Cellular receptors of gonadotropic hormones. Immunocytochemical evidence in rat testicle]

We have performed topical applications of HCG and LH on fixed paraffin-embedded sections of adult rat testes. Thereafter, immuno-cytochemical demonstration of the peptides was carried on and demonstrated that HCG and LH specifically bound to the cell membrane of Leydig cells. This technique is highly reproductible provided that adequate fixation (with formalin containing fixatives) as been performed.     

Immunocytochemical detection of ricin. I. Preliminary immunofluorescence studies

Summary Ricin injected into rat musclein vivo can be localized within a few hors using routine immunofluorescence techniques on formalin-fixed tissues. However, the level of sensitivity decreases with increase in size of animal injected, time after administration and decrease in dose given. These findings are discussed in relation to the known chemistry and subcellular mode of action of ricin.     

The midgestational human fetal pancreas contains cells coexpressing islet hormones.

In the fetal development of the mouse pancreas, endocrine cells have been found that express more than one hormone simultaneously. Our objective was to evaluate the existence of such cells in the human fetal pancreas. We found cells coexpressing two of the major pancreatic hormones (insulin, glucagon, and somatostatin) in sections of eight midgestational (12-18 weeks) pancreata and in 0-7% of cells in single-cell suspensions from midgestational pancreata. By electron microscopy, using granule morphology and immunoelectron microscopic techniques, we could confirm these findings and even detect cells containing three hormones. Morphologically different granules contained different immunoreactivities, suggesting parallel regulation of hormone production and packaging. In six newborn pancreata (born after 22-40 weeks of gestation), we could not find any multiple-hormone-containing cells. Subsequently, we evaluated whether multiple-hormone-containing cells proliferate by using pancreatic fragments and single-cell preparations at the light and electron microscopic level (six pancreata). No endocrine hormone-containing cells incorporated bromodeoxyuridine during a 1-hr culture period, indicating that these cells have lost the ability to proliferate under the conditions chosen. We conclude that, as in mice, the human fetal pancreas of 12-18 weeks of gestation contains endocrine cells that express multiple hormones simultaneously. These (multiple) hormone-containing cells do not seem to proliferate under basal conditions.     

Immunocytochemical detection of ricin. II. Further studies using the immunoperoxidase method

Summary Radio-iodinated ricin was injected into rat musclein vivo to establish the distribution of the toxin at various time intervals after injection. Injection site muscle and para-aortic lymph nodes were selected for localization of ricin by the immunoperoxidase technique. Sections of snap-frozen tissues were fixed using a variety of methods to establish the best protocol for the immunodetection method. This was found to be with an ether—ethanol mixture. Ricin was detected in tissue at the site of injection taken from rats sacrificed 1, 4, 8 and 24 h after injection and in tissue from animals dying from ricin intoxication after about 30 h. This method, however, failed to demonstrate unequivocally the presence of ricin in lymphoid tissue which had been indicated by the radiotracer study. The significance of these findings and their relevance to forensic diagnosis are discussed.     

Immunocytochemical studies of hamster oocyte activation

By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non-physiologically (microsurgically) activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two- and four-cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP-dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations.     

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.     

Immunocytochemical studies on the zona pellucida of cow blastocysts

Labeling of the zona pellucida of cow blastocysts with zona-specific anti-serum shows that antigenicity is unaffected by abnormal cleavage, in vitro culture, or frozen storage. The uniform labeling in thin sections indicates that the zona pellucida is homogeneous antigenically. Heavier labeling of the inner and outer surfaces of the zona pellucida in thick sections appers to be due to greater porosity of these regions, in which the zona material becomes highly dispersed, or even partly solubilized, thereby permitting the formation of an antigen-antibody matrix.     

