Claudin-3, claudin-7, and claudin-10 show different distribution patterns during decidualization and trophoblast invasion in mouse and human.

Implantation of the mammalian embryo requires profound endometrial changes for successful pregnancy, including epithelial-mesenchymal transition of the luminal epithelium and stromal-epithelial transition of the stromal cells resulting in decidualization. Claudins (Cldn) determine the variability in tight junction paracellular permeability and may play a role during these epithelial and decidual changes. We here localized Cldn3, Cldn7 and Cldn10 proteins in the different compartments of murine endometrium up to day 8.5 of pregnancy (dpc) as well as in human endometrium and first trimester decidua. In murine estrous endometrium, luminal and glandular epithelium exhibited Cldn3 and Cldn7, whereas Cldn10 was only detectable in glandular epithelium. At 4.5 dpc, Cldn3 protein shifted to an apical localization, whereas Cldn7 vanished in the epithelium of the implantation chamber. At this stage, there was no stromal signal for Cldn3 and Cldn7, but a strong induction of Cldn10 in the primary decidual zone. Cldn3 proteins emerged at 5.5 dpc spreading considerably from 6.5 dpc onward in the endothelial cells of the decidual blood sinusoids and in the decidual cells of the compact antimesometrial region. In addition to Cldn3, Cldn10 was identified in human endometrial epithelia. Both proteins were not detected in human first trimester decidual cells. Cldn3 was shown in murine trophoblast giant cells as well as in human extravillous trophoblast cells and thus may have an impact on trophoblast invasion in both species. We here showed a specific claudin signature during early decidualization pointing to a role in decidual angiogenesis and regulation of trophoblast invasion.

Vesicular transfer of membrane components to bovine epididymal spermatozoa.

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture.

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as "mini tissues" to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D(3) induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D(3) has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D(3) and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Early activation of sperm by HCO(3) (-) is regulated hormonally in the murine uterus.

Sperm deposited in the female genital tract receive signals for capacitation. Past work indicates that HCO(3) (-) is the initiating signal that the female reproductive tract contains the HCO(3) (-) -permeant anion channel cystic fibrosis transmembrane conductance regulator (CFTR) and that mutations in CFTR cause subfertility in both sexes. In this study, we examined whether CFTR controls uterine HCO(3) (-) content and sperm responses to it. Both CFTR protein and mRNA were absent in prepubertal murine uterus, but appeared in pubertal and adult tissues. Thus, CFTR is upregulated during development. Uterine CFTR mRNA additionally increased upon induced oestrus, most abundantly in uterus body and distal horns. Uterine fluid of oestrous females contained two-, and nearly fourfold more HCO(3) (-) than that of dioestrous and prepubertal animals, correlating with increased CFTR expression. For sperm incubated in and recovered from prepubertal uteri, flagellar beat frequency was no different from that before incubation. However, for sperm recovered from dioestrous and oestrous uteri, beat frequency was two- and fourfold higher, respectively. Thus, uterine HCO(3) (-) content may have physiological consequences for sperm motility. The male reproductive tract showed no regional distributions or developmental dependence of CFTR expression. Although the sperm flagellum showed CFTR immunoreactivity, CFTR blockers GlyH-101 or CFTR(inh) -172 did neither diminish HCO(3) (-) -evoked increases in sperm motility nor protein tyrosine phosphorylation. Our results indicate that in the uterus, both CFTR expression and the supply of HCO(3) (-) are upregulated hormonally. We propose that these changes coordinate ovulation with increases in sperm motility and promote other components of capacitation by pathways that do not require CFTR in sperm.

Adhesion of fibroblasts on micro- and nanostructured surfaces prepared by chemical vapor deposition and pulsed laser treatment.

The development of micro- and nanostructured surfaces which improve the cell-substrate interaction is of great interest in today's implant applications. In this regard, Al/Al2O3 bi-phasic nanowires were synthesized by chemical vapor deposition of the molecular precursor (tBuOAlH2)2. Heat treatment of such bi-phasic nanowires with short laser pulses leads to micro- and nanostructured Al2O3 surfaces. Such surfaces were characterized by scanning electron microscopy (SEM), electron dispersive spectroscopy and x-ray photoelectron spectroscopy. Following the detailed material characterization, the prepared surfaces were tested for their cell compatibility using normal human dermal fibroblasts. While the cells cultivated on Al/Al2O3 bi-phasic nanowires showed an unusual morphology, cells cultivated on nanowires treated with one and two laser pulses exhibited morphologies similar to those observed on the control substrate. The highest cell density was observed on surfaces treated with one laser pulse. The interaction of the cells with the nano- and microstructures was investigated by SEM analysis in detail. Laser treatment of Al/Al2O3 bi-phasic nanowires is a fast and easy method to fabricate nano- and microstructured Al2O3-surfaces for studying cell-surface interactions. It is our goal to develop a biocompatible Al2O3-surface which could be used as a coating material for medical implants exhibiting a cell selective response because of its specific physical landscape and especially because it promotes the adhesion of osteoblasts while minimizing the adhesion of fibroblasts.

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2.

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene.

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.

Purification and characterization of macrophage migration inhibitory factor as a secretory protein from rat epididymis: evidences for alternative release and transfer to spermatozoa.

BACKGROUND: The cytokine macrophage migration inhibitory factor (MIF), originally described as a T cell product, has recently been identified to mediate cellular interactions in several endocrine organs. Western blots analysis of rat epididymal homogenates using an anti-MIF antibody indicated the presence of substantial amounts of an immunoreactive protein with the apparent Mr of 12 kDa. Our study aimed to characterize the molecular nature of this immunoreactive factor. MATERIALS AND METHODS: The purified 12 kDa protein and a cloned cDNA fragment were characterized by sequence analysis. Furthermore, expression pattern and localization of the 12 kDa protein were investigated using in situ hybridization, immunohistochemistry, immunoelectron microscopy, and western blots experiments on epididymal sections, isolated epididymal vesicles, and outer dense fibers from spermatozoa. RESULTS: The N-terminal amino acid sequence analysis over 10 amino acids revealed a 100% homology of the 12 kDa protein to the N-terminus of the cytokine MIF. These data were confirmed by sequence analysis of a reverse transcription polymerase chain reaction (RT-PCR) amplified cDNA fragment from rat epididymis, which also showed complete homology to the MIF cDNA sequence. MIF protein and mRNA were localized in the epithelial cells of the epididymis in a regional distribution manner, with the expression maximal in the caput. Immune cells were not labeled. MIF is the first classical cytokine identified to be expressed by the epididymal epithelial cells. Immunoelectron microscopy detected MIF immunoreactivity in the cytoplasm, with no reaction visible in the Golgi complex and the cisternae of the endoplasmic reticulum. At the apical cell surface, MIF accumulated in stereocilia and vesicles that were pinched off from the plasma membrane. MIF detection in vesicles isolated from epididymal secretion together with the lack of a N-terminal signal sequence for translocation in the endoplasmic reticulum strongly suggested a nonclassical secretion mode. Furthermore, MIF was identified as a new component of the outer dense fibers (ODF), a cytoskeletal element of the mid- and principal piece of the sperm tail. CONCLUSION: The cytokine MIF was identified in substantial amounts in the epithelial cells of rat epididymis and in the outer dense fibers of rat epididymal spermatozoa. Our results indicate a nonclassical secretion mode for MIF and suggest a cell-to-cell transfer of MIF via vesicles to the sperm cells.

Assessment of fibronectin as a potential new clinical tool in andrology.

In this prospective clinical study, 892 patients with normal and impaired semen were examined in order to investigate the correlation between the concentration of fibronectin in seminal plasma and the motility of spermatozoa. The fibronectin concentration in seminal plasma, total sperm motility and linear sperm motility were measured. We report here a significant negative correlation between the fibronectin concentration in seminal plasma and total sperm motility (r=-0.3474). There was no link between varicocele and vasectomy, or between varicocele and variation in the concentration of fibronectin. It is concluded that higher concentrations of the acute-phase protein fibronectin may be a cause of severe reduction in sperm motility. The investigation of fibronectin concentrations in seminal fluid could be a new and helpful clinical tool in the assessment of male fertility.

Macrophage migration inhibitory factor-induced Ca(2+) response in rat testicular peritubular cells.

Macrophage migration inhibitory factor (MIF), originally described as a T-cell product, has recently been identified in several endocrine organs. In the rat testis, MIF is secreted by the Leydig cells into testicular interstitial fluid that directly contacts Sertoli and peritubular cells. To investigate whether MIF is involved in calcium-dependent signal transduction, we have isolated rat Sertoli and peritubular cells. Despite progress in understanding functional properties of MIF, the molecular mechanism of MIF action in target cells is almost completely unknown. Here we find that recombinant MIF evokes a transient increase in calcium levels in peritubular cells but not in Sertoli cells from dissociated rat testis. Concentrations in the range between 12.5 ng/ml and 120 ng/ml of recombinant MIF were found to be effective, with 50 ng/ml yielding the largest increase in intracellular calcium. Preincubation of MIF with a neutralizing monoclonal antibody specifically blocked the response. Incubation of the peritubular cells in calcium-free buffer clearly decreased the evoked response in intracellular calcium concentration. However, the calcium response was greatly decreased by thapsigargin, an inhibitor of the Ca(2+) ATPase of the endoplasmic reticulum. The results strongly indicate that calcium is mobilized from reticulum stores during MIF-mediated signal transduction in the testis. In conclusion, our results provide the first characterization of MIF signal transduction in the testis and suggest that signaling from Leydig cells to peritubular cells through MIF is mediated by receptors coupled to release of intracellular calcium.

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm.

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.

Macrophage migration inhibitory factor (MIF) as a paracrine mediator in the interaction of testicular somatic cells.

Originally the macrophage migration inhibitory factor (MIF) was described as a classical T-cell cytokine. Recently, a much broader tissue distribution for MIF has been revealed. We demonstrated that MIF protein and mRNA are present in the Leydig cells of the normal adult rat testis. Addition of recombinant MIF to cultures of rat seminiferous tubules resulted in decreased secretion of inhibin, whereas follistatin and activin levels remained unchanged, suggesting a paracrine role for MIF in Sertoli cell regulation. Furthermore, MIF showed unique compensatory production in the rat testis. Depletion of the original MIF source, the Leydig cells, by the specific toxin EDS prompted MIF expression by the previously negative Sertoli cells. Leydig cell re-population of the interstitial tissue by precursor cells resulted in a switch back to production by Leydig cells. Therefore, testicular MIF appears to be under very tight paracrine control. MIF has thus been identified as a new mediator in the cross-talk between Leydig cells and the somatic cells of the seminiferous tubules of the rat testis.

Distribution and function of angiotensin II receptors in mouse spermatozoa.

Previous work indicates that angiotensin II (AngII) stimulates sperm motility and acrosomal exocytosis. Here we determined the distribution of AngII receptors on mouse sperm by immunocytochemistry and used Ca2+ probe photometry to examine their coupling to sperm regulatory pathways. We found both AT1 and AT2 receptors localized on the acrosomal region of the sperm head. The AT1 receptor, but not the AT2 receptor, is found also on the principal piece of the sperm tail. Local perfusion of motile but nonprogressive sperm with 0.1-1 microM of AngII evokes a rapid, substantial rise in intracellular [Ca2+]. This response is blocked by losartan, a specific antagonist of the AT1 receptor. These results indicate that sperm possess functional AT1 receptors that are distributed to sites that may allow selective control of motility and exocytosis. They also suggest that the AT2 receptors detected by immunoreactivity are either nonfunctional or are not coupled to Ca(2+)-mediated signalling mechanisms.

Fibronectin in human prostatic cells in vivo and in vitro: expression, distribution, and pathological significance.

In the present study we examined the expression and release of the extracellular matrix glycoprotein fibronectin (FN) in a prostate cancer cell line (LNCaP) and in primary prostatic stromal cells using the reverse transcription-polymerase chain reaction (RT-PCR) and by an enzyme-linked immunosorbent assay. Perturbation experiments in vitro using antibodies directed against FN and the FN receptor were also performed. Immunohistochemistry was used to show the in vivo distribution of FN and the FN receptor in tissue sections of normal human prostate, benign prostatic hyperplasia, and prostate carcinoma. The expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue was investigated by RT-PCR. The FN mRNA was expressed by LNCaP and primary prostatic stromal cells, respectively. Both cell types released FN into the medium in a time-dependent manner, whereby FN secretion was about 2.5-fold higher in cultures of stromal cells relative to LNCaP cells. Blocking FN with anti-FN antibodies resulted in a significant decrease in cell adhesion for LNCaP cells and a change in morphology for the primary stromal cells. FN was located mainly in the stromal compartment of the prostate, showing a distinct distribution pattern in prostate carcinoma, whereas the FN receptor was detectable only in the prostate epithelia. RT-PCR experiments showed the expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue, with a 3.5-fold higher expression in the prostate carcinoma probes. Our data point to an important role for FN in cell adhesion of prostatic cells and show that an alternatively spliced FN mRNA is upregulated in the pathologically altered human prostate.

Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread.

Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

Vulvar amyloidosis mimicking giant condylomata acuminata in a patient with multiple myeloma.

We report a case of unusual cutaneous amyloidosis involving the vulva in a patient with multiple myeloma. Genital examination revealed a dense agglomeration of verrucous papules and pedunculated condyloma-like tumors. The correct diagnosis was established by immunohistochemical examinations that visualized large amounts of lambda light chains, whereas no reaction was detected for kappa light chains or human papilloma virus. In this way, the differential diagnosis of condylomata acuminata could be ruled out. Condyloma-like lesions have been described in patients suffering from multiple myeloma, but the present case is unusual because of the extensive involvement. Vulvar amyloidosis should be added to the list of possible presentations of myeloma-associated systemic amyloidoses.

Expression and enzymic activity of ecto 5'-nucleotidase in the human male genital tract.

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.

Measurement of calcium influx in surface-fixed single sperm cells: efficiency of different immobilization methods.

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.

Immunohistochemistry of a prostate membrane specific protein during development and maturation of the human prostate.

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.