The release of 15-hydroxyeicosatetraenoic acid by human placental trophoblast is increased in preeclampsia.

Objective: We tested the hypothesis that trophoblast produces 15-hydroxyeicosatetraenoic acid, and its level is elevated in trophoblast from preeclamptic women compared with normal. We also used selective enzymatic inhibitors to determine the relative contributions of 15-lipoxygenase and the two isozymes of prostaglandin H synthase to 15-hydroxyeicosatetraenoic acid levels.

Study Design: Cytotrophoblasts isolated from placentas of normal or preeclamptic women were cultured in the presence or absence of enzyme inhibitors.

The expression and activity of prostaglandin H synthase-2 is enhanced in trophoblast from women with preeclampsia.

Preeclampsia is associated with altered biosynthesis of vasoactive prostanoids in placental villi. The two isozymes of prostaglandin H synthase (PGHS) are essential for prostanoid synthesis. We tested the hypothesis that PGHS-2 expression is elevated in trophoblast from preeclamptic women, compared with trophoblast from healthy women.

Aspirin induces increased expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 in cultured human placental trophoblast.

Objective: We tested the hypothesis that aspirin affects trophoblast like other epithelial cells do, by inhibiting prostanoid production, inducing prostaglandin H synthase-2 expression, and enhancing secretion of 15-hydroxyeicosatetraenoic acid.

Study Design: Cytotrophoblast from placentas (n = 15) of uncomplicated singleton pregnancies were cultured in medium 199 for 4 to 72 hours in the presence or absence of aspirin.

Results: Aspirin (10(-4) M) inhibited (p < 0.

Characterization of the heat shock protein P70 in rat spermatogenic cells.

A number of hsp70-like proteins are associated with developing male germ cells. One of these molecules, P70, is not sensitive to heat stress and is germ cell-specific, and its expression is developmentally regulated. We have characterized the association of the rat P70(rP70) with differentiating germ cells in the testis and with posttesticular sperm.

The effects of cocaine on neutral amino acid uptake by human placental basal membrane vesicles.

Objective: Prior studies have demonstrated that cocaine binds to human placental microvillous membrane vesicles at a single high-affinity site and that both 10 and 500 nmol/L cocaine inhibit sodium-dependent alanine uptake. The purpose of this study was to characterize cocaine binding to human placental basal plasma membrane and to determine the effects of cocaine on basal vesicle uptake of alanine and leucine.

Study Design: Basal vesicles were isolated from the placentas of uncomplicated human pregnancies with no history of cocaine use.

Cocaine inhibits alanine uptake by human placental microvillous membrane vesicles.

Objective: The aim of this study was to determine the effects of cocaine on alanine uptake by human placental microvillous membrane vesicles and to characterize cocaine binding to the microvillous membrane.

Study Design: Microvillous vesicles were isolated from the placentas of 10 human pregnancies with no history of cocaine use. The binding of tritiated cocaine to microvillous vesicle membrane and uptake of tritiated cocaine and tritiated alanine were determined with the use of filtration assays.

Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis.

We demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 micrograms/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC.

Distribution of tritiated cocaine in selected genital and nongenital organs following administration to male mice.

Study Objective: To determine the distribution of cocaine administered to male mice in selected extragenital and genital organs and to investigate its possible binding to sperm.

Design: Twenty-seven sexually mature virus-free albino male mice were used in various experiments whereby following intravenous injection of tritiated cocaine hydrochloride, radioactivity was determined in several extragenital and genital organs, as well as sperm.

Results: Radioactivity was detected in all of the organs that were tested, and the highest concentrations per milligram of tissue were found in the kidney and epididymis.

Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies.

A testicular protein band has been identified and shown to be immunoreactive to both of the proacrosin (53-55 kd) and the proacrosin binding protein (28 kd) antibodies. pH 4.5 extracts of boar testis were prepared and subjected to Western blot analysis using polyclonal antibodies of the proacrosin and the proacrosin binding protein.

Proacrosin binding protein: immunocomparative studies in boar, bovine, hamster, human and ram.

The cross reactivities of acidic extracts from boar, bovine, hamster, human and ram spermatozoa to a polyclonal antibody of the boar proacrosin binding protein has been investigated. The pH 3.0 extracts of the washed spermatozoa from each species were subjected to Western blot analysis using a polyclonal antibody developed against the 28 kDa boar proacrosin binding protein.

Protein transport and organization of the developing mammalian sperm acrosome.

Experiments indicate that the mammalian acrosome develops as a result of a time-dependent sequence of events which involves protein incorporation into distinct regions or acrosomal domains. These domains can be characterized by electron microscopy and their isolation and partial purification are being accomplished. Recent success in isolating and characterizing major proteins that compromise the Golgi apparatus should accelerate knowledge of the interaction of the Golgi with the developing acrosome.

Partial characterization of a proacrosin binding protein.

All of the acid (pH 4.0) extracted proacrosin from porcine epididymal spermatozoa was found to be tightly associated with a specific protein referred to as the binding protein. A combination of gel filterations and gel electrophoresis revealed that the binding protein is composed of a major 28 kd and a minor 29 kd protein.

Demonstration of specific binding of cocaine to human spermatozoa.

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis.

Identification of a proacrosin precursor in the cell-free translation of boar testicular poly(A)(+)-mRNA.

A cell-free translation system was used to determine the molecular mass of the protein component of precursor(s) to boar proacrosin. Poly(A)(+)-mRNA was extracted from freshly excised boar testis into phenol/chloroform, precipitated in chilled (-20 degrees C) ethanol, then translated in a cell-free, reticulocyte lysate system with Tran 35S-label. Analysis of the resulting products by SDS-PAGE followed by autoradiography demonstrated multiple bands of translated proteins.

Diisopropyl fluorophosphate labeling of sperm-associated proteinases.

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP.

Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems.

Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species.

Comparison between proteinases of human seminal plasma and of sperm origin.

A comparison of the alkaline proteinase activity of human seminal plasma, the seminal non-gamete cellular material and spermatozoa was made by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymography. Several major (molecular weights = greater than 56,000) and minor (35,000 to 44,000) bands of proteinase activity were seen in the seminal plasma samples from nonvasectomized and vasectomized, healthy donors. Similar activity profiles were observed in the nongamete cellular material of vasectomized donor ejaculates.

Partial purification and characterization of human sperminogen.

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column.

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system.

A rapid and efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized.

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system.

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography.

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis.

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically.

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa.

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes.

Acrosin inhibition. Comparisons of membrane-associated and -solubilized enzyme.

Acrosin is an extrinsic membrane proteinase from spermatozoa which functions in the fertilization process. Liposomes were utilized as a model system to determined possible effects of membrane association on acrosin's enzymatic activity. By comparison with solubilized enzyme, liposome-bound acrosin had a substantial reduction in the apparent affinity for "progressive" inhibitors such as leupeptin, lima bean trypsin inhibitor, soy bean trypsin inhibitor, and for a proteinase inhibitor from sperm extracts.

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin.

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.