Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis
TL;DR: The results suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis.
Abstract: During the maturation process spermatozoa undergo a series of changes in their lateral and horizontal lipid profiles. However, lipid metabolism in spermatozoa is not clearly understood for two reasons: i) the mature spermatozoa are devoid of endoplasmic reticulum, which is the major site of phospholipid (PL) synthesis in somatic cells, and ii) studies have been superficial due to the difficulty in culturing spermatozoa. We hypothesize that spermatozoa contain biogenic membrane flippases since immense changes in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes. In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP-independent flip-flop movement of phosphatidylcholine (PC), phosphatidylserine, and phosphatidylglycerol. Half-life time of PC flipping was found to be ∼3.2±1 min for whole sperm membrane, which otherwise would have taken ∼11-12 h in the absence of protein. Further biochemical studies confirm the flip-flop movement to be protein-mediated, based on its sensitivity to protease and protein-modifying reagents. To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP-independent flippase activity. Interestingly, mitochondrial membranes showed flip-flop movement but were specific for PC with half-life time of ∼5±2 min. Our results also suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis.
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TL;DR: The reactome enrichment analysis allowed the reconstruction of the biochemical pathways involved in sperm epididymal maturation and STRING analysis permitted the identification of molecular events possibly involved in that process.
Abstract: To become fertile, mammalian spermatozoa require completing a complex biochemical maturation that begins in the testis and ends within the female oviduct. Here, we paid attention to the events occurring at the membrane level during the epididymal transit. Indeed, in the epididymis, the molecular composition and the physical-chemical proprieties of sperm membranes markedly change, with functional cross talking among the spermatozoa, the epithelium, and the luminal content (particularly the epididymosomes). To study this process, we undertook a biological networks study, representing the involved molecules as nodes and their interactions as links. The analysis of network topology revealed that it has a scale free and small world architecture and it is robust against random failure. That assures a fast and efficient transmission of information and it leads to identifying the molecules exerting a higher level of control on the system, among which cholesterol plays a pivotal role. The reactome enrichment analysis allowed the reconstruction of the biochemical pathways involved in sperm epididymal maturation and STRING analysis permitted the identification of molecular events possibly involved in that process. In conclusion, this approach allows inferring interesting information, thus contributing to the knowledge on this process and suggesting staring points for further research.
8 citations
Cites background from "Biochemical evidence for energy-ind..."
...This molecule plays a key role in pre- and post-ejaculatory life of spermatozoa [Sheriff and Ali 2010; Schwarz et al. 2013; Saez et al. 2011; Rajasekharan et al. 2013]....
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...molecule plays a key role in pre- and post-ejaculatory life of spermatozoa [Sheriff and Ali 2010; Schwarz et al. 2013; Saez et al. 2011; Rajasekharan et al. 2013]....
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11 Aug 2021
TL;DR: It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro.
Abstract: Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro. Lay summary Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules – called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.
4 citations
DOI•
01 Jan 2013
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3 citations
References
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01 Jan 1975
TL;DR: The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts.
Abstract: A B S T R A C T Superoxide anion production by liver microsomes from intact, adrenalectomized, and cortisoltreate(l alrenalectoinized rats has been determined. The amount formed was roughly proportionate to the amiount of cortisol given, and a similar response was seen in the activity of NADPH-cytochrome c reductase. The amount of measurable superoxide anion was markedly reduced by the addition of superoxide dismutase. The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts.
11 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...…during the development process while DNA synthesis, transcription, and translation are virtually nonexistent in spermatozoa; however, proteins involved in transcription and translation are abundantly present (Travis & Kopf 2002, MartinezHeredia et al. 2006, Baker et al. 2007, de Mateo et al. 2007)....
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TL;DR: It was shown by pulsed-field gel electrophoresis that the DNA from the isolated mitochondria ranged in size from 45 to 100 kb, suggesting that different types of mitochondria with different physiological status, mass, and genomic DNA size probably coexist and carry out different physiological functions throughout the whole process of maize leaf growth and development.
