Biological activity assessment of a novel contraceptive antimicrobial agent.

Microbicides are a new category of compounds being developed as a prophylactic approach for the prevention of transmission of sexually transmitted diseases (STDs), including the human immunodeficiency virus (HIV). These are primarily being developed as women-controlled methods, with the target of designing new compounds or formulations that can be used without the knowledge of a male partner. Microbicide screening can be initially based on their hyaluronidase-inhibiting (HI) activity, as this enzyme plays a major role in the sperm and microbe penetration into the substrate. Derivatives of hesperidin, a citrus flavonoid glycoside, have been reported in the literature for their HI effects. Hesperidin was thereby sulphonated under strictly controlled conditions and the active fraction isolated and characterized, based on its HI activity. This derivative was screened for antimicrobial and enzyme-inhibitory activities, specifically for the reproductive tract. Sulphonated hesperidin (SH) was found to completely inhibit the sperm enzymes hyaluronidase, giving an indication toward its contraceptive effects. It was also been found to inhibit various sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoea, HIV, and Herpes Simplex virus type 2 (HSV-2). Its safety assessment was based on its noninterference in sperm motility and its penetration through the cervical mucus, and no effect on the growth of lactobacilli, the normal vaginal flora. It was also found to be nontoxic to the HIV substrate cells (MT2 cells). The study concludes that sulphonated hesperidin can be developed as a potential microbicide for a dual prophylaxis of contraception and transmission of STDs and AIDS.

Poly(sodium 4-styrene sulfonate): evaluation of a topical microbicide gel against herpes simplex virus type 2 and Chlamydia trachomatis infections in mice.

OBJECTIVE: To investigate the in vivo activity of poly(sodium 4-styrene sulfonate) (T-PSS) gel formulations as topical microbicides. METHODS: The ability of the gel formulations to reduce the incidence of infection when applied prior to pathogen challenge was examined in mouse models of vaginal herpes simplex type 2 (HSV-2) and Chlamydia trachomatis infection, and rectal HSV-2 infection. RESULTS: In the vaginal HSV-2 challenge studies, 10% T-PSS gel provided significant protection against infection, even when administered 60 min prior to virus challenge (P < 0.0001). Both 5% and 10% T-PSS gel formulations significantly reduced the incidence of upper genital tract C. trachomatis infection in animals treated up to 5 min before challenge (P < 0.001). However, no protection against C. trachomatis infection was seen in animals treated 30 min before challenge. In mice challenged rectally with HSV-2, both the 5% and 10% T-PSS gels significantly reduced infection at 20 s (P < 0.01 for both). However, only the 10% gel provided significant protection when administered 5 min before challenge (P < 0.01). CONCLUSIONS: T-PSS gel formulations have promising in vivo activity as topical microbicides.

Mandelic acid condensation polymer: novel candidate microbicide for prevention of human immunodeficiency virus and herpes simplex virus entry.

Presently marketed vaginal barrier methods are cytotoxic and damaging to the vaginal epithelium and natural vaginal flora when used frequently. Novel noncytotoxic agents are needed to protect men and women from sexually transmitted diseases. One novel candidate is a mandelic acid condensation polymer, designated SAMMA. The spectrum and mechanism of antiviral activity were explored using clinical isolates and laboratory-adapted strains of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). SAMMA is highly effective against all CCR5 and CXCR4 isolates of HIV in primary human macrophages and peripheral blood mononuclear cells. SAMMA also inhibits infection of cervical epithelial cells by HSV. Moreover, it exhibits little or no cytotoxicity and has an excellent selectivity index. SAMMA, although not a sulfonated or sulfated polymer, blocks the binding of HIV and HSV to cells by targeting the envelope glycoproteins gp120 and gB-2, respectively, and also inhibits HSV entry postattachment. SAMMA is an excellent, structurally novel candidate microbicide that warrants further preclinical evaluation.

Chemistry and pharmacology of the Citrus bioflavonoid hesperidin.

Hesperidin, a bioflavonoid, is an abundant and inexpensive by-product of Citrus cultivation. A deficiency of this substance in the diet has been linked with abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and night leg cramps. No signs of toxicity have been observed with the normal intake of hesperidin or related compounds. Both hesperidin and its aglycone hesperetin have been reported to possess a wide range of pharmacological properties. This paper reviews various aspects of hesperidin and its related compounds, including their occurrence, physical and chemical properties, analysis, pharmacokinetics, safety and toxicity and the marketed products available. A special emphasis has been laid on the pharmacological properties and medicinal uses of these compounds.

Papillomavirus microbicidal activities of high-molecular-weight cellulose sulfate, dextran sulfate, and polystyrene sulfonate.

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.

Properties of a new acid-buffering bioadhesive vaginal formulation (ACIDFORM).

Vaginal prophylactic methodology may prevent heterosexual transmission of the HIV and other sexually transmitted disease-causing organisms as well as unplanned pregnancies. A new delivery system (ACIDFORM) was designed with acid-buffering, bioadhesive, and viscosity-retaining properties to (1) maintain the acidic vaginal milieu (the low pH inactivates many pathogens and spermatozoa), (2) form a protective layer over the vaginal/cervical epithelium (minimizing contact with pathogenic organisms), and (3) provide long-term vaginal retention. A Phase I clinical study with ACIDFORM provided initial information about its safety and showed the formation of a layer over the vaginal/cervical epithelium [1; Amaral et al., Contraception 1999;60:361-6]. To study the properties of the gel (without active ingredient) in more detail, ACIDFORM's acid-buffering, bioadhesive, viscosity-retaining, and spermicidal properties were compared in vitro to marketed formulations, and its long-term stability was assessed. ACIDFORM, either when titrated with NaOH or when mixed directly with semen, is highly acid buffering and much more effective than Aci-Jel, a commercial acid-buffering vaginal product. ACIDFORM adheres well to two model membranes (excised sheep vagina and cellophane) and is more bioadhesive than Conceptrol, Advantage S, Replens, Aci-Jel, and K-Y jelly. On dilution, ACIDFORM also retains its viscosity better than these marketed products. ACIDFORM is spermicidal and is stable for at least 2 years. These results suggest that ACIDFORM has advantages over presently marketed vaginal delivery systems. The gel may either be useful by itself as an antimicrobial contraceptive product or as a formulation vehicle for an active ingredient with antimicrobial and/or contraceptive properties.

Properties of a new, long-lasting vaginal delivery system (LASRS) for contraceptive and antimicrobial agents.

In view of the need for improved vaginal formulations that are contraceptive, that may prevent transmission of sexually transmitted infections, or both, a new delivery system (base formulation; called Long Acting, Sustained Release of Spermicide, or LASRS) was developed that contains bioadhesive and other ingredients with a long history of safety, and was designed to provide long-lasting vaginal retention of the formulation and to minimize possible vaginal irritation caused by incorporated active ingredients. Nonoxynol-9 (N-9) was added as an active ingredient to study the vaginal irritating properties of the formulation and to assess its long-term effectiveness by postcoital spermicidal tests. In the first series of experiments, in vitro studies showed that the formulation spreads rapidly over a cellulose membrane, forming a bioadhesive layer that remained for at least 12 hours. The second series of experiments addressed the safety of the LASRS suppository in rabbits and primates. Even with a very high concentration of N-9 (20.5%), LASRS caused only mild/moderate but acceptable irritation in the rabbit. No vaginal irritation occurred in the primate at an even higher concentration (22.5%). During the third series of experiments, the long-lasting vaginal retention properties were evaluated by postcoital spermicidal tests in the primate. LASRS with N-9 was highly spermicidal even when mating was delayed for 12 hours after placement of the formulation. Spermicidal activity was also observed when 1) mating was delayed for 24 hours after insertion of the formulation, and 2) if the females were mated 2 or even 3 times without reinsertion of the suppository before collection of the vaginal contents. In the final series of tests, the postcoital spermicidal properties of menfegol, another cytotoxic spermicide, were evaluated as were several modifications in the base formulation. Menfegol produced essentially the same results as N-9. Altering the base formulation proved to be nonbeneficial because a decrease in the long-term spermicidal effectiveness was obtained. These results suggest that the LASRS suppository has good vehicle properties for the delivery of active ingredients to the vagina.

[Prevention of sexually transmitted diseases in women: association with socioeconomic and demographic variables]. / Prevenção de doenças sexualmente transmissíveis em mulheres: associação com variáveis sócio-econômicas e demográficas.

Sexually transmitted diseases (STDs) have been a subject of discussion both among scientists and in the mass media, especially because of their association with the human immunodeficiency virus (HIV). We studied the adoption of specific protective behaviors for the prevention of STDs among women, as well as the associations between these behaviors and socioeconomic and demographic variables. This was a descriptive study based on secondary data from a previous study carried out in Campinas, São Paulo State, Brazil. A total of 635 women were selected using the social network ("snowball") technique. Subjects were classified into four groups: adolescents and adults of upper middle and lower socioeconomic status, respectively. Condoms were the STD prevention method most frequently mentioned by interviewees. A negative association was observed between having a steady partner and condom use in all the groups. The main reason mentioned for not using condoms was "having a single partner and trusting him". Among adolescents, a positive association was observed between schooling above the 8th grade and condom use, and a negative association was observed between age and condom use. Among adults, only condom use in general was also positively associated with socioeconomic status.

A new vaginal antimicrobial contraceptive formulation: phase I clinical pilot studies.

Pilot clinical trials were performed with a new vaginal suppository called "Long Acting, Sustained Release of Spermicide" ("LASRS"). No visual or colposcopic lesions or patient complaints occurred as a result of using LASRS with increasing doses of nonoxynol-9 (up to 20%) for 5 days or of applying the highest dose of nonoxynol-9 (20%; total 400 mg) for 8 h. Colposcopic or visual lesions were also not induced when LASRS with 20% nonoxynol-9 was used for 7 consecutive days by the study participants except for those who developed symptomatic monilia vaginitis. Symptoms were reported although these were mostly minor. A long-lasting, bioadhesive, translucent layer (film) of formulation formed over the vaginal and cervical surfaces. Postcoital spermicidal studies showed LASRS to be highly effective for prolonged periods of time. Although intercourse was delayed for 5 to 8.5 h after insertion of the formulation, an average of only 0. 2 motile sperm/HPF could be found in cervical mucus. These studies suggest LASRS to possess advantages over presently marketed formulations by having long-term efficacy and by forming a bioadhesive, presumably protective layer over the genital tract epithelium. The results also suggest the formulation to decrease the vaginal irritation caused by nonoxynol-9 as noted by colposcopy. These pilot data support a more extensive study with the LASRS suppository.

Evaluation of poly(styrene-4-sulfonate) as a preventive agent for conception and sexually transmitted diseases.

A commercial preparation of a sodium polystyrene sulfonate (designated as N-PSS; its molecular weight is 500000 daltons) was tested as an inhibitor of sperm function and as a preventive agent for conception and the transmission of sexually transmitted diseases. The polymer is an irreversible inhibitor of hyaluronidase and acrosin; its IC50 values are 5.7 microg/mL and 0.5 microg/mL, for hyaluronidase and acrosin, respectively. N-PSS is also a stimulus of human sperm acrosomal loss. It produces maximal acrosomal loss at 2.5 microg/mL. Contraception in rabbits is nearly complete when rabbit spermatozoa are pretreated with 0.5 mg/mL of N-PSS before artificial insemination; however, N-PSS does not immobilize spermatozoa at concentrations as high as 50 mg/mL. N-PSS has broad spectrum antiviral and antibacterial activities. Infection by human immunodeficiency virus and herpes simplex virus are inhibited by N-PSS; 3-log reductions are produced by 7 microg/mL and 3 microg/mL, respectively. N-PSS is active against Chlamydia trachomatis and Neisseria gonorrhoeae. At 1 mg/mL, N-PSS inhibits chlamydial infectivity by more than 90%. N-PSS produces a 3-log reduction in gonococcal growth at 15 microg/mL. In contrast, N-PSS (5 mg/mL) does not affect the growth of Lactobacillus (normal component of the vaginal flora). N-PSS can be classified as a noncytotoxic contraceptive antimicrobial agent. These properties justify bringing a polystyrene sulfonate into clinical trials for its evaluation as a preventive agent for conception and several sexually transmitted diseases.

Poly(sodium 4-styrene sulfonate): an effective candidate topical antimicrobial for the prevention of sexually transmitted diseases.

Presently marketed vaginal barrier agents are cytotoxic and damage the vaginal epithelium and natural vaginal flora with frequent use. Novel noncytotoxic agents are needed to protect women from sexually transmitted diseases. One candidate compound is a high-molecular-mass form of soluble poly(sodium 4-styrene sulfonate) (T-PSS). The antimicrobial activity of T-PSS was evaluated in primary culture systems and in a genital herpes murine model. Results obtained indicate that T-PSS is highly effective against herpes simplex viruses, Neisseria gonorrhoeae, and Chlamydia trachomatis in vitro. A 5% T-PSS gel protected 15 of 16 mice from vaginal herpes, compared with 2 of 16 mice treated with a placebo gel. Moreover, T-PSS exhibited little or no cytotoxicity and has an excellent selectivity index. T-PSS is an excellent candidate topical antimicrobial that blocks adherence of herpes simplex virus at low concentrations, inactivates virus at higher concentrations, and exhibits a broad spectrum of antimicrobial activity.

Women's preferences for vaginal antimicrobial contraceptives. I. Methodology.

Sexually transmitted diseases, including AIDS, and unplanned pregnancies continue to be a serious worldwide problem. A number of organizations are developing woman-controlled vaginal formulations to prevent these problems. However, little information is available regarding the types of products women prefer even though such knowledge is essential to obtain widespread use. This is the first of several articles that describe the results of a consumer preference study for such vaginal formulations performed in Campinas, São Paulo State, Brazil. Because no published methodology was available, the instruments and interview techniques were developed first and procedures established for the identification and participation of research subjects. After preparation of a questionnaire, a pilot study was performed to evaluate it, to establish the interview technique, and to determine the optimal method for subject recruitment. Based on the results, the approach was selected and applied to 635 subjects from different age and socioeconomic groups. The developed methodology and questionnaire, the advantages and the problems encountered, are presented.

Women's preferences for vaginal antimicrobial contraceptives. II. Preferred characteristics according to women's age and socioeconomic status.

A study was carried out to identify characteristics that women would want for an idealized vaginal contraceptive, and the possible association of these characteristics with age and socioeconomic status. The study was done in Campinas, São Paulo State, Brazil. A total of 635 women were selected by age and socioeconomic status, using the "social network" technique. Almost half were adolescents (15-19 years old) and the rest were adults (20-45 years old). Half were of low socioeconomic status and the rest of medium-high status. The data were analyzed with SPSS-PC and EPI-INFO 6.0. Logistic regression and chi 2 were used for the analysis. Despite some differences found between age and socioeconomic status in regard to the characteristics desired for the idealized method, most of the participants expressed the same preferences. The results indicate that women would like the idealized method to be a cream, rather than a suppository, with no odor or flavor, to be colorless, to be placed in the vagina with an applicator well before coitus, and to offer protection against sexually transmitted diseases including AIDS.

Women's preferences for vaginal antimicrobial contraceptives. III. Choice of a formulation, applicator, and packaging.

Novel vaginal formulations are under development to combat the increasing incidence of sexually transmitted diseases, including AIDS, and also unplanned pregnancies. A study was performed to determine women's preferences for different dosage forms (gel, cream, ovule/suppository, film, foam, tablet), width, length, and color of an applicator, and various types of packages. The study was conducted in Campinas, Brazil. A total of 635 women were interviewed, including both adolescents and adults and low and middle-high socioeconomic groups. The large majority of the women preferred a gel over a cream; both were preferred over the other methods. When asked which method they would not use, the film was most frequently identified, followed by the tablet and ovule. The primary reasons for selecting a particular dosage form were ease of use, absence of odor or the presence of a pleasant one, absence of color, and insertion with an applicator. The major reasons for not using a method were discomfort, "plastic" appearance, distrust of effectiveness, difficulty with insertion, messiness, and rigidity/hardness. The majority of the women liked the applicator shown. The prefilled single dose applicator was by far the preferred packaging. This information should aid in the development of consumer-friendly, vaginal formulations.

Women's preferences for vaginal antimicrobial contraceptives. IV. Attributes of a formulation that would protect from STD/AIDS.

Vaginal formulations may have "dual" protective activity, against sexually transmitted diseases/AIDS and unplanned pregnancy. The attributes that women find acceptable or unacceptable for such dual protective methods were investigated. More than 50% of the women would not accept messiness, but it was more accepted for dual protective methods than for contraceptives. Very few women would use a dual protective method if it caused vaginal irritation, itching, swelling, or burning, problems associated with presently marketed methods. More than half of the women would use it if it appeared on the penis of their partner or required refrigeration. Use of an applicator to insert the formulation was generally preferred over a manual method. Most women preferred the formulation to be colorless or white, about 16% liked light colors, and about 10% liked darker colors. Almost half of the women were willing to pay up to $5.00 per application of a dual protective formulation, about 15% $3.00, and 30% $1.00. Dual protective methods seem highly acceptable and women would pay much more for them than for condoms. However, these methods should be free of problems usually associated with presently marketed formulations.

Characterization of cysteamine as a potential contraceptive anti-HIV agent.

Cysteamine (beta-mercaptoethylamine, or MEA) is a thiol-reducing agent and has anti-HIV activity. Because of these properties, cysteamine was evaluated as a vaginal contraceptive and tested for its effects on sperm function and on other sexually transmitted microbes. Cysteamine was contraceptive in the rabbit. Conception was inhibited completely when sperm were pretreated with 500 microg/ml cysteamine and was inhibited by more than 60% when 7.5 mg cysteamine was applied vaginally as a suspension in 50% K-Y Jelly. Cysteamine had multiple effects on spermatozoa. Both acrosin (EC 3.4.21.10) and hyaluronidase (EC 3.2.1.35) were reversibly inhibited by cysteamine. Calculated IC50 values were 370 microg/ml and 150 microg/ml for acrosin and hyaluronidase, respectively. Cysteamine behaved as a poor spermicide when activity was measured by the 30-second Sander-Cramer test. However, sperm motility was inhibited completely when cysteamine was preincubated for 10 minutes prior to motility evaluation, at concentrations as low as 50 microg/ml. The calcium ionophore A23187-induced human acrosome reaction was inhibited by cysteamine (IC50 = 0.5 microg/ml). Neither herpes simplex virus nor Neisseria gonorrhoeae was affected by cysteamine at concentrations as high as 500 microg/ml and 100 microg/ml, respectively. Cysteamine appears to have no effect on normal vaginal flora (i.e., lactobacillus). These results, together with published data, strongly support the further development of cysteamine as a topical contraceptive anti-HIV agent.

Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy.

The acrosome reaction is an important marker for human sperm function. Since different laboratory techniques may be used for the detection of this exocytotic process, the purpose of the present study was to compare three common markers [Pisum sativum agglutinin (PSA), concanavalin A (ConA), double staining] and transmission electron microscopy for identification of acrosomal changes. Preliminary findings had demonstrated that similar results were achieved with Trypan Blue and Hoechst 33258 staining. Therefore, supravital stainings were omitted. In various experiments, human spermatozoa were treated with two concentrations (10 and 3.3 microM) of calcium ionophore A23187 for 15, 30 and 60 min after capacitation for 3 and 6 h at 37 degrees C. The percentages of spermatozoa with acrosomal loss detected by fluorescein isothiocyanate (FITC)-ConA were consistently lower than those obtained by double staining or FITC-PSA, which showed comparable results. Following 6 h of capacitation and incubation with 10 microM ionophore for 1 h at 37 degrees C, 25.9 +/- 15.7% of all spermatozoa showed almost complete loss of the acrosomal content. Binding of FITC-ConA to the acrosomal region was observed in 27.0 +/- 13.2% of spermatozoa obtained from the same sample. FITC-ConA and double staining or FITC-PSA detect different stages of the acrosome reaction and may be helpful for a differentiated evaluation of this sperm function.

Paramagnetic beads coated with Pisum sativum agglutinin bind to human spermatozoa undergoing the acrosome reaction.

For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction. Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 microM A23187 (20.3 +/- 6.7% [mean +/- SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1 +/- 8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1 +/- 12.5%, n = 10). Bead binding was significantly reduced by pre-incubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with alpha-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.

Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma.

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.

Atrial natriuretic peptide: a chemoattractant of human spermatozoa by a guanylate cyclase-dependent pathway.

Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as egg-derived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half-maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5-27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)