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E L Calvo, G Bernatchez, G Pelletier, J L Iovanna, and J Morisset

ABSTRACT

Insulin-like growth factors (IGFs) are important peptides involved in the regulation of cell growth and differentiation in many tissues. The ontogeny of IGF-I was examined in pancreata from 19-day rat fetuses, newborns and 5-, 11-, 26- and 70-day-old rats. For the regeneration studies two models were used: (i) 90% pancreatectomy was carried out and the rats were killed at 1, 2, 3 and 6 days after resection; (ii) acute pancreatitis was induced with caerulein (12μg/body weight three times a day every 8 h for 2 days) and the rats were killed at 1, 2, 5, 7 and 9 days after the first injection. Total RNA was extracted by the guanidinium isothiocyanate method and Northern blots were performed using total RNA and labeled cRNA probes. Abundance of the different mRNA transcripts was estimated by densitometric scanning and normalized to the abundance of 18 S rRNA for each time point. Northern blot analysis during ontogeny showed four (0·8–1·2, 1·9, 4·7 and 7·5 kb) major transcripts in the rat pancreas and liver. Total IGF-I mRNA was 40-fold higher in the adult liver than in the adult pancreas. Moreover, in the liver, IGF-I mRNA levels were higher in the adult than in the fetus, whereas in the pancreas, the highest levels were observed around birth. During the first 3 days after pancreatectomy, a peak of maximal expression was observed after the second day. Densitometric analysis of each IGF-I mRNA species showed concomitant increases in all transcripts. After 6 days, all transcripts had returned to near-control values. IGF-I mRNA expression 2 days after pancreatectomy was 3·5-fold higher than in the newborn. During the first 2 days of acute pancreatitis induction, overexpression of IGF-I mRNA was observed. However, soon after the second day of caerulein treatment, the 7·5 kb transcripts remained elevated whereas those of the others regressed toward control values. Our results show that IGF-I mRNA is overexpressed in both models of pancreatic regeneration but downregulated in the normal adult pancreas.

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G. Pelletier, C. Labrie, J. Simard, M. Duval, M. G. Martinoli, H. Zhao, and F. Labrie

ABSTRACT

Prostatic steroid-binding protein (PBP) is the most abundant protein synthesized in the rat ventral prostate. The protein is under strict androgenic control and is made of two subunits containing the polypeptides Cl, C2 and C3. Using an 35S-labelled cDNA probe, we have used quantitative in-situ hybridization to assess the regulation of polypeptide Cl mRNA levels by sex steroids in the adult male rat. Densitometric quantification of autoradiographic hybridization signals revealed that a significant decrease in Cl mRNA levels could be detected 5 h after castration. Levels of Cl mRNA decreased by 50% 2·5 days after castration, while undetectable levels were reached within 7 days. Administration of the potent androgen 5α-dihydrotestosterone to castrated rats caused a progressive increase in Cl mRNA levels which became significant 5 h after the first injection, while prolonged treatment, for 3 and 7 days, caused 50 and 100% reversals respectively of the effect of castration on Cl mRNA levels. Similar results were obtained by dot-blot hybridization using the same 32P-labelled cDNA probe, thus confirming the specificity and quantification achieved by in-situ hybridization. Administration of oestradiol-17β to orchiectomized adult rats for 14 days had no effect on steady-state Cl mRNA levels. Progesterone, on the other hand, at the dose used (2 mg twice daily) caused a marked increase in Cl mRNA levels, measured by in-situ hybridization, which was completely reversed by concomitant administration of the pure antiandrogen flutamide.

The present data clearly demonstrate that the expression of PBP Cl peptide mRNA is under strict androgenic control and is a very sensitive and specific parameter of androgenic activity. They also indicate that quantitative in-situ hybridization is a powerful, sensitive and most efficient tool to study the regulation of gene expression while, in addition, providing precise information about the site of mRNA localization as well as information about the histology of the tissue, particularly the heterogeneous nature of the acinar response to androgenic stimulation and deprivation.

