Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein

Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.     

Combined positive/negative purging and transplantation of peripheral blood progenitor cell autografts in breast cancer patients: a pilot study

Capacitation is an important process in bovine sperm maturation and is an obligatory step prior to fertilization. Two capacitating agents, namely heparin and high-density lipoprotein (HDL), have been shown to induce sperm capacitation. A family of major proteins of bovine seminal plasma designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) bind to the sperm surface upon ejaculation via their membrane choline phospholipids. Our previous studies with bovine epididymal sperm showed that BSP proteins potentiate sperm capacitation induced by heparin and HDL. This study was undertaken to clarify the mechanism of capacitation induced by heparin and HDL in the presence of BSP proteins. Washed bovine ejaculated sperm were incubated with heparin (12 microg/ml) or HDL (10-160 microg/ml) in the presence of polyclonal antibodies against purified BSP proteins (anti-BSP proteins). The percentage of capacitated sperm was evaluated after the induction of the acrosome reaction (AR) with lysophosphatidylcholine. When sperm were incubated for 5 h with heparin and anti-BSP proteins (40 microg/ml), the AR level was not significantly different from control levels (16. 8 +/- 0.9% vs. 12.9 +/- 0.9%). In contrast, incubation of sperm for 8 h with HDL and anti-BSP proteins did not inhibit the AR (42.4 +/- 1.1% vs. 17.1 +/- 1.6 for the control samples). We also investigated the effect of heparin and HDL on protein tyrosine phosphorylation associated with capacitation. The tyrosine phosphorylation of a group of proteins was increased in the presence of heparin. However, HDL did not significantly stimulate protein phosphorylation. The increase in phosphorylation was correlated with an increase in the AR after the incubation with heparin but not with HDL. These results indicate that heparin and HDL mediate capacitation via different mechanisms.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Cellular cholesterol transport and efflux in fibroblasts are abnormal in subjects with familial HDL deficiency

Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre-beta-migrating HDLs but are deficient in alpha-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL3- and apolipoprotein (apo) A-I-mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [3H]cholesterol and [3H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an approximately 25% reduction in HDL3-mediated cellular cholesterol efflux and an approximately 50% to 80% reduction in apoA-I-mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL3 in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL3 binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in 125I-HDL3 binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated ( approximately 2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL3- and apoA-I-mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL3 binding or a defect in a putative HDL binding protein.     

Race/ethnicity misclassification of persons reported with AIDS. The AIDS Mortality Project Group and The Supplement to HIV/AIDS Surveillance Project Group

Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (delta A234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [3H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 37% at lipoprotein concentrations of 10 to 200 micrograms protein/ml. Cholesterol efflux correlated negatively with TBARS production (r = -0.97, P < 0.003) and delta A234 (r = -0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.     

Differential rate of cholesterol efflux from the apical and basolateral membranes of MDCK cells

Epithelial cells contain two distinct membrane surfaces, the apical and basolateral plasma membranes, which have different lipid and protein compositions. In order to assess the effect of the compositional differences of the apical and basolateral membranes on their ability to undergo cholesterol efflux, MDCK cells were radiolabeled with [3H]cholesterol and grown as a polarized monolayer on filter inserts, that separate the upper apical compartment from the lower basolateral compartment. The rate of cholesterol efflux from the basolateral membrane into media containing HDL in the basolateral compartment was 6.3%/h +/-0.7, whereas HDL-mediated efflux from the apical membrane was approximately 3-fold slower (1.9%/h +/-0.3). In contrast, Fu5AH cells, which do not form distinct polarized membrane domains, had a similar rate of HDL-mediated cholesterol efflux into the apical and basolateral compartments. Similar to HDL, other cholesterol acceptors, namely LDL, bovine serum albumin, and a lipid emulsion, also showed a decreased rate of cholesterol efflux from the apical membrane surface versus the basolateral membrane. Compared to the basolateral membrane, the apical membrane was also found to be more resistant to cholesterol oxidase treatment, to bind less HDL, and to take up less cholesterol from the medium. In conclusion, cholesterol efflux occurred less readily from the apical membrane than from the basolateral membrane for all types of acceptors tested. These results suggest that differences in the composition of the apical and basolateral membrane lead to a relative decrease in cholesterol desorption from the apical membrane and hence a reduced rate of cholesterol efflux.     

Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice

The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. The contribution of specific lipoprotein components to this process remains to be established. In this study, the role of human apolipoproteins (apo) A-I and A-II in the efflux of cellular cholesterol was investigated in transgenic mouse models in which the expression of murine apoA-I was abolished due to gene targeting (A-IKO). Serum from A-IKO mice and from mice expressing human apoA-I and/or human apoA-II was incubated with [3H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours at 37 degrees C. The cholesterol efflux to the serum of A-IKO mice was markedly lower than that to the serum of mice transgenic for human apoA-I (5.0 +/- 1.5% versus 25.0 +/- 4.0%). Expression of human apoA-II alone did not modify the cholesterol efflux capacity of A-IKO mouse serum. Cholesterol efflux to serum of mice expressing human apoA-II together with human apoA-I was significantly lower than that to human apoA-I mouse serum (20.0 +/- 2.3% versus 25.0 +/- 4.0%). Regression analysis of cholesterol efflux versus the lipid/apolipoprotein concentrations of mouse serum suggested that 3 independent factors contribute to determine the cholesterol efflux potential of serum: the apolipoprotein composition of HDL, the serum concentration of HDL phospholipids, and the presence of a small fraction of particles containing apoA-I.     

The effects of political and economic transitions on health and safety in Estonia: an Estonian-Swedish comparative study

This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [3H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/ microg) than did cellular FC, and several potential causes of this finding were investigated. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) during loading decreased cell [3H]EC by 95+/-1.4% but decreased cell EC mass by only 66.0+/-4.0%, indicating that some intracellular undegraded AcLDL-derived EC was present in these cells. Esterification of [3H]oleate to EC in THP-1 cells loaded with AcLDL was 2.0 nmol x mg-1 x h-1, consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.0+/-0.07%, 80.0+/-0.5%, and 18.9+/-0.3% of cell [3H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-beta-cyclodextrin (hp-ss-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified <8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP-1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL3 respectively removed 46.6+/-3.7%, 61. 3+/-3.4%, and 76.4+/-10.1% of [3H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T1/2 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles.     

Decreased cholesterol efflux from fibroblasts of a patient without Tangier disease, but with markedly reduced high density lipoprotein cholesterol levels

A 51-yr-old woman without clinical evidence of Tangier disease, but with an extremely low high density lipoprotein (HDL) cholesterol level, was studied. No defect in the major structural protein of HDL, apolipoprotein AI (apo AI), was detected. A preponderance of small HDL particles in the patient's plasma suggested defective uptake of cellular cholesterol. Efflux of [3H]cholesterol from patient fibroblasts to normal apo AI was decreased 50%. Cholesterol efflux to HDL was also decreased, but efflux to trypsin-modified HDL was not. The patient's cells partitioned more exogenously provided [3H]cholesterol into free cholesterol and synthesized greater amounts of phosphatidylcholine than did normal or Tangier fibroblasts. Her fibroblasts did not differ from normal fibroblasts in sterol synthesis rate, cellular cholesterol and cholesterol ester content, or incorporation of oleate into cholesterol ester. The data indicate the presence of a defect in apolipoprotein-dependent cellular cholesterol efflux that differs from that seen in Tangier disease. These findings are the first evidence that other low HDL cholesterol syndromes, besides Tangier disease, may also be associated with cholesterol efflux abnormalities. The identification of mutant genes responsible for apolipoprotein-mediated efflux abnormalities should provide valuable insights into cellular mechanisms involved in the reverse cholesterol transport pathway.     

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.     

Impaired cellular cholesterol efflux by oxysterol-enriched high density lipoproteins

One of the proposed antiatherogenicity role of high-density lipoproteins (HDL) is believed to stimulate removal of cholesterol from the peripheral cells back to the liver for excretion. We have investigated the effects of oxidation-related modifications of HDL on their ability to stimulate cholesterol efflux from cultured cells. Human HDL (HDL3, 1.13 < d < 1.21 g/ml) have been modified either by malondialdehyde or by copper-mediated oxidation (Ox-HDL3). Compared with native HDL3, the modified HDL3 resulted in a significantly reduced efflux of labeled cholesterol from preloaded macrophages (P388D1 cell line). Analysis of lipid composition of Ox-HDL3 by gas chromatography revealed the presence of oxysterols (OS). Enrichment of native HDL3 with oxysterols resulted in a reduced capacity to stimulate cholesterol efflux. The reduced ability of OS-enriched HDL3 to elicit cholesterol efflux may contribute to cellular cholesterol accumulation and subsequently to atherosclerosis.     

