89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.

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79 EFFECT OF PRE-FREEZE ADDITION OF PLATELET-ACTIVATING FACTOR AND PLATELET-ACTIVATING FACTOR:ACETYLHYDROLASE ON THE POST-THAW INTEGRITY OF FROZEN - THAWED BOAR SPERM

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab79 ◽

2005 ◽

Vol 17 (2) ◽

pp. 189

Author(s):

R. Bathgate ◽

B.M. Eriksson ◽

W.M.C. Maxwell ◽

G. Evans

Keyword(s):

Platelet Activating Factor ◽

Egg Yolk ◽

Large White ◽

Boar Sperm ◽

Thawing Process ◽

Potential Benefits ◽

Boar Semen ◽

Acrosome Integrity ◽

And Control

The use of frozen-thawed boar sperm is not widespread, owing to reduced fertility rates and high cost per dose (Eriksson et al. 2004 Proc. Aust. Assoc. Pig Vet., 61–69). Improvements in post-thaw sperm survival are required for commercialization. Platelet-activating factor (PAF) is a phospholipid involved in regulating sperm function. PAF:acetylhydrolase (PAF:AH) regulates PAF activity by conversion to its inactive isoform. Both occur naturally in boar semen (Kordan et al. 2003 Pol. J. Vet. Sci. 6, 55–60). Removal of PAF and PAF:AH along with seminal plasma during the cryopreservation process may inhibit the ability of sperm to withstand the freeze-thawing process. The aim of this study was to assess the effect of PAF and PAF:AH, added to boar semen pre-freeze, on the post-thaw motility and acrosome integrity of sperm. The sperm rich fraction was collected from a mature Large White × Landrace boar, diluted with Androhep (1:2, semen:Androhep; Minitube, Verona, WI, USA), cooled to 17°C over 2 h, and then centrifuged (10 min, 800g). The sperm pellet was resuspended in cooling extender (11% (w/v) lactose solution with 20% (v/v) egg yolk; control), cooling extender plus 100 ng/mL PAF (PAF), or cooling extender plus 0.4% (v/v) PAF:AH (Pafase; ICOS Corporation, Seattle, Washington, USA), and cooled to 5°C over 2.5 h. Sperm were further diluted with cooling extender plus 9% (v/v) glycerol and 1.5% (v/v) Equex STM (freezing extender), loaded into 0.5-mL straws, and frozen. Straws were thawed (20 s, 42°C) and the motility and acrosome integrity (FITC-PNA; Mortimer etal. 1990 Hum. Reprod. 5, 99–103) assessed at 0, 3, and 6 h post-thaw after incubation at 37°C. Data from three replicates were analyzed by ANOVA and a Tukey test applied where significant differences were found. Post-thaw motility (0 and 3 h) was higher for PAF (60.0 ± 0.0% and 25.0 ± 2.9%) than for control (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05), but was similar for Pafase (41.7 ± 1.7% and 16.7 ± 1.7%; P > 0.05). By 6 h post-thaw, motility was similar for PAF (1.7 ± 1.7%), Pafase (6.7 ± 6.8%), and control (1.7 ± 1.7%, all respectively; P > 0.05). Acrosome integrity was higher at 0, 3 and 6 h post-thaw for Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%) than for control (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%, all respectively; P < 0.05), but was similar between control and PAF (P > 0.05). Supplementation of cooling extender with 100 ng/mL PAF increased initial post-thaw motility, but this benefit was lost after 6 h post-thaw. Pafase in the cooling extender improved the proportion of intact acrosomes, even after 6 h post-thaw. In vitro studies investigating the interaction between Pafase-treated frozen-thawed sperm and oviducal epithelial cells would be of interest to further establish the potential benefits of pre-freeze addition of Pafase on the fertilizing potential of frozen-thawed boar sperm.

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Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0499 ◽

2006 ◽

Vol 17 (1) ◽

pp. 56-66 ◽

Cited By ~ 77

Author(s):

John H. Evans ◽

Diana Murray ◽

Christina C. Leslie ◽

Joseph J. Falke

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Membrane Binding ◽

Membrane Lipid ◽

C2 Domain ◽

Plasma Membrane Lipid ◽

Protein Kinase Cα ◽

Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

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INFLUENCE OF GLYCEROL CONCENTRATION AND FREEZING TO −79 C ON OXYGEN UPTAKE AND LIVABILITY OF BOAR AND BULL SPERMATOZOA

