Biomedical subjects
Publications and source records attributed to H Renneberg.
Direct implanting of fetal urogenital sinus (UGS) tissue into the ventral prostate gland of adult mice led to a 4-fold weight increase of the manipulated prostatic lobe. The induced growth could be reduced by the polysaccharide fraction (POLY-M) of the 20% methanolic extract of stinging nettle roots by 33.8%.
OBJECTIVES: To determine the efficacy, safety and feasibility of intermittent androgen deprivation (IAD) in patients with prostate-specific antigen (PSA) relapse after radical prostatectomy or with an incidental prostate cancer (pT1B) after transurethral resection of the prostate (TURP). METHODS: Open, nonrandomized, prospective pilot study using the luteinizing hormone-releasing hormone analogue (LH-RHa), leuprorelin acetate (1-month depot) and cyproterone acetate. RESULTS: Forty-four patients have been enrolled. After a 30-64 months' follow-up no progression to androgen-independent status has been observed. Of the entire observation period, 26.6 months (44-58%) remained treatment-free. During the treatment-free periods, normal testosterone levels were obtained, resulting in a cessation of the symptoms of androgen suppression and an improvement in quality of life. CONCLUSIONS: These results indicate that IAD is an effective and feasible therapy in patients with early stages of prostate cancer. Larger trials are necessary to confirm these encouraging results. Therefore, a European prospective, randomized, multicenter study (RELAPSE study) has been started to compare IAD with continuous androgen blockade in terms of time to tumor progression, safety and quality of life in patients with PSA relapse after radical prostatectomy.
Angiotensin-converting enzyme (ACE) is known to be released from human spermatozoa during capacitation. However, it has not yet been localized ultrastructurally in ejaculated sperm cells. Therefore, the purpose of the present study was to demonstrate the location of ACE by means of immunoelectron microscopy and direct immunofluorescence. In addition, ACE activity of spermatozoa was correlated with standard semen parameters. The activity of angiotensin-converting enzyme was measured in spermatozoa from 115 donors and patients attending the andrological outpatient department. Progressive motility was negatively correlated with sperm ACE activity (Spearman rank correlation r=-0.364, P < 0.0001), whereas no statistically significant correlations with sperm concentration, total motility and morphology were observed. Immunoelectron microscopy demonstrated that ACE is mainly located at the plasma membrane of the acrosomal region, equatorial segment, postacrosomal region and midpiece. In contrast, only weak ACE-like immunoreactivity was found at the flagellum. In cases of cells with missing plasma membranes ACE seems also to be located at the surface of the outer acrosomal membrane. By means of immunohistochemical methods, different patterns of ACE-like immunofluorescence were observed: (i) fluorescence of the acrosome or the entire sperm head, midpiece and flagellum; (ii) fluorescence of the postacrosomal region, midpiece and flagellum; (iii) bright fluorescence of the equatorial segment with less intensive labelling of the postacrosomal region and flagellum. Induction of the acrosome reaction by calcium ionophore A23187 resulted in an increase of spermatozoa with weak acrosomal fluorescence, indicating loss of the plasma membrane.
Human 5'-nucleotidase (5'-NT, EC 3.1.3.5) is an enzyme that hydrolyzes nucleotides such as AMP or IMP (inosine 5'-monophosphate) into inorganic phosphate and the respective nucleoside. It has been suggested that the enzyme acts as a scavenger of injured cell or membrane components or as a supplier of adenosine. We have purified to homogeneity human 5'-NT, a 69-kDa glycoprotein containing a glycosylphosphatidylinositol anchor, present in human seminal fluid. With use of a polyclonal rabbit antiserum against the protein, a strong immunoreaction was detected in prostatic epithelium, exceeding that in placental syncytiotrophoblast and amnion cells. A slightly less intense immunoreaction was present in some cells of seminal vesicle epithelium and in vesicular intraluminal secretion. In the epididymis, only the apical cell portion and particularly the stereocilia of the epididymal principal cells, as well as clusters of small nonciliated cells in the efferent ductules, were immunoreactive. In the testis, no immunoreactive cells at all were detected, and likewise no clear-cut signal was observed in testicular and epididymal spermatozoa. The immunohistochemical results were coincident with Western blots prepared from homogenates of the respective tissues. Reverse transcription-polymerase chain reaction studies were performed with primers derived from the sequence of human placental ecto 5'-NT. Using human placenta as a reference tissue, positive results were obtained in the epididymis, seminal vesicle, and prostate, but not in the testis. On Northern blots, we determined the size of the mRNA at 2.4 kilobases. The relatively strong expression of 5'-NT in the human male accessory sex glands points to a potential regulatory role of the enzyme during posttesticular modification of the sperm surface.
Objectives: To determine the efficacy, safety and feasibility of intermittent androgen deprivation (IAD) in patients with prostate-specific antigen (PSA) relapse after radical prostatectomy or with an incidental prostate cancer (pT1B) after transurethral resection of the prostate (TURP). Methods: Open, nonrandomized, prospective pilot study using the luteinizing hormone-releasing hormone analogue (LH-RHa), leuprorelin acetate (1-month depot) and cyproterone acetate. Results: Forty-four patients have been enrolled. After a 30-64 months' follow-up no progression to androgen-independent status has been observed. Of the entire observation period, 26.6 months (44-58%) remained treatment-free. During the treatment-free periods, normal testosterone levels were obtained, resulting in a cessation of the symptoms of androgen suppression and an improvement in quality of life. Conclusions: These results indicate that IAD is an effective and feasible therapy in patients with early stages of prostate cancer. Larger trials are necessary to confirm these encouraging results. Therefore, a European prospective, randomized, multicenter study (RELAPSE study) has been started to compare IAD with continuous androgen blockade in terms of time to tumor progression, safety and quality of life in patients with PSA relapse after radical prostatectomy.
BACKGROUND: To elucidate the origin of so-called "prostasomes" in human semen, a polyvalent rabbit antiserum was produced against a highly purified preparation of these secretory particles. METHODS: An immunoaffinity selected fraction of the antiserum contained one antibody which in Western blots of prostate extracts showed a 100 kDa band. RESULTS: Semithin sections processed for immunohistochemistry by using this immunoselected antibody presented a distinct immunoreaction at the apical plasma membrane of secretory cells. The immunoreaction was not organ specific, but was present also in seminal vesicles, epididymis, salivary glands, and kidney. As spermatozoa present in epididymis and seminal vesicles were likewise immunoreactive, a polytopic origin of the respective antigen was concluded. CONCLUSIONS: Prostatsomes therefore should rather be designated as "seminosomes" which more clearly refers to the composite origin of these particles.
A unifying hypothesis is presented postulating an apocrine release of several seminal proteins which mix and reaggregate in seminal fluid, thereby eventually forming particles designated either as "prostasomes", "vesiculosomes" or "seminosomes". The term "aposomes" should be restricted to the blebs released from secretory cells in the rat dorsal prostate and coagulating gland. Three different proteins present in human seminosomes along with the respective antibodies have been used to identify the localization, function and hypothetical interaction with spermatozoa. The proteins were (1) seminal vesicle-derived fibronectin, (2) prostate-derived 5'-nucleotidase and (3) a hitherto unidentified 100 kD membrane protein from epididymis, seminal vesicle and prostate. I. Fibronectin is an extracellular matrix protein which is also secreted from the seminal vesicles participating in the formation of the seminal clot. Immunofluorescence and immunoelectron microscopy revealed a relatively broad distribution pattern of fibronectin immunoreactivity on spermatozoa from different donors. Adding a fibronectin antiserum at a moderate dilution to vital spermatozoa in vitro resulted in a significant increase in sperm motility. Purified plasma fibronectin added at various concentrations to a vital sperm preparation was found to inhibit sperm motility in a dose-dependent manner. Measurement of calcium fluxes in individual sperm in the presence of fibronectin showed a significant increase. These findings point to a possible post-testicular regulatory function of seminal fibronectin. 2.5'-Nucleotidase (5'-NT) is an enzyme that hydrolyzes nucleotides such as AMP or IMP into inorganic phosphate and the respective nucleoside. The highest amount and activity of 5'-nucleotidase was present in glandular cells of the prostate; much less was detected in seminal vesicles and epididymis. On spermatozoa, the enzyme was localized on the outer leaflet of the plasma membrane covering the acrosomal region. Addition of purified enzyme to an in vitro incubation system of spermatozoa had no effect on sperm motility. A slight reduction of overall motility, however, was observed after addition of 5'-NT antibody to the spermatozoa. When 5'-nucleotidase inhibitors and adenosine channel antagonists were added to the sperm incubation system, a clear-cut inhibition of sperm motility occurred in a dose-dependent manner. This result is interpreted as indicating a significant role of ecto-5'-nucleotidase in the regulation of sperm motility. 3. A polyvalent antiserum against native human prostasomes recognized antigens in the range of 10-14 kD and of approximately 100 kD, respectively, in seminal fluid and prostate homogenates. Immunohistochemical studies revealed the presence of respective antigens in the epididymis, seminal vesicles and the prostate. Immunoelectron microscopy of ultracryo-sections showed labeling both of the apical plasma membrane in the prostate, as well as intraluminal secretory particles indicating the apocrine i.e. plasma-membrane bounded release of these particles. The secretory elements are termed "seminosomes". An affinity-purified fraction within the antiserum recognizes a 100 kD protein which is present both in the apical plasma membrane of the male genital glands, but also in the sperm head and principal piece of human spermatozoa. Incubation of spermatozoa with seminosomes and the respective purified antiserum had no effect on sperm motility. This is in contradistinction to former reports on motility increase induced by the so-called prostasomes.
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The tissue distribution, preferentially in the human male genital system, and the subcellular localization of the lysosome-associated membrane protein 2 (lamp 2) was studied immunohistochemically using a mouse monoclonal antibody, 2D5. Strong immunoreactivity was present in the tubular system of the kidney, in acinar cells of salivary glands and pancreas, prostate, mammary glands, placenta and in cutaneous sweat glands. Moderate immunoreactivity was observed in cerebral neuronal cells, epidermal cells, testis, epididymis, seminal vesicle and endometrium. Very low immunoreactivity was found in liver. In some of the tissues mentioned, the distribution pattern of immunoreactivity is smooth and homogeneous, while in others it is granular and concentrated in the supra- or perinuclear cytoplasm. The subcellular distribution was studied on ultracryosections and on pre-embedding-processed chopper sections of human prostate. In the latter gland, the protein is not restricted to epithelium, but is also present in stromal cells. Ultrastructurally, the immunoreactivity in secretory cells was localized in electron-translucent vacuoles and granules, including the secretory granules. A close association with cell membranes was not generally the case. Only part of the immunoreactive material was linked to the apical plasma membrane pointing to a biosynthesis independent from an association step with the apical plasma membrane. As shown by immunoelectron microscopy and Western blotting, a high amount of lamp 2 is secreted and is found in so-called prostasomes. The findings indicate that in the human prostate most of the membrane-bound lamp 2 is released from the secretory cells, presumably in an apocrine fashion.
An antiserum against secretory vesicles from human seminal fluid (prostasomes) was used to study the localisation and distribution of the respective antigen(s) during prenatal development and pubertal maturation of the human prostate. The crude antiserum stained both secretory and membrane proteins in the adult prostate and other glands, such as pancreas and parotid gland. An immunoaffinity purified fraction from the antiserum selectively reacted with the apical plasma membrane of prostatic epithelium adluminal cells, recognizing a 100 kDa antigen (PMS). Even in the earliest stages of embryonic prostate specimens studied, the adluminal plasma membrane of the epithelial cells from developing glandular anlagen reacted strongly. The occurrence of PMS immunoreactivity in prostatic anlagen was directly correlated with lumen formation. As the antigen is an androgen-independently synthesised membrane protein of the prostate, it may possibly be used as a marker of cell polarity in the normal and pathologically altered prostate.
To contrast the previously described distribution of 5 alpha-reductase isoenzyme 2 in human organs we have raised synthetic C-terminal peptides of human 5 alpha-reductase isoenzyme 1 (h 5 alpha r1) and isoenzyme 2 (h 5 alpha r2) respectively, and studied the cellular and subcellular distribution of both isoforms of this key enzyme of testosterone metabolism using the human prostate gland as a reference. h 5 alpha r1, which in Western blots of human prostatic proteins had an apparent molecular weight of 23 kDa, was localized immunohistochemically in the nucleus of prostatic epithelial and stromal cells. Ultrastructurally, it was closely associated with the nuclear matrix. The apparent molecular weight of h 5 alpha r2 was 26 kDa in Western blotting of human prostatic proteins. In immunohistochemically processed sections, it was seen in the cytoplasm of prostatic epithelial as well as stromal cells. An organ screening with genital and extragenital tissues of males and females was performed and to elucidate the distribution of the isoenzymes. Presence of both isoenzymes was demonstrated for a number of tissues, including the central nervous system, the urogenital tract, the respiratory tract, the gastrointestinal tract, skin, and the endocrine system. The divergent localization of 5 alpha-reductase isoenzymes points to potentially different functions in various organs. In view of the nuclear localization of isoenzyme 1, its close spatial relationship with the androgen receptor is presumed to indicate a close association with the receptor mechanism.
Secretory particles, designated as 'vesiculosomes', were isolated and purified from bovine seminal vesicle fluid. The protein pattern was characterized using one- and two-dimensional gel electrophoresis. Native vesiculosome fractions were used for immunization of female New Zealand rabbits. The resulting vesiculosome antiserum was used for immunohistochemical studies, designed to obtain data on the intracellular formation, the localization and the potential release from secretory cells of the respective proteins present in these particles. The specificity and sensitivity of the antiserum was tested by Western blot analysis and by an ELISA system. Western blot analysis of isolated vesiculosomes separated on SDS-PAGE showed several heterogeneous immunoreactive bands in the range of 16 kD and above 66 kD, indicating a polyvalent mixture of immunoglobulins. Immunohistochemical staining of different bovine tissues, nevertheless, revealed a strong reaction exclusively at the apical portion of secretory cells of the seminal vesicle and the ampulla of the vas deferens and of intraluminal secretion. The antiserum recognizes both secretory granules and plasma membrane proteins of seminal vesicle epithelium, supporting the concept of vesiculosomes to represent cytoplasmic particles formed from plasma membranes surrounding apical 'blebs' of the cells that are released physiologically during secretion.
Hydrogen peroxide (H(2)O(2)) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50 degrees C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K(m) = 40 millimolar) and H(2)O(2) (apparent K(m) = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.
Calcium fluxes across the plasma membrane of spermatozoa are part of the signal transduction pathway during sperm capacitation. To identify the topography, the time sequence and frequency of calcium fluxes in motile human spermatozoa, individual spermatozoa have to be locally immobilized in a measurement chamber to allow the quantification of ionized calcium by use of a microspectro-photometric method (FURA-2AM). In this study, we compared different immobilization methods using agarose-, gelatin-, laminin- and poly-L-lysine-coated glass slides. Optimal results were obtained with poly-L-lysine coating, which permitted the adhesion of motile spermatozoa that could be analysed microspectro-photometrically. The loss of adherent spermatozoa during the washing procedures was below 10%. However, a major disadvantage of poly-L-lysine coating is that this polymer itself induces a low calcium flux in spermatozoa. Comparing all tested variations, laminin offered the best adhesion result without any detectable effects on calcium fluxes. Our method allowed the rapid change of incubation fluid and calcium concentrations around individual motile spermatozoa and the reproducible quantification of calcium fluxes in single motile spermatozoa.