Vesicular transfer of membrane components to bovine epididymal spermatozoa.

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.

Arrangement of PMCA4 in bovine sperm membrane fractions.

The plasma membrane Ca(2+) -ATPase (PMCA) is the main restorer of Ca(2+) balance in sperm. Particularly, PMCA isoform 4 has an essential function in sperm fertility by its participation in gaining sperm hypermotility. PMCA activity is influenced by its lipid environment. Sperm membranes exhibit lipid raft microdomains or detergent-resistant membrane domains, enriched in sphingolipids and cholesterol, forming functional specialized areas. Lipid and protein composition of lipid rafts alters during the capacitation process, which is characterized by a cholesterol efflux. In this study, the localization of PMCA4 in lipid membrane fractions of the sperm plasma membrane was investigated. We identified PMCA4 in both the detergent-resistant membrane (DRM) and in the detergent-soluble (DS) fraction of caput and cauda sperm, respectively. Capacitation did not influence PMCA4 localization. In immunocytochemical studies PMCA4 was co-localized with the lipid raft/DRM marker caveolin in the mid piece of caput and cauda sperm. Functional studies with seminal vesicle major protein PDC-109 showed that the Ca(2+) -ATPase activity in DS fractions of cauda sperm and capacitated cauda sperm was stronger enhanced than in the DRMs. In both fractions the effect was statistically significant. In contrast, in lipid overlay experiments PDC-109 interacted stronger with the lipids extracted from DRMs than with lipids extracted from DS. Our results indicate a possible functional compartmentalization of PMCA in bull sperm membranes and point to a presumptive, yet unknown interaction partner of Ca(2+) -ATPase and PDC-109, mediating the PDC-109 action from DRMs to the DS fraction of sperm plasma membrane.

The heart: a target organ for estradiol.

Autoradiographic studies of rat heart reveal that tritiated estradiol concentrates in cell nuclei of the myocardium of the atria and auricles, similar to the myometrium of the uterus. This suggests that estrogen has a direct effect on atrial myocytes through which its "protective" action may be mediated. Cardiac glycosides that are known to exert estrogen-like effects on classical estrogen target tissues, such as uterine muscle, endometrium, vagina, and mammary gland, probably act on atrial muscle through a genomic, steroid hormone-like mechanism of action.

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells.

BACKGROUND AND PURPOSE: Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands. EXPERIMENTAL APPROACH: Tissue specimens of the right submandibular gland taken from 18 Wistar rats after intraglandular injection of BoNT A, BoNT B, or a combination of both were examined. As a sham control, an equivalent volume of saline was injected into the left submandibular gland. Morphometric measurements, immunohistochemistry, electron microscopy and western blot analysis were used to analyse the morphological and functional changes of the denervated glands. KEY RESULTS: Morphological and ultrastructural analyses of the cell organelles and secretory granula showed a clear atrophy of the acini, which was more prominent in glands injected with the combination of BoNT/A and B. Morphometric measurements of the glandular acini revealed a significant reduction of the area of the acinar cells after injection of BoNT (P=0.031). The expression of amylase was significantly reduced in BoNT treated glands. CONCLUSIONS AND IMPLICATIONS: Intraglandular application of BoNT induces structural and functional changes of the salivary glands indicated by glandular atrophy. These effects may be due to glandular denervation induced by the inhibition of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) involved in acetylcholine release at the neuroglandular junction and also specially inhibition of those involved in exocytosis of the granula of the acinar cells.

Calcyclin is differentially expressed in rat testicular cells.

Immature rat Sertoli cells aggregate and form tubule-like structures when cultured on a monolayer of peritubular myoid cells. In this study, differential gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells were examined. One of the cDNA clones isolated showed high homology to calcyclin and a microvascular differentiation gene, CEC5, which was reported to be highly homologous to CASK, a membrane-associated guanylate kinase homolog. Sequencing and mRNA analysis of rat calcyclin demonstrated that the gene was differentially expressed and was found only in peritubular cells and cocultures with increased levels. In contrast, CASK was expressed by Sertoli cells, peritubular cells, and cocultures, whereas CEC5 was never found in the testicular somatic cells. Our findings point to a paracrine regulation of calcyclin expression in testicular peritubular fibroblasts which seems to be related to tubular growth.

Solubilization of nuclear steroid 5 alpha-reductase from rat ventral prostate.

delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.

Purification and molecular characterization of a secretory transglutaminase from coagulating gland of the rat.

