primary-culture-rat-adrenocortical-cells-assays-steroidogenic

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

National Cheng Kung University

The effects of Cordyceps sinensis (CS) and its extracted fractions on steroidogenesis in MA-10 cells were determined. Different concentrations of CS and 3 fractions of CS (F1, a water-soluble polysaccharide; F2, a water-soluble protein; and F3, a poorly water-soluble polysaccharide and protein) were added to MA-10 mouse Leydig tumor cells with or without human chorionic gonadotropin (hCG), and the production of steroid and the expression of steroidogenic acute regulatory protein (StAR) were examined. The results showed that CS alone (2-10 mg/mL) stimulated MA-10 cell progesterone production in a dose-dependent relationship. Fractions F1 and F3 (2-10 mg/mL) also had significant (P < .05) stimulatory effects on MA-10 cell steroidogenesis with a dose-dependent relationship. However, fraction F2 did not have an effect on MA-10 cells. CS and F3, but not F1, significantly induced more steroid production in hCG-stimulated MA-10 cells (P < .05). As a temporal relationship, F1 and F3 (2 mg/mL) maximally stimulated progesterone production between 1 and 3 hours after stimulation in MA-10 cells. In addition, CS and F3 significantly enhanced MA-10 cell StAR protein expression, which indicates that CS and F3 may use a cyclic adenosine monophosphate signal transduction pathway to activate MA-10 Leydig cell steroidogenesis in a manner to that of luteinizing hormone.

Cordyceps sinensis and its fractions stimulate MA-10 mouse Leydig tumor cell steroidogenesis.

Lead (Pb) and lipopolysaccharide (LPS) affect not only neurons but also glial cells, and cause neurological impairment in the brain. We have previously shown an enhancement of tumor necrosis factor-alpha (TNF-alpha) in U-373MG glial cells after exposure of Pb and LPS. To determine whether Pb- or LPS-induced TNF-alpha triggers glial cell death, the apoptotic response was investigated in glial cells both in vitro and in vivo. In vitro, Pb ranging from 0.1 to 10 microM increased the expression of TNF-alpha in a dose-dependent manner in U-373MG cells. Similarly, LPS (500 ng/ml) stimulated TNF-alpha expression in the same cells. A combination of LPS and Pb did not cause higher mRNA TNF-alpha expression than LPS or Pb alone. However, exposure to Pb and LPS did not affect apoptosis in U-373MG cells detected by flow cytometric analysis with merocyanine 540 staining and propidium iodine staining. In B6 mice, glial cells in the brain were immunostained positive for TNF-alpha after an intraperitoncal administration of Pb (12.5 mg/kg) or LPS (10 mg/kg). Consistent with the findings in the cell culture system, few apoptotic cells were detected in glial and neurons by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) in the brain of B6 mice. We conclude that induction of TNF-alpha alone by Pb and LPS does not cause cell apoptosis in cells of glial origin.

Enhancement of TNF-alpha expression does not trigger apoptosis upon exposure of glial cells to lead and lipopolysaccharide.

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells.

The development and differentiation of steroidogenic organs are controlled by Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1). Besides, lysosomal activity is required for steroidogenesis and also enables adrenocortical cell to survive during stress. However, the role of lysosomal activity on steroidogenic cell growth is as yet unknown. Here, we showed that lysosomal activity maintained Ad4BP/SF-1 protein stability for proper steroidogenic cell growth. Treatment of cells with lysosomal inhibitors reduced steroidogenic cell growth in vitro. Suppression of autophagy did not affect cell growth indicating that autophagy was dispensable for steroidogenic cell growth. When lysosomal activity was inhibited, the protein stability of Ad4BP/SF-1 was reduced leading to reduced S phase entry. Interestingly, treatment of cells with lysosomal inhibitors reduced glycolytic gene expression and supplying the cells with pyruvate alleviated the growth defect. ChIP-sequence/ChIP studies indicated that Ad4BP/SF-1 binds to the upstream region of Ccne1 (cyclin E1) gene during G1/S phase. In addition, treatment of zebrafish embryo with lysosomal inhibitor reduced the levels of the interrenal (adrenal) gland markers. Thus lysosomal activity maintains steroidogenic cell growth via stabilizing Ad4BP/SF-1 protein.

Lysosomal activity maintains glycolysis and cyclin E1 expression by mediating Ad4BP/SF-1 stability for proper steroidogenic cell growth.

Cordyceps sinensis has various biological and pharmacological functions, and it has been claimed as a tonic supplement for sexual and reproductive dysfunctions for a long time in oriental society. In this article, the in vitro and in vivo effects of C. sinensis and cordycepin on mouse Leydig cell steroidogenesis are briefly described, the stimulatory mechanisms are summarized, and the recent findings related to the alternative substances regulating male reproductive functions are also discussed.

Bu-Miin Huang

Department of Cell Biology and Anatomy,
College of Medicine,
Department of Cell Biology and Anatomy, College of Medicine

National Cheng Kung University

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

Bu-Miin Huang

Department of Cell Biology and Anatomy,College of Medicine,Department of Cell Biology and Anatomy, College of Medicine

National Cheng Kung University

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

Department of Cell Biology and Anatomy,
College of Medicine,
Department of Cell Biology and Anatomy, College of Medicine