Synthesis and inhibition of human acrosin and trypsin and acute toxicity of aryl 4-guanidinobenzoates.

The aryl 4-guanidinobenzoate, 4'-nitrophenyl 4-guanidinobenzoate (NPGB), is a potent inhibitor of sperm acrosin, an enzyme with an essential function in the fertilization process. NPGB prevents fertilization in a number of animal species and is a good lead compound for the development of contraceptive agents. In order to assess the efficacy of other aryl 4-guanidinobenzoates as acrosin inhibitors, 24 of these compounds were synthesized. Their inhibitory activity toward human acrosin was determined and compared with their activity toward human pancreatic trypsin in order to assess whether inhibitor sensitivity differed between these similar enzymes. Nine of the inhibitors were synthesized from phenols approved by the FDA for therapeutic use. The acute toxicity of these inhibitors in mice was determined and compared to that of nonoxynol-9, the most commonly used active ingredient in today's vaginal contraceptive preparations. All of the compounds proved to be potent inhibitors of human acrosin although 3 orders of magnitude difference were observed between the most and least effective inhibitors. Little specificity was present in regard to their inhibition of acrosin and trypsin. All the aryl 4-guanidinobenzoates synthesized from FDA-approved phenols were less toxic than nonoxynol-9, and it is concluded that these 4-guanidinobenzoates are of interest for further development and testing as nonhormonal contraceptive agents.

Objective assessment of meconium content of amniotic fluid.

The amount of meconium in amniotic fluid is subjectively estimated by visual inspection and classified as thin (light), moderate, or thick (heavy). This estimate may be important for assessing the neonatal risk of perinatal asphyxia and meconium aspiration syndrome. This study reports on the "meconium-crit," a simple, rapid, inexpensive, and reproducible method of quantifying meconium concentration in amniotic fluid. The purpose of the study was to determine the relationship between the meconium-crit and meconium concentration. Specimens were prepared by placing 3.0 g of fresh neonatal meconium into clear amniotic fluid and vortexing for 15 minutes to obtain a stock solution of 15.0 g meconium/100 mL amniotic fluid. Stock solutions were then diluted with clear amniotic fluid to obtain concentrations of 10.0, 7.5, 5.0, 3.0, and 1.5 g/100 mL. One-tenth milliliter of the amniotic fluid/meconium mixture was drawn into a standard hematocrit tube and centrifuged. The meconium-crit was then measured directly as with a hematocrit. Regression analysis indicated that meconium-crit values were linearly related to meconium concentration (r = 0.901-0.995). This method provides a reproducible means of quantifying meconium in amniotic fluid.

Synthesis of myelin glycosphingolipids by isolated oligodendrocytes in tissue culture.

Isolated lamb oligodendrocytes in tissue culture were tested for their ability to express differenciated functions related to the synthesis of myelin. Galactocerebrosides and sulfatides are highly enriched in myelin and are recognized as markers for myelination. Hence, we followed the synthesis of these glycosphingolipids. At selected times, monolayer cultures of cells were double labeled for 60-72 h with [3H]galactose and H2 35SO4. Glycosphingolipids were separated and identified by thin layer chromatography. During the first week in tissue culture, there was gradual decline in incorporation of both isotopes relative to the values, obtained for freshly isolated cells. However, by 3 weeks high levels of H2 35SO4 incorporation into sulfatide and [3H]galactose into cerebrosides were found. A complex pattern of incorporation ensued after this peak which varied for each particular glycosphingolipid. It is concluded that: (1) cultured oligodendrocytes retain the capacity to synthesize components of myelin; (2) since the isolated cells have already been involved in myelination, these cultures afford a unique model to study remyelination' amd (3) these cells provide a good system to study the properties of oligodendrocytes.

Are capacitation or calcium ion influx required for the human sperm acrosome reaction?

