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Sodium aurothiomalate, a low molecular weight inhibitor of hyaluronidase, blocked in-vitro fertilization in hamsters at the level of the zona pellucida: concentrations of 25-250 micrograms inhibitor/ml prevented fertilization of cumulus-intact and cumulus-denuded eggs. Fertilization of zona-free ova was not affected. In-vivo fertilization was also reduced (from 100% controls to 37.5%) by 10 mg inhibitor/ml added to an epididymal sperm suspension before artificial insemination into the uterus. These findings suggest that hyaluronidase may play a role in zona penetration or that sodium aurothiomalate blocks sperm penetration of the zona by inhibiting an enzyme(s) other than hyaluronidase.

Chemicals can interfere with hormonal control of the male reproductive tract and/or directly alter male reproductive tract function. A review is presented of those chemicals developed and tested as male contraceptive agents which have a direct effect on the male reproductive tract with minimal disturbance of the hormonal milieu. Such chemicals can have one or more sites of action: 1) the testis, disturbing spermatogenesis; 2) the epididymis, altering sperm maturation; 3) the vas deferens, affecting sperm transport; and 4) the accessory sex glands, entering the ejaculate and changing the functional activity of the spermatozoa. Examples of each mode of action are presented.

Previous experiments with inbred mice showed that chronic ethanol treatment delays male pubertal development. An initial event in sexual maturation in the rat is a transient increase in 5 alpha-reductase. The present study was conducted to determine whether similar ethanol effects occur in outbred mice (Swiss-Webster), to determine the ontological profile of testicular 5 alpha-reductase in the mouse, and to evaluate the effect of ethanol treatment on this enzyme. After 29 days of treatment with a liquid diet (beginning at age 20 days), reductions in the ethanol-treated mice as compared with the controls were noted in testicular weight (55.0 +/- 2.0 vs. 63.0 +/- 2.4 mg; P less than 0.01), epididymal sperm content (6.8 X 10(5) vs. 14.4 X 10(5); P less than 0.05), and sperm motility (45% vs. 57%; P less than 0.05). After 43 days, differences no longer existed. In chow-fed mice, a substantial rise in 5 alpha-reductase (1 unit = 1 pmole DHT formed/45 min/mg testis) began at age 24 days. Activity peaked at approximately 65 units at 25 to 30 days and gradually declined to 6.4 +/- 0.8 units at 63 days. After 29 days treatment, 5 alpha-reductase of the pair-fed control group was 26.8 +/- 4.9 units, which decreased to a baseline value of 7.0 +/- 2.1 units after 43 days treatment. In contrast, 5 alpha-reductase of the ethanol-treated group remained at baseline levels after 29 days (7.7 +/- 2.3 units) and 43 days of treatment (7.6 +/- 2.3 units).(ABSTRACT TRUNCATED AT 250 WORDS)

Penetration of zona-free hamster oocytes by human spermatozoa after preincubation with BWW medium (standard technique) or a buffer system containing egg yolk (TESTY) was compared by applying both treatments to aliquots of 60 ejaculates from 34 patients. The TESTY-treated spermatozoa penetrated the oocytes much more successfully than the spermatozoa treated by the standard technique. Although the penetration levels achieved by the two methods were significantly correlated, the correlation coefficient was too low to predict the penetration outcome from one technique to the other. More consistent results were obtained when different ejaculates from the same donor were treated by TESTY than by the standard technique. Spermatozoan penetrating ability was more strongly correlated with sperm concentration, motility, and morphology after treatment by the standard technique than after TESTY treatment. Much larger differences in penetration were present between normal and abnormal ejaculates when the standard technique was used than TESTY.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.

Lysozyme, alpha-amylase, neutral proteinase and plasminogen activator were most concentrated in the initial portion of the ejaculate that consists mostly of Cowper's gland and prostate gland fluids as well as spermatozoa. The concentration of the high molecular weight proteinase inhibitors, alpha1-antitrypsin and alpha1X-antichymotrypsin, was essentially unaltered throughout the ejaculate fractions, although their absolute amounts showed an increase towards the final fraction. By contrast, the total inhibitory activity towards pancreatic trypsin was highest both in concentration and amount in the last fraction, thus indicating that the seminal vesicles are its primary source. Plasminogen, prothrombin, Factor XIII, and the proteinase inhibitors antithrombin III, alpha2-macroglobulin, inter-alpha-trypsin inhibitor and C1S-inactivator could not be detected immunochemically in whole ejaculates, and indicates the dissimilarity between the coagulation/liquefaction processes of semen and blood.

