Lourens J.D. Zaneveld

Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa

Summary:u2002 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.

Hormonal imbalance and alterations in testicular morphology induced by chronic ingestion of ethanol

Abstract Physical correlates of chronic ethanol consumption and measures of in vitro fertilization were examined in male C57B1 mice after a treatment period of 34 days with a total liquid nutriment diet which contained 5%/95% (v/v) ethanol. This diet represented relatively low levels of ethanol ingestion by the C57B1 mouse, as evidenced by the low peak levels of blood ethanol (160 mg/100 ml), the lack of behavioral signs of intoxication during treatment, and the absence of overt signs of physical dependence after withdrawal from the ethanol-containing diet. Plasma testosterone levels of experimental animals were depressed during the treatment period, as compared to testosterone levels of pair-fed controls. No evidence of hepatic injury was observed following the treatment period. Although fertility, as measured by the ability of spermatozoa to penetrate mouse ova in vitro , was unaffected by the chronic ethanol treatment, signs of testicular dysfunction were evident. Abnormal testicular morphology included disruption of the basement membranes of the seminiferous tubules, decreased tubular diameter, and desquamation of immature germ cells into the lumina of the tubules. The present study provides convincing evidence of the adverse effects upon male reproductive functions of relatively low levels of ethanol, when administered chronically, under controlled conditions.

Male reproductive tract sensitivity to ethanol: a critical overview.

While endocrinological effects of acute and chronic ethanol ingestion have been extensively reviewed, a survey of ethanol induced functional and physical perturbations of the male reproductive tract remains lacking. A brief overview of recent literature concerned with ethanol sensitivity of various components of the reproductive tract is presented. Clinical findings are reviewed as they relate to the possible pathogenesis of alcohol-related testicular atrophy. Currently available animal models for the study of ethanol-induced male reproductive failure are discussed. Attempts have been made to separate the contribution of ethanol per se from secondary factors, such as hepatic dysfunction and nutritional deficiency, to manifestations of male infertility. Studies directed toward elucidating the mechanism(s) by which ethanol exerts its inhibitory effect on testicular steroidogenesis are discussed. Finally, evidence suggesting an effect of ethanol on the functional integrity of other components of the reproductive tract is reviewed. It is concluded that ethanol is a male reproductive tract toxin. Future clinical studies of alcoholics afflicted with testicular atrophy, but having normal liver histology, will be of great value in efforts to identify the mechanisms by which chronic ethanol ingestion results in reproductive impairment. Similar benefits will be realized in laboratory experimentation, in which models are employed that describe ethanol-induced infertility, while minimizing nutritional factors and hepatic involvement, and control for the reproductive maturity of the organism.

Release of Acrosin and Acrosin Inhibitor from Human Spermatozoa

On the basis of results of studies on spermatozoal acrosin, the following can be concluded: (1) The total amount of acrosin in human spermatozoa averages about 5 mU/10 6 spermatozoa. The large majority of the enzyme is bound to acrosin inhibitor (acrostatin) when extracted. (2) Acid extraction of human spermatozoa consistently yields a higher amount of acrosin than does detergent extraction. (3) Only a small amount of acrosin is spontaneously released from human spermatozoa when these are incubated at approximately physiologic pH, the highest release rate occurring during the 1st hour. All or most of the liberated acrosin forms a complex with inhibitor, so that the highly lytic proteinase is not released in the active form. This is of importance in the genital tract, where the death of spermatozoa and the subsequent release of acrosin could otherwise cause tissue damage. A physiologic function for the acrosin inhibitor is thus established. (4) Acrostatin cannot be removed by washing or aging the spermatozoa, indicating that its dispersal in the female genital tract during capacitation is an active process. (5) A zymogen form of acrosin (proacrosin) is not present in the material that is spontaneously released from human spermatozoa at physiologic pH. (6) p-Nitrophenyl-p′-guanidinobenzoate readily penetrates the plasma membrane and outer acrosomal membrane of human spermatozoa. (7) The gelatin-plate (substrate film) test should not be used to measure the amount of acrosin in human spermatozoa. (8) The hypothesis is made that both acrosin and acrostatin are associated with two sites on the human spermatozoon: (a) the outer acrosomal membrane and (b) the inner sperm head membranes.

Chemical Constituents of Human Seminal Plasma: Relationship to Fertility/Chemische Bestandteile des menschlichen Spermaplasmas: Beziehungen zur Fertilität

Summary: Eighteen different chemical constituents of seminal plasma from 52 patients in an in vitro fertilization (IVF) program were quantitated. The ejaculates were divided into “fertile” and “infertile” groups depending on whether the spermatozoa did or did not fertilize oocytes. Glycerylphosphorylcholine showed a significant difference between the two groups and was also significantly correlated with fertility, suggesting that GPC may influence the fertilizing ability of the spermatozoa. No such differences/correlations were found for the other constituents although a number of these components were lowest in the “infertile” group. In no case was the correlations between a seminal constituentns and fertilization high enough to predict whether an ejaculate is fertile or infertile.

Vaginal contraceptive activity of hyaluronidase and cyclooxygenase (prostaglandin synthetase) inhibitors in the rabbit

Seven inhibitors of the sperm enzyme hyaluronidase were tested at nonspermicidal concentrations for their vaginal contraceptive activity in rabbits. Five of these compounds are marketed antiinflammatory agents and the other two were synthesized. Most of the agents showed contraceptive activity. Of the antiinflammatory agents, phenylbutazone and oxyphenbutazone were particularly potent. Besides being hyaluronidase inhibitors, these compounds are also cyclooxygenase (prostaglandin synthetase) inhibitors; based on other in vitro data, their primary effect on spermatozoa is probably by the inhibition of that enzyme.

Hemorrhage induced by intrauterine devices: control by local proteinase inhibition.

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.

Transcervical tubal occlusion with a steerable hysteroscope: Implantation of devices into extirpated human uteri

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.

Orally administered ketoconazole rapidly appears in seminal plasma and suppresses sperm motility

Ketoconazole has been shown to exert spermatostatic effectsin vitro on ejaculated dog, monkey, and human spermatozoa. Oral administration of the compound to adult male beagle dogs (50–246 mg/kg) or rhesus monkeys (85–100 mg/kg) was associated with a decline in motility of sperm in ejaculates obtained after dosing. In dogs the decline in sperm motility was correlated with the presence of ketoconazole in the seminal plasma, although the measured concentrations of ketoconazole were no more than one tenth that needed forin vitro activity. The serum levels of testosterone in the dogs receiving oral ketoconazole were profoundly suppressed but the extreme rapidity of onset of theex vivo effect on sperm motility, which was noted within 4 hours of dosing, makes it unlikely that testosterone withdrawal plays more than a minor role in the spermatostasis. The results in animals invite further pursuit of this novel, rapid onset, reversible, single dose use of spermatostatic agents for their potential as male contraceptives.ResuméIl a été démontré que la cétoconazole exerce des effets spermatostatiques in vitro sur les spermatozoïdes éjaculés du chien, du singe et de lhomme. Ladministration de ce produit par la voie orale à des beagles mâles adultes (50–246 mg/kg) ou à des macaques rhésus (85–100 mg/kg) a entraîné un déclin de motilité du sperme dans les éjaculats obtenus suite à cette administration. Chez les chiens, la réduction de motilité du sperme a été mise en corrélation avec la présence de cétoconazole dans le fluide séminal, bien que les taux de concentration de cétoconazole naient pas excédé le dixième du taux requis pour lactivité in vitro. Les niveaux de sérum de testostérone chez les chiens recevant des doses orales de cétoconazole avaient subi une suppression considérable; néanmoins, la rapidité extrême des premiers signes de leffet ex vivo sur la motilité du sperme, que lon avait remarqués dans les quatre heures suivant ladministration de la dose, semble indiquer, quil est fort peu probable que la disparition de la testostérone joue un rôle important dans la spermatostase. Les résultats obtenus chez les animaux encouragent une étude plus approfondie du potentiel de ces agents spermatostatiques nouveaux, rapides, réversibles et administrés en une seule dose, comme stérilisants mâles.ResumenSe ha visto que el ketoconazol posee un efecto espermatostático in vitro en eyaculados de perro, de mono y en esperma humano. la administración oral del compuesto a pequeños sabuesos adultos (50–246 mg/kg) o a monos rhesus (85–100 mg/kg) estuvo asociada con una declinación en la motilidad del espermatozoide en los eyaculados obtenidos despues del dosaje. En los perros, la declinación de la motilidad del espermatozoide estuvo relacionada con la presencia del ketoconazol en el plasma seminal aunque la concentración de ketoconazol fué no mas de una décima de lo necesario para su actividad in vitro. Los niveles de testosterona en suero en perros recibiendo ketoconazol oral estuvieron muy suprimidos, pero la extremada rapidez del comienzo del efecto ex vivo en la motilidad del espermatozoide, que se notó dentro de las 4 horas de recibida la dosis, hace poco probable que la supresión de la testosterona juegue mas que un rol menor en la espermatostasis. Los resultados en animales estimulan a que se continuen los estudios de este nuevo agente espermatostatico de acción rápida, reversible, de dosis única, por su potencial como anticonceptivo masculino.

Postcoital, vaginal, spermicidal potency of formulations: the macaca arctoides (stumptailed macaque) as animal model*

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.