Isolation and characterization of rat pituitary endothelial cells

Most previous studies that determined the effect of estradiol on angiogenesis used endothelial cells from nonpituitary sources. Because pituitary tumor tissue receives its blood supply via portal and arterial circulation, it is important to use pituitary-derived endothelial cells in studying pituitary angiogenesis. We have developed a magnetic separation technique to isolate endothelial cells from pituitary tissues and have characterized these cells in primary cultures. Endothelial cells of the pituitary showed the existence of endothelial cell marker, CD31, and of von Willebrand factor protein. These cells in cultures also showed immunoreactivity of estrogen receptors alpha and beta. The angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, significantly increased proliferation and migration of the pituitary-derived endothelial cells in primary cultures. These results suggest that a magnetic separation technique can be used for enrichment of pituitary-derived endothelial cells for determination of cellular mechanisms governing the vascularization in the pituitary.     

Isolation and characterization of epithelial progenitor cells from human fetal liver

Aim: Hepatic progenitor cells can serve as an alternative source of hepatocytes for the treatment of liver diseases. Methods: We isolated and expanded the epithelial progenitor cells (EPC) from the human fetal liver and investigated the differentiation of EPC into hepatic cells by fluorescence-activated cell sorter (FACS), real-time polymerase chain reaction (PCR), immunofluorescence assay, western blotting, and periodic acid-Schiff staining. Results: Isolated EPC possessed highly proliferative ability and subpassaged for more than 25 passages. Real-time PCR showed that EPC expressed liver epithelial markers (cytokeratin [CK]8 and CK18) and biliary-specific markers (CK7 and CK19). FACS analysis indicated that these cells were positive for CD117, CD147, CD90, CD44, human leucocyte antigen class I and CD71, but negative for CD34 and CD45. The EPCpossessed multipotential indicated by differentiating into osteoblasts and adipocytes; when subjected to the hepatic differentiation condition, EPC could be induced to hepatocyte-like cells, which expressed albumin, alpha-fetoprotein, and CK18 proteins. Two months after EPC transplantation, we observed that the grafted cells differentiated into hepatocyte-like cells and there was no observable tumor mass. Conclusion: We have isolated and characterized the human fetal liver-derived EPC and these cells may serve as an ideal cell source for cell-replacement therapy of diseased livers.     

Partial characterization of a unique mitogenic activity secreted by rat Sertoli cells

Sertoli cell conditioned medium (SCCM) contains a potent mitogen, Sertoli cell secreted growth factor (SCSGF). A431 cells, derived from a human epidermoid carcinoma have provided an excellent model cell line for the study of this apparently unique activity secreted by rat Sertoli cells in vitro. Previously, it was shown that SCCM contained an epidermal growth factor (EGF)-like activity which was thought to be the mitogen for A431 cells. The present study showed that these two factors are distinct entities. The secretion of the EGF-like activity decreased with increasing number of culture days, while that of SCSGF and of another Sertoli cell specific protein, transferrin remained constant. The addition of SCCM stimulated whereas 2.5 ng/ml EGF inhibited the A431 cell growth. The proliferative response of A431 cells to a wide variety of growth factors and known Sertoli cell secretions was investigated. SCSGF was the only growth factor of known Sertoli cell secretions tested (transforming growth factors (TGF alpha, TGF beta), EGF, bombesin, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), prostaglandins E-1 and E-2, insulin, transferrin and lactate) which stimulated A431 cell proliferation. SCSGF was mitogenic for A431 cells even in the presence of serum in the culture medium. The partially purified SCSGF was heat- and acid-stable, protease-sensitive with a molecular weight of 14,000. It did not bind to heparin or concanavalin A-Sepharose. The secretion of a mitogenic activity by the Sertoli cell which is different from other previously identified growth factors and which coincides with active spermatogenesis could have important implications in the regulation of spermatogenesis.     

Isolation and characterization of islet stellate cells in rat

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs. PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-I, col-III and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.     

Isolation and characterization of islet stellate cells in rat

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs. PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-I, col-III and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.     

