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Xiumin Wang, Li Liang and Lizhong Du

Ghrelin has a correlation with insulin secretion, β-cell development, and diabetes in crucial development period. The aim of this study was to compare the changes in plasma ghrelin, insulin, and glucose concentrations, and variation of ghrelin expression in the pancreas in response to intrauterine malnutrition in newborn rats. Pregnant rats at day 2 were randomly divided into two groups: nourished (fed ad libitum; NR) and undernourished rats (UR). The offspring of NR were defined as normal-birth-weight group (NBW, n = 79) and those of UR were defined as low-birth-weight group (LBW, n = 74). Plasma glucose, ghrelin, and serum insulin of both dams and their pups were analyzed at the first day after birth. The entire pancreas was collected for determination of ghrelin and insulin mRNAs, and quantification of pancreas ghrelin and insulin. Immunohistochemical double staining and confocal microscopy were performed on rat pancreas. Birth weight was 5.81 ± 0.64 and 4.76 ± 0.23 g in NBW group and LBW group respectively. Fasting plasma ghrelin concentrations in UR group (1382 (1287–1513) pg/ml) were higher than that of NR group (1072 (974–1205) pg/ml). Plasma ghrelin concentrations in the LBW group (2176 (2031–2384) pg/ml) were significantly lower than that of the NBW group (2493 (2311–2675) pg/ml). Undernutrition caused a decrease in plasma insulin concentrations in both UR dams and LBW pups (P < 0.001). Ghrelin mRNA and total ghrelin of pancreas were significantly affected by intrauterine nutrition state. Pancreas insulin concentrations were significantly affected by intrauterine nutrition (P = 0.007). The majority of ghrelin-producing cells were present at the periphery of islets in the NBW group. Ghrelin was colocalized with insulin in ß-cells in LBW group. The percentage of ghrelin-positive cells in the islets of LBW group was significantly higher than that of the NBW group (P < 0.01). Intrauterine undernutrition may affect the birth weight, plasma insulin and ghrelin levels, islet ghrelin expression, and ghrelin cell distribution. It will be interesting to investigate intrauterine nutrition which is involved in islet ghrelin expression and ghrelin cell distribution.

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Z Li, FA Karlsson and S Sandler

The aim of this study was to investigate the alpha cell population during the development of type 1 diabetes following multiple low-dose streptozotocin administration in mice. For this purpose C57BL/Ks male mice were injected with streptozotocin (40 mg/kg body weight for 5 days). Development of hyperglycemia was monitored over 28 days and a morphometric analysis of islet endocrine cells was performed. A reduction of islet cell area was observed after two injections of streptozotocin. The subsequent decrease of the area throughout the study period averaged 35%. Insulin-positive beta cells gradually disappeared from the identified islets. Hyperglycemia was present from day 7 onwards and in parallel with hyperglycemia, insulitis developed. An analysis of the alpha cell number per islet area revealed a 2- to 3-fold increase in this cell population, with the highest value on day 21. Confocal microscopy analysis of the ICA 512 protein tyrosine phosphatase revealed strong expression in the alpha cells at day 21, suggesting high secretory activity in the diabetic state. It is concluded that multiple low-dose streptozotocin treatment of C57BL/Ks male mice causes the disappearance of a fraction of the islets of Langerhans. In the remaining islet tissue an expansion of alpha cells occurs, reflecting a loss of intraislet beta cells as well as a regeneration of alpha cells.

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A Janossy, JY Li and JM Saez

In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [(3)H]quinuclidinyl benzilate ([(3)H]QNB) demonstrated binding sites of high affinity (K(d)=0.45 nM) and low capacity ( approximately 8000 sites/cell). Pharmacological characterisation of muscarinic receptors was assessed by evaluating the effects of the M(3)>M(1)>>M(2) antagonist 4-DAMP (4-diphenylacetyl-N-methylpiperidine) and the M(1)=M(4)> M(3)>>M(2) antagonist pirenzepine on the binding of [(3)H]QNB and carbachol-induced cortisol production. For both parameters, the potency of 4-DAMP was about two orders of magnitude higher than that of pirenzepine. Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M(2), M(3) and M(4) receptors revealed the expression of only M(3) mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M(3) receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells.

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D. Schams, R. Koll and C. H. Li

ABSTRACT

The effect of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF) and nerve growth factor (NGF) on production of oxytocin and progesterone by cultured bovine granulosa and luteal cells was studied. Secretion of oxytocin was stimulated, in a dose-dependent manner, by IGF-I at 48 and 120 h of culture to levels much higher than those after stimulation with LH, FSH, EGF, FGF or NGF. A similar effect of IGF-I was observed for progesterone but, in contrast to oxytocin, secretion of progesterone was not increased by EGF, NGF or FGF. During primary culture, for 4 h, of dispersed bovine luteal cells obtained from corpora lutea between days 4 and 10 of the oestrous cycle, all the growth factors tested failed to stimulate secretion of oxytocin or progesterone. The data suggest the relevance of growth factors (especially IGF-I) for ovarian physiology and their possible importance for differentiation of follicles and luteinization.

J. Endocr. (1988) 116, 97–100

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MA De Bortoli, MH Garraza and LI Aguado

The present study investigates the acute consequences of central adrenergic stimulation on the release of steroids from the ovary. The influence of the superior ovarian nerve (SON) and the relationship between the neural effect and peripheral LH levels were also examined. The intracerebroventricular (i.c.v.) injection of 5 microg epinephrine in SON-intact rats on day 1 of dioestrus (D1) increased progesterone levels in ovarian vein blood from 7 to 21 min after injection but the same injection in SON-intact rats on day 2 of dioestrus (D2) decreased progesterone levels in ovarian vein blood from 1 to 25 min. A smaller dose (0.5 microg) of epinephrine injected i.c.v. in SON-intact rats produced a decrease in progesterone levels in ovarian vein blood of shorter duration. In SON-transected (SONt) animals, 0.5 microg epinephrine i.c.v. caused a smaller decrease in progesterone levels compared with SON-intact rats (P<0.05). On the other hand, in SON-intact rats on D2, the i.c. v. injection of 0.5 microg epinephrine did not modify the peripheral LH levels during 25 min, but 5 microg epinephrine injected i.c.v. raised the peripheral LH level from the third minute after injection (P<0.05). Oestradiol levels in the ovarian vein blood did not change after epinephrine i.c.v. injection in rats on D2. To avoid any humoral influence, SONt and SON-intact rats on D2 were injected i.c.v. with 5 microg epinephrine or with vehicle, and 5 min later the ovaries were incubated in vitro with or without LH. Under these conditions, it was demonstrated that the previous injection of epinephrine in SON-intact rats resulted in a diminished release of progesterone from ovaries incubated with or without LH. These results suggest that a central adrenergic stimulus increases progesterone release from the ovary on D1 and decreases it on D2. Also, this neural input would arrive at the ovary through the SON, and would condition the ovarian response to LH on D2. Ovarian progesterone changes could be attributed to signals coming from ganglionar neurons, which are affected by the central adrenergic stimulation.

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R Wang, J Li and N Yashpal

The limitation of available islets for transplantation is a major obstacle for the treatment of diabetes through islet therapy. However, islet monolayers expanded ex vivo may provide a source of progenitor cells and a model to help understand islet development from precursor cell types. The existence of progenitor cells within the islets is highly likely, yet, to date, no fully defined or characterized postnatal stem cell has been isolated, expanded or marked. Our study evaluates the expression of progenitor markers, including the haematopoietic stem cell marker c-Kit, in epithelial monolayers derived from postnatal rat islets through immunofluorescence and RT-PCR, and the ability of precursor-rich monolayers to reform islet-like structures. Islets formed confluent monolayers when cultured on a type I collagen gel which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating c-Kit-expressing cells, with the proportion reaching a maximum of 45+/-6% at 8 weeks of culture. Evaluation of transcription factors at the mRNA level revealed constant PDX-1, ngn3 and Pax4 expression, while undifferentiated cell markers, such as Oct4 and alpha-fetoprotein, were also detected frequently after 4 weeks of culture. Changing the extracellular matrix protein to laminin-rich Matrigel, the monolayers re-formed islet-like clusters that secreted insulin in a glucose-responsive fashion. Our data show that islets can be expanded ex vivo to form epithelial monolayers with rich undifferentiating cell populations that are characterized by cells expressing the progenitor markers. These monolayers are capable of extensive proliferation and retain plasticity to form new islet cells, and c-Kit-expressing cells may play an important role in new islet cluster formation.

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S You, W Li and T Lin

Gap junctions are intercellular protein channels which provide a pathway for the exchange of ions and small molecules. This exchange of materials allows metabolic coupling of cells. Gap junction channels are made up of connexins, integral membrane proteins encoded by a multigene family. Rat testes contain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown that Cx43 assembles gap junctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combination of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll gradient centrifugation. Leydig cells from 20- and 30-day-old rats were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plateau at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were cultured overnight and then treated with human chorionic gonadotropin (hCG) for variable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein mRNA levels and testosterone formation. However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2 h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx43 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells stained strongly positive with anti-Cx43 monoclonal antibody. Treatment with hCG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the periphery of the cells. To evaluate the regulation of Cx43 in vivo, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by histochemical staining and were positive for Cx43 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx43. Twenty-four hours after hCG treatment, 3beta-HSD activity increased while Cx43 immunostaining of Leydig cells was reduced. In conclusion, gap junction channels of Leydig cells are regulated by hCG both in vivo and in vitro. hCG increased Leydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.

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SJ Zhu, Y Li, H Li, YL Wang, ZJ Xiao, P Vihko and YS Piao

The biosynthesis of 17beta-estradiol (E(2)) in human placenta involves the actions of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). Aromatase, an enzyme complex comprised of P450aromatase (P450arom) and NADH-cytochrome P450 reductase, converts androgens to estrogens, whereas 17HSD1 catalyzes the reduction of estrone to E(2). In the present study, the effects of retinoic acids (RAs) on P450arom and 17HSD1 expression in placental cells were investigated. Treatment with all-trans-RA (at-RA) or 9cis-RA increased E(2) production in JEG-3 choriocarcinoma cells and cytotrophoblast (CTB) cells isolated from normal early placentas. Meanwhile, the activity of aromatase and expression of P450arom mRNA were induced by at-RA in JEG-3 cells. Northern blot analysis showed that the effect on P450arom mRNA expression occurs in a dose- and time-dependent fashion. Similar to at-RA and 9cis-RA, Ro40-6055, the retinoic acid receptor alpha (RARalpha)-selective activator, increased the expression of P450arom and 17HSD1 mRNA in JEG-3 cells. On the other hand, Ro41-5253 (Ro41), the RARalpha-selective antagonist, blocked the stimulatory effect of RAs on P450arom expression. Surprisingly, Ro41 induced the activity and mRNA expression of 17HSD1 in JEG-3 cells, which is in contrast to the expected inhibitory effect and, moreover, remarkably potentiated the induction by at-RA and 9cis-RA. However, reporter gene analysis revealed that the influence of Ro41 on the transcription of the HSD17B1 gene, which encodes 17HSD1, is considerably milder in JEG-3 cells, and it only additively enhanced the effect of at-RA. Finally, it was found that at-RA and 9cis-RA increased the expression of P450arom and 17HSD1 mRNA in CTB cells, but to a lesser extent. The data suggest that RAs may play a role in promoting the biosynthesis of E(2 )in the placenta. In addition, Ro41 has divergent effects on gene expression in JEG-3 cells.

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Caiyun Sun, Da Duan, Bo Li, Chaobin Qin, Jirong Jia, Bin Wang, Haiyan Dong and Wensheng Li

Urotensin II (UII) is a cyclic peptide that was originally extracted from the caudal neurosecretory system (CNSS) of fish. UII is well known to exhibit cardiovascular, ventilatory, and motor effects in vertebrates. Studies have reported that UII exerts mitogenic effects and can act as an autocrine/paracrine growth factor in mammals. However, similar information in fish is limited. In this study, the full-length cDNAs of UII and its receptor (UT) were cloned and characterized in the orange-spotted grouper. UII and UT were expressed ubiquitously in various tissues in grouper, and particularly high levels were observed in the CNSS, CNS, and ovary. A functional study showed that UT was coupled with intracellular Ca2 + mobilization in HEK293 cells. Studies carried out using i.p. injections of UII in grouper showed the following: i) in the hypothalamus, UII can significantly stimulate the mRNA expression of ghrh and simultaneously inhibit the mRNA expression of somatostatin 1 (ss1) and ss2 3 h after injection; ii) in the pituitary, UII also significantly induced the mRNA expression of gh 6 and 12 h after injection; and iii) in the liver, the mRNA expression levels of ghr1/ghr2 and igf1/igf2 were markedly increased 12 and 3 h after the i.p. injection of UII respectively. These results collectively indicate that the UII/UT system may play a role in the promotion of the growth of the orange-spotted grouper.

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Shan-Jin Wang, Xin-Feng Li, Lei-Sheng Jiang and Li-Yang Dai

Regulation of the physiological processes of endochondral bone formation during long bone growth is controlled by various factors including the hormones estrogen and leptin. The effects of estrogen are mediated not only through the direct activity of estrogen receptors (ERs) but also through cross talk with other signaling systems implicated in chondrogenesis. The receptors of both estrogen and leptin (OBR (LEPR)) are detectable in growth plate chondrocytes of all zones. In this study, the expression of mRNA and protein of OBR in chondrogenic ATDC5 cells and the effect of 17β-estradiol (E2) stimulation were assessed using quantitative PCR and western blotting. We have found that the mRNA of Obr was dynamically expressed during the differentiation of ATDC5 cells over 21 days. Application of E2 (10−7 M) at day 14 for 48 h significantly upregulated OBR mRNA and protein levels (P<0.05). The upregulation of Obr mRNA by E2 was shown to take place in a concentration-dependent manner, with a concentration of 10−7 M E2 having the greatest effect. Furthermore, we have confirmed that E2 affected the phosphorylation of ERK1/2 (MAPK1/MAPK3) in a time-dependent manner where a maximal fourfold change was observed at 10 min following application of E2. Finally, pretreatment of the cells with either U0126 (ERK1/2 inhibitor) or ICI 182 780 (ER antagonist) blocked the upregulation of OBR by E2 and prevented the E2-induced phosphorylation of ERK. These data demonstrate, for the first time, the existence of cross talk between estrogen and OBR in the regulation of bone growth whereby estrogen regulates the expression of Obr in growth plate chondrocytes via ERs and the activation of ERK1/2 signaling pathways.