The Marburg Museum Anatomicum and its unusual gynecological specimens: A brief history and ethical contextualisation.

INTRODUCTION: The Marburg Museum Anatomicum displays a number of unique specimens related to obstetric problems. An ethically intensely disputed example is the bisected body of a pregnant woman and her fetus. Current information stemming from previous publications relates it to a fictional young woman who, who, having got pregnant by a student, committed suicide. This narrative was derived from a novel by the author Walter Bloem (1868-1951), orally transmitted without further proof of reliability. The present study attempts to uncover the true background beyond this narrative and to clarify the acquisition of the body by the anatomical collection and its personal background. SOURCES AND METHODS: Archival material as well as contemporary publications of professors of obstetrics and of anatomy along with data derived from civil and ecclesiastic registry offices were evaluated and compared with observations on the specimen. FINDINGS: Comparison of data derived from the fictional description and observations on the specimen showed significant differences, excluding the narrative as a reliable source. Closer examination of the scientific output of former chairs of obstetrics showed that Professor Wilhelm Zangemeister (1871-1930), head of the clinics of gynecology and obstetrics between 1910 and 1925, published several studies on the clinical significance of narrow pelvis during delivery. In his textbook of obstetrics, published in 1927, he showed an illustration of a frozen section of a pregnant woman with kyphosis who had died from myocarditis. The drawing clearly represents the specimen, having been mounted in a large glass vessel in 1922 and included in the collection of the Anatomical Institute. CONCLUSIONS: The current narrative on the bisected body of a pregnant woman and her fetus preserved in the Marburg Museum Anatomicum has nothing to do with the specimen in the collection. In fact, the latter was prepared in 1922 by order of the former professor of obstetrics, Wilhelm Zangemeister, who later published the case in his textbook of obstetrics. The ethical consequences of the changing ontological status and origin of the specimen and its public display are discussed.

[Historical (cultural) view of the heart and cardiovascular system]. / Herz und Herz-Kreislauf-System in der (kultur-)historischen Betrachtung.

Who discovered the cardiovascular and capillary systems? When students in advanced semesters are asked about historical matters that have decisively influenced the path to present day medicine, as a rule no answer or a false answer is forthcoming. Whoever wants to understand scientific thinking and action, cannot do better than to grapple with the historical and cultural developments in medicine; however, more than any other science the natural sciences and medicine provide evidence that new ways and knowledge must be consistently sought for the benefit of patients. The aim of this article is to make a contribution to remembering how the cardiovascular system was discovered and the cultural and historical importance of the heart. Last but not least, however, the article aims to convey the impression of the huge personal sacrifice, including one's own life, and the stony path which led to the acquisition of this knowledge.

[The Discovery of the Cardiac Atrioventricular Node by Sunao Tawara and Ludwig Aschoff]. / Die Entdeckung des AV-Knotens des Herzens durch Sunao Tawara und Ludwig Aschoff.

Already in 1664, the Danish anatomist and naturalist Niels Stensen proved that the heart is a muscle. But for a long time it remained unclear what triggered the heart contractions.The Dutch physiologist Willem Einthoven registered the electrical processes in the contraction of the heart muscle and thus provided the first electrophysiological basis of cardiac muscle activity. Since 1903, Sunao Tawara was assistant to Ludwig Aschoff in Marburg. Both left Marburg in 1906: Tawara went back to Japan and Aschoff to Freiburg. In 1905, Tawara discovered the connections of the His' bundle to the AV node and the Purkinje fibers. At that time, there was no thought of a functional interpretation. Tawara discovered a kind of "knot" that linked to the adjacent myocardial cells, as well as the "Tawara thighs", which frayed and went into structures known as Purkinje fibers. Tawara detected the tree-like structure he had discovered as a muscle-fiber system that controlled the arousal of the heart's musculature. Thus the old dispute between myogenic and neurogenic arousal of the heart was decided in favor of the myogenic excitation conduction. The atrioventricular node described by Tawara was given the eponym "Aschoff-Tawara node". Tawara's groundbreaking work on the conduction system was the basis for the discovery of the sinus node and the interpretation of the heart's electrophysiology.

[The Hessian Landgrave Philipp the Magnanimous (1504-1567) : His diseases and their political consequences]. / Der hessische Landgraf Philipp der Großmütige (1504­1567) : Seine Krankheiten und ihre politischen Folgen.

Landgrave Philipp of Hesse was one of the leading figures of the reformation period. His morganatic marriage to a young Saxonian lady resulted in a massive impairment of his importance and initiated the division of his territory among his four legitimate sons. Triorchidism ascribed to him was more likely a misdiagnosed spermatozele, and also, there is no reliable account for his syphilis infection. The foundation of the Hessian Grand Hospitals, housing the poor, sick and disabled from the countryside, was his major social-medical achievement.

Neuroendocrine Cells of the Prostate Derive from the Neural Crest.

The histogenesis of prostatic neuroendocrine cells is controversial: a stem cell hypothesis with a urogenital sinus-derived progeny of all prostatic epithelial cells is opposed by a dual origin hypothesis, favoring the derivation of neuroendocrine cells from the neural crest, with the secretory and basal cells being of urogenital sinus origin. A computer-assisted 3D reconstruction was used to analyze the distribution of chromogranin A immunoreactive cells in serial sections of human fetal prostate specimens (gestation weeks 18 and 25). Immunohistochemical double labeling studies with YFP and serotonin antisera combined with electron microscopy were carried out on double-transgenic Wnt1-Cre/ROSA26-YFP mice showing stable YFP expression in all neural crest-derived cell populations despite loss of Wnt1 expression. 3D reconstruction of the distribution pattern of neuroendocrine cells in the human fetal prostate indicates a migration of paraganglionic cells passing the stroma and reaching the prostate ducts. Double-transgenic mice showed 55% double labeling of periurethral neuroendocrine cells expressing both serotonin and YFP, whereas single serotonin labeling was observed in 36% and exclusive YFP labeling in 9%. The results favor the assumption of a major fraction of neural crest-derived neuroendocrine cells in both the human and murine prostates.

Plasma membrane calcium ATPase expression in human colon multistep carcinogenesis.

The expression of the plasma membrane Ca(2+) ATPase (PMCA) was analyzed in a series of 84 formalin-fixed and paraffin embedded colon samples including normal mucosa (n = 32), adenoma (n = 19), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) using (i) immunohistochemistry, (ii) mRNA in situ hybridization, and (iii) quantitative reverse-transcriptase PCR. A marked reduction of PMCA4 protein was observed in high-grade adenoma, colon cancer as well as lymph node metastasis, pointing to its potential role in the progression of cancer. However, PMCA4 RNA transcripts were unchanged or even increased in colon carcinomas, suggesting posttranscriptional regulation of PMCA4 during carcinogenesis.

Regional distribution of neuroendocrine cells in the urogenital duct system of the male rat.

BACKGROUND: Neuroendocrine (NE) cells are frequently present in the human prostate and urethra, whereas they are lacking in the other urogenital organs. This study was undertaken as there are only few detailed studies available on the distribution, form and function of NE cells and the structure of excretory ducts of the accessory sex organs in the male rat. METHODS: Systematic gross anatomical dissections were combined with immunohistochemical and electron microscopic studies of the excretory ducts of the urogenital glands in male rats, with particular focus on the distribution and ultrastructure of the NE cells. RESULTS: The topography and structure of the excretory ducts of the different glands were characterized in detail and analyzed for the distribution of NE cells. These are present (in falling frequencies) in the ducts of seminal vesicles and ventral and lateral prostate and are rare in ducts of coagulating gland, dorsal prostate, urethral epithelium, and excretory ducts of the (bulbo) urethral glands. They are absent in the respective glands proper, the deferent duct and ejaculatory ampulla. Approximately 40% of the NE cells of the ventral prostate ducts are of the "open" type, whereas these are less frequent (14%) in the seminal vesicle ducts, where the "closed" type prevails. CONCLUSIONS: NE cells are present in unequal quantities in the excretory ducts of the accessory sex glands, but they are absent in the glands proper and the deferent ducts. This distribution pattern points to a strictly localized function and differentiation potency of NE precursor cells.

Switch of PMCA4 splice variants in bovine epididymis results in altered isoform expression during functional sperm maturation.

Ca(2+) and Ca(2+)-dependent signals are essential for sperm maturation and fertilization. In mouse sperm the plasma membrane Ca(2+)-ATPase (PMCA) isoform 4 plays a crucial role in Ca(2+) transport. The two major splice variants of PMCA4 are PMCA4a and PMCA4b. PMCA4a differs from PMCA4b in the mechanism of calmodulin binding and activation. PMCA4a shows a much higher basal activity and is more effective than PMCA4b in returning Ca(2+) to resting levels. Knock-out mice carrying a PMCA4-null mutation are infertile because their sperm cannot achieve a hyperactivated state of motility. As sperm reach functional maturity during their transit through the epididymis, the expression of PMCA4a and 4b was assessed in bull testis and epididymis. Quantitative PCR revealed that PMCA4b is the major splice variant in testis, caput, and corpus epididymidis. In contrast, PMCA4a is the major splice variant in cauda epididymidis, whereas sperm are transcriptionally silent. Immunohistochemical staining using a new antibody against bovine PMCA4a located the PMCA4a to the apical membrane of the epithelium of cauda epididymidis, whereas testis, caput, and corpus epididymidis were negative. Western blotting of testis, epididymis, and sperm isolated from caput and cauda epididymidis showed a much higher level of PMCA4a in cauda epididymidis and sperm from cauda epididymidis compared with testis membranes and sperm from caput epididymidis. These findings suggest that PMCA4a is transferred to bovine sperm membranes in cauda epididymidis. This isoform switch may facilitate a higher calcium turnover in sperm necessary to traverse the female genital tract.

Expression of proteinase-activated receptor-2 (PAR2) is androgen-dependent in stromal cell line (hPCPs) from benign prostatic hyperplasia.

BACKGROUND: Growth properties of the prostate are regulated by a variety of hormones and growth factors. Benign prostatic hyperplasia (BPH) is characterized by abnormal epithelial and stromal proliferation. Varying androgen hormone levels in elderly men are correlated with abnormal proliferations of the prostate. Proteinase-activated receptor-2 (PAR2), a subtype of G-protein-coupled receptors, is known to induce multiple biological processes. It could also play a key role in the proliferation and metastasis of prostate cancer, but its effect on BPH pathogenesis is to a great extent unknown. METHODS: Localization of PAR2 was determined both in pathologically altered and in normal prostate tissues by using immunohistochemical techniques. PAR2 activity was assessed by measuring changes in intracellular calcium [Ca(2+)](i) following stimulation of cultured stromal cells with a PAR2 agonist (trypsin) and a synthetic PAR2-activating peptide (AP). DHT-dependence of PAR2 expression in prostate cancer and prostatic stromal cell lines was examined with semi-quantitative and quantitative PCR. Cultured stromal cells (hPCPs) were stimulated with PAR2 AP and cell proliferation was determined through [(3)H]-thymidine incorporation. RESULTS: In comparison to normal prostate, PAR2 expression was increased in BPH stroma. DHT induced a higher expression of PAR2 when sub-physiological DHT-levels were used. Higher levels of DHT produced reduced PAR2 expression. A mitogenic effect was induced by applying PAR2 AP to hPCPs-cells. CONCLUSIONS: In conclusion, we found that PAR2 expression is hormone-dependent in prostatic stromal cells with a negative correlation and we consider it to be an important factor in mitogenesis in BPH.

The localization of PMCA1b in epithelial cells and aposomes of the rat coagulating gland is influenced by androgens.

BACKGROUND: Rat coagulating gland epithelial cells export proteins by an apocrine secretion mode within membrane blebs arising from the apical plasma membrane. Using a pan-PMCA antibody, we have recently shown the plasma membrane Ca(2+)-ATPase (PMCA) being part of the apical plasma membrane of epithelial cells and incorporated into the aposomal membrane. The mRNA of PMCA isoforms 1 and 4 respectively, have been detected by RT-PCR in rat coagulating gland. METHODS: In order to identify which PMCA isoform is integrated into aposomes during apocrine secretion and whether or not PMCA export is influenced by androgens RT-PCR, in situ hybridization, Western blotting, and immunofluorescence experiments were performed. RESULTS: PMCA1b is the isoform which is expressed and located in the apical plasma membrane of coagulating gland epithelial cells and is integrated into the aposomal membrane. In contrast, PMCA4 mRNA and protein are restricted to the stroma. Androgen deprivation by castration within 14 days leads to an accumulation of PMCA1b in coagulating gland epithelium, while aposomes are not detected anymore. CONCLUSIONS: We showed for the first time that PMCA isoform 1b is released via aposomes of the epithelial cells of the rat coagulating gland and that the localization of PMCA1b in the epithelial cells is influenced by androgens.

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.

Expression and localization of PMCA4 in rat testis and epididymis.

It has recently been shown in mice that the plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.

Alternative splicing of TGF-betas and their high-affinity receptors T beta RI, T beta RII and T beta RIII (betaglycan) reveal new variants in human prostatic cells.

BACKGROUND: The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2 and TGF-beta 3, and their receptors [T beta RI, T beta RII, T beta R III (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants T beta RIB, T beta RIIB and TGF-beta 2B in human prostatic cells. RESULTS: Interestingly, a novel human receptor transcript T beta RIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant T beta RIB with four additional amino acids was identified also in human. Expression of the variant T beta RIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of T beta RIIC and TGF-beta 2B mainly in the epithelial cells with a preferential localization of TGF-beta 2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-beta ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-beta2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of T beta RII. CONCLUSION: In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants T beta RIB and the novel isoform T beta RIIC in man clearly contributes to the growing complexity of the TGF-beta family.

Localization and regulation of plasma membrane Ca(2+)-ATPase in bovine spermatozoa.

Calcium (Ca(2+)) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca(2+) removal from the sperm, required to restore resting [Ca(2+)](i), include plasma membrane Ca(2+)-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na(+)-Ca(2+) exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca(2+)-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.

Analysis of the mRNA expression of the TGF-Beta family in testicular cells and localization of the splice variant TGF-beta2B in testis.

The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2, and TGF-beta3, and their receptors [TbetaRI, TbetaRII, TbetaRIII (betaglycan)] elicit many functions in the testis, for example, they perturb the blood testis barrier (BTB). Although expression of the ligands and receptors have been investigated, the alternative splice variants are incompletely examined. We therefore have analyzed all ligands, the receptors, and the splice variants TbetaRIB, TbetaRIIB, and TGF-beta2B in testicular cells from rat and mouse. In mouse, the novel transcript variant TGF-beta2B was identified and was found in Leydig cells, spermatogonia, pachytene spermatocytes, and in the apical regions of the Sertoli cells in adult testis. Even though expression of the splice variant TbetaRIB could be shown in mouse and rat, we never found the isoform TbetaRIIB in the rat cell lines studied. Whereas in all testicular cells expression of all TGF-beta ligands could be shown, receptor mRNA expression was slightly more diverse. Furthermore, expression pattern of the splice variants was more heterogeneous, for example, TbetaRIB was not detectable in adult Sertoli cells, primary peritubular cells, and immortalized peritubular cells. The heterogeneous expression of the receptors and especially of the splice variants might provide possible clues for the different functions of the TGF-beta ligands in testicular cells.

Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40.

A new immortal Sertoli cell line from pubertal rat testis was established and characterized. We have generated the clonal line SCIT-C8 expressing established markers for Sertoli cells (SC) like transferrin, clusterin and steel factor/stem cell factor (SCF). Additionally, the immortalized cells express afadin, a protein which is a member of tight and adherens junctions, therefore the cells may be useful for studies of the blood-testis barrier (BTB) in vitro. In contrast to primary SC, the immortalized cells lost expression of androgen receptor and responsiveness to androgens and follicle-stimulating hormone. Surprisingly, we found mRNA expression and protein secretion of the mesenchymal markers, fibronectin and entactin-1, which we also observed for the immortalized SC lines, ASC-17D and 93RS2. In comparison to primary SC, the immortalized cells demonstrated enhanced adhesion in vitro. This correlated with the expression of entactin-1 because adhesion was strongly reduced by antibody perturbation experiments. Additionally, we found the alternatively spliced and primarily muscle cell-specific long variant of TGF-beta2 not only in peritubular cells (PC), but also in the primary and immortalized SC. Furthermore, all immortalized cell lines secreted higher amounts of TGF-beta2 than primary SC. In conclusion, the immortalized SC lines from different developmental stages showed a similar pattern of epithelial and mesenchymal markers.

Angiotensin II-mediated calcium signals and mitogenesis in human prostate stromal cell line hPCPs.

Western blots and immunocytochemistry were used to detect angiotensin 1 (AT(1)) and angiotensin 2 (AT(2)) receptors in human primary cultures of the prostate stromal compartment (hPCPs). Immunohistochemistry was performed on human prostate tissue-embedded paraffin. In addition, pharmacological tools were applied in combination with photometry experiments to characterize the physiological activity of AT(1) and AT(2) receptors in hPCPs cell culture. A proliferation assay was used to describe the mitogenic activity of angiotensin II (Ang II) on hPCPs cells. Only the AT(1) receptor was detected in Western blot analysis. Immunocytochemistry of hPCPs cells showed that the AT(1) receptor is present in both the smooth muscle type and the fibroblastic type. In the stromal compartment of human prostate tissue, immunoreaction with antibodies against the AT(1) receptor was detectable.Fura-2-loaded hPCPs cells showed an instantaneous and linear rise in free intracellular calcium ion concentration ([Ca(2+)](i)) after local perfusion with Ang II in concentrations of 10 nM. Removing of external calcium or emptying intracellular calcium stores before Ang II application diminished or abolished this [Ca(2+)](i) response. The response to Ang II was also diminished when hPCPs cells were perfused with the AT(1) receptor inhibitor losartan prior to Ang II application. No inhibition of the [Ca(2+)](i) increase was detectable after perfusion with PD 123319, a specific inhibitor of the AT(2) receptor.hPCPs cells were stimulated with Ang II in various concentrations over a period of 2 days. The subsequently performed proliferation assay revealed a mitogenic effect of Ang II on hPCPs in concentrations starting at 10 nM. This effect could be inhibited by losartan.

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.

Cell specific expression of CD10/neutral endopeptidase 24.11 gene in human prostatic tissue and cells.

BACKGROUND: Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloproteinase that functions as part of a regulatory loop controlling local concentrations of peptide substrates and associated peptide-mediated signal transduction processes. In contrast to the encouraging data dealing with NEP activity and regulation in prostate epithelial cells, only a few studies are available on the cellular expression and localization of neutral endopeptidase in the prostatic stromal and cancer cells. Here, we describe the cellular localization of NEP in human prostatic tissue and cells using in situ RT-PCR as a novel molecular biological approach. METHODS: Immunofluorescence and Western blot experiments were performed to control the expression and distribution of the NEP in normal and malignant human prostatic tissues and cell lines. NEP gene expression was monitored by RT-PCR, NEP mRNA was detected in paraffin tissue sections and cultured cells of human prostate by the highly sensitive method of one step-in situ reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: NEP mRNA was detected in human prostatic tissue and in cultured cells by means of in situ RT-PCR. Prostatic tissue showed strong signals in the glandular epithelium and weak signals in the stroma, cultured cells displayed strong signals in prostate cancer cells (LNCaP) and weak signals in stromal cells (hPCPs). Western blot experiments were performed using whole cell extracts to proof the presence of NEP protein in LNCaP and hPCPs. The experiments confirm the expression of NEP by both cell types, however, the experiment with hPCPs cells showed two bands. NEP-immunofluorescence was strong in normal prostatic epithelium and confined to the apical plasma membrane. In dedifferentiated prostate cancer specimens, immunofluorescence of apical plasma membranes was lost, and both the cytoplasm and portions of the plasma membrane were immunoreactive for NEP. Prostate cancer cells (LNCaP) showed a strong immunoreaction of the plasma membrane and the cytoplasm. In comparison with LNCaP cells, only a weak cytoplasmic immunofluorescence was found in some stromal cells (hPCPs). CONCLUSIONS: In normal prostatic tissue and specimens derived from human prostate cancer, NEP mRNA and protein are expressed mainly by the epithelial cells and to a minor extent by the stromal cells of human prostate glands. In situ RT-PCR is a powerful and straightforward approach for the routine and rapid detection of cellular specific expression of low copy genes.