Regulation of expression of Sertoli cell glucose transporters 1 and 3 by FSH,IL1β, and bFGF at two different time-points in pubertal development

Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1β (IL1β) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1β, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1β, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones. The work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (PICT 25365) and CONICET (PIP 5479), Argentina. R.M.F., C.H.E., C.S.B., and M.S.B. are established investigators of CONICET. G.M.N. is a recipient of a CONICET fellowship and a teaching assistant of Departamento de Bioquímica Humana, Facultad de Medicina, UBA. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.     

Cytochemical and immunocytochemical localization of Na,K-ATPase α subunit isoenzymes in the rat heart

In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidasediaminobenzidine methods, demonstrated ₁ isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For ₂ isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For ₃ isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent ₁ isoenzyme of Na,K-ATPase. A differential as well as unique localization of subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites.     

Requirement of Cl− and Na+ for the ouabain-resistant control of cell volume in slices of rat liver

Summary The ability of liver cells to control their volume in the presence of ouabain has been studied in tissue slices that were recovering at 38°C from a period of swelling at 1°C. Morphological observations were made in conjunction with measurements of the net movements of water and ions. Extrusion of water in the presence of ouabain (2mm) was accompanied by a net loss of Na⁺ and Cl^– and by the formation of characteristic, rounded vesicles in the peri-canalicular regions of the hepatocytes; bile canaliculi were patent. When incubation was carried out in a medium in which either NO ₃ ^– or SO ₄ ^2– replaced Cl^–, ouabain-resistant water extrusion was prevented and the cytoplasmic vesicles normally found with ouabain were almost totally absent. When these slices were subsequently transferred to Cl^– medium with oubain, extrusion of intracellular water was initiated and cytoplasmic vesicles reappeared. Replacement of medium Na⁺ by Li⁺ mimicked the effects of ouabain on water and ion movements and ultrastructure. In addition, the ouabain-resistant extrusion of water and Cl^– was reduced and there was some diminution in the number of vesicles induced by ouabain. Furosemide (2mm) had little effect on water movement or ultrastructure in the absence of ouabain, but it slowed the net water loss and substantially reduced the formation of cytoplasmic vesicles in the presence of ouabain. The results show a close relationship between ouabain-resistant water extrusion and the formation of the cytoplasmic vesicles that are characteristic of treatment with ouabain. They further suggest that a cotransport of Na⁺ and Cl^– forms an important part of the mechanism underlying ouabain-resistant water extrusion and, specifically, that this cotransport may take place across the membranes of the cytoplasmic vesicles.     

Immunohistochemical localization of β1-adrenergic receptors in the liver of male and female F344 rat

The distribution of beta(1)-adrenergic receptors in the liver of Fischer 344 (F344) rat has been examined by an immunohistochemical method. The study was carried out on formalin-fixed and paraffin-embedded livers from young adult, middle-aged, and old female and male F344 rats. An antibody specific for the beta(1)-adrenoreceptor subtype was used. A positive reaction was found in the liver parenchyma of female and male rats from all age groups. Within the liver lobule, a clear zonation is observed, with the beta(1)-adrenoreceptor positivity most evident in pericentral zone hepatocytes and a gradual fading of the immunostaining from pericentral to periportal zone hepatocytes, which may be completely negative. Immunoreactivity is localized on the cell membrane and on the membrane of peripheral cytoplasmic vesicles, and is mostly confined to the cell side facing vascular space. The intensity of immunostaining seems to be slightly higher in the 6- and 10-month-old female rats as compared to the matched male rats and to the senescent female rats. No age-related changes in the intensity of immunostaining are appreciable in male rats. However, no definite conclusion could be drawn about the existence of gender-related differences or age-related changes in the density of beta(1)-adrenoreceptors. A low density of beta1-adrenoreceptor was observed in the spontaneous preneoplastic lesions of the livers from senescent rats.     

Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines

The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein–protein interactions in medulloblastoma cells.     

Effect of luminal pH on the absorption of water,Na+ and Cl− by rat intestine in vivo

The effects of luminal pH on the net absorption of water, Na⁺ and Cl⁻ from in vivo loops of ileum and colon of rat have been studied. Using HCO₃⁻-containing solutions, absorption of water, Na⁺ and Cl⁻ takes place at an initial pH of 7.6 in the ileum, though it is negligible (water and Na⁺), or reduced (Cl⁻) at initial pH's of 6.6 and 5.6. In the colon, absorption takes place between pH's 5.6 and 7.6 with an optimum pH of 6.6. At pH 4.2 there is negligible absorption of water and Na⁺ but significant absorption of Cl⁻.     

Expression of the α-subunit of glycoprotein hormones in the pars tuberalis-specific glandular cells in rat,mouse and guinea-pig

The nature of the hormone(s) secreted by the pars tuberalis (PT) is still unknown. This pituitary lobe is mainly formed by specific glandular cells that differ in their ultrastructural features from the other adenohypophysial cell types. Data from the literature indicate the presence of thyroid-stimulating hormone immunoreactivity in the PT-specific cells of the rat and the Djungarian hamster but not of other species, including the mouse and guinea-pig. The PT also encloses variable numbers of pars distalis cells, essentially gonadotrophs that are mainly dispersed in its caudal area. We studied the expression of the glycoprotein hormone -subunit in the PT of the rat, mouse and guinea-pig by in situ hybridization and immunocytochemistry. In situ hybridization, using an oligonucleotide probe complementary to rat cDNA sequence 196–237 revealed the expression of the -subunit gene throughout the PT of the rat and the mouse; in the guinea-pig, the probe labelled no pituitary cells. Light-and electron-microscopic immunocytochemistry demonstrated -subunit immunoreactivity in the secretory granules of the PT-specific cells in the three species examined. These cells did not react with a specific antibody against the -subunit of luteinizing hormone, an antibody that labelled scattered gonadotrops. The present data suggest that hormone(s) produced by the PT-specific glandular cells are, at least partly, related to glycoprotein hormones.     

Studies on the localization and properties of rat duodenal HCO3−-ATPase with special relation to alkaline phosphatase

Brush-border membrane fractions were isolated from rat duodenum. Purity and integrity of the fraction was confirmed by electron microscopy, enzymic analysis and demonstration of Na⁺-dependent glucose uptake. The membranes were enriched 15-fold in alkaline phosphatase and α-glucosidase and 6-fold in HCO₃⁻-ATPase activities. Assays of latent activity indicated that these enzymes were predominantly localised to the external aspect of the microvillus membrane. The enzymes were solubilised and subjected to analysis by gel filtration, ion exchange and phenylboronate chromatography. No separation of alkaline phosphatase and HCO₃⁻-ATPase was obtained and it is suggested that they reflect the same enzyme activity. The apparent activation by HCO₃⁻ was investigated, and was found to be due to shifts in the pH dependency of the activity due to changes in ionic strength.     

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ

The renin-angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10ng/ml) for 48h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.     

Simultaneous determination of leu-enkephalin localization and [3H]γ-aminobutyric acid uptake in rat striatal cell cultures

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.     

Expression of hepatic 3β-hydroxysteroid dehydrogenase and sulfotransferase 2A1 in entire and castrated male pigs

The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(?) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3β-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3β-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3β-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3β-HSD and thereby influence steroid metabolism, e.g. of androstenone.