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R. WONG
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C. B. C. WHITESIDE
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In a recent study reported in this journal Reiss, Davis, Sideman & Plichta (1963) had shown that the s.c. injection of protein-free pineal extract inhibits the wheelrunning activity of female rats. The present study was designed to examine the effect of injections of melatonin (N-acetyl-5-methoxytryptamine) on the wheelrunning activity of male rats. Male rats were used because the possibility of an interaction of the oestrous cycle with melatonin could thus be avoided. After 3 days in which the rats received daily i.p. injections of 0·9% NaCl solution and were then placed in the wheel in order to adapt to the apparatus, they were injected with either melatonin or saline and tested for 13 days. This procedure was adopted in order to assess the reliability of the inhibitory effect reported by Reiss et al. and also to satisfy the criticism of Eayrs (1954) that measurements of activity before an animal

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K P Lai Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, People’s Republic of China

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M H Wong Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, People’s Republic of China

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C K C Wong Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, People’s Republic of China

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Polychlorinated dibenzo-p-dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been recognized as highly potent developmental and reproductive toxins. We have previously demonstrated effects of TCDD in modulating the expression of rat Sertoli cell secretory products and markers for cell–cell interaction. In this study, we examined the direct biological effects of TCDD in rat Leydig cell primary cultures. Mature rat Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining and a testosterone induction assay. To examine TCDD-induced biological consequences, we measured the changes in the secretion of progesterone and testosterone, as well as transcript levels of some selected steroidogenic enzymes (i.e. StAR, P450scc, 3β-HSD and CYP17α), in TCDD/human chorionic gonadotropin (hCG) co-treated cells. Our results indicated that TCDD (0.2 or 2 ng/ml) treatment significantly suppressed hCG (5 or 10 ng/ml)-induced testosterone secretion. The suppressive effect aligned with a reduction of progesterone secretion (P<0.05), as well as a decrease of P450scc mRNA and protein expression (P<0.05). The mechanistic action of TCDD was found to be via the reduction of cellular cAMP levels in the hCG-treated cells. This observation was further confirmed, as the TCDD-mediated suppressive effect could be reversed by dibutyryl cAMP co-treatment. The data indicate that TCDD can modulate cAMP signaling in rat Leydig cells to affect the process of steroidogenesis.

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C. C. WONG
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K. -D. DÖHLER
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A. VON ZUR MÜHLEN
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Replacement of the 3′-halogen of the tri-iodothyronine (T3) molecule by a propyl-group produces a thyromimetic analogue, 3′-isopropyl-3,5-di-iodo-l-thyronine (T2iPr), with high biological potency. A serum thyroid-stimulating hormone (TSH) suppression test with one single intraperitoneal injection of 3 or 30 nm-T3 or T2iPr or with 30 or 300 nm-thyroxine (T4) per kg body weight was performed on 56 adult male Lewis rats which were maintained for 3 weeks on an iodine-deficient diet containing 0·2% 6n-propyl-2-thiouracil (PTU). Blood was withdrawn from each rat by cardiac puncture 24 h before and 3, 7, 24 and 48 h after application of the iodothyronines.

Raised serum levels of TSH, due to the treatment with PTU, were significantly reduced within 3 h of treatment with 30 nm-T3, 300 nm-T4, 3 or 30 nm-T2iPr and they remained low throughout the observation period. Treatment with 3 nm-T3 or 30 nm-T4 per kg body weight was less effective. Pituitary concentrations of growth hormone, TSH, prolactin and FSH were significantly reduced by the treatment with PTU. There was also a slight, but insignificant reduction of pituitary concentrations of LH. Treatment with T3, T4 or T2iPr stimulated the reaccumulation of growth hormone, TSH, prolactin, LH and FSH in the pituitary gland.

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P. Y. D. Wong
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C. N. Uchendu
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ABSTRACT

In order to investigate the role of renin angiotensin in the epididymis, angiotensin-converting enzyme (ACE) activity and angiotensin I (AI) and angiotensin II (AII) concentrations were measured in the male reproductive tract and blood serum of the rat. High ACE activity was detected in the rat epididymis, with a major part of the activity being associated with epididymal spermatozoa. When spermatozoa were prevented from entering the epididymis by efferent duct ligation, the ACE activity in the epididymis was greatly reduced. The epithelial cells lining the epididymal duct were also shown to possess ACE activity which was dependent upon circulating androgens.

