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Biomedical subjects

W B Schill

Publications and source records attributed to W B Schill.

At least 19 recordsLinked to original sources
PubMed1997

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.

Animals↗
PubMed1997

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

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

Acrosome↗
PubMed1997

A new combined in-vitro test model for the identification of substances affecting essential sperm functions.

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.

Acrosome↗
PubMed1997

[Therapy of male subfertility].

The possibilities of treating male subfertility are still limited. Approaches at medical therapy include stimulation of spermatogenesis at the testicular level, improvement of epididymal function (sperm maturation), influence on sperm transport and activation of sperm metabolism with improvement of sperm motility. Causal therapy has been most successful in patients with hormonal insufficiency and male adnexitis, while microsurgical reconstructive measures have yielded best results in cases of occlusion within the efferent seminal ducts. New therapeutic approaches include the use of mast cell blockers and alpha blockers as well as vitamin C/E as an antioxidative treatment to reduce reactive oxygen species. If medical or surgical therapy has failed, methods for improvement of sperm quality in vitro must be considered (swim-up technique, glass wool filtration, migration/sedimentation technique, density gradient centrifugation). In cases of severe male sterility factor, intracytoplasmic sperm injection (ICSI) has been a breakthrough in the therapy of childlessness. A further progress is the collection of spermatozoa from the epididymis (MESA = microsurgical epididymal sperm aspiration) or testis (TESE = testicular sperm extraction). Finally, pressure in terms of time and organization can now be avoided by the use of cryopreserved spermatozoa from the ejaculate, epididymis or testicular tissue so that microinjection may be planned independently of the partner. In any case, a close cooperation between gynecologist and andrologist is of utmost importance.

Epididymis↗
PubMed1997

The zona pellucida "receptors".

Binding of mammalian sperm to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. In the mouse model, the zona pellucida consists of three sulfated glycoproteins, ZP1, ZP2, and ZP3. Zona pellucida proteins are secreted to form a filamentous zona matrix in which ZP2 and ZP3 complex into co-polymers cross-linked by ZP1. ZP3 is the ligand for primary sperm binding and important for the induction of the acrosome reaction. The zona pellucida glycoprotein ZP2 is also crucially involved in the process of fertilization. Previous reports suggest that ZP2 mediates secondary binding of spermatozoa and that cleavage of ZP2 by proteases released through cortical granule reaction causes zona "hardening" and thus prevents polyspermy. Human and mouse ZP2 proteins differ in the primary structure as derived from cDNA clones. We designed an immunological approach to search for ZP2 domains with functional relevance. Antisera were generated against synthetic peptides derived (a) from ZP2 amino acid sequences that are homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or (b) from human ZP2 amino acid sequences that differ from the mouse ZP2 sequence (AS ZP2-26). Immunochemical studies with microbisected bovine zonae pellucidae demonstrated that both antisera, AS ZP2-20 and AS ZP2-26, specifically detected ZP2 protein. Using the competition-hemizona-assay, sperm binding to antibody treated bovine hemizonae pellucidae were compared with control hemizonae (given as hemizona index). Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae (p < 0.0001), whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-egg interaction. Our results show that antibodies against ZP2 peptides react with bovine zonae pellucidae and can be used as markers for ZP2. Furthermore, AS ZP2-20 identifies a ZP2 epitope that is possibly of functional relevance for sperm-egg interaction.

Animals↗
PubMed1996

Sperm selection methods for intracytoplasmic sperm injection (ICSI) in andrological patients.

PURPOSE: To improve the chances of successful in vitro fertilization, spermatozoa have to be separated from semen before insemination. Therefore, sperm preparation methods are of great importance. METHODS: To obtain sufficient numbers of spermatozoa from patients with cryptozoospermia or severe OAT syndrome, only Minipercoll centrifugation and migration-sedimentation (MS) are practicable methods. The present study was performed to compare these two methods with regard to sperm concentration, motility, vitality, morphology, and chromatin condensation. The number of spermatozoa obtained after minipercoll was higher than that after MS, but sperm quality in all parameters examined was clearly better after MS than after Minipercoll. In the second stage of this study, the MS method was used for preparation of the spermatozoa for intracytoplasmic sperm injection (ICSI). RESULTS: Over a period of 13 months, 159 cycles were treated by ICSI. Of 1045 aspirated oocytes, 790 were injected. The fertilization rate was 70.4% of injected oocytes (556 oocytes with clearly visible pronuclei). In 146 cases, embryonic transfer was achieved; 58 patients became pregnant (39.7% per transfer and 36.5% per cycle). CONCLUSIONS: Although the abortion rate was very high (18 women lost their embryos), the results demonstrate that the microinjection method can be successfully used in combination with a MS method for preparation of spermatozoa.

