Endocytic activity of male germ cells during puberty

It is well known that numerous germ cells degenerate during the first meiotic division and spermatid elongation is due mostly to an adverse physiological microenvironment in relation to hormonal deficiency. The present study is aimed at investigating the endocytic activity of germ cells, during the first wave of mouse spermatogenesis, using transferrin coupled to gold particles, in order to study the efficiency of this possible pathway of communication between Sertoli cells and germ cells. Labelling experiments in control animals confirmed a receptor-mediated pathway in all germ cells during puberty. Furthermore, our morphological and quantitative data revealed that during spermatid elongation, degenerating germ cells possessed a highly developed endocytic apparatus which contained twice as many transferrin gold particles than normal adult cells. The fact that endocytosis of transferrin was increased in degenerating germ cells indicates that, most probably, germ cell degeneration during the first wave of spermatogenesis did not result from a deficiency in iron transport. The higher endocytic activity of degenerating germ cells, compared to adult control cells, could not only be the result of a simple process of plasma membrane internalization but also a complex mechanism which could be involved in the degradation of the cells.     

Freeze-fracture of Rana pipiens gonad anlage: study of primordial germ cells and other cellular types

Freeze-fracture was used to study Anura Amphibia primordial germ cells (PGCs) from the time when they have invaded genital ridges until the time when sexual differentiation has begun. We observed tight junctions with a variety of configurations including linear, macular, and extensive occluding cross-linking complexes. True gap junctions were not observed. Rod-shaped particles were found disseminated among particles on the P fracture faces of the germ cells.     

Morphogenesis of Avian Infectious Bronchitis Virus and a Related Human Virus (Strain 229E)

Avian infectious bronchitis virus (IBV) and strain 229E, a virus recently recovered from patients with colds, have been shown to possess a similar distinctive morphology in negatively stained preparations. An electron microscopic study of the morphogenesis of IBV in the chorioallantoic membrane and of strain 229E in WI-38 cells was performed. In infected cells, round electron-dense particles 82 mmu in diameter were observed to form by a process of budding from membranes of the endoplasmic reticulum and cytoplasmic vesicles. The particles in IBV-infected cells were similar in size and shape to those in strain 229E-infected cells but showed certain differences in internal structure. The evidence that the particles represent virions and the implications of these findings in the classification of this virus group are discussed.     

Semliki Forest Virus in HEp-2 Cell Cultures

The growth and development of Semliki Forest virus (SFV), an arbovirus of serological group A, in HEp-2 cells in tissue culture was examined by various techniques at frequent intervals. Infectivity and fluorescent-antibody studies demonstrated the presence of infective virus and viral antigens within the cells at 8 hr after infection. The antigen was particulate and distributed throughout the cytoplasm. Thereafter, there was rapid progression of virus production and cell destruction. By electron microscopy, tubular structures bounded by a fine membrane were observed in cytoplasm at 12 hr. Rows of small (25 mmu) virus particles were often present on the outer surface of these membranes, and at later times they became progressively more encrusted with the small virus particles. These structures subsequently increased rapidly in number, size, and complexity, and the space between the membrane and the tubules increased, thus forming vacuoles which contained tubules and were covered with the small particles. At later times (24 hr and later) larger (42 to 50 mmu) particles were observed, usually inside of the vacuoles. These larger particles (and occasionally the smaller ones) were also seen at the cell periphery and in the extracellular space. The large SFV particles appear to form by three distinct processes: (i) from the smaller particles, (ii) by development on an intravacuolar membrane, and (iii) at the ends of the tubules. The mode of development of SFV is unique among viruses studied to date, but in some characteristics it resembles that of other group A arboviruses. Its development differs from that of most arboviruses of group B and other serological groups.     

Receptor-mediated and adsorptive endocytosis by male germ cells of different mammalian species

Summary The routes for adsorptive and receptor-mediated endocytosis were studied in vivo after microinjection of tracers into the lumen of the seminiferous tubules, and in vitro in isolated germ cells of different mammals. Cationic ferritin was located on the plasma membrane, in vesicles, in tubules, in multivesicular bodies and in lysosome-like granules of mouse spermatocytes. In these cells the number of multivesicular bodies varied during spermatogenesis. Spermatids and to a lesser extent residual bodies also performed adsorptive endocytosis. In the rat and monkey (Macaca fascicularis) diferric transferrin was specifically taken up by germ cells via receptor-mediated endocytosis. The labelling was observed subsequently in membrane pits, vesicles, endosome-like bodies and pale multivesicular bodies. A progressive decrease in the frequency of the labelling of the germ cells by transferrin-gold particles was observed from spermatogonia to spermatocytes and to early spermatids, which could indicate that iron is particularly required by germ cells during the mitotic and meiotic processes. Adsorptive and receptor-mediated endocytosis therefore occurs in all classes of germ cells. These endocytic processes are most probably required for germ cell division, differentiation and metabolism.     

Structure and Composition of a Small Particle Prepared from a Simian Adenovirus.

Mayor, Heather D. (Baylor University College of Medicine, Houston, Tex.), Richard M. Jamison, Liane E. Jordan, and Joseph L. Melnick. Structure and composition of a small particle prepared from a simian adenovirus. J. Bacteriol. 90:235-242. 1965.-When tissue-culture fluids infected with simian adenovirus SV15 are examined in an electron microscope, either as fresh harvests or after treatment with Genetron, typical mature adenovirus particles are found. These are 65 to 70 mmu in diameter, with an icosahedral capsid built from 252 capsomeres. Also present is a population of small polyhedral particles approximately 20 mmu in diameter. These small particles can be separated from the mature virions by ultrafiltration or density gradient centrifugation. The small particles have a density of 1.43 in cesium chloride. They contain protein and double-stranded deoxyribonucleic acid. They appear to possess cubic symmetry of the icosahedral type, with a coat composed of 12 subunits each at the vertex of an icosahedron.     

Changes in Intensity and Spectral Distribution of Fluorescence: Effect of Light Treatment on Normal and DCMU*-Poisoned Anacystis nidulans‡

The intensity of the "steady-state" fluorescence of "aerobic" Anacystis nidulans is variable under prolonged illumination with orange (590 mmu) or blue (440 mmu) light for both normally photosynthesizing and DCMU-poisoned cells. In general, orange light illumination causes an increase of the fluorescence intensity followed by a decrease, while blue light causes an increase until a steady level is reached. Poisoned Anacystis cells show four to eight times larger changes in fluorescence intensity than the normal cells; the detailed time course of fluorescence changes is also different in poisoned and normal cells. When algae are cooled to -196 degrees C in light, the light-induced changes in the "steady-state" fluorescence disappear in both types of cells. Difference fluorescence spectra, constructed by subtracting the fluorescence spectra taken after 5-15 min of illumination from those after 60-90 min of illumination, show a doublet structure of the difference band with a major peak coinciding with the Anacystis emission maximum (685 mmu) and a minor peak located at about 693 mmu.     

Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells,bind Tudor and protect from transposable elements

Germ cells give rise to all cell lineages in the next‐generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single‐particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.     

Recovery of Division Ability in Ultraviolet-irradiated Escherichia coli Induced by Photoreactivation, Photoprotection, and Liquid Holding Treatment

Small doses of ultraviolet light (UV, 265 mmu) cause Escherichia coli B to grow into long, multinucleate, nonseptate, filamentous cells. This UV-induced filament formation can be prevented by irradiating with photoprotecting light (335 mmu) prior to UV irradiation, and by irradiating with photoreactivating light (406 mmu), or by liquid holding treatment, after UV irradiation. It is concluded that UV-induced division inhibition in E. coli B is initially induced by repairable lesions in the deoxyribonucleic acid, probably pyrimidine dimers.     

In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

The genes encoding the major 42S storage particle proteins are expressed in male and female germ cells of Xenopus laevis.

As components of the 42S storage particles (thesaurisomes), thesaurin a and thesaurin b are involved in the long-term storage of tRNA and 5S RNA in previtellogenic oocytes of Xenopus laevis. Thesaurin a and thesaurin b are among the most abundant proteins in previtellogenic oocytes. We show here that the mRNAs encoding thesaurin a and thesaurin b are present not only in previtellogenic oocytes but also in pre-meiotic germ cells (oogonia). These mRNAs can also be detected in spermatogonia and early spermatocytes, and are translated into protein in testis, as they are in ovary. We conclude that male germ cells mimic female germ cells in several aspects of gene activity related to RNA accumulation and metabolism.     

Biophysical studies of vesicular stomatitis virus

McCombs, Robert M. (Baylor University College of Medicine, Houston, Tex.), Matilda Benyesh-Melnick, and Jean P. Brunschwig. Biophysical studies of vesicular stomatitis virus: J. Bacteriol. 91:803-812. 1966.-The infectivity and morphology of vesicular stomatitis virus (VSV) were studied after density gradient centrifugation in cesium chloride (CsCI), potassium tartrate (KT), and sucrose. Centrifugation in CsCl revealed two equally infectious bands corresponding to densities of 1.19 and 1.22 g/ml, and a third (density, 1.26 g/ml) band of low infectivity. Two bands (densities of 1.16 and 1.18 g/ml) were observed in the KT gradient, in which the lighter band contained most of the infectivity. Centrifugation in sucrose resulted in a single broad infectious band (density, 1.16 g/ml). The typical rod-shaped VSV particles were found mainly in the lighter bands obtained in CsCl (1.19 g/ml) and KT (1.16 g/ml) and in the single sucrose gradient band (1.16 g/ml). Bent particles equally as infectious as the rod-shaped particles were a constant finding in the CsCl preparations, and were observed mainly in the second band (density, 1.19 g). Numerous strands 15mmu wide were found in the third CsCl (density, 1.26 g/ml) and the second KT (1.18 g/ml) bands. Similar strands could be liberated from VSV particles after treatment with deoxycholate. Internal transverse striations were found to be a regular feature of VSV particles examined with the pseudoreplication negative-staining technique. For crude virus stocks, the physical particle-to-infectivity ratio ranged from 73 to 194. Several morphological similarities between VSV and myxoviruses were observed, including 10 mmu surface projections, pleomorphic morphological forms, and 15 mmu seemingly nucleoprotein strands.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells

Germ cells normally differentiate in the context of encapsulating somatic cells. However, the mechanisms that set up the special relationship between germ cells and somatic support cells and the signals that mediate the crucial communications between the two cell types are poorly understood. We show that interactions between germ cells and somatic support cells in Drosophila depend on wild-type function of the stet gene. In males, stet acts in germ cells to allow their encapsulation by somatic cyst cells and is required for germ cell differentiation. In females, stet function allows inner sheath cells to enclose early germ cells correctly at the tip of the germarium. stet encodes a homolog of rhomboid, a component of the epidermal growth factor receptor signaling pathway involved in ligand activation in the signaling cell. The stet mutant phenotype suggests that stet facilitates signaling from germ cells to the epidermal growth factor receptor on somatic cells, resulting in the encapsulation of germ cells by somatic support cells. The micro-environment provided by the surrounding somatic cells may, in turn, regulate differentiation of the germ cells they enclose.     

Possible sequential transformation of a series of acidophils in the pituitary autografts in the renal capsule of male rats in association with ACTH cell.

In our electron microscopy, acidiphils in the pituitary autografts placed in the renal capsules of immature male rats underwent a sequential transformation with the lapse of time: Within 3 and 6 days, all the somaotrophs packed with the large granules of about 350 mmu diameter dispersed. The size and number of the granules in somatotrophs were quickly and markedly reduced with severe modification of cell shape. There was evidence during this time course that Siperstein's or Moriarty's corticotrophs might be synonymous with the stellate shape of acidophils with the arrangement of small granules 150-200 mmu in diameter along the cell acidophils. The "acidophils of the small granule type" possibly related to ACTH production according to Yoshimura et al. (1974) were frequently detected in the grafts as elongated or irregularly shaped cells. Their minute granules 100-150 mmu in diameter were also distributed in row in the cytoplasmic peripheral area. Gradual loss of the minute granules below 100 mmu in diameter eventually made the acidophils to transform into agranular cells. Our own idea that ACTH secretion might correlate with a series of cells transforming along the acidophil-axis was indirectly supported by the present observation on pituitary grafts. On the other hand, basophils rapidly degenerated and died away. Ten and 20 days after autografting, the graft cells which might be principally composed of the cells of acidophil origin enormously proliferated through mitotic division, showing the homologous fine structure, without the normal cell individuality. They always contained three different size and shape of granules simulataneously. Significance of such a rapid and strong response of acidophils to the ectopic replacement in the immature male rats was discussed from the view-point of hypothalamic regulation to simple protein hormones.     

Hybridization of testis-derived stem cells with somatic cells and embryonic stem cells in mice

Somatic cell hybridization is widely used to study the control of gene regulation and the stability of differentiated states. In contrast, the application of this method to germ cells has been limited in part because of an inability to culture germ cells. In this study, we produced germ cell hybrids using germ-line stem (GS) cells and multipotent germ-line stem (mGS) cells. While GS cells are enriched for spermatogonial stem cell (SSC) activity, mGS cells are similar to embryonic stem (ES) cells and originally derived from GS cells. Hybrids were successfully obtained between GS cells and ES cells, between GS cells and mGS cells, and between mGS cells and thymocytes. All exhibited ES cell markers and a behavior similar to ES cells, formed teratomas, and differentiated into somatic cell tissues. However, none of the hybrid cells were able to reconstitute spermatogenesis after microinjection into seminiferous tubules. Analyses of the DNA methylation patterns of imprinted genes also showed that mGS cells do not possess a DNA demethylation ability, which was found in embryonic germ cells derived from primordial germ cells. However, mGS cells reactivated the X chromosome and induced Pou5f1 expression in female thymocytes in a manner similar to ES cells. These data show that mGS cells possess ES-like reprogramming potential, which predominates over-SSC activity.     

Germ cell binding to rat Sertoli cells in vitro

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.     

Concanavalin A-induced attachment of spermatogenic cells to Sertoli cells in vitro

Adhesive contacts between developing germ cells and Sertoli cells may play an important role in mammalian spermatogenesis. Adhesion between isolated spermatogenic cells (pachytene spermatocytes and round spermatids) and Sertoli cells was studied. The attachment of single mouse germ cells to mouse Sertoli cell layers was weak, but the rate of attachment was stimulated by Concanavalin A (conA; 5 μg/ml). ConA-induced attachment was largely stable during a subsequent incubation for 30 min in the presence of 20 mM α-methyl- -mannoside (an inhibitor of conA). The cellular specificity of the stable attachment of germ cells to Sertoli cells was inferred from the observations that a comparable inhibitor-resistant attachment could not be obtained between germ cells and kidney cells, and between mouse myeloma cells and Sertoli cells. Juxtaposition of male germ cells and Sertoli cells through conA bridges may lead to the subsequent formation of strong and specific cell-cell adhesive bonds.     

Cytochromes of aquatic fungi

The cytochrome systems of two classes of aquatic fungi, the Oomycetes and Chytridiomycetes, were studied by means of reduced-minus-oxidized difference spectra at room and at low temperature. At room temperature, all of these fungi have a c-type cytochrome with an absorption maximum at 551 mmu and a b-type cytochrome at 564 mmu. The Oomycetes have a-type cytochromes at 605 mmu, and the Chytridiomycetes have a-type cytochromes at 606 mmu (Blastocladiales) or at 609 mmu (Monoblepharidales). Additional b-type cytochromes are found at 557 mmu in the Oomycetes and at approximately 560 mmu in the Chytridiomycetes. The data obtained from spectra at low temperature are consistent with these conclusions. Thus, the difference spectra reveal variation between the cytochrome systems of these two classes of aquatic fungi.