A transglutaminase (TGase, EC 2.3.2.13) was isolated from the secretion of rat coagulating gland (CGS-TGase). The protein consists of a single polypeptide chain and has a molecular mass of 65 kDa. During purification the net charge changes from pI 7.6 in the crude extract to pI 8.5-8.7 for the purified protein. Nearly equal numbers of glutamyl- and lysyl-residues were detected by amino acid analysis. The protein therefore represents an appropriate substrate of autocatalytic crosslinking. The total number of cysteine residues is 18-19, six of which being present in free form. One of the thiol groups is essential for the enzymic activity. The protein core is glycosylated with mannosyl residues and in addition substituted with saturated acyl residues and phosphoinositol. The phosphoinositol anchor was demonstrated by use of a specific antibody. Removal of the acyl- and glycosyl-residues or of the total anchor group results in autoaggregation and decrease of enzymic activity. In contrast to tissue-type TGases, Ca2+ dependent enzymic activity of CGS-TGase is not inhibited by GTP. The secretory TGase shows no immunological cross-reactivity to tissue-type enzyme or blood factor XIII.

Intracellular distribution of adenine and 5-hydroxytryptamine in megakaryocytes isolated by density gradient and velocity sedimentation from bone marrow.

Megakaryocytes were isolated from rat and guinea pig bone marrow by Percoll density and/or elutriator centrifugation. As compared with the suspension of bone marrow an enrichment of factor 530 with a recovery of 34% can be achieved if a combination of both methods is applied.--The pattern of DNA percentage distribution in isolated cells paralleled the polyploidy found in megakaryocytes of the bone marrow. As determined by scanning cytophotometry and impulse cytophotometry the percentage distribution was maximal for the 16 C cells.--Exogenous [3H]adenine was incorporated by isolated megakaryocytes via a time and concentration dependent process showing two different affinities, Kt1 = 11 nM, V1 = 0.07 pmol/min per 10(5) cells; Kt2 = nM, V2 = 0.21 pmol/min per 10(5) cells.--Autoradiographic studies quantificated by means of the "probability circle" and "percent density" analysis showed that [3H]5-hydroxytryptamine (5-HT) accumulated in the osmiophilic dense bodies in contrast to [3H]adenine (metabolite) which was found mainly in the cytoplasm.

Morphological and functional characteristics of isolated acini used for in vitro studies of prostatic secretion.

Acini of the rat ventral prostate were isolated by interstitial injection of a collagenase-containing medium, subsequent incubation in the same medium and repeated aspiration through pipette tips with decreasing gauge of the tip opening. Functional integrity of the isolated acini was assessed by morphological studies, including transmission and scanning electron microscopy, freeze-fracturing, and immunocytochemistry. Incubation studies with different incubation media were performed monitoring O2-consumption as a parameter of functional activity, in addition to the incorporation rate of radioactively labeled amino acids into newly synthesized proteins. Optimal incubation conditions (shaking water bath, 20 strokes/min, 37 degrees C, gassing with carbogen at 15 min intervals) were found with M 199 medium supplemented with dihydrotestosterone. Stimulation of prostatic secretion was maximal with 10(-7) M of pilocarpin, which was more effective than carbamylcholine. Incorporation of precursors into prostatic proteins proceeded for about 2 h at a linear rate. Thereafter a rapid loss of functional and morphological integrity of the isolated acini was observed including disintegration, vacuolation and lysis of individual cells. The system of isolated prostatic epithelium developed is a useful tool in the study of prostatic secretion in vitro in short term experiments.

SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.

Hormonally induced changes in apocrine secretion of transglutaminase in the rat dorsal prostate and coagulating gland.

Coagulating gland and dorsal prostate of the rat are peculiar in secreting transglutaminase, a protein-cross linking enzyme that is released in an apocrine fashion. To elucidate whether or not the intracellular pathway and the unusual extrusion mechanism proceed constitutively or were differentially regulated, transglutaminase immunoreactivity was studied both at the light and electron microscopic levels. In addition, ultrastructural morphometry and scanning densitometry were applied to quantitate hormone-dependent distribution of transglutaminase. Coagulating glands and dorsal prostate, respectively, from sexually active rats were compared to those from sexually inactive, castrated, estradiol-treated or testosterone-substituted castrated animals. In intact, sexually active animals, no labeling of the cisternae of rough endoplasmic reticulum was seen, but instead the hyaloplasm was labeled. In the supranuclear portions of the cells an increase in labeling density of the hyaloplasm subjacent to the plasma membrane was found, whereas no labeling of either Golgi stacks or vesicles was observed. Apical blebs projecting into the acinar lumen were densely labeled. In castrated animals, epithelium showed a reduction of rough endoplasmic reticulum, loss of secretory blebs, and a decrease in cell size. Morphometric analysis of immunolabeling of coagulating gland epithelium from experimental animals resulted in a highly significant reduction of labeling of the hyaloplasm and apical blebs which was reversed by testosterone supplementation of castrated animals. After estrogen treatment, the reduction in immunolabeling was less pronounced, but morphology of apical blebs was obviously changed. Results from scanning densitometry of Western blots correlated with quantitative immunoelectron microscopical findings. Northern blot analysis using a secretory transglutaminase cDNA probe showed characteristic changes at the RNA levels. Our results indicate that apocrine secretion of transglutaminase in rat coagulating gland and dorsal prostate is a hormonally controlled process, where androgen deprivation results in impaired biosynthesis and release of transglutaminase, whereas estradiol treatment only partially inhibits secretion, but changes morphological features of the glandular epithelium, especially apocrine bleb formation.

