Protein–glycolipid interactions during spermatogenesis. Binding of specific germ cell proteins to sulfatoxygalactosylacylalkylglycerol, the major glycolipid of mammalian male germ cells

1985 ◽  
Vol 63 (10) ◽  
pp. 1077-1085 ◽  
Author(s):  
C. A. Lingwood
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Immune Serum ◽  
Spermatogenic Cells ◽  
Molecular Weights ◽  
Testicular Spermatozoa ◽  
Antibody Staining ◽  
A New Technique

Specific binding of membrane proteins extracted from rat spermatogenic cells to the major glycolipid of the male germ cell has been demonstrated by affinity chromatography. A new method for the production of affinity matrices, using photoactivatable heterobifunctional cross-linking agents, has been used to immobilize sulfatoxygalactosylacylalkylglycerol. Three proteins of apparent molecular weights 68 000, 34 000, and 24 000 from spermatogenic cells have been shown to selectively and reversibly bind to this affinity matrix. Antiserum raised against the major of these species (68 000) was demonstrated to be specific for this protein by immunoblotting. A new technique for the reduction of background nonspecific antibody staining using this method is described. The immune serum has been used to localize the antigen in frozen testicular sections. The protein is present in the plasma membranes of all germ cells, but the expression is elevated for testicular spermatozoa and cells in or near the basal compartment of the seminiferous epithelium. The relevance of these findings to intercellular communication and spermatogenesis is discussed.

scholarly journals Molecular signatures to define spermatogenic cells in common marmoset (Callithrix jacchus)

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 597-609 ◽  
Author(s):  
Zachary Yu-Ching Lin ◽  
Masanori Imamura ◽  
Chiaki Sano ◽  
Ryusuke Nakajima ◽  
Tomoko Suzuki ◽  
...  
Keyword(s):  
Germ Cell ◽  
Reproductive Biology ◽  
Germ Cells ◽  
Methylation Status ◽  
Common Marmoset ◽  
Methylation Pattern ◽  
Callithrix Jacchus ◽  
Molecular Signatures ◽  
Spermatogenic Cells ◽  
Human Reproductive

Germ cell development is a fundamental process required to produce offspring. The developmental program of spermatogenesis has been assumed to be similar among mammals. However, recent studies have revealed differences in the molecular properties of primate germ cells compared with the well-characterized mouse germ cells. This may prevent simple application of rodent insights into higher primates. Therefore, thorough investigation of primate germ cells is necessary, as this may lead to the development of more appropriate animal models. The aim of this study is to define molecular signatures of spermatogenic cells in the common marmoset, Callithrix jacchus. Interestingly, NANOG, PRDM1, DPPA3 (STELLA), IFITM3, and ZP1 transcripts, but no POU5F1 (OCT4), were detected in adult marmoset testis. Conversely, mouse testis expressed Pou5f1 but not Nanog, Prdm1, Dppa3, Ifitm3, and Zp1. Other previously described mouse germ cell markers were conserved in marmoset and mouse testes. Intriguingly, marmoset spermatogenic cells underwent dynamic protein expression in a developmental stage-specific manner; DDX4 (VASA) protein was present in gonocytes, diminished in spermatogonial cells, and reexpressed in spermatocytes. To investigate epigenetic differences between adult marmoset and mice, DNA methylation analyses identified unique epigenetic profiles to marmoset and mice. Marmoset NANOG and POU5F1 promoters in spermatogenic cells exhibited a methylation status opposite to that in mice, while the DDX4 and LEFTY1 loci, as well as imprinted genes, displayed an evolutionarily conserved methylation pattern. Marmosets have great advantages as models for human reproductive biology and are also valuable as experimental nonhuman primates; thus, the current study provides an important platform for primate reproductive biology, including possible applications to humans.

sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species

1996 ◽  
Vol 109 (4) ◽  
pp. 851-858
Author(s):  
G. Berruti ◽  
B. Borgonovo
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Germ Cell ◽  
Molecular Mass ◽  
Germ Cells ◽  
Mammalian Species ◽  
Tissue Expression ◽  
Spermatogenic Cells ◽  
Male Germ Cells ◽  
Boar Spermatozoa

sp42, a tyrosine kinase of 42 kDa originally found in ejaculated boar spermatozoa, is so far the only tyrosine protein kinase to have been purified from mature male germ cells. We have developed and characterized rabbit polyclonal antibodies specifically directed against the boar sperm enzyme, which has been here purified to homogeneity. Anti-sp42 serum and sp42 affinity-purified antibodies work very well in western blot, immunoprecipitation and immunocytochemistry, and do not inhibit sp42 catalytic activity. Immunoblotting analyses reveal the presence of sp42 both in maturing boar epididymal (caput, corpus and cauda segment) spermatozoa and in testicular spermatogenic cells, thus establishing that the protein is effectively expressed in the germ cells and is not a sperm-associated protein secreted by the epididymal epithelium or male accessory glands. This finding is further strengthened by the fact that sp42 is not glycosylated, since different lectins fail to bind to sp42 and treatment of sp42 with different deglycosylation enzymes does not result in a reduction of the molecular mass of sp42. When different boar tissues are immunoscreened in western blot analysis, the results are all sp42-negative. The extension of the study to other mammalian species (human, mouse and rat) demonstrates that proteins immunologically related to boar sp42, which share the same molecular mass and tyrosine kinase activity, are both expressed in spermatogenic cells and maintained in mature sperm cells. Intriguingly, when a wide spectrum of somatic mouse and rat tissues is probed with sp42-antiserum, no tissue presents anti-sp42 immunoreactivity. Immunocytochemistry shows that in boar spermatozoa sp42 is confined to the tail mid-piece, while by immunohistochemistry carried out on sections of adult rat testis the appearance time of the kinase appears to be consistent with a post-meiotic synthesis in haploid spermatids. Altogether, these results demonstrate that boar sp42 is a new male germ cell-specific gene product, with highly conserved tissue expression extended to other mammalian species, and suggest a possible role played by the cytoplasmic tyrosine kinase in the cell signalling network specific to haploid male germ cells.

scholarly journals The incorporation in vitro of sulphate ions into synaptic-membrane glycoproteins

1981 ◽  
Vol 200 (2) ◽  
pp. 373-377 ◽  
Author(s):  
C J B White
Keyword(s):  
Plasma Membranes ◽  
Specific Binding ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Membrane Glycoproteins ◽  
Synaptic Plasma Membranes ◽  
Molecular Weights ◽  
Carrier Free ◽  
Sheep Brain

Synaptosomes from sheep brain cortex were incubated with carrier-free Na235SO4 and the synaptic plasma membranes were isolated. The membranes were free of contamination from cytosol, mitochondria and microsomal material and accounted for 30% of the radioactivity present in the synaptosomal particulate fraction. Control experiments demonstrated that the radioactivity present in the preparation was not due to non-specific binding of sulphate ions. The synaptic membranes contained at least six 35S-containing protein bands with molecular weights between 160 000 and 16 000. Analysis showed that the radioactivity was located in the carbohydrate moiety of a glycopeptide.

Isolation of plasma membranes from purified mouse spermatogenic cells

1980 ◽  
Vol 43 (1) ◽  
pp. 279-299
Author(s):  
C.F. Millette ◽  
D.A. O'Brien ◽  
C.T. Moulding
Keyword(s):  
Cell Surface ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Lectin Binding ◽  
Round Spermatid ◽  
Ultrastructural Observation ◽  
Spermatogenic Cells ◽  
Cell Surface Antigens ◽  
Total Recovery

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5′-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.

Electron microscope study of the effects of radiation on insect fertility

1990 ◽  
Vol 48 (3) ◽  
pp. 72-73
Author(s):  
Judy Ju-Hu Chiang ◽  
Robert Kuo-Cheng Chen
Keyword(s):  
Electron Microscopy ◽  
Germ Cell ◽  
Germ Cells ◽  
Electron Microscope Study ◽  
Radiation Treatment ◽  
Light And Electron Microscopy ◽  
Stem Borer ◽  
Metabolic Energy ◽  
Rice Stem Borer ◽  
Effects Of Radiation

Germ cells from the rice stem borer Chilo suppresalis, were examined by light and electron microscopy. Damages to organelles within the germ cells were observed. The mitochondria, which provide the cell with metabolic energy, were seen to disintegrate within the germ cell. Lysosomes within the germ cell were also seen to disintegrate. The subsequent release of hydrolytic enzymesmay be responsible for the destruction of organelles within the germ cell. Insect spermatozoa were seen to lose the ability to move because of radiation treatment. Damage to the centrioles, one of which is in contact with the tail, may be involved in causing sperm immobility.

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

2007 ◽  
Vol 30 (4) ◽  
pp. 90
Author(s):  
Kirsten Niles ◽  
Sophie La Salle ◽  
Christopher Oakes ◽  
Jacquetta Trasler
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Chromosome 9 ◽  
Specific Gene ◽  
Global Methylation ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

A New Technique for the Enumeration of Rotaviruses in Wastewater Samples

1989 ◽  
Vol 21 (3) ◽  
pp. 99-104 ◽  
Author(s):  
J. I. Oragui ◽  
D. D. Mara ◽  
S. A. Silva ◽  
A. M. Konig
Keyword(s):  
Inorganic Ions ◽  
Assay Method ◽  
Toxic Substances ◽  
Waste Stabilization Ponds ◽  
Virus Removal ◽  
Molecular Weights ◽  
Large Numbers ◽  
Waste Stabilization ◽  
A New Technique ◽  
Wastewater Samples

Rotaviruses are generally excreted in large numbers in diarrhoeal stools, but in wastewaters their numbers are subject to variations. Detection and enumeration of these viruses involve a concentration step followed by an assay method. Enumeration in wastewater concentrates is complicated by the presence of toxic substances which are often concentrated with the viruses. These toxic substances often cause the destruction of cells during rotavirus assay, thus leading to underestimation of viral numbers. Such concentrates were detoxified by a simple and effective method using polyacrylamide (Biogel P-6DG) or dextran (Sephadex G50) beads. Concentrates (10 ml) were mixed with 0.5 g gel and the mixtures were allowed to stand for 2 h at room temperature during which time the beads swell by the passage of water into them along with inorganic ions and substances with molecular weights of less than 30,000. The supernatants were then decontaminated with antibiotics and assayed for rotaviruses by the indirect immunofluorescent technique. Most untreated ultrafiltrates of raw sewage and those from anaerobic ponds were found to be too toxic to MA104 and LLC MK2 cells, whereas the above treatment rendered over 90% of wastewater concentrates non-toxic to cells. This technique was used to study virus removal in samples from deep waste stabilization ponds in northeast Brazil.

scholarly journals Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2540
Author(s):  
Teresa Chioccarelli ◽  
Marina Migliaccio ◽  
Antonio Suglia ◽  
Francesco Manfrevola ◽  
Veronica Porreca ◽  
...  
Keyword(s):  
Bisphenol A ◽  
Germ Cell ◽  
Germ Cells ◽  
Estrogenic Activity ◽  
Endocannabinoid System ◽  
Perinatal Period ◽  
Cell Junction ◽  
Cell Content ◽  
Site Specific ◽  
Epididymal Fat

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3β-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell–cell junction genes (i.e., zonula occcludens protein-1, vimentin and β-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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