Canadian Journal of Animal Science ◽

10.4141/cjas72-007 ◽

1972 ◽

Vol 52 (1) ◽

pp. 65-72 ◽

Cited By ~ 2

Author(s):

L. M. SANFORD ◽

G. J. KING ◽

J. W. MACPHERSON

Keyword(s):

Oxygen Uptake ◽

Incubation Period ◽

Skim Milk ◽

Egg Yolk ◽

Freezing Process ◽

Glycerol Concentration ◽

Motile Cells ◽

Boar Semen ◽

Boar Spermatozoa

Boar and bull spermatozoa were diluted in a skim milk–egg yolk–glucose extender containing 0, 7.5, or 15% glycerol (v/v) and incubated aerobically for 6 hr at 37 C. Other partially diluted boar semen samples were cooled to 5 C. Glycerol was added to a final concentration of 0, 7.5, and 15%. Samples were frozen to −79 C, rewarmed, and incubated for 3 hr at 37 C. The presence of glycerol in the extender depressed (P < 0.01) the oxygen uptake by nonfrozen boar and bull spermatozoa during the 6-hr incubation period. The reduction of oxygen uptake by semen samples increased as the level of glycerol in the extender increased. There was a corresponding decrease (P < 0.01) in the number of motile cells at the conclusion of the incubation period. Glycerol appeared to have more of a detrimental effect on boar spermatozoan oxygen uptake. The rate of oxygen uptake by boar semen samples postfreezing was extremely depressed, suggesting that spermatozoa surviving the freezing process metabolize at a much lower rate than normal. Active progressive motility of most of the surviving boar spermatozoa ceased within 1–2 hr of incubation under the in vitro conditions of this experiment.

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Studies on the Effect of Supplementing Boar Semen Cryopreservation Media with Different Avian Egg Yolk Types on in Vitro Post-thaw Sperm Quality

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.2006.00623.x ◽

2006 ◽

Vol 41 (1) ◽

pp. 68-73 ◽

Cited By ~ 41

Author(s):

R Bathgate ◽

WMC Maxwell ◽

G Evans

Keyword(s):

Sperm Quality ◽

Egg Yolk ◽

Semen Cryopreservation ◽

Boar Semen ◽

Avian Egg

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Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytes in vitro

Bioscience Reports ◽

10.1007/bf01117242 ◽

1990 ◽

Vol 10 (3) ◽

pp. 263-270 ◽

Cited By ~ 9

Author(s):

J. Pascal Zimmer ◽

Hans A. Lehr ◽

Christoph Hübner ◽

Stephan G. Lindner ◽

Ralf Ramsperger ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Fluidity ◽

Lipid Composition ◽

Membrane Lipid ◽

Serum Factors ◽

Membrane Lipid Composition ◽

Plasma Membrane Lipid ◽

Hiv 1

Although most non-human primates, except the chimpanzee and the gibbon in vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1 in vitro.In order to investigate whether the in vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.We found a correlation between increased HIV-1 in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.

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309 SEX SORTED BOAR SPERMATOZOA: TIME COURSE AND PROFILE OF THEIR IN VITRO PENETRATION ABILITY AFTER STORAGE IN THE PRESENCE OF HOMOLOGOUS SEMINAL PLASMA

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab309 ◽

2005 ◽

Vol 17 (2) ◽

pp. 305

Author(s):

I. Parrilla ◽

J.M. Vazquez ◽

M.A. Gil ◽

I. Caballero ◽

C. Almiñana ◽

...

Keyword(s):