OBJECTIVE: To determine if the acrosome reaction of human spermatozoa can occur without prior incubation to induce capacitation or in calcium-free medium. DESIGN: Noncapacitated (washed ejaculated) or capacitated (incubated for 3 hours in the presence of albumin) human spermatozoa were treated with either solubilized zonae pellucidae (ZP); a calcium ionophore (A23187); or activators of protein kinases A, G, and C, and the acrosomal status was monitored by the double stain technique. During agonist treatment, the capacitated spermatozoa were in medium either with or without calcium ions (Ca2+). The noncapacitated spermatozoa were always in Ca(2+)-containing medium. Sperm motility was monitored throughout the experiments. RESULTS: Solubilized ZP and kinase activators induced acrosomal exocytosis of capacitated spermatozoa (both in Ca(2+)-containing and Ca(2+)-free medium) and of noncapacitated spermatozoa. The lack of added Ca2+ or capacitation reduced the percentage of spermatozoa that reacted in response to solubilized ZP but not to the kinase activators. Ionophore A23187 stimulated acrosomal loss from noncapacitated spermatozoa to the same extent as capacitated spermatozoa in Ca(2+)-containing medium but had no effect on capacitated spermatozoa in Ca(2+)-free medium. CONCLUSIONS: Human spermatozoa do not require incubation under capacitating conditions or the presence of extracellular Ca2+ before they can undergo the acrosome reaction in response to certain agonists. Therefore, Ca2+ influx and/or preincubation to induce capacitation are not absolute requirements for the in vitro agonist-induced acrosome reaction. However, these conditions can optimize the acrosome reaction response to zona proteins. The intracellular mechanisms leading to the acrosome reaction appear to be functional in noncapacitated spermatozoa but membrane changes probably are required before certain extracellular ligands such as zona proteins can exert their maximal effect.

Inhibition of the human sperm acrosome reaction by a high molecular weight factor from human seminal plasma.

Human seminal plasma possesses a factor (acrosome reaction-inhibitory factor) that is precipitated by high speed centrifugation and that inhibits the ionophore- and dbc AMP-induced acrosome reaction of capacitated human spermatozoa but only if it is added toward the end of the capacitation period. Acrosome reaction-inhibitory factor can be partially purified by cation exchange chromatography and appears to differ from another factor that can be obtained by ultracentrifugation of human seminal plasma and that prevents the fertilization of mouse gametes.

Synchronous assay for human sperm capacitation and the acrosome reaction.

A synchronous acrosome reaction system was established for human spermatozoa. Seminal plasma is removed from the spermatozoa by centrifugation and the washed spermatozoa are capacitated in a modified BWW medium (without exogenous substrates) containing 35 mg/ml human serum albumin for 3 h at 37 C. Subsequently, 10 microM ionophore A23187 (final concentration) is added, the mixture incubated for 15 min at 37 C and the percent acrosome reaction determined by a modified triple stain technique (trypan blue stain omitted). Since no significant decrease in sperm motility occurs during incubation or after ionophore treatment, a vital stain does not need to be employed, allowing the use of any acrosome detection technique. The average percentage of acrosome-rejected spermatozoa after ionophore treatment (40 +/- 10%) was about 2- to 3-fold higher than that seen without ionophore treatment. Ionophore treatment for 15 min failed to stimulate the acrosome reaction in spermatozoa incubated for less than 3 h. Additionally, the presence of substrates in the BWW medium, higher sperm numbers, increased ionophore concentrations or longer incubation periods did not enhance the induction of the acrosome reaction. Ionomycin, a more specific calcium ionophore than A23187, produced essentially the same results as A23187 but tended to decrease sperm motility. This synchronous acrosome reaction system for human spermatozoa is relatively simple and can be used to study the effect of modulators on capacitation and/or the acrosome reaction.

The human sperm acrosome reaction does not depend on arachidonic acid metabolism via the cyclooxygenase and lipoxygenase pathways.