Serum albumin as a potential carrier for the apocrine secretion of proteins in the rat coagulating gland.

A protein of 66k was purified to homogeneity from the total secretion of rat coagulating gland. Its close structural relationship to serum albumin was demonstrated by N-terminal amino acid sequence analysis, proteolytic fingerprinting and Western blotting studies using polyclonal antibodies raised against the 66k protein and rat serum albumin. Immunofluorescence staining showed that the 66k protein was localised in the cytoplasm of coagulating gland epithelial cells from which it is released via apocrine blebs. Performing immunoelectron microscopy, the 66k protein was by no means detectable in the endoplasmic reticulum and the Golgi apparatus. Reverse transcription-PCR, Northern blotting studies and in situ hybridisation experiments demonstrated that mRNA of albumin is not expressed by coagulating gland epithelial cells. Therefore, intravascular albumin should be transferred into the epithelial cells of the rat coagulating gland followed by secretion via aposomes. Furthermore, overlay blots proved that the 66k protein binds to the apocrine proteins carbonic anhydrase II and secretory transglutaminase and vice versa. In contrast, no binding was evident to the merocrine 115k protein and to cytoplasmic resident proteins e.g. lactate dehydrogenase. These findings point to the assumption that serum albumin taken up from extracellular sources could function as a selective carrier for cytoplasmic proteins destined for apocrine secretion.

Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro.

Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.

Change in location and processing of inhibin alpha-subunit precursors during sexual maturation of the Djungarian hamster testis.

Immunohistochemical location and immunoblot of inhibin alpha-subunit peptides were analyzed in the testis of the Djungarian hamster from days 0-31 of postnatal development using a specific antibody. An intense immunoreaction was observed in the centrally located T1 prespermatogonia at day 0. The staining intensity decreased gradually in the spermatogonia when they make contact with the basal lamina at days 8-10. At days 13 and 15 there is no staining. Thereafter the immunoreactivity in Sertoli cells as well as in A spermatogonia gradually increased, being highest in sexually mature animals. The intensity of alpha-subunit staining in the seminiferous tubules was stage specific, being strongest at stages III and IV. Immunoblot analysis of testis homogenates with the anti-INH alpha 1-32 antibody showed several bands: 88K, 80K, and 43K in immature hamster testis (0-, 2-, 6-, 8-, or 10-day-old). In the adult hamster (31-day-old) 88K, 80K, 28K, and 20K bands were seen, but no 43K band. Dimeric inhibin was not detected. The 43-44K band most likely corresponds to the pro-alpha N alpha C, the 28K band to intermediate forms between alpha N alpha C and alpha C (alpha I alpha C), and the 20K band to mature alpha-subunit (alpha C). The shift from the immature pattern to mature occurs at about 20 days of age. Freezing of the samples was deleterious to alpha C, since it could be detected only in freshly homogenized samples. The results suggest that prespermatogonia produce predominantly monomeric alpha-subunit precursor pro-alpha N alpha C, whereas the mature Sertoli cells as well as A spermatogonia contain mainly monomeric alpha I alpha C. The alpha-inhibin precursors may act as auto-/paracrine regulators of spermatogenesis. Our results suggest that different alpha-subunit precursors, pro-alpha N alpha C and alpha I alpha C, might be involved in the differentiation and maintenance of spermatogenesis, respectively. The posttranslational processing of alpha-subunit precursors seems to play an important role in the physiology of reproduction.

Transforming growth factor-beta2 mediates mesenchymal-epithelial interactions of testicular somatic cells.

Transforming growth factor-beta2 (TGFbeta2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFbeta2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFbeta2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFbeta2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFbeta2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFbeta2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFbeta2 secretion by peritubular cells was increased after stimulation with glucocorticoids and after addition of recombinant TGFbeta2, indicating an autoregulation of TGFbeta2. Furthermore, application of recombinant TGFbeta2 to cocultures resulted in an enhanced aggregation and cell clustering of Sertoli cells, pointing to an important role of TGFbeta2 in the paracrine interaction of peritubular and Sertoli cells of the developing rat testis.

