Immunohistochemical localization of insulin-like growth factor I in the adult rat

Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradecapeptide, representing the carboxyterminal amino acids 57-70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I. High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoietic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoietic, thrombocytopoietic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine. The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Y;autosome translocations and mosaicism in the aetiology of 45,X maleness: assignment of fertility factor to distal Yq11

Summary Three 45,X males have been studied with Y-DNA probes by Southern blotting and in situ hybridization. Southern blotting studies with a panel of mapped Y-DNA probes showed that in all three individuals contiguous portions of the Y chromosome including all of the short arm, the centromere, and part of the euchromatic portion of the long arm were present. The breakpoint was different in each case. The individual with the largest portion (intervals 1–6) is a fertile male belonging to a family in which the translocation is inherited in four generations. The second adult patient, who has intervals 1–5, is an azoospermic, sterile male. These phenotypic findings suggest the existence of a gene involved in spermatogenesis in interval 6 in distal Yq11. The third case, a boy with penoscrotal hypospadias, has intervals 1–4B. In situ hybridization with the pseudoautosomal probe pDP230 and the Y chromosome specific probe pDP105 showed that Y-derived DNA was translocated onto the short arm of a chromosome 15, 14, and 14, respectively. One of the patients was a mosaic for the 14p+ translocation chromosome. Our data and those reported by others suggest the following conclusions based on molecular studies in eight 45,X males: The predominant aetiological factor is Y;autosome translocation observed in seven of the eight cases. As the remaining case was a low-grade mosaic involving a normal Y chromosome, the maleness in all cases was due to the effect of the testis determing factor, TDF. There is preferential involvement of the short arm of an acrocentric chromosome (five out of seven translocations) but other autosomal regions can also be involved. The reason why one of the derivative translocation chromosomes becomes lost may be that it has no centromere.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

Inhibitory effect of 11-ketoandrostenedione and androstenedione on spermatogenesis in the three-spined stickleback, Gasterosteus aculeatus L.

Stickleback males were implanted with Silastic capsules filled with 11-ketoandrostenedione or androstenedione at the end of the breeding season. 11-Ketoandrostenedione prevented the natural decline in secondary sexual characters and testes weight. It also completely inhibited the commencement of spermatogenesis, which normally takes place in late summer after having been quiescent during the breeding season. Androstenedione also exerted these effects, but to a lesser degree.     

Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion

Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg^-1·h^-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.     

The gene for dominant white color in the pig is closely linked to ALB and PDGRFRA on chromosome 8.

White is a widespread coat color among domestic pig breeds and is controlled by an autosomal dominant gene I. The segregation of this gene was analyzed in a reference pedigree for gene mapping developed by crossing the European wild pig and a Large White domestic breed. The gene for dominant white color was shown to be closely linked to the genes for albumin (ALB) and platelet-derived growth factor receptor alpha (PDGFRA) on chromosome 8. An unexpected phenotype with patches of colored and white coat was observed among the F1 and F2 animals. The segregation data indicated that the phenotype was controlled by a third allele, denoted patch (Ip), most likely transmitted by one of the Large White founder animals. It is shown that the ALB, PDGFRA, I linkage group shares homologies with parts of mouse chromosome 5, human chromosome 4, and horse linkage group II, all of which contain dominant genes for white or white spotting. Candidate genes for the dominant white and patch mutations in the pig are proposed on the basis on these linkage homologies and the recent molecular definition of the dominant white spotting (W) and patch (Ph) mutations in the mouse.     

A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings

A simple method for the preparation of paraffin-embedded cell blocks from cytologic specimens obtained by fine needle aspiration, by brushing or from effusions is described. The cells are fixed in suspension in 50% ethanol for one hour and pelleted by centrifugation in a 50-mL plastic tube. The fixative is removed, and the pellet is suspended in 3 mL of acetone for dehydration for ten minutes and thereafter repelleted. The acetone is then removed, and the cell pellet is dried at 60 degrees C for one hour. Melted paraffin is added onto the dry warmed cell mass and allowed to solidify at room temperature. A conical paraffin block with the cells in the top is obtained and can be handled as a routine tissue block.     

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.