Enzymes have been implicated in facilitating cervical mucus penetration by spermatozoa. One of these enzymes in the neutral proteinase acrosin, which is associated with the sperm acrosome. To determine the validity of this hypothesis, human spermatozoa were incubated with the following acrosin inhibitors: p-aminobenzamidine (AB), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and p-nitropheyl-p'-guanidino benzoate (NPGB). An in vitro slide test system was developed which allowed inhibitor-treated and control spermatozoa to be evaluated against the same human cervical mucus sample. At inhibitor concentrations far exceeding those necessary for the inhibition of human acrosin, there was no effect on spermatozoal penetration into or through the mucus. These findings indicate that, in man, acrosin activity is neither necessary nor facilitory to sperm penetration of cervical mucus. Evidence is also presented that demonstrates the superiority of the newly developed double-interface slide test, especially for comparative purposes, over the tests currently in use.

An improved substrate-film technique has been developed for the assay of released proteinase from individual human spermatozoa. During the preparation of the thin gelatin membrane, it is pretreated with formaldehyde and NaOH. These agents alter the plasma membrane and the outer acrosomal membrane of the spermatozoon, facilitating the release of acrosomal enzymes. This effect is further enhanced by the addition of albumin to the incubation mixture. More than 90% of the spermatozoa in a normal ejaculate give a reaction by this method. It reaches a maximum after 4 hours of incubation and does not increase further even up to 12 hours. No difference in the reaction between washed and unwashed ejaculated spermatozoa can be found. The nature of the proteolytic activity and its possible significance in infertility are discussed.

Treatment of midcycle human cervical mucus with the lectins Ulex europaeus agglutinin I (UE), wheat germ agglutinin (WGA), and peanut agglutinin (PA) did not alter the ability of spermatozoa to enter and migrate through the mucus. These lectins form glycoconjugates by binding to L-fucose (UE), N-acetylglucosamine and sialic acid (WGA), and D-galactose and D-galactose-(1-3)-D-N-acetylgalactosamine (PA), sugars that are present in the carbohydrate side chains of the mucus glycoproteins (mucins). Ricinus communis, with high affinity for D-galactose and N-acetylgalactosamine, did cause a significant decrease in sperm migration but only within the mucus (not at the sperm-mucus interface); this may have been because of an effect on sperm motility. These sugars are thought to be important for the secondary structure of the glycoproteins and for the cross linking between the mucins that produce the rheologic properties of cervical mucus. However, it appears that interference with the sugars by lectin binding does not significantly alter the ability of spermatozoa to migrate through cervical mucus.

A high molecular weight antifertility factor (AF-1) was obtained in a high degree of purity from human seminal plasma by ultracentrifugation, CM-cellulose and concanavalin A chromatography, and Sepharose 6B gel filtration. A final purification step involving preparative disc electrophoresis was occasionally required. AF-1 showed a dose-dependent inhibition of the in vitro fertilizing ability of capacitated mouse spermatozoa, causing 50% inhibition of fertilization at approximately 27.5 micrograms/10(6) spermatozoa. Removal of the follicle cell layer of the oocyte did not decrease the antifertility effect of AF-1 but inhibition of fertilization was no longer observed after dispersal of the zona pellucida. The effect of AF-1 was on the spermatozoa and not on the oocytes. These results show that AF-1 treatment prevents capacitated spermatozoa from penetrating the zona pellucida and possibly the follicle cell layer. The mechanism by which AF-1 does so is not known because AF-1 did not prevent the in vitro acrosome reaction of guinea pig spermatozoa, nor did it inhibit the activity of human acrosin or bovine testicular hyaluronidase. The antifertility activity of AF-1 is reversed after recapacitation of the AF-1 treated spermatozoa and it can be assumed that AF-1 is either dissipated or loses activity during the transport of the spermatozoa through the female genital tract when capacitation takes place. AF-1 is heat labile. The properties of AF-1 are the same as those found previously for the pellet obtained by high-speed centrifugation of human seminal plasma, indicating that this is the primary, nonparticulate, high molecular weight factor with antifertility activity in human semen.

A good animal model that simulates the human subject has not been available for the evaluation of the in vivo effectiveness of vaginal contraceptives. After careful consideration. The stumptailed macaque (Macaca arctoides) was studied for its applicability since it has a reproductive tract similar to that of the woman, is easy to handle, does not require tranquilization or anesthesia when the contraceptive is deposited, and breeds and conceives readily under caged conditions. The reported observations show the usefulness of this animal. Both postcoital sperm motility studies and breeding experiments were performed with the use of Delfen vaginal cream and K-Y jelly. K-Y jelly had no effect on the motility of vaginal spermatozoa or on the conception rate of the primates. Although Delfen vaginal cream consistently immobilized all spermatozoa in the postcoital test, half of the animals became pregnant within an average of 3.7 breeding cycles. These results illustrate the discrepancy between spermicidal tests and fertility measurements, and it is recommended that primate-breeding experiments be performed before a spermicide is evaluated in women as a contraceptive.

