Atomic force microscopy analysis of central nervous system cell morphology on silicon carbide and diamond substrates.

Brain machine interface (BMI) devices offer a platform that can be used to assist people with extreme disabilities, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease. Silicon (Si) has been the material of choice used for the manufacture of BMI devices due to its mechanical strength, its electrical properties and multiple fabrication techniques; however, chronically implanted BMI devices have usually failed within months of implantation due to biocompatibility issues and the fact that Si does not withstand the harsh environment of the body. Single crystal cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD) are semiconductor materials that have previously shown good biocompatibility with skin and bone cells. Like Si, these materials have excellent physical characteristics, good electrical properties, but unlike Si, they are chemically inert. We have performed a study to evaluate the general biocompatibility levels of all of these materials through the use of in vitro techniques. H4 human neuroglioma and PC12 rat pheochromocytoma cell lines were used for the study, and polystyrene (PSt) and amorphous glass were used as controls or for morphological comparison. MTT [3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide] assays were performed to determine general cell viability with each substrate and atomic force microscopy (AFM) was used to quantify the general cell morphology on the substrate surface along with the substrate permissiveness to lamellipodia extension. 3C-SiC was the only substrate tested to have good viability and superior lamellipodia permissiveness with both cell lines, while NCD showed a good level of viability with the neural H4 line but a poor viability with the PC12 line and lower permissiveness than 3C-SiC. Explanations pertaining to the performance of each substrate with both cell lines are presented and discussed along with future work that must be performed to further evaluate specific cell reactions on these substrates.

Development of Sertoli cell binding competency in the peripubertal rat.

The peripheral distribution of Sertoli cell F-actin, a cytoskeletal protein found in Sertoli cell ectoplasmic specializations, is associated with enhanced spermatid binding to the Sertoli cell and, as such, serves as a functional marker for its acquisition of binding competency. Previous studies suggest that the peripheral distribution of actin is dependent on follicle-stimulating hormone (FSH). To investigate the developmental pattern of Sertoli cell actin distribution in relation to peripubertal FSH and testosterone levels, we examined epithelial sheets from 2-8 week-old-rats. Tissues were processed for light microscopy and for the visualization of rhodamine-labeled F-actin. At 2 weeks, actin staining was diffuse throughout most of the Sertoli cells and was similar to that observed in binding-incompetent Sertoli cells. By 4 weeks, actin distribution was peripheral, acquiring the same staining pattern as observed in binding-competent Sertoli cells. Serum levels of FSH peaked at 4 weeks and declined to adult levels thereafter. Testosterone levels did not increase significantly until 6 weeks. Results show that Sertoli cell actin undergoes peripheral reorganization concurrent with the peripubertal peak of FSH but prior to the peripubertal rise of testosterone. The study demonstrates a temporal correlation between the peripubertal FSH rise and the actin redistribution in Sertoli cells that is consistent with an induction of this redistribution by FSH. These results suggest that FSH induces binding competency in Sertoli cells.

Opioid inhibition of adrenergic and dopaminergic but not serotonergic stimulation of luteinizing hormone releasing hormone release from immortalized hypothalamic neurons.

Opioids are known to have an inhibitory effect on the secretion of luteinizing hormone releasing hormone (LHRH) when administered to whole animals in vivo or when applied to hypothalamic fragments in vitro. Whether opioids have this effect by acting directly on the LHRH secreting neurons or require the mediation of an interneuron is controversial. To examine this question, a clonal cell line derived from a hypothalamic neuron (GT1-7) was perfused and fractions collected every 6 min. Morphine treatment had no effect on basal secretion of LHRH, nor on the spontaneous, pulsatile release of LHRH. Isoproterenol, dopamine, and serotonin all produced significant increments in LHRH secretion. Pretreatment of GT1-7 cells for 2 h with morphine, suppressed the LHRH response to isoproterenol and dopamine but had no apparent effect on serotonin-induced LHRH release. These data indicate that morphine has a direct effect on GT1-7 cells that alters their responsiveness to some, but not all, LHRH secretagogues. These results suggest that, in vivo, the inhibitory effects that opioids have on LHRH release may not require an interneuron.

Effects of follicle-stimulating hormone on the junction-related Sertoli cell cytoskeleton and daily sperm production in testosterone-treated hypophysectomized rats.

Specialized junctional binding of step 8 spermatids to Sertoli cells is an important spermiogenic event. In the hypophysectomized (Hypox) rat, the daily sperm production (DSP) is reduced and Sertoli cells become binding incompetent. Delayed replacement of testosterone (DT-Hypox) does not restore the normal DSP and Sertoli cells remain binding incompetent. In this study, DT-Hypox rats received FSH daily for 2 days to 3 wk concurrent with delayed testosterone replacement and were killed 8 wk after the initiation of hormone treatment. The DT-Hypox rat treated with FSH for 2 days to 2 wk had a significantly reduced DSP. Sertoli cells remained binding incompetent as evidenced by structurally abnormal ectoplasmic specializations and an abnormal pattern of f-actin and vinculin immunostaining. The DT-Hypox rat treated with FSH for 3 wk had a normal DSP. Sertoli cells were binding-competent as evidenced by structurally intact ectoplasmic specializations and the normal pattern of f-actin and vinculin immunostaining. The results indicate that the "priming" effect of FSH necessary to restore normal spermiogenesis is associated with restoration of the junction-related Sertoli cell cytoskeleton (i.e., FSH induction of binding competency) expressed as intact ectoplasmic specializations and peripheral distribution of f-actin and vinculin.

Junction-related Sertoli cell cytoskeleton in testosterone-treated hypophysectomized rats.

Testosterone is the principal hormone necessary for insuring the completion of normal spermatogenesis. However, its precise role in spermatid maturation is not clear. In hypophysectomized rats, testosterone can maintain spermiogenesis if replaced soon after surgery. Delaying treatment results in a reduction in the number of mature spermatids. In culture, testosterone and FSH are required to maximize spermatid attachment to binding-competent Sertoli cells. This binding event is an essential step in the process of spermiogenesis and is dependent on components of the Sertoli cell cytoskeleton. The present study was undertaken to determine the binding competency of Sertoli cells and their junctional interaction with spermatids in the hypophysectomized rat after immediate testosterone replacement and after delayed testosterone replacement. Hypophysectomized rats treated with immediate testosterone replacement had peripheral distribution of Sertoli cell f-actin and vinculin, structurally intact Sertoli ectoplasmic specializations facing step 8 spermatids, and daily sperm production similar to these parameters as observed in intact controls. In the delayed treatment group, these parameters were abnormal and were similar to those observed in the untreated hypophysectomized animals. The results suggest that testosterone can maintain binding competency of the Sertoli cell and normal Sertoli-spermatid junctional interaction but cannot restore them.

Reduced testosterone during puberty results in a midspermiogenic lesion.

The aim of this study was to determine the role of testosterone, as reflected in the testicular interstitial fluid, in the completion of the first wave of spermatogenesis and to further elucidate its role in spermiogenesis. At weekly intervals beginning with 26-day-old rats, body and testis weights were obtained, testicular interstitial fluid testosterone (TIF-T) was assayed, daily sperm production (DSP) was determined, and testicular tissue was structurally analyzed by light and electron microscopy. At 40 days postpartum, half the rats were treated with ethane dimethanesulphonate (EDS) to temporarily reduce Leydig cells. The other half served as controls and were treated with the vehicle. The timing of EDS treatment was just prior to the elongation of spermatids. At Day 47 (1 week after EDS treatment), TIF-T, testis weight, DSP, and number of Leydig cells were significantly reduced. At Day 54 (2 weeks after treatment), TIF-T had returned to the normal adult level, Leydig cell repopulation was apparent, and testis weight was normal. The DSP returned to normal by Day 61 (3 weeks after treatment). At 1 and 2 weeks after treatment, Step 8-9 spermatids were partially or completely detached from Sertoli cells. Results indicate that a temporary reduction of testosterone during the peripubertal period leads to a temporary reduction of the DSP approximately 1 week later. It is suggested that reduced testosterone is associated with a mid-spermiogenic lesion interfering with stable attachment of Step 8-9 spermatids to Sertoli cells during Stage VIII-IX of the spermatogenic cycle.

Structural remodeling of cardiac myocytes in patients with ischemic cardiomyopathy.

BACKGROUND: Chronic ischemic heart disease may lead to ventricular dilation and congestive heart failure (ischemic cardiomyopathy [ICM]). The changes in cardiac myocyte shape associated with this dilation, however, are not known. METHODS AND RESULTS: Left ventricular myocyte dimensions were assessed in cells isolated from explanted human hearts obtained from patients with ICM (n = 6) who were undergoing heart transplantation. Cells were also examined from three nonfailing donor hearts with normal coronary arteries (NCA). Compared with cells from patients with NCA, myocyte length was 40% longer in hearts from patients with ICM (197 +/- 8 versus 141 +/- 9 microns, p less than 0.01), cell width was not significantly different, and cell length/width ratio was 49% greater (11.2 +/- 0.9 versus 7.5 +/- 0.6, p less than 0.01). Sarcomere length was the same in myocytes from both groups. The extent of myocyte lengthening is comparable to the increase in end-diastolic diameter commonly reported in patients with ICM. CONCLUSIONS: These data suggest that increased myocyte length (an intracellular event), instead of myocyte slippage (an extracellular event), is largely responsible for the chamber dilation in ICM. Furthermore, maladaptive remodeling of myocyte shape (e.g., increased myocyte length/width ratio) may contribute to the elevated wall stress (e.g., increased chamber radius/wall thickness) in ICM.

Hormonal regulation of spermatid binding.

A Sertoli-spermatid coculture model is described in which a large percentage (greater than 76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD = the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH + testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described 'Sertoli ectoplasmic specialization' observed adjacent to post-step 8 spermatids in vivo. FSH + testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH + testosterone and is associated with the peripheral distribution of actin and vinculin.

Proenkephalin products are stored in the sperm acrosome and may function in fertilization.

Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells.

Vectorial (transcellular) transport of potassium (86Rb+) by cultured Sertoli cells.

Sertoli cells from rats aged 25 days were grown on Millipore filters (pore diameter 0.5 micron) for 7 days and were then used for determination of transport of 86Rb+ through the cells (base to apex); this procedure is referred to as measuring transcellular or vectorial transport. Sertoli cells were also used to measure apical efflux of 86Rb+ by loading the cells with the isotope to steady state and then incubating cells so that the apical surfaces were in contact with medium not containing 86Rb+, from which samples were taken. Basal efflux was measured in the same way except that the opposite surface of the cells was in contact with the medium. Cells grown on filters treated with collagen IV plus fibronectin showed transcellular transport of 86Rb+; t1/2 for equilibration across the cells was 9-12 min. The rate of transport was accelerated by addition of (Bu)2cAMP, forskolin, or FSH to the incubation medium. Half-maximal responses were seen with (Bu)2cAMP at 0.2 mM and with forskolin at 20 microM. Apical efflux (t1/2 9.8 +/- 2.1 min) was not influenced by the presence or absence of K+ in the medium nor by azide or (Bu)2cAMP. Basal efflux showed similar values for t1/2 in the presence of K+ (9.7 +/- 1.9 min) and values of 21.4 +/- 4.2 min in the absence of K+. Vectorial transport of 86Rb+ by these cells may account for the K+ gradient seen in the seminiferous tubule and appears to result from a basolateral potassium pump together with an apical membrane that is permeable to K+.

Gonadal steroids regulate proenkephalin gene expression in a tissue-specific manner within the female reproductive system.

