Shyi-Wu Wang

Differential effects of nonylphenol on testosterone secretion in rat Leydig cells

Nonylphenol (NP), a final metabolite of nonylphenol polyethoxylate, has been reported to interfere with male reproduction. However, its mechanisms are not fully understood. In the present study, we examined the effects of NP on steroidogenesis of testosterone in rat Leydig cells. The testosterone concentrations in rat plasma were examined after intravenous injection of NP (100 microg/kg) at different time intervals. In addition, rat Leydig cells were challenged with different concentrations of NP (4.25-127.5 microM) to evaluate its influences on testosterone steroidogenesis. Administration of NP showed a decrease of hCG-induced plasma testosterone. Moreover, in vitro experiments revealed that NP (127.5 microM) alone stimulated testosterone release through increase of both protein levels and activities of the StAR and P450(SCC). In contrast, NP inhibited hCG-induced testosterone release in rat Leydig cells. The inhibitory effect was also observed after incubation of the Leydig cells in the presence of different precursors. These results suggested that NP had differential effects on testosterone synthesis.

Effects of estradiol on aldosterone secretion in ovariectomized rats.

The effects of estradiol benzoate (EB) on steroidogenesis in rat zona fasciculata‐reticularis (ZFR) cells were studied. Female rats were ovariectomized (Ovx) for 2 weeks and then injected subcutaneously with oil or EB for 3 days before decapitation. ZFR cells were isolated and incubated with adrenocorticotropin (ACTH) or prolactin (PRL) for 1 h. Corticosterone concentrations in plasma and cell media, and adenosine 3′,5′‐cyclic monophosphate (cAMP) production in ZFR cells were determined by radioimmunoassay. The effects of EB replacement in vivo on the activities of steroidogenic enzymes in ZFR cells were measured by the amounts of intermediate steroidal products separated by thin‐layer chromatography. Replacement of EB in vivo resulted in a dose‐dependent increase of plasma PRL and corticosterone in Ovx rats. The basal, ACTH‐, and PRL‐stimulated release of corticosterone by ZFR cells was greater in EB‐ than in oil‐treated animals. Forskolin‐induced production of cAMP was greater in the EB‐replaced rats than in oil‐treated animals, which correlated with the increase of corticosterone production. The 3‐isobutyl‐l‐methylxanthine (IBMX) plus ACTH‐, IBMX plus PRL‐, and forskolin plus PRL‐stimulated productions of cAMP were higher in EB‐ than in oil‐treated rats. The enzyme activities of postpregnenolone were not affected by EB replacement in Ovx rats. These results suggest that the EB‐related increase of corticosterone production in Ovx rats is associated with an increase of cAMP generation and the stimulatory effect of PRL on ZFR cells. J. Cell. Biochem. 77:560‐568, 2000.

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)‐independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 × 105 cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10−5 M), or 8‐bromo‐adenosine‐3′:5′‐cyclic monophosphate (8‐Br‐cAMP, 10−4 M), SQ22536 (an adenylyl cyclase inhibitor, 10−6–10−5 M), steroidogenic precursors (25‐hydroxy‐cholesterol, pregnenolone, progesterone, and androstenedione, 10−5 M each), nifedipine (a L‐type Ca2+ channel blocker, 10−5–10−4 M), or nimodipine (a potent L‐type Ca2+ channel antagonist, 10−5–10−4 M) in the presence or absence of lactate at 34°C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5–20 mM dose‐dependently increased the basal testosterone production by 63–187% but did not alter forskolin‐ and 8‐Br‐cAMP‐stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25‐hydroxy‐cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30‐ nor 60‐min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate‐stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L‐type Ca2+ channel. J. Cell. Biochem. 83: 147–154, 2001.

Anti-Proliferative Effects of Evodiamine on Human Thyroid Cancer Cell Line ARO

The incidence of thyroid cancer increases with age, and it is twice in women as common as in men. The undifferentiated thyroid cancer (UTC) is the most aggressive of all thyroid cancers. Unfortunately, there are almost no efficacious therapeutic modalities. It is important to develop some new effective therapies. Evodiamine is a chemical extracted from a kind of Chinese herb named Wu‐Chu‐Yu and has been demonstrated to be effective in preventing the growth of a variety of cancer cells. In the present study, the mechanism by which evodiamine inhibited the undifferentiated thyroid cancer cell line ARO was examined. Based on 3‐(4,5‐dimethylthiazol ‐2‐yle)2,5‐diphenyltetrazolium bromide (MTT) assay, cell proliferation rate was reduced dose‐dependently by evodiamine, but not by rutaecarpine. According to the flow cytometric analysis, evodiamine treatment resulted in G2/M arrest and DNA fragmentation in ARO cells. The G2/M arrest was accompanied with an increase of the expression of cdc25C, cyclin B1, and cdc2‐p161 protein, and it was also with a decrease of the expression of cdc2‐p15. Furthermore, by using the TUNEL assay, evodiamine‐induced apoptosis was observed at 48 h and extended to 72 h. Western blotting demonstrated that evodiamine treatment induced the activation of caspase‐8, caspase‐9, caspase‐3, and the cleavage of poly ADP‐ribose polymerase (PARP). These results suggested that evodiamine inhibited the growth of the ARO cells, arrested them at M phase, and induced apoptosis through caspases signaling. J. Cell. Biochem. 110: 1495–1503, 2010.

The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine

1 The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2 Administration of amphetamine (10−12–10−6 M) in vitro resulted in a dose‐dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone. 3 Amphetamine (10−9 M) enhanced the basal and hCG‐increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4 Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5 Nifedipine (10−6 M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10−10–10−6 M). 6 Amphetamine (10−10–10−6 M) significantly (P<0.05 or P<0.01) decreased the activities of 3β‐hydroxysteroid dehydrogenase (3β‐HSD), P450c17, and 17‐ketosteroid reductase (17‐KSR) as indicated by thin‐layer chromatography (t.l.c.). 7 These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3β‐HSD, P450c17, and 17‐KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

Disturbed Small Intestinal Motility in the Late Rat Pregnancy

We investigated whether various periods of pregnancy might disturb rat gastrointestinal motility. When the proestrus of female rats occurred, they were housed with male rats. Motility studies were conducted on day 7 (first period), day 14 (second) and day 21 (third) of pregnancy, respectively. After the orogastric feeding of radiochromium marker, rats were sacrificed 15 min later. Gastric emptyings of pregnant rats measured at various periods did not differ from the nonpregnant diestrus controls. The geometric center represented intestinal transits in the first, second and third periods of pregnancy and controls were (mean ±SEM) 4.54 ± 0.25, 4.47 ± 0.17, 3.61 ± 0.27 and 4.98 ± 0.13, respectively (p < 0.01) while their plasma progesterone levels were 15.6 ± 2.6, 18 ± 1.4, 7.1 ± 0.5 and 8.6 ± 0.4 ng/ml, respectively (p < 0.01). This shows that late pregnancy inhibits small intestinal transit, whereas gastric emptying remains unchanged. Altered progesterone during pregnancy is not a main mediator to disturb intestinal transit.