The intrauterine application of proteinase inhibitors, tranexaminc acid and the pancreatic trypsin inhibitor (Trasylol), reduces or eliminates menorrhagia and intermenstrual bleeding (spotting) produced by an intrauterine device (IUD). A decrease in pain and vaginal (cervical) discharge is also frequently observed. A single application is usually sufficient, more than three never being required. The effect lasts for an average of three cycles. In addition to the clinical use of these agents for the treatment of uterine hemorrhage, the slow release of proteinase inhibitors from an IUD may well be useful in minimizing its side effects without interfering with its contraceptive activity.

A polymeric delivery system consisting of ethylene-vinyl acetate copolymer (EVAc) was developed for 2'-carbomethoxyphenyl 4-guanidinobenzoate (MSGB), a potent inhibitor of the sperm enzyme acrosin. The optimal device consists of copolymer with 40% vinyl acetate by weight (EVAc/40), 65% drug loading and MSGB with a particle size of 250-499 micron. This formulation yields a device that is highly flexible and can be shaped to many forms and sizes. Construction of the device does not alter the properties of MSGB. Well controlled release of MSGB from the device occurs in vitro and in the uteri of rats. The in vitro release rate under "infinite sink" conditions is essentially the same as the in vivo release rate. The contraceptive effect of the MSGB-releasing device was tested in rabbits by placing a blank (control) device in one uterine horn and an MSGB-releasing device in the contralateral horn. In contrast to blank devices, MSGB-releasing devices completely prevent pregnancy, not only by inhibiting fertilization but also by decreasing implantation. MSGB possesses high in vitro antifibrinolytic activity. These results indicate that a very flexible device can be constructed for uterine application which retains its contraceptive effect by release of MSGB. The antifibrinolytic activity of MSGB may further decrease the menorrhagia that can be associated with IUD use.

The standard sperm parameters, and a newly developed assay, the hypoosmotic swelling (HOS) test, of 208 ejaculates were correlated with in vitro fertilization (IVF) outcome to assess their predictive value of the fertilizing capacity of spermatozoa. One hundred fifty-three (73.6%) of the ejaculates possessed spermatozoa that fertilized at least one oocyte and were considered fertile. Among the semen parameters studied, only the HOS test had a relatively high (r = 0.46, p less than 0.001) association with the fertilizing capacity of the spermatozoa. This test was also able to predict the outcome of IVF more reliably than the other semen parameters studied.

In vitro studies have shown that acetaldehyde is a more potent inhibitor of testicular steroidogenesis than ethanol. The present study examined the in vivo role of acetaldehyde in ethanol-induced reduction of testosterone by (1) determining the levels of acetaldehyde to which the testes were exposed subsequent to acute ethanol administration to mice; and (2) examining the effect of ethanol on testosterone in animals subsequent to drug pretreatment which decreased or increased ethanol-derived acetaldehyde. Ethanol-induced (3 g/kg) depression of testosterone was dependent upon gonadotropin stimulation. The increase in hCG-induced testosterone was suppressed (P less than 0.01) in ethanol- as compared to saline-treated animals [39.8 +/- 2.6 (S.E.M.) vs 28.1 +/- 2.3 ng/ml]. Pargyline (100 mg/kg) or cyanamide (8.4 mg/kg) increased (P less than 0.05) plasma and testicular acetaldehyde, while having no effect on the testosterone response to ethanol. Similarly, 4-methylpyrazole (25 mg/kg) reduced blood and testicular acetaldehyde to nondetectable levels, while having no effect on testosterone. Testicular acetaldehyde was lower (P less than 0.001) than plasma levels (14 +/- 2 vs 2.0 +/- 0.2 microM). This functional blood-testis barrier to acetaldehyde could be explained by testicular aldehyde dehydrogenases in the mitochondria (Km for acetaldehyde = 1.5 microM) and in the cytosol (Km = 123 microM) whose maximal activities totaled to more than 25-fold greater than that of testicular alcohol dehydrogenase (ADH). ADH was concentrated in the Leydig cells, while aldehyde dehydrogenase was evenly distributed in the testis. Ethanol prevented further hCG-induced rises in testosterone rather than inhibiting testosterone production to below pre-ethanol values. The above data argue against a significant role of acetaldehyde in the in vivo response of testosterone to ethanol. Ethanol appears to impair gonadotropin-testicular receptor interaction in vivo.

