Glycerylphosphorylcholine Research Articles

Preparation of phosphorylcholine derivatives of bovine serum albumin and their application to the affinity chromatography of C-reactive protein

A simple method for the preparation of phosphorylcholine derivatives of bovine serum albumin (PC-BSA) by reductive alkylation of the amino groups of bovine serum albumin with choline phosphoryl glycoaldehyde is described. Choline phosphoryl glycoaldehyde was generated by periodate oxidation of glyceryl phosphorylcholine. PC-BSA was immobilized on SH-derivatized Toyopearl HW 65 by reacting the single SH group of PC-BSA with a bismaleimido reagent and then coupling maleimidated PC-BSA to the thiolated gel. The affinity purification of C-reactive protein (CRP), which is based on the Ca 2+-dependent affinity of CRP for the phosphorylcholine residue of PC-BSA, was readily accomplished using the PC-BSA Toyopearl HW 65 column. The resulting CRP preparation from serum and pleural fluid was homogenous as assessed by native polyacrylamide gel electrophoresis. PC-BSA derivatives were also shown to be reactive with Limulus polyphemus CRP.

Stallion sperm and seminal plasma phospholipids and glycerylphosphorylcholine

Total phospholipid concentration in stallion sperm was 1.17 ± 0.05 μmole/10 9 spermatozoa (mean ± S.D., n = 33). Six identified phospholipid components included phosphatidyl choline (27%), sphingomyelin (13%), choline plasmalogen (12%), phosphatidyl ethanolamine (13%), ethanolamine plasmalogen (6%) and cardiolipin (5%). Of the saturated fatty acids, palmitic acid was in highest concentration (22%) with lower concentrations of tridecanoic (6%), myristic (7%), pentadecanoic (11%), stearic (6%), arachidic (1%) and behenic (1%). Unsaturated fatty acids in highest concentration were arachidonic (18%), docosapentaenoic acid (17%), oleic (5%) and docosahexaenoic (8%). The overall ratio of unsaturated to saturated fatty acids in the phospholipids of stallion sperm was 0.90. The concentration (mean ± S.D.) of total phospholipid and glycerylphosphorylcholine (GPC) in stallion seminal plasma were 28.4 ± 20.2 μmole/100 ml and 55.8 ± 30.3 mg/100 ml, respectively.

Physiology of the epididymis and spermatozoa

The epididymis is an ideal extragonadal target site to inhibit fertility in the male. Synthesis and secretion of constituents like sialic acids, protein and glycerylphosphoryl choline by the epididymal epithelium under androgen control provide an ideal fluid environment for sperm maturation. An optimal level of sialic acid secretion by the epididymal epithelium is needed to maintain functional integrity of sperm. The existence of specific androgen receptors in the epididymis and spermatozoa are related to their ability to metabolise androgens.

A study of phospholipase in albumin and its role in inducing the acrosome reaction of guinea pig spermatozoa in vitro.

Phospholipase activity associated with guinea pig spermatozoa during capacitation and the acrosome reaction (AR) was determined by measuring the production of [14C] glycerylphosphorylcholine (GPC) from [14C] phosphatidylcholine (PTC) over a 2-hour period at the beginning and at the end of a 6-hour incubation. Spermatozoa converted 13 X more [14C] PTC to [14C] GPC during the last 2 hours of incubation than during the first 2 hours which corresponded to an increase in AR from 7% at 2 hours to 55% at 6 hours. In vitro studies also were performed to assess the role of phospholipase activity in bovine serum albumin (BSA) in inducing the AR of guinea pig spermatozoa. Spermatozoa, obtained by backflushing the cauda epididymidis, were incubated for up to 6 hours in Biggers, Whitten, and Whittinghams' (BWW) medium alone or containing various additives. These additives were: 0.1% BSA, phospholipase A2, p-bromophenacyl bromide, DMSO, 0.1% BSA plus p-bromophenacyl bromide, or 0.1% BSA plus DMSO. Each hour of incubation, samples were assessed for the percentage of AR sperm and sperm motility. The percentage of AR sperm after 6 hours in BWW supplemented with phospholipase A2, at the level of PTC hydrolyzing ability detected in BWW with 0.1% BSA, was similar to that of BWW and BSA. However, the percentage of AR sperm in BWW with BSA and the phospholipase inhibitor p-bromophenacyl bromide was significantly lower than that in BWW with BSA or phospholipase A2 and similar to that in BWW without BSA. Sperm motility was significantly less in incubations containing phospholipase A2, but lacking BSA. It was concluded that phospholipase activity in BSA may contribute to capacitation and the AR of guinea pig sperm in vitro by mimicking the action of native sperm phospholipase.