Proenkephalin gene expression undergoes marked changes within the female reproductive system of rodents during the estrous cycle and in pregnancy. In order to define the factors responsible for this regulation, the effects of 17-beta-estradiol (E2) and progesterone (P4) have been examined in the ovary and uterus. In the ovary of the rat and hamster, E2 and P4 were without effect on proenkephalin RNA levels when injected individually. However, P4 increased ovarian transcript abundance 2- to 3-fold after pretreatment of animals with E2. In the uterus of either species, E2 had little effect but P4 alone stimulated both proenkephalin RNA abundance and total content severalfold. Glucocorticoids and androgen reproduced this stimulatory effect on proenkephalin transcript levels. The interaction between E2 and P4 on proenkephalin gene expression in the uterus varied with species. In the rat, E2 inhibited stimulation by P4, while in the hamster uterus the two hormones had a synergistic effect, producing a 15-fold elevation of proenkephalin RNA abundance and a 50-fold increase in total uterine content. These distinct steroid responses appear to account for tissue- and species-related differences in the variation of proenkephalin gene expression during the estrous cycle in the rodent ovary and uterus. The stimulatory effect of P4 was shown to involve direct steroid action on the uterus and to be inhibited both by the steroid antagonist RU-486 and the transcriptional inhibitor actinomycin D. These data are consistent with receptor-mediated activation of proenkephalin gene transcription in uterine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrous cycle- and pregnancy-related differences in expression of the proenkephalin and proopiomelanocortin genes in the ovary and uterus.

Expression of the genes for two opioid peptide precursors, proenkephalin and POMC, was examined within the female reproductive system of rodents as a function of the estrous cycle and during pregnancy. Proenkephalin RNA was found to change markedly during the estrous cycle in both the ovary and uterus (approximately 6- and 3-fold, respectively). The highest concentrations occurred at estrus in the rat ovary and at metestrus and diestrus in the rat uterus. In sharp contrast to proenkephalin RNA, the abundance of POMC RNA remained relatively constant throughout the estrous cycle in both tissues. Similar results were obtained in the cycling hamster ovary. During pregnancy, the concentrations of proenkephalin RNA in the rat ovary showed little variation, while in the uterus a 4-fold increase in this transcript was observed. The effects of pregnancy on POMC RNA were the reverse of this pattern; its abundance increased 2-fold in the ovary and did not vary substantially in the uterus. These differences in the expression of proenkephalin and POMC genes during the estrous cycle and pregnancy suggest that these two opioid peptide precursors are associated with distinct functional roles within the female reproductive system.

Plasma membranes from rat Sertoli cells: purification and properties.

A method is reported for preparing surface (plasma) membranes from rat Sertoli cells. The procedure is based upon homogenization in hypotonic buffer, extraction in a two-phase system, and sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by electron microscopy, enzyme markers, and functional activities associated with the membranes (binding of follicle-stimulating hormone [FSH] and production of cyclic adenosine 5'-monophosphate [cAMP]. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside of the membrane sheets. Marker enzymes for plasma membrane (5'-nucleotidase and alkaline phosphatase) showed more than 16- and 6-fold enrichment, respectively, and other enzymes showed that contamination by nuclei, mitochondria, endoplasmic reticulum, or cytosol was negligible. Binding of FSH was found to be specific, with KD 1.2 nM and the equivalent of 7500 sites per cell. This binding was enriched 20-fold compared to whole homogenate. Production of cAMP by membranes was increased by addition of FSH and by forskolin to the purified membranes in vitro.

Preparation and characterization of submitochondrial fractions from adrenal cells.

A method for preparing submitochondrial fractions from adrenocortical cells was developed by adapting a procedure that has been successful with yeast mitochondria. The method is based upon osmotic swelling, sonication and centrifugation in sucrose. The preparation yields highly purified fractions of outer membrane, intermembrane space, inner membrane and a less purified fraction of matrix. Recoveries are good so that 10(7) cells yield approximately 170 micrograms of inner membrane protein and 12 micrograms of outer membrane protein. Electron microscopy shows that the outer membrane fraction consists of vesicles (0.2-0.6 micron diameter) while inner membrane appears as densely packed sheets of membranous material. Two-dimensional polyacrylamide gels (isoelectric focusing followed by electrophoresis) of all the fractions give highly reproducible patterns of protein spots with Coomassie staining. Steroidogenic proteins were found only in inner membrane fractions which were shown to contain cytochrome P-450 C27 side-chain cleavage and P-450 11 beta-hydroxylase together with adrenodoxin and adrenodoxin reductase. Inner membrane catalyzes side-chain cleavage of cholesterol (conversions to pregnenolone) and 11 beta-hydroxylation (DOC----corticosterone) when substrate and NADPH are added. The preparation yields highly purified submitochondrial fractions from Y-1 mouse adrenal tumor cells and from porcine and bovine adrenocortical mitochondria. The method has the virtue of yielding highly purified intermembrane fluid which is not true of other methods for fractionation of adrenal mitochondria. The procedure also yields cleaner preparations of the two membranes than two other published methods currently used to prepare submitochondrial fractions from adrenocortical cells.

