326 COMPARING CHANGES IN MEMBRANE LIPID ORDER IN EPIDIDYMAL AND EJACULATED BOAR SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 277
Author(s):  
C. Matas ◽  
F. Garcia-Vazquez, ◽  
M. Sansegundo ◽  
S. Ruiz ◽  
J. Gadea
Keyword(s):  
Plasma Membrane ◽  
Seminal Plasma ◽  
Lipid Membrane ◽  
Calcium Influx ◽  
Membrane Lipid ◽  
Treatment Groups ◽  
Lipid Disorder ◽  
Lipid Order ◽  
Merocyanine 540 ◽  
Boar Spermatozoa

The diffusion of lipids in the plasma membrane of ejaculated spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium (Wolfe et al. 2001 Mol. Reprod. Dev. 59, 306–313). Merocyanine 540 (M540) is a hydrophobic dye that has been shown to stain cell membranes more intensely if their lipid components are in a higher state of disorder, as is the case of capacitated spermatozoa. It is believed that the membrane fluidity changes detected by M540 precede the calcium influx, making M540 a method for evaluating the early events of capacitation. The aim of this study was to determine if there are differences in the dynamics of lipid disorder in the plasma membrane of ejaculated and epididymal boar spermatozoa under different conditions of capacitation. The sperm capacitation treatments were: washed in Delbucco's PBS supplemented with 0.1 % BSA (PBS-BSA), washed on a Percoll gradient (PG), and unwashed (UW: Control). During measurement, the samples were kept at 38�C and 5 % CO2 to maintain constant incubation conditions. Membrane lipid order and sperm viability were determined by flow cytometry with M540 (2.7 �M) and Yo-Pro-1 (25 nM), respectively. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter Co., Inc., Fullerton, CA, USA). A total of 10 000 gated events were collected per sample, with sample running rates of approximately 600 events/s. Data were analyzed by analysis of variance (ANOVA). For the epidydimal vs. ejaculated results, the percentage of low lipid disorder spermatozoa was higher in the epididymal (19.23%) than in the ejaculated (5.84%) groups, and the proportion of high disorder (42.85%) and dead cells (48.59%) was higher in the ejaculated group. In relation to sperm treatment (UW, PBS-BSA, and PG), the percentage of high disorder was similar in all of the treatment groups (UW: 44.62 %; PBS-BSA: 43.08%; PG: 43.41%). Finally, the percentage of low disorder was lower in the PBS-BSA and PERCOLL (10.68% and 12.83%, respectively) groups, and the highest was obtained for the UW group (14.09%). In conclusion, the staining with M540 revealed that the lipid disorder was affected by the source of the sperm and the sperm treatment. A significant increase in membrane lipid low disorder and decrease in high disorder and dead cells were detected when epididymal sperm were compared with ejaculated sperm, so the seminal plasma and the sperm treatment to eliminate disorder have an important effect in the lipid membrane order. Supported by MEC (AGL2006-03495/GAN) and Fundaci�n S�neca (03018/PI/05).

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2407-2420 ◽  
Author(s):  
B.M. Gadella ◽  
R.A. Harrison
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipid ◽  
Lipid Disorder ◽  
Outer Leaflet ◽  
Flow Cytometric ◽  
Merocyanine 540 ◽  
Sperm Plasma Membrane ◽  
Dependent Protein ◽  
Boar Spermatozoa

A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 154
Author(s):  
J. Gadea ◽  
S. Martínez-Miró ◽  
G. Decuadro-Hansen ◽  
C. Matás
Keyword(s):  
Seminal Plasma ◽  
Acrosome Reaction ◽  
Sperm Viability ◽  
Lipid Packing ◽  
Lipid Disorder ◽  
Merocyanine 540 ◽  
Centrifuge Tube ◽  
Before And After ◽  
Centrifugation Technique

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

scholarly journals Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2128
Author(s):  
Marion Papas ◽  
Jaime Catalán ◽  
Sandra Recuero ◽  
Jane M. Morrell ◽  
Marc Yeste ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Mitochondrial Membrane ◽  
Single Layer ◽  
Polymorphonuclear Neutrophils ◽  
Lipid Disorder ◽  
Seminal Plasma Proteins

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

ADH modulates plasma membrane lipid order of living MDCK cells via a cAMP-dependent process

1990 ◽  
Vol 259 (1) ◽  
pp. F95-F103
Author(s):  
M. C. Giocondi ◽  
G. Friedlander ◽  
C. Le Grimellec
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Membrane Lipid ◽  
Target Cells ◽  
Similar Response ◽  
Solute Permeability ◽  
Lipid Order ◽  
Plasma Membrane Lipid ◽  
Darby Canine Kidney

Using 1-[4-(trimethylamino)phenyl]-6-phenyl-hexa-1,3,5-triene, a fluorescent probe that specifically labels the external leaflet of the plasma membrane of living cells, we examined the effects of antidiuretic hormone (ADH) and various agents known to raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on the physical state of the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In polarized cells grown as a monolayer, [desamino-Cys1, DArg8]-vasopressin (V2-agonist) elicited a biphasic decrease in the lipid order as estimated from the decrease in fluorescence anisotropy (from r = 0.317 to r = 0.304, 37 degrees C) of the apical domain of the plasma membrane, equivalent at the peak response (t = 5 min) to that produced by an upward shift in temperature of 5-6 degrees C. A similar response was obtained by adding dibutyryl cAMP to the monolayers. Experiments on cell suspensions further indicated that the biphasic decrease in lipid order could also be evoked by forskolin, prostaglandin E2, and bradykinin but not by bradykinin plus indomethacin and was inhibitable by the protein kinases inhibitor compound H7. These data demonstrate that the lipid order of the plasma membrane of MDCK cells can be modulated in situ by cAMP-dependent processes probably involving protein kinase A activity, i.e., that membrane “fluidity” might act in the regulation of the cellular function of living epithelial cells. They provide a rationale for the changes in lipophilic solute permeability that accompany the increase in water permeability of target cells on ADH administration.

scholarly journals Elucidating the Role of K+ Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

2019 ◽  
Vol 20 (24) ◽  
pp. 6330 ◽  
Author(s):  
Marc Yeste ◽  
Marc Llavanera ◽  
Guillermo Pérez ◽  
Fabiana Scornik ◽  
Josep Puig-Parri ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Channel Blocker ◽  
Physiological Role ◽  
Membrane Lipid ◽  
K Channel ◽  
Sperm Capacitation ◽  
Lipid Disorder ◽  
Principal Piece ◽  
Boar Spermatozoa

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.

scholarly journals Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ariadna Delgado-Bermúdez ◽  
Marc Llavanera ◽  
Leira Fernández-Bastit ◽  
Sandra Recuero ◽  
Yentel Mateo-Otero ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Sperm Quality ◽  
Membrane Lipid ◽  
Lipid Disorder ◽  
Long Term Storage ◽  
Boar Sperm ◽  
Functional Relevance ◽  
Positive Effect

Abstract Background Aquaporins (AQPs) are a family of transmembrane water channels that includes orthodox AQPs, aquaglyceroporins (GLPs) and superAQPs. AQP3, AQP7, AQP9 and AQP11 have been identified in boar sperm, and they are crucial for sperm maturation and osmoregulation. Water exchange is an important event in cryopreservation, which is the most efficient method for long-term storage of sperm. However, the freeze-thaw process leads to sperm damage and a loss of fertilizing potential. Assuming that the quality of frozen-thawed sperm partially depends on the regulation of osmolality variations during this process, AQPs might play a crucial role in boar semen freezability. In this context, the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors. Results Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance. Whereas the use of 1,3-propanediol (PDO), an inhibitor of orthodox AQPs and GLPs, decreased total motility (P < 0.05), it increased post-thaw sperm viability, lowered membrane lipid disorder and increased mitochondrial membrane potential (MMP) (P < 0.05). When acetazolamide (AC) was used as an inhibitor of orthodox AQPs, the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels (P < 0.05). Finally, the addition of phloretin (PHL), a GLP inhibitor, had detrimental effects on post-thaw total and progressive sperm motilities, viability and lipid membrane disorder (P < 0.05). Conclusions The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability. Moreover, the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

scholarly journals Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

2020 ◽  
Author(s):  
Iztok Urbančič ◽  
Lisa Schiffelers ◽  
Edward Jenkins ◽  
Weijian Gong ◽  
Ana Mafalda Santos ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Cell Receptor ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Membrane Properties ◽  
Plasma Membrane Vesicles ◽  
Aggregation State ◽  
Lipid Order

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins − in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.

scholarly journals The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis

2020 ◽  
Vol 21 (12) ◽  
pp. 4520
Author(s):  
Ana Paula Pinoti Pavaneli ◽  
Sandra Recuero ◽  
Bruna Resende Chaves ◽  
Estela Garcia-Bonavila ◽  
Marc Llavanera ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Seminal Plasma ◽  
Glycogen Synthase ◽  
Mammalian Species ◽  
Membrane Lipid ◽  
Mitochondrial Activity ◽  
Lipid Disorder ◽  
Liquid Storage ◽  
Acrosomal Exocytosis

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/β were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/β after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/β. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

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