Leang-Shin Wu

Cortisol content of eggs and larvae of teleosts.

The whole-animal content of the cortisol was measured in embryos and larvae of tilapia (Oreochromis mossambicus), rainbow trout (Oncorhynchus mykiss), ayu (Plecoglossus altivelis), milkfish (Chanos chanos), and yellowfin bream (Acanthropagrus latus) by radioimmunoassay following the validation of an extraction method. The total cortisol content in tilapia was 50.3 +/- 19.1 pg immediately following fertilization, then decreased abruptly and maintained a lower level of 10-17 pg until larval hatching; after hatching the cortisol content increased to 47.2 +/- 11.9 pg by the seventh day. Newly hatched rainbow trout had 60.3 +/- 6.4 pg cortisol and then increased their cortisol level slowly to 83.0 +/- 7.2 pg by the fifth day after hatching. Ayu larvae contained 5.2 pg cortisol immediately following hatching. On the other hand, pelagic milkfish revealed a much lower cortisol level, being undetectable from hatching until the second day and ranging from 0.4 to 3.7 pg from the third to seventh day after hatching. Yellowfin bream, demonstrating a similarity to milkfish, were not found to have any detectable cortisol from hatching until the third day, but presented 1.6-7.7 pg from the fifth to seventh day after hatching. The presence and clearance of cortisol during early development of fertilized eggs of tilapia suggest a maternal origin of the hormone. The amount of cortisol deposited in the larval body of tilapia increased after hatching from 25% to nearly 100% of the total cortisol in whole larvae, while that in the larval yolk sac decreased to an undetectable level, implying that the increased cortisol may be produced or secreted by the larva. The possible role of cortisol in larval development is discussed.

Ginkgo biloba extract enhances male copulatory behavior and reduces serum prolactin levels in rats

The aim of this study was to investigate the effects of Ginkgo biloba extract (EGb 761) on male copulatory behavior in rats. EGb 761 (1 mg/ml) induced significant production of testosterone (T) in rat Leydig cells in vitro. Its effects on sexual behavior were then tested in Long-Evans male rats after 7, 14, 21, or 28 days of oral gavage of vehicle (distilled water) or EGb 761 at doses of 10, 50, or 100 mg/kg. Administration of 50 mg/kg of EGb 761 for 28 days and of 100 mg/kg for 14 or 21 days significantly increased intromission frequency compared to controls on the same day. An increase in ejaculation frequency was seen after treatment with 50 mg/kg of EGb 761 for 14, 21, or 28 days when compared to either the control group on the same day or the same group on day 0. A reduction in ejaculation latency was only seen after administration of 50 mg/kg of EGb 761 for 14 days compared to the vehicle-treated group. After treatment for 28 days, no significant difference was seen in mount latency, intromission latency, serum T levels, reproductive organ weight, sperm number, or levels of the metabolite of dopamine, 3,4-dihydroxyphenylacetic acid in the brain with any dose of EGb 761, but significantly reduced serum prolactin levels and increased dopamine levels in the medial preoptic area and arcuate nucleus were seen at the dose of 50 mg/kg. These findings show that EGb 761 (especially at the dose of 50 mg/kg) enhances the copulatory behavior of male rats and suggest that the dopaminergic system, which regulates prolactin secretion, may be involved in the facilitatory effect of EGb 761.

Herbal Medicine Research in Taiwan *Appropriate person among the authors to be in contact for any further information on the status of CHM research in Taiwan and for opportunities to study and conduct research in the field of TCM in Taiwan

Of all the countries in the world, why did you choose Taiwan to pursue your study? It is a question that I (comments of the first author) have answered a thousand times. My first visit to a laboratory at National Taiwan University opened my eyes to the possibilities of herbal medicine research, especially in the area of veterinary medicine. It became my ambition to link the knowledge of Traditional Chinese Medicine (TCM) and Ayurveda from the Indian subcontinent and their integration with other systems of medicine, including Western medicine (WM), to achieve the concept of Sustainable Medicine, firstly for animals and then for humans. The Ministry of Economic Affairs (MOEA) has implemented a technology development program to quickly establish the key technologies, and this is a moment of opportunity for Taiwans traditional herbal medicine industry to upgrade and transform itself. This paper, initially intended to be a students narration, has evolved into a multi-author treatise on the present state and likely future scenario of herbal medicine research in Taiwan.

Comparison of Substance P Concentration in Acupuncture Points in Different Tissues in Dogs

Histologically, acupuncture points show neuronal innervation. One hundred seventy-seven tissue samples of acupuncture points and control points from 4 dogs were collected. Concentrations of substance P, a neurotransmitter, in the skin, muscle and subcutis of acupuncture points and control points were measured by means of enzyme immunoassay. The determined tissue concentrations of substance P (mean +/- SEM) in acupuncture points and control points were 1.81 +/- 0.13 and 1.51 +/- 0.1 ng/g in the muscle, 3.33 +/- 0.29 and 2.63 +/- 0.28 ng/g in the skin and 1.59 +/- 0.22 and 1.32 +/- 0.16 ng/g in the subcutis, respectively. The results indicate that there is a difference in the neurochemical profile between acupuncture points and control points.

Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens

We investigated the roles of estrogens and androgens in the progesterone biosynthesis of bovine luteal cells. The responsiveness of primary luteal cells to the stimulation of tropic agents was observed in a dose-dependent manner. Estrogens and androgens significantly inhibited tropic agent-induced progesterone secretions, but glucocorticoids did not, which indicated the inhibitions were specific. The failure of exogenous 8-Br-cAMP to prevent these inhibitions suggested that took place at the post-cAMP steps. The immunoblot showed that testosterone remarkably decreased the amount of induced P450scc protein after 6-hour treatment, yet 17beta-estradiol did not. The 3beta-HSD activity assays demonstrated that both 17beta-estradiol and testosterone efficiently blocked induced 3beta-HSD activities. Both inhibitory effects of E2 and T on progesterone synthesis were observed one hour after treatment and accompanied with suppressed 3beta-HSD activities. This study presents that estrogens and androgens specifically inhibit bovine luteal function through different mechanisms.

Acupuncture Effects on Cardiac Functions Measured by Cardiac Magnetic Resonance Imaging in a Feline Model

The usefulness of acupuncture (AP) as a complementary and/or alternative therapy in animals is well established but more research is needed on its clinical efficacy relative to conventional therapy, and on the underlying mechanisms of the effects of AP. Cardiac magnetic resonance imaging (CMRI), an important tool in monitoring cardiovascular diseases, provides a reliable method to monitor the effects of AP on the cardiovascular system. This controlled experiment monitored the effect electro-acupuncture (EA) at bilateral acupoint Neiguan (PC6) on recovery time after ketamine/xylazine cocktail anesthesia in healthy cats. The CMRI data established the basic feline cardiac function index (CFI), including cardiac output and major vessel velocity. To evaluate the effect of EA on the functions of the autonomic nervous and cardiovascular systems, heart rate, respiration rate, electrocardiogram and pulse rate were also measured. Ketamine/xylazine cocktail anesthesia caused a transient hypertension in the cats; EA inhibited this anesthetic-induced hypertension and shortened the post-anesthesia recovery time. Our data support existing knowledge on the cardiovascular benefits of EA at PC6, and also provide strong evidence for the combination of anesthesia and EA to shorten post-anesthesia recovery time and counter the negative effects of anesthetics on cardiac physiology.

Effects of Aqueous Extracts of "Betel Quid" and Its Constituents on Testosterone Production by Dispersed Mouse Interstitial Cells

Betel quid (BQ) is a favorite chewing item among many communities in different parts of Asia where it is popular by different names. BQ is a unique combination of nut or fruit from the Areca catechu Linn. (AN) tree, leaf from the Piper betle Linn. (BL) vine, slaked lime, paste of bark from the Acacia catechu tree and other spices. AN has been used successfully in various traditional medicines by different civilizations over several ages. Initially condemned by the medical communities for its health hazards, identification and application of potent pharmacologically bioactive compounds from different constituents of BQ have rekindled growing interest in related investigations. Curious about the stimulating role of BQ, we investigated the potential steroidogenic activity of hot water extract from BQ and its constituents and arecoline on testosterone producing ability in an in vitro experiment. Enzyme dissociated interstitial cells from adult mouse testes (ICR strain) were cultured with/without different doses of the extracts and the level of testosterone produced was assayed by an enzyme immunoassay (EIA) technique. It was found that at lower doses of arecoline, AN and BL extracts had significantly stimulated testosterone production over the basal level (p < 0.05). BQ extract, on the other hand, did not show any significant effect on testosterone production. Combinations of arecoline at low doses with 10 ng/ml ovine leutinizing hormone (oLH) showed increases in testosterone produced, while cyclic adenosine monophosphate (cAMP) co-culture showed dose-related inhibition. Our current finding hints at the possible dose-dependent dualistic role of AN and BL extracts and arecoline for testosterone production employing possible non-cAMP-dependent pathway of steroidogenesis. However, the identity of the active compounds besides arecoline and the exact mechanism involved remains to be further investigated.

Effects of Ginseng on the Blood Chemistry Profile of Dexamethasone-treated Male Rats

Ginseng, a panacea in the Orient, has been widely investigated in the last two decades and found to possess a wide range of pharmacological activities including anti-fatigue properties, a transient regulatory action on metabolism and blood pressure, and an increase in the hypothalamo-pituitary-adrenocortical activities. However, a panoramic clinical chemistry study including adrenal and thyroid functions has never been done before. Two experiments with the same design but different concentrations of dexamethasone were performed in this study. The results obtained from the two experiments indicated that ginseng administration at this regime did not influence the blood chemistry profiles in normal rats, but significantly decreased AST and ALT levels from those in dexamethasone-treated ones. It implies that ginseng has a liver-protective effect. Meanwhile, ginseng therapy restores the adrenal and thyroid functions of rats inhibited by dexamethasone treatment.

CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle‐stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis

In vitro and in vivo studies implicate that follicle‐stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFβ1 greatly enhances FSH‐stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium‐sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFβ1‐stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin‐primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH + TGFβ1 additionally induced late‐phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA‐ and calcineurin‐dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)‐modulated FSH action in a calcineurin‐mediated and PKA‐independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1‐stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSH ± TGFβ1‐increased progesterone synthesis and protein levels of StAR, P450scc, and 3β‐HSD enzyme. Furthermore, chromatin‐immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFβ1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFβ1‐stimulated steroidogenesis in ovarian granulosa cells. J. Cell. Physiol. 227: 2430–2440, 2012.

Astaxanthin Protects Steroidogenesis from Hydrogen Peroxide-Induced Oxidative Stress in Mouse Leydig Cells

Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress.