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J E Parks

Publications and source records attributed to J E Parks.

35 records · Page 2Linked to original sources
PubMed1991

Two genes encoding 1-aminocyclopropane-1-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated.

A 17-kilobase (kb) region of the zucchini (Cucurbita pepo) genome has been sequenced and contains two genes, CP-ACC1A and CP-ACC1B, encoding 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14). The genes are transcribed convergently and are separated by a 5.7-kb intergenic region. Their coding regions are interrupted by four introns located in identical positions. While the DNA identity in their coding regions is 97%, their 5' and 3' flanking regions are highly divergent. Transcription of CP-ACCIA is rapidly induced by wounding in fruit and etiolated hypocotyls and by indoleacetic acid (IAA)/benzyladenine/LiCl only in fruit tissue. Conditions that induce CP-ACC1B expression have not been found. Protein synthesis inhibition derepresses the expression of CP-ACC1A and other unidentified ACC synthase genes, suggesting that they may be under negative control. The amino acid sequences deduced from the nucleotide sequences of the genes are 493 and 494 residues long with 95% identity. The most notable feature of the amino acid sequence is the presence of 11 of the 12 invariant amino acid residues involved in the binding of the substrate and pyridoxal-5'-phosphate in various aminotransferases. We conclude that ACC synthase is encoded by a multigene family of which certain members are differentially induced by auxin in a tissue-specific manner. Furthermore, ACC synthase, a pyridoxal-containing enzyme, may have an evolutionary relationship with the superfamily of aminotransferases.

Amino Acid Sequence↗
PubMed1990

Bovine oviductal fluid components and their potential role in sperm cholesterol efflux.

Bovine oviductal fluid (OF) was collected and analyzed throughout the estrous cycle, and the capacity of the protein and lipoprotein components to support cholesterol efflux from bovine sperm was evaluated. Blood was collected and assayed for progesterone (P4) to monitor the estrous cycle. Protein and lipoprotein separation was achieved by density gradient centrifugation. Two major bands were identified. The first (1.056 less than delta 20 less than 1.140 g/ml) corresponded to bovine and rabbit plasma high-density lipoprotein (HDL) based on distribution in the density gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second band (1.235 less than delta 20 less than 1.243 g/ml) consisted predominantly of oviductal fluid albumin (OFA). Oviductal fluid protein concentration increased as serum P4 decreased around the time of estrus. Mean OF protein concentration was 21.3 mg/ml when serum P4 was lower than 0.5 ng/ml and 6.9 mg/ml when serum P4 was greater than 0.5 ng/ml. An inverse log relationship was found between HDL protein concentration and serum P4. Unesterified cholesterol (UC), cholesteryl ester, and phospholipid (PL) content of HDL for HDL protein concentrations of 3-56.1 micrograms/ml were 1.35-46.2 micrograms/ml, 1.91-44.48 micrograms/ml, and 1.69-59.8 micrograms/ml, respectively. Phosphatidylcholine and -ethanolamine were the major PLs present in the HDL fraction and their molar ratio (4:1 mol/mol) was relatively constant through the estrous cycle. The OFA fraction of the same samples accounted for more than 90% of total protein and for most of the variation in OF protein. To determine the ability of OF components to serve as sperm cholesterol acceptors, OF samples were incubated 1:1 (v/v) with and without 4 X 10(8) bovine sperm in 1.0 ml of modified Tyrode's solution and OF for 2 hr at 39 degrees C. After incubation, HDL and OFA fractions were isolated and analyzed for changes in protein and lipid content. After OF, samples were incubated with sperm, an increase in UC was found in the HDL fractions. UC in HDL increased by 12.1 +/- 1.0 micrograms/ml (means +/- SE) when serum P4 was less than or equal to 0.5 ng/ml. For samples corresponding to higher serum P4, the increase in UC was 3.60 +/- 0.89 micrograms/ml. Values for UC in HDL were corrected for the contribution of UC from OFA of OF samples. Cholesterol efflux from sperm has been implicated in the process of sperm capacitation. These results indicate that HDL from OF is elevated during the follicular phase of the estrous cycle and can serve as an acceptor for bovine sperm cholesterol.

Animals↗
PubMed1990

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa.

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.

Journal Article↗
PubMed1990

Platelet activating factor activity in the phospholipids of bovine spermatozoa.

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

Animals↗
PubMed1988

Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol.

Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 x 10(8)) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [14C]-Chol (35:35:30 molar ratio, 300 nmol total PL). [3H]-triolein was included as a nonexchangeable marker. After 90 min at 39 degrees C, a 13% net exchange of [14C]-Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 x 10(6) sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [14C]-Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol (+Chol), and with Chol-free liposomes (-Chol) were washed and resuspended in TS with 0.2% BSA and 30 micrograms lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC-BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC-BSA at 39 degrees C, the mean (+/- SE) percent motile sperm for control, +Chol, and -Chol treatments was 57.0 +/- 4.9, 60.0 +/- 4.7, and 57.0 +/- 6.8, respectively. Corresponding values for percent AR were 14.0 +/- 3.4, 20.3 +/- 4.4, and 39.7 +/- 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.

Acrosome↗
PubMed1988

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova.

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39 degrees C) in a modified Tyrode's solution (TALP) containing 1) Chol-free liposomes (-Chol, 50 x 10(6) sperm and 600 nmol phospholipid/ml); 2) liposomes containing 30 mol% Chol (+Chol, 2 x 10(8) sperm and 300 nmol total lipid/ml); or 3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with -Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 x 10(6)) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-microliters droplets of TALP under medical fluid in an atmosphere of 5% CO2 in air (3 h, 39 degrees C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa.

Plasma membrane (PM), primarily from the anterior sperm head, and outer acrosomal membrane (OAM), were isolated from ejaculated bovine spermatozoa, and the major lipid classes were characterized. Whole sperm (WS) lipids were analyzed for comparison. PM was removed by nitrogen cavitation and purified by sucrose density-gradient centrifugation. The OAM was removed by centrifugation through hyperosmotic sucrose and recovered by sucrose density-gradient centrifugation. The PM contained primarily spherical vesicles from the region overlying the OAM and was enriched 9- and 13-fold in 5'-nucleotidase and alkaline phosphatase activity, respectively, compared to the original cavitate. The OAM was recovered as caplike structures with associated ground substance. Protein, phospholipid, and cholesterol (PR, PL, and CH as micrograms/5 x 10(9) sperm) were 300, 467, and 93 for PM and 276, 111, and 25 for OAM, respectively. Corresponding values for WS (mg/5 x 10(9) sperm) were 31.4, 6.63, and 0.72. The PR/PL (w/w) and CH/PL (mol/mol) ratios were 0.66 and 0.38 for PM; 2.48 and 0.26 for OAM; and 4.39 and 0.22 for WS. Cholesterol was the only free sterol detected by gas/liquid chromatography in WS, PM, and OAM, with traces of CH sulfate present in all three preparations. Glycolipid tentatively identified as sulfogalactolipid was detected by thin-layer chromatography (TLC) in PM but not OAM. Phospholipid composition of WS and membranes was determined by TLC. Cardiolipin (3% of total PL) was present in WS only. Choline, ethanolamine, and inositol phosphoglycerides (CP, EP, PI, PIP, PIPP); sphingomyelin (SP); phosphatidylserine (PS); and lysophosphatidylcholine (LPC) were present in WS, PM, and OAM. Approximately 50% of total PL was CP in all preparations; SP was 13% of PL in PM and 17% in OAM (p less than 0.05); EP was 7% of PL in PM and 10% in OAM (p less than 0.05). The differences in composition between PM and OAM is discussed with respect to capacitation and ability of sperm to undergo the acrosome reaction.

5'-Nucleotidase↗
PubMed1985

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane.

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.

Animals↗
PubMed1985

Counting mammalian spermatozoa in biological fluids containing particulate matter.

Accurate counting of spermatozoa in biological fluids by particle counters requires elimination of competing background. This was accomplished by dissolving cell organelles and other lipid and proteinaceous material with sodium dodecyl sulfate to leave the sperm nucleus. Progress of selective dissolution of interfering background versus sperm nuclei was monitored by phase contrast microscopy. Rabbit semen was diluted in 2.5% sodium dodecyl sulfate (wt/vol) and then 1:1 (vol/vol) with .5 M sodium hydroxide. Suspensions were incubated, diluted 1:200 in .1 M sodium citrate-.1% Triton-X, and counted after 5, 10, and 20 min with a Coulter Counter. All treatment times resulted in similar mean counts, ranging from 367 to 369 X 10(6) sperm/ml. These means were slightly higher than the 350 X 10(6) sperm/ml for hemocytometer counts, but the correlation with hemocytometer counts was r greater than or equal to .98. Bull semen was diluted in either 1) citrate-Triton-X, 2) .25 M Tris-20% egg yolk, or 3) heated whole milk and further treated with 10% sodium dodecyl sulfate and .5 M sodium hydroxide. After 15 min, treated samples were diluted 1:200 in citrate-Triton-X and counted. Following treatment, estimated sperm concentration in the three diluents was not different from that of untreated sperm in citrate-Triton-X. The procedure was successfully applied to bull sperm diluted in milk and packaged in .5-ml French straws used routinely for artificial insemination.

Animals↗
PubMed1979

Synthesis and therapeutic testing of mono- and dialkyl esters of pentetic (diethylenetriaminepentaacetic) acid for decorporation of polymeric plutonium.

The synthesis, characterization, and therapeutic evaluation of a series of partially esterified derivatives of pentetic (diethylenetriaminepentaacetic) acid are reported. These compounds were prepared in an attempt to promote increased decorporation of insoluble colloidal forms of plutonium, which are removed by pentetic acid alone. The dimethyl, diethyl, dibutyl, dioctyl, monoethyl esters were synthesized by reaction of the appropriate alcohol with the dianhydride of pentetic acid. These esters were injected intravenously into mice as their calcium chelates in saline. None of the esters was effective in removing plutonium from the liver. All esters removed approximately 20% of the plutonium in the skeleton. However, when the esters were given together with pentetic acid, only the dioctyl ester showed enhanced removal of plutonium compared to pentetic acid alone. The small increase in effectiveness and the increased acute toxicity make these esters of limited practical interest in plutonium decorporation therapy.

Animals↗
PubMedDate not supplied

Platelet-activating factor acetylhydrolase activity in bovine seminal plasma.

Platelet-activating factor (PAF) is a potent signaling molecule that has been detected in mammalian sperm from several species. The biological function of sperm-derived PAF and mechanisms controlling its production have not been clearly defined. In the remodeling pathway for PAF biosynthesis, PAF is produced by phospholipase A2 hydrolysis of 1-O-alkyl phospholipids followed by acetylation by PAF acetyltransferase. PAF is inactivated by PAF acetylhydrolase. PAF acetylhydrolase activity has been detected recently in human seminal plasma, where it may play a role in regulating PAF production or content by sperm. The purpose of this study was to measure and partially characterize PAF acetylhydrolase in bovine seminal plasma. Acetylhydrolase activity was detected in seminal plasma, was linear with time and protein concentration, and had a specific activity of 122 nmol/minute/mg protein. The enzyme was cation independent and was not inhibited by phosphatidylcholine but was inhibited by p-bromophenacylbromide and partially inhibited by phenylmethylsulfonylfluoride. Very little acetylhydrolase activity was detected in caudal epididymal fluid or caudal epididymal sperm. Enzyme activity associated with ejaculated sperm was largely removed by their centrifugation through Percoll and subsequent washing. These results demonstrate very high PAF acetylhydrolase activity in bovine seminal plasma. The enzyme appears to be of accessory gland origin and has properties similar to those of the enzyme from other sources.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMedDate not supplied

Partial purification and localization of platelet-activating factor acetylhydrolase from bovine seminal plasma.

Platelet-activating factor (PAF) is a potent lipid mediator that is inactivated by platelet-activating factor acetylhydrolase (PAF-AH). Platelet-activating factor bioactivity has been detected in bovine sperm phospholipids and PAF-AH activity is extraordinarily high in bovine seminal plasma. The purpose of this study was to purify and characterize partially the PAF-AH in bovine seminal plasma. Platelet-activating factor acetylhydrolase was partially purified from bovine seminal plasma using gelatin-agarose and ion-exchange chromatography and nondenaturing polyacrylamide gel electrophoresis (PAGE). Enzyme activity was increased 11-fold over seminal plasma with a yield of 11%. Platelet-activating factor acetylhydrolase activity was eluted from a single band with a R(f) of 0.258 from a nondenaturing preparative PAGE gel along with several other proteins of varying molecular weights. Following separation by sodium dodecyl sulfate (SDS)-PAGE under reducing conditions, PAF-AH was identified as a approximately 60-kD band by western blotting using antiserum directed against human blood PAF-AH. N-terminal sequencing of the approximately 60 kD band, followed by amino acid-sequence similarity searching, demonstrated a single-sequence match with PAF-AH from bovine blood. Based on western blotting, a approximately 60-kD band corresponding to PAF-AH was detected in seminal vesicle fluid but not in samples of washed, sonicated sperm or sperm plasma membranes where activity was low (<5% and <0.3%, respectively, of that in seminal plasma), suggesting that seminal plasma PAF-AH does not bind tightly to sperm. Specific PAF-AH activity measured in seminal vesicle fluid was in the lower range of that in seminal plasma. These results demonstrate that PAF-AH activity in bovine seminal plasma is due to PAF-AH secreted by the seminal vesicles with sequence homology to the enzyme in human blood.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