Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70–140 nmol/l and maximal, carbacholinduced peaks reached 1·1 µmol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells

Presence and localization of a 30-kDa basic fibroblast growth factor-like protein in rodent testes

We have used a recently characterized rabbit antiserum against basic fibroblast growth factor (bFGF), which recognizes various forms of bFGF, to examine the presence and localization of bFGF in the testes of adult rats and mice and the 5-day-old rat. In Western blots of testicular homogenates of adult rats and mice and immature rats, immunoreactive single bands at approximately 30 kDa were detected. Immunocytochemistry revealed specific staining restricted to the tubular compartment. In 5-day-old rat testes, prespermatogonia were immunoreactive. The cytoplasm of pachytene spermatocytes was heavily stained in the adult testes of both species. Staining of these cells became evident around stage IV/V, was prominent in stage VII through IX and declined about stage XII/XIII (rat) or X-XI (mouse). Staining was seen in type A spermatogonia and in elongating spermatids in their cytoplasmatic lobes and along their flagellae. Sertoli cells were unstained. We propose that the pluripotential growth factor bFGF could be involved in the regulation of germ cell proliferation and differentiation in the adult and immature testis

Concerted action of human chorionic gonadotropin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells

Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta- adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone

Villous Mucinous Cystadenoma of the Appendix in a Postmenopausal Woman

Because a significant number of mucoceles are caused by mucinous cystadenocarcinoma, the authors stress that a general surgeon be consulted in cases of right lower quadrant “dumbbell shaped” abdominal cysts

The wave equation on singular space-times

We prove local unique solvability of the wave equation for a large class of weakly singular, locally bounded space-time metrics in a suitable space of generalised functions.Comment: Latex, 19 pages, 1 figure. Discussion of class of metrics covered by our results and some examples added. Conclusion more detailed. Version to appear in Communications in Mathematical Physic

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man

Identification of Basophils as a Major Source of Hepatocyte Growth Factor in Chronic Myeloid Leukemia: A Novel Mechanism of BCR-ABL1-Independent Disease Progression

AbstractChronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML

Insights into replicative senescence of human testicular peritubular cells

There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced beta-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target