Biochemical components of stallion seminal plasma before and after the breeding season

Seasonal and breed differences were determined for glyceryl-phosphoryl-choline (GPC), ergothioneine (EGT), fructose and total protein in the seminal plasma of 24 stallions from three breed groups. Significant differences were found between the GPC, EGT, fructose and total protein levels before and after the breeding season. During the post-breeding period, the concentrations of EGT, fructose and total protein increased more than 100% while that for GPC decreased by about 50%. This phenomenon was found in each of the breed groups investigated. Breed differences in the GPC level during the pre- and post-seasonal periods were not significant. Differences were found only during the pre-seasonal period in EGT, fructose and total protein levels.

Isolation of epithelial cells from the corpus epididymidis and analysis for glycerylphosphorylcholine, sialic acid, and protein.

Enzymatically dispersed cells from the rat corpus epididymidis were separated by unit gravity sedimentation and centrifugal elutriation. Based on differential cell counts, principal cells isolated by centrifugal elutriation were 55% pure, while basal cells and fibroblasts were 88% and 61% pure, respectively. Compared to unit gravity separation, purity was an average of 7% higher in principal and basal cell fractions obtained by elutriation. Viability was greater than 87% by either method, but yields following elutriation averaged 23% higher for basal cells, 78% higher for principal cells, and 290% for fibroblasts than corresponding yields following unit gravity separation. Analyses for epididymal secretory products indicated that cellular content (mumoles/10(9) cells) of glycerylphosphorylcholine (GPC), sialic acid, and protein was greater in principal cells than other cell types (p less than .05). When expressed on the basis of cellular volume, concentrations of GPC and protein also were significantly greater in basal and principal cells than fibroblasts (p less than .05). Caput sperm contained significantly more GPC than either corpus or cauda sperm (p less than .005). Protein concentrations in caput and corpus sperm were similar, but concentrations were lower in cauda sperm (p less than .005). Levels of sialic acid did not vary among sperm from different epididymal regions. It was concluded that principal cells from the corpus epididymidis contain major quantities of three epididymal secretory products and that regional differences in the function of the epithelium are present.

Antifertility effects of chronically administered Malvaviscus conzattii flower extract on male albino mice.

The antifertility effects of alcoholic extract of Malvaviscus conzattii flowers were observed on male albino mice after oral administration of 30 and 50 doses (one dose = 50 mg/day/mouse). The genital degenerative changes were found to be more pronounced when the results were observed after oral administration of 50 doses. The drug administration resulted in mass atrophy of the spermatogenic elements and the testicular stages were left with only 1-2 cell layers. Epididymal epithelium was regressed and the lumina were either devoid of spermatozoa or contained its debris. The flower extract treatment produced a significant decrease in absolute weights of testes, epididymes, vas deferens and seminal vesicles. Administration of 50 doses produced a significant reduction in levels of protein (P < 0.001; P < 0.001 and P < 0.001) and sialic acid (P < 0.005; P < 0.005 and P < 0.001) of testes, epididymes and seminal vesicle respectively. Glyceryl phosphoryl choline of epididymes also reduced (P < 0.001) significantly at 50 doses. In contrast, the contents of cholesterol (P < 0.001) and glycogen (P < 0.001) were elevated when observed after 50 doses. Alkaline phosphatase activity in testes, epididymes and seminal vesicles remained within normal range. Fructose of seminal vesicles was reduced (P < 0.001) significantly at 30 doses, then attained normaly after 50 doses. Reduced androgen production was reflected in low levels of sialic acid and protein in testes, epididymes and seminal vesicles. In conclusion, the administration of M. conzattii flower extract caused an effective impairment of spermatogenesis in albino mice after 50 doses, thus inducing an antifertility state.

The effects of carnitine, glycerylphosphorylcholine, caffeine, and egg yolk on the motility of rat epididymal spermatozoa

AbstractExperiments were performed to further the understanding of epididymal processes involved in the acquisition of sperm motility. Samples of luminal contents were collected by micropuncture from four regions of the rat epididymis. These samples were incubated in various diluents to observe the effects of the diluents on sperm motility. Consonant with previous reports, 40 mM glycerylphosphorylcholine (GPC) and 60 mM DL‐carnitine reduced overall motility scores of cauda epididymidal spermatozoa but did not prevent normal initiation of motility. Additionally, control sperm cells and cells treated with carnitine could reinitiate full motility after becoming immotile. Spermatozoa treated with GPC could not reinitiate motility. The sperm cells in our system thus react to GPC and carnitine in fundamentally different ways, the exact nature of which remains to be determined.Spermatozoa from the distal caput epididymidis evidenced high motility scores when diluted in a 5% egg yolk + 10 mM caffeine diluent. It was demonstrated, however, that the subjective appearance of full motility in these immature cells was not supported by actual progressive motility as measured in an assay of linear distance traveled. It was concluded that neither 10 mM caffeine, 5% egg yolk, nor their combination was sufficient to induce progressive motility in immature rat spermatozoa.

