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

Dopamine receptor repertoire of human granulosa cells

<p>Abstract</p> <p>Background</p> <p>High levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.</p> <p>Methods</p> <p>Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4) were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system.</p> <p>Results</p> <p>We found members of the two DA receptor families (D₁- and D₂ -like) associated with different signaling pathways in human GCs, namely D₁ (as expected) and D₅ (both are Gs coupled and linked to cAMP increase) and D₂, D₄ (Gi/Gq coupled and linked to IP3/DAG). D₃ was not found. The presence of the trophic hormone hCG (10 IU/ml) in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR) or protein levels (immunocytochemistry/Western blotting) of D_1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D₁ and D₂, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D₂ expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D₂L, D₂S). Irrespective of these variants, D₂ proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed in the absence of extracellular calcium and was abolished by a D₂ blocker (L-741,626). DA treatment (48 h) of human GCs resulted in slightly, but significantly enlarged, viable cells.</p> <p>Conclusion</p> <p>A previous study showed D₂ in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D₂-like receptors, linked to calcium. Ovarian DA can act thus via D_1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.</p

The Unintended Consequences of Education Policies on South African Participation and Unemployment

In the late 1990s the South African Department of Education implemented two policies that were meant to reduce the large number of over-age learners in the school system: schools were no longer allowed to accept students who were more than two years older than the correct grade-age and students could not be held back more than once in each of four schooling phases. Our analysis uses school administrative data and household survey data to show that these policies coincided with a decrease in school enrolment of at least 400,000 and possibly more than 900,000 learners. These policies appear to have pushed many students into the labour market at earlier ages than was observed for previous generations, which explains much of the sudden increase in labour force participation and unemployment during this period. However, since these individuals would probably have entered the labour market sooner if not for their poor employment prospects, we argue that the resulting increase in unemployment signifies a more accurate reflection of disguised unemployment that already existed in the mid-1990s rather than a deterioration of labour market conditions

A rapid and robust method for the cryopreservation of human granulosa cells

Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript~levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell-cell contact genes (GJA1) between the two groups in cells cultured for 1-5~days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells

The impact of the international economic crisis on child poverty in South Africa

This paper reports on a study to provide insights into the magnitude of the shocks associated with the recent global economic crisis in macroeconomic terms in South Africa, the country’s capacity to withstand or cushion these shocks, and the extent of fragility in terms of poverty levels and child wellbeing. The analysis combines macro-economic and micro-economic tools to assess the extent of the crisis’ impact on the country. The study finds that the poverty headcount ratio increases little in the moderate crisis scenario, but substantially under the severe scenario. However, under both scenarios there is a relatively successful return to close to the business as usual trend. It is important to note though that under both scenarios, more poverty sensitive measures (the poverty gap ratio and the poverty severity ratio) decline more, and remain in negative territory longer, showing that the major impact of the crisis is on the poorest, and that this impact is most difficult to overcome.ECONOMIC CRISIS, COMPUTABLE GENERAL EQUILIBRIUM, FORECASTING AND SIMULATION, ALMOST IDEAL DEMAND SYSTEMS, CHILD POVERTY MEASUREMENT, SOUTH AFRICA

The impact of the international economic crisis on child poverty in South Africa

This paper reports on a study to provide insights into the magnitude of the shocks associated with the recent global economic crisis in macroeconomic terms in South Africa, the country’s capacity to withstand or cushion these shocks, and the extent of fragility in terms of poverty levels and child wellbeing. The analysis combines macro-economic and micro-economic tools to assess the extent of the crisis’ impact on the country. The study finds that the poverty headcount ratio increases little in the moderate crisis scenario, but substantially under the severe scenario. However, under both scenarios there is a relatively successful return to close to the business as usual trend. It is important to note though that under both scenarios, more poverty sensitive measures (the poverty gap ratio and the poverty severity ratio) decline more, and remain in negative territory longer, showing that the major impact of the crisis is on the poorest, and that this impact is most difficult to overcome.Economic crisis, Computable general equilibrium, Forecasting and simulation, Almost ideal demand system, Child poverty measurement, South Africa

Thresholds for Intersecting D-branes Revisited

Gauge threshold corrections for intersecting D6-brane string models on toroidal orbifold backgrounds are reconsidered. Both by dimensionally regularising the appearing open string one-loop diagrams in tree-channel as well as by zeta-function regularisation of the corresponding loop-channel one-loop diagrams, we arrive at a result which takes into account the infrared divergence from the contribution of the massless states in the running of the gauge coupling constant as well as the contribution of states, which become light in certain regions of the moduli space.Comment: 14 page