Effect Of Cholesterol Addition Research Articles

22 EFFECT OF CHOLESTEROL ADDITION TO EQUINE SPERM MEMBRANE ON FERTILITY

Artificial insemination with cooled-shipped semen has been widely used in horse breeding. However, some stallions, referred to as poor coolers, present abrupt fertility decrease when their semen is processed, cooled, and transported. Cholesterol incorporation into sperm membranes improves the quality of cryopreserved semen by increasing the sperm membrane stability and fluidity at low temperatures. Despite the beneficial effect of cholesterol addition on sperm quality, studies demonstrate that the presence of large amounts of cholesterol in the plasma membrane interferes with the physiological process of sperm capacitation and is detrimental to frozen equine sperm fertility. The aim of this study was to assess the fertility of cooled semen from good-cooler and poor-cooler stallions after adding cholesterol to sperm membranes. Two stallions were used and classified as good cooler (n = 1) and poor cooler (n = 1) based on the ability to maintain sperm progressive motility after 24 h of cooling at 5°C. For classification of the stallions, the fertility history was also taken into account through the results of pregnancy per cycle using inseminations with cooled semen (<50% for poor cooler and >70% for good cooler). Ejaculates of these stallions were subjected to 2 treatments: control (CON) and cholesterol (CLC). In the CON group, the semen was extended to 30 × 106 sperms mL–1 with skim milk-based extender (BotuSemen™). In the CLC group, the semen was extended as in the CON group plus 0.25 µL/1 × 106 sperms of 6.1 mM cholesterol-loaded cyclodextrin was added. Afterwards, both treatments were cooled at 5°C for 24 h. To test the fertility of poor-cooler and good-cooler stallions, 2 cycles from 25 mares and 2 cycles from 10 mares were respectively used. For both stallions, randomly for each mare, the inseminations were performed by alternating both treatments. If the mare was first inseminated with the CLC treatment, in the next cycle the CON treatment was used and vice versa. After 24 h of ovulation induction, the inseminations were done in the uterine body with 1 × 109 viable cells. Statistical analyses were performed using the Fisher exact test and significance was set at P < 0.05. For the poor cooler, the CON treatment presented 44% pregnancy/cycle compared to 76% for the CLC treatment (11/25a v. 19/25b). For the good cooler, both treatments presented 80% (8/10) pregnancy/cycle. The results suggest that the fertility capability of stallions is not affected by incorporation of cholesterol-loaded cyclodextrin to the sperm plasma membrane. Additionally, the utilization of cholesterol-loaded cyclodextrin may be an option to enable the utilization of cooled-shipped semen from poor cooler stallions for AI programs.

The Effect of Cholesterol Addition, Buffer, and pH on Equine Sperm Stored at 5°C

Cooled stallion semen has a short viable life, which ranges with acceptable motility and viability from 24 up to 48 hours. The purpose of this study was to compare the effects of storage pH, the ability of three different zwitterionic buffers, and cholesterol-loaded cyclodextrins (CLC) to preserve the motility and integrity of stallion sperm cooled to 5°C for 48 hours. Fourteen ejaculates were collected and split to receive CLC or not (control group). After incubation, each sample was split into six subsamples and diluted in KMT extender containing 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), or 2-(N-morpholino)ethanesulfonic acid (MES) buffers, and the final pH was adjusted to either 7.0 or 6.6, totalizing 12 experimental groups as a function of CLC, buffer, and pH variables (2 × 3 × 2 factorial). The motility parameters and integrity of plasma and acrosome membranes (live cell index) were determined using computer-automated semen analysis and epifluorescence microscopy at 3, 6, 24, and 48 hours of cooling period. According to results, pH was not a significant source of variation for motility and live sperm over different cooling periods. However, samples diluted in BES exhibited higher progressive motility within 3 hours and higher percentages of total motile cells after 48 hours of incubation at 5°C (P < .05). After 24 hours of storage, CLC-treated sperm samples presented higher motility than control group (P < .05), and after 48 hours of incubation, CLC-treated sperm exhibited higher percentages of live, motile, and progressively motile sperms (P < .05). We inferred that equine semen diluted in KMT containing BES as buffer and CLC treatment improve the equine sperm survival during storage at 5°C for 48 hours.