Immunocytochemical localization studies of myelin basic protein

The location of myelin encephalitogenic or basic protein (BP) in peripheral nervous system (PNS) and central nervous system (CNS) was investigated by immunofluorescence and horseradish peroxidase (HRP) immunocytochemistry. BP or cross-reacting material could be clearly localized to myelin by immunofluorescence and light microscope HRP immunocytochemistry. Fine structural studies proved to be much more difficult, especially in the CNS, due to problems in tissue fixation and penetration of reagents. Sequential fixation in aldehyde followed by ethanol or methanol provided the best conditions for ultrastructural indirect immunocytochemical studies. In PNS tissue, anti-BP was localized exclusively to the intraperiod line of myelin. Because of limitations in technique, the localization of BP in CNS myelin could not be unequivocally determined. In both PNS and CNS tissue, no anti-BP binding to nonmyelin cellular or membranous elements was detected.     

Binding studies of polypeptide hormones to bovine neurophysins.

Experimental binding isotherms of [9-glycinamide-1-(14)C]oxytocin and [9-glycinamide-1-(14)C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6 degrees, 22 degrees, and 37 degrees in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4-glycine-1-(14)C]oxytocin to neurophysin II and I in aqueous buffer, and [9-glycinamide-1-(14)C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37 degrees the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.     

Immunocytochemical demonstration of steroid hormones in the granulosa cells of the medaka, Oryzias latipes

Immunocytochemical identification of steroid hormones was performed in granulosa cells isolated from large ovarian follicles of the medaka. The granulosa cells recovered from daily spawning females, 8.5 h after the onset of light, reacted with anti-progesterone and anti-17 alpha-hydroxyprogesterone antibody, while the granulosa cells treated 2.5 h after the onset of light did not. After 6 h stimulation with gonadotropin (PMS) in vitro, the isolated granulosa cells reacted with anti-progesterone antibody. The granulosa cells examined 8.5 h after the onset of light did not react with anti-estradiol or anti-estriol antibody. These results demonstrate that, in Oryzias latipes, progestins (progesterone and 17 alpha-hydroxyprogesterone) are synthesized in the granulosa cells which are directly stimulated by gonadotropin.     

Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle.

In muscle and adipocytes, glucose transport is regulated by the translocation of insulin regulatable glucose transporters (GLUT4) between an intracellular compartment and the cell surface. In these studies we have characterized the cellular compartments containing GLUT4 in rat skeletal muscle. Immunocytochemical studies showed that in unstimulated muscle, GLUT4 was not present in surface membranes. Tubulo-vesicular structures clustered in the trans Golgi reticulum were enriched in GLUT4. GLUT4 underwent translocation to the sarcolemma in response to combined stimulation with insulin and exercise. Using immunoisolation, the intracellular GLUT4 vesicles (IRGTV) were purified 300-fold over the cell homogenate. IRGTV from unstimulated muscle were not enriched in markers specific for the sarcolemma, transverse tubules, sarcoplasmic reticulum or mitochondria; this was confirmed using gel filtration chromatography. Insulin resulted in a 40% decrease in GLUT4 levels in IRGTV confirming that this represents the intracellular compartment of GLUT4. GLUT4 is a major component of the IRGTV, constituting at least 5% of total vesicle protein. A subset of polypeptides are also markedly enriched in the muscle IRGTV. In conclusion, these data suggest that translocation of GLUT4 from intracellular tubulo-vesicular structures is the major mechanism by which insulin and exercise regulate muscle glucose transport.     

Immunocytochemical and in situ hybridization studies of gastrin after cisplatin treatment.

Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in regulating gastric acid production. To study the role of gastrin in this increased gastric acid production after cisplatin treatment, male Wistar rats (100-150 g) were treated with cisplatin (9 mg/kg) in five divided doses over 5 consecutive days. The rats were sacrificed 1, 6, 10, or 15 days after the last treatment. As measured by immunocytochemistry, in situ hybridization, Northern blot, and dot-blot techniques, gastrin was found to be below detectable limits just 1 day after cisplatin treatment. However, 10-15 days after the last injection, the levels for both gastrin and its mRNA gradually recovered to normal. Northern blot studies showed that decreased somatostatin mRNA parallels the changes of gastrin and its mRNA. These results suggest that after cisplatin treatment the increased gastric acid production in rat stomach is independent of gastrin. This decrease of gastrin production is not under the influence of somatostatin, which also decreased after cisplatin treatment.