Abstract: Flow cytometric analysis of mitochondria isolated from maize leaves revealed two distinct rhodamine-123-stained fluorescence populations distinguishable by their main fluorescence channel. Further microscopic observation of mitochondria stained with Janus Green B and rhodamine-123 revealed the occurrence of differently sized mitochondrial particles. It was shown by pulsed-field gel electrophoresis that the DNA from the isolated mitochondria ranged in size from 45 to 100 kb. These results suggest that different types of mitochondria with different physiological status, mass, and genomic DNA size probably coexist and carry out different physiological functions throughout the whole process of maize leaf growth and development.
10 citations
"Biochemical evidence for energy-ind..." refers methods in this paper
...Protein and PL recovery in the reconstituted vesicles was w40 and w80% respectively as reported by many groups following this protocol (Gummadi & Menon 2002, Yang et al. 2007)....
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...Images were procured using a bright-field microscope (Nikon Eclipse Ti) at 40! magnification (Yang et al. 2007)....
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TL;DR: The transbilayer movement and distribution of spin-labelled phospholipid analogues were studied in the plasma membrane of ram sperm cells isolated from functionally different regions of the epididymis (caput, cauda) and from the ejaculate, suggesting an asymmetric steady state transbilayers distribution ofospholipids.
Abstract: The transbilayer movement and distribution of spin-labelled phospholipid analogues were studied in the plasma membrane of ram sperm cells isolated from functionally different regions of the epididymis (caput, cauda) and from the ejaculate. As already shown for ejaculated cells, (i) a rapid movement of aminophospholipid analogues phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet, and (ii) a slow transbilayer movement of phosphatidylcholine were observed in the plasma membrane of maturating ram sperm cells. This suggests an asymmetric steady state transbilayer distribution of phospholipids, with a preferential enrichment of aminophospholipids on the cytoplasmic leaflet of those cells. The fast inward redistribution of the aminophospholipid analogues is consistent with the presence of an aminophospholipid translocase activity in all three sperm cell preparations. The translocase activity was enhanced slightly during the epididymal transit of spermatozoa. However, compared with epididymal sperm cells, a marked increase in the aminophospholipid translocase activity and a more pronounced phospholipid asymmetry for phosphatidylethanolamine was established for ejaculated spermatozoa. The physiological relevance of the rapid removal of aminophospholipids from the exoplasmic leaflet of the plasma membrane of ram sperm cells is discussed. The quality of sperm cell fractions was characterized by the cell morphology, membrane integrity and the cellular ATP concentration.
8 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...Another report showed transbilayer motion of spin-labeled PLs in sperm membrane preparation from ram caput, cauda, and ejaculated fractions (Müller et al. 1997)....
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TL;DR: Data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.
Abstract: Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.
7 citations
"Biochemical evidence for energy-ind..." refers methods in this paper
...Mitochondria were isolated according to previously reported methods (Nagdas et al. 2006)....
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...Isolation of mitochondria from whole cell Mitochondria were isolated according to previously reported methods (Nagdas et al. 2006)....
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...The pellet containing sedimented tissue fragments was discarded and supernatant centrifuged at 1500 g for 10 min at 4 8C to pellet spermatozoa (Nagdas et al. 2006, Chakrabarty et al. 2007)....
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TL;DR: It is shown that intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner and the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.
Abstract: Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.
7 citations
"Biochemical evidence for energy-ind..." refers background or methods in this paper
...It was then ultracentrifuged in a MLA 130 rotor at 175 000 g for 30 min at 4 8C (Rajasekharan & Gummadi 2011)....
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...…to be in the order of few minutes with reports indicating their presence in ER, Golgi complex and chloroplasts (Bishop & Bell 1985, Backer & Dawidowicz 1987, Hrafnsdóttir et al. 1997, Menon et al. 2000, Gummadi & Menon 2002, Kubelt et al. 2002, Chang et al. 2004, Rajasekharan & Gummadi 2011)....
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...This experiment was performed to verify the reconstitution of membrane proteins from TE as described previously (Rigaud et al. 1988, Rajasekharan & Gummadi 2011)....
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...The assay was performed using Perkin Elmer LS-55 Fluorescence Spectrophotometer as reported previously (Sahu & Gummadi 2008, Rajasekharan & Gummadi 2011)....
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