Development of ethosome-like catanionic vesicles for dermal drug delivery

A carrier for enhanced skin delivery of drugs has ever been discovered and named “ethosomes,” which are phospholipid vesicular systems embodying ethanol in relatively high concentrations. This work aims at developing competent ethosome-like catanionic vesicles for dermal drug delivery. The preparation of double-chained catanionic surfactant (or ion-pair-amphiphile, IPA) DeTMA-DS from single-chained cationic surfactant (decyltrimethylammonium bromide, DeTMAB) and anionic surfactant (sodium dodecyl sulfate, SDS) by the precipitation method was demonstrated first. This lipid-like surfactant was thereafter used as the material to prepare the catanionic vesicles with the aid of ethanol as the cosolvent in aqueous buffer solution by a simple semispontaneous process. Formability and physical stability of the as-prepared catanionic vesicles were discussed based on the viewpoint of mixed solvent dielectric constant. The potential application of the catanionic vesicles as nano-carriers in dermal drug delivery was demonstrated by the encapsulation of vitamin E acetate (α-tocopherol acetate, α-TA). Furthermore, effects of cholesterol addition on physical stability, bilayer membrane rigidity, and encapsulation efficiency of the catanionic vesicles with 20% ethanol, in which the most stable vesicle can be formed, was systematically studied. The experimental results revealed that vesicle stability can be enhanced further by the addition of cholesterol. The vesicle membrane rigidity and encapsulation efficiency are both increased with the increase of cholesterol addition. It is, therefore, suggested that the encapsulation efficiency of α-TA in the catanionic vesicles is dependent on the vesicle membrane rigidity. Finally, the results of gelation of catanionic vesicles by a water soluble polymer with hydrophobical modification were also reported and the role played by the vesicular bilayer rigidity in the interaction between catanionic vesicles and polymer molecules were discussed. The phase maps and the rheological properties obtained for mixtures of catanionic vesicles and polymer molecules may provide useful information for practical use of the catanionic vesicles in dermal delivery of drugs.

Phase Behavior and Hydrated Solid Structure in Lysophospholipid/Long-chain Alcohol/Water System and Effect of Cholesterol Addition

Phase behavior in lysophospholipid/long-chain alcohol/water system at 80°C was investigated using hexanol and oleyl alcohol as the long-chain alcohol. Similarly to hydrophilic surfactant, a micellar phase in a lysophospholipid/water system transitioned to a lamellar liquid-crystalline phase by the addition of long-chain alcohol. In the oleyl alcohol system the lamellar liquid-crystalline phase was observed in wider region compared to the hexanol system. The effect of cholesterol addition on the phase behavior was also studied. The region of liquid-crystalline phase and (reverse micellar + liquid-crystalline + water) phase shifted towards higher lysophospholipid concentrations. The structure of hydrated solid as well as the transition between lamellar liquid-crystalline phase and hydrated solid was analyzed by X-ray scattering measurement and differential scanning calorimetry measurement. It was revealed that the hydrated solid was α-type crystals with lamellar structure. The hydrated solid (gel)-liquid crystal transition temperature gradually decreased with increasing oleyl alcohol concentration and the decrement was enhanced by the addition of cholesterol.

Kinetic and equilibrium studies of incorporation of di-sulfonated aluminum phthalocyanine into unilamellar vesicles

The interactions of cis-di-sulfonated aluminum phthalocyanine (PcS 2Al) with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles have been investigated by fluorescence spectroscopy. At pH 7.0, PcS 2Al incorporates into the vesicles with a high affinity constant (2.7×10 6 M −1, in terms of phospholipid concentration). The fluorescence changes following rapid mixing of PcS 2Al with vesicles are biphasic. The first phase is attributed to the entry of PcS 2Al into the vesicles, as deduced from the linear dependence of the rate upon lipid concentration. More surprisingly, this rate is strongly pH dependent with a marked maximum around pH 7.3, a result interpreted in terms of the coordination state of the aluminum ion in aqueous solutions. At this pH, a hydroxide ion neutralizes the residual positive charge of the metal ion that remains unbalanced after coordination by the phthalocyanine cycle. A water molecule is likely to complete the metal coordination sphere. Only this form, PcAl +(OH −)(OH 2), with an uncharged core is quickly incorporated into the vesicles. The protonation of OH − or the deprotonation of the coordinated H 2O leading to a positively or negatively charged core, respectively, account for the observed pH effect. Studies on the effect of cholesterol addition and exchange of PcS 2Al between vesicles and albumin all indicate the absence of transfer of the phthalocyanine between the vesicle hemileaflets, a result expected from the presence of the two negatively charged sulfonated groups at the ring periphery. Instead, the slower kinetic phase is likely due to the movement of the phthalocyanine becoming more buried within the outer leaflet upon the loss of the water molecule coordinated to the aluminum ion.

Effect of cholesterol addition on growth kinetics and shear stress sensitivity of adherent mammalian cells

The growth kinetics of adherent baby hamster kidney cells cultivated with additional cholesterol as well as the effect of cholesterol addition on shear stress sensitivity were investigated. The influence of various cholesterol preparations was tested, whereby dimethylsulfoxide and ethanol show negative effects at higher concentrations. With addition of cholesterol in the range of 90 μg ml −1, a positive effect on the shear stress resistance was achieved.