Partial characterization of a somatomedin-like peptide from the medium of cultured rat Sertoli cells

A peptide that is recognized by antibodies to human somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) has been partially purified from cultured Sertoli cells prepared from sexually immature rats. The mol wt of this peptide is about 25,000, as determined by gel filtration chromatography and immunoblot analysis of samples resolved by polyacrylamide gel electrophoresis. Isoelectric focusing indicated that the isoelectric point of this peptide was near neutrality. However, a smaller peptide of mol wt 8,000 that cross-reacted with antibodies to Sm-C/IGF-I, was released after gel filtration in acetic acid. Similarly, reverse phase HPLC on a C18 column under acidic conditions released a Sm-C/IGF-I immunoreactive peptide of 8,000 mol wt. This smaller species apparently resulted from the dissociation of this peptide from a binding protein. Unlike the larger neutral form, the isoelectric point of the smaller peptide was 9.8. This pI is similar to the GH-dependent Sm-C/IGF-I peptide isolated from rat serum. The small peptide, unlike the larger form, reacted in a parallel manner to human Sm-C/IGF-I in the Sm-C/IGF-I RIA and radioreceptor assays. In addition, the 8,000 mol wt peptide behaved as a progression factor in the BALB/c-3T3 assay and competed with [125I]Sm-C/IGF-I for binding to the type I Sm-C/IGF-I receptor from cultured rat Sertoli cells. In summary, results of this study demonstrate that rat Sertoli cells in culture secrete a peptide that is the rat equivalent of human Sm-C/IGF-I.     

Pituitary hormones inhibit the function and differentiation of fetal Sertoli cells

Although the role of pituitary hormones in fetal Sertoli cell proliferation is well understood, their involvement in fetal Sertoli cell differentiation is poorly documented. In this study, we evaluated rat fetal Sertoli cell function by measuring basal transferrin secretion ex vivo and transferrin and anti-Müllerian hormone (AMH) mRNA levels in vivo. The differentiation state of the Sertoli cells was estimated from the amount of transferrin secreted ex vivo after acute stimulation with FSH. Surprisingly, we found that the amount of transferrin secreted by each Sertoli cell in basal condition and after acute FSH stimulation decreased between 18.5 and 21.5 day post coitum (dpc), which corresponds to the onset of pituitary hormone secretion. All of the Sertoli cell parameters measured (basal and FSH-stimulated transferrin secretion ex vivo, transferrin and AMH mRNA levels in vivo) were higher in 21.5-dpc fetuses that had been decapitated on 16.5 dpc than in control littermates. Furthermore, immunostaining for AMH was strongly increased after decapitation. Taken together, these results suggest that pituitary hormones in the fetus and in the immature or adult rat differently regulate Sertoli cells, which suggests that fetal Sertoli cells have their own particular physiology.     

Isolation and characterization of biliary epithelial cells from normal rat liver

A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.     

胰岛与Sertoli细胞体外共同培养

目的 研究胰岛体外长期培养的新方法。方法 采用绿色荧光蛋白（GFP）标记成年SD大鼠胰岛，与Sertoli细胞共同培养20周，应用形态学方法和透射电镜观察胰岛细胞单独培养和共同培养的生长特性；放射免疫法测定胰岛素分泌量。结果 胰岛单独培养3周。细胞存活率显著下降，胰岛阳性细胞数目减少，胰岛素24h累积分泌量和对葡萄糖刺激的反应性下降，大部分胰岛β细胞超微结构破坏，分泌颗粒减少；与Sertoli细胞共同培养的胰岛存活时间显著延长，细胞存活率和胰岛素阳性细胞数目较单独培养显著增加，胰岛素分泌量始终处于高水平状态，培养20周胰岛β细胞超微结构基本正常，结论 大鼠胰岛与Sertoli细胞共同培养可以促进胰岛生长，显著延长存活时间，是一种新的体外长期培养胰岛的方法。     

Nuclear triiodothyronine receptors in rat Sertoli cells

The existence of specific triiodothyronine (T3) receptors in cultured rat Sertoli cells was investigated by evaluating the affinity and capacity of nuclear binding for T3. The results demonstrate the presence of high affinity (Ka = 0.15 +/- 0.02 X 10(10) M-1), low capacity (1.35 +/- 0.07 pmol T3/mg DNA) binding sites for T3 in rat Sertoli cell nuclei. It is demonstrated that, within the developing testis, the major localization of nuclear T3 receptors is in Sertoli cells.     

