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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 ability to capacitate sperm in vitro has been of great importance to both scientists and clinicians, and knowledge of how cholesterol efflux occurs in these cells, as well as how this efflux is integrated with transmembrane signaling to regulate sperm function, may reveal much about the fertilization process and may also provide insights into the role and dynamics of membraneolesterol efflux in somatic cell function.
Abstract: Following spermatogenesis and spermiogenesis, mammalian spermatozoa leaving the testis appear to be morphologically mature but clearly are immature from a functional standpoint; that is, they have acquired neither progressive motility nor the ability to fertilize a metaphase II‐arrested egg. Although progressive motility is acquired and signaling pathways mature during sperm transit through the epididymis, complete fertilization capacity in vivo is conferred only during residence in the female reproductive tract. Similar observations have been made using a variety of in vitro assays, suggesting that a series of events, some initiated by environmental cues, confer on sperm the ability to fertilize the egg. This acquired capacity to fertilize was first observed by Austin (1) and Chang (2), who demonstrated that freshly ejaculated sperm cannot fertilize eggs until they reside in the female reproductive tract for a finite period of time. All of the cellular events that allow the ejaculated sperm to fertilize an egg were subsumed into a single phenomenon that was termed “capacitation.” The ability to capacitate sperm in vitro has been of great importance to both scientists and clinicians, who have used it to study the basic biology of fertilization and to develop various assisted reproductive technologies for humans and other species. Work by many investigators has established that the process of fertilization, not surprisingly, represents a series of elegant intercellular communication and cellular activation events (3‐5). Sperm functions such as motility and capacitation in the female reproductive tract are likely modulated by environmental cues in the luminal fluid, as well as by interactions with oviductal epithelium or other female tissues (6). When sperm arrive in the oviduct and encounter the ovulated, metaphase II‐arrested egg enclosed in its cumulus cell matrix, a complex series of cell-cell and cell-ECM interactions ensues, initiating cellular signaling events that permit the fusion of the sperm and egg plasma membranes. Several of these cell-matrix and cell-cell interactions involve novel gamete surface proteins and matrices. Signal transduction events leading to gamete activation, in particular sperm acrosomal exocytosis and egg cortical granule secretion, share some features with signaling events described in somatic cells. Sperm membrane cholesterol efflux contributes to one such novel signaling mechanism that controls sperm capacitation, and the details of this effect are now beginning to be understood at the molecular level. Knowledge of how cholesterol efflux occurs in these cells, as well as how this efflux is integrated with transmembrane signaling to regulate sperm function, may reveal much about the fertilization process and may also provide insights into the role and dynamics of membrane cholesterol efflux in somatic cell function. Here, we offer a short overview of the role of cholesterol efflux in regulating sperm capacitation, with an aim toward identifying areas of future investigation that may ultimately pro

225 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: It is concluded that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.
Abstract: Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F1 β-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CFoF1 activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

190 citations


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

  • ...The major contaminant in isolation of mitochondria from cells is PM and its contamination was checked using a PM ATPase assay (Bunney et al. 2001)....

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Journal ArticleDOI
TL;DR: The latest insights about sperm lipid raft research are covered and how sperm lipid buoy dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction is discussed.
Abstract: The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.

189 citations


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

  • ...Neutral lipids such as diacylglycerol (DAG) and ether lipids are present in unusually high proportions and a majority of PLs are composed of unsaturated fatty acids (22:6 and 22:5) (Gadella et al. 2008, Petcoff et al. 2008)....

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Journal ArticleDOI
TL;DR: The results are interpreted as broadly supporting the previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro and compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.

187 citations


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

  • ...However, it is continuously removed from the membrane on arrival of spermatozoa in the female reproductive tract, in preparation for the fusion steps (Davis et al. 1979, Apel-Paz et al. 2003)....

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Journal ArticleDOI
TL;DR: The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual‐oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa.
Abstract: A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X-100 soluble and insoluble proteins were recovered and separated by SDS-PAGE. The separation lanes were dissected into 96 fractions and analyzed individually by LC-MS(n) . A comprehensive protocol, involving LC-MS/MS analysis eventually down to the ninth most intense peak found in the MS-survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual-oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven-pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate-gated ion channel receptors, transient receptor potential cation channels, and a non-genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC-MS/MS analysis.

186 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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