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A heat treatment for a vanadium-containing steel involving high-temperature austenitisation followed by two-stage isothermal holding was deliberately conducted to produce allotriomorphic ferrite (ALF), idiomorphic ferrite (IDF) and martensite. The resulting microstructures and crystallographic relationships with respect to the parent austenite grains were examined using the electron backscatter diffraction technique. Results indicated that IDF grains did not possess a specific orientation relationship (OR) with respect to the surrounding austenite matrix. The detailed substructures of IDF were also examined. In contrast, the ALF grains had an approximate Kurdjumov–Sachs OR with respect to one or both of the adjacent austenite grains; the latter case was termed a dual OR and was analysed in detail. [ABSTRACT FROM AUTHOR]

Cordyceps sinensis has been used as nutritious food and medicine in Chinese society. CS can inhibit tumor growth and induce tumor cell apoptosis. CS induced MA-10 mouse Leydig tumor cell death, but the anti-tumor mechanisms are not fully understood. Thus, the aim of this study was to investigate the apoptotic effect of CS on MA-10 cells and determine the molecular mechanism. CS (2–10 mg/ml) was added to MA-10 cells at different time scales (0–24 h). The condensation of DNA chromatin and apoptotic nuclear fragmentation increased in CS-treated MA-10 cells. Western blot analysis showed that 3 hours of CS treatment caused an increase in caspase-3 and -8 expressions only, which provided further evidence for the involvement of caspase-3 and -8 in CS-induced MA-10-cell apoptosis. CS blocked NF-?B protein expression in a dose-dependent relationship. CS induces MA-10 cell apoptosis by activating caspase-8-dependent and caspase-9-independent pathways and downregulating NF-?B protein expression. [ABSTRACT FROM AUTHOR]

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as food and crude medicine to improve several kinds of symptoms in Chinese society for a long time. Recent studies have illustrated that the fractions of fruiting bodies of TM exhibit a significant hypoglycemic activity in diabetic mouse models, which usually suffer from sexual dysfunction. In a previous study, we showed that TM reduced plasma testosterone production in normal rats without any positive effect in diabetic rats. It evolved a question of TM directly regulating Leydig cell steroidogenesis. In this study, MA-10 mouse Leydig tumor cells were treated with vehicle, different dosages of TM with or without human chorionic gonadotropin (hCG 50 ng/ml) to clarify the effects. Results showed that TM at different dosages (0.01–10 mg/ml) did not have any effect on MA-10 cell steroidogenesis (p > 0.05). In the presence of hCG, there was an inhibitory trend that TA suppressed MA-10 cell progesterone production at 3 hr treatment with a statistically significant difference by the 10 mg/ml TM (p 0.05). However, TM did reduce hCG-treated MA-10 cell progesterone production at 1, 2 and 3 hr (p [ABSTRACT FROM AUTHOR]

The in vitro effect of extracted fractions of Cordyceps sinensis (CS) mycelium on hCG-treated testosterone production from purified normal mouse Leydig cells was examined. Different fractions extracted from CS (F1-water soluble polysaccharide, F2- water soluble protein and F3- poorly water soluble polysaccharide, and protein) were added to Leydig cells with hCG, and the production of testosterone was determined by radioimmunoassay (RIA). Testosterone productions stimulated by hCG in mouse Leydig cells were suppressed by F2 at 10 mg/ml and F3 at doses from 3 to 10 mg/ml, respectively. F2 and F3 at 10 mg/ml did inhibit dbcAMP-stimulated testosterone productions which indicated that F2 and F3 might affect steroidogenesis at the site after the formation of cyclic AMP. Finally, cycloheximide inhibited F2- and F3-treated mouse Leydig cell testosterone production. [ABSTRACT FROM AUTHOR]

Objective: To establish an intramedullary transplantation model of primary megakaryocytes to evaluate the platelet-producing capacity of megakaryocytes and explore the underlying regulatory mechanisms. Methods: Donor megakaryocytes from GFP-transgenic mice bone marrow were enriched by magnetic beads. The platelet-producing model was established by intramedullary injection to recipient mice that underwent half-lethal dose irradiation 1 week in advance. Donor-derived megakaryocytes and platelets were detected by immunofluorescence staining and flow cytometry. Results: The proportion of megakaryocytes in the enriched sample for transplantation was 40 to 50 times higher than that in conventional bone marrow. After intramedullary transplantation, donor-derived megakaryocytes successfully implanted in the medullary cavity of the recipient and produce platelets, which showed similar expression of surface markers and morphology to recipient-derived platelets. Conclusion: We successfully established an in vivo platelet-producing model of primary megakaryocytes using magnetic-bead enrichment and intramedullary injection, which objectively reflects the platelet-producing capacity of megakaryocytes in the bone marrow.

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.

Studies in the mouse have demonstrated for the first time in vivo regulation of gonadotropin-releasing hormone (GnRH) on the minute-to-minute dynamics of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release and the effects of testosterone on this regulation. Intact and castrated mice with different testosterone levels (3-9 ng/mL) were challenged with exogenous GnRH while under general anesthesia to block endogenous GnRH release. Plasma concentrations of LH and FSH were determined by radioimmunoassay from sequential blood samples collected from anesthetized mice with in-dwelling catheters. The release of LH was correlated with the infusion of different doses of GnRH (0.35, 3.5, and 35 ng) in both intact and castrated mice (r = 0.942, approximately 0.999). GnRH-stimulated LH release was significantly lower in intact mice and in castrated mice with high testosterone levels than in castrated mice with low testosterone levels (P < .05). However, GnRH did not induce FSH release except in castrated males with low testosterone levels and at the highest dose of GnRH. The profiles of FSH release in intact mice and castrated mice with the highest testosterone levels were significant lower than the other groups (P < .05). In conclusion, release of LH, but not FSH, was correlated with increasing dosages of GnRH (r = 0.970), and testosterone significantly suppressed GnRH-stimulated LH release in the mouse (P < .05).

The effects of melatonin on steroidogenesis and steroidogenic acute regulatory (StAR) protein expression were investigated in MA-10 mouse Leydig tumor cells. MA-10 cells were treated with human chorionic gonadotropin/cyclic adenosine monophosphate (hCG/cAMP) analogue alone or with hCG/cAMP analogue plus melatonin in different dosages (0.1 nM to 10 microM). Steroid production and the expression of StAR protein were measured. Melatonin directly inhibited hCG- or dbcAMP-stimulated progesterone production in MA-10 cells within 3 hours. The inhibitory effects of melatonin on hCG- or dbcAMP-stimulated steroid production in MA-10 cells were abolished by a comparative melatonin receptor antagonist, luzindole. 22R-hydroxycholesterol reversed melatonin's inhibitory effects, which illustrated that melatonin did not suppress P450scc enzyme activity. Moreover, StAR protein expression stimulated by hCG and dbcAMP was maximally reduced by 10 nM of melatonin treatment for 3 hours. The effects of prolonged exposure (12 h) to melatonin with dbcAMP stimulation in MA-10 cells were also examined. The expression of StAR protein and steroid production were reduced by melatonin concentrations from 1 nM to 10 microM. However, melatonin at a dose of 1 nM had no effect in 3-hour treatment. Our results indicate that melatonin suppressed MA-10 mouse Leydig cell steroidogenesis through specific binding sites by blocking StAR protein expression without altering the activity of P450scc enzyme.

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.

Extracts from the mycelium of Cordyceps sinensis (CS) were tested to determine the in vitro effect on Leydig cell function. MA-10 mouse Leydig tumor cells were used to conduct the experiments. Results showed that progesterone production gradually increased as the dosage of combined water and ethanol extracted CS increased, and there was a statistically significant difference in progesterone production stimulated by 20 mg/ml of CS extracts compared to the control. The combined water and ethanol extracted CS significantly stimulated MA-10 cell steroid production at 12 and 24 h of incubation. In addition, a protein synthesis inhibitor, cycloheximide, did not block the stimulatory effects of CS extracts on MA-10 cell steroid production or total protein expression. Moreover, the expression of steroidogenic acute regulatory (StAR) protein, which is a critical protein for steroidogenesis, stimulated by CS extracts, could not be detected by Western blot analysis. These data indicate that CS extracts might not induce StAR protein and/or other protein expressions to stimulate steroidogenesis in MA-10 mouse Leydig tumor cells.