Glycerylphosphorylcholine, Sialic Acid and Protein in Epithelial Cells Isolated from the Rat Caput Epididymidis by Elutriation

Fractions containing principal cells (72%), basal cells (88%) and fibroblasts (75%) were isolated from enzymatically dispersed rat caput epididymidis by centrifugal elutriation. Viability of cells in all fractions was >89%. Biochemical analyses of separated cells for suspected epididymal secretory products revealed that total cellular contents of glycerylphosphorylcholine (GPC), sialic acid and protein were significantly greater in principal cells than in basal cells or fibroblasts. When data were expressed as μmoles/g cellular protein, levels of sialic acid were similar among cell types, while principal cells contained two times more GPC than did other cell types. The cellular concentration (μmoles/cm3 cellular volume) of GPC was significantly greater in basal and principal cells than in fibroblasts, while the protein concentration in basal cells was greater than in other somatic cell types. We concluded that epididymal secretions of GPC, sialic acid and protein are derived primarily from principal cells.

Origin of glycerylphosphorylcholine, inositol, N-acetylaminosugar, and prostaglandins in human seminal plasma and their effects on sperm metabolism.

The origin of glycerylphosphorylcholine (GPC), N-acetylaminosugar, inositol, and prostaglandins in human seminal plasma was investigated by correlating the concentration of these components in split ejaculates with known marker constituents. Fructose and acid phosphatase were selected as markers of the secretory activity of the seminal vesicles and prostate gland, respectively, and spermatozoa indicated epididymal origin. The concentration of fructose was lowest in the first fraction of the semen and increased to a maximum in the final portion. Prostaglandins E and F and N-acetylaminosugar values closely followed this pattern, indicating that these components originate in the seminal vesicles. The concentration of spermatozoa was high in the first two fractions, decreasing to a minimum in the final fraction. The distribution of GPC was similar to that of the spermatozoa, indicating that the epididymis secretes this compound. Inositol levels were similar in all fractions, indicating that it is probably present in epididymal, vesicular, and prostatic fluid. Human spermatozoa were unable to utilize N-acetylglucosamine or inositol. High concentrations of some prostaglandins (100 micrograms/ml of PGF1 alpha, 15S 15 met. F2 alpha, PGA1, and PGA2) depressed the endogenous oxygen uptake of human spermatozoa.

Seminal Plasma Concentration of Glycerylphosphorylcholine Before and After Vasectomy and Vas Reanastomosis

The concentration of glycerylphosphorylcholine (GPC) was determined in semen obtained from normal fertile, vasectomized, and vas-reanastomosed subjects. Concentrations of GPC were markedly lower in the semen of vasectomized men. GPC levels observed in vas-reanastomosed subjects were similar to those found in normal fertile men. Vasectomy may not affect GPC synthesis significantly.

The Role of Antiandrogenic Action in Cyproterone Acetate-Induced Morphologic and Biochemical Changes in Human Semen

Following the daily administration of 10 mg of cyproterone acetate to three normal fertile human volunteers for 12 to 16 weeks, there was a marked decrease in the count, motility, and cervical mucus-penetrating ability of spermatozoa, with a concomitant increase in abnormal and immature forms. The levels of seminal acid phosphatase and glycerylphosphoryl choline were also significantly decreased. Subsequently, concurrent daily administration of 75 mg of mesterolone increased the count, motility, and cervical mucus-penetrating ability of spermatozoa and stimulated the seminal biochemical constituents. The results indicate that the effects of a low dose of cyproterone acetate are due mainly to peripheral androgen deprivation.

The Role of Phosphate Esters in Male Fertility

Spermatozoa do not achieve full maturation and fertilizing capacity until passage through the epididymis. During this time they also gain motility, although spermatozoa do not move until after ejaculation. The organic fraction of human seminal plasma contains phosphate esters, particularly glycerylphosphorylcholine (GPC), phosphorylcholine (PCh), and inorganic phosphate (Pi). GPC is found in relatively high concentrations in the semen of many male animals, including man. GPC is synthesized by the epithelial cells of the epididymis, apparently under androgenic control. Consequently, it has been suggested that GPC might be a useful indicator of epididymal function. We have measured GPC, Pi, and PCh in fresh and frozen semen samples, using phosphorus nuclear magnetic resonance (31P NMR). All samples were assayed for phosphate esters. It was found that PCh was totally hydrolized to Pi. The average ratio of GPC to total phosphate (TP = GPC + Pi) remained constant at a value of about 0.1 for sperm counts over 20 million/ml. The ratio for azoospermic specimens was 0.02 or less; the same results were obtained from vasectomy specimens. This finding indicates that most of the GPC comes from the epididymis. There was a significant correlation between motility, progression, and the GPC ratio. Poor motility and progression in the specimens were accompanied by low GPC ratios regardless of the sperm counts.

Composition of rabbit semen and the origin of several constituents.

Semen was collected twice a day every other day (2X/48 h) for 36 days from 12 Dutch-belted rabbits. Sex drive, ejaculate volume, microscopic appearance of the semen and sperm counts were recorded. Seminal plasma was analyzed for itcontent of fructose, citric acid, glycerylphos- phorylcholine (GPC) and the minerals Na, K, P, Mg, Ca and Zn. Six males were exsanguinated 24 h after the last semen collection and 6 were exsanguinated 5 weeks later. The organs of the reproductive tract were weighed and analyzed for their contents of spermatozoa, fructose, citric acid and GPC. Rabbits differed in libido and several characteristics of semen. Second ejaculates had lower concentrations of fructose, citric acid, K and Zn than did the first ejaculates. Fructose was found chiefly in the different portions of the prostate gland and major concentrations of citric acid were found in the ampulla of the ductus deferens and in the vesicular gland. GPC concentrations were highest in the caput and cauda epididymides. Correlations among different constituents were calculated and their significance is discussed.

Functional maturation of the epididymis in the rat.

Weight, histological and biochemical changes in the rat epididymis were investigated during prepubertal, pubertal and postpubertal periods. The phase of most rapid growth of the epididymis commenced at 21 days and extended to 60 days of age; this period corresponded closely to the onset of androgen production at 3 weeks and stabilization of the leydig cell number at 60 days. Histological differentiation in the caput epididymis started before sperm entry and was complete in the cauda only several days after the spermatozoa had appeared. The presence of appreciable quantities of glycerophosphorylcholine (GPC) and sialic acid in the epididymis of 21-day-old rats suggested inherent secretory ability of the epididymal epithelium. The concentrations of GPC, sialic acid, phospholipids and glycogen in the epididymis gradually increased with age, but each came under the influence of androgen at a different age. There was no evidence to suggest that the presence of spermatozoa has a stimulatory effect on the epididymis. Maximal secretory activity of the epididymis became established only by 90 days of age.

Effect of storage temperatures on accumulation of glycerylphosphorylcholine and decomposition of phosphatidylcholine in fish muscle during cold storage.

The accumulation rate of glycerylphosphorylcholine (GPC) in carp ordinary muscle at -20°C was significantly lower than the rates at 0 and -10°C. The rate at -10°C, on the contrary, was slightly higher than that at 0°C. In frozen fish, the accumulation of GPC proceeded more appreciably than the increase in K value. At these low temperatures the accumulation of GPC corresponded closely to the decomposition of phosphatidylcholine (PC). However, this was not the case at 40°C at which GPC decreased after an initial increase and the amounts of accumulated GPC were much less than expected from the amounts of decreased PC. In muscle suspensions of a pacific mackerel and a carp, bacteria decomposed not only GPC but PC without forming GPC at 37°C, pH 7.5. However, no bacterial decomposition was observed at 0°C for 10 days in pacific mackerel muscle. Consequently, the accumulation rate of GPC at 0°C which was slightly lower than that at -10°C could not be attributed to bacterial action.

Comparative physiology of the mammalian epididymis

The mammalian epididymis is a highly metabolically active organ which synthesizes and/or secretes sialic acids (sialomucoproteins), lipids, glycerylphosphoryl chlorine, carnitine and steroids. The secretory activity of the different segments of the epididymis varies and is affected by age, levels of circulating androgens, testicular fluid and its androgens and spermatozoa. The caput epididymidis of rat, hamster, bull, monkey and human show higher concentrations of total lipids and phospholipid phosphorous than the cauda epididymidis. During their transit through the epididymis, the spermatozoa lose phospholipids which may be due to their utilization as a substrate for energy. Glycerylphosphoryl choline is a secretory product of the caput epididymidis in the rabbit and is accumulated in the cauda epididymidis. Carnitine is present in high concentration in the cauda epididymidis of rat and bull; bovine spermatozoa acquire carnitine during their epididymal transit which may be involved in fatty acid oxidation. Sialic acids (free or bound to proteins as sialomucoproteins) are secreted by the epididymis of the rat, hamster, monkey and human. Bound sialic acid in the spermatozoa decreases markedly during their transit through the epididymis in the rat, hamster and monkey. An inverse relationship exists between levels of bound sialic acid in the luminal plasma and that of spermatozoa in the cauda epididymidis. The epididymis actively synthesizes steroids and probably secretes them. The ep didymis is also capable of metabolizing the androgens. Hormonal homeostasis for the maintenance of the functions of the epididymis may involve not only free testosterone and its metabolites but also estosterone bound to androgen binding proteins.

