Intracellular Ca 2C stores play a central role in the regulation of cellular [Ca 2C ] i and the generation of complex [Ca 2C ] signals such as oscillations and waves. Ca 2C 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 Ca 2C 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 Ca 2C stores of somatic cells and ii) the evidence for the existence of functional Ca 2C 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 Ca 2C at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.Reproduction (

Background: Ca2+ signals, elicited by cues from the oocyte and female tract, regulate human sperm behavior.Results: CatSper channel activation (flagellum) and Ca2+ store mobilization (neck) caused similar [Ca2+]i elevation but induced functionally different behaviors.Conclusion: Sperm motility pattern is determined by the site of Ca2+ mobilization.Significance: Selection of Ca2+ signaling components and/or regulation of their availability for activation controls human sperm behavior.

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.

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.

STUDY QUESTIONDoes 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 ANSWERhFF 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 ALREADYCatSper, 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, DURATIONThis 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, METHODSSemen 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 CHANCEhFF 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 DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis was an in vitro study. Caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis 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 modulat...

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...

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. Video LinkThe video component of this article can be found at http://www.jove.com/video/1996/ ProtocolSperm from healthy fertile males, with a normal semen analysis, are normally prepared for imaging as follows. .8 Na lactate) supplemented with 0.3% charcoal de-lipidated/fatty acid free Fraction V BSA (quality of the BSA is crucial for successful capacitation of sperm). 1 ml of sEBBS is pipetted into each of a series of 5 ml tubes and gently underlayered with 0.3 ml of semen. After incubation for 1 hour (37°C; 6% CO 2 ) the top 0.7 ml is gently removed from each tube and pooled. 10 μl of the sperm suspension is diluted with 90 μl of 1% (v/v) formalin to immobilize the cells, then sperm are counted in a Neubauer chamber. Cell density in the suspension is then adjusted (with sEBSS) to 6 million cells/ml. 2. The sample is then divided into aliquots of 200 μl in loosely-capped tubes and incubated (37°C; 6% CO 2 ) in for 5-6 h to allow capacitation. 3. Coverslips (22x50 mm) have previously been treated with poly-D-lysine. 10 μl of poly-D-lysine solution (10% w/v) is applied as a number of small drops to the centre of the coverslip. The poly-D-lysine is then allowed to air dry. This can be on a heated stage and should be to complete dryness. A coverslip is attached with vacuum grease to an enclosed, purpose-built, perfusable, polycarbonate imaging chamber (dimensions 35 mm x 20 mm x 5 mm; capacity ≈ 180 μl) similar to the Warner RC20 chamber .The poly-D-lysine-coated coverslip forms the base ...

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