SKF96365 modulates activity of CatSper channels in human sperm.

Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.

Transcription Factor Pit-1 Affects Transcriptional Timing in the Dual-Promoter Human Prolactin Gene.

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains 2 promoters, a pituitary-specific promoter that requires the transcription factor Pit-1 and displays dramatic transcriptional bursting activity and an alternate upstream promoter that is active in nonpituitary tissues.

Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription.

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions.

Multiplexing information flow through dynamic signalling systems.

We consider how a signalling system can act as an information hub by multiplexing information arising from multiple signals. We formally define multiplexing, mathematically characterise which systems can multiplex and how well they can do it. While the results of this paper are theoretical, to motivate the idea of multiplexing, we provide experimental evidence that tentatively suggests that the NF-κB transcription factor can multiplex information about changes in multiple signals.

Asymmetry between Activation and Deactivation during a Transcriptional Pulse.

Transcription in eukaryotic cells occurs in gene-specific bursts or pulses of activity. Recent studies identified a spectrum of transcriptionally active "on-states," interspersed with periods of inactivity, but these "off-states" and the process of transcriptional deactivation are poorly understood. To examine what occurs during deactivation, we investigate the dynamics of switching between variable rates.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state.

Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation.

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators.

Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

Prolactin (PRL) is mainly expressed in the pituitary in rodents, whereas in humans, expression is observed in many extrapituitary sites, including lymphocytes. Due to the lack of adequate experimental models, the function of locally produced PRL in the immune system is largely unknown. Using transgenic rats that express luciferase under the control of extensive human PRL regulatory regions, we characterized immune cell responses to thioglycollate (TG)-induced peritonitis.

Pulsatile patterns of pituitary hormone gene expression change during development.

Important questions in biology have emerged recently concerning the timing of transcription in living cells. Studies on clonal cell lines have shown that transcription is often pulsatile and stochastic, with implications for cellular differentiation. Currently, information regarding transcriptional activity at cellular resolution within a physiological context remains limited.

Dynamic analysis of stochastic transcription cycles.

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h.

Wnt signaling in estrogen-induced lactotroph proliferation.

Prolactinomas are the most common type of functioning pituitary adenoma in humans, but the control of lactotroph proliferation remains unclear. Here, using microarray analysis, we show that estrogen treatment increased expression of Wnt4 mRNA in adult Fischer rat pituitary tissue. Dual immunofluorescence analysis revealed that Wnt4 expression was not confined to lactotrophs, but that it was expressed in all anterior pituitary cell types.

Physiological levels of TNFalpha stimulation induce stochastic dynamics of NF-kappaB responses in single living cells.

Nuclear factor kappa B (NF-kappaB) signalling is activated by cellular stress and inflammation and regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses to different doses of tumour necrosis factor alpha (TNFalpha). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-kappaB nuclear translocation and associated IkappaBalpha degradation.

Population robustness arising from cellular heterogeneity.

Heterogeneity between individual cells is a common feature of dynamic cellular processes, including signaling, transcription, and cell fate; yet the overall tissue level physiological phenotype needs to be carefully controlled to avoid fluctuations. Here we show that in the NF-kappaB signaling system, the precise timing of a dual-delayed negative feedback motif [involving stochastic transcription of inhibitor kappaB (IkappaB)-alpha and -epsilon] is optimized to induce heterogeneous timing of NF-kappaB oscillations between individual cells. We suggest that this dual-delayed negative feedback motif enables NF-kappaB signaling to generate robust single cell oscillations by reducing sensitivity to key parameter perturbations.

Dynamic organisation of prolactin gene expression in living pituitary tissue.

Gene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP).

Bayesian inference of biochemical kinetic parameters using the linear noise approximation.

Background: Fluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires the development of effective statistical methods to calibrate such models against available data. Given the prevalence of stochasticity and noise in biochemical systems inference for stochastic models is of special interest.

Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription.

The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals.

Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states.

Reconstruction of transcriptional dynamics from gene reporter data using differential equations.

Motivation: Promoter-driven reporter genes, notably luciferase and green fluorescent protein, provide a tool for the generation of a vast array of time-course data sets from living cells and organisms. The aim of this study is to introduce a modeling framework based on stochastic differential equations (SDEs) and ordinary differential equations (ODEs) that addresses the problem of reconstructing transcription time-course profiles and associated degradation rates. The dynamical model is embedded into a Bayesian framework and inference is performed using Markov chain Monte Carlo algorithms.

Spatio-temporal protein dynamics in single living cells.

The development and application of single cell optical imaging has identified dynamic and oscillatory signalling processes in individual cells. This requires single cell analyses since the processes may otherwise be masked by the population average. These oscillations range in timing from seconds/minutes (e.

Dynamic resolution of acrosomal exocytosis in human sperm.

An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) - exocytosis of a single large vesicle (the acrosome) that surrounds the nucleus at the apical sperm head. The acrosomal and plasma membranes fuse, resulting in both the release of factors that might facilitate penetration of the zona pellucida (which invests the egg) and the externalisation of membrane components required for gamete fusion. Exocytosis in somatic cells is a rapid process - typically complete within milliseconds - yet acrosomal enzymes are required throughout zona penetration - a period of minutes.

Ca2+ signalling in the control of motility and guidance in mammalian sperm.

Ca2+ signalling in the sperm plays a key role in the regulation of events preceding fertilisation. Control of motility, including hyperactivation and chemotaxis, is particularly dependent upon [Ca2+]i signalling in the principal piece of the flagellum and the midpiece. Here we briefly review the processes that contribute to regulation of [Ca2+]i in mammalian sperm and then examine two areas: (i) the regulation of hyperactivation by [Ca2+]i and the pivotal roles played by CatSpers (sperm-specific, Ca2+-permeable membrane channels) and intracellular Ca2+ stores in this process and (ii) the elevation of [Ca2+]i and consequent modulation of motility caused by progesterone including the ability of progesterone at micromolar concentrations to cause sperm hyperactivation and/or accumulation and the recent discovery that progesterone, at picomolar concentrations, acts as a chemoattractant for mammalian sperm.

[Ca2+]i signalling in sperm--making the most of what you've got.

Thanks to a worrying decrease in male fertility, understanding how sperm 'work' is a matter both of interest and great importance. Sperm of all animals detect various environmental cues. The 'behavioural' and physiological responses of sperm must be specific, appropriate and correctly timed.

Kinetics of the progesterone-induced acrosome reaction and its relation to intracellular calcium responses in individual human spermatozoa.

Progesterone at 3 microM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca(2+)](i)) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca(2+)](i) signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca(2+)](i) signal is not the critical determinant of AR.

Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling.

Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-alpha in GH3 cells was confirmed using real-time PCR.

Reassessing the role of progesterone in fertilization--compartmentalized calcium signalling in human spermatozoa?

Progesterone is present at micromolar concentrations in the vicinity of the oocyte. Human spermatozoa generate a biphasic rise in intracellular calcium concentration ([Ca(2+)](i)) and undergo the acrosome reaction upon progesterone stimulation, suggesting that the hormone acts as a secondary inducer or 'primer' of the acrosome reaction in association with the zona pellucida. However, the sensitivity of human spermatozoa to progesterone is such that many cells may undergo the acrosome reaction prematurely, compromising their ability to fertilize.