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

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

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

Expression and alternative splicing of the neural cell adhesion molecule NCAM in human granulosa cells during luteinization

Freshly aspirated human granulosa cells from pre-ovulatory follicles and granulosa cells luteinized in culture possess the neural cell adhesion molecule (NCAM) of approximate molecular mass of 140,000 and NCAM mRNA as confirmed by S1-nuclease protection assays and RT-PCR. Moreover, in the process of luteinization the NCAM isoform pattern is modified. Isoforms containing an insert of 10 amino acids (termed VASE) in the extracellular domain of NCAM were supplemented by alternatively spliced isoforms without this insert. NCAM immunoreactivity, at light and electron microscope levels, was associated with the cell membrane of most granulosa cells which formed clusters. During time in culture an increasing subpopulation of granulosa cells, devoid of NCAM immunoreactivity, spread out and formed monolayers. This differential expression and the alternative splicing of NCAM during luteinization of granulosa cells raise the possibility that NCAM could be involved in folliculogenesis and the formation of the corpus luteum in the human

Синусоїдальні зубчасті передачі як альтернатива традиційним передачам та новий метод їх виготовлення

Дана характеристика синусоидальных зубчатых колес и передач, особенности их построения и преимущества по сравнению с традиционными передачами. Описан новый универсальный высокоэффективный способ нарезания синусоидальных профилей, обеспечивающий изготовление большинства известных типов зубчатых колес одним инструментом на одном станке.The sine type gears and transmissions takes many advantages in the comparison with the standard transmissions. The new universal high-efficiency method of sine curve cogs cutting, providing makes the most known types of gear-wheels by one instrument on one machine-tool , is described

A spectroscopic survey of the youngest field stars in the solar neighbourhood. I. The optically bright sample

We present the first results of a ground-based programme conducted on 1-4m class telescopes. Our sample consists of 1097 active and presumably young stars, all of them being optical counterparts of RASS X-ray sources in the northern hemisphere. We concentrate on the 704 optically brightest (V_Ticho<=9.5 mag) candidates. We acquired high-res spectroscopy in the Halpha/Li spectral regions for 426 of such stars without relevant literature data. We describe the sample and the observations and we start to discuss its physical properties. We used a cross-correlation technique and other tools to derive accurate radial/rotational velocities and to perform a spectral classification for both single and SB2 stars. The spectral subtraction technique was used to derive chromospheric activity levels and Li abundances. We estimated the fraction of young single stars and multiple systems in stellar soft X-ray surveys and the contamination by more evolved systems, like RS CVn's. We classified stars on the basis of Li abundance and give a glimpse of their sky distribution. The sample appears to be a mixture of young Pleiades-/Hyades- like stars plus an older Li-poor population (~1-2 Gyr). 7 stars with Li abundance compatible with the age of IC 2602 (~30 Myr) or younger were detected as well, although 2 appear to be Li-rich giants. The discovery of a large number of Li-rich giants is another outcome of this survey. The contamination of soft X-ray surveys by old systems in which the activity level is enhanced by tidal synchronisation is not negligible, especially for K-type stars. 5 stars with Li content close to the primordial abundance are probably associated with known moving groups in the solar neighbourhood. Some of them are PTTS candidates according to their positions in the HR diagram.Comment: 16 pages, 12 figures, 6 tables; 2 figures and 2 tables in electronic form only. Paper accepted by Astronomy and Astrophysic

A Photometric Study of the Young Stellar Population Throughout the lambda Orionis Star-Forming Region

We present VRI photometry of 320,917 stars with 11 < R < 18 throughout the lambda Orionis star-forming region. We statistically remove the field stars and identify a representative PMS population throughout the interior of the molecular ring. The spatial distribution of this population shows a concentration of PMS stars around lambda Ori and in front of the B35 dark cloud. Few PMS stars are found outside these pockets of high stellar density, suggesting that star formation was concentrated in an elongated cloud extending from B35 through lambda Ori to the B30 cloud. We find a lower limit for the global stellar mass of about 500 Mo. We find that the global ratio of low- to high-mass stars is similar to that predicted by the field initial mass function, but this ratio varies strongly as a function of position in the star-forming region. Locally, the star-formation process does not produce a universal initial mass function. We construct a history of the star-forming complex. This history incorporates a recent supernova to explain the distribution of stars and gas today.Comment: 42 pages, 11 figures; to appear in the Astronomical Journa

Отказоустойчивый вентильный электро-привод для гибридного транспортного средства

Выпускная квалификационная работа содержит 79 страниц, 10 рисунка, 30 таблиц, 24 источников. Ключевые слова: гибридное транспортное средство, отказоустойчивый вентильный электропривод, математическая модель вентильного двигателя, неполнофазный режим работы, обрыв фазы двигателя, отказ ключа преобразователя частоты. Был проведён обзор аккумуляторов, обоснован выбор ёмкости аккумуляторной батареи. Цель работы – разработка отказоустойчивого вентильного электропривода для гибридного транспортного средства, обеспечение алгоритма восстановления работоспособности. Выпускная квалификационная работа выполнена в текстовом редакторе Microsoft Office Word 2010 с использованием пакетов: Microsoft Visio 2010, Matlab Simulink.Final qualifying work consists of 79 pages, 10 figure, 30 tables, 24 sources. Key words: hybrid vehicle, failover the valve actuator, the mathematical model of the brushless DC motor, open-phase mode, output phase loss, failure of a key of the frequency Converter. A review was undertaken of batteries and justifies the choice of battery capacity. The work purpose – development of fault-tolerant brushless drive for a hybrid vehicle, provision of algorithm restore functionality. Final qualifying work is executed in a text editor Microsoft Office Word 2010 packages: Microsoft Visio 2010, Matlab Simulink