Production of components of extracellular matrix by cultured rat Sertoli cells.

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.

Contraction of collagen lattice by peritubular cells from rat testis.

Peritubular cells from 15- and 25-day-old rat testis trapped in collagen lattices caused those lattices to contract. Contraction proceeded more rapidly and to a greater extent using cells from younger rats. When 36,000 cells from 15- and 25-day-old rats were trapped in 800 mm2 lattices, the areas were reduced to 28 mm2 and 170 mm2, respectively, within 24 h. The cells from older rats were less effective at contracting the lattice than cells from younger rats. Cytochalasin B (5 micrograms ml-1) inhibited lattice contraction and caused disruption of actin filaments as seen by fluorescent staining with Rh-phalloidin. Cholera toxin (10 micrograms ml-1), and 1 mM-dibutyryl cAMP inhibited lattice contraction, as did 10 microM-trifluoperazine, commonly an inhibitor of calmodulin. The total intracellular concentration of cAMP was greater in peritubular cells from 25-day-old rats than in those from 15-day-old rats: 427 +/- 34 and 120 +/- 16 pmol mg-1 cell protein, respectively. When peritubular cells in monolayer were permeabilized with glycerol, the addition of ATP caused the cells to contract. Cell contraction was greater in cells from 15-day-old rats than 25-day-old rats. When cells were grown on silicone rubber, they caused that surface to wrinkle. Peritubular cells from 15-day-old rats caused the onset of wrinkling at 4 h. At the same time, no wrinkling was observed with cells from 25-day-old rats. Studies of lattice contraction and cell contraction were also made using cells from 20-day-old rats. In each case, contraction was intermediate between that of cells from 15-and 25-day-old rats. The possibility exists that lattice contraction, cell contraction and wrinkling of silicone film result from a mechanism of actin filament sliding, generated by myosin ATPase activity, and is inhibited by cAMP. The reduced rate of contraction in cells from 25-day-old rats may be related to their higher intracellular levels of cAMP. Evidence exists to show that cAMP blocks myosin ATPase activity by inhibiting the phosphorylation of its regulatory peptide, myosin light chain.

Content of K+ and Na+ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes.

Seminiferous tubules of rats exposed to x-irradiation before birth were subjected to micropuncture in situ at 50 days of age to obtain samples of fluid 4 h after ligation of efferent ducts. The concentrations of cations in this fluid were: potassium, 39.7 +/- 1.2 mM, and sodium, 136.3 +/- 1.2 mM (means and standard errors, n = 5). Histologic examination revealed that germ cells constitute less than 1% of the cell population within the seminiferous tubules of these rats; the remaining cells were all Sertoli cells. Sertoli cells showed efflux of 86Rb+ with t1/2 of approximately 11 min and an active ATPase in plasma membranes. These activities were similar to those of Sertoli cells from normal rats. Germ cells from normal rats showed less rapid efflux of 86Rb+ (t1/2 greater than 60 min) and less active Na+/K+ ATPase in plasma membranes. It is concluded that Sertoli cells are responsible for the high concentration of potassium in seminiferous tubule fluid and that plasma membranes of these cells contain an active K+ pump that is not inhibited by ouabain (1 mM).

The absence of premalignant changes in the cryptorchid testis before adulthood.

Previous investigators have reported that premalignant changes in the form of abnormal germ cells can be detected in the cryptorchid and infertile adult testis. These cells are characterized by a thin rim of pale-staining cytoplasm, a large hyperchromatic nucleus and prominent nucleoli. We studied 113 biopsies from 102 patients to determine if these abnormal germ cells occur in the nonadult cryptorchid testis. The patients ranged in age from 3 months to 16 years. Evaluation of testicular tissue by light microscopy disclosed histological alterations in the undescended testis, which were those commonly ascribed to the undescended testis. However, none of the changes could be interpreted as premalignant. The abnormal germ cells described by other investigators were not found in any of these specimens.