Fine structure of the atrial cardiocytes in the house musk shrew (Suncus murinus).

The atrial and ventricular cardiocytes of the house musk shrew were examined by immunohistochemistry, and the right atrium containing the auricle was studied by transmission electron microscopy. The atrial natriuretic peptide (ANP)-granules of the cardiocytes in the auricle and the rest of the atrium were also analyzed by ultrastructural morphometry. On immunohistochemistry, ANP immunoreactivity was detected in the atria, with the most intensely reacting cardiocytes being localized in the right auricular part of the atrium. ANP immunoreactivity was not detected in the ventricular muscles. On ultrastructure, in most of the atrial cardiocytes including the auricle, ANP-granules, well-developed Golgi apparatus and rough endoplasmic reticulum were observed, and the nuclei were characteristically situated in the periphery of the cardiocytes, being different from many other mammalian hearts. The ANP-granules were classified into two types (A and B), with most of these granules being located in the paranuclear region in association with the Golgi apparatus, and a few ANP granules being observed throughout the sarcoplasmic layers intervening between the myofibrilar bundles. On ultrastructural morphometry, the total number of granules in the right auricular cardiocytes was significantly greater than those in the atrial cardiocytes, and the diameter of the A-granules was significantly greater than that of the B-granules in both the atrial and auricular cardiocytes.     

Fine structure of atrial natriuretic peptide (ANP)-granules in the atrial cardiocytes of the mouse, rat and Mongolian gerbil.

Mouse, rat and Mongolian gerbil atrial and ventricular cardiocytes were examined by immunohistochemistry, and the right atrium including the auricle was examined by transmission electron microscopy. In addition the ANP granules of both right atrial and auricular cardiocytes were analyzed by ultrastructural morphometry. ANP immunoreactivity was detected in the atria of all three species, and the most intensely reacting cardiocytes were localized in the right auricular part of the atrium. These reactions were more prominent in the mouse and rat than in the Mongolian gerbil. ANP immunoreactivity was not detected in the ventricular myocardium of any of the three species, but was occasionally seen in the subendocardium of the ventricular septum. Ultrastructurally, the ANP granules in the auricular and atrial cardiocytes were observed to be variable in size and number, and these granules were located principally in the paranuclear region in association with the Golgi apparatus, and found throughout the sarcoplasmic layers in all three species. The ANP granules were classified into two types: A-granules containing a conspicuous electron-dense core possessing a membrane, and B-granules having profiles with a fibrillogranular, less electron-dense core than the A-granules and an indistinct membrane. The features of these granules were similar in all three species. When examined by ultrastructural morphometry, the number of each type granule and the total number of granules in the right auricular and atrial cardiocytes of the mouse and rat were significantly greater than in the Mongolian gerbil. The total number of granules in the right auricular cardiocytes was significantly greater than in the cardiocytes of the right atrium exclusive of the auricle, however, there was no significant difference between the number of A-granules and B-granules in the three species. The diameter of each type of granule in the right auricular and atrial cardiocytes of the mouse and rat was significantly greater than in the Mongolian gerbil, and the diameter of the A-granules was significantly greater than the diameter of the B-granules in all three species.     

Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands

Antibodies produced in the mouse by repeated intraperitoneal injections of partly purified atrial natriuretic factor (low molecular weight peptide (LMWP) and high molecular weight peptide (HMWP)) have been used to localize these factors by immunohistochemistry (immunofluorescence and immunoperoxidase method) and by immunocytochemistry (protein A-gold technique) in the heart of rats and of a variety of animal species including man and in the rat salivary glands. Immunofluorescence and the immunoperoxidase method gave identical results; in the rat, atrial cardiocytes gave a positive reaction at both nuclear poles while ventricular cardiocytes were consistently negative. The cardiocytes of the right atrial appendage were more intensely reactive than those localized in the left appendage. A decreasing gradient of intensity was observed from the subpericardial to the subendocardial cardiocytes. The cardiocytes of the interatrial septum were only lightly granulated. Sodium deficiency and thirst (deprivation of drinking water for 5 days) produced, as already shown at the ultrastructural level, a marked increase in the reactivity of all cardiocytes from both atria with the same gradient of intensity as in control animals. Cross-reactivity of intragranular peptides with the rat antibodies allowed visualization of specific granules in a variety of animal species (mouse, guinea pig, rabbit, rat, dog) and in human atrial appendages. No reaction could be elicited in the frog atrium and ventricle although, in this species, specific granules have been shown to be present by electron microscopy in all cardiac chambers. With the protein A-gold technique, at the ultrastructural level, single labeling (use of one antibody on one face of a fine section) or double labeling (use of two antibodies on the two faces of a fine section) showed that the two peptides are localized simultaneously in all three types (A, B and D) of specific granules. In the rat salivary glands, immunofluorescence and the immunoperoxidase method showed reactivity exclusively in the acinar cells. The reaction was most intense in the acinar cells of the parotid gland. In the sublingual gland, only the serous cells, sometimes forming abortive "demi-lunes", were reactive. In the submaxillary gland, the reaction was weaker and distributed seemingly haphazardly in the gland. The most constantly reactive cells were localized near the capsule while many cells did not contain visible reaction product.     

Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands

Summary Antibodies produced in the mouse by repeated intraperitoneal injections of partly purified atrial natriuretic factor (low molecular weight peptide (LMWP) and high molecular weight peptide (HMWP)) have been used to localize these factors by immunohistochemistry (immunofluorescence and immunoperoxidase method) and by immunocytochemistry (protein A-gold technique) in the heart of rats and of a variety of animal species including man and in the rat salivary glands. Immunofluorescence and the immunoperoxidase method gave identical results: in the rat, atrial cardiocytes gave a positive reaction at both nuclear poles while ventricular cardiocytes were consistently negative. The cardiocytes of the right atrial appendage were more intensely reactive than those localized in the left appendage. A decreasing gradient of intensity was observed from the subpericardial to the subendocardial cardiocytes. The cardiocytes of the interatrial septum were only lightly granulated. Sodium deficiency and thirst (deprivation of drinking water for 5 days) produced, as already shown at the ultrastructural level, a marked increase in the reactivity of all cardiocytes from both atria with the same gradient of intensity as in control animals. Cross-reactivity of intragranular peptides with the rat antibodies allowed visualization of specific granules in a variety of animal species (mouse, guinea pig, rabbit, rat, dog) and in human atrial appendages. No reaction could be elicited in the frog atrium and ventricle although, in this species, specific granules have been shown to be present by electron microscopy in all cardiac chambers. With the protein A-gold technique, at the ultrastructural level, single labeling (use of one antibody on one face of a fine section) or double labeling (use of two antibodies on the two faces of a fine section) showed that the two peptides are localized simultaneously in all three types (A, B and D) of specific granules. In the rat salivary glands, immunofluorescence and the immunoperoxidase method showed reactivity exclusively in the acinar cells. The reaction was most intense in the acinar cells of the parotid gland. In the sublingual gland, only the serous cells, sometimes forming abortive demi-lunes, were reactive. In the submaxillary gland, the reaction was weaker and distributed seemingly haphazardly in the gland. The most constantly reactive cells were localized near the capsule while many cells did not contain visible reaction product.     

Atrial natriuretic peptide (ANP)-granules of auricular cardiocytes in aging Mongolian gerbils.

The atrial natriuretic peptide (ANP)-granules of right auricular cardiocytes were studied in aging Mongolian gerbils by immunohistochemistry and transmission electron microscopy, and analyzed by ultrastructural morphometry. Immunohistochemically, the ANP immunoreactions were weaker in 3 and 4-year-old animals than in 90-day-old, 1- and 2-year-old animals. There was no difference in the reaction among 90-day-old, 1 and 2-year-old animals, or between 3 and 4-year-old animals. Ultrastructurally, the morphological features in 1 and 2-year-old animals were similar to those of 90-day-old animals, but distinct in 3 and 4-year old animals by the presence of a few lysosomal structures in the cell. By ultrastructural morphometry, number and diameter of ANP-granules of 3 and 4-year-old animals were significantly smaller than those in the 90-day-old, 1 and 2-year-old. There was no significant difference in number and diameter among the 90-day-old, 1 and 2-year-old, or between the 3 and 4-year-old.     

Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles

Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.     

Speciesabhängigkeit der Monoaminoxydase-Aktivität in verschiedenem Lebensalter

Summary The monoamine oxidase activity in ten species (man, dog, cat, rabbit, guinea pig, rat, hamster, mouse, chicken, goose) was histochemically studied in the myocardium, liver, kidney and psoas muscle in newborn and older individuals.An age-dependent increase of monoamine oxidase activity is established in the liver and kidney of man, dog, cat, guinea pig and hamster. In the psoas muscle of the rat the monoamine oxidase activity is consistently weak. In the myocardium only the rat shows an increase with age.     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

The activity of hepatic gamma-glutamyltranspeptidase in various animal species

The activity of gamma-glutamyltranspeptidase was determined in liver homogenates derived from various animals belonging to different phylogenetic groups. Although gamma-glutamyltranspeptidase was present in all livers studied, the activity varied greatly, with the order of activity being: guinea pig much greater than frog much greater than rabbit greater than toad greater than dog congruent to cat = duck greater than hamster = budgerigar = goldfish congruent to rat = mouse. There was no trend with respect to enzyme activity, phylogeny, diet, or habitat.     

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.     

Comparative studies on the gastric glycopeptide in eleven animal species.

1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.     

Localization of neurotensin-immunoreactive cells in the small intestine of man and various mammals.

Antibodies against synthetic bovine neurotensin were raised in rabbits and used to demonstrate neurotensin-immunreactive cells by immunohistochemical methods. In the jejunum and ileum of all species investigated (man, dog, monkey, cat, rabbit, sheep, rat, mouse, hamster, chinese hamster, gerbil, pig and guinea pig) cells were present in the mucosa, which reacted specifically with antineurotensin serum using the indirect immunofluorescence and peroxidase-antiperoxidase methods. In the monkey Tupaia the distribution of neurotensin-immunoreactive cells was examined by investigating serial sections through the entire gastro-entero-pancreatic (GEP) endocrine system, again showing most neurotensin-immunoreactive cells in the jejunum and ileum. The functional role of the presence of neurotensin immunoreactivity in the gut is discussed.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

The relation between structure and function of bile ducts in man, some laboratory animals and the Adelie penguin.

The biliary trees of man, dog, cat, rabbit, rat, guinea pig and penguin were examined in histological sections and by latex casts. The trees of man, dog, and cat were similar with only minor differences. Tubulo-alveolar glands were present in all three species around large intrahepatic ducts and in large portal tracts there were zones of ductules (areas with many small bile ducts), surrounded by small vessels with no apparent relation to hepatocytes. Both these features were present in the guinea pig and tubulo-alveolar glands were present in the penguin liver. The biliary epithelium of the rat was comparatively simple but that of the rabbit appeared to be highly specialized. An estimation of the complexity of the biliary tree was obtained in the mammals by comparing the circumference of small portal venous branches with the circumference of the accompanying bile ducts, and obtaining a ratio. Man, dog, and cat had fewer and smaller bile ducts than the other species. The literature on the rate of formation and composition of bile in the species studied here was reviewed and it appears that the physiology of bile secretion can be related to the morphology of the biliary tree.     

Localization of neurotensin-immunoreactive cells in the small intestine of man and various mammals

Summary Antibodies against synthetic bovine neurotensin were raised in rabbits and used to demonstrate neurotensin-immunreactive cells by immunohistochemical methods. In the jejunum and ileum of all species investigated (man, dog, monkey, cat, rabbit, sheep, rat, mouse, hamster, chinese hamster, gerbil, pig and guinea pig) cells were present in the mucosa, which reacted specifically with antineurotensin serum using the indirect immunofluorescence and peroxidase-antiperoxidase methods. In the monkey Tupaia the distribution of neurotensin-immunoreactive cells was examined by investigating serial sections through the entire gastro-entero-pancreatic (GEP) endocrine system, again showing most neurotensin-immunoreactive cells in the jejunum and ileum. The functional role of the presence of neurotensin immunoreactivity in the gut is discussed.Supported by the German Research Foundation     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.     

DIFFERENTIATION OF MONOCYTES : Origin, Nature, and Fate of Their Azurophil Granules

The origin, content, and fate of azurophil granules of blood monocytes were investigated in several species (rabbit, guinea pig, human) by electron microscopy and cytochemistry. The life cycle of monocytes consists of maturation in bone marrow, transit in blood, and migration into tissues where they function as macrophages. Cells were examined from all three phases. It was found that: azurophil granules originate in the Golgi complex of the developing monocyte of bone marrow and blood, and ultimately fuse with phagosomes during phagocytosis upon arrival of monocytes in the tissues. They contain lysosomal enzymes in all species studied and peroxidase in the guinea pig and human. These enzymes are produced by the same pathway as other secretory products (i.e., they are segregated in the rough ER and packaged into granules in the Golgi complex). The findings demonstrate that the azurophil granules of monocytes are primary lysosomes or storage granules comparable to the azurophils of polymorphonuclear leukocytes and the specific granules of eosinophils. Macrophages from peritoneal exudates (72–96 hr after endotoxin injection) contain large quantities of lysosomal enzymes throughout the secretory apparatus (rough ER and Golgi complex), in digestive vacuoles, and in numerous coated vesicles; however, they lack forming or mature azurophil granules. Hence it appears that the monocyte produces two types of primary lysosomes during different phases of its life cycle—azurophil granules made by developing monocytes in bone marrow or blood, and coated vesicles made by macrophages in tissues and body cavities.     

The interglobular spaces in the endochondral layer of the osseous labyrinth. Comparative anatomical study

The endochondral layer of the osseous labyrinth in the rat, golden hamster, mouse, guinea pig, pig, rabbit, cat, dog and monkey was studied and compared with that of man. (1) With the exception of the mouse and golden hamster, interglobular spaces were found. (2) In all species but the rat, the interglobular spaces contain acid mucopolysaccharides. An analogy between these structures and the 'basophilic islands' (basophile Inseln) is discussed. (3) Extension, arrangement, direction, occurrence and frequency of interglobular spaces vary within each species so that no constant relations could be found, which are also lacking in man. Possible reasons for the persistence throughout life of interglobular spaces in the osseous labyrinth of man and some mammals are discussed.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I, IIA and IIB fibres of mammalian diaphragms

Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p greater than 0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p less than 0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

I. Ultrastructure of acinar cell granules in mammalian submaxillary glands

Summary Granules in acinar cells show considerable variations in size, shape, electron-density and molecular organisation of secretory material. Discrete organelles with an electron-dense homogenous matrix are seen in the guinea-pig and to some extent in the male hamster. Similar organelles with moderate electron-density are seen in the cat and dog. Acinar cells of the cat, hamster, guinea-pig and to a lesser extent the rat, contain discrete, pale granules and also confluent organelles arising from two to three of the pale particles. Composite, electron-pale secretory units are observed in the dog and rabbit.No correlation could be elicited between the histochemical reactivity of the acinar cells and the content of the secretory enzymes. The relationship between the histochemical reactivity and ultrastructural appearance of acinar cell granules and the organelles containing kallikrein, trypsin-like proteases and amylase seems much more important and functionally relevant. Morphological aspects of the intracellular transport and secretion of granules are discussed.We wish to thank The Wellcome Trust for a travel grant to attend the International Symposium on Vasopeptides (Florence, July, 1971) where this work was briefly reported.