scholarly journals Dynamics of intracellular calcium induced by lactate and glucose in rat pachytene spermatocytes and round spermatids

Reproduction ◽  
2002 ◽  
pp. 701-710 ◽  
Author(s):  
JG Reyes ◽  
E Herrera ◽  
L Lobos ◽  
K Salas ◽  
N Lagos ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Carbohydrate Metabolism ◽  
Adenine Nucleotides ◽  
Differential Modulation ◽  
Glycolytic Metabolism ◽  
Spermatogenic Cells ◽  
Lactate Metabolism ◽  
The Media ◽  
Hexose Phosphorylation ◽  
Pachytene Spermatocytes

Glycolytic metabolism in meiotic and post-meiotic spermatogenic cells shows differentiation-related changes. The developmental and physiological significance of these metabolic changes is not known. The aim of the present study was to test the hypothesis that glucose and lactate metabolism can modulate intracellular calcium [Ca2+](i) in spermatogenic cells in an opposing and dynamic manner. Fluorescent probes were used to measure [Ca2+](i) and pH(i), and HPLC was used to measure intracellular adenine nucleotides and mitochondrial sensing of ATP turnover. [Ca2+](i) in pachytene spermatocytes and round spermatids was modulated by changes in lactate and glucose concentrations in the media. The kinetics and magnitude of the [Ca2+](i) changes induced by lactate and glucose were different in meiotic and post-meiotic spermatogenic cells. The presence of glucose in the medium induced a decrease in pH(i) in spermatogenic cells. This glucose-induced pH(i) decrease occurred later than the changes in [Ca2+](i), which were also observed when the pH(i) decrease was inhibited, indicating that the glucose-induced [Ca2+](i) increase was not a consequence of pH(i) changes. Hexose phosphorylation in glycolysis was part of the mechanism by which glucose metabolism induced a [Ca2+](i) increase in spermatogenic cells. The sensitivity of [Ca2+](i) to carbohydrate metabolism was higher in round spermatids than in pachytene spermatocytes. Thus, differentiation-related changes in carbohydrate metabolism in spermatogenic cells determine a dynamic and differential modulation of their [Ca2+](i) by glucose and lactate, two substrates secreted by the Sertoli cells.

Adenine nucleotides and other factors indicative of glycolytic metabolism in murine spermatozoa

1987 ◽  
Vol 78 (6) ◽  
pp. 407-409
Author(s):  
R. P. Erickson ◽  
K. J. Harper ◽  
S. R. Hopkins ◽  
G. J. Brewer
Keyword(s):  
Adenine Nucleotides ◽  
Glycolytic Metabolism

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

1990 ◽  
Vol 166 (2) ◽  
pp. 909-915 ◽  
Author(s):  
Roger C. Ashmore ◽  
Richard F. O'Brien ◽  
Thomas J. Stelzner ◽  
Ira M. Dauber ◽  
Lawrence D. Horwitz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Adenine Nucleotides

scholarly journals DNA analysis and sorting of viable mouse testis cells.

1981 ◽  
Vol 29 (6) ◽  
pp. 738-746 ◽  
Author(s):  
W M Grogan ◽  
W F Farnham ◽  
J M Sabau
Keyword(s):  
Dna Content ◽  
Adult Mouse ◽  
Dna Analysis ◽  
Cell Types ◽  
Mouse Testis ◽  
Spermatogenic Cells ◽  
Hoechst 33342 ◽  
Cell Sorter ◽  
Pachytene Spermatocytes

The dye Hoechst 33342 and a 2-parameter cell sorter have been used to measure DNA content in viable testis cells and to sort pachytene spermatocytes and round spermatids from adult mouse testis to virtually 100% homogeneity. Early diploid spermatogenic cells were enriched to 90%, a 10-fold purification. The capability for viable sorting of most testis cell types to homogeneity in numbers suitable for many biochemical applications is demonstrated.

scholarly journals PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Junya Suzuki ◽  
Sadaki Yokota
Keyword(s):  
Sertoli Cells ◽  
Rna Silencing ◽  
Binding Proteins ◽  
Spermatogenic Cells ◽  
P Bodies ◽  
Cytoplasmic Rna ◽  
Dsrna Binding ◽  
Chromatoid Bodies ◽  
Pachytene Spermatocytes ◽  
Ectoplasmic Specialization

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including PRKRA, TRBP, and Dicer. RISC localizes to P-bodies. The nuage of the spermatogenic cells has function similar to the P-bodies. We study whether PRKRA localizes to nuage of spermatogenic cells of rat and mouse. PRKRA localized to four types of nuage structures, including aggregates of 60–90 nm particles, irregularly-shaped perinuclear granules, and intermitochondrial cement of pachytene spermatocytes, and chromatoid bodies of round spermatids. In addition, PRKRA is associated with dense material surrounding tubulobulbar complexes and with the ectoplasmic specialization. The results suggest that PRKRA functions in the nuage as an element of RNA silencing system and plays unknown role in the ectoplasmic specialization and at the tubulobulbar complexes of Sertoli cells attaching the head of late spermatids.