Previous reports have demonstrated that corticotropin-releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of steroidogenesis, probably by acting through the cAMP/protein kinase A second messenger pathway. Based on this observation, the mechanism of CRH-stimulated steroidogenesis is now further investigated and compared to trophic hormone stimulation. Both cell types were treated with human chorionic gonadotropin (hCG) or CRH in the absence and presence of the following agents: the translation inhibitor cycloheximide, the transcription inhibitor actinomycin D, the protonophore carbonyl cyanide m-chlorophenylhydrozone (mCCCP), which disrupts the mitochondrial electrochemical gradient or the phorbol ester, phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C. Cortico-releasing hormone-stimulated steroidogenesis was completely blocked by cycloheximide in both cell types, indicating that CRH-stimulated steroidogenesis in mouse Leydig cells requires ongoing protein synthesis. Actinomycin D had profound inhibitory effects on CRH-stimulated steroidogenesis in MA-10 cells, and this inhibition was greater than that seen in mouse primary Leydig cells. mCCCP severely inhibited CRH-stimulated steroid production in both cell types, indicating that an electrochemical gradient across the inner mitochondrial membrane is required for CRH-stimulated steroidogenesis. In addition, PMA inhibited hCG- and CRH-stimulated steroidogenesis in MA-10 cells and CRH-stimulated steroidogenesis in primary Leydig cells, suggesting that activation of the protein kinase C pathway can influence protein kinase A stimulated steroidogenesis. Results of these studies suggest that the mouse Leydig cell steroidogenic response to CRH shares many similarities to that of the LH response.

We have previously demonstrated that corticotropin-releasing hormone (CRH) treatment of MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of progesterone production. In view of this observation we wished to determine the effects of CRH on the synthesis of the steroidogenic acute regulatory (StAR) protein in these cells. StAR is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage enzyme. Treatment of MA-10 cells with the cAMP analogue dibutyryl cAMP (dbcAMP) resulted in a dose- and time-dependent increase in the levels of StAR protein that reached a maximum at 800 microM dbcAMP and within a time period of 6 h. Further, treatment of MA-10 cells with CRH also resulted in a dose-dependent increase in the synthesis of the StAR protein with a maximal response observed at 1 microM. Slightly different from that observed with dbcAMP, the maximal response to 1 microM CRH was seen at 4 h following stimulation. These results indicate that the observed increase in steroid production in response to CRH in MA-10 Leydig tumor cells is similar to that previously seen with trophic hormone stimulation acting through the cAMP second messenger pathway, and that it occurs as a result of an increase in the synthesis of the StAR protein.

The actions of corticotropin-releasing hormone (CRH) on steroidogenesis in enriched preparations of mouse and rat Leydig cells were investigated. Primary cultures of purified Leydig cells as well as a Leydig tumor cell line were used in these studies. CRH had a stimulatory effect on steroid production in both isolated preparations of mouse Leydig cells (80-90% Leydig cells) and MA-10 cells (a mouse Leydig tumor cell line). In primary cultures of mouse Leydig cells, CRH was effective over a range of 1 nM-100 nM, while MA-10 cells were responsive over a wider range (10 nM-100 microM). When a submaximal dosage of CRH was given together with a maximal dosage of hCG, steroid production was stimulated even more highly in MA-10 cells. However, when primary cultures of mouse Leydig cells were treated with CRH and hCG, no similar response was observed. In addition, a CRH antagonist, alpha-helical CRH9-41, reversed the CRH stimulatory effect on steroidogenesis in both mouse Leydig cells and MA-10 cells. The accumulation of intracellular cAMP after CRH treatment was dose-responsive to CRH in both cell types, a finding similar to the results described above for steroid production. CRH had no effect on steroidogenesis in rat Leydig cells (60-80% Leydig cells) in the present study. These results indicate that mouse Leydig cells respond to CRH through specific receptors with increased production of cAMP and steroids.