Treatment of male rats with captopril at a single oral dose (20 mg/kg) significantly inhibited the ACE activity in the blood serum but had no effect on the activity of the epididymal fluid. The intraluminal ACE was protected from the circulating captopril by the blood–epididymis barrier. Long-term treatment with captopril (20 mg/kg per day, 8 weeks), however, caused an increase in blood serum ACE activity but was without effect on intraluminal ACE. The fertility and fecundity of male rats after treatment were apparently normal.

The concentrations of AI and AII were high in the epididymal plasma and epididymal cell when compared with the respective concentrations in blood serum. The intraluminal AII concentration found (13 nmol/l) was close to the threshold concentrations that stimulate anion (and fluid) secretion in cultured epididymal epithelium in vitro. The high intraluminal AII concentration could not have been derived from the testicular fluid or spermatozoa since the rete testis fluid and sperm contained little AII. When spermatozoa were prevented from entering the epididymis by efferent duct ligation, the AII concentration of the epididymal plasma was almost completely abolished, indicating that intraluminal AII was formed endogenously in the epididymal lumen by sperm ACE. We propose that ACE released by epididymal spermatozoa converts AI (formed from the epididymal epithelial cells) to AII which plays a paracrine role in regulating electrolyte and fluid transport through apical membrane angiotensin receptors.

Journal of Endocrinology (1990) 125, 457–465

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P. Y. D. Wong
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C. N. Uchendu
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ABSTRACT

Monolayer cell cultures formed from the rat cauda epididymidis exhibited renin-like and angiotensin-converting enzyme (ACE) activities and contained immunoreactive angiotensin I (AI) and angiotensin II (AII). Renin-like activity, determined indirectly by radioimmunoassay of generated AI at a near-neutral pH of 6·0, was demonstrated in the cell lysate but was almost undetectable in the serum-free cell culture medium, suggesting that renin expression in epididymal cells is an intracellular phenomenon. In contrast, both AI and AII were detected in the cell lysate and cell culture medium. The level of AI was enhanced by pretreating the cells with the ACE inhibitor captopril (100 nmol/l). Incubating the cell monolayers with thoroughly washed sperm cells obtained from the intact cauda epididymides of rats increase (P < 0·01) the AII content of the cell culture medium, with a parallel decline (P < 0·01) in the AI concentration. However, adrenaline (0·23 μmol/l), which was found to stimulate electrogenic anion secretion by cell monolayers grown on pervious supports, was without effect on the renin-like activity or concentration of angiotensins.

The ACE activity in cells was confirmed by its strong dependence on chloride ion and its susceptibility to inhibition by captopril (100 nmol/l). Enzyme activity was significantly (P < 0·005) higher in the culture medium than in the cell lysate and cell membrane fragments. Angiotensinogen, which is obligatory for an intrinsic renin-angiotensin system, is present in epididymal cells. Presumably, it is synthesized and processed in the cell cytosol by intracellular renin.

These findings in cultured cells provide further evidence for a local renin–angiotensin system in epididymal tissue and suggest a possible role of endogenous AII in the regulation of epididymal function.

Journal of Endocrinology (1991) 131, 287–293

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PS Leung
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WP Chan
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TP Wong
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C Sernia
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The possibility of an intrinsic renin-angiotensin system (RAS) in the pancreas has been raised by previous studies in which immunohistochemical examination showed the presence of angiotensin II and its receptor subtypes, type 1 (AT1) and type 2 (AT2). In the present study, gene expression of several key RAS components was investigated by reverse-transcription PCR. mRNA expression for angiotensinogen, renin and angiotensin II receptor subtypes, AT1a, AT1b and AT2 was shown. The presence of angiotensinogen protein, the mandatory component for an intrinsic RAS, was demonstrated by Western blotting and localized by immunohistochemistry to the epithelia and endothelia of pancreatic ducts and blood vessels respectively. Immunoblot analysis detected a predominant protein band of about 60 kDa in the pancreas. This was consistent with the predicted value for angiotensinogen as reported in other tissues. Together with previous findings, the present study shows that the rat pancreas expresses the major RAS component genes, notably angiotensinogen and renin, required for intracellular formation of angiotensin II. The data support the notion of an intrinsic RAS in the rat pancreas which may play a role in the regulation of pancreatic functions.

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A. D. Mooradian
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L. Deebaj
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N. C. W. Wong
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ABSTRACT

To determine the age-related alterations in tissue-responsiveness to thyroid hormone action, the mRNA levels and the enzyme activities of hepatic cytosolic malate dehydrogenase (ME) and mitochondrial α-glycerophosphate dehydrogenase (α-GPD) were determined in groups of euthyroid, hypothyroid and hyperthyroid male Fischer 344 rats at various ages. The basal α-GPD level (change in optical density/min per mg) in 2-month-old rats (0·163 ±0·003) (s.e.m.) was significantly (P < 0·001) higher than that in 6-month-old and 26-month-old rats (0·116±0·012 and 0·098±0·013 respectively). The basal ME activity (mU/mg) was significantly (P < 0·001) reduced in aged rats (9·2±0·89) compared with younger rats (54·2±3·4 and 17·1±2·3 for 2-month-old and 6-month-old rats respectively). The response of ME to tri-iodothyronine (T3) in aged rats compared with young rats was reduced approximately 50%. This age-related reduction in ME response to T3 could not be attributed to reduced food intake with ageing. Northern blot analysis showed that ME mRNA levels in hyperthyroid aged rats were 50% of those of young hyperthyroid rats. There were no significant differences in either basal or T3-stimulated α-GPD mRNA levels. The proportional reduction in steady-state ME mRNA levels and ME activity in aged rats indicates that this age-related change is modulated at a pretranslational level.

Journal of Endocrinology (1991) 128, 79–84

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C. L. AU
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H. K. NGAI
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C. H. YEUNG
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P. Y. D. WONG
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Department of Physiology, Faculty of Medicine, University of Hong Kong, Li Shu Fan Building, Sassoon Road, Hong Kong

(Received 22 November 1977)

The rat testis secretes fluid (Tuck, Setchell, Waites & Young, 1970; Cheung, Hwang & Wong, 1977) which is reabsorbed by the epididymis (Crabo & Gustafsson, 1964; Levine & Marsh, 1971). In the cauda epididymidis, sodium chloride is reabsorbed isotonically with water and potassium is secreted into the ductal lumen (Wong & Yeung, 1977, 1978); these transport processes seem to have many characteristics typical of the processes occurring in the distal tubule of the kidney. Apart from electrolyte and water transport, proteins are also secreted into the ductal lumen. The epididymal epithelium actively maintains a definite milieu within the tubule in which the spermatozoa are maturing. In several transporting epithelia such as those of the toad bladder, frog skin, salivary and sweat glands, intestine and renal tubule (for references,

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K.-D. DÖHLER
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C. C. WONG
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D. GAUDSSUHN
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A. VON ZUR MÜHLEN
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K. GÄRTNER
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URSULA DÖHLER
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Abteilung für Klinische Endokrinologie, Department für Innere Medizin und Zentrales Tierlabor, Medizinische Hochschule, 3000 Hannover 61, Federal Republic of Germany

(Received 8 May 1978)

The application of a blood sampling technique which by itself has little or no influence on the concentrations of hormones in the circulation of the rat is of utmost importance in experimental hormone research. It has recently been demonstrated that the type of anaesthesia and probably also the site of withdrawal of blood from the body may become critical parameters when the determination of unaltered hormone levels is required (Döhler, von zur Mühlen, Gärtner & Döhler, 1977b). That the site of blood sampling may act as an interfering parameter is questionable, since cage transport, handling and possible differences in the intensity of anaesthesia may also influence the variability of individual hormone levels (Baldwin, Colombo & Sawyer, 1974; Krulich, Hefco, Illner & Read, 1974; Euker, Meites &

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P S Leung
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H C Chan
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L X M Fu
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P Y D Wong
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Abstract

Previous studies have demonstrated the existence of several key components of the renin–angiotensin system in the pancreas. In the present study, the localization of angiotensin II receptor subtypes, type I (AT1) and type II (AT2), in the mouse and the rat pancreas was studied by immunocytochemistry using specific antipeptide antibodies against the second extracellular loops of AT1 and AT2 receptors in conjunction with confocal laser scanning microscopy. In the mouse, immunoreactivity for AT1 and AT2 was observed predominantly in the endothelia of the blood vessels and the epithelia of the pancreatic ductal system. Similar distribution of immunoreactivity for AT1 and AT2 was also observed. However, the intensity of immunoreactivity for AT1 and AT2 was stronger in the rat than that found in the mouse pancreas. Much weaker immunostaining for both AT1 and AT2, as compared with that found in ductal regions, was also found in the acini of the rodent pancreas. Together with the previous findings, the present results suggest that AT1 and/or AT2 receptors may play a role in regulating pancreatic functions in the rodent.

Journal of Endocrinology (1997) 153, 269–274

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