pharmacology ; Mammary Neoplasms, Experimental ; chemically induced ; pathology ; Nitriles ; administration & dosage ; pharmacology ; Ovariectomy ; Postmenopause ; Rats ; Rats, Sprague-Dawley ; Triazoles ; administration & dosage ; pharmacology

OBJECTIVETo investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.

METHODSA549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.

RESULTSThe expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.

CONCLUSIONDRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.

20-Hydroxysteroid Dehydrogenases ; genetics ; metabolism ; Alpha-Amanitin ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Dichlororibofuranosylbenzimidazole ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Nucleic Acid Synthesis Inhibitors ; pharmacology

OBJECTIVETo explore the mechanism of inhibitory effect of SLW on estrogen production by endometrial cells of endometriosis.

METHODAfter the model of eutopic primary cultured endometrial cells of endometiosis and hysteromyoma in vitro was successfully established, the changes of steroidgenic factor-1 (SF-1), chicken ovalbumin upstream-transcription factor (COUP-TF), 17-beta-hydroxysteroid dehydrogenase 1 (17-beta-HSD1) and 17-beta-hydroxysteroid dehydrogenase 2 (17-beta-HSD2) mRNA were detected by RT-PCR before and after treatment of medicated serum of SLW. The changes of SF-1 and COUP-TF protein were also observed by western blot synchronously according to the same treatment method mentioned-above. Meanwhile ,the data of hysteromyoma group was obtained from the above experiments.

RESULTThe expression of SF-1 mRNA and protein, 17-beta-HSD1 mRNA was weak, but COUP-TF mRNA and protein, 17-beta-HSD2 mRNA was remarkable in Hysteromyoma endometrium, as compared with those of endometiosis ,which was taken as control group (P<0.01). After the 48 hours' treatment of medicated serum of 5.0, 2.5 g kg(-1) d(-1) of SLW , the expression of COUP-TF mRNA and protein, 17beta-HSD2 mRNA was found significantly increased, but SF-1 mRNA and protein, 17-beta-HSD 1 mRNA was decreased in contrast to the control group (P <0.01 or P <0.05). Although the expresson of COUP-TF mRNA and protein was increased, SF-1 protein and 17-beta-HSD1 mRNA was decreased in 1.25 g kg(-1) d(-1) medicated serum group ,compared with those of the control group (P <0.01), the low dose group had no apparent inhibitory effect on the expression of SF-1, 17-beta-HSD2 mRNA.

CONCLUSIONThe medicated serum of SLW could inhibit the secretion of estradiol in eutopic endometrial cells of endometiosis, and its mechanism might be associated with combined action of inhibiting expression of SF-1, 17-beta-HSD1 and up-regulating expression of COUP-TF, 17-beta-HSD2.

17-Hydroxysteroid Dehydrogenases ; genetics ; Adult ; Animals ; COUP Transcription Factors ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endometriosis ; blood ; metabolism ; pathology ; Endometrium ; drug effects ; metabolism ; pathology ; Estradiol Dehydrogenases ; Estrogens ; biosynthesis ; Female ; Gene Expression Regulation ; drug effects ; Humans ; In Vitro Techniques ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Rats ; Serum ; chemistry ; Steroidogenic Factor 1 ; genetics

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

OBJECTIVETo investigate the effects of Ginkgo biloba extract (EGB) on the testosterone synthesis in the Leydig cells of type 2 diabetic rats.

METHODSThirty male SD rats were equally randomised into a normal control, a type 2 diabetic and an EGB group. Morphological changes of Leydig cells were observed by light microscopy (LM) and transmission electron microscopy (TEM), concentrations of serum luteinizing hormone (LH) and testosterone (T) were determined by enzyme linked immunosorbent assay (ELISA), and the mRNA levels in the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17a-hydroxylase (P450c17), 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) from the Leydig cells were examined by RT-PCR.

RESULTSCompared with the normal control, there was a significant decrease in the number and volume of Leydig cells, the levels of serum LH and T and the expression of mRNA in StAR, P450scc, 17beta-HSD3 and 3beta-HSD1 in the type 2 diabetes group. And the expression of the P450c17 gene showed a tendency of descending, but with no significance. Compared with the type 2 diabetes group, 12 weeks of EGB treatment caused very slight pathological changes in the Leydig cells, significantly increased the concentrations of blood LH and T, markedly elevated the levels of mRNA in StAR and P450scc and induced an ascending tendency of the expressions of P450c17, 17beta-HSD3 and 3beta-HSD1.

CONCLUSIONEGB enhances testosterone synthesis and secretion of Leydig cells by reducing the impairment of the testis in type 2 diabetic rats.

17-Hydroxysteroid Dehydrogenases ; genetics ; Animals ; Cholesterol Side-Chain Cleavage Enzyme ; genetics ; Diabetes Mellitus, Type 2 ; blood ; genetics ; physiopathology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Ginkgo biloba ; chemistry ; Hydroxysteroid Dehydrogenases ; genetics ; Leydig Cells ; drug effects ; metabolism ; ultrastructure ; Luteinizing Hormone ; blood ; Male ; Microscopy, Electron, Transmission ; Phosphoproteins ; genetics ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; biosynthesis ; blood

OBJECTIVEMany studies have shown that glucocorticoids play a crucial role in the development of obesity and insulin resistance. This study investigated the therapeutic effects of long-term inhibition of glucocorticoid activity on obesity and insulin resistance.

METHODSFour-week-old male Sprague-Dawley (SD) rats were randomly fed with a high-fat diet (fat content accounting for 20% of total calorie) (control group, n=8) or with a high-fat diet along with glycyrrhetic acid (GE, 800 mg/L), an inhibitor of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) for 24 weeks (GE-treated group, n=9). The body weights and the amount of food intake were monitored weekly and daily, respectively. After 24 weeks of GE treatment, oral glucose tolerance tests were performed. Blood glucose was measured by glucose oxidase method. The levels of plasma glucocorticoids, insulin and leptin were measured with radioimmunoassay. The levels of serum cholesterol and triglyceride were determined with an automatic measuring analyzer.

RESULTSThe food intake amount decreased significantly in the GE-treated group from 6 weeks and body weight gain was markedly less from 8 weeks after GE administration compared with the control group. After 24 weeks of treatment, the plasma levels of leptin and insulin in GE-treated rats were significantly reduced compared with the control group. The serum levels of cholesterol and triglyceride decreased markedly compared with the control group and the levels of blood glucose were significantly lower 15, 30, 60 and 120 minutes after oral glucose load in the GE-treated group compared with the control group.

CONCLUSIONSLong-term GE treatment may contribute to resisting diet-induced obesity and insulin resistance.

11-beta-Hydroxysteroid Dehydrogenases ; antagonists & inhibitors ; Animals ; Body Weight ; drug effects ; Dietary Fats ; administration & dosage ; Enzyme Inhibitors ; pharmacology ; Glucocorticoids ; physiology ; Glucose Tolerance Test ; Glycyrrhetinic Acid ; pharmacology ; therapeutic use ; Insulin ; blood ; Insulin Resistance ; Leptin ; blood ; Male ; Obesity ; drug therapy ; Rats ; Rats, Sprague-Dawley

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