The effects of prolonged pacifier use on language development in infants and toddlers.

Pacifiers are a common soothing tool used by parents to calm and comfort infants and toddlers. While pacifiers can provide temporary relief, there is growing concern about the potential long-term effects of prolonged pacifier use on language and cognitive development. Previous studies have suggested that prolonged use of pacifiers may have negative consequences on language outcomes in infants and toddlers, especially during the first few years of life known to be a critical period for language development. Previous studies have shown that children who use pacifiers extensively have smaller vocabulary sizes at 1 and 2 years of age which can have subsequent effects on socioemotional. In addition, significant association between greater frequency of daytime pacifier use and worsening of cognitive outcomes was shown. Furthermore, research has shown a strong dose-response association between intense pacifier use up to 4 years of age and lower IQ at 6 years. Recently, the importance of oral motor movements and sensorimotor production for speech perception in infants as young as 6 months has been highlighted, raising important questions on the effect of oral motor movement restrictions at an early age. Together, these findings raise concern about the potential long-term effects of prolonged pacifier use on language and cognitive development at a critical time in child development. However, it is still debatable within the scientific field the potential relationship between pacifier use and language development in early life most likely due to the complexity of studying child development. This mini review aims to provide valuable insights for parents, caregivers, and healthcare professionals in making informed decisions and understand regarding pacifier use for infants and toddlers.

Skin-to-Skin Contact and Breastfeeding After Planned Cesarean Birth Before and During the COVID-19 Pandemic.

Background: Benefits of early skin-to-skin contact (SSC) between mother and newborn are widely documented, including improved breastfeeding outcomes. While promoting immediate SSC is standard practice for vaginal birth, it happens less often after cesarean birth. It is not known how changes in hospital practices and staffing shortages during the COVID-19 pandemic have influenced the practice of SSC in the operating room (OR). This study aims to identify the relationship between SSC after cesarean birth and breastfeeding and compare SSC before and during the COVID-19 pandemic at a single institution. Materials and Methods: This was a retrospective cohort study of 244 subjects who had scheduled cesarean births during 2019 and 2020. The primary outcome was newborn feeding at hospital discharge. Secondary outcomes were time to initiate breastfeeding, newborn feeding at 4-8-weeks postpartum, and location of SSC initiation in 2019 versus 2020. Results: SSC within 3 days of birth was significantly associated with feeding type on discharge and/or 4-8 weeks postpartum. More subjects intending to exclusively breastfeed met this intention at discharge with SSC in the OR. Newborns who had SSC in the OR had significantly earlier initiation of breastfeeding. There was an increase in SSC in the OR between 2019 (27%) and 2020 (39%). Conclusion: SSC in the OR was associated with improved short-term breastfeeding outcomes in our study. If immediate SSC is not possible, SSC within 3 days of birth may have breastfeeding benefits. The increase in SSC in the OR during the COVID-19 pandemic indicates that SSC practices can be implemented, despite challenging circumstances.

Dynamic Interplay between Social Brain Development and Nutrient Intake in Young Children.

Myelination of the brain structures underlying social behavior in humans is a dynamic process that parallels the emergence of social-emotional development and social skills in early life. Of the many genetic and environmental factors regulating the myelination processes, nutrition is considered as a critical and modifiable early-life factor for establishing healthy social brain networks. However, the impact of nutrition on the longitudinal development of social brain myelination remains to be fully understood. This study examined the interplay between childhood nutrient intake and social brain development across the first 5 years of life. Myelin-sensitive neuroimaging and food-intake data were analyzed in 293 children, 0.5 to 5 years of age, and explored for dynamic patterns of nutrient-social brain myelin associations. We found three data-driven age windows with specific nutrient correlation patterns, 63 individual nutrient-myelin correlations, and six nutrient combinations with a statistically significant predictive value for social brain myelination. These results provide novel insights into the impact of specific nutrient intakes on early brain development, in particular social brain regions, and suggest a critical age-sensitive opportunity to impact these brain regions for potential longer-term improvements in socio-emotional development and related executive-function and critical-thinking skills.