Abortion, Spontaneous↗
PubMed1996

Glass wool filtration reduces reactive oxygen species by elimination of leukocytes in oligozoospermic patients with leukocytospermia.

PURPOSE: Two types of glass wool were used to remove leukocytes in semen for evaluation of reactive oxygen species production by spermatozoa in oligozoospermic patients with leukocytospermia. METHODS: Semen samples were prepared using fine-structure glass wool (SpermFertil) and coarse-structure glass wool. In each treatment group, native semen was evaluated for sperm concentration, percentage motility, viability, leukocyte concentration, and production of reactive oxygen species. RESULTS: Electron microscopically, SpermFertil showed a higher number of leukocytes attached to the fibers compared to coarse-structure glass wool. Leukocytes in native semen and after glass wool filtration as determined by peroxidase cytochemistry confirmed this observation. Reactive oxygen species decreased from 45.303 counts/10(7) viable cells in native semen to 15.806 counts/10(7) cells in coarse structure wool and 7.465 counts/10(7) cells in Spermfertil, respectively. CONCLUSIONS: Removal of leukocytes from semen of oligozoospermic patients by means of glass wool filtration is a useful method to distinguish production of reactive oxygen species by leukocytes versus sperm cells.

Glass↗
PubMed1996

[Diagnosis, clinical aspects and therapy of early chromoblastomycosis in a case example].

Despite the availability of modern antimycotics, which produce high cure rates in early infections, the therapy of advanced chromoblastomycosis is still unsatisfactory. An initial chromoblastomycosis caused by a hitherto unidentified species of the genus Phialophora was diagnosed in a 46-year-old teacher. The organism was isolated twice at an interval of 6 weeks from a partly psoriasiform, partly verrucous lesion on the 4th toe. The infection was apparently acquired 4 years ago during a holiday at Cape Verde. Treatment with itraconazole (Sempera). 200 mg/day, and amphotericin B (Ampho-Moronal) cream for 6 weeks initially resulted in rapid regression. However, 4 weeks after cessation of therapy, the Phialophora species was cultured again from skin scrapings. Complete healing was achieved after re-treatment with itraconazole for 20 weeks at the same dosage in combination with topical amorolfine and local hyperthermia. Until now, no relapse has occurred. The present case demonstrates that this rare disease, which mainly occurs as a traumatic mycosis in the rural population of tropical regions, must be included in the differential diagnosis of psoriasiform or verrucous skin lesions and also included in the list of diseases which may be acquired while on vacation in exotic locations.

Administration, Topical↗
PubMed1996

[Skin infections caused by Mycobacterium gordonae. Case report and review of the literature].

Mycobacterium gordonae is an atypical mycobacterium of very low pathogenic potential. It is widely distributed in soil and water and often detected on the mucous membranes of healthy persons. In recent years, there have been numerous reports of infections by M. gordonae in immunocompromised patients. In contrast, only four cases of skin infections by M. gordonae in immunocompetent patients have been published. We report on another patient without evidence of immunodeficiency who developed an atypical mycobacteriosis after a thorn injury during gardening. M. gordonae was isolated by tissue culture. The skin lesion cleared completely after treatment with doxycycline for three months.

Bacteriological Techniques↗
PubMed1996

Characterization of kininases in testicular cells.

Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminifereous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the Pro7-Phe8, Phe5-Ser6 and Gly4-Phe5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located in Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin (1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the Ph5-Ser6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16.

Animals↗
PubMed1996

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function.

OBJECTIVE: To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. DESIGN: Prospectively designed, blinded, internally controlled study. SETTING: Tertiary care academic center. PATIENTS: Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. INTERVENTIONS: Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. RESULTS: A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. CONCLUSIONS: These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Adult↗
PubMed1996

Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11.

In a large collaborative screening project, 370 men with idiopathic azoospermia or severe oligozoospermia were analysed for deletions of 76 DNA loci in Yq11. In 12 individuals, we observed de novo microdeletions involving several DNA loci, while an additional patient had an inherited deletion. They were mapped to three different subregions in Yq11. One subregion coincides to the AZF region defined recently in distal Yq11. The second and third subregion were mapped proximal to it, in proximal and middle Yq11, respectively. The different deletions observed were not overlapping but the extension of the deleted Y DNA in each subregion was similar in each patient analysed. In testis tissue sections, disruption of spermatogenesis was shown to be at the same phase when the microdeletion occurred in the same Yq11 subregion but at a different phase when the microdeletion occurred in a different Yq11 subregion. Therefore, we propose the presence of not one but three spermatogenesis loci in Yq11 and that each locus is active during a different phase of male germ cell development. As the most severe phenotype after deletion of each locus is azoospermia, we designated them as: AZFa, AZFb and AZFc. Their probable phase of function in human spermatogenesis and candidate genes involved will be discussed.

Chromosome Mapping↗
PubMed1996

Ageing and sperm function.

To evaluate the fertilizing capacity of spermatozoa from elderly men, ejaculates from 29 older fathers (mean age 50.3 years) were compared with those from 35 younger fathers (mean age 32.2 years). In addition to conventional semen parameters, sperm functions were studied that have been reported to be positively correlated with the fertilization rate: progressive motility, acrosin activity, inducible acrosome reaction, and chromosome condensation. Sperm concentration and follicle stimulating hormone concentration differed significantly in both groups. With regard to sperm functions there were no differences between older men and younger men, except for decreased sperm motility in the older group which, however, reached nearly normal values according to World Health Organization criteria. Decreased fertility of older couples is obviously more dependent on the age of the female partner. The significance of genetic risks remains to be clarified, especially when methods of assisted reproduction are applied.

Acrosin↗
PubMed1996

Low expression of adhesion molecules and matrix proteins in patients showing poor penetration in zona-free hamster oocytes.

The expression of adhesion molecules and matrix proteins by human spermatozoa as well as their binding to and penetration of zona-free hamster eggs were investigated in 17 patients by means of flow cytometry. Both binding and penetration of hamster oocytes and expression of alpha- and beta-chains of beta 1, beta 3 and beta 4 integrins were determined before and after induction of the acrosome reaction. The expression of the integrin ligands, fibronectin and laminin were also analysed. Significant differences in the expression of very late antigen (VLA) integrins, VLA alpha 4-chain (CD49d), the classical fibronectin receptor VLA alpha 5-chain (CD49e), leukocyte function-associated molecule-3 (LFA-3; CD58) and fibronectin were observed between patients showing good (> 10%) or poor (< 10%) penetration in the sperm penetration assay (P = 0.0068). It is concluded that these adhesion molecules are intimately involved in the sperm-oolemma interaction. Since no differences were observed in either spermiogram parameters or the percentages of acrosome-reacted spermatozoa in the two groups, sperm-oolemma binding and penetration should be regarded as discrete parameters of sperm function.

Animals↗
PubMed1996

Melanoma in situ versus melanocytic hyperplasia in sun-damaged skin. Assessment of the significance of histopathologic criteria for differential diagnosis.

For differentiation of melanoma in situ (MIS) from melanocytic hyperplasia (MH) in sun-damaged skin, several criteria have been proposed. To assess sensitivity and specificity of those criteria, we examined the epidermis adjacent to 50 consecutive basal cell carcinomas and 50 MISs in skin with significant solar elastosis. The most valuable criteria for the diagnosis of MIS, as opposed to MH, were presence of nests of melanocytes, irregular distribution of melanocytes, descent of melanocytes far down adnexal epithelial structures, irregular distribution of pigment, presence of melanocytes above the junction, a high number of melanocytes, pleomorphism of melanocytes, and atypical nuclei of melanocytes. Other criteria, e.g., collapse of cytoplasm around nuclei of melanocytes; flattening of rete ridges; differences in the area, shape, and contour of nuclei of melanocytes as assessed by nuclear morphometry; and presence of melanocytes stained by HMB-45 and Ki-67/MIB-1 monoclonal antibodies, were found to be of low or no value for differential diagnosis.

Antigens, Neoplasm↗
PubMed1996

Andrological work-up of patients undergoing microsurgical epididymal sperm aspiration or testicular sperm extraction.

Microsurgical epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) require close cooperation between andrologists, urologists and gynaecologists. Intracytoplasmic sperm injections were established in Giessen in March 1994 and embryo transfer (ET) was performed in 342 of 375 patients (91.2%). The percentage of pregnancies and ongoing pregnancies are 35.4% per ET (32.3% per cycle) and 25.1% per ET (22.9% per cycle), respectively. Microsurgical procedures such as epididymovasostomy or vasovasostomy and cryopreservation of human semen are also established methods. The purpose of the present study was to describe the andrological work-up for patients before MESA and TESE. Experiments demonstrate that incubation of testicular tissue samples in IVF medium and treatment with 1 mg ml-1 pentoxifylline increase the number of extracted motile spermatozoa. Centrifugation of the medium results in a further concentration of sperm cells. If no motile spermatozoa can be found in the supernatant medium, they may be extracted directly from the testicular tissue samples by means of a micromanipulator.

Cell Separation↗
PubMed1995

Identification of heterotrimeric G proteins in human sperm tail membranes.

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein alpha-subunits, and A 86, which detects all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein alpha-subunits failed to detect any specific antigens in enriched tail membranes. AS 36, recognizing the beta 2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive alpha-subunits. However, membrane preparations of nonpurified human spermatozoa contained alpha i2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein alpha-subunits; the putative beta-subunit was identified as a beta 2-subunit.

Cell Membrane↗