ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process

Complex CatSper-dependent and independent [Ca2⁺]i signalling in human spermatozoa induced by follicular fluid

STUDY QUESTION: Does progesterone in human follicular fluid (hFF) activate CatSper and do other components of hFF modulate this effect and/or contribute separately to hFF-induced Ca2+ signaling?SUMMARY ANSWER: hFF potently stimulates CatSper and increases [Ca2+]i, primarily due to high concentrations of progesterone, however,other components of hFF also contribute to [Ca2+]i signaling, including modulation of CatSper channel activity and inhibition of [Ca2+]i oscillations.WHAT IS KNOWN ALREADY: CatSper, the principal Ca2+ channel in spermatozoa, is progesterone-sensitive and essential for fertility. Both hFF and progesterone, which is present in hFF, influence sperm function and increase their [Ca2+]i.STUDY DESIGN, SIZE, DURATION: This basic medical research study used semen samples from >40 donors and hFF from >50 patients who were undergoing surgical oocyte retrieval for IVF/ICSI.PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service REC1. Activities of CatSper and KSper were assessed by patch clamp electrophysiology. Sperm [Ca2+]i responses were examined in sperm populations and single cells. Computer-assisted sperm analysis (CASA) parameters and penetration into viscous media were used to assess functional effects.MAIN RESULTS AND THE ROLE OF CHANCE: hFF and progesterone significantly potentiated CatSper currents. Under quasiphysiologicalconditions, hFF (up to 50%) failed to alter membrane K+ conductance or current reversal potential. hFF and progesterone (at an equivalent concentration) stimulated similar biphasic [Ca2+]i signals both in sperm populations and single cells. At a high hFF concentration (10%), the sustained (plateau) component of the [Ca2+]i signal was consistently greater than that induced by progesterone alone. In single cell recordings, 1% hFF-induced [Ca2+]i oscillations similarly to progesterone but with 10% hFF generation of [Ca2+]i oscillations was suppressed. After treatment to ‘strip’ lipid-derived mediators, hFF failed to significantly stimulate CatSper currents but induced small [Ca2+]i responsesthat were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+]i responses were observed when sperm pretreated with 3 μM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped hFF. hFF stimulated viscous media penetration and was more effective than the equivalent does of progesterone.LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGS: This study directly demonstrates that hFF activates CatSper and establishes that the biologically important effects of hFF reflect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [Ca2+]i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution

Single-cell analysis of [Ca²⁺]i signalling in sub-fertile men:characteristics and relation to fertilization outcome

STUDY QUESTIONWhat are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+]i signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability?SUMMARY ANSWERSingle cell analysis of progesterone-induced (CatSper-mediated) [Ca2+]i showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate.WHAT IS KNOWN ALREADYStimulation with progesterone is a widely used method for assessing [Ca2+]i mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability.STUDY DESIGN, SIZE, DURATIONThis was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA).MAIN RESULTS AND THE ROLE OF CHANCEFor analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve).LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study and caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies

Beyond a warming fingerprint: individualistic biogeographic responses to heterogeneous climate change in California.

Understanding recent biogeographic responses to climate change is fundamental for improving our predictions of likely future responses and guiding conservation planning at both local and global scales. Studies of observed biogeographic responses to 20th century climate change have principally examined effects related to ubiquitous increases in temperature - collectively termed a warming fingerprint. Although the importance of changes in other aspects of climate - particularly precipitation and water availability - is widely acknowledged from a theoretical standpoint and supported by paleontological evidence, we lack a practical understanding of how these changes interact with temperature to drive biogeographic responses. Further complicating matters, differences in life history and ecological attributes may lead species to respond differently to the same changes in climate. Here, we examine whether recent biogeographic patterns across California are consistent with a warming fingerprint. We describe how various components of climate have changed regionally in California during the 20th century and review empirical evidence of biogeographic responses to these changes, particularly elevational range shifts. Many responses to climate change do not appear to be consistent with a warming fingerprint, with downslope shifts in elevation being as common as upslope shifts across a number of taxa and many demographic and community responses being inconsistent with upslope shifts. We identify a number of potential direct and indirect mechanisms for these responses, including the influence of aspects of climate change other than temperature (e.g., the shifting seasonal balance of energy and water availability), differences in each taxon's sensitivity to climate change, trophic interactions, and land-use change. Finally, we highlight the need to move beyond a warming fingerprint in studies of biogeographic responses by considering a more multifaceted view of climate, emphasizing local-scale effects, and including a priori knowledge of relevant natural history for the taxa and regions under study

Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle

Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin-sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose-stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR-AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole-bodyGUR were not different from controls.Of importance, ADRβ2 stimulation with β1/β3 inhibition increases both insulin sensitivity and whole-body glucose utilization in IUGR lambs. In IUGR and IUGR-AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR-AR skeletal muscle than in controls but GLUT1 was greater in IUGR-AR. ADRβ2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR-AR lambs heart rates were greater, which was independent of cardiac ADRβ1 activation. We conclude that targeted ADRβ2 stimulation improved whole-body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch-up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes

Islet Adaptations in Fetal Sheep Persist Following Chronic Exposure to High Norepinephrine

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion following chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. Following an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129±1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were again performed on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P\u3c0.01) fetal plasma NE concentrations and lowered (P\u3c0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P\u3c0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P\u3c0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P\u3c0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P\u3c0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity

Fragmentation of a Circular Disc by Impact on a Frictionless Plate

The break-up of a two-dimensional circular disc by normal and oblique impact on a hard frictionless plate is investigated by molecular dynamics simulations. The disc is composed of numerous unbreakable randomly shaped convex polygons connected together by simple elastic beams that break when bent or stretched beyond a certain limit. It is found that for both normal and oblique impacts the crack patterns are the same and depend solely on the normal component of the impact velocity. Analysing the pattern of breakage, amount of damage, fragment masses and velocities, we show the existence of a critical velocity which separates two regimes of the impact process: below the critical point only a damage cone is formed at the impact site (damage), cleaving of the particle occurs at the critical point, while above the critical velocity the disc breaks into several pieces (fragmentation). In the limit of very high impact velocities the disc suffers complete disintegration (shattering) into many small fragments. In agreement with experimental results, fragment masses are found to follow the Gates-Gaudin-Schuhmann distribution (power law) with an exponent independent of the velocity and angle of impact. The velocity distribution of fragments exhibit an interesting anomalous scaling behavior when changing the impact velocity and the size of the disc.Comment: submitted to J. Phys: Condensed Matter special issue on Granular Medi

Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction

In pregnancies complicated by placental insufficiency and intrauterine growth restriction (IUGR), fetal glucose and oxygen concentrations are reduced, whereas plasma norepinephrine and epinephrine concentrations are elevated throughout the final third of gestation. Here we study the effects of chronic hypoxemia and hypercatecholaminemia on β-cell function in fetal sheep with placental insufficiency-induced IUGR that is produced by maternal hyperthermia. IUGR and control fetuses underwent a sham (intact) or bilateral adrenal demedullation (AD) surgical procedure at 0.65 gestation. As expected, AD-IUGR fetuses had lower norepinephrine concentrations than intact-IUGR fetuses despite being hypoxemic and hypoglycemic. Placental insufficiency reduced fetal weights, but the severity of IUGR was less with AD. Although 2 basal plasma insulin concentrations were lower in intact-IUGR and AD-IUGR fetuses compared with intact-controls, glucose-stimulated insulin concentrations were greater in AD-IUGR fetuses compared with intact-IUGR fetuses. Interestingly, AD-controls had lower glucose- and arginine-stimulated insulin concentrations than intact-controls, but AD-IUGR and AD-control insulin responses were not different. To investigate chronic hypoxemia in the IUGR fetus, arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction. Oxygenation of IUGR fetuses enhanced glucose-stimulated insulin concentrations 3.3-fold in intact-IUGR and 1.7-fold in AD-IUGR fetuses but did not lower norepinephrine and epinephrine concentrations. Together these findings show that chronic hypoxemia and hypercatecholaminemia have distinct but complementary roles in the suppression of β-cell responsiveness in IUGR fetuses. Placental insufficiency restricts the supply of oxygen and nutrients to the fetus and causes intrauterine growth restriction (IUGR) (1, 2). The resulting fetal hypoxemia and hypoglycemia provoke endocrine responses that lower plasma insulin concentrations (3,–5). High norepinephrine and epinephrine concentrations are a hallmark of both human and animal IUGR fetuses (6,–11). These high concentrations of catecholamines inhibit insulin secretion from pancreatic β-cells and may contribute to very low insulin concentrations in the IUGR fetus (12, 13). In addition, chronic elevation of norepinephrine has been shown to slow fetal growth and induce asymmetric growth of fetal tissues (14, 15)