Effect of intensified training on cognitive function, psychological state & performance in trained cyclists.

Athletes often undertake intensified training loads prior to competition with the goal of functionally overreaching for temporary performance enhancement; however, little is known about the impact of this on cognitive function. The aim of this study was to investigate the effect of intensified training induced fatigue on cognitive function, psychological state and performance in trained cyclists. Twenty-three trained male cyclists were randomly assigned to an intensified training group or a control group for two-weeks, followed by a two-week taper period. At baseline, one-week, two-weeks and post-taper, participants undertook a series of cognitive, performance, mood and recovery-stress assessments. The training intervention significantly increased training volume, load and strain by 108%, 116% and 151% respectively. Peak and mean power output on a maximal test and time trial significantly decreased by 4.8% and 9.4% following the two-week training intervention compared to baseline, in addition to a 169% change in total mood disturbance and significant disruption to recovery-stress balance. No change in any cognitive measure was observed across the study period. Following a two-week taper, performance, mood and well-being measures returned to baseline. Two weeks of intensified training resulted in overreaching as identified by performance and psychological measures. Cognitive function was not sensitive to intensified training promoting caution with its use as a measure for the early identification of overreaching.HighlightsTwo-weeks of intensified training significantly increased training volume, load and strain eliciting a state of overreaching in trained male cyclists.Intensified training caused deteriorations in physical performance but did not influence cognitive measures.Mood and recovery-stress balance were negatively affected by intensified training but recovered back to baseline following a two-week taper at a reduced training volume.A two-week taper period following two-weeks of intensified training did not result in improved physiological measures, physical performance parameters or mood above initial baseline values highlighting the need for careful consideration over the purpose, desired outcomes and necessity of intensified training on an individualised basis.

The Matrix Matters: Beverage Carbonation Impacts the Timing of Caffeine Effects on Sustained Attention.

Both caffeine and the perception of refreshment delivered by cooling, tingling, and mouth-watering flavors have individually been shown to positively impact cognitive performance and mood, though presently there is limited evidence on their possible combined effects. This study explored the contribution of refreshing compounds in beverages, namely, carbon dioxide and citric acid, on the acute effects of caffeine on sustained attention and self-rated physical and mental energy. A randomized, controlled crossover trial was conducted by testing three products: a carbonated caffeinated beverage; a comparator caffeinated beverage; and a flavor-matched control beverage. Findings from 24 healthy adults revealed product-dependent variations in cognitive performance during a 60-min visual sustained-attention task, suggesting that the carbonated-caffeinated beverage led to faster, greater and more consistent levels of accuracy, compared to the control beverage. Specifically, significant differences were found between: (1) the carbonated-caffeinated beverage and the caffeinated beverage, and (2) between the caffeinated beverage and the control beverage for number of hits, reaction time and false alarm scores. Both caffeinated beverages led to higher physical and mental energy, and lower physical and mental fatigue 60-min post-consumption. These findings suggest beneficial effects on sustained attention through the combination of caffeine with refreshing compounds.

Detrimental effects on executive function and mood following consecutive days of repeated high-intensity sprint interval exercise in trained male sports players.

Intensified periods of competition are common in many team sports, potentially leading to increased fatigue and reduced performance. The purpose of this study was to investigate the effect of repeated high-intensity sprint interval exercise on cognitive function, mood and perceptions of energy and fatigue. Twenty-four trained rugby players completed multiple bouts of repeated sprints across two consecutive days. Prior to and following each set of maximal effort sprints or equivalent control duration, a battery of cognitive tasks assessing simple and choice reaction time, visuo-spatial working memory and inhibition were completed as well as visual analogue scales that assessed mood, energy, and fatigue. Accuracy of incongruent Stroop responses was significantly lower across day 2 compared to day 1 and the control condition. Four-choice reaction time was slower across day 2 whilst feelings of alertness, contentedness, and physical and mental energy were reduced while ratings of physical and mental fatigue increased. These findings suggest that intensified periods of high-intensity sprint interval exercise have detrimental effects on executive function, mood and perceptions of physical and mental energy, and fatigue. These deleterious effects have the potential to impact performance and may increase the propensity for injury/accidents in certain sporting and non-sporting contexts.