Different types of rigid, reversible, vas deferens occlusive devices were developed and evaluated in 14 unilaterally vasectomized dogs. All prosthetic devices had molded silicone rubber bodies, and rigid inflow and outflow tubes. Various techniques for the removal of the vas luminal epithelium, and for the preparation of porous ceramic and etched stainless steel surfaces to encourage tissue ingrowth into the prosthetic device end tubues were attempted. The devices differed in their methods of achieving occlusion. One device used a "rotary stem valve" which had a C-section rotating mechanism; the others used the "shuttle stem valve" which possessed an occlusive element that moved transverse to the axis of flow in the device, thus occluding the device when the stem was depressed. The rotarystem valve was implanted by means of a longitudinal incision. The remaining 13 shuttle stem devices were placed in the vas using either a longitudinal or a transverse implantation. Inno case was sperm transport through the prosthetic devices obtained for more than a few ejaculations.

The secretory proteins of the mucosa of the cervix, uterus, and fallopian tubes were investigated by measuring the proteins that were released by isolated mucosal areas. Initial screening disclosed that the immunoglobulins IgG and IgA were released in measurable quantities, but that IgM and the secretory (T) piece of IgA were either absent or present only in trace amounts. Relatively low levels of diffusable total complement activity and the C3 component of complement were present, whereas the C1q, C1r, and C4 components were either absent or present only in trace quantities. No neutral proteinase activity was present, but lysozyme, plasminogen activator, alpha 1-antitrypsin, and alpha 1x-antichymotrypsin could be found in reasonable amounts. The site of secretion, concentration, and cyclic variation of the proteins that diffused from the mucosal sites in measurable quantities were studied. The types and amounts of protein secreted by a particular site in the cervix, uterus, or fallopian tube varied from those of protein from other sites, even within the same organ. During the menstrual cycle, variations occurred in the amount of protein secreted by each mucosal site. However, whether an increase or a decrease in the release of a particular protein took place varied with each protein, even at the same site. The mucosal sites also differed from each other in their response to the phase of the menstrual cycle, that is, whether more or less protein was released, even sites within the same organ. The conclusion is that each organ and even different sites within an organ can respond independently from each other to changes in hormone levels, producing different types and amounts of secretory proteins. The amount of diffusable protein produced by an individual site during the menstrual cycle depends on the type of protein as well as the mucosal site.

Sperm without acrosome of two males--spouses of infertile marriages--were studied. No acrosome could be identified in the Papanicolaou stained smears of either patient. Electron microscopy study confirmed the absence of acrosome and post-acrosomal sheath. The physical and functional integrity of the sperm membrane was determined by vital staining technique (72% unstained) and hypoosmotic stress test (69% swollen). Acrosin assay showed a five-fold decrease, and proacrosin values were almost eight-fold lower than the normal values. Nonzymogen acrosin level was normal. Zone-free hamster oocytes were not penetrated by the acrosomeless sperm showing 46% progressive motility, but postcoital tests in both female spouses showed active sperm. The results would indicate that the sperm devoid of acrosome may be considered as a primary cause of male infertility.

Stained sperm morphology slides from an in-vitro fertilization programme were evaluated by the modified Williams system, the World Health Organization (WHO) system and morphometric size measurements (image analysis) to determine which system(s) were better predictors of fertility. Amorphous sperm head shape was the best predictor of fertility in both the modified Williams system and the WHO system, predicting the proper fertility status in 65% of the cases. Using only normal sperm in the statistical model, normal sperm became a correct predictor in 66% of the cases in the modified Williams system. Morphometric measurements were not useful in predicting the fertility status of these samples. These studies indicate that morphology can not be used as a sole predictor of fertilizing capacity of human spermatozoa.

Lippes Loop and Multiload intrauterine contraceptive devices (IUDs) were fitted with silicone rubber sleeves which either did not contain any compound (blank) or contained the antifibrinolytic agent, epsilon-aminocaproic acid (EACA. In vitro measurements showed that the antifibrinolytic agent was released from the sleeves for a period of 20 days. After insertion into women, the blank Lippes Loop IUDs and Multiload IUDs as well as copper-containing Multiload IUDs caused a significant increase in blood loss beginning with the first menstrual cycle after insertion. The enhanced blood loss was generally retained during subsequent menstrual cycles. By contrast, the menstrual blood loss induced by IUDs containing EACA sleeves was not significantly greater during the first menstrual cycle after insertion that the preinsertion levels. When no more EACA was released, menstrual blood loss increased to approximately the same level as that observed with the blank and copper IUDs. Thus, release of EACA from an IUD retains menstrual blood loss at approximately physiologic levels. These results encourage the development of IUDs that are capable of releasing antifirbrinolytic agents over a long period so that the antimenorrhagic effect is maintained.

