ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l -phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7-day old), immature (14-day old), and adult (42-day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine-induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline-treated as well as phenylalanine-treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally-administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine-treated and saline-treated infant rats by the intracisternal administration of [³H] or [¹⁴C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline-treated rats were more highly aggregated than those derived from phenylalanine-treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell-free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.     

Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

ONTOGENY OF CHICKEN HEMOGLOBIN II. CHROMATOGRAPHIC STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

Some properties of the carbonmonoxyhemoglobin (HbCO) from chicken embryos of ages 5, 10 and 15 days of incubation, from 1-day posthatching and from adult chickens have been investigated by chromatography on carboxymethylcellulose (CM-cellulose) column and by starch gel electrophoresis.
Chromatogram of the hemoglobin (Hb) from 5-day chicken embryos has shown that it consists of at least 6 components. Starch gel electrophoresis of each isolated component from the column in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown later that there are 3–4 embryonic type Hb components in 5-day embryos.
Chromatogram of the hemoglobin from adult chickens has shown that it consists of at least 4 components, but the examination of each isolated component from the column by electrophoresis in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown that there are 4–6 adult type Hb components in adults.
In ontogenic process, embryonic Hb type is detectable in embryos up to 15 days of incubation. Fetal Hb type, which is not detectable in adult chickens, can be first found in 10-day embryos.     

Myelin proteolipid protein is not esterified at the site of synthesis

Brain slices prepared from 20-day old rats were incubated with [³H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [³H]palmitic acid into PLP of fraction SN₄ (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type ‘precursor-product’ between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [³H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.     

The effect of culture medium composition and incubation time on synthesis of gibberellin-like substances by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.)

It was found that synthesis of gibberellin-like substances by ten strains of Coryneform bacteria isolated from the roots of pine seedlings depended on both the composition of the medium and incubation time. More of these substances were produced in mineral medium with glucose in complex medium with casamino acids and yeast extract. Most gibberellin-like substances were found in 7 or 14-day old cultures. Culture supernatant fluids of most of the bacteria tested contained several gibberellin-like substances which on chromatograms run with the solvent system benzene, acetic acid (10:3, v/v) were located at Rf 0.0-0.3; 0.4-0.6 and 0.8-1.0.     

Characteristics of L-proline and sodium transport in renal brush border membranes isolated from 7-day-old and adult rats

To explore the nature of differences in uptake by renal brush border vesicles from animals of different ages, vesicles were isolated from 7-day old and adult rats by a Mg-aggregation method. A number of criteria were compared in vesicles from the young and mature animals. The vesicles isolated from animals of both ages appear similar on electron microscopy, in response to osmotic changes, and in uptake kinetics for L-glucose. Despite these parameters which indicate no basic differences between the membranes of young and mature kidney, differences in proline and sodium handling are seen. When compared to the uptake pattern seen in vesicles from adult animals, the height of the sodium gradient-stimulated proline overshoot is diminished and sodium entry is faster in vesicles of the 7-day old rats. These are the same differences which were found in vesicles prepared by differential centrifugation from 7-day old animals. In addition, although sodium efflux was faster from vesicles of immature kidney and mirrored the faster sodium entry, proline efflux was slower. The data indicate a dissociation of proline and sodium fluxes in brush borders of the young rat kidney.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Developmental changes of myelin-associated glycoprotein in rat brain: study on experimental hyperphenylalaninemia

We examined developmental changes of myelin-associated glycoprotein (MAG), basic protein (BP), abd proteolipid protein (PLP) in central nervous system myelin isolated from experimental hyperphenylalaninemic rats (PKU rats) and controls. Higher amounts of MAG, including high-molecular-weight MAG in myelin, were found in 12- to 21-day-old control rats than in adult rats. MAG in developing myelin was at a maximum in 18-day-old rats and began to decrease in 21-day-old rats, while PLP and BP in developing myelin increased at these developmental stages. The level of high-molecular-weight MAG decreased in myelin prepared from 21-day-old rats. These results suggest that the decreasing high-molecular-weight MAG is important for compaction of myelin in the early stage of myelination. In myelin from 12- to 18-day-old PKU rats, the ratio of each protein such as MAG, PLP, or BP to that of control was about 0.5 at 12 days, and increased to almost 1.0 at 18 days. The myelination seems to be initially delayed but to be close to that of controls in PKU rats about 18 days old.     

KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE1

Brain slices were prepared from 17-day old rats, and incubated with [³H]glycine or [³H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [³H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.     

MEMBRANE PROTEINS OF RAT BRAIN: COMPOSITIONAL CHANGES DURING POSTNATAL DEVELOPMENT

Abstract— Membrane fractions from forebrain of rat were isolated at ages ranging from 5 to 93 days. Among these fractions were total membranes, three fractions isolated by density gradient centrifugation, and three subfractions which consisted of purified myelin and of two supernatant fractions. All membrane fractions showed an increase in protein content during the first postnatal month; however, only the myelin fraction and one of its supernatant fractions showed a prolonged accumulation. Myelin protein increased continually from 0.17 mg/g brain at 15 days to 8.3 mg/g brain at 93 days.
All fractions were analysed for protein composition by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Characteristic changes in protein composition were noted during postnatal development, most of which were pronounced up to the age of 20 days. Among others was a decrease in histones as compared to other proteins, with a concomitant shift in preponderance from the slow- to the fast-migrating histone band. In parallel, other proteins of high molecular weight became more prominent. No myelin could be isolated at 5 and 10 days. The deposition of myelin proteins was parallelled by the appearance of the Wolfgram protein which points to a close correlation of the Wolfgram protein to the process of myelination.     

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.     

A comparison of sciatic nerve neuropathy in diabetic and aged rats

We compared the development of sciatic nerve neuropathy in young diabetic rats with that in non-diabetic aged rats. Diabetes was induced in six-month old rats by injection with alloxan and was moderately controlled by single daily injections of insulin. Blood insulin levels in diabetic rats were significantly reduced compared to the aged animals, and glucose was significantly higher in diabetic rats. Sciatic nerve conduction velocities were measured monthly. Both motor and sensory conduction velocities decreased in the diabetic rats to a level that was similar to those in 36-month old rats. The decreases in conduction velocities in the diabetic rats were most dramatic during months 6 through 12 of diabetes. After 6 and 12 months of diabetes, sciatic nerves were examined by electron microscopy and compared to nerves from 24- and 36-month old rats respectively. Ultrastructural changes in the sciatic nerves of diabetic rats at 6 months included disruptions of myelin and dense axoplasm. In comparison, the 24-month old rats only had distorted contours of the nerve fibres. After 12 months of diabetes, the axoplasm had large spaces and the myelin was thickened and deformed. The axoplasm of 36-month old rats was normal in appearance; however the myelin sheath was thickened and split into layers. The Schwann cells were vacuolated and irregular in shape. These observations indicate that diabetes results in the early onset of age-like changes in the sciatic nerve. It suggests that the control of hyperglycemia in humans may preserve sciatic nerve structure and function.     

Characterization of myelin of chick sciatic nerve during development.

Myelin was isolated from the sciatic nerves of chicks of ages 18-day embryonic, 1-day, 4-day, 7-day post-hatch, and adult to study developmental changes in lipid composition of this structure. The yield of myelin increased throughout the early stages of development and the preparations were of high purity. Although the lipid content of the myelin did not change, significant changes took place in lipid composition during development. The most significant changes were a relative increase in cerebrosides, phosphatidalethanolamine and long-chain fatty acids of cerebrosides, and a relative decrease in the content of phosphatidylserine and phosphatidylethanolamine. A second fraction ("lower band") was obtained during the isolation procedure. This "lower band" was present at all developmental stages and layered consistently at the interface of 1.2 and 0.8 M sucrose on a discontinuous gradient. The quantity of this fraction did not change during development and it differed from myelin in electron microscopic appearance. Its lipid composition, which did not change, resembled that of 18-day embryonic myelin in its high phospholipid:cholesterol ratio and low galactolipid content. The enzyme, 2':3'-cyclic-nucleosidemonophosphate phosphodiesterase was found to be present in both the myelin and "lower band" fractions; however there was no enrichment of this enzyme in purified myelin.     

Role of Myelin-Associated Neuraminidase in the Ganglioside Metabolism of Rat Brain Myelin

The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.     