The objective of this study was to determine whether the metabolism of arachidonic acid via the cyclooxygenase pathway, the lipoxygenase pathway, or both has a pivotal role in the human sperm acrosome reaction. To do so, the stimulatory effect of arachidonic acid and a number of its metabolites, as well as the inhibitory effect of cyclooxygenase and lipoxygenase inhibitors on the acrosome reaction, was evaluated. Arachidonic acid, prostaglandin E2, and prostacyclin (PGI2) induced the acrosome reaction when added to 3-hour preincubated (capacitated) spermatozoa. The arachidonic acid-induced acrosome reaction was dependent upon extracellular calcium. Leukotriene B4 and 15-HPETE only induced the acrosome reaction when present throughout the preincubation period, indicating that they may enhance the capacitation process rather than the acrosome reaction. Thromboxane did not affect the acrosome reaction under any of the conditions tested. Inhibitors of cyclooxygenase (indomethacin, phenylbutazone) and lipoxygenase (phenidone, nordihydroguiaretic acid) or FPL 55712 (a leukotriene antagonist) did not prevent the arachidonic acid-stimulated acrosome reaction. Furthermore, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), the acetylenic analog of arachidonic acid that inhibits arachidonic acid metabolism, induced an acrosome reaction equivalent to that of arachidonic acid. These results strongly suggest that the acrosome reaction induced by exogenous arachidonic acid is not mediated via either the cyclooxygenase pathway or the lipoxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitative effect of pulsed addition of dibutyryl cAMP on the acrosome reaction of noncapacitated human spermatozoa.

The in vitro acrosome reaction of noncapacitated human spermatozoa was induced by both calcium ionophore (A23187) and dibutyryl adenosine cyclic monophosphate (Bu2cAMP), a membrane permeant cyclic nucleotide analog, in a dose-dependent manner. Maximal frequencies of acrosome-reacted spermatozoa above baseline values (12%; 90% confidence limits = 10.6 to 14.2%) were similar for Bu2cAMP and A23187 (24.5% and 25.1%, respectively). The concentration of Bu2cAMP required for a half-maximal response was 14.3 mumol/L, while that for A23187 was 24.5 pmol/L. The ability of A23187 to induce the acrosome reaction depended on the presence of calcium ion in the incubation medium. The A23187-induced reaction was prevented by the inclusion of human serum albumin in the medium; the inhibitory effect of albumin was partially reversed after preincubation of spermatozoa for 3 hours under capacitating conditions. In contrast, the Bu2cAMP-induced acrosome reaction was unaffected by either Ca2+ or albumin. Pulsed addition of Bu2cAMP enhanced the frequency of acrosome-reacted spermatozoa. This effect appeared to be influenced by pulse frequency: additions made every 5 minutes produced a greater maximal response than additions made every 2 minutes or every 15 minutes. The maximum theoretical acrosome reaction above baseline values (12%) was 88% of the total number of cells, accounting for almost the entire sperm population. Pulsed addition of A23187 did not increase the frequency of acrosome-reacted spermatozoa above values obtained from single equimolar additions of this agent. These data indicate that: (1) intracellular mechanisms for the human acrosome reaction are functional in noncapacitated spermatozoa; (2) the acrosome reaction can be separated from the process of capacitation; and (3) the acrosome reaction is affected by the pattern, as well as the type, of activation.

Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction.

The acrosome reaction of spermatozoa may be analogous to various somatic cell exocytotic events that incorporate cascade reactions. One such cascade system involves the hydrolysis of a membrane-bound phospholipid; generation of the intracellular second messenger, diacylglycerol; and activation of protein kinase C, followed by the phosphorylation of a number of intracellular proteins. Stimulators of protein kinase C, phorbol diesters and synthetic diacylglycerols, were evaluated to determine if this system functions in the human sperm acrosome reaction. Phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-didecanoate caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa. Conversely, an inactive phorbol diester had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. The synthetic diacylglycerols, 1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-dioleoyl-sn-glycerol caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa, and to a similar extent as the phorbol diesters. A nonactivating isomer of 1,2-dioleoyl-sn-glycerol, 1,3-diolein, had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. Protein kinase C activation is a diacylglycerol-dependent and Ca2(+)-dependent process, and stimulation of the acrosome reaction by 1,2-dioctanoyl-sn-glycerol required the presence of calcium ions in the capacitation medium. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), prevented the diacylglycerol-induced acrosome reaction (P less than 0.01). These results support the hypothesis that protein kinase C, via activation by the intracellular second messenger diacylglycerol, has a role in the human sperm acrosome reaction.

Human sperm acrosin. Further studies with the clinical assay and activity in a group of presumably fertile men.

The goals of this study were to determine the effect of the nonionic detergent, Triton X-100, on the recovery of acrosin in the clinical assay (Kennedy et al, 1989), since previous investigations have used higher concentrations for acrosin extraction from spermatozoa; and to establish the minimal acrosin activity in fertile men. The recovered acrosin activity was dependent on the concentration of Triton. A peak in acrosin activity was obtained at 0.01% to 0.02% Triton, the approximate critical micelle concentration (CMC; 0.015%). The bimodal effect of Triton was not due to substrate/buffer alterations, to the degree of acrosomal disruption as assessed by light and transmission electron microscopic examination, or to its effect on the kinetic properties of acrosin, as determined by spectrophotometric analysis of acid-extracted enzyme. However, Triton affected the conversion of proacrosin to acrosin, with peak activation occurring at 0.01% to 0.02% detergent. The acrosin activity of a group of presumably fertile men (as established by the production of offspring under natural conditions) varied from 18 to 42 microIU/10(6) spermatozoa, as assessed by the clinical assay containing 0.01% Triton. Furthermore, men who had initial acrosin values in the low normal range (18 to 25 microIU/10(6) sperm) were observed for 11 months. The acrosin activity of their ejaculates never fell below 17 microIU/10(6) sperm. Thus, it can be tentatively assumed that the minimal levels of acrosin for naturally fertile men are 17 to 18 microIU acrosin/10(6) sperm in this assay.

Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome.

Atrial natriuretic peptides (ANPs) from several species induced the human acrosome reaction. The maximal response was highest for human ANP (18.6% above unstimulated or baseline values) and decreased progressively for peptides derived from animals lower on the phylogenetic scale. ANP concentrations required for a half-maximal effect in noncapacitated spermatozoa ranged from 0.07 to 0.38 nM. ANP induced the acrosome reaction in capacitated spermatozoa, but the concentration required was higher than in noncapacitated cells. The response in noncapacitated spermatozoa was independent of added extracellular Ca2+ and was completely inhibited by 1 microM LY83583 (inhibits particulate guanylate cyclase). However, 10 microM N omega-nitro-L-arginine (inhibits soluble guanylate cyclase) had no effect. ANP (80 pM) and 3 microM 1,2-dihexanoyl-sn-glycerol each induced a nearly half-maximal acrosome reaction. Added in combination, they produced no increased response, suggesting antagonism. Follicular fluid had variable levels of immunoreactive ANP. Average ANP content was nearly zero in samples that contained no oocyte at the time of aspiration but was higher (6.9 pM; 90% confidence limits = 1.67-28.72 pM) in follicular fluid containing oocytes that did not fertilize in vitro. Highest concentrations of ANP were present in follicular fluid containing oocytes that fertilized in vitro (72.8 pM; 90% confidence limits = 38.1-139.1 pM). These data suggest that noncapacitated spermatozoa can acrosome react without added extracellular Ca2+ in response to an extracellular ligand. Also, human spermatozoa appear to contain receptors for ANP similar to those found in other cell types. The ANP content of follicular fluid might partly explain the ability of follicular fluid to induce the acrosome reaction.

Development of the polyurethane sponge as a delivery system for aryl 4-guanidinobenzoates.

The Today Contraceptive Sponge was evaluated as a vehicle for the delivery of aryl 4-guanidinobenzoates (AGs) which are highly active sperm acrosin inhibitors. Studies in animals have shown that several AGs are more potent vaginal contraceptives and less irritating to the vagina than nonoxynol-9 (N-9), the most frequently used active ingredient in commercial vaginal contraceptive formulations. Neither nonoxynol-9 nor the material that could be solubilized from the sponge matrix altered the enzyme-inhibitory activity of 4'-acetamidophenyl 4-guanidinobenzoate HCl (AGB), 4'-carboxyphenyl 4-guanidinobenzoate HCl (EGB) or 4'-carbomethoxyphenyl 4-guanidinobenzoate HCl (MSGB). Besides being acrosin inhibitors, all three AGs exhibited antimotility activity towards human spermatozoa, EGB being as potent as N-9. The antimotility effects of the AGs and N-9 were additive. For subsequent studies, AGB was used as the model compound. Manufacture of the AGB-containing sponges did not affect the chemical structure of AGB. Good release rates of AGB were obtained from the sponges over a 7-day period. The release rates were 20-50% higher when the sponges also contained N-9. These results indicate that certain AGs exert a dual contraceptive action on spermatozoa by inhibiting both the sperm enzyme acrosin and sperm motility. Furthermore, the polyurethane sponge appears to be a convenient and satisfactory long-term delivery system for the AGs. A mixture of N-9 and AG can be used clinically because these compounds have no adverse effects on each other.

