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
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TL;DR: The results suggest for the plasma membrane of ram sperm cells the presence of an aminophospholipid translocase and an asymmetric transversal lipid distribution with aminolysis preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet.
Abstract: We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an aminophospholipid translocase and an asymmetric transversal lipid distribution with aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.
85 citations
TL;DR: The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured, and Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes.
Abstract: The incorporation of 14C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.
84 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...Spermatozoa were able to incorporate radiolabeled precursors of palmitic acid, glycerol, choline, and arachidonic acid into DAG and phosphatidylcholine (PC), indicating that they possess active PL-synthesizing capability (Neill & Masters 1972, Vasquez & Roldan 1997)....
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TL;DR: Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane.
79 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...But in somatic and yeast cells, the transbilayer movement of lipids is reported to be independent of their head group and a t1/2 of 10–15 min was reported for spin-labeled PE, PC, and cardiolipin in purified beef heart mitochondrial inner membranes (Simbeni et al. 1990, Gallet et al. 1999)....
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TL;DR: Though formation of MZRCs raises questions regarding the nature of the block to polyspermy, formation and assembly of such a structure would no doubt explain the strenuous maturation process that sperm endure on their sojourn to functional maturity.
Abstract: For mammalian spermatozoa to exhibit the ability to bind the zona pellucida (ZP) they must undergo three distinct phases of maturation, namely, spermatogenesis (testis), epididymal maturation (epididymis) and capacitation (female reproductive tract). An impressive array of spermatozoa surface remodeling events accompany these phases of maturation and appear critical for recognition and adhesion of the outer vestments of the oocyte, a structure known as the ZP. It is becoming increasingly apparent that species-specific zona adhesion is not mediated by a single receptor. Instead, compelling evidence now points toward models implicating a multiplicity of receptor-ligand interactions. This notion is in keeping with emerging research that has shown that there is a dynamic aggregation of proteins believed to be important in sperm-ZP recognition to the regions of sperm that mediate this binding event. Such remodeling may in turn facilitate the assembly of a multimeric zona recognition complex (MZRC). Though formation of MZRCs raises questions regarding the nature of the block to polyspermy, formation and assembly of such a structure would no doubt explain the strenuous maturation process that sperm endure on their sojourn to functional maturity.
78 citations
"Biochemical evidence for energy-ind..." refers background in this paper
...In boar spermatozoa during transit from epididymis, the membrane proportion of PE, PS, and phosphatidylinositol decreased along with an increase in the amount of PC, sphingomyelin, and polyphosphoinositides (Nikolopoulou et al. 1985, Reid et al. 2011)....
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TL;DR: Investigation of the detailed changes due to cryopreservation in the plasma membrane lipid composition of the goat epididymal sperm cells indicates that profound increase of the hydrophobicity of the cell membrane is one of the major mechanisms by which spermatozoa acquire potential to resist or combat stress factors like cryodamage.
75 citations
"Biochemical evidence for energy-ind..." refers methods in this paper
...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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