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Research activities - Dept. of Dermatology / Andrology |
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The basic and clinical research program has included research on: 1. Investigations on transport of monosaccharides and ions across spermatozoal membranes using radioactive tracers D-glucose uptake by human spermatozoa showed a hyperbolic kinetic like the D-fructose uptake. The affinities to the transport system were similar, but the maximum capacity of D-glucose transport exceeded the D-fructose transport. Both transport processes were inhibited by monoiodoacetate and ouabain and influenced by Na+. The influence of the extracellular glucose-fructose-ratio on the D-glucose as well as on the D-fructose uptake suggested that both systems interact with another. The influence of the cryopreservation process on the sperm netto-86 Rb+-uptake as an analogous of the 42K+-uptake was examined. On this way conclusions about the functional impairment of the plasma membran should be characterized. The 86Rb+-uptake was an active event depending on the cellular metabolism. The cryopreservation modified the kinetic of Rb+-uptake in a sigmoid manner. The transformation from the liquid to solid phase caused the strongest alteration of Rb-uptake during the cryopreservation, whereas, the introduction of an equilibration time decreased the impairing effects of cryopreservation on ion transport. 2. The DNA-content and integrity of human spermatozoa and its relation to other semen qualities. The DNA-content of human spermatozoa quantitatively evaluated by the detection of deoxyribose in a defined cell number showed higher values in teratozoospermic and asthenozoospermic semen samples. More recently methods to detect DNA integrity are established in conjunction with the research programme of apoptosis related signalling in human sperm. 3. Analysis of nuclearproteins of spermatozoa by polyacrylgel-electrophoresis The nucleoproteins (NP) of cryopreserved human spermatozoa of fertile men and of infertility patients were investigated and compared with the NP of somatic hepatocytes of rat and mouse. The protamines of fertile men did not significantly differ from that of infertility patients with normal spermiogram parameters but they showed a significantly higher value compared with the samples with pathological spermiograms. On average the protamines of all infertile patients were significantly lower than the protamines of fertile semen donors. 4. Investigations on structure and function of spermatozoal membranes by micro-electrophoresis, electrode sensitive against lipophilic cations and measurements of membrane-bound enzymes. The net surface charge of spermatozoa was indirectly evaluated by the sperm electrophoretic mobility (EPM) in the microelectrophoresis. The net surface charge of spermatozoa represents a characteristic of the plasma membrane. The spermatozoa separated by „swim up“-technique did not differ from the remaining spermatozoa except they were demembranated. The investigation demonstrated that the internal structure of the spermatozoa selected by „swim up“ exhibit an additional dimension of supriority in electrophoretic mobility to nonselected spermatozoa. The cryopreservation did not cause a significant change of sperm EPM. A significant correlation between EPM and the cryotolerance was found. After contact with egg yolk spermatozoa achieved a electrophoretic behaviour that corresponded to a better cryotolerance. The activities of the categories of cyclic nucleotide phosphodiesterases (cNPDE) of human spermatozoa were detected and compared with the enzyme activities in the seminal plasma. The enzymes differed in the influence of type 3 by cyclic GMP and the pattern of activities. The acrosome reaction activated type 2 and 3. An attempt was made to measure the transmembrane potential of human spermatozoa by an electrode sensitive to the lipophilic cation tetraphenyl phosphonium (TPP+). This method was able to determine the negatively charged groups on the sperm surface as a hint to an impairment of the plasma membrane structure but could not detect the active transmembrane potential. 5. Fibronectin on human spermatozoa and influencing factors on its expression Spermatozoa separated by „swim up“-technique and incubated with polyclonal anti-fibronectin antibodies in the immunofluorescence technique showed a fluorescence band on the equatorial segment. The fluorescence pattern decreased on spermatozoa with pathological shape and after contact with microorganisms. This might go some way to explaining the connection between genital infection and sterility. 6. HLA-Expression on human spermatozoa The expression of human leukocyte antigens (HLA) on ejaculated spermatozoa and lymphocytes was compared by flow cytometry using monoclonal antibodies towards HLA class I (pan-HLA-A, -B, -C) and class II (DR) antigens. No antibodies were bound to spermatozoa to any significant extent neither after modifications of sperm preparation (density gradient centrifugation, „swim up“-technique, addition of acide, foetal calf serum or benzamidine chloride) nor after treatment of spermatozoa with detergens. In blood plasma the concentrations of soluble HLA-A, -B, -C were around fourfold higher than in seminal plasma using an ELISA technique. 7. Examination of aerobic metabolism of human spermatozoa by Clark-electrode and comparison of the cell vitality by sperm metabolic parameters after various freezing programmes Various cryopreservation protocols were compared to find out the optimum method of sperm cryopreservation. The post-freezing vitality was evaluated by motility, surface charge, ion-fluxes, cAMP-concentration, equatorial fibronectin band and aerobic metabolism. The oxygen consumption of human spermatozoa and its modification by rotenone, succinate, digitonine and 2,4-dinitrophenole was measured before and after cryopreservation. The optimum cryopreservation protocol included glycerol as cryoprotective agent in the medium with a final concentration of 7% (v/v), an equilibration time, the one step dilution and a frozen volume of 0.2 mL. 8. The spermiological prerequisites for the assisted fertilization of human oocytes. In co-operation with the Division of Human Reproduction and Endocrinology (Professor Alexander) various preparation techniques („swim up“, centrifugation protocols, media) were tested especially after cryopreservation. The criteria of evaluation were the motility, sperm count, acrosome intactness and membrane integrity. 9. Inhibin concentration in the serum and seminal plasma of normo- and oligozoospermic patients There was no relation of the serum inhibin concentration to the sperm concentration as well as to the concentrations of FSH, LH and testosterone in serum. The inhibin concentration in the seminal plasma and blood serum did not significantly differ from each other. There was no hint to a direct mutual influence between FSH and inhibin. 10. Testicular function after bone marrow transplantation (BMT) The long-term effects of bone marrow transplantation on the testicular functions over an 8-year period revealed the indication of andrological examinations after BMT. In our analysis 9% of the BMT patients (n=59) showed spermatozoa in their ejaculates. The earliest recovery of spermatogenesis was seen at 34 months after BMT. 11. Multicenter-study about criteria to evaluate the male fertility In eight andrological centres of the former GDR including Leipzig more than 2000 childless married men were examined catamnestically. Fertility was compared with the results of the routine spermiogram. Conception rate was significantly reduced if the sperm concentration was lower than 10 Mill/mL and less than 50% of the spermatozoa were progressively motile and less than 60% had a normal morphology. 12. Psychological investigations of infertile couples before and after therapeutic insemination of cryopreserved donor semen In the period from 1978 to 1990 762 infertile couples were treated in 3087 cycles by therapeutic insemination of cryopreserved donor semen. Four hundred sixty six patients (61,1 %) had at least one course of successful treatment. A psychological follow up-study did not show any significant difference or higher level of problems compared with the control group. 13. Socio-psychological investigations on patients with erectile dysfunctions This study is performed by Dr. Seikowski in cooperation with the Department of Urology as well as Prof. Brähler and co-workers. 14. Investigation of influence of reactive oxygen species on transduction and execution of apoptosis signals in immature native and cryopreserved human sperm using the relational workflow based database system (Winsperm® 2003). 15. Study of influence of gradient centrifugation on apoptosis related signal transduction and DNA – fragmentation in ejaculated human sperm. 16. Development of relational workflow based database system (Winsperm® 2003). 17. Investigation of executers and regulators of apoptosis in human spermatogenesis of infertility patients German Research Council (Deutsche Forschungsgemeinschaft, DFG) Since January 1992 research project (Gl 199/1-1, Gl 199/1-2 and Gl 199/1-3) „Adhesion molecules (AM) on human spermatozoa and their relation to fertility“ has been performed in cooperation with the Department of Dermatology of St. Barbara Hospital Duisburg (Docent Dr. J. Schaller), the Institute of Microbiology of the University of Bochum (Dr. Rohwedder) and with the Centre of Andrology and Dermatology of the University of Gießen (Dr. Henkel). Spermatozoa from patients who showed normal semen parameters expressed very late antigen (VLAa3, 4, 5, 6)-integrins and corresponding matrix proteins laminin and fibronectin. Semen samples with teratozoospermia were characterized by a reduced expression of AM. After artificial induction of the acrosomal reaction changed the pattern of AM on the sperm surface. Investigations of human testicular tissue by immunohistology using monoclonal antibodies against ß1-integrins and fibronectin suggested a production of these adhesion molecules during the spermatogenesis. Spermatozoa with higher expression of very-late-antigen (VLAa3,4,5 and 6)- integrins after the acrosome reaction have a better fertilization rate in vitro, i.e. a role of these molecules in adhesive mechanisms of spermatozoa is very likely. The percentage of spermatozoa with detectable AM differed only slightly between fresh and cryopreserved semen samples. The ingredients of the cryoprotective medium did not cause a significant difference to the control assay. The amounts of soluble AM detected by enzyme immunoassays after an extraction procedure of washed spermatozoa were low and not affected by the cryopreservation. In contrast, the motility parameters were stronger altered by the cryopreservation than the AM expression. The in vitro binding capacity of spermatozoal integrins to matrix components after disintegration of sperm membranes suggested the conclusion that cells with fragile plasma membrane are more prone to functional inactivation of their integrins by the seminal plasma. In ejaculated human spermatozoa the messenger ribonucleic acid transcripts of ß1 integrins could be detected by nested reverse transcription polymerase chain reaction. Human spermatozoa with low expression of adhesion molecules and matrix proteins also showed a poor penetration in in zona-free hamster oocytes. Since May 2002 Signaltransduction in capacitation and apoptosis in human spermatozoa, role of lipids for the fertilising process DFG Gl 199/4-1: Comparison of transmembraneous signal transduction and phosphorylation in human spermatozoa during capacitation and apoptosis Mechanisms of apoptosis and capacitation in human spermatozoa are compared to search for correspondent and different pathways in both of these physiological processes. To clarify this problem phosphorylation of proteins, the lipid composition of spermatozoa, generation of reactive oxygen species, sperm motility, and the characteristics of apoptosis are investigated after specific modification design of several pathways of signal transduction in apoptosis and capacitation (cAMP-, phosphoinositol 3-kinase-, proteinkinase C-, phospholipase A2- and caspases-mediated pathways and others). As aside effect the project evaluates the validity and usefulness of the matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) as a tool for the studying of the lipid composition of human spermatozoa as well as of the corresponding degradation products.
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Research activities - Dept. of Urology |
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The basic and clinical research programme has included research on:
1. At present the Department of Urology deals with uro-oncological and immunological research as well as with studies on the field of minimalinvasive operative techniques. 2. Additionally, the research work is focused on the anatomy and physiology of the lower urinary and sexual tract (see publication list). A new hitherto unknown muscular system has been proved. 3. At 17 January 1995 a research group was established to study the physiology and pharmacology of the continent function of the lower urinary tract. The examinations of the nerval system of the lower urinary tract by neurobiological methods and the investigations of the muscular physiology are the main tasks. The research project includes a close collaboration with the Paul-Flechsig-Institute of brain research. A computer assisted technique for urodynamical measurements is available in the Urological Clinic according to the international standard. These new results are very useful for investigations on the anatomy and physiology of ejaculation. This scientific work is supported by the University of Leipzig and sponsored by industry. · Physiology and neurophysiology of the lower urinary tract · Thesis (Alexander Winkler): Signaltransduction and modulation of calcium transients in cultured smooth muscle cells evoked by cholinergic stimulation · Thesis (Karel Handrik): Signaltransduction and modulation of calcium transients in cultured smooth muscle cells evoked by adrenergic stimulation · Histamine receptors in cultured smooth muscle cells of the human lower urinary tract · Anatomy and histology of the lower urinary tract · Thesis (Alexandra Kirseck): Anatomie des unteren Harntraktes der Katze / Anatomy of the lower urinary tract of the cat Dr. Jochen Neuhaus (neuj@medizin.uni-leipzig.de), Alexandra Kirseck, Professor Dr. Wolfgang Dorschner · Thesis (Cordula Globig): Neuroanatomy, Histology and morphology of the pelvic ganglia of the rat · Thesis (Michaela Panhofer): Comparative 3-dimensional visualization of the ventral longitudinal musculature of the human bladder neck using histological section series · Thesis (Andreas Kunze): Innervation of the lower urinary tract in the guinea-pig · Comparative anatomy of the canine urogenital tract · Comparative anatomy of the urogenital tract of the Rhesus Monkey (Macaca mulatta)
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