A spermicidal agent called "741" is used in the People's Republic of China as a substitute for nonoxynol-9 in vaginal contraceptive preparations. The relative spermicidal activity of these two agents was evaluated using dog, rabbit, monkey and human semen. Acute LD50 determinations in mice were also performed. The spermicidal activity and acute toxicity of both agents were either identical or very similar in all species. Since agent 741 is less expensive than nonoxynol-9 to manufacture and equally as potent in vitro, it is an alternative spermicide for vaginal contraceptive formulations.

In at least 4 of 7 cases, fertilization of intact human oocytes was more successful when spermatozoa were pretreated with TEST yolk medium at 5 degrees C for 2 hours as compared with the standard treatment with Ham's F-10 only. Both pregnancies that were obtained after the transfer of the fertilized oocytes resulted from oocytes fertilized by TEST yolk-treated spermatozoa. No decrease in fertilization occurred in any of the cases after TEST yolk treatment. If these results hold true for a larger series of patients, it may be worthwhile for the standard IVF incubation system of spermatozoa to include TEST yolk.

Three cyclooxygenase (prostaglandin synthetase) inhibitors, indomethacin, phenylbutazone, and oxyphenbutazone, decreased fertilization in vitro when mixed with capacitated mouse spermatozoa before addition of the treated gametes to oocytes. Fertilization was inhibited whether the oocytes were intact, follicle cell-free, or both follicle cell-free and zona-free. At various concentrations of inhibitor, no effect was observed on the motility or forward progression of the spermatozoa. These cyclooxygenase inhibitors also decreased the guinea pig acrosome reaction. Inhibition of the acrosome reaction did not occur when a mixture of the prostaglandins (PGE2 or PGF2 alpha) and one of the inhibitors was added to the spermatozoa. Alone, these prostaglandins tended to enhance the rate at which the acrosome reaction took place. Lowered calcium levels reduced the occurrence of the acrosome reaction, an effect that could be reversed at least partially by the addition of PGE2. Even in the nominal absence of calcium, some acrosome reaction took place when PGE2 was present in the medium. These results support an essential role for cyclooxygenase and arachidonic acid metabolites, including prostaglandins, in the events leading to the acrosome reaction and fertilization.

Scanning electron microscopy was used to examine surface ultrastructural characteristics of cells of the epithelium of female reproductive tract, cervical mucus, and amniotic fluid cells. The female epithelium undergoes hormone-dependent cyclical morphological alterations in cell shape, apical microvilli, ciliation, and secretory patterns. The frequency distribution of ciliated cells varies in different parts of the female reproductive tract and different segments of the same organ. In the endocervix, a few ciliated cells are randomly distributed. The density of ciliated cells is variable in several areas of the endometrium. The sparse distribution of ciliated cells in the central portion of the uterine wall may indicate a possible implantation site. According to surface ultrastructure of cells, three segments are noted in the uterotubal junction: cornual endometrium, interstitial tubal epithelium, and transitional area. The vaginal epithelium is made of flat polygonal cells with interdigitating borders and a fine interlacing network of microridges. After the menopause, the microridge pattern and distinct cell borders are lost or severely modified. Two types of fibrous structures can be found in cervical mucus: a) microfibrils that vary in diameter from 500 to 1,500 A and from bundles or networks, and b) long, thick fibers that vary in diameter from 0.5 to 5 micrometer run parallel to each other, and appear to be made up of microfibrils. Amniotic fluid cells, derived from amniotic epithelium and fetal ectoderm, consist of non-nucleated and nucleated cells with extensive microplicae.

Temperature and ion sensitivity of human acrosin (EC 3.4.21.10) was compared to that of human trypsin. With the exception of zinc, no ion tested had significant effects on either enzyme. Zinc behaved as a noncompetitive inhibitor of both enzymes, with inhibition constants of 1.8 and 1.7 mM for acrosin and trypsin respectively. Trypsin was inhibited by the chelators EDTA and EGTA, a specific effect reversed by either calcium or magnesium. EDTA inhibited acrosin in a nonspecific manner, while EGTA was without effect. Unlike acrosin from the other species, human acrosin was unaffected by calcium or the polyamines, spermine and spermidine. Acrosin was sensitive to inhibition by preincubation temperatures above 5 degrees C; trypsin, however, was stable to preincubation temperatures up to 60 degrees C. Hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester was more efficiently catalyzed by trypsin (6800 cal/mol) than by acrosin (9511 cal/mol).