The split ejaculate of the boar: contributions of the epididymides and seminal vesicles.

The epididymal and seminal vesicular contributions to split-ejaculate fractions from boars were analysed for sperm concentration, glycerylphosphorylcholine (GPC), total-N, ethanol-soluble and insoluble N, citrate, zinc and haemagglutinin. The same components were also determined in epididymal plasma (EP), vesicular secretion (VS) and whole seminal plasma (SP). Isoelectric focusing of protein patterns was studied in the fractions. With the exception of haemagglutinin, the components were present to a major extent in either VS or EP and in lower concentrations in the other secretion. The parameters in VS or EP were positively correlated among themselves and negatively correlated with most of the parameters of the other fluid. The correlation coefficients were not significant in all cases for individual animals, but the degree of significance was greater for the over-all correlations. The EP components were mainly secreted in the first three or four fractions, but occasionally from fraction four onwards. Those of VS were emitted during the entire ejaculation, the maximum occurring in the sperm-rich fraction or the immediately succeeding fraction. The first fractions were devoid of VS components in only one case. The majority of the EP proteins could be identified electrophoretically in the sperm-rich fractions, but the protein patterns in the other fractions were similar to those of VS. The results are discussed and compared with previous findings.

Studies on Human Serum High-Density Lipoproteins (HDL) IV. Isolation of Lipoprotein Families after Incubation of HDL

The high-density lipoproteins (HDL) of human serum appear to be unstable and easily exposed to chemical changes during isolation. In earlier studies we have isolated and purified HDL subfractions either in the presence of an SH-blocking agent, DTNB, or in the cold. By both procedures reproducible lipoprotein subfractions could be recovered by hydroxyl apatite column chromatography at the elution steps 0.03-0.05 mol/l (subfraction II) and 0.05-0.15 mol/l phosphate buffer (subfraction III). The protein moiety of both lipoprotein subfractions contained polypeptides A-I , A-II, thin line (TL), C-I and C-II, and the protein moiety of subfraction III contained also C-III. The incubation at 37 degrees C of these HDL subfractions gave reproducible daughter lipoprotein fractions that could be recovered by subsequent rechromatography on hydroxyl apatite. At each of the elution steps 0.05-0.075 mol/l and 0.075-0. mol/l one daughter fraction was recovered, the protein moiety of which was composed of polypeptide A-I, as judged by polyacrylamide gel electrophoresis, immunodiffusion, and amino acid analysis. The incubation of parent subfractions II and III caused also the appearance at elution step 0.001-0.01 mol/l of a daughter lipoprotein fraction - lipoprotein A (Lp-A) - that was characterized by a protein moiety with polypeptides A-I and A-II in equal amounts. The 'release' of lipoprotein A-I (Lp-A-I) and Lp-A was shown to be due rather to the incubation than to the column chromatography as such. The chemical changes occurring during the incubation of HDL suggested a degradation of phosphatidylcholine (PC) to lysophosphatidylcholine (lyso-PC) and glycerylphosphorylcholine (GPC). It is suggested that the degradation of PC might interfere with the interaction between the lipoprotein families composing HDL.

コイ普通肉におけるphosphatidylcholineの酵素分解によるglycerylphosphorylcholineの生成

The formation of glycerylphosphorylcholine (GPC) by enzymatic hydrolysis of phosphatidylcholine (PC) was studied in carp Cyprinus carpio ordinary muscle by using PC-Me-14C and GPC-Me-14C as the substrates.Nearly all the decomposition products of PC were GPC, and the rate of choline liberated from GPC was only about five percent of that from PC. Moreover, there was scarcely any accumulation of lysophosphatidylcholine in the decomposition pathway of PC to GPC. Thus it was concluded that the formation of GPC by enzymatic hydrolysis of PC was nearly quantitative in carp ordinary muscle.During storage of carp and yellow tail Serbia quinqueradiata ordinary muscle, the formation of GPC increased progressively for 12 days at 0°C. The amount of GPC in yellow tail reached a plateau of approximately 68 μmoles/100g muscle after 30 days at -10°C.