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 1. Collection procedure and physical characteristics.

Hemolymph was collected from adult female Anopheles stephensi by centrifugation of incised mosquitoes. Approximately 0.1 muliter was collected from each recently emerged mosquito, although smaller amounts were recovered with increasing age of the mosquito. Determinations were made of the pH, osmotic pressure, and specific gravity of this hemolymph at various times during the life of the adult mosquito. The values obtained were within the ranges found for other insects. Hemolymph collected from mosquitoes fed on hamsters infected with Plasmodium berghei had different values than hemolymph from mosquitoes fed on noninfected hamsters. This probably was due to differences between the quality of these 2 types of blood meals, rather than to the direct effects of the malaria parasite on the infected mosquito itself.

Hemolymph of Anopheles stephensi from uninfected and Plasmodium berghei-infected mosquitoes. 2. Free amino acids.

Determinations were made of free amino acids in hemolymph collected from adult female Anopheles stephensi mosquitoes. The hemolymph first was fractionated by extraction and precipitation procedures, after which qualitative determinations of free amino acids were made by high voltage thin layer electrophoresis, and thin layer chromatography. Subsequent quantitative determinations were made with an automatic amino acid analyzer. The concentration of total free amino acids in the hemolymph rose 60--70% after the mosquito took a blood meal, and remained relatively constant thereafter. When mosquitoes took a blood meal infected with the rodent malaria parasite Plasmodium berghei, the rise in total free amino acids was only 15--25%. The chief differences that occurred with individual free amino acids was that infected mosquitoes had greater increases in arginine, greater decreases in valine and histidine, and a total loss of detectable methionine.

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.

Comparative activation studies with extracted and purified human proacrosin.

Proacrosin was purified from acid extracts of human spermatozoa by concanavalin A precipitation and Bio-Gel P-100 chromatography. Two molecular weight forms of proacrosin were obtained, a major one with a Mr of 70,000-71,000 and a minor one with a Mr of 47,000-53,000. In contrast to sperm extracts, the purified forms of proacrosin were free of acrosin inhibitor(s) and nonzymogen acrosin. By modulating pH, ionic strength and temperature, the activation of proacrosin in sperm extracts was compared to only the major form of purified proacrosin, since it seemed to be the source of the lower molecular weight form of proacrosin. In both preparations, proacrosin activation occurred maximally over a broad pH range (7.6-8.8 for purified proacrosin and 7.6-9.6 for extract). Additionally, an ionic strength of 0.1 and above caused a decrease in proacrosin activation in both preparations. Similarly, proacrosin was sensitive to short incubation periods at 45 degrees C and above which caused a decrease in the amount of proacrosin found in both preparations.

Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa.

Pooled semen judged to be normal in all parameters was divided into a number of aliquots which were either 1) kept untreated; 2) mixed with glycerol (10% v/v); 3) washed by centrifugation and resuspended to the original volume with buffer; or 4) washed and resuspended in buffer with glycerol (10% v/v). The progressive motility, viability, ultrastructure, and acrosomal enzyme activity (8 different hydrolases) were studied before and after cryotreatment. The described washing procedure effectively removed seminal plasma, and did not alter sperm motility, sperm viability, sperm ultrastructure, or the acrosomal enzymes studied. Glycerol (10%, v/v) had a deleterious effect on most parameters evaluated before cryotreatment. Cryotreatment severely altered the motility and viability of the spermatozoa and their acrosomal morphology but did not cause significant decreases in most of the acrosomal hydrolases measured. However, acrosin/proacrosin levels decreased by 50-80% and were correlated to the acrosomal damage. A simple assay for the measurement of acrosin/proacrosin enzyme levels in whole sperm is presented which could be used as a monitor for acrosomal integrity. No significant differences were seen between the samples cryotreated in the absence or presence of seminal plasma.

Isolation and partial characterization of the plasma membrane from human spermatozoa.

Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions. The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM.