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K D Roberts

Publications and source records attributed to K D Roberts.

At least 19 recordsLinked to original sources
PubMed1995

Purification of lysophospholipase of human spermatozoa and its implication in the acrosome reaction.

The lysophospholipase of human spermatozoa was purified to homogeneity by sequential ion-exchange, gel filtration, and hydrophobic chromatography. The final preparation exhibited a single protein band on SDS-PAGE. The molecular mass of the enzyme was estimated to be 51 kDa by SDS-PAGE and 52 kDa by gel filtration. The optimal pH of this enzyme is 8.0. Polyclonal antibodies against lysophospholipase were prepared by placing the enzyme adsorbed on nitrocellulose directly into the spleen of rabbits. These antibodies were purified by protein A-agarose and by affigel-lysophospholipase chromatography. The purified antibodies and enzyme were used to study the possible role of lysophospholipase in the acrosome reaction. The addition of these antibodies led to an increase in the acrosome reaction, thus suggesting that inhibition of lysophospholipase produces a higher lysophosphatidylcholine concentration and results in an acrosome reaction level similar to that obtained by the calcium ionophore A23187. Immunofluorescence localization of the enzyme indicated that the enzyme is located on the head of spermatozoa. The purified sperm lysophospholipase and its specific antibodies represent important tools for the study of the regulation of this enzyme in reproductive processes. Furthermore, the study of this enzyme will allow evaluation of the mechanisms underlying the acrosome reaction.

Acrosome↗
PubMed1994

Major proteins of bovine seminal plasma inhibit phospholipase A2.

We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.

Animals↗
PubMed1994

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravital stain.

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction. Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.

Acrosome↗
PubMed1993

Partial characterization of galactosyltransferase in human seminal plasma and its distribution in the human epididymis.

Galactosyltransferase activity has been partially characterized in human seminal plasma. Km values of 130 mumol l-1 for UDP-galactose and 2.25 mmol l-1 for N-acetylglucosamine were calculated and the enzyme was found to be dependent on temperature and manganese and present as a highly active component of human seminal plasma. Galactosyltransferase was inhibited by nucleotides, glycosylated nucleotides, bovine and human alpha-lactalbumin but not by monosaccharides. Radiation inactivation studies revealed that the biologically active unit of seminal plasma galactosyltransferase has a molecular mass of 45 kDa. Although the majority of galactosyltransferase activity found in seminal plasma is probably of prostatic origin, we report for the first time that it is also present in human epididymal intraluminal fluid. Low activity was detected in the proximal caput region but activity increased to maximum values in the adjacent downstream segment, the intermediate caput region. Specific activity was relatively constant albeit at a lower value in the following epididymal segments and vas deferens. The significance of the epididymal and seminal plasma galactosyltransferase activities is unknown, but the enzyme could be implicated in glycosylation events that are known to be important in gamete interaction.

Animals↗
PubMed1992

The zona pellucida binds the mature form of an oviductal glycoprotein (oviductin).

The use of a monoclonal antibody (MAb) specific for the oviductal zona pellucida (ZP) of the hamster has demonstrated that a new antigen (oviductin) is acquired by the ZP during transit of the oocyte in the oviduct. The epitope that is recognized by the MAb bears a terminal N-acetyl-D-galactosamine residue. We conducted a study in order to determine whether this immunoreactivity of the oviductal ZP results from the addition of the terminal sugar residue to a preformed ZP protein or from the transfer of the mature glycoprotein produced by oviductal secretory cells. We measured the incorporation of [35S]methionine into proteins using four different incubation systems: cumulus oophorus (CO) alone, CO in the presence of oviductal fluid, CO co-incubated with empty oviducts, and CO within intact oviducts. At the end of the incubation period, the ZP, vitelli, dispersed cumulus without oocyte, oviducts, and culture medium were isolated and analyzed for their protein content by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and immunodetection. The cumulus cells synthesized several proteins, independently of the oviductal environment; however, none of these proteins corresponded to oviductin. The ZP and the vitelli of cumulus oophorus that were incubated either alone or in the presence of oviductal fluid did not contain radioactive oviductin. When the oviduct (empty or intact) was present in the incubation system, radiolabeled oviductin was synthesized and secreted into the incubation medium. The ZP picked up a detectable amount of radioactive antigen only in the system in which intact oviducts were incubated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The phospholipase A2 of human spermatozoa; purification and partial sequence.

In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution.

Amino Acid Sequence↗
PubMed1992

Platelet-activating factor acetylhydrolase in human seminal plasma.

