Analysis of the progression of meiosis in dispersed rat testicular cells by flow cytofluorometry.

Initiation and progression of meiosis was followed in dispersed rat testicular cells by flow cytofluorometry and cytology. The DNA content of dissociated testicular cells of rats 6--30 days old, killed at daily intervals, was analysed by flow cytofluorometry using propidium iodide as a DNA-specific and quantitative fluorochrome. Testicular cells of a 6-day-old rat showed one peak of fluorescence. A second peak, at twice the modal channel number, appeared in testicular cells of 9-day-old animals. The number of cells under this peak increased progressively with age. A third peak, at half the channel number of the original one, appeared at 20 days and accounted for an increasing proportion of cells in testes taken from older rats. Cytological examination of the testicular tissue used for flow cytofluorometric analysis showed that preleptotene spermatocytes first appeared at 8 days after birth. Spermatids were first observed cytologically at 20 days after birth. The close temporal appearance of the fluorescence peaks with that of spermatocytes and spermatids, and the close association of the frequency of diploid and tetraploid cells as derived by flow cytofluorometry and cytology, indicated that the fluorescence peaks correspond--in order of increasing fluorescence--to spermatids, spermatogonia and somatic cells, and to spermatocytes. This conclusion was re-examined by analysing the ploidy levels of testicular cells of hypophysectomized or estradiol-treated by flow cytofluorodmetry. There was a loss of the haploid and tetraploid peaks subsequent to hypophysectomy. Estradiol dipropionate-treated rats, given weekly injections starting at 7 days of age, showed no appearance of the haploid peak and the regression of the tetraploid peak after an initial and transitory appearance. These results indicate that changes in ploidy levels that accompany the progression of meiosis in the testis were reflected in the sequential appearance of three fluorescence peaks as detected by flow cytofluorometry. The close correlation between the frequency of cell types as obtained by cytology and flow cytofluorometry indicates that the latter is a sensitive method for studying selected aspects of spermatogenesis in dissociated testicular cells.     

Catabolism of thyroliberin by rat adenohypophyseal tissue extract

Rapid fragmentation of thyroliberin (less than Glu-His-Pro-NH2) by rat adenohypophyseal tissue enzymes could be demonstrated. Based on the identification of the metabolic products and by the demonstration that the individual enzymatic reactions can be preferentially blocked by enzyme inhibitors, specific and sensitive biochemical tests could be developed in order to monitor the enzymatic activities after gel chromatographic fractionation of the tissue extracts. These findings are in agreement with the interpretation that the observed degradation of thyroliberin by hypophyseal tissue extracts may follow the proposed pathways. The primary enzymatic cleavage of thyroliberin is either initiated by the action of a 'thyroliberin-deamidating enzyme' (thyroliberin leads to less than Glu-His-Pro-OH + NH3), or by the action of a pyroglutamate aminopeptidase (thyroliberin leads to less than Glu + His-Pro-NH2). While the pyroglutamate aminopeptidase also catalyzes the subsequent degradation of deamidated thyroliberin (less than Glu-His-Pro-OH leads to less than Glu + His-Pro-OH), the enzymatic deamidation of His-Pro-NH2 is not catalyzed by the 'thyroliberin-deamidating enzyme; but by a post-proline dipeptidyl aminopeptidase. Hydrolysis of the common intermediary metabolite His-Pro-OH to the free amino acids is apparently catalyzed by a proline dipeptidase. In addition to these enzymatic events rapid cyclization of His-Pro-NH2 to histidyl-proline-diketopiperazine His-Pro could be observed. This reaction however is mainly due to the non-enzymatic intramolecular condensation reaction which is characteristic for proline-containing dipeptide derivatives. An enzymatic activity which catalyzes this reaction could not be observed when the enzyme fractions were tested. Enzymatic degradation of His-Pro by hypophyseal tissue extracts could also not be observed.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

Ultrastructural study of the Ca2+-dependent ATPase activity in rat adenohypophyseal cells

Ca-dependent ATPase activity in the rat anterior pituitary was demonstrated in 50-microns tissue slices of aldehyde-fixed tissue with the medium of Takano et al. (Cell Tissue Res. 243:91. 1986).--The outer surface of the plasma membrane of the parenchymal as well as the folliculo-stellate cells was lined with lead precipitate. The reaction deposit was particularly well localized in intercellular spaces both between two parenchymal cells, and between a parenchymal and a folliculo-stellate cell. A fine reaction deposit was also seen in the endoplasmic reticulum and Golgi apparatus of some parenchymal cells. Elimination of Ca2+ from the tissue and the substrate medium drastically reduced the amount of reaction product. If ATP was omitted or replaced by sodium beta-glycerophosphate, no reaction product was seen. Changing the Ca2+ concentration or addition of Mg2+ to the standard medium caused a decrease in reaction intensity. Substitution of Mg2+ for Ca2+ resulted, again in well-localized lead deposition which we attribute to the activity of another enzyme. We suggest that the activity we described in the membrane of glandular cells may correspond to the enzyme involved in the long-term regulation of intracellular Ca2+ level.     

Ultrastructural study of the Ca2+-dependent ATPase activity in rat adenohypophyseal cells

Summary Ca-dependent ATPase activity in the rat anterior pituitary was demonstrated in 50-m tissue slices of aldehyde-fixed tissue with the medium of Takano et al. (Cell Tissue Res. 243:91. 1986). — The outer surface of the plasma membrane of the parenchymal as well as the folliculo-stellate cells was lined with lead precipitate. The reaction deposit was particularly well localized in intercellular spaces both between two parenchymal cells, and between a parenchymal and a folliculo-stellate cell. A fine reaction deposit was also seen in the endoplasmic reticulum and Golgi apparatus of some parenchymal cells. Elimination of Ca²⁺ from the tissue and the substrate medium drastically reduced the amount of reaction product. If ATP was omitted or replaced by sodium -glycerophosphate, no reaction product was seen. Changing the Ca²⁺ concentration or addition of Mg²⁺ to the standard medium caused a decrease in reaction intensity. Substitution of Mg²⁺ for Ca²⁺ resulted again in well-localized lead deposition which we attribute to the activity of another enzyme. We suggest that the activity we described in the membrane of glandular cells may correspond to the enzyme involved in the long-term regulation of intracellular Ca²⁺ level.     