Nutrients for executive function development and related brain connectivity in school-aged children.

Executive functions refer to a set of higher-order cognitive processes involved in the control and organization of information to serve goal-directed behaviors. Skills in executive functioning are developed throughout childhood and adolescence and have been shown to be predictive of academic achievement. The coordination of these complex processes is critically dependent on brain maturation and connectivity, including key neurodevelopmental processes like myelination and synaptogenesis. Among other factors, research highlights the influential effect of nutrition and diet on these neurodevelopmental processes, which may impact executive function performance in healthy and deficient populations. This review considers the research to date on the role of key nutrients that have been identified for executive function development and their underlying neurophysiological processes in school-aged children.

Aminoglycoside-modifying enzyme and 16S ribosomal RNA methyltransferase genes among a global collection of Gram-negative isolates.

OBJECTIVES: The prevalence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases among 200 Gram-negative clinical isolates resistant to different aminoglycosides and collected worldwide during 2013 was evaluated. METHODS: Selected AMEs and 16S rRNA methyltransferase genes were screened by PCR/sequencing among 49 Acinetobacter spp., 52 Pseudomonas aeruginosa and 99 Enterobacterales. RESULTS: In total 72 isolates carried aac(6')-lb variants (36.0% overall; 55.6% Enterobacterales): 30 aac(6')-Ib-cr, 21 aac(6')-Ib and 21 aac(6')-Ib-like displaying substitutions L119S (alone or in combination with V71A or R173K) or S100G. Ten aph(3')-VI variants were detected among 35 isolates (46.9% of Acinetobacter spp.). Nineteen isolates carried variants of aac(3)-I, with aac(3)-Ia (n=13, mostly Acinetobacter spp.) being the most prevalent. Other AME genes detected were ant(3″)-Ia (n=41), ant(2″)-Ia (n=24), aac(3)-IIe (n=23), aac(3)-IId (n=21), aac(6')-Im (n=13, mostly P. aeruginosa), aacA8 (n=3), aac(3)-IIf (n=1) and aac(3)-IVa (n=1). Among 42 isolates resistant to amikacin, gentamicin and tobramycin tested for 16S rRNA methyltransferase genes, 21 (50.0%) tested positive; armA was most common (n=14), but 4 isolates carried rmtB1, 2 rmtF1 and 1 new variant rmtB4. Over 60 gene combinations, consisting of one to four AMEs and 16S rRNA methyltransferases, were observed. Cloning genes not previously characterised revealed diverse aminoglycoside resistance patterns for some AMEs, but expected results for rmtB4. CONCLUSIONS: Studies broadly evaluating these aminoglycoside resistance genes are needed. Using agents stable in the presence of these resistance genes might help overcome resistance.

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

STUDY QUESTION: What 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 ANSWER: Single 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 ALREADY: Stimulation 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, DURATION: This 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, METHODS: Semen 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 CHANCE: For 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 CAUTION: This is an in vitro study and caution must be taken when extrapolating these results in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a MRC project grant (MR/M012492/1; MR/K013343/1). Additional funding was provided by Chief Scientist Office/NHS research Scotland.

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 quasi-physiological conditions, 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 responses that 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. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P., C.L.R.B.) and University of Abertay (sabbatical for S.G.B.). Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest.

Mechanisms of Resistance, Clonal Expansion, and Increasing Prevalence of Acinetobacter baumannii Strains Displaying Elevated Tigecycline MIC Values in Latin America.

The aim of the study was to characterize forty-eight Acinetobacter baumannii (ACB) isolates with confirmed tigecycline MIC values >2 mg/L observed in six Latin American (LATAM) hospitals (four countries) in 2011. During 2005-2011, 6,923 ACB isolates were collected as part of the SENTRY Program, and tigecycline susceptibility was quantified using the reference broth microdilution method. A total of 102/1881 ACB from LATAM hospitals displayed tigecycline minimum inhibitory concentration (MIC) values >2 mg/L, showing an increase from 4.3% in 2010 to 10.5% in 2011, which is considerably high when compared to other geographical regions. Forty-eight ACB from 2011 displaying elevated tigecycline MICs were typed by pulsed-field gel electrophoresis, which showed multiple clusters in Sao Paulo, Brazil, and a major clone in Guadalajara, Mexico. Eighteen unique isolates had the expression of adeA and adeF determined and results compared to a group of tigecycline-susceptible strains, which demonstrated that 18/18 strains had significantly increased expression of AdeABC and three isolates overexpressed AdeFGH. Sequencing of adeS and adeR revealed that 11 isolates displayed adeS mutations, and 5 isolates had mutations in adeR. Sequencing of trm showed frameshift mutations in eight isolates and insertion sequences leading to nonfunctional proteins in three isolates. TetX-encoding genes were not detected. We documented the recent increase of ACB displaying elevated tigecycline MICs in LATAM hospitals, dominantly due to the clonal expansion of isolates in Brazil and Mexico. Control of tigecycline usage in those countries and more strict infection control practices in the involved hospitals should be considered to reduce such outbreaks.

Ceftazidime-avibactam activity tested against Enterobacteriaceae isolates from U.S. hospitals (2011 to 2013) and characterization of ß-lactamase-producing strains.

Ceftazidime-avibactam (MIC50/90, 0.12/0.25 µg/ml) inhibited 99.9% (20,698/20,709) of Enterobacteriaceae isolates at ≤8 µg/ml. This compound was active against resistant subsets, including ceftazidime-nonsusceptible Enterobacter cloacae (MIC50/90, 0.25/0.5 µg/ml) and extended-spectrum ß-lactamase (ESBL) phenotype isolates. An ESBL phenotype was noted among 12.4% (1,696/13,692 isolates from targeted species) of the isolates, including 776 Escherichia coli (12.0% for this species; MIC50/90, 0.12/0.25 µg/ml), 721 Klebsiella pneumoniae (16.3%; MIC50/90, 0.12/0.25 µg/ml), 119 Klebsiella oxytoca (10.3%; MIC50/90, 0.06/0.25 µg/ml), and 80 Proteus mirabilis (4.9%; MIC50/90, 0.06/0.12 µg/ml) isolates. The most common enzymes detected among ESBL phenotype isolates from 2013 (n = 743) screened using a microarray-based assay were CTX-M-15-like (n = 307), KPC (n = 120), SHV ESBLs (n = 118), and CTX-M-14-like (n = 110). KPC producers were highly resistant to comparators, and ceftazidime-avibactam (MIC50/90, 0.5/2 µg/ml) and tigecycline (MIC50/90, 0.5/1 µg/ml; 98.3% susceptible) were the most active agents against these strains. Meropenem (MIC50/90, ≤0.06/≤0.06 µg/ml) and ceftazidime-avibactam (MIC50/90, 0.12/0.25 µg/ml) were active against CTX-M-producing isolates. Other enzymes were also observed, and ceftazidime-avibactam displayed good activity against the isolates producing less common enzymes. Among 11 isolates displaying ceftazidime-avibactam MIC values of >8 µg/ml, three were K. pneumoniae strains producing metallo-ß-lactamases (all ceftazidime-avibactam MICs, >32 µg/ml), with two NDM-1 producers and one K. pneumoniae strain carrying the bla(KPC-2) and bla(VIM-4) genes. Therapeutic options for isolates producing ß-lactamases may be limited, and ceftazidime-avibactam, which displayed good activity against strains, including those producing KPC enzymes, merits further study in infections where such organisms occur.

Evaluation of clonality and carbapenem resistance mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus complex and Enterobacteriaceae isolates collected in European and Mediterranean countries and detection of two novel ß-lactamases, GES-22 and VIM-35.

We evaluated doripenem-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ACB; n = 411) and Enterobacteriaceae (n = 92) isolates collected from patients from 14 European and Mediterranean countries during 2009 to 2011 for the presence of carbapenemase-encoding genes and clonality. Following susceptibility testing, carbapenem-resistant (doripenem MIC, >2 µg/ml) isolates were screened for carbapenemases. New ß-lactamase genes were expressed in a common background and susceptibility was tested. Class 1 integrons were sequenced. Clonality was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing (Pasteur scheme). Relative expression of ß-lactam intrinsic resistance mechanisms was determined for carbapenemase-negative Enterobacteriaceae. ACB and Enterobacteriaceae displayed 58.9 and 0.9% doripenem resistance, respectively. bla(OXA-23), bla(OXA-58), and bla(OXA-24/OXA-40) were detected among 277, 77, and 29 ACB, respectively (in 8, 6, and 5 countries). Ten Turkish isolates carried bla(GES-11) or bla(GES-22). GES-22 (G243A and M169L mutations in GES-1) had an extended-spectrum ß-lactamase profile. A total of 33 clusters of ≥ 2 ACB isolates were observed, and 227 isolates belonged to sequence type 2/international clone II. Other international clones were limited to Turkey and Israel. Doripenem-resistant Enterobacteriaceae increased significantly (0.7 to 1.6%), and 15 blaKPC-2- and 22 blaKPC-3-carrying isolates, mostly belonging to clonal complexes 11 and 258, were observed. Enterobacteriaceae isolates producing OXA-48 (n = 16; in Turkey and Italy), VIM-1 (n = 10; in Greece, Poland, and Spain), VIM-26 (n = 1; in Greece), and IMP-19, VIM-4, and the novel VIM-35 (n = 1 each from Poland) were detected. VIM-35 had one substitution compared to VIM-1 (A235T) and a similar susceptibility profile. One or more resistance mechanisms were identified in 4/6 carbapenemase-negative Enterobacteriaceae. This broad evaluation confirms results from country-specific surveys and shows a highly diverse population of carbapenemase-producing ACB and Enterobacteriaceae in Europe and Mediterranean countries.

Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 µm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

2-APB-potentiated channels amplify CatSper-induced Ca(2+) signals in human sperm.

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pre-treatment with 5 µM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 µM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50-100 µM 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50-100 µM 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.

Techniques for imaging Ca2+ signaling in human sperm.

Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca(2+)-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca(2+) response as % change in fluorescence is obtained.

Ca2+-stores in sperm: their identities and functions.

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+](i) and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca2+ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the probable identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally, we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.

Mobilisation of stored calcium in the neck region of human sperm--a mechanism for regulation of flagellar activity.

Calcium signalling plays a pivotal role in sperm physiology, being intimately involved in the regulation of acrosome reaction, chemotaxis and hyperactivation. Here we describe briefly the mechanisms of calcium regulation in somatic cells and the ways in which these mechanisms have been adapted to function in mature spermatozoa. We then consider recent data from this and other laboratories on the responses of sperm to three compounds: progesterone and nitric oxide (both products of the cumulus oophorus) and 4-aminopyridine. All of these compounds induce calcium signals in the posterior sperm head and neck region and, when applied at appropriate concentrations, modify flagellar activity, causing asymmetric bending of the proximal flagellum. We argue that these effects reflect a common mode of action, mobilisation of calcium stored in the sperm neck region. Finally we consider the nature of calcium signalling pathways in sperm. We suggest that this highly specialised and extremely polarised cell, though working with the same calcium signalling 'tools' as those of somatic cells, employs them to generate unusually 'hard-wired' calcium signals that do not act to integrate stimuli. 'Leakage' between these calcium signalling pathways will generate inappropriate responses, compromising functioning of the cell.