Basis for rapid efflux of biosynthetic desmosterol from cells

Previous work shows that the efflux of biosynthetic desmosterol from cells is three times more efficient than that of cholesterol. To explain this difference, we labeled CHO-K1 cells with [3H]acetate precursor and measured sterols in the whole cells, plasma membranes and caveolae, and those released to high density lipoprotein (HDL3). The [3H]desmosterol-to-[3H]cholesterol ratio was similar in the plasma membrane and whole cells but was greater in HDL3, suggesting that the more efficient efflux of desmosterol is due to more rapid desorption from the plasma membrane. The ratio in caveolae was similar to that in whole cells, arguing against selective delivery of desmosterol to caveolae as an explanation for the more rapid efflux of this sterol. Additionally, to demonstrate that the enhanced release of desmosterol was not due to enhanced intracellular cycling, we made vesicles from CHO-cell plasma membranes labeled with [3H]desmosterol or [14C]cholesterol, and the rapid release of desmosterol was demonstrated in this system. To characterize sterol efflux from a simple lipid bilayer system, we measured the transfer of cholesterol and desmosterol between large unilamellar vesicles (LUV), and found that desmosterol transferred two to three times more rapidly than cholesterol. A similar differential was seen when HDL3 or low density lipoprotein (LDL) served as the acceptor. These results show that the greater efflux efficiency of biosynthetic desmosterol can be attributed to more efficient desorption from the plasma membrane, and that this difference is a property of the sterols' association with the lipid bilayer. In vivo, the rapid efflux of biosynthetic sterol intermediates, followed by efficient delivery to the liver, may constitute an important mechanism for preventing various types of pathology associated with these materials.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.     

Expression of ubiquitin protein in each organ at death from hypothermia

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.     

pH regulation in mouse sperm: identification of Na(+)-, Cl(-)-, and HCO3(-)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation

Intracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na(+)-dependent Cl(-)-HCO3- exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates in N-methyl-D-glucamine+ Cl- or in 0.25 M sucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na(+)-H+ antiport pathway and the Na(+)-independent Cl(-)-HCO3- exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHi increases (from 6.54 +/- 0.08 to 6.73 +/- 0.09) that require a functional Na(+)-, Cl(-)-, and HCO3(-)-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHi regulator.     

Potencies of lipoproteins in fasting and postprandial plasma to accept additional cholesterol molecules released from cell membranes

To investigate the role of various lipoproteins in plasma to promote cholesterol efflux from cell membranes, potencies of lipoproteins in normolipidemic fasting and postprandial (PP) plasmas to accept additional cholesterol molecules from cell membranes were determined. We used red blood cells (RBCs) and lipoproteins in fresh blood as donors and acceptors of cell membrane cholesterol, respectively. When fresh fasting plasma (n=24) containing active lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer proteins (CETP) was incubated with a 3-fold excess of autologous RBCs at 37 degrees C for 18 hours, plasma cholesterol levels increased by 19.6% (38.5+/-14.2 mg/dL) owing to an exclusive increase in the CE level. Very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions retained 48.1%, 26.3%, and 25.6% of the net cholesterol mass increase in fasting plasma, resulting in 91%, 8%, and 21% increases in their cholesterol contents, respectively. The PP plasma was 1.3-fold more potent than fasting plasma in promoting cholesterol efflux from RBCs by associating excess cholesterol with chylomicrons, resulting in a 356% increase in the cholesterol content of chylomicrons. These increases in lipoprotein cholesterol content indicate that chylomicrons were about 3.9x, 44x, and 17x more potent than fasting VLDL, LDL, and HDL, respectively, in accepting additional cholesterol molecules released from RBCs. The capacity of PP plasma to promote cholesterol efflux from RBCs was significantly correlated with plasma cholesterol levels (r=0.60, P<0.005), triglycerides (r=0.68, P<0.001), chylomicrons (r=0.90, P<0.001), VLDL (r=0.65, P<0.001), and LDL (r=0.47, P<0.025) but not with the levels of HDL (r= -0.34, P<0.20). In fasting plasma containing a low level of VLDL and HDL, isolated chylomicrons supplemented to the plasma were approximately 9x more potent than HDL in boosting the capacity of plasma to promote cholesterol efflux from RBCs. This study indicates that chylomicrons in PP plasma are the most potent ultimate acceptors of cholesterol released from cell membranes and that a low HDL level is not a factor that limits the ability of PP plasma to promote cholesterol efflux from cell membranes. Our data obtained from an in-vitro system suggest that PP chylomicrons may play a major role in promoting reverse cholesterol transport in vivo, since the transfer of cholesterol from cell membranes to chylomicrons will lead to the rapid removal of this cholesterol by the liver. HDL in vivo may promote reverse cholesterol transport by enhancing the rapid removal of chylomicrons from the circulation, since the rate of clearance of chylomicrons is positively correlated with the HDL level in plasma.     

Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.     

Cholesterol transport between cells and high density lipoprotein subfractions from obese and lean subjects

We studied the pathway of cholesterol efflux from fibroblasts by testing plasma samples from obese and lean subjects. Plasma samples were incubated with [3H]cholesterol-labeled human skin fibroblasts for 1 h to ensure uniform labeling of all of the high density lipoprotein (HDL) subfractions. Supernatants were then transferred to unlabeled cells and the displacement of labeled cholesterol within HDL subfractions by unlabeled cellular cholesterol was analyzed in short-term experiments. Plasma samples of obese subjects were characterized by a lower content of total apolipoprotein A-I (apoA-I) and alpha1-HDL and a lower overall capacity to take up labeled cholesterol. In plasma of lean subjects, pre beta2-HDL and alpha1-HDL appeared to be the most active particles in the initial uptake of unlabeled cellular cholesterol. By contrast, in plasmas of obese subjects, the pre beta1-HDL appeared to be most active in taking up unlabeled cellular cholesterol and transferring [3H]cholesterol. There were negative correlations between body mass index (BMI) and apoA-I and alpha1-HDL concentrations, and with the apparent increments of cellular cholesterol uptake within pre beta2-HDL and alpha1-HDL, as well as with the overall capacity to promote cholesterol efflux. By contrast, BMI was positively correlated with the apparent increment in cellular cholesterol within pre beta1-HDL. While cholesterol efflux was correlated with total plasma apoA-1, there were no such correlations with the concentration of any individual HDL subfraction. We conclude that the pattern of cholesterol transfer between fibroblasts and high density lipoprotein particles is influenced by body fatness and may be a factor in the abnormal metabolism of HDL in obesity.     

A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formation of alpha-high density lipoproteins (HDL) but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma

ApoA-I(L141R)Pisa is a naturally occurring apolipoprotein A-I variant that causes virtual absence of HDL in hemizygotes and hypoalphalipoproteinemia with half-normal levels of HDL-cholesterol in heterozygotes. In this study we analyzed the distribution of HDL subclasses in plasmas of four hemizygotes for this mutation. We also investigated the abilities of these plasmas to esterify cholesterol and to promote cholesterol efflux. Residual apoA-I-containing lipoproteins in plasmas of hemizygotes for apoA-I(L141R)Pisa correspond to pre beta 1-LpA-I and small alpha-LpA-I. Unlike normal pre beta 1-LpA-I, pre beta 1-LpA-I of apoA-I(L141R)Pisa hemizygotes was not converted into a larger alpha-migrating particle. Plasmas of apoA-I(L141R)Pisa hemizygotes were significantly reduced in their activity to esterify cholesterol in either endogenous or exogenous lipoproteins. Cholesterol efflux capacity was significantly lower than that of normal plasma. Efflux of [3H] cholesterol from radiolabeled fibroblasts into apoB-depleted plasma of normal probands was biphasic with fast cholesterol efflux occurring in the first minute. Thereafter, cholesterol efflux was slow and unsaturable. After incubation with radiolabeled fibroblasts, efflux values of [3H]cholesterol into apoB-depleted plasma from normal controls and from apoA-I(L141R)Pisa hemizygotes were indistinguishable at 1 min. Longer incubations with apoB-free plasma from apoA-I(L141R)Pisa hemizygotes did not, however, lead to the unsaturable increase in cholesterol efflux that was observed during incubations with apoB-free plasma of normolipidemic probands. Pre beta 1-LpA-I of apoA-I(L141R)Pisa hemizygotes took up significantly less cell-derived [3H]cholesterol than pre beta 1-LpA-I of normal donors. We conclude that apoA-I(L141R)Pisa interferes with the formation of lipid-rich alpha-HDL but not with that of lipid-poor pre beta 1-LpA-I. Very low concentrations of alpha-HDL in plasmas of apoA-I(L141R)Pisa hemizygotes combined with reduced LCAT activity cause a decrease of the slow, unspecific, and LCAT-dependent components of cholesterol efflux into plasma.