Electron spin resonance line-shape analysis of x-doxylstearic acid spin probes in dihexadecyl phosphate vesicles and effects of cholesterol addition

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTElectron spin resonance line-shape analysis of x-doxylstearic acid spin probes in dihexadecyl phosphate vesicles and effects of cholesterol additionPeter J. Bratt and Larry KevanCite this: J. Phys. Chem. 1993, 97, 28, 7371–7374Publication Date (Print):July 1, 1993Publication History Published online1 May 2002Published inissue 1 July 1993https://doi.org/10.1021/j100130a041RIGHTS & PERMISSIONSArticle Views83Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (477 KB) Get e-Alerts

Electron spin echo modulation studies of doxylstearic acid spin probes in frozen dihexadecyl phosphate and dioctadecyldimethylammonium chloride vesicles: interaction of the spin probe with deuterated water and effects of cholesterol addition

Electron spin echo studies have been carried out for a series of x-doxylstearic acid (x=5, 7, 10, 12, and 16) and 3-doxyl-5α-cholestane spin probes in frozen deuterated aqueous solutions of anionic dihexadecyl phosphate (DHP) vesicles and cationic dioctadecyldimethylammonium chloride (DODAC) vesicles. Modulation effects due to interactions of the spin probe nitroxide group with D 2 O at the vesicle interface give distance-based information which can be used to draw conclusions about the ordering and chain packing of the vesicles

Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron spin echo modulation studies of doxylstearic acid spin probes in frozen vesicles: interaction of the spin probe with water-d2 and effects of cholesterol addition

Electron spin echo studies have been carried out for a series of x-doxylstearic acid (x = 5, 7, 10, 12 and 16) spin probes in frozen deuteriated aqueous solutions of phospholipid vesicles and cationic dioctadecyldimethylammonium chloride (DODAC) vesicles. Modulation effects due to interactions of the nitroxide group of the spin probes with D{sub 2}O give information about the conformations of the probes and the degree of hydration of the surfactant headgroups as well as about the degree of packing of the alkyl chain. We show that DODAC headgroups are more hydrated than choline headgroups and that the doxylstearic acid probes show a larger tendency for bending in DODAC vesicles than in phospholipid vesicles. Upon addition of cholesterol into phospholipid vesicles, the headgroups are separated and their degree of hydration increases.

Delipidation of a beta-adrenergic receptor preparation and reconstitution by specific lipids.

The role of lipids in the function of membrane receptors for hormones and neurotransmitters is still obscure. To gain information on this subject, a delipidated receptor preparation was developed. The beta-adrenergic receptor from turkey erythrocyte membranes was solubilized in deoxycholate and was freed extensively of phospholipids and of cholesterol by gel filtration. The delipidated preparation, after removal of the detergent, showed little, if any, ligand binding to the receptor as measured with the beta-adrenergic antagonist [125I] iodocyanopindolol. Readdition of soybean lipids restored specific radioligand binding. The lipid reconstituted receptor demonstrated agonist and antagonist binding affinities which were not very different from those of the native receptor. The receptor also retained its ability to function, as demonstrated by transfer to a foreign adenylate cyclase system. The delipidated receptor preparation lent itself conveniently to study the requirement for specific lipids in restoration of agonist and antagonist binding. Phosphatidylethanolamine restored maximal binding. Acidic phospholipids and sphingomyelin were inefficient in reconstitution of the receptor. The effect of cholesterol addition was also investigated. Binding was dramatically increased when a cholesterol ester was added in mixture with the acidic phospholipids, cardiolipin or phosphatidylinositol. Further studies unexpectedly revealed that reconstitution of the delipidated receptor is not exclusively dependent on the addition of a phospholipid; a mixture of 1-monooleylglycerol with cholesteryl hemisuccinate restored binding as efficiently as phosphatidylethanolamine. The presently described preparation should be useful in elucidating the part played by lipids in the action of the receptor in the adenylate cyclase system.

Free radical label: new approach to the study of super-slow molecular dynamics of lipid systems

A new method of EPR-spectroscopy, the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation, was applied to the study of molecular dynamics of dimyristoyl phosphatidylcholine in mass and in the structure of liposomes above and below the transition temperature. It was shown that the mobility of lipid molecules in crystalline liposomes was lower than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreased the lateral molecular motion of lipids in crystalline and liquid-crystalline states; in the latter case, the effect of cholesterol addition was more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown that the lipid matrix possesses a high degree of heterogeneity. The solubility of oxygen in the lipid bilayer and the mechanism of lipid diffusion are discussed.

Effect of cholesterol on the non-electrolyte permeability of planar lecithin membranes.

IT is well known that the addition of cholesterol to artificial lipid membranes decreases their permeability. Most of the studies on artificial systems have been performed on liposomes1–4, whereas the effect of the cholesterol on planar lipid bilayers was investigated on water transport only. In particular Finkelstein and Cass5 found that addition of cholesterol to membrane of lecithin resulted in a decrease of water permeability. In the present work we studied the effect of cholesterol addition on the non-electrolyte permeability of lecithin planar membranes. The artificial membranes were formed with egg lecithin dissolved in decane to form a 0.5% solution. Lecithin was extracted from egg yolks6 and purified by alumina chromatography. Cholesterol, twice crystallised, was added to lecithin solution to obtain cholesterol–lecithin molar ratios of 1:1 or 2:1.