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G Pelletier, V Luu-The, M El-Alfy, S Li, and F Labrie

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.

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G Pelletier, V Luu-The, S Li, L Ren, and F Labrie

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 catalyzes the conversion of estrone (E1) into 17β estradiol (E2). To gain information about the cellular localization of 17β-HSD mRNA type 1 expression, we performed in situ hybridization using a 35S-labeled cRNA probe in several tissues of adult mice of both sexes. In the ovary, high expression was found in granulosa cells of growing follicles. No specific labeling could be observed in corpora lutea or interstitial cells. In the pituitary gland of animals of both sexes, 17β-HSD type 1 mRNA was expressed in the intermediate lobe melanotrophs while no specific signal could be detected in the anterior or posterior lobes of the pituitary. In the prostate, 17β-HSD type 1 mRNA was exclusively found in the epithelial cells. In both male and female mouse dorsal skin, a specific hybridization signal was seen in the sebaceous glands while the epidermis, stroma, hair follicles and sweat glands were unlabeled. In the testis, a hybridization signal was detected in germ cells of the seminiferous tubules, Leydig cells being unlabeled. The present data indicate that E2 can be formed through the action of 17β-HSD type 1 in specific cells of the gonads and peripheral tissues. In the testes and peripheral tissues, the action of E2 is probably limited to the cells involved in its formation in an intracrine fashion.

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V Compère, D Lanfray, H Castel, F Morin, J Leprince, B Dureuil, H Vaudry, G Pelletier, and M C Tonon

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.

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M. Lamacz, M. C. Tonon, E. Louiset, L. Desrues, L. Cazin, J. Guy, G. Pelletier, and H. Vaudry

ABSTRACT

The effect of modifications of extracellular calcium concentrations on α-MSH release has been studied using perifused frog neurointermediate lobes. Increasing concentrations of calcium (from 2 to 10 mmol/l) gave rise to a dose-related stimulation of α-MSH secretion, whereas reduction of Ca2+ from 2 to 15 mmol/l partially inhibited α-MSH release. The direct effect of extracellular Ca2+ on α-MSH secretion was confirmed by the dose-dependent stimulation of α-MSH release induced by the calcium ionophore A23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mmol Co2+/l both resulted in an inhibition of spontaneous and TRH-induced α-MSH release. Conversely, administration of verapamil or methoxy-verapamil (10 μmol/l each) did not alter basal secretion and had no effect on the response of the glands to TRH. Nifedipine (10 μmol/l), which was able to block KCl (20 mmol/l)-evoked α-MSH release, induced a slight inhibition of basal α-MSH secretion, indicating that extracellular Ca2+ levels may regulate α-MSH release in part by Ca2+ influx through voltage-dependent Ca2+ channels. In contrast TRH-induced α-MSH release was not affected by nifedipine or dantrolene (10 μmol/l), and BAY-K-8644 (1 μmol/l) did not significantly modify the response of neurointermediate lobes to TRH. Taken together, these results suggest that TRH-induced α-MSH secretion is associated with calcium influx across the plasma membrane and that calcium entry caused by TRH may occur through nifedipine/verapamil-insensitive Ca2+ channels.

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F Labrie, V Luu-The, SX Lin, J Simard, C Labrie, M El-Alfy, G Pelletier, and A Belanger

In women and men, an important proportion of estrogens and androgens are synthesized locally at their site of action in peripheral target tissues. This new field of endocrinology has been called intracrinology. In postmenopausal women, 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors while, in adult men, approximately 50% of androgens are made locally in intracrine target tissues. The last and key step in the formation of all estrogens and androgens is catalyzed by members of the family of 17beta-hydroxysteroid dehydrogenases (17 beta-HSDs) while different 17 beta-HSDs inactivate these steroids in the same cell where synthesis takes place. To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.