Effects of hypoxia on testosterone release in rat Leydig cells

The aim of this study was to explore the effect and action mechanisms of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12% O(2) + 88% N(2), 1.5 l/ml) 8 h/day for 4 days. Normoxic rats were used as control. In an in vivo experiment, hypoxic and normoxic rats were euthanized and the blood samples collected. In the in vitro experiment, the enzymatically dispersed rat Leydig cells were prepared and challenged with forskolin (an adenylyl cyclase activator, 10(-4) M), 8-Br-cAMP (a membrane-permeable analog of cAMP, 10(-4) M), hCG (0.05 IU), the precursors of the biosynthesis testosterone, including 25-OH-C (10(-5) M), pregnenolone (10(-7) M), progesterone (10(-7) M), 17-OH-progesterone (10(-7) M), and androstendione (10(-7)-10(-5) M), nifedipine (L-type Ca(2+) channel blocker, 10(-6)-10(-4) M), nimodipine (L-type Ca(2+) channel blocker, 10(-5) M), tetrandrine (L-type Ca(2+) channel blocker, 10(-5) M), and NAADP (calcium-signaling messenger causing release of calcium from intracellular stores, 10(-6)-10(-4) M). The concentrations of testosterone in plasma and medium were measured by radioimmunoassay. The level of plasma testosterone in hypoxic rats was higher than that in normoxic rats. Enhanced testosterone production was observed in rat Leydig cells treated with hCG, 8-Br-cAMP, or forskolin in both normoxic and hypoxic conditions. Intermittent hypoxia resulted in a further increase of testosterone production in response to the testosterone precursors. The activity of 17β-hydroxysteroid dehydrogenase was stimulated by the treatment of intermittent hypoxia in vitro. The intermittent hypoxia-induced higher production of testosterone was accompanied with the influx of calcium via L-type calcium channel and the increase of intracellular calcium via the mechanism of calcium mobilization. These results suggested that the intermittent hypoxia stimulated the secretion of testosterone at least in part via stimulatory actions on the activities of adenylyl cyclase, cAMP, L-type calcium channel, and steroidogenic enzymes.

Effects of ovarian steroid hormones and thyroxine on calcitonin secretion in pregnant rats

In the present study, the roles of ovarian steroid hormones and thyroxine (T4) in regulating the secretion of calcitonin (CT) in pregnant rats were examined. The levels of plasma progesterone, pre- and post-CaCl2 plasma CT, and recovery time of plasma CT and calcium after calcium challenge were greatest in midterm pregnant rats. The levels of basal plasma progesterone, CT, calcium, and recovery time of plasma CT after calcium challenge were less in late pregnant rats, but basal plasma estradiol was highest in late pregnancy. The concentrations of plasma T4 were gradually decreased in rats during pregnancy. Regardless of the presence of estradiol, administration of progesterone in ovariectomized (Ovx) rats resulted in an increase of plasma T4 as well as the basal and calcium-induced secretion of CT. Administration of estradiol alone did not alter the CaCl2-induced levels but decreased the post-CaCl2 levels of plasma calcium in Ovx rats. The basal levels of plasma CT were decreased in Ovx rats treated with T4. These results suggest that the hypercalcitoninemia in midterm pregnant rats is due to an increased secretion of progesterone. Hypocalcitoninemia in late pregnant rats, however, is due in part to lower plasma calcium.

Regulation of testosterone secretion by prolactin in male rats.

The goal of this study was to characterize the mechanism by which hyperprolactinemia alters testosterone production in rat testicular interstitial cells (TICs). Hyperprolactinemia was induced by grafting 2 anterior pituitary (AP) glands under the subcapsular space of the kidney in experimental rats. Control rats were grafted with brain cortex (CX). Six weeks post‐grafting, rats were challenged with human chorionic gonadotropin (hCG) then, the changes in either plasma testosterone or luteinizing hormone was measured. Additionally, TICs were isolated and challenged in vitro with hCG or prolactin, and the testosterone release measured by radioimmunoassay. Further investigation in signal transduction as intracellular 3′:5′ cyclic adenosine monophosphate (cAMP) production was observed under a regulation of forskolin or SQ22536. After the challenge of hCG or GnRH, the AP‐grafted rats showed a suppressed response in testosterone release as compared to those in the CX‐grafted group. The in vitro data from the AP‐grafted rats compared to the CX‐grafted animals showed a diminished response in testosterone release upon hCG stimulation. Administration of forskolin or SQ22536 disclosed dysfunction of adenylate cyclase in TICs from the AP‐grafted rats. When 8‐Br‐cAMP was incubated with TICs, the testosterone production was lower in the AP‐grafted compared to the CX‐grafted group. These results suggest that in addition to adenylate cyclase dysfunction, inefficiency of post‐cAMP pathways are also involved in the hypogonadism elicited by hyperprolactinemia in rats. J. Cell. Biochem. 74:111–118, 1999.

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)‐stimulated secretion of testosterone and the production of adenosine 3′,5′‐cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin‐releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH‐stimulated LH release as well as hCG‐stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17β‐HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats. J. Cell. Biochem. 73:554–562, 1999.