Sperm from Hyh Mice Carrying a Point Mutation in αSNAP Have a Defect in Acrosome Reaction

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of αSNAP has been detected in these animals. αSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had αSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type αSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant αSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of αSNAP, and that a fully functional αSNAP is necessary for acrosomal exocytosis, a key event in fertilization

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4) and Endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H+)-ATPases (V-ATPases) to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes

Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance: Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract

C mobilisation in disturbed tropical peat swamps: old DOC can fuel the fluvial efflux of old carbon dioxide, but site recovery can occur

Southeast-Asian peat swamp forests have been significantly logged and converted to plantation. Recently, to mitigate land degradation and C losses, some areas have been left to regenerate. Understanding how such complex land use change affects greenhouse gas emissions is essential for modelling climate feedbacks and supporting land management decisions. We carried out field research in a Malaysian swamp forest and an oil palm plantation to understand how clear-felling, drainage, and illegal and authorized conversion to oil palm impacted the C cycle, and how the C cycle may change if such logging and conversion stopped. We found that both the swamp forest and the plantation emit centuries-old CO2 from their drainage systems in the managed areas, releasing sequestered C to the atmosphere. Oil palm plantations are an iconic symbol of tropical peatland degradation, but CO2 efflux from the recently-burnt, cleared swamp forest was as old as from the oil palm plantation. However, in the swamp forest site, where logging had ceased approximately 30 years ago, the age of the CO2 efflux was modern, indicating recovery of the system can occur. 14C dating of the C pool acted as a tracer of recovery as well as degradation and offers a new tool to assess efficacy of restoration management. Methane was present in many sites, and in higher concentrations in slow-flowing anoxic systems as degassing mechanisms are not strong. Methane loading in freshwaters is rarely considered, but this may be an important C pool in restored drainage channels and should be considered in C budgets and losses

A TEM-traceable physiologically functional gold nanoprobe that permeates non-endocytic cells

Maria Victoria Berberian,1 Cristian A Pocognoni,2 Luis S Mayorga1,2 1Institute of Histology and Embryology of Mendoza – CONICET, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina; 2Institute of Histology and Embryology of Mendoza – CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina Background: Nanoparticles’ intracellular fate requires proper internalization. Most cells make use of a battery of internalization pathways, but some are practically sealed, as they lack the biochemical machinery for cellular intake. Non-endocytic cells, such as mammals’ spermatozoa, challenge standard drug-delivery strategies. Purpose: In this article, we present a gold nanoprobe that permeates the external and internal membranes of human sperm. Methods: Our design makes use of a gold nanoparticle functionalized with a membrane-permeable cysteine-rich recombinant protein. The chimeric protein contains two units of physiologically active metallothioneins (MT) that also provide binding motifs to gold and a cell-penetrating-peptide sequence (CPP) that confers cell permeability to the nanoparticle. Results: Transmission electron microscopy, indirect immunofluorescence, and functional assays show that the nanoprobe is readily internalized in sperm, without compromising cell integrity, while preserving MT’s physiological activity. Our findings highlight the potential of CPP-functionalized nanogold for investigating the physiology of otherwise impermeable non-endocytic cells. Keywords: human sperm, metallothionein, gold nanoparticles functionalization, cell-penetrating peptides, transmission electron microscop

The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. LTV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP3)-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP3-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP3-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis