Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

On structural patterns of the lamina propria of human seminiferous tubules

Summary The ultrastructure of the lamina propria of human seminiferous tubules was analyzed in normal specimens and compared to biopsies showing great thickenning of this area in light microscopy.The contractile cells are stellate in shape, the intercellular gaps between their branchings being less than 150 Å. The cytoplasmic features of these cells are similar to those described by Ross and Long (1966) and do not differ significantly in the pathological cases examined.The intercellular components, namely collagen fibers, microfibrils and an incomplete basement membrane-like coating of the contractile cells, are strikingly increased in the thickenned lamina propria, although the number of layers making up this structure needs not be increased. Occasionally, the intercellular space is occupied by only one of these materials.The distribution of collagen permits identification of two main patterns in the thickenned lamina propria: a) one where the basement membrane of the seminiferous epithelium is separated from the first layer of contractile cells by a wide collagen zone, and b) another case where the layer displaying greater thickness because of increased collagen deposition is located further away from the germinal epithelium.The functional activity of the contractile cells, the physiological implication of structural alterations of the lamina propria and the necessity to correlate these observations to andrological findings, are discussed.Presented in part at the 68. Meeting of the Anatomische Gesellschaft, Lausanne, April 1973.Fellow of the Alexander von Humboldt Foundation, on leave of absence from Depto. de Biología y Genética, Sede Norte, Universidad de Chile, Santiago.Supported by Grants from the Deutsche Forschungsgemeinschaft.     

Concretions in the human testis are derived from the basal lamina of seminiferous cords

Summary We provide evidence that testicular concretions form exclusively in association with seminiferous cords. Testicular material obtained from 6 patients with various disorders of spermatogenesis was studied by means of light-and electron microscopy. Seminiferous cords in the testes examined form so-called hypoplastic zones. They contain immature Sertoli cells and are surrounded by an unusually thick basal lamina and one or two layers of myofibroblasts. They display extensive coiling and form ring-like structures. Concretions are located at the periphery or in the center of the cords and consist of a hyaline material with an occasional granular core. They are enveloped by Sertoli cells or myofibroblasts. Data presented indicate that concretions may develop within the above-mentioned ring-like structures or in deep indentations of the surrounding connective tissue. Computer-assisted 3-D reconstructions of a seminiferous cord based on 24 serial semithin sections revealec that the majority of concretions communicate with the interstitial tissue through a broad opening comparable to a meso. On the basis of our observations we could demonstrate that concretions are accumulations of basal lamina that occasionally contain remnants of degenerated myofibroblasts. They form outside the cords and belong to the connective tissue compartment. As the components of the basal lamina are thought to be produced by the interaction of Sertoli cells and myofibroblasts we tentatively assume that the development of concretions reflects a disturbance in this complex interaction. We suggest that the presence of concretions refers to a developmental disorder and may be pathognomonic for the occurrence of defective seminiferous cords.Supported by Deutsche Forschungsgemeinschaft (Ho 388/6-1) and Verein zur Förderung der Forschung auf dem Gebiet der Fortpflanzung e.V; Hamburg     

Enhanced nerve growth factor efficiency in neural cell culture by immobilization on the culture substrate

Nerve growth factor (NGF) immobilization on a culture substrate may dramatically reduce the amount of NGF required for pheochromocytoma (PC12) cell culture. Coverslips on which NGF had been immobilized, or with NGF added to the culture medium daily, were used to culture PC12 cells. We examined the effects of adding 5, 10, or 100 ng of NGF to cultures daily, and compared them to the effects of immobilizing 5, 10, or 100 ng of NGF on culture substrates in a single dose. Cultures with 10 or 5 ng NGF added daily showed dramatically decreased cell viability, mitochondrial metabolic activity, and neuronal differentiation compared to cultures with 100 ng NGF added daily, while also exhibiting increased apoptosis. In contrast, a single dose of 100 ng immobilized NGF yielded results similar to 100 ng NGF added daily (total: 300 ng over 3 days), and 10 or 5 ng immobilized NGF showed far better results than 10 or 5 ng NGF added daily. These results demonstrate that NGF immobilization can dramatically reduce the amount of NGF required in neuronal cell culture.     

