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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Abstract: ................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of
3 citations
References
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TL;DR: Assays are used to show that proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphate-based transport proteins, and that the functionally critical sulfhydryl group in the flipp enzyme protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Trit on 100.
Abstract: Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.
67 citations
"Biochemical evidence for energy-ind..." refers background or methods in this paper
...…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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...Liposomes and proteoliposomes were prepared as described previously (Chang et al. 2004, Sahu & Gummadi 2008); 4.5 mmol ePC and 0.3 mol% of labeled lipids such as NBD-PC, NBD-PS, NBD-PG, and NBD-PE were dried under a stream of nitrogen and solubilized in reconstitution buffer....
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...The proportion of functional flippases eliminated by treatment with protein-modifying reagents (% inhibition) was calculated from the percentage change in Pred after subtracting Pred for liposomes (Chang et al. 2004): Percentage inhibition Z 1K Aprotein -modifying reagent Amock !...
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TL;DR: It is concluded that Bacillus vesicles possess the ability to promote rapid transbilayer movement of phospholipids, and that the transport is probably protein (flippase)-mediated.
Abstract: We investigated the transbilayer movement or flip-flop of phospholipids in vesicles derived from the cytoplasmic membrane of Bacillus megaterium. Since common assay techniques were found to be inapplicable to the Bacillus system, we exploited and elaborated a newly described method in which fluorescent phospholipids (1-myristoyl-2-C6-NBD phospholipids) are used as tracers to monitor flip-flop. These lipids were introduced into Bacillus vesicles from synthetic donor vesicles containing a fluorescence quencher. Transport was measured by monitoring the increase in fluorescence as the tracers departed the quenched environment of the donor vesicle and entered first the outer membrane leaflet and subsequently the inner leaflet of Bacillus vesicles. Independent experiments involving cobalt quenching of NBD fluorescence provided results consistent with the existence of pools of fluorescent phospholipid in the outer and inner leaflets of Bacillus vesicles at the completion of transport. Using the assay we show that phospholipid flip-flop in Bacillus vesicles occurs rapidly (half-time approximately 30 s at 37 degrees C) with no preference for a particular phospholipid headgroup and that it is sensitive to proteolysis. We also establish that flip-flop does not occur in synthetic phospholipid vesicles or vesicles made from Bacillus phospholipids. We conclude that Bacillus vesicles possess the ability to promote rapid transbilayer movement of phospholipids, and that the transport is probably protein (flippase)-mediated.
65 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...…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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TL;DR: A method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid is described, developed from the amido black 10 B methods and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference.
Abstract: Publisher Summary This chapter describes a method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid. This procedure was developed from the amido black 10 B methods of Schaffner and Weissmann and Newman et al. and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference. One approach has been to remove an interfering lipid by extraction with organic solvents. However, because certain proteins display a limited solubility in such solvents, this strategy often fails. Another widely used approach involves the inclusion of sodium dodecyl sulfate (SDS) in a modified Lowry procedure to reduce lipid (and detergent) interference. As oxidized lipid continues to react to produce a substantial amount of color in the Lowry assay and as most lipid samples are partially oxidized, this procedure is not suitable for the accurate measurements of a protein in samples containing excess of lipid.
59 citations
"Biochemical evidence for energy-ind..." refers methods in this paper
...The amount of protein and PLs in the fractions was estimated as described previously (Bligh & Dyer 1959, Kaplan & Pedersen 1989)....
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...The protein:PL ratio (PPR) of proteoliposomes was determined as described previously (Bligh & Dyer 1959, Kaplan & Pedersen 1989)....
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TL;DR: An assay to measure the transbilayer translocation of dipalmitoylphosphatidylcholine, a membrane-embedded phospholipid, in proteoliposomes generated from detergent-solubilized rat liver endoplasmic reticulum is described.
56 citations
"Biochemical evidence for energy-ind..." refers background or methods in this paper
...…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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...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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...Previous studies on biogenic membrane flippases reported low saturation values for rat liver microsomes (60 mg/mmol), yeast (10 mg/mmol), Bacillus subtilis (40 mg/mmol), and spinach ER (40 mg/mmol) (Gummadi & Menon 2002, Kubelt et al. 2002)....
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...It has been widely reported that cysteine is a critical amino acid present in the class of flippases (Gummadi & Menon 2002, Kubelt et al. 2002, Sahu & Gummadi 2008)....
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TL;DR: Transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E. coli) wild-type strain, as well as in proteoliposomes reconstituted from detergent extracts of the IIMV, indicated flip-flop of M-C6-NBD-PE occurred similarly to IIMv and could be largely eliminated by proteinase K treatment.
Abstract: We investigated the transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E. coli) wild-type strain (MG1655), as well as in proteoliposomes reconstituted from detergent extracts of the IIMV. The transbilayer movement of 1-myristoyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (M-C6-NBD-PE) and -phosphocholine (M-C6-NBD-PC) was measured by a fluorescence stopped-flow back-exchange assay. Both analogues were rapidly translocated across the IIMV membrane, with half-times of <1 min (outward movement) and approximately 3 min (inward movement). No flip-flop was detected in protein-free liposomes, but in IIMV-derived proteoliposomes flip-flop of M-C6-NBD-PE occurred similarly to IIMV and could be largely eliminated by proteinase K treatment.
50 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...…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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...Previous studies on biogenic membrane flippases reported low saturation values for rat liver microsomes (60 mg/mmol), yeast (10 mg/mmol), Bacillus subtilis (40 mg/mmol), and spinach ER (40 mg/mmol) (Gummadi & Menon 2002, Kubelt et al. 2002)....
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...It has been widely reported that cysteine is a critical amino acid present in the class of flippases (Gummadi & Menon 2002, Kubelt et al. 2002, Sahu & Gummadi 2008)....
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