A new steerable fiberoptic hysteroscope particularly useful in visualizing the tubal ostia has been evaluated in 61 extirpated uteri. The efficacy and safety of this hysteroscope were shown. The feasibility of deploying occlusive devices into the intramural portion of the tubes with the steerable hysteroscope was demonstrated. Whether these devices or modifications thereof will prove to be superior to other currently tested methods of occlusion, e.g., caustics, plastics, or cautery under in vivo conditions, remains to be established. The steerability of the hysteroscope has advantage irrespective of the method to achieve transcervical tubal occlusion as it allows coaxial alignment of the delivery.

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.

The Macaca arctoides (stumptailed macaque) was found to be a good animal model for determining the postcoital spermicidal activity of vaginal preparations. The stumptailed macaque is easy to handle, so formulations can be inserted correctly into the vagina just before coitus. The male mates rapidly, and the entire test can be completed within 5 to 10 minutes, minimizing all extraneous factors other than those inherent to the reproductive tract and the coital act. Data from postcoital breeding experiments were found to be reliable and consistent when results of six primate mating tests for a single dose level of a test formulation were averaged. Dose-response curves can be prepared from these average results, and the relative in vivo effectiveness of the spermicides can be determined. The Sander-Cramer test proved to be a good assay with which to quantitate the in vitro spermicidal potency of formulations. The spermicidal preparations tested immobilized human and primate spermatozoa in vitro to the same extent with the exception of one formulation (possibly due to the vehicle in which the active ingredient was incorporated). The relative spermicidal effectiveness of preparations differs with in vitro and postcoital testing. Because the latter is a more realistic indicator of the contraceptive potency of a formulation, it is recommended that postcoital primate experiments be performed with newly developed vaginal spermicides before extensive animal breeding experiments clinical trials are initiated.

Flexible prosthetic devices developed previously to reversibly block sperm transport through the vas degerens were implanted in the vasa deferentia of dogs or kept in a semen bath for a 6-month period. Dimensional measurements, flow characteristics, leakage tests, and detent action force tests to displace the valve stem were performed with the valves. No changes occurred in any of these parameters with the exception of a decrease in the force required to change the position of the valve stem. This explains the occasional passage of spermatozoa through certain closed devices after they have been implanted for long periods of time. Histologic studies indicated that the same types of genital tract changes are associated with an implanted device as with vasectomy, and that they occur with approximately equal frequencies. Scanning electron microscope studies showed (1) excellent tissue ingrowth into the Dacron velour material of the implanted device; (2) the build-up of material, consisting in part of spermatozoa, in the device lumen and on the value stem; and (3) the absence of surface degradation of either the Silastic material or the valve stem of the device. It is concluded that no irreversible tissue damage is caused by the long-term implantation of flexible prosthetic devices into the vas deferens.

Fifty-three dogs received implants of several types of flexible devices containing valving mechanisms. These devices were constructed entirely of silicone rubber with the exception of the valve stem, which was made of stainless steel. Generally, the devices were (1) implanted in the open mode and left this way for 27 to 44 months, (2) implanted in the closed mode and kept this way for 11 to 12 months before the valves were reopened, (3) implanted in either the closed or open mode and cycled to the opposite mode every 3 months (four or five cycles), or (4) implanted in either the closed or open mode and cycled to the opposite mode every 6 months (two or three cycles). Different implant methods were also evaluated. Semen analyses were regularly performed on all of the dogs, and a number of the animals were bred during the experiments. It can be concluded that (1) the devices can be opened and closed successfully over long periods, respectively allowing and preventing sperm transport; (2) the breeding ability of the animals is not impaired while the devices are in the open mode, independent of the type of device; (3) the devices do not result in an enhanced incidence of congenital abnormalities in the offspring; and (4) the success rate of device performance does not depend on the method of implanatation used. Thus, the results clearly indicate that the basic mechanism of a soft, reversible valve is a feasible approach to conception control.

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.

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.

A poor penetration rate of glycerol-treated, cryopreserved human spermatozoa as compared to untreated fresh control, was observed in the zona-free hamster oocyte test. Similarly, glycerol treatment of freshly ejaculated spermatozoa depressed the penetration rate unless the culture medium also contained glycerol. Immediately after thawing, glycerol-treated, cryopreserved spermatozoa possessed adequate progressive motility, but their incubation in glycerol-free culture medium caused a severe reduction in motility. Even if the same number of progressively motile, cryopreserved, glycerol-treated spermatozoa as unfrozen spermatozoa were added to the eggs, a much lower penetration rate was obtained by the treated spermatozoa. It is concluded that spermatozoa develop a glycerol dependence and that removal of glycerol from the surrounding medium, as most likely occurs when spermatozoa pass through the cervix, reduces both the motility and the ability of spermatozoa to become capacitated and fuse with oocytes. Thus, glycerol is not an optimal cryopreservative agent. Further, the decreased oocyte penetration rate of glycerol-treated, cryopreserved spermatozoa is due to other factors besides the decrease in sperm motility.