Identification of insulin receptors on rat Sertoli cells

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75-80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of Kd = 1.8 X 10(-9) M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10-50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30-40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of Mr greater than 300,000 or Mr = 130,000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of Mr 130,000 may represent the alpha-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.     

Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells

In this report we demonstrate glucocorticoid receptors in seminiferous tubules of the rat testis, and that these receptors are localized in Sertoli cells and peritubular cells. The receptors had high affinity for [3H]dexamethasone (Kd = 0.5 - 1 x 10(-9) M), and similar Kd values were calculated from equilibrium analysis and from rate studies (k1 = 1.5 x 10(6) M-1 min-1 and k-1 = 1.4 x 10(-3) min-1, O C). Binding specificity was typical for glucocorticoid receptors (affinity: dexamethasone greater than corticosterone greater than cortisol approximately R5020 approximately progesterone greater than aldosterone = R1881 greater than 17 beta-estradiol approximately cortisone approximately testosterone greater than 5 alpha-dihydrotestosterone). The concentration of glucocorticoid receptors in rat seminiferous tubules revealed an age-dependent decrease, coinciding with the increase in the number of germ cells. Glucocorticoid receptor levels were higher in Sertoli cells from immature rats than in cells from adult rats. Cultured peritubular cells from immature rats contained levels of glucocorticoid receptors similar to cultured Sertoli cells from rats of the same age. With a nick-translated human glucocorticoid receptor complementary DNA probe, a messenger RNA (mRNA) species of approximately 7 kilobase was clearly detected in both Sertoli cells and peritubular cells. In peritubular cells, a smaller mRNA species (5 kilobase) was also clearly detectable. In mRNA from whole testis tissue, a similar developmental pattern as for dexamethasone binding was found. Dexamethasone caused a concentration-dependent stimulation of mRNA levels for androgen binding protein and for the cAMP-dependent protein kinase regulatory subunit type II beta in cultured immature rat Sertoli cells. On the other hand, mRNA levels for glucocorticoid receptor decreased, whereas mRNA levels for beta-actin remained constant. This report documents for the first time the presence of glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells, and is also the first demonstration of glucocorticoid receptors in peritubular cells of the rat testis.     

Isolation, purification and culture of Sertoli cells from immature piglet testes

Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.     

Isolation and characterization of rat pancreatic somatostatin

A peptide representing the major form of somatostatin-like immunoreactivity was isolated from 600 rat pancreata by using anti-somatostatin affinity chromatography, gel permeation chromatography and reverse-phase high-performance liquid chromatography (HPLC). The isolated peptide elutes with the same retention time as synthetic somatostatin-14 in isocratic HPLC and its amino acid composition is in agreement with that of the tetradecapeptide. We propose that the structure of the major rat pancreatic somatostatin is identical to that of somatostatin-14 characterized in other species.     

Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver

To study directly the functions of the cells that line the bile ducts inside the liver, we developed a new technique for isolating intrahepatic bile duct epithelial cells (IBDECs) from normal rat liver. Parenchymal and nonparenchymal cells were separated from whole liver by enzymatic digestion and mechanical disruption; subpopulations of individual nonparenchymal cells then were isolated by serial counter-flow elutriation, isopycnic centrifugation, and immunoaffinity separation with a specific monoclonal antibody against an antigen on the plasma membrane of IBDECs. Using this approach, we isolated 1.2 +/- 0.2 x 10(6) (mean +/- SE) viable (greater than 95% trypan blue exclusion) cells, greater than 95% of which were identified as IBDECs by morphologic appearance and specific cytochemical markers. The IBDECs averaged 7.4 +/- 0.16 microM in diameter and retained their in situ appearance, including morphologic polarity. They appeared as single cells or as cell doublets attached by tight junctions that excluded ruthenium red. Microvilli were abundant and were restricted to the apical (i.e., luminal) domain of the plasma membrane. Coated pits were observed on both apical and basolateral cell surfaces. Internally, IBDECs contained a well-developed system of organelles, including mitochondria, Golgi, and discrete types of vesicles, such as coated vesicles, multivesicular bodies, and lysosomes. These results indicate that a highly purified suspension of viable, morphologically intact, and polar IBDECs can be prepared from normal rat liver using a novel approach that separates liver cells on the basis of size, density, and specific membrane components. The availability of such a model will allow experimental studies to be performed directly on IBDECs, an approach that has not previously been possible.     

Transforming growth factor-alpha stimulates proliferation of rat Sertoli cells

The number of Sertoli cells is positively correlated with the number of germ cells produced in the testis, but the regulation of Sertoli cell proliferation and final density is poorly understood. Using non-aggregated Sertoli cells from 8 to 9-day-old rat testes, highly enriched by lectin binding, we explored effects of Sertoli cell growth factor candidates in vitro. Proliferation was assessed by 3H-thymidine incorporation, bromodeoxyuridine labeling and supravital staining, and FSH was used as positive control. Transforming growth factor-alpha (TGF-alpha) was found to stimulate Sertoli cell proliferation in a dose-dependent manner. Epidermal growth factor (EGF) and betacellulin mimicked the effect, demonstrating specificity of the response as they share receptors with TGF-alpha. Insulin-like growth factor I and II, acidic and basic fibroblast growth factor and stem cell factor lacked significant stimulatory effects. We conclude that EGF/TGF-alpha is a growth factor for Sertoli cells in vitro, possibly contributing to paracrine regulation of Sertoli cell proliferation in vivo.     

Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae

Cultured bones have been shown to secrete local regulators of bone remodeling, such as beta 2-microglobulin, transforming growth factor-beta, and insulin-like growth factor (IGF), but the IGF secreted has not been characterized. In the present study, IGF from medium conditioned by 21-day-old fetal rat calvariae was isolated and characterized. IGF was purified using dialysis, gel filtration, and reverse phase HPLC. Amino acid composition was compatible with that of IGF I (somatomedin-C), and amino-terminal sequence analysis revealed homology with IGF-I. The concentration of IGF-I in the calvarial culture medium was 1 nM and was suppressed by cycloheximide. Calvaria-derived rat IGF I at 20 nM stimulated DNA and collagen synthesis by 42% and 26%, respectively, in monolayer cultures of osteoblast-rich rat parietal bone cells. This study indicates that locally produced IGF-I regulates bone formation in cultures of 21-day-old fetal rat calvariae.     

Metabolism of amino acids by cultured rat Sertoli cells

Sertoli cells support spermatogenesis both spatially and energetically; for this reason, these cells have important adaptations. The energetic metabolism of Sertoli cells was adapted to provide lactate and pyruvate to developing germ cells, because these substrates are essential for spermatocytes and spermatids. In this study, we investigated whether Sertoli cells use alanine, leucine, valine, and glycine as energetic substrates and whether the simultaneous addition of other nutrients, such as glucose and glutamine, might affect the metabolism of these amino acids. Alanine, leucine, valine, and glutamine are almost totally oxidized to CO2 by these cells. In contrast, glycine has been demonstrated to be a poor energetic substrate, being mainly incorporated into proteins, and their metabolism did not change in the presence of palmitic acid, glucose, and/or glutamine. The metabolism of the 3 other amino acids was modified by palmitic acid; besides, glucose changed alanine, leucine, and valine oxidation. Glutamine decreased the oxidation of alanine, leucine, and valine to CO2. Conversely, both alanine and leucine decreased the oxidation of glutamine. Our present findings show that Sertoli cells can adapt its energy metabolism to the oxidative substrates available to fulfill their role in spermatogenic energetic supply.