Seminal Plasma ◽

Time Course ◽

Egg Yolk ◽

Vitro Fertilization ◽

Penetration Ability ◽

And Control ◽

Pig Oocyte ◽

Boar Spermatozoa ◽

Penetration Time

Addition of seminal plasma (SP) to the collection medium has been shown to be beneficial for motility and viability of sex-sorted and stored spermatozoa. However, SP could not only delay but also decrease the in vitro fertilization rates of IVM pig oocytes. In the present study, the time-course of IVM pig oocyte penetration of sex sorted boar spermatozoa stored in the presence or absence of SP was evaluated. Spermatozoa were sex-sorted following the Beltsville sperm sexing technology (Johnson and Welch 1999 Theriogenology 52, 1323–1342) and collected in TEST-egg yolk buffer (2%) with (10%) or without (control) SP. Sex-sorted spermatozoa were stored at 20°C during 0, 2, 5, and 10 h after sorting. Oocytes were matured in vitro in NCSU23 (Peters and Wells 1663 J. Reprod. Fertil. 48, 61–73) for 44 h in 5% CO2 in air at 39°C. The in vitro penetration time-course was determined by co-incubating the sex-sorted and stored spermatozoa with IVM oocytes during 3, 6, and 18 h in modified TRIS-buffered medium (mTBM) (Abeydeera and Day 1997 Theriogenology 48, 537–544) at 39°C in an atmosphere of 5% CO2 in air. Penetration rates and number of spermatozoa per oocyte were assessed after fixation and staining of the oocytes. Statistical analyses were conducted by ANOVA. Presence of SP did not delay the onset of the oocyte penetration. Moreover, at 3 h of co-incubation, SP increased (P < 0.05) both penetration rates and mean number of spermatozoa per oocyte in sorted and stored boar spermatozoa when compared with control (45 vs. 20, 50 vs. 32, 38 vs. 23, 15 vs. 8, at 0, 2, 5, and 10 h of storage with SP and control, respectively). High penetration rates were reached after 6 h of co-incubation (82 vs. 51, 96 vs. 76, 83 vs. 48, 31 vs. 24, at 0, 2, 5, and 10 h of storage with SP and control, respectively) in sorted and stored samples, with no further increase at 18 h (70 vs. 63, 92 vs. 79, 87 vs. 53, 55 vs. 40, at 0, 2, 5, and 10 h of storage with SP and control, respectively). Spermatozoa stored 2 h in the presence of SP showed the best penetration rate and highest mean number of spermatozoa per oocyte. The mean number of spermatozoa per oocyte increased as the co-incubation time increased (ranging from 2.1 to 5.8 for sorted spermatozoa stored 2 h in the presence of SP at 3 h and 18 h of co-incubation, respectively). In conclusion, the presence of SP during the storage of sex-sorted spermatozoa improves their in vitro fertilizing ability without affecting the onset of the oocyte penetration time. This work was supported by DGICYT (AGL 2001-0471), Fundación Seneca (PB174/FS/02) and CTIC (2103SIU0040).

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An in vitro1H-MRS Model of Oncogene Transfection

Acta Radiologica ◽

10.1080/02841859709172136 ◽

1997 ◽

Vol 38 (6) ◽

pp. 1083-1086

Author(s):

T. Nakai ◽

R. Ishima ◽

H. Sakahara ◽

K. Endo ◽

J. Konishi ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Lipid ◽

Ras Gene ◽

Membrane Viscosity ◽

Nih3t3 Cells ◽

Transfected Cells ◽

H Nmr ◽

Plasma Membrane Lipid ◽

Reduced Mobility

Purpose: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes. This note describes the in vitro1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells Material and Methods: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR. the peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra Results: the 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules. No other differential spectra for characterizing metabolic change was detected Conclusion: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression

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Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse.

The Journal of Cell Biology ◽

10.1083/jcb.102.4.1372 ◽

1986 ◽

Vol 102 (4) ◽

pp. 1372-1377 ◽

Cited By ~ 89

Author(s):

D E Wolf ◽

S S Hagopian ◽

S Ishijima

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Plasma Membranes ◽

Membrane Lipid ◽

Anterior Region ◽

Plasma Membrane Lipid ◽

Sperm Plasma Membrane ◽

Induced Changes

We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.

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Pseudomonas syringae effector AvrE associates with plant membrane nanodomains and binds phosphatidylinositides in vitro

10.1101/2021.07.08.451616 ◽

2021 ◽

Author(s):

Xiufang Xin ◽

Lisa Kinch ◽

Boying Cai ◽

Bradley C. Paasch ◽

Brian Kvitko ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Transgenic Arabidopsis ◽

Biochemical Properties ◽

Membrane Lipid ◽

Effector Proteins ◽

Host Cells ◽

Plant Proteins ◽

In Planta ◽

Plasma Membrane Lipid

Bacterial phytopathogens deliver effector proteins into host cells as key virulence weapons to cause disease. Extensive studies revealed diverse functions and biochemical properties of different effector proteins from pathogens. In this study, we show that the Pseudomonas syringae effector AvrE, the founding member of a broadly conserved and pathologically important bacterial effector family, binds to phosphatidylinositides (PIPs) in vitro and shares some properties with eukaryotic PROPPINs (β-propellers that bind polyphosphoinositides). In planta pull down experiments with transgenic Arabidopsis plants expressing AvrE revealed that AvrE is associated with several plant proteins including plasma membrane lipid-raft proteins. These results shed new light on the properties of a bacterial effector that is crucial for bacterial virulence in plants.

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