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Journal ArticleDOI

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis

01 Sep 2013-Reproduction (Society for Reproduction and Fertility)-Vol. 146, Iss: 3, pp 209-220
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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Journal ArticleDOI
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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Journal ArticleDOI
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
TL;DR: ...................................................................
Abstract: ................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of

3 citations

References
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Journal ArticleDOI
TL;DR: The expression of an important number of proteins (58 different 2‐D spots) is correlated in independent sperm samples at high statistical significance and this is the first report describing the correlation between proteomics, DNA integrity and protamine content.
Abstract: The present work was started to explore whether a correlation could be detected among proteomic expression, protamine content and DNA integrity in human sperm cells. Towards this goal, we extracted the proteins present in the sperm cells from 47 sperm samples from infertile patients and from ten semen donors, analysed each sample by 2-D gel electrophoresis, and quantified the expression of 101 spots identified by MALDI-TOF analysis. Additionally, the protamine content and DNA integrity were also determined. Several interesting proteins such as transcription factors, prohibitin, heat shock and proteasome proteins have been identified. We have found that the expression of an important number of proteins (58 different 2-D spots) is correlated in independent sperm samples at high statistical significance (p 0.5). Additionally, eight proteins have also been found to correlate with DNA integrity and seven with protamine content (p<0.05). To our knowledge, this is the first report describing the correlation between proteomics, DNA integrity and protamine content. It also sheds new light into the fundamental aspects of the human sperm and points to new potential proteins involved in male infertility.

124 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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Journal ArticleDOI
TL;DR: In this article, the presence of cholesterol in the bilayer modulated the basal and drug-stimulated ATPase activity of reconstituted P-glycoprotein (Pgp) in a modest fashion.
Abstract: Resistance to a broad spectrum of structurally diverse chemotherapeutic drugs (multidrug resistance; MDR) is a major impediment to the treatment of cancer. One cause of MDR is the expression at the tumor cell surface of P-glycoprotein (Pgp), which functions as an ATP-powered multidrug efflux pump. Since Pgp interacts with its substrates after they partition into the lipid bilayer, changes in membrane physicochemical properties may have substantial effects on its functional activity. Various interactions between cholesterol and Pgp have been suggested, including a role for the protein in transbilayer movement of cholesterol. We have characterized several aspects of Pgp−cholesterol interactions, and found that some of the previously reported effects of cholesterol result from inhibition of Pgp ATPase activity by the cholesterol-extracting reagent, methyl-β-cyclodextrin. The presence of cholesterol in the bilayer modulated the basal and drug-stimulated ATPase activity of reconstituted Pgp in a modest fashion...

106 citations


"Biochemical evidence for energy-ind..." refers background in this paper

  • ...100%: Methyl-b-cyclodextrin treatment Many reports have shown that exposing cells to b-cyclodextrin results in solubilization and removal of cellular cholesterol, and it is widely used in cell culture experiments for the same purpose (Yancey et al. 1996, Eckford & Sharom 2008)....

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Journal ArticleDOI
TL;DR: Reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive showed that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane.

94 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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Journal ArticleDOI
01 Jan 1987-Nature
TL;DR: Reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles suggests involvement in the endoplasmic reticulum of a phosphate-trans-locating protein, as was first proposed by Bretscher7 who called it 'flippase'.
Abstract: The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface. To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface. Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4). Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis. The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids. These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase'. Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles.

93 citations


"Biochemical evidence for energy-ind..." refers background in this paper

  • ...…was calculated 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 &…...

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Journal ArticleDOI
TL;DR: Results indicate that phospholipase A2 plays a fundamental role in the exocytosis of the acrosome elicited by Ca2+ and ionophore stimulation and it is possible that activation of this enzyme constitutes an essential Ca2-dependent event underlying exocyTosis in response to physiological stimuli.

87 citations