Immunohistochemical localization of the glucocorticoid receptor in pancreatic beta-cells of the rat.

We used a monoclonal antibody against an epitope located in the N-terminal moiety of the rat glucocorticoid receptor to identify the glucocorticoid receptor-containing cells in the rat pancreas. Monospecific polyclonal antisera against insulin, glucagon, somatostatin, and amylase were applied to serial sections in colocalization studies to identify the respective endocrine and exocrine cells. Glucocorticoid receptor immunoreactivity was exclusively present in nuclei and cytoplasm of the beta-cells of pancreatic islets. Western blots using the glucocorticoid receptor antibody resulted in identical 94K immunoreactive proteins in both liver and pancreas. After adrenalectomy, the glucocorticoid receptor immunoreactivity of beta-cells decreased significantly. A computer-assisted method of semiquantitative evaluation of the glucocorticoid receptor immunoreactivity demonstrated a significant decrease in the staining intensity of the beta-cells by 23.5% and in that of insulin antibodies by 10.4%, while amylase immunoreactivity was only slightly decreased. Serum levels of corticosterone determined by RIA decreased from 225 micrograms/ml in sham-operated animals to 55 micrograms/ml in animals 14 days after adrenalectomy, while the tissue content of amylase decreased by 45%. The immunohistochemical findings give circumstantial evidence of the presence of glucocorticoid receptor in beta-cells. We interpret our data as indicating an indirect effect of glucocorticoids on amylase synthesis via a glucocorticoid-insulin-exocrine cell pathway.

Immunocytochemical localization of human 5 alpha-reductase 2 with polyclonal antibodies in androgen target and non-target human tissues.

We studied the tissue distribution and cellular localization of 5 alpha-reductase 2, the human prostatic isoenzyme, in different human tissues, both cryostat-sectioned and paraffin-embedded. Polyclonal antibodies raised in rabbits against a native peptide (C-terminal amino acids 229-254) or synthetic peptides (amino acids 234-245), either as carrier-conjugated linear peptides or multiple antigen peptides (MAP), were assayed for specificity and sensitivity with Western blotting and an ELISA system. One antibody showing monospecificity on Western blots and in ELISA was used for immunohistochemical detection of the respective antigen in tissues from male and female subjects. Positive cells were found (with decreasing intensity) in inner epithelial sheath of hair follicles, pyramidal cells of the cerebral cortex, hepatocytes and bile duct cells, prostate epithelial cells, seminal vesicle epithelial cells, endothelial cells of small vessels, fat cells, fibrocytes of genital and extragenital organs, and smooth muscle cells of prostate and seminal vesicles. Some variation in the immunoreactivity of testis and ovary tissue was seen with different antibodies. 5 alpha-Reductase 2 is obviously not restricted to androgen target organs in the male, but is present in a large number of cells and tissues in both males and females.

Cytoplasmic carbonic anhydrase II of rat coagulating gland is secreted via the apocrine export mode.

Two different pathways for protein secretion are described for epithelial cells of rat coagulating gland and dorsal prostate: the classical merocrine and the alternative apocrine release mode. Apocrine-secreted proteins are synthesized on cytoplasmic polyribosomes and are subsequently exported in protrusions on the apical cell surface (aposomes). In this article we report the identification and purification to homogeneity of a 29-kD protein from the secretion of rat coagulating gland. N-terminal amino acid sequence analyses revealed 100% identity to rat brain carbonic anhydrase II (CAH II). In addition, the 29-kD protein showed CAH enzyme activity. On Western blot analysis, a polyclonal anti-CAH II antibody raised in rabbit reacted specifically with the rat and human but not bovine CAH II isoforms. Immunohistochemical studies on rat coagulating gland showed strong labeling for CAH II protein in aposomes. Immunoelectron microscopy confined CAH II protein to the cytoplasm and aposomes, whereas no staining was visible in the compartments of the classical merocrine route, the endoplasmic reticulum and Golgi apparatus. The resident cytoplasmic protein lactate dehydrogenase, however, was not found in the secretion. Taken together, the morphological and biochemical data clearly indicate that cytoplasmic CAH II from rat coagulating gland is specifically selected and then secreted via the apocrine pathway.

Establishment, growth properties, and morphological characteristics of permanent human small cell lung cancer cell lines.

Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in establishing and permanently growing SCLC. Serum-free SIT medium and SIT2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R 10 medium was superior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.

Intermediate filaments in Sertoli cells.

Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre- and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone-dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycle-dependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cell-supporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad.