Rapid sperm capture: high-throughput flagellar waveform analysis.

STUDY QUESTION: Can flagellar analyses be scaled up to provide automated tracking of motile sperm, and does knowledge of the flagellar waveform provide new insight not provided by routine head tracking? SUMMARY ANSWER: High-throughput flagellar waveform tracking and analysis enable measurement of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses, which are not possible by tracking the sperm head alone. WHAT IS KNOWN ALREADY: The clinical gold standard for sperm motility analysis comprises a manual analysis by a trained professional, with existing automated sperm diagnostics [computer-aided sperm analysis (CASA)] relying on tracking the sperm head and extrapolating measures. It is not currently possible with either of these approaches to track the sperm flagellar waveform for large numbers of cells in order to unlock the potential wealth of information enclosed within. STUDY DESIGN, SIZE, DURATION: The software tool in this manuscript has been developed to enable high-throughput, repeatable, accurate and verifiable analysis of the sperm flagellar beat. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the software tool [Flagellar Analysis and Sperm Tracking (FAST)] described in this manuscript, we have analysed 176 experimental microscopy videos and have tracked the head and flagellum of 205 progressive cells in diluted semen (DSM), 119 progressive cells in a high-viscosity medium (HVM) and 42 stuck cells in a low-viscosity medium. Unscreened donors were recruited at Birmingham Women's and Children's NHS Foundation Trust after giving informed consent. MAIN RESULTS AND THE ROLE OF CHANCE: We describe fully automated tracking and analysis of flagellar movement for large cell numbers. The analysis is demonstrated on freely motile cells in low- and high-viscosity fluids and validated on published data of tethered cells undergoing pharmacological hyperactivation. Direct analysis of the flagellar beat reveals that the CASA measure 'beat cross frequency' does not measure beat frequency; attempting to fit a straight line between the two measures gives ${\mathrm{R}}^2$ values of 0.042 and 0.00054 for cells in DSM and HVM, respectively. A new measurement, track centroid speed, is validated as an accurate differentiator of progressive motility. Coupled with fluid mechanics codes, waveform data enable extraction of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses. We provide a powerful and accessible research tool, enabling connection of the mechanical activity of the sperm to its motility and effect on its environment. LARGE SCALE DATA: The FAST software package and all documentation can be downloaded from www.flagellarCapture.com. LIMITATIONS, REASONS FOR CAUTION: The FAST software package has only been tested for use with negative phase contrast microscopy. Other imaging modalities, with bright cells on a dark background, have not been tested but may work. FAST is not designed to analyse raw semen; it is specifically for precise analysis of flagellar kinematics, as that is the promising area for computer use. Flagellar capture will always require that cells are at a dilution where their paths do not frequently cross. WIDER IMPLICATIONS OF THE FINDINGS: Combining tracked flagella with mathematical modelling has the potential to reveal new mechanistic insight. By providing the capability as a free-to-use software package, we hope that this ability to accurately quantify the flagellar waveform in large populations of motile cells will enable an abundant array of diagnostic, toxicological and therapeutic possibilities, as well as creating new opportunities for assessing and treating male subfertility. STUDY FUNDING/COMPETING INTEREST(S): M.T.G., G.C., J.C.K-B. and D.J.S. gratefully acknowledge funding from the Engineering and Physical Sciences Research Council, Healthcare Technologies Challenge Award (Rapid Sperm Capture EP/N021096/1). J.C.K-B. is funded by a National Institute of Health Research (NIHR) and Health Education England, Senior Clinical Lectureship Grant: The role of the human sperm in healthy live birth (NIHRDH-HCS SCL-2014-05-001). This article presents independent research funded in part by the NIHR and Health Education England. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The data for experimental set (2) were funded through a Wellcome Trust-University of Birmingham Value in People Fellowship Bridging Award (E.H.O.).The authors declare no competing interests.

CASA: tracking the past and plotting the future.

The human semen sample carries a wealth of information of varying degrees of accessibility ranging from the traditional visual measures of count and motility to those that need a more computational approach, such as tracking the flagellar waveform. Although computer-aided sperm analysis (CASA) options are becoming more widespread, the gold standard for clinical semen analysis requires trained laboratory staff. In this review we characterise the key attitudes towards the use of CASA and set out areas in which CASA should, and should not, be used and improved. We provide an overview of the current CASA landscape, discussing clinical uses as well as potential areas for the clinical translation of existing research technologies. Finally, we discuss where we see potential for the future of CASA, and how the integration of mathematical modelling and new technologies, such as automated flagellar tracking, may open new doors in clinical semen analysis.

Hyaluronic Acid Binding Sperm Selection for assisted reproduction treatment (HABSelect): study protocol for a multicentre randomised controlled trial.

INTRODUCTION: The selection of a sperm with good genomic integrity is an important consideration for improving intracytoplasmic sperm injection (ICSI) outcome. Current convention selects sperm by vigour and morphology, but preliminary evidence suggests selection based on hyaluronic acid binding may be beneficial. The aim of the Hyaluronic Acid Binding Sperm Selection (HABSelect) trial is to determine the efficacy of hyaluronic acid (HA)-selection of sperm versus conventionally selected sperm prior to ICSI on live birth rate (LBR). The mechanistic aim is to assess whether and how the chromatin state of HA-selected sperm corresponds with clinical outcomes-clinical pregnancy rate (CPR), LBR and pregnancy loss (PL). METHODS AND ANALYSIS: Couples attending UK Centres will be approached, eligibility screening performed and informed consent sought. Randomisation will occur within 24 hours prior to ICSI treatment. Participants will be randomly allocated 1:1 to the intervention arm (physiological intracytoplasmic sperm injection, PICSI) versus the control arm using conventional methods (ICSI). The primary clinical outcome is LBR ≥37 weeks' gestation with the mechanistic study determining LBR's relationship with sperm DNA integrity. Secondary outcomes will determine this for CPR and PL. Only embryologists performing the procedure will be aware of the treatment allocation. Steps will be taken to militate against biases arising from embryologists being non-blinded. Randomisation will use a minimisation algorithm to balance for key prognostic variables. The trial is powered to detect a 5% difference (24-29%: p=0.05) in LBR ≥37 weeks' gestation. Selected residual sperm samples will be tested by one or more assays of DNA integrity. ETHICS AND DISSEMINATION: HABSelect is a UK NIHR-EME funded study (reg no 11/14/34; IRAS REF. 13/YH/0162). The trial was designed in partnership with patient and public involvement to help maximise patient benefits. Trial findings will be reported as per CONSORT guidelines and will be made available in lay language via the trial web site (http://www.habselect.org.uk/). TRIAL REGISTRATION NUMBER: ISRCTN99214271; Pre-results.

2016 Laboratory guidelines for postvasectomy semen analysis: Association of Biomedical Andrologists, the British Andrology Society and the British Association of Urological Surgeons.

Post-vasectomy semen analysis (PVSA) is the procedure used to establish whether sperm are present in the semen following a vasectomy. PVSA is presently carried out by a wide variety of individuals, ranging from doctors and nurses in general practitioner (GP) surgeries to specialist scientists in andrology laboratories, with highly variable results.Key recommendations are that: (1) PVSA should take place a minimum of 12 weeks after surgery and after a minimum of 20 ejaculations. (2) Laboratories should routinely examine samples within 4 h of production if assessing for the presence of sperm. If non-motile sperm are observed, further samples must be examined within 1 h of production. (3) Assessment of a single sample is acceptable to confirm vasectomy success if all recommendations and laboratory methodology are met and no sperm are observed. Clearance can then be given. (4) The level for special clearance should be <100 000/mL non-motile sperm. Special clearance cannot be provided if any motile sperm are observed and should only be given after assessment of two samples in full accordance with the methods contained within these guidelines. Surgeons are responsible both preoperatively and postoperatively for the counselling of patients and their partners regarding complications and the possibility of late recanalisation after clearance. These 2016 guidelines replace the 2002 British Andrology Society (BAS) laboratory guidelines and should be regarded as definitive for the UK in the provision of a quality PVSA service, accredited to ISO 15189:2012, as overseen by the United Kingdom Accreditation Service (UKAS).

The mechanics of hyperactivation in adhered human sperm.

Hyperactivation is an important phenomenon exhibited by mammalian sperm during the process of acquiring fertilization capacity. The majority of studies have focused on incubation-induced hyperactivation in non-human species, which typically differ in size, shape, and are more homogeneous than human sperm. We develop an alternative approach via drug-induction, using high-speed imaging and analysis of same-cell changes in the flagellar movement of adhered cells. Following stimulation with 4-aminopyridine, approximately two-thirds (21 of 34) of the cells analysed exhibited a waveform with a single characteristic frequency; in all cases, the frequency was lower than before stimulation. The remaining cells (13 of 34) exhibited a more complex motility with multiple-frequency modes. The lowest mode in all cases was lower than the frequency prior to stimulation. Flagellar bending increased in all cells following stimulation and was significantly greater in the multiple-frequency responders. Despite the increased bending, time-averaged hydrodynamic power dissipation decreased significantly when assessed across all cells, the effect being significantly greater in the multiple-frequency responders than single frequency. These results reveal the heterogeneity of responses of human sperm to a hyperactivating stimulus, the methodology being potentially useful for assessing dynamic responses to stimuli in human sperm, and physiological selection of cells for assisted reproduction.

Blast injury to the perineum.

Recent military operations have resulted in a small but significant number of military personnel suffering severe perineal injuries. In association with lower limb amputation and pelvic fracture, this complex is described as the 'signature injury' of the current conflict in Afghanistan. There are significant consequences of surviving severe perineal injury but the experience of managing these casualties is limited. This article gives an overview of the processes developed to meet these challenges and introduces a series of articles which examine the subject in finer detail.

Modelling a tethered mammalian sperm cell undergoing hyperactivation.

The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions.

Comment on the article by J. Elgeti, U. B. Kaupp, and G. Gompper: hydrodynamics of sperm cells near surfaces.

A recent study by Elgeti et al. used multiparticle collision dynamics to simulate a long-standing problem: the approach of sperm to surfaces, and subsequent accumulation. The authors highlight differences in their predictions with those of the earlier Stokes flow simulations of Smith et al. attributing the differences to methodological flaws in the earlier article. In this Comment, we discuss the criticisms leveled in detail, and review some recently published work that shows how species-specific details of cell morphology provides a more likely explanation for the differing predictions of the two studies. We also highlight experimental work that supports the study of Smith et al.

Nonlinear instability in flagellar dynamics: a novel modulation mechanism in sperm migration?

Throughout biology, cells and organisms use flagella and cilia to propel fluid and achieve motility. The beating of these organelles, and the corresponding ability to sense, respond to and modulate this beat is central to many processes in health and disease. While the mechanics of flagellum-fluid interaction has been the subject of extensive mathematical studies, these models have been restricted to being geometrically linear or weakly nonlinear, despite the high curvatures observed physiologically. We study the effect of geometrical nonlinearity, focusing on the spermatozoon flagellum. For a wide range of physiologically relevant parameters, the nonlinear model predicts that flagellar compression by the internal forces initiates an effective buckling behaviour, leading to a symmetry-breaking bifurcation that causes profound and complicated changes in the waveform and swimming trajectory, as well as the breakdown of the linear theory. The emergent waveform also induces curved swimming in an otherwise symmetric system, with the swimming trajectory being sensitive to head shape-no signalling or asymmetric forces are required. We conclude that nonlinear models are essential in understanding the flagellar waveform in migratory human sperm; these models will also be invaluable in understanding motile flagella and cilia in other systems.

Bend propagation in the flagella of migrating human sperm, and its modulation by viscosity.

A pre-requisite for sexual reproduction is successful unification of the male and female gametes; in externally-fertilising echinoderms the male gamete is brought into close proximity to the female gamete through chemotaxis, the associated signalling and flagellar beat changes being elegantly characterised in several species. In the human, sperm traverse a relatively high-viscosity mucus coating the tract surfaces, there being a tantalising possible role for chemotaxis. To understand human sperm migration and guidance, studies must therefore employ similar viscous in vitro environments. High frame rate digital imaging is used for the first time to characterise the flagellar movement of migrating sperm in low and high viscosities. While qualitative features have been reported previously, we show in precise spatial and temporal detail waveform evolution along the flagellum. In low viscosity the flagellum continuously moves out of the focal plane, compromising the measurement of true curvature, nonetheless the presence of torsion can be inferred. In high viscosities curvature can be accurately determined and we show how waves propagate at approximately constant speed. Progressing waves increase in curvature approximately linearly except for a sharper increase over a distance approximately 20-27 microm from the head/midpiece junction. Curvature modulation, likely influenced by the outer dense fibres, creates the characteristic waveforms of high viscosity swimming, with remarkably effective cell progression against greatly increased resistance, even in high viscosity liquids. Assessment of motility in physiological viscosities will be essential in future basic research, studies of chemotaxis and novel diagnostics.

Pregnancy and perinatal outcomes after assisted reproduction: a comparative study.

BACKGROUND: Increasing use of fertility therapy has elicited concerns regarding adverse effects for expectant mothers and the health of children thus conceived. AIMS: To study the risk of adverse perinatal outcomes, birth defects and pregnancy complications following assisted reproductive technology (ART). METHODS: Questionnaire-based study involving 1,524 children and 1,182 pregnancies conceived following in vitro fertilisation (IVF) in two units. Outcomes were compared with the general population. RESULTS: In the study group versus the general population; multi-foetal gestations, 26 versus 2%; singleton preterm delivery and low birth weight, 8.7 and 6.4 versus 4.3 and 4%, respectively; non-lethal congenital malformation rate, 2.6 versus 2.1%; placenta praevia, 2.8 versus 0.5%. CONCLUSIONS: Multi-foetal gestations remain the principal cause of adverse perinatal outcomes after ART. Singleton ART pregnancies have an increased risk of preterm delivery and low birth weight at term. Non-lethal congenital malformation rates are not increased following ART. Placenta praevia is increased following ART.

Understanding the physiology of pre-fertilisation events in the human spermatozoa--a necessary prerequisite to developing rational therapy.

Sperm dysfunction is the single most common defined cause of infertility. One in 15 men is sub-fertile and the condition is increasing in frequency. However, the diagnosis is poor and, excluding assisted conception, there is no treatment. The reason for this is our limited understanding of the biochemical, molecular and genetic functions of the spermatozoon. The underlying premise of our research programme is to establish a rudimentary understanding of the processes necessary for successful fertilisation. In this manuscript, we detail advances in our understanding of calcium signalling in the cell and outline genetic and proteomic technologies that are being used to improve the diagnosis of the condition.

Patch-clamp 'mapping' of ion channel activity in human sperm reveals regionalisation and co-localisation into mixed clusters.

Ion channels are pivotal to many aspects of sperm physiology and function. We have used the patch clamp technique to investigate the distribution of ion channels in the plasma membrane of the head of human spermatozoa. We report that three types of activity are common in the equatorial and acrosomal regions of the sperm head. Two of these (a chloride-permeable anion channel showing long stable openings and a second channel which flickered between open and closed states and was dependent upon cytoplasmic factors for activity) were localised primarily to the equatorial segment. A third type, closely resembling the flickering activity but with different voltage sensitivity of P(open), was more widely distributed but was not detectable over the anterior acrosome. In the anterior acrosomal area channels were present but showed very low levels of spontaneous activity. A unique feature of channel activity in the sperm equatorial region was co-localisation into mixed clusters, most patches were devoid of activity but 'active' patches typically contained two or more types of activity (in a single 200-300 nM diameter patch). We conclude that ion channels in the sperm membrane show regionalisation of type and activity and that the channels are clustered into functional groups, possibly interacting through local effects on membrane potential.

Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone.

Human spermatozoa stimulated with progesterone (a product of the cumulus and thus encountered by sperm prior to fertilization in vivo) apparently mobilize Ca(2+) and respond very differently according to the way in which the steroid is presented. A progesterone concentration ramp (0-3 microM) induces [Ca(2+)](i) oscillations (repetitive store mobilization) which modify flagellar beating, whereas bolus application of micromolar progesterone causes a single large transient (causing acrosome reaction) which is apparently dependent upon Ca(2+) influx. We have investigated Ca(2+)-mobilization and functional responses in human sperm exposed to 3 muM progesterone. The [Ca(2+)](i) response to progesterone was abolished by 4 min incubation in 0 Ca(2+) medium (2 mM EGTA) but in nominally Ca(2+)-free medium (no added Ca(2+); 0 EGTA) a smaller, slow response occurred. Single cell imaging showed a similar effect of nominally Ca(2+)-free medium and approximately 5% of cells generated a small transient even in the presence of EGTA. When cells were exposed to EGTA-containing saline (5 min) and then returned to nominally Ca(2+)-free medium before stimulation, the [Ca(2+)](i) transient was greatly delayed (approximately 50 s) and rise time was doubled in comparison to cells not subjected to EGTA pre-treatment. We conclude that mobilization of stored Ca(2+) contributes a 'slow' component to the progesterone-induced [Ca(2+)](i) transient and that incubation in EGTA-buffered saline is able rapidly to deplete this store. Analysis of flagellar activity induced by 3 muM progesterone showed an effect (modified beating) associated with the [Ca(2+)](i) transient, in >80% of cells bathed in nominally Ca(2+)-free medium. This was reduced greatly in cells subjected to 5 min EGTA pre-treatment. The store-mediated transient showed a pharmacological sensitivity similar to that of progesterone-induced [Ca(2+)](i) oscillations (consistent with filling of the store by an SPCA) suggesting that the transient induced by micromolar progesterone is a 'single shot' activation of the same store that generates Ca(2+) oscillations.

Development of a novel home sperm test.

BACKGROUND: The majority of men find the production of a semen sample an embarrassing and stressful experience. Consequently, the availability of an over-the-counter home sperm test, which would reliably and accurately allow the patient to obtain an assessment of fertility potential at their convenience, would be a major benefit. Our objective was to develop and evaluate a home sperm test that provides a visual estimate of the concentration of progressively motile sperm in a semen sample. METHODS: Three particular challenges are described (i) developing a visualization system; (ii) optimization of the detection limit; and (iii) controlling variation due to changes in ambient temperature. The accuracy of the device was tested against two reference methods: computer-assisted sperm analysis (CASA) and a hyaluronate migration test (HMT). RESULTS: In 129 semen samples, where both reference methods agreed (positive or negative), the accuracy of the device was 95%. The observed likelihood ratio of 8.8 indicated that a sample showing a red line in the device was over eight times more likely to have a positive (normal) result in CASA and HMT than a sample without a red line. CONCLUSIONS: The final device provides a visual estimate of the concentration of progressively motile sperm in a semen sample using a test that is completed within approximately 1 h of production of the sample and can be used by the man in the comfort of his own home.

Multi-state, 4-aminopyridine-sensitive ion channels in human spermatozoa.

Although ion channels are known to be pivotal to sperm function, the technical difficulty of applying electrophysiological techniques to spermatozoa has prevented significant progress in this area. This is due to the cell size and angular shape in combination with their motility. Using a refined technique, specifically for patch clamping spermatozoa, we have made recordings from human cells. This technique permitted approaches which enable functional analysis of sperm ion channels, including acquisition of inside-out patches, generation of averaged currents, and observation of the effects of pharmacological manipulation during prolonged recordings. As well as a low conductance (7 pS) channel and a 25-pS channel, the most striking finding was the presence of very high conductance, 4-aminopyridine-sensitive multistate channels resembling the non-selective cation channel of sea urchin and mouse spermatozoa. Application of 2 mM 4-aminopyridine (a dose sufficient to cause channel blockade) caused an instant and dramatic transition of motility in the sperm population increasing hyperactivated motility by more than 10-fold as assessed by computer-assisted semen analysis. Combined application of patch clamp and pharmacological investigation of mature sperm cells and will permit rapid advances in our understanding the role of ion channels in sperm function.

Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa.

The steroid progesterone, an agonist of acrosome reaction, induces a biphasic [Ca(2+)](i)-signal in human sperm comprising an initial transient [Ca(2+)](i) elevation, and a subsequent ramp or plateau. Nifedipine, an inhibitor of voltage-operated Ca(2+) channels, inhibits progesterone-induced acrosome reaction in human sperm, but fluorimetric studies have detected no effect of this compound on the progesterone-induced [Ca(2+)](i) signal. We have used single-cell imaging to study the effects of nifedipine on [Ca(2+)](i) signalling in human sperm. Analysis of mean responses from large numbers of cells showed that treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced [Ca(2+)](i) transient. In control cells, the latency of the transient peak (maximum fluorescence) fell within the range of 30-105 s. In the presence of nifedipine, very few cells peaked "late" (>60 s after application of progesterone). Analysis of transient responses in control cells revealed characteristic "early" and "late" responses, most cells showing both "early" and "late" transients, whereas "late" transients were rare and smaller in the presence of nifedipine. Sustained responses showed strong association with late transients, and occurrence and amplitude of sustained responses were significantly reduced in nifedipine pretreated cells. These findings are consistent with the occurrence of a discrete, nifedipine-sensitive component of the progesterone-induced [Ca(2+)](i) transient that peaks 1-2 min after exposure to the hormone and is crucial for the induction of the sustained [Ca(2+)](i) signal.

Inhibitors of receptor tyrosine kinases do not suppress progesterone-induced [Ca2+]i signalling in human spermatozoa.

Previous studies have implicated receptor tyrosine kinases in progesterone-induced [Ca2+]i signalling, and consequent induction of the acrosome reaction, in human spermatozoa. We have investigated the effects of tyrosine kinase inhibition on [Ca2+]i responses in large numbers of individual human spermatozoa. Genistein (5, 50 and 250 micromol/l), an inhibitor of receptor-linked tyrosine kinases, significantly inhibited the progesterone-induced acrosome reaction (P < 0.05). However, we could detect no effect of genistein on progesterone-induced [Ca2+]i signalling. In control experiments, application of progesterone induced a significant transient [Ca2+]i response in approximately 77% of cells and a sustained [Ca2+]i ramp/plateau in approximately 48% of cells (n = 26; 5411 cells). In preparations pretreated with genistein (50 micromol/l), significant transient and sustained responses were detected in 69.5 and 39.1% of cells respectively (n = 5; 1109 cells). The amplitudes of both transient and sustained [Ca2+]i responses were similar in control and genistein-pretreated preparations. Tyrphostin A47 (100 micromol/l), another receptor tyrosine kinase inhibitor, also failed to inhibit either the transient or sustained [Ca2+]i response (n = 3; 468 cells). Assessment of tyrosine phosphorylation of two sperm proteins (p105/81) showed greatly increased levels of phosphotyrosine in response to capacitation but a negligible increase in response to progesterone stimulation. Pretreatment with genistein (50 and 250 micromol/l) decreased capacitation-induced tyrosine phosphorylation and resulted in a loss of phosphorylation in response to progesterone treatment. We conclude that neither the transient nor sustained phases of the progesterone-induced [Ca2+]i response require receptor tyrosine kinase signalling. Previous reports of modulation of the progesterone-induced [Ca2+]i signal by tyrosine kinase inhibition probably reflect inhibition of the acrosome reaction.

Biphasic elevation of [Ca(2+)](i) in individual human spermatozoa exposed to progesterone.

Fluorimetric studies on progesterone-induced [Ca(2+)](i) signalling in mammalian spermatozoa show both the well-characterised [Ca(2+)](i) transient and a subsequent sustained phase. However, the sustained phase is thought to reflect release of the fluorochrome during the acrosome reaction and has not been subject to critical investigation. We have used single-cell imaging of [Ca(2+)](i) to analyse the progesterone-induced [Ca(2+)](i) response in large numbers (>2000) of capacitated, human spermatozoa. In 70% of cells, treatment with progesterone induced a transient increase, which typically peaked within 1 min and decayed with a similar time