Werner Miska

Molecular Mechanism for Human Sperm Chemotaxis Mediated by Progesterone

Sperm chemotaxis is a chemical guiding mechanism that may orient spermatozoa to the egg surface. A picomolar concentration gradient of Progesterone (P), the main steroidal component secreted by the cumulus cells that surround the egg, attracts human spermatozoa. In order to elucidate the molecular mechanism of sperm chemotaxis mediated by P, we combine the application of different strategies: pharmacological inhibition of signaling molecules, measurements of the concentrations of second messengers and activation of the chemotactic signaling. Our data implicate a number of classic signal transduction pathways in the response and provide a model for the sequence of events, where the tmAC-cAMP-PKA pathway is activated first, followed by protein tyrosine phosphorylation (equatorial band and flagellum) and calcium mobilization (through IP3R and SOC channels), whereas the sGC-cGMP-PKG cascade, is activated later. These events lead to sperm orientation towards the source of the chemoattractant. The finding proposes a molecular mechanism which contributes to the understanding of the signal transduction pathway that takes place in a physiological process as chemotaxis.

Relevance of zinc in human sperm flagella and its relation to motility

OBJECTIVE To measure the zinc content of human sperm flagella and to analyze its relation to sperm motility. DESIGN Prospective study. SETTING Center of Dermatology and Andrology. PATIENT(S) Semen samples collected from 90 andrology patients and healthy donors after 3-5 days of sexual abstinence. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Sperm morphology after Shorr staining, sperm motility, and patient age were recorded. In addition, zinc concentrations in the seminal plasma, sperm heads, and flagella were determined with the use of atomic absorption spectrometry. RESULT(S) The mean zinc concentration was 144.3 mg/L in the seminal plasma and 146.9 mg/L in the whole ejaculate and was significantly correlated with parameters of motility. The sperm heads contained only 6.7% of the zinc that was present in the whole spermatozoon. The zinc concentration in the flagella was negatively correlated with sperm motility and velocity. In addition, it was positively correlated with the percentage of abnormally blue-stained flagella and the age of the patients. CONCLUSION(S) Our results clearly demonstrate the importance of zinc elimination during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, generation of motility.

Possible effects of the kallikrein-kinin system on male reproductive functions.

Summary. All four components of the kallikrein‐kinin system—kininogens, tissue kallikreins, kinins, and kininases—have been found in human male genital secretions. Kinins are continuously released from seminal plasma kininogens through limited proteolysis by kininogenases like tissue kallikrein from prostate and sperm acrosin. Kinins are the terminal effectors of the kallikrein‐kinin system and increase sperm motility and sperm metabolism at nanomolar concentrations. Recent investigations indicate that these effects are possibly mediated by a specific sperm membrane integrated bradykinin receptor, subtype B2. The two major kininases that are present in seminal plasma are kininase II and neutral metallo‐endopeptidase. Kininase II, which is identical with angiotensin‐converting enzyme, is also involved in the renin‐angiotensin system as it converts angiotensin I into angiotensin II and thus is the connecting enzyme of both systems. Apart from the observed effects of kinins on sperm motility, the kallikrein‐kinin system is thought to be involved in the regulation of spermatogenic functions of the testis: in the rat, kallikrein activates Sertoli cell function, increases the relative number of spermatocytes and the [3H] thymidine incorporation of testicular tissue, enhances glucose‐intake, and increases testicular blood flow. Clinical trials showed that systemic administration of kallikrein may be particularly useful for treatment of infertile men suffering from asthenozoospermia and/or oligozoospermia. During kallikrein therapy, the number of spermatozoa and both quantitative and qualitative sperm motility increased, and a significant improvement of the conception rate was achieved. An increased sperm number was also observed after application of the specific kininase II inhibitor captopril. Recent investigations proved intestinal absorption of porcine pancreatic kallikrein in men, supporting the clinical effectiveness of oral kallikrein therapy. Furthermore, the results indicate that absorbed kallikrein may be enzymatically active for hours in serum. Thus, according to the various in vitro experiments, animal studies, and clinical trials, there is increasing evidence that the kallikrein‐kinin system is involved in male reproductive functions.

Reactive oxygen species induce reversible capacitation in human spermatozoa

Summary. Leucocytospermia has been associated with reduced sperm motility and decreased capacity for sperm–egg interaction. This effect could be mediated through reactive oxygen species (ROS), which, at high concentrations, induce lipid peroxidation and cellular death. The high impact on sperm capacitation reported in other mammalians should be more accurately assessed in the human because premature activation could affect sperm fertilizing capacity. The aim of this study was to evaluate both the effect of ROS on sperm capacitation and the protective role of seminal plasma. Spermatozoa selected by Percoll gradient were incubated with polymorphonuclear (PMN) granulocytes isolated from blood and activated by phorbol‐12‐myristate 13‐acetate (PMA). Different seminal plasma concentrations were added immediately or after 3‐h incubation. Afterwards, ROS production was evaluated by luminescence and sperm capacitation by chlortetracycline stain. In PMN granulocytes and sperm suspensions, the basal ROS production was <32 × 103 relative luminescence units (RLU). After stimulation with PMA, the rate of ROS production by PMN increased to 1287 × 103 RLU. Incubation of sperm with activated PMN resulted in an increase of sperm capacitation (37% versus 19% in the control). Immediate addition of seminal plasma caused a significant reduction in ROS (P < 0.01) and prevented sperm from capacitating. A higher effect in inhibition of sperm capacitation was observed when seminal plasma had been added after 3‐h incubation. The results suggest that human sperm capacitation can prematurely be induced by exogenous ROS and this effect can be reversed by seminal plasma. Thus, human sperm capacitation is another functional parameter that may be affected by nonphysiological ROS production.

Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk.

Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.

Differentiation of ejaculates showing reactive oxygen species production by spermatozoa or leukocytes

Summary. Differences between subgroups and correlations between reactive oxygen species (ROS), sperm motility, concentration of leukocytes and viability in semen samples from 143 men were investigated. Patients with azoospermia or leuko‐cytospermia were excluded from the study. Spermatozoa were separated by means of glass wool nitration. Reactive oxygen species were determined by means of luminol chemiluminescence before and after sperm separation; thereafter, normozoospermic and oligozoospermic patients were divided into three subgroups using the mean of all patients investigated (17462 count 10−7 viable spermatozoa) as cut‐off value as follows: G1—high reactive oxygen species production in native semen and after glass wool nitration; G2—high production of reactive oxygen species only in native semen; G3—low levels of reactive oxygen species before and after glass wool filtration. In general, reactive oxygen species were significantly higher in oligozoospermic samples than in normozoospermic samples. In men with normal sperm count, ROS production correlated significantly with the number of leukocytes in the ejaculate before glass wool nitration, but not thereafter. Glass wool nitration is useful to distinguish between sperm samples in which reactive oxygen species are generated by leukocytes and those in which reactive oxygen species are excessively generated by the spermatozoa themselves.

Indirect immunofluorescence using monoclonal antibodies for the detection of leukocytospermia: comparison with peroxidase staining.

Summary. The presence of increased number of leukocytes in semen is indicative of inflammation in the male genital tract. Inflammatory processes at this level may lead to marked impairment of sperm function, and finally to a reduction in their fertilizing capability. An immunocytological technique for the detection of seminal leukocytes was evaluated in this study. As part of the standardization technique, different fixation methods were tested to ascertain whether samples could be stored and examined later. It was found that fixation with cold acetone at freezing temperatures retained immunoreactivity until day 11 of storage. All other methods showed a significant loss of immunoreactivity, from as little as day 2 of storage. In 46 specimens with elevated numbers of round cells, number of peroxidase‐positive cells and number and type of leukocytes were evaluated by means of indirect immunofluorescence. Determination of peroxidase‐positive cells to detect leukocytospermia, the standard procedure recommended by the WHO, was compared with the indirect immunofluorescence technique using monoclonal antibodies. While 19 of 46 patients showed high numbers of leukocytes in the ejaculate, as determined by the immunocytological method, only 9 of these were identified to be leukocytospermic, according to the WHO (standard) procedure. This difference was statistically significant (P < 0.01) and indicates that the standard method of detection of seminal leukocytes may be inaccurate.

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application

Summary.  α‐Glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of α‐glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of α‐glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean ± SEM): normozoospermia (control): 20.2 ± 1.4 mU ml−1; azoospermia: normal value: 17.6 ± 2.2 mU ml−1, low value: 7.4 ± 1.8 mU ml−1; oligozoospermia: normal value: 22.3 ± 2.5 mU ml−1, low value: 7.3 ± 0.7 mU ml−1; leucocytospermia: increase value: 38.9 ± 3.7 mU ml−1, low value: 11.1 ±1.3 mU ml−1. This study suggests that determination of α‐glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.

Migration/sedimentation sperm selection method used in bovine in vitro fertilization: Comparison with washing/centrifugation

Sperm selection methods are usually considered for in vitro fertilization (IVF) programs. To achieve a population of viable, motile and morphologically normal spermatozoa, seminal plasma and contaminants must be removed. In this study 2 sperm selection methods were compared with regard to conventional parameters: 1) repeated washing/centrifugation, and 2) migration/sedimentation. Their effect on the fertilization of oocytes aspired from ovaries of slaughtered cows was determined. Frozen-thawed semen was used for all assays (n = 8). The sperm concentration was adjusted to 1.0 × 106 cells/ml for in vitro insemination. Selected oocytecumulus complexes were matured in vitro for 24 h and were co-incubated with spermatozoa prepared by the 2 different methods. Presumptive zygotes were co-cultered in TCM-199. The percentage of motile, viable and morphologically normal spermatozoa was markedly higher (p < 0.05) with the migration-sedimentation method. Triple stain showed that the integrity of the acrosome was significantly better maintained after migration/sedimentation (68.3%) than after washing/centrifugation (53.2%; p < 0.05). In accordance with these results, a high fertilisation rate was achieved after application of the migration/sedimentation method (92.5 vs 69.1%;p < 0.05). It is concluded, that this method is very promising for obtaining spermatozoa with optimal fertilization capacity.

Evidence for the synthesis and secretion of a CBG-like serpin by human cumulus oophorus and fallopian tubes.

Summary The acrosome reaction (AR)‐inducing effect of follicular cells, like that of the cumulus oophorus and granulosa cells, has been described previously. In addition to the well known steroid secreting activity of cumulus cells, the results obtained here demonstrate the secretion of a corticosteroid‐binding globulin (CBG)‐like protein. An AR‐inducing effect was shown with the culture medium of human cumulus oophorus. This effect could be eliminated by treating the sample with monoclonal antibodies against CBG. Moreover, Western blotting after SDS‐PAGE of the culture medium strongly indicates that human cumulus cells actively express and secrete a CBG‐like protein. This might give an indication as to the origin of the acrosome reaction‐inducing substance found in follicular fluid. Furthermore, AR‐inducing activity and the elimination of this activity by antibodies against CBG was shown for oviductal fluid. With immunohistochemical techniques the CBG‐like protein was localized in the epithelial lining of the fallopian tubes, giving possible evidence for the involvement of this molecule in fallopian tube function.