Uridine Transport and Metabolism in the Central Nervous System

Myelin and myelin-containing (P3) fractions were prepared from human white matter by discontinuous sucrose gradient centrifugation. The myelin isolated from each of the fractions of different densities was morphologically and biochemically distinct. Light myelin fractions consisted of compact, multilamellar myelin, whereas the denser fractions consisted predominantly of loose myelin with fewer lamellae. The amounts of both basic protein and lipophilin (proteolipid protein) were reduced in the denser fractions. In contrast, the high-molecular-weight components were elevated in the dense fractions. The lipid composition was similar in all the fractions studied. Analysis of basic protein by gel electrophoresis at pH 10.6 revealed differences in basic protein microheterogeneity among the fractions. The light myelin fraction was enriched in the more positively charged basic protein components (components 1, 2, and 3), whereas these components were reduced in the denser fractions. Myelin in the dense fractions was enriched in the more modified forms of basic protein (components 6, 7, and 8). The pattern of microheterogeneity was different for basic protein isolated from myelins of a 2-year-old and an adult brain; the former showed fewer components and mainly the most cationic species. On the other hand, the pattern of microheterogeneity of basic protein isolated from the different density gradient fractions was similar for both ages.     

Disappearance of isocitrate lyase enzyme from cells of Chlorella pyrenoidosa

1. Mitochondria isolated from livers of fed adult, starved adult, and embryonic rats can be separated into three distinct bands by isopycnic density centrifugation on a sucrose density gradient. The least dense band (B1) has a mean buoyant density of 1.162 and consists mainly of disrupted mitochondria. The middle band (B2) has a mean buoyant density of 1.184. The most dense band (B3) has a mean buoyant density of 1.216. B2 and B3 consist of intact mitochondria. 2. The mitochondria in B2 and B3 have very similar protein/phospholipid ratios, virtually identical phospholipid and fatty acid compositions and similar specific activities for cytochrome oxidase, malate dehydrogenase and monoamine oxidase. Both fractions have very low glucose 6-phosphatase and acid phosphatase activities. 3. As isolated, adult rat liver mitochondria have electron-dense matrices (condensed forms); some embryonic liver mitochondria are condensed, but a significant proportion have dilated matrices. All B2 mitochondria are in the condensed form. B3 mitochondria from adult rats are condensed if fixed in their equilibrium-density sucrose, but when this is diluted rapidly to 0.25m they become swollen. Some B3 mitochondria from embryonic rats are condensed, the others have dilated matrices. They all swell if rapidly diluted to 0.25m-sucrose. B2 mitochondria retain their condensed form on dilution of the sucrose. 4. It is concluded that the matrix space of B2 mitochondria is almost totally inaccessible to sucrose, but that of B3 mitochondria is readily accessible to sucrose. 5. In liver from normally fed adult rats the B2 mitochondria predominate, whereas in starved rats B2 and B3 are present in approximately equal proportions. Mitochondrial preparations from embryonic liver consist predominantly of B3 mitochondria, but the proportion of these decreases progressively as development proceeds. 6. The B2 mitochondria from livers of fed adult rats can be converted into B3 mitochondria by incubation with 10mm-succinate and 10mm-phosphate. 7. Some B2 mitochondria are converted into B3 mitochondria by exposure to high concentrations of sucrose.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.     

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

The aim of the present work was to study the effect of muscarinic agonist on cell proliferation and muscarinic acetylcholine receptors (mAChRs) regulation in rat Sertoli cells. Primary cultures of Sertoli cells were obtained from 8-day and 15-day old male Wistar rats. In proliferation assays, [methyl-3H]thymidine incorporation in Sertoli cells from 8-day and 15-day old rats reached a plateau after 60 min of carbachol incubation and decreased after 120 min of agonist incubation. Binding studies with [N-Methyl-3H]scopolamine ([3H]NMS) indicated a rapid loss of cell surface mAChRs when Sertoli cells from 15-day old rats were incubated with carbachol at 35 degrees C for 2 min. This effect was temperature-dependent. When the incubation of the cells was prolonged at 35 degrees C or at 4 degrees C, after the agonist had been washed away, 94% of mAChRs were present in the cell surface after 120 min incubation at 35 degrees C. At 4 degrees C, however, a low percentage of mAChRs was detected in the cell surface. In the presence of cycloheximide, the recycling of mAChRs to the cell surface was not changed, suggesting that the appearance of mAChRs on cell surface was not dependent on de novo receptor synthesis. In conclusion, our studies indicate that the activation of mAChRs may play a role in rat Sertoli cell proliferation. These receptors may be under regulation (internalization and recycling) when cells are exposed to muscarinic cholinergic agonist.