Uptake and turnover of dopamine in rat mast cells studied by cytofluorometry and high performance liquid chromatography

Summary Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injectedl-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.     

The role of lysosomal enzymes in protein degradation in different types of rat liver cells

Highly purified suspensions of parenchymal, endothelial and Kupffer cells were prepared from the rat liver. The respective roles of these cell classes in the degradation of proteins was investigated by analysing the cellular distribution of two lysomal proteases. The specific arginine naphthylamidase activity was 2 times higher in Kupffer cells compared with the nearly equal activities in endothelial and parenchymal cells. The specific activity of the important endopeptidase cathepsin D in endothelial and Kupffer cells was about 12 and 36 times higher, respectively, than the activity in parenchymal cells. These results are in agreement with an important role of Kupffer and endothelial cells in the degradation of proteins and protein containing material of exogenous origin.     

Characterization of the major sialidases of various types of rat blood cells: their comparison with rat liver sialidases

The substrate specificity and subcellular location of the major sialidases of three types of rat blood cells were characterized and compared with those of the known three types of rat liver sialidase, which have been designated intralysosomal, cytosolic, and plasma membrane-associated sialidases. Platelets and leucocytes contain mainly an acid sialidase, which is highly active towards oligosaccharides and 4MU-NeuAc, and erythrocytes possess a high level of a sialidase acting on gangliosides. A Percoll gradient centrifugation study showed that the former is located in lysosomes and the latter in plasma membrane. When the sialidase was solubilized and partially purified from erythrocyte ghosts, the enzyme was found to hydrolyze actively gangliosides but only poorly other substrates such as 4MU-NeuAc, oligosaccharides, and glycoproteins. The sialidase partially purified from rat liver membrane fraction exhibited the same substrate specificity. It is concluded that the major sialidase of platelets and leucocytes corresponds to hepatic intralysosomal sialidase while erythrocytes contain almost exclusively a ganglioside sialidase which corresponds to hepatic plasma membrane sialidase.     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Kinematics of different types of locomotor movements in the deafferented rat

The kinematics of rat hindlimb movements were assessed and compared pre- and post-deafferentation during swimming, forelimb treadmill locomotion plus hindlimb swimming motion, and walking using all four limbs. All types of locomotion were characterized by an increase in the frequency of locomotor rhythm and reduced amplitude of motion at the hindlimb joints following deafferentation. The reduced change observed in the angle of the coxofemoral joint, indicative of a horizontal component in locomotor motion, was mainly brought about by less marked extension. This would confirm evidence indicating that increased load on the extremities, with its ensuing naturally-occurring afferent outflow, is accompanied by a reduced locomotor motion rate and a rise in the amplitude of the latter due to intensified extension of the limb. The increased forward carriage of the hind limb seen during the transition to four-legged locomotion persisted after deafferentation; this may be considered a sign of coordination amongst the limbs. Deafferentation led to a reduction in the MEG of muscle activity, which was found to be lowest in swimming and highest during walking. The role of the afferent inflow in shaping different types of locomotor motion is evaluated.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 520–525, July–August, 1987.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (beta-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X(7) receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Two different molecular types of rat mitochondrial DNAs

Experiments with restriction endonucleases showed that Sprague-Dawley strain rats have two types of mitochondrial DNAs (mtDNAs), named α- and β-types, which differ in primary structure. Individual rats have only one type. Individuals of other strains, such as Wistar, Fischer, and Donryu, also all have one of these types of mtDNA. Thus irrespective of their strain, all rats can be classified into one of two groups according to the type of their mtDNA.     

Two tinctorial types of gonadotrophic cells in the rat hypophysis

Summary A modified PAS-methyl blue staining method is described which, in conjunction with aldehyde fuchsin, enables the tinctorial differentiation of two types of gonadotrophs in the pituitary gland of the rat. That these two cell types are both gonadotrophic in function is indicated by their development into distinct PAS-red and PAS-purple castration cells following gonadectomy. In addition to these two kinds of castration cells, thyroidectomy cells which are PAS-negative, aldehyde fuchsin-negative, and have an affinity for orange G are seen in the pituitary glands of animals that have been both gonadectomized and thyroidectomized. Thus, in such glands the three types of modified basophiles are tinctorially different. Differences in the distribution of the PAS-red and PAS-purple gonadotrophs are characteristic of the glands of normal animals. Following gonadectomy, however, extensive shifts in the distribution of these cells occur so that the normal pattern is soon lost.This study was supported by research grants B-410 and RG-4723 from the U. S. Public Health Service.     

Collodion membrane secures cells sorted by flow cytofluorometry onto microscope slides

Quantitative microscopic cytology of cells previously sorted by flow cytofluorometry has been hindered by the loss of cells from the microscope slide during staining procedures. The simple application of a semi-permeable membrane of collodion over fixed or unfixed cells sorted directly onto a microscope slide secured virtually 100% of the cells onto the slide. Cells covered with the collodion membrane studied with Papanicolaou's stain as well as routine clinical cervical cytologic preparations. In contrast, fewer than one half of the cells sorted onto uncoated or albumin coated slides were retained after staining.