scholarly journals Intracellular Calcium Levels Determine Differential Modulation of Allosteric Interactions within G Protein-Coupled Receptor Heteromers

2014 ◽  
Vol 21 (11) ◽  
pp. 1546-1556 ◽  
Author(s):  
Gemma Navarro ◽  
David Aguinaga ◽  
Estefania Moreno ◽  
Johannes Hradsky ◽  
Pasham P. Reddy ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
G Protein ◽  
Differential Modulation ◽  
G Protein Coupled Receptor ◽  
Allosteric Interactions ◽  
Receptor Heteromers ◽  
G Protein Coupled

Adenosine metabolism in human skin fibroblasts

1985 ◽  
Vol 248 (1) ◽  
pp. C21-C26 ◽  
Author(s):  
M. J. Holland ◽  
E. Murphy ◽  
J. K. Kelleher
Keyword(s):  
Initial Rate ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Product Formation ◽  
Human Skin Fibroblasts ◽  
Metabolic Fate ◽  
Normal Cells ◽  
Extracellular Adenosine ◽  
Adenosine Concentration ◽  
The Media

When normal fibroblasts were incubated in media containing various initial concentrations of [8-14C]adenosine, ranging from 0.25 to 400 microM, under conditions where product formation was linear, greater than 90% of the intracellular label was found in adenine nucleotides, largely in the form of ATP, less than 1% of the intracellular label appeared in the nucleic acids, the remaining intracellular label was found in adenosine, inosine, and hypoxanthine, and the media contained two labeled products, inosine and hypoxanthine. Production of labeled inosine and hypoxanthine from adenosine was considerably lower in adenosine deaminase (ADA)-deficient cells than in normal cells and virtually eliminated in normal cells by the presence of 1 microM deoxycoformycin (a potent ADA inhibitor), suggesting that labeled inosine and hypoxanthine production requires ADA activity. Initial rates of deamination (inosine and hypoxanthine formation) and phosphorylation (adenine nucleotide formation) were estimated by examining the metabolic fate of [8-14C]adenosine in hypoxanthine phosphoribosyltransferase-deficient cells, which cannot recycle hypoxanthine. The estimate of the initial rate of phosphorylation exceeded that of deamination only at the lowest adenosine concentration examined (0.25 microM). The ratio of deamination to phosphorylation rose from approximately 1 at 0.41 microM to approximately 15 at 400 microM extracellular adenosine.

403. Differential effect of hexoses on sperm metabolism and function in culture

2008 ◽  
Vol 20 (9) ◽  
pp. 83
Author(s):  
H. W. Bakos ◽  
M. Lane
Keyword(s):  
Lipid Peroxidation ◽  
Dna Damage ◽  
Carbohydrate Metabolism ◽  
Culture Media ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Function ◽  
Ros Production ◽  
Sperm Dna Damage ◽  
The Media

Currently there is lack information regarding how human spermatozoa regulate their energy metabolism. This is surprising considering that carbohydrate metabolism is a vital point for the understanding of sperm function. This coupled with the increased use of assisted reproductive technology and the importance of a well balanced culture media has led us to hypothesise that an imbalance of carbohydrate presence in the media may alter sperm function, particularly in relation to oxidative stress, DNA damage and lipid peroxidation. Sperm samples were obtained from three healthy normospermic donors for this study. Motile sperm were separated from semen samples using density gradient separation. Samples were incubated at different media conditions with varying glucose or fructose concentrations (0, 2.5, 25 mM) for 6–24hrs. Reactive oxygen species (ROS) were measured using 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (DCFDA). Sperm DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). Lipid peroxidation was assessed using the probe BODIPY (581/591) C11. Carbohydrate uptake from the media was measured using a fluorometric procedure. Statistical differences between treatments were assessed by ANOVA and Bonferroni post-hoc test. No significant motility differences were found following treatments. Results showed an increased level of ROS production as glucose concentration increased (P < 0.05). This was accompanied by an increased number of TUNEL positive cells (P < 0.05). Furthermore, lipid peroxidation of spermatozoa was significantly increased when incubated under high glucose concentrations (P < 0.01). In contrast, increases in fructose concentrations did not alter ROS levels or the number of TUNEL positive cells. Sperm metabolised both glucose and fructose in vitro and the removal of one carbohydrate resulted in a compensatory increase in the metabolism of the other. To our knowledge, this is the first report providing evidence that altered carbohydrate metabolism may induce ROS production, lipid peroxidation and increase the number of sperm exhibiting DNA damage.

Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis

1994 ◽  
Vol 107 (10) ◽  
pp. 2887-2898
Author(s):  
M. Morse-Gaudio ◽  
M.S. Risley
Keyword(s):  
Dna Damage ◽  
Xenopus Laevis ◽  
Topoisomerase Ii ◽  
Spermatogenic Cell ◽  
Spermatogenic Cells ◽  
Dna Breaks ◽  
Whole Cells ◽  
Topo Ii ◽  
Pachytene Spermatocytes ◽  
Antipeptide Antibody

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.

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