Taenia crassiceps: A secretion-substance of low molecular weight leads to disruption and apoptosis of seminiferous epithelium cells in male mice

The present research was performed to isolate and study the effects of a low molecular weight (<1300 Da) parasite-associated substance, obtained from peritoneal fluids of female mice infected with Taenia crassiceps cysticerci, on seminiferous epithelium cells of male mice testis. The results showed an intense disruption of Sertoli cells and germ cells within the seminiferous tubules of experimental mice, along with the destruction of their gap junction (GJ). Significant generalized apoptosis of germ cells within seminiferous tubules was determined by TUNEL staining (P = 0.0159). In addition, a significant number of infiltrating macrophages were found in the luminal space of these seminiferous tubules (P < 0.0001). Finally, electron microscopy studies revealed structural and morphological abnormalities in the somatic cells (Sertoli and Leydig cells) and in the germ cells, primarily in the round and elongate spermatids.     

Quantitation of the cells of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant

Summary Two variants of a method for determining the quantitative structure of the human seminiferous epithelium are described. Both versions (counting cells in whole tubules and in CA's) utilize specific criteria for the identification of cells, tubules and /or CA's. Both methods utilize the Sertoli cell as a constant with the cell types expressed as numbers of cells per Sertoli cell. One hundred biopsies were quantitated by the tubular method and 100 by the CA method. Counts were found to be reproducible and consistent. Comparisons were made between the two methods, different individuals doing the quantitation, quantitations of the right and left testes of the same subject and between biopsies from the same subject taken at different times. No difference was found between the right and left testes or biopsies from the same subject taken at different times. Quantitation of biopsies from one individual before and after testicular X-ray irradiation show how the method can be applied.This investigation was supported in part by U.S. Atomic Energy Commission Contract AT(45-1)1780 and Grant No. 680-0806 from The Ford Foundation.The authors wish to express their thanks and appreciation to Dr. Yves Clermont (Montreal) and Dr. Eugene Oakberg (Oak Ridge) for their aid in the conception of the project and the general outline of the protocol, and again to the latter for personally performing some of the tubular counts once the method had been established, to Mr. Malcom Hobbs for assistance with the subjects, to Dr. Daniel DiIaconi for performing the biopsy technique and to Dr. Kathleen O'Keefe for the statistical analysis.     

Computer-aided three-dimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules

Summary With the use of a digital image-processing method three-dimensional reconstructions of the arrangement of spermatocytes in human seminiferous tubules were performed. With this method it was possible to investigate the cellular distribution in the tubule in nearly any given perspective and projection. In addition, by means of simple mathematical procedures, such as by transformation of Cartesian coordinates into cylindrical coordinates, it was possible to vary the shape of a reconstruction, i.e., to convert the cylindrical image of a tubular portion into a right-angled r--z-representation.The present work not only confirms the existence of a complex helical plan of organization of the human seminiferous epithelium but also provides further aspects of the phenomenon of physiological germ-cell loss and its integration into the kinetics of spermatogenesis.Dedicated to Prof. E.C. Roosen-Runge, Seattle, on the occasion of his 75th birthday     

A method for the isolation of intact Sertoli cell-germ cell associations from rat seminiferous tubules and their further partition into Sertol cell and germ cell fractions

A technique is described for obtaining a Sertoli cell-enriched and a germ cell-enriched fraction from immature rat testes. Sertoli cell-germ cell associations were obtained by incubating washed seminiferous tubule fragments with Collagenase and Pancreatin. They were then manually dissociated into a suspension comprising Sertoli cells as well as the various germ cell types characteristic for a given day of ontogeny. Fractionation into a Sertoli cell-enriched fraction and a germ cell-enriched fraction was effected by centrifugation following layering over a stepwise gradient of Ficoll-400. While the time-span compares favourably with other procedures reported in the literature, it is believed this is the first time a method is described that enables the simultaneous recovery of both the Sertoli cells and the germ cells.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Taenia crassiceps: Infections of male mice lead to severe disruption of seminiferous tubule cells and increased apoptosis

This research was carried out to study the effects of infection with Taenia crassiceps cysticerci on the seminiferous epithelium histoarchitecture in the testes of male mice. Our results showed a severe disruption of the histoarchitecture of the testis epithelium in infected mice. In these animals, a significant infiltration of macrophages within seminiferous tubules was observed (P < 0.001). Generalized apoptosis of germ cells within the seminiferous tubules was observed, as assessed by TUNEL assay and apoptotic nuclei were quantified. The total number of fluorescent objects (DNA) (including clusters, singles, and objects in clusters) was significantly higher in the infected cells than in the control group (P = 0.0286). Observation of the interstitial tissue showed disorder and deterioration of many Leydig cells of infected mice, as well as intense vacuolization and destruction of their inter-cellular junctions. Several ultrastructural abnormalities were observed through electron microscopy as well. The observed pathology could lead to a state of infertility.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role

Background

The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors.

Scope of review

In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used.

Major conclusions

Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca²⁺.

General significance

Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.     

Sertoli cells are capable of proliferation into adulthood in the transition region between the seminiferous tubules and the rete testis in Wistar rats

Sertoli cells (SCs) play a crucial role in testis differentiation, development and function, determining the magnitude of sperm production in sexually mature animals. For over 40 years, it has been considered that these key testis somatic cells stop dividing during early pre-pubertal phase, between around 10 to 20 days after birth respectively in mice and rats, being after that under physiological conditions a stable and terminally differentiated population. However, evidences from the literature are challenging this dogma. In the present study, using several important functional markers (Ki-67, BrdU, p27, GATA-4, Androgen Receptor), we investigated the SC differentiation status in 36 days old and adult Wistar rats, focusing mainly in the transition region (TR) between the seminiferous tubules (ST) and the rete testis. Our results showed that SCs in TR remain undifferentiated for a longer period and, although at a lesser degree, even in adult rats proliferating SCs were observed in this region. Therefore, these findings suggest that, different from the other ST regions investigated, SCs residing in the TR exhibit a distinct functional phenotype. These undifferentiated SCs may compose a subpopulation of SC progenitors that reside in a specific microenvironment capable of growing the ST length if needed from this particular testis region. Moreover, our findings demonstrate an important aspect of testis function in mammals and opens new venues for other experimental approaches to the investigation of SC physiology, spermatogenesis progression and testis growth. Besides that, the TR may represent an important site for pathophysiological investigations and cellular interactions in the testis.     

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support

Summary Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine--hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if -nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber net-works and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.     

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.     

Processing of nerve growth factor: the role of basic amino acid clusters in the pro-region

Neurotrophins are synthesized first as precursors called pro-neurotrophins, and their propeptides are then proteolytically removed to form mature neurotrophins. However, a significant proportion of total neurotrophins has been shown to be secreted as pro-neurotrophins. Furthermore, pro- and mature neurotrophins have been shown to elicit opposite effects on cell survival. Thus, the processing step of neurotrophins is very important. In order to understand the mechanism of neurotrophin processing, we focused on the two basic amino acid clusters in the pro-region of nerve growth factor (NGF). Various NGFs mutated at basic amino acids in the pro-region were introduced in COS7 and PC12 cells. The results indicated that these basic amino acid clusters were actually cleaved in the cells by furin, but that their cleavage contributed little to the production of mature NGF. However, one of the two sites was considered to contribute to mature NGF production depending on conditions used.     

Large scale purification and immunological characterization of human placental nerve growth factor

Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which crossreared characterization. A protein species of approximately 26 kDa was obtained, which crossreacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF.     

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium

Culture medium with elevated K⁺ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (K _d13×10^–11 M) and the other of low-affinity (K _d22×10^–9 M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K⁺. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca²⁺, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K⁺ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K⁺ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.Abbreviations NGF nerve growth factor - 7S NGF the high molecular weight form of NGF - NGF the -subunit of 7S NGF - ¹²⁵I-NGF ¹²⁵I-labeled NGF - mNGF–_rAb polyclonal rabbit IgG raised against mouse NGF - DRG dorsal root ganglia - K_d the equilibrium dissociation constant - N the maximal number of binding sites for the ligand NGF - NGFR the biologically relevant receptor through which the neurite outgrowth and neuron survival are mediated - GBS Gey's balanced salts - HKGBS high K⁺ GBS - PBG phosphate buffered GBS - HKPBG high K⁺ PBG - CFHKPBG Ca⁺² free high K⁺ PBG - PBG-cyt c PBG containing 2 mg/ml cytochrome c - HKPBG-cyt c HKPBG containing 2 mg/ml cytochrome c - AbU antibody unit - BU biological unit PBS, phosphate buffered saline - HKPBS high K⁺ PBS Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.