OBJECTIVE: To search for the presence of platelet-activating factor (PAF) acetylhydrolase activity in human seminal plasma. DESIGN: Experimental. SETTING: Reproductive laboratory in a university-affiliated hospital research center. PARTICIPANTS: Human male volunteers were selected on the basis of apparent normal health. RESULTS: Human seminal plasma contained significant levels of PAF-acetylhydrolase activity. Enzymatic hydrolysis of PAF displayed typical kinetics and was a calcium-independent process. Platelet-activating factor acetylhydrolase in seminal plasma was associated with a very high-density lipoprotein fraction. Enzymatic activity was independent of sperm count and motility. CONCLUSION: Human seminal plasma contains significant levels of PAF acetylhydrolase. This enzyme may be an important factor in the regulation of PAF concentration and activity.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1991

Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.

The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies.

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.

Adult↗
PubMed1990

Identification of heparin-binding proteins in bovine seminal plasma.

A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Safety of protamine sulfate administration in vasectomized men.

The majority of vasectomized men develop antibodies against different sperm antigens, including protamine. Due to the fact that salmon protamine is used clinically for heparin reversal and that a cross-reactivity has been observed between human and salmon protamine, vasectomized men may be at risk for adverse reactions to protamine sulfate. In order to explore this possibility, serum samples were analyzed for the presence of anti-sperm and anti-protamine antibodies in 20 vasectomized and 20 non-vasectomized men (controls) about to undergo major heart surgery requiring heparin reversal. The patients were closely monitored for any possible reaction following the injection of protamine. Anti-sperm antibodies were detected in the serum of 15 of the vasectomized men whereas only two of the non-vasectomized subjects had this type of antibody. Twenty-five percent of the vasectomized men presented with anti-protamine antibodies; in the control group, none of the patients had developed this type of antibody. Following the administration of protamine sulfate, none of the patients in either group showed any adverse reaction to the drug. It is concluded that vasectomized men are not at increased risk towards adverse reactions following the injection of protamine sulfate.

Adult↗
PubMed1989

Colon interposition in children.

Since 1960 we have performed 80 colonic interpositions on 79 children. We present a retrospective review of their progress. Sixty-nine interpositions were for long-gap oesophageal atresia, while the remainder followed oesophagectomies, eight for strictures and two others. Overall, the transthoracic route was used in 69 per cent of cases, while 29 per cent were retrosternally placed and one was retropleural. The mean follow-up was 11.08 years. The mortality rate was 12 per cent, with one-third of deaths unrelated to the operation. Graft failure, which occurred on eight occasions (10 per cent), was due to ischaemia in four, intractable stricture in two and stomal ulceration in two. Leakage from the proximal anastomosis occurred in 31 per cent of cases and stricture formation in 27 per cent. Thirty per cent of patients complained of acid reflux, of whom one-third developed stomal ulceration. We present data concerning other complications, including long-term recurrent respiratory infections, malabsorption, gastrointestinal haemorrhage, diarrhoea, intestinal obstruction and redundancy of the graft. Staging the procedure did not affect the outcome, however the higher incidence of serious complications encountered following retrosternal interposition finally led us to abandon this procedure in favour of the transthoracic route. There has been little improvement in the growth rate, but in terms of swallowing ability the outcome was satisfactory in 94 per cent of cases.

Adolescent↗
PubMed1989

Detection of prophospholipase A2 in human spermatozoa.

Prophospholipase A2 (proPA2) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C4) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A2 (PA2) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C4 column. The proenzyme was separated from active PA2 on the C4 column. Human sperm proPA2 exhibited a less cationic charge than active PA2 on the SP 5PW column. Porcine pancreatic proPA2 had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA2. The purification procedure resulted in the isolation of proPA2 which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA2 of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA2 exists in human spermatozoa.

Chromatography, High Pressure Liquid↗
PubMed1988

Activation of phospholipase A2 of human spermatozoa by proteases.

The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A2 activity (PA2: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA2 activity optimally with trypsin concentrations of 1.0-1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA2 activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA2 activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA2 activation upon the addition of proteases, thus indicating that the increase in PA2 activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A2 that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A2 could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.

Acrosome↗
PubMed1988

Evidence for two forms of phospholipase A2 in human semen.

The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 degrees C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at -20 degrees C for 48 hr followed by a heating period of 10 min at 60 degrees C. Long-term storage of spermatozoa at -20 degrees C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 degrees C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 degrees C or -20 degrees C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature.

Drug Stability↗
PubMed1988

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation.

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), as well as the albumin fraction (d greater than 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60-70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2-3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.

Acrosome↗