In contrast to the rabbit: 1) no hyaluronidase could be detected in fresh or frozen human seminal plasma, all color formation on assay being due to a dialyzable, heat stable factor; 2) almost no hyaluronidase could be extracted from frozen-thawed human testicles; 3) the hyaluronidase from human spermatozoa was rapidly inactivated upon release, either spontaneously or on extraction; and 4) a large decrease in hyaluronidase activity occurred when human spermatozoa were stored under various conditions. Rabbit spermatozoa contained six to 13 times more hyaluronidase than human spermatozoa. These results show that distinct species differences exist in the hyaluronidase associated with spermatozoa. None of the human genital tract sources studied could be used to obtain adequate amounts of hyaluronidase for the further isolation and purification of the enzyme. For both human and rabbit spermatozoa, it was optimal to add the cells directly to the assay system for the quantitation of hyaluronidase on spermatozoa rather than extracting the spermatozoa and testing the extracts. The spermatozoa of both species appear to be capable of digesting hyaluronic acid directly. As had previously been found for acrosin, a higher amount of hyaluronidase was maintained when human spermatozoa were cryopreserved in a zwitter buffer (TESTCY) rather than in glycerol.

The capacity of glass wool-filtered spermatozoa to penetrate zona-free hamster oocytes was studied. As compared to prefiltered sperm samples, oocyte penetration was significantly increased. A significant increase in the penetration rate for the filtered sperm population was noted even after the sperm motility in the filtrate was adjusted with medium equal to that of the prefiltered sample. However, no significant differences in oocyte penetration were seen between the prefiltered and the filtered sperm population when the filtered sperm samples were diluted with nonviable spermatozoa. These results show that glass wool filtration yields a sperm population with a greater penetrating capacity. It was concluded that motility alone could not account for the improved penetrability and that the removal of nonviable spermatozoa may at least, in part, be responsible for this effect.

Ethanol is generally regarded as a reproductive toxin. However, the mechanism(s) of ethanol-induced infertility remain poorly understood. As male fertility depends upon the ability of spermatozoa to fertilize ova, it was the purpose of the present study to examine the effects of chronic ethanol treatment on several parameters related to sperm fertility. Male C57Bl/6J mice of proven fertility were administered liquid diets as follows: 5% (v/v) ethanol for either 1) 5 weeks; 2) 10 weeks; 3) 20 weeks; or 4) 6% (v/v) ethanol for 5 weeks. After each treatment, epididymal spermatozoa were evaluated with respect to quantity, motility, morphology and the ability to fertilize. A biphasic effect on sperm content was noted: 5- and 10-week treatments with 5% ethanol increased content by 80 and 65%, respectively, whereas 20-week treatment with 5% ethanol and 5-week treatment with 6% ethanol decreased content by 52 and 71%, respectively. Although the proportion of motile spermatozoa was unaffected by ethanol, average forward progression velocity was reduced, the effect being dependent on ethanol dose and duration of exposure. Similarly, the frequency of abnormal spermatozoa was increased; 20-week treatment with 5% ethanol and 5-week treatment with 6% ethanol increased the frequency of sperm morphological anomalies by 50 and 40%, respectively. Fertility of spermatozoa was reduced as a function of ethanol dose and duration of exposure. The ability of sperm to fertilize mouse ova in vitro was reduced by 34% (P less than .02) and 62% (P less than .001) subsequent to 20-week treatment with 5% ethanol and 5-week treatment with 6% ethanol, respectively. An animal model has been developed which describes ethanol-induced male infertility. The degree of reproductive impairment varies with the amount of ethanol ingested, and the duration of ethanol exposure. The continuum of effects should make possible the evaluation of putative mechanisms of male sterility resulting from chronic ethanol consumption.

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. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.

This study compared the cryoprotective effect of glycerol with that of a zwitter ion buffer system (TESTCY). Spermatozoa that are cryopreserved in the presence of glycerol possess a somewhat higher progressive motility immediately after thawing than those preserved in the presence of TESTCY. However, after a 1-hour incubation in glycerol-free medium, the progressive motilities of the glycerol- and TESTCY-treated spermatozoa become essentially identical. After 2 hours in culture medium, TESTCY-treated spermatozoa possess a higher motility than glycerol-treated spermatozoa, indicating that TESTCY is a better preservative than glycerol for the long-term motility of human spermatozoa. The fertilizing potential of the cryopreserved spermatozoa was assessed by their ability to penetrate zona-free hamster oocytes in vitro. Spermatozoa that are cryopreserved in the presence of TESTCY produce three- to four-fold higher penetration rates than glycerol-treated, cryopreserved spermatozoa. Cryopreservation in the presence of TESTCY also results in a higher stability of the acrosin/proacrosin system than when the spermatozoa are preserved in glycerol, since about two- to three-fold higher levels of proacrosin are retained. These results indicate that TESTCY is a better cryopreservative for human spermatozoa than glycerol.

Antifibrinolytic agents when released into the uterine cavity decrease menorrhagia associated with IUD use. Our objective was to develop a matrix that could be incorporated onto an IUD and release anti-fibrinolytic agents. The copolymer ethylene-vinyl acetate (EVA) was selected for detailed study because it has the advantage over other materials in that it can release large molecular weight substances for more than 100 days, and allows incorporation of large amounts of anti-fibrinolytic agents with different molecular weights. Two compounds, tranexamic acid (AMCA, MW=157) and Trasylol (Kunitz pancreatic trypsin inhibitor, (MW=6,500) were incorporated into the EVA matrix and their release rates measured. In vitro studies with AMCA showed that after the initial burst, a constant high release rate was obtained over a prolonged period of time. The in utero release rate of AMCA from the EVA matrix in rabbits was similar to that obtained in vitro. By contrast, the release rate of Trasylol decreased to low levels during incubation in vitro. The release rate of Trasylol in utero however, appeared to be higher than that in vitro.

Ejaculates were obtained from C57BL mice by applying two successive series of electrical stimuli which were delivered via a bipolar rectal probe. The ejaculates thus collected contained fertile spermatozoa as indicated by results from in-vitro fertilization. Once separated from the seminal plasma, ejaculated spermatozoa possessed the same in-vitro fertilization rate as epididymal sperm. Ejaculates were analysed for coagulum weight, ejaculate volume, sperm count, sperm motility, acid phosphatase content and fructose content. Significant differences were present between several of these values for fertile and infertile mice, and values were therefore empirically assigned to represent minimal amounts for 'normal' fertility (1.5 microliters ejaculate volume; 10.2 mg coagulum weight; 2.5 x 10(6) spermatozoa/ml; 2.3 x 10(3) motile spermatozoa/ejaculate). One half of the fertile animals had no deficiencies in any of the characteristics measured, whereas 97% of the infertile animals had at least one deficiency. No fertile male had more than 2 deficiencies. These data show that the characteristics of mouse semen obtained by the present method of electroejaculation are related to the fertility status of the animal.

The present study was performed to evaluate the involvement of reduced testosterone in ethanol-induced impairment of male reproductive tract development. In vivo and in vitro gonadotropin (hCG)-stimulated steroidogenesis were examined in CFW mice as a function of chronic ethanol treatment during pubertal development. Chronic ethanol treatment from ages 20 to 49 days impaired testicular growth from ages 35 days (29 +/- 2 mg vs 37 +/- 2 mg for pair-fed controls) to 50 days (42 +/- 2 mg vs 63 +/- 2 mg for pair-fed controls). Consistent with a reduction in testicular weight, testicular content of androstenedione, testosterone, and dihydrotestosterone (DHT) was depressed in ethanol-treated mice. At age 50 days, the content (expressed as pg/testis) of androstenedione, testosterone, and DHT was reduced in ethanol-treated animals by 49%, 31%, and 38%, respectively, as compared to that of their respective controls. However, no difference in plasma (ng/mL) or testicular (pg/mg protein) concentrations of steroids was observed. Except for the DHT response at ages 35 to 40 days, neither in vivo nor in vitro steroidogenesis was impaired by chronic ethanol treatment at ages 26 to 50 days; similarly, the acute ethanol effect on steroidogenesis was unaffected. However, an adaptive increase (54%-173%) in the in vivo testosterone response to hCG was seen at ages 26 to 40 days. The data indicate that 1) chronic ethanol treatment does not impair gonadotropin-stimulated steroidogenesis or result in tolerance to acute ethanol effects on steroidogenesis in older animals; and 2) ethanol-induced reduction in testosterone is not a likely mechanism for delayed sexual maturation.

The in vitro spermicidal effect of gossypol, gossypol acetic acid and gossypol-polyvinylpyrrol idone was investigated. Gossypol-polyvinylmulations. Thus, gossypol in an appropriate chemical form may be useful as a spermicide.

The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.