Downregulation of testosterone production through luteinizing hormone receptor regulation in male rats exposed to 17α-ethynylestradiol.

The pharmaceutical 17α-ethynylestradiol (EE2) is considered as an endocrine-disrupting chemical that interferes with male reproduction and hormonal activation. In this study, we investigated the molecular mechanism underlying EE2-regulatory testosterone release in vitro and in vivo. The results show that EE2 treatment decreased testosterone release from rat Leydig cells. Treatment of rats with EE2 reduced plasma testosterone levels and decreased the sensitivity of human chorionic gonadotropin (hCG). EE2 reduced luteinizing hormone receptor (LHR) expression associated with decreased cAMP generation by downregulation of adenylyl cyclase activity and decreased intracellular calcium-mediated pathways. The expression levels of StAR and P450scc were decreased in Leydig cells by treatment of rats with EE2 for 7 days. The sperm motility in the vas deferens and epididymis was reduced, but the histopathological features of the testis and the total sperm number of the vas deferens were not affected. Moreover, the serum dihydrotestosterone (DHT) level was decreased by treatment with EE2. The prostate gland and seminal vesicle atrophied significantly, and their expression level of 5α-reductase type II was reduced after EE2 exposure. Taken together, these results demonstrate an underlying mechanism of EE2 to downregulate testosterone production in Leydig cells, explaining the damaging effects of EE2 on male reproduction.

Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10-9 -10-4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc.

Induction of renal senescence marker protein-30 (SMP30) expression by testosterone and its contribution to urinary calcium absorption in male rats.

The aim of this study was to investigate the involvement of androgen, mainly testosterone, in the expression of renal senescence marker protein-30 (SMP30) in male rats. We found that the renal SMP30 expression was up-regulated by endogenous testosterone stimulation during puberty. Interestingly, androgen-deficient orchidectomized (ORX) rats exhibited lower SMP30 mRNA and protein expression in the kidney, and that was restored by testosterone propionate (TP) replacement. Abrogation of androgen receptor (AR) activity by co-treatment with flutamide abolished testosterone-induced SMP30 expression in the kidney as well as in the NRK52E cells. However, SMP30 expression was unaltered in the liver of ORX rats. We also showed a positive correlation between renal SMP30 expression and plasma testosterone level during the aging process. TP-induced SMP30 expression in ovariectomized (OVX) rats was observed and was an evidence to explain the gender difference of SMP30 levels. Immunofluorescence assay showed that renal SMP30 was specifically expressed in the proximal tubular segments of the kidney. The urinary Ca(2+) level was increased in both ORX and male aging rats. Taken together, our results indicate a novel role of testosterone in regulating SMP30 expression specifically in the kidney to contribute to urinary calcium absorption.

Attenuation of exercise effect on inflammatory responses via novel role of TLR4/PI3K/Akt signaling in rat splenocytes.

Moderate exercise diminishes proinflammation cytokine production in various types of immune cells, but the intracellular signaling pathways involved are not completely understood. Phosphoinositide 3-kinase (PI3K)/Akt, a crucial downstream protein of toll-like receptor 4 (TLR4), may modulate inflammation. The present study aimed to investigate the effects of exercises on lipopolysaccharide (LPS)-stimulated inflammatory response in splenocytes and to explore potential mechanisms of the PI3K/Akt pathway. Male rats were divided into sedentary and exercise groups. Animals in the exercise group underwent endurance training 30 min/day, 7 days/wk, at the speed of 20 m/min on a treadmill for 1 wk. Here, we showed that exercise 1) attenuated TLR4, 2) increased PI3K/phospho-Akt (p-Akt), and 3) diminished phospho-nuclear factor-κB (p-NF-κB) expression. In addition, administration of splenocytes isolated from trained rats with LPS in vitro showed 1) reduced tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and nitric oxide secretion and 2) decreased splenocyte proliferation. The plasma corticosterone (CCS) level in the exercise group was higher than that in the sedentary group. We confirmed that CCS down-regulated TNF-α and IL-6 secretion in response to LPS in rat splenocytes. Dexamethasone also significantly attenuated LPS-evoked release of TNF-α and IL-6 in a dose-dependent manner. These findings suggested that exercise dampened the secretion of inflammation mediators probably through partial inhibition of TLR4 and p-NF-κB and activation of PI3K/p-Akt expression in the spleen.

Role of testosterone in regulating induction of TNF-α in rat spleen via ERK signaling pathway.

Spleen is a pivotal organ for regulating immune homeostasis. It has been shown that testosterone diminishes secretion of various inflammatory molecules under multiple conditions. However, the mechanisms of action of endogenous testosterone affecting immune responses in the spleen remain unknown. The aim of the present study was to evaluate the immune functions of the spleen in response to testosterone withdrawal after orchidectomy, and the impact of splenocytes on the bacterial endotoxin lipopolysaccharide (LPS)-induced secretion of inflammatory molecules. Male rats were divided into 3 groups, i.e. intact, orchidectomized (Orch) and orchidectomized plus replacement of testosterone propionate (TP) (Orch+TP). The Orch and Orch+TP rats underwent bilateral orchidectomy one week before TP replacement (2mg/kg body weight) or sesame oil in intact rats as controls for seven days. Orch resulted in a significant increase of spleen weight and basal secretion of nitric oxide (NO) from splenocytes. Additionally, LPS up-regulated cell proliferation and the secretion of tumor necrosis factor-alpha (TNF-α) in splenocytes of Orch rats. Orch further up-regulated phosphorylation of extracellular signal-regulated kinases. Interestingly, the plasma corticosterone concentration in the Orch group was higher than that in the intact and Orch+TP groups. Deficiency of testosterone-elevated TNF-α and NO secretion in response to LPS were confirmed in the rat splenocytes. Testosterone also significantly attenuated LPS-elicited release of TNF-α and NO in a dose-dependent manner. However, testosterone did not suppress splenic blastogenesis at doses in the 10(-10)-10(-7)M concentration range. In this context, testosterone might have a protective role against inflammatory responses in the spleen. The present study provides evidence to indicate that testosterone might modulate the immune system.

Effect of swimming on the production of aldosterone in rats.

It has been demonstrated that exercise is one of the stresses known to increase the aldosterone secretion. Both potassium and angiotensin II (Ang II) levels are shown to be correlated with aldosterone production during exercise, but the mechanism is still unclear. In an in vivo study, male rats were catheterized via right jugular vein (RJV), and divided into four groups namely water immersion, swimming, lactate infusion (13 mg/kg/min) and pyruvate infusion (13 mg/kg/min) groups. Each group was treated for 10 min. Blood samples were collected at 0, 10, 15, 30, 60 and 120 min from RJV after administration. In an in vitro study, rat zona glomerulosa (ZG) cells were challenged by lactate (1-10 mM) in the presence or absence of Ang II (10(-8) M) for 60 min. The levels of aldosterone in plasma and medium were measured by radioimmunoassay. Cell lysates were analyzed by immunoblotting assay. After exercise and lactate infusion, plasma levels of aldosterone and lactate were significantly higher than those in the control group. Swimming for 10 min significantly increased the plasma Ang II levels in male rats. Administration of lactate plus Ang II significantly increased aldosterone production and enhanced protein expression of steroidogenic acute regulatory protein (StAR) in ZG cells. These results demonstrated that acute exercise led to the increase of both aldosterone and Ang II secretion, which is associated with lactate action on ZG cells and might be dependent on the activity of renin-angiotensin system.