Effect of oxytocin on free intracellular Ca2+ levels and progesterone release by human granulosa-lutein cells

Oxytocin and its receptor are found in the corpus luteum in a variety of species, including the human. In the present study we used fura-2 microfluorimetry to investigate whether activation of the oxytocin receptor of cultured human granulosa-lutein cells causes intracellular calcium (Ca2+) signals and affects progesterone release. Although after 1 day in culture, cells were not responsive to oxytocin, the number of responsive cells increased steadily during the first 3 days in culture, reaching a maximum on days 4 and 5 (59-66%) and then declined again until day 8. Effective oxytocin concentrations were apparently independent of the culture day, and concentrations as low as 10 nmol/L increased intracellular free Ca2+ levels from 70-140 nmol/L (basal levels) to maximal peak levels of 800 nmol/L. The oxytocin-induced Ca2+ signal was not affected by removal of extracellular Ca2+ with EGTA. Moreover, depletion of intracellular Ca2+ stores by ionomycin treatment rendered the cells unresponsive to oxytocin, pointing also at the intracellular source of the oxytocin-inducible Ca2+ signal. Interestingly, after one single stimulation with oxytocin, cells became refractory to additional stimuli, and only extremely high concentrations of oxytocin induced a second increase in intracellular free Ca2+. To examine the possible effects of oxytocin on progesterone release by cultured cells, we incubated cells on culture day 2 (20% responsive cells in the fura measurements) and culture day 5 (66% responsive cells in the fura measurements) for 24 h with oxytocin (10 nmol/L) and hCG (10,000 IU/L). Although hCG significantly stimulated progesterone release, oxytocin alone was without a stimulatory effect on either day. However, a significant augmentation of the effect of hCG on progesterone release was found in incubations of cells on day 5. Interestingly, the effects of hCG also included stimulation of oxytocin release by cultured granulosa-lutein cells into the culture medium, as determined by RIA. In summary, our data indicate the presence of a functional oxytocin receptor on human granulosa-lutein cells that is linked to Ca2+ as a second messenger released from intracellular Ca2+ stores. The number of oxytocin-responsive cells increases during differentiation in culture. Moreover, oxytocin release induced by hCG and a stimulatory effect of oxytocin on the hCG-induced progesterone production during the period of maximal responsiveness of cultured cells were found. We, therefore, propose that oxytocin may have autocrine and/or paracrine functions in human granulosa-lutein cells, including fine-tuning of progesterone release

An autocrine role for pituitary GABA: Activation of GABA-B receptors and regulation of growth hormone levels

There is increasing evidence suggesting that the neurotransmitter gamma-aminobutyric acid (GABA) is a local factor involved in the regulation of endocrine organs. Examples of such functions are documented in the pancreas, but recent results suggest that GABA may act in a similar way in the pituitary, in which GABA receptors are expressed and pituitary growth hormone (GH) cells provide a source of GABA. We hypothesised that GABA secreted in somatotropes may act as an autoregulatory signaling molecule. To test this hypothesis we first examined the nature of GABA receptors expressed by GH cells. RT-PCR analysis demonstrated that GABA-B receptor subunits R1 and R2 are present in the whole rat pituitary. Laser microdissection of immunostained GH cells, followed by RT-PCR as well as immunoelectron microscopy, showed that GABA-B receptors are expressed on somatotropes. To investigate GABA-B receptor function in somatotropes, we used rat GH3 adenoma cells, which, like pituitary GH cells, express GABA-B R1 and R2 (as assessed by RT-PCR and immunoelectron microscopy) and produce GABA (checked by high performance liquid chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by baclofen, a chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by bactofen, a GABA-B receptor agonist. By contrast, blocking GABA-B receptors by an antagonist, phaclofen, decreased GH levels. We conclude that in GH-producing cells, GABA acts as an autocrine factor via GABA-B receptors to control GH levels. Copyright (C) 2002 S. KargerAG, Basel

Insights into GABA receptor signalling in TM3 Leydig cells

gamma-Aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A) receptor subunits, but also bind the GABA agonist {[}H-3] muscimol with a binding affinity in the range reported for other endocrine cells (K-d = 2.740 +/- 0.721 nM). However, they exhibit a low B-max value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl- currents, changes in resting membrane potential, intracellular Ca2+ or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an untypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Base

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

<p>Abstract</p> <p>Background</p> <p>Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺levels. In this study, we aimed at characterizing 1. expression and functions of K_Cachannels (including BK_Cabeta-subunits), and 2. biophysical properties of BK_Cachannels.</p> <p>Methods</p> <p>GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique.</p> <p>Results</p> <p>We identified two K_Catypes in human GCs, the intermediate- (IK) and the small-conductance K_Ca(SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K_Cablockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K_Cachannels with distinctive features. Both, IK and BK_Cachannels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BK_Cachannel revealed the presence of mRNAs encoding several BK_Cabeta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca²⁺dependence of individual BK_Cachannels which we observed in electrophysiological recordings.</p> <p>Conclusion</p> <p>Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK_Ca, they express all three K_Catypes known. We suggest that the plurality and co-expression of different K_Cachannels and BK_Cabeta-subunits might allow differentiated responses to Ca²⁺signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian K_Cachannel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.</p

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

Continuous Equilibrium in Affine and Information-Based Capital Asset Pricing Models

We consider a class of generalized capital asset pricing models in continuous time with a finite number of agents and tradable securities. The securities may not be sufficient to span all sources of uncertainty. If the agents have exponential utility functions and the individual endowments are spanned by the securities, an equilibrium exists and the agents' optimal trading strategies are constant. Affine processes, and the theory of information-based asset pricing are used to model the endogenous asset price dynamics and the terminal payoff. The derived semi-explicit pricing formulae are applied to numerically analyze the impact of the agents' risk aversion on the implied volatility of simultaneously-traded European-style options.Comment: 24 pages, 4 figure

Harnessing the Microbiomes of Suppressive Composts for Plant Protection: From Metagenomes to Beneficial Microorganisms and Reliable Diagnostics

Soil-borne diseases cause significant yield losses worldwide, are difficult to treat and often only limited options for disease management are available. It has long been known that compost amendments, which are routinely applied in organic and integrated farming as a part of good agricultural practice to close nutrient cycles, can convey a protective effect. Yet, the targeted use of composts against soil-borne diseases is hampered by the unpredictability of the efficacy. Several studies have identified and/or isolated beneficial microorganisms (i.e., bacteria, oomycetes, and fungi) from disease suppressive composts capable of suppressing pathogens (e.g., Pythium and Fusarium) in various crops (e.g., tomato, lettuce, and cucumber), and some of them have been developed into commercial products. Yet, there is growing evidence that synthetic or complex microbial consortia can be more effective in controlling diseases than single strains, but the underlying molecular mechanisms are poorly understood. Currently, a major bottleneck concerns the lack of functional assays to identify the most potent beneficial microorganisms and/or key microbial consortia from complex soil and compost microbiomes, which can harbor tens of thousands of species. This focused review describes microorganisms, which have been isolated from, amended to or found to be abundant in disease-suppressive composts and for which a beneficial effect has been documented. We point out opportunities to increasingly harness compost microbiomes for plant protection through an integrated systems approach that combines the power of functional assays to isolate biocontrol and plant growth promoting strains and further prioritize them, with functional genomics approaches that have been successfully applied in other fields of microbiome research. These include detailed metagenomics studies (i.e., amplicon and shotgun sequencing) to achieve a better understanding of the complex system compost and to identify members of taxa enriched in suppressive composts. Whole-genome sequencing and complete assembly of key isolates and their subsequent functional profiling can elucidate the mechanisms of action of biocontrol strains. Integrating the benefits of these approaches will bring the long-term goals of employing microorganisms for a sustainable control of plant pathogens and developing reliable diagnostic assays to assess the suppressiveness of composts within reach

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

Luteinizing hormone and androstendione are independent predictors of ovulation after laparoscopic ovarian drilling: a retrospective cohort study

<p>Abstract</p> <p>Background</p> <p>Our objective was to investigate luteinizing hormone, follicle-stimulating hormone, testosterone, and androstenedione as predicitve markers for ovulation after laparoscopic ovarian drilling.</p> <p>Methods</p> <p>We retrospectively analyzed 100 clompihen-resistant patients with the polycystic ovary syndrome who underwent laparoscopic ovarian drilling at our department. The main outcome measure was spontaneous postoperative ovulation within three months after laparoscopic ovarian drilling. In order to predict spontaneous ovulation, we tested the following parameters by use of a univariate followed by a multivariate regression model: Preoperative serum levels of LH, FSH, testosterone, and androstenedione as well as patients' age and body mass index. In addition, we focused on pregnancy and life birth rates.</p> <p>Results</p> <p>Spontaneous ovulation was documented in 71/100 patients (71.0%). In a univariate and multivariate analysis, luteinizing hormone (OR 1.58, 95%CI: 1.30-1.92) and androstenedione (OR 3.03, 95%CI: 1.20-7.67), but not follicle-stimulating hormone and testosterone were independent predictors of ovulation. Using a cut-off for luteinizing hormone and androstenedione of 12.1 IU/l and 3.26 ng/ml, respectively, spontaneous ovulation was observed in 63/70 (90.0%) and 36/42 patients (85.7%) with elevated and in 8/30 (26.7%) and 35/58 (60.3%) patients with low luteinizing hormone and androstenedione levels, respectively. The sensitivity, specificity, positive and negatvie predictive values for luteinizing hormone and androstendione as predictors of spontaneous ovulation after ovarian drilling were 88.7% (95%CI: 79.0-95.0%), 75.9% (95%CI: 56.5-89.7%), 90.0% (95%CI: 80.5-95.8%), and 73.3% (95%CI: 54.1-87.7%) for luteinizing hormone, and 50.7% (95%CI: 38.6-62.8%), 79.3% (95%CI: 60.3-92.0%), 85.7% (95%CI: 71.5-94.6%), and 39.7% (95%CI: 27.0-53.4%) for androstenedione, respectively. Complete one-year follow-up was available for 74/100 patients (74%). We observed a one-year pregnancy rate and a resulting life-birth rate of 61% and 51%, respectively.</p> <p>Conclusions</p> <p>Luteinizing hormone and androstenedione prior to laparoscopic ovarian drilling are independent predictors of spontaneous ovulation within three months of surgery. We suggest to preferentially performing laparoscopic ovarian drilling in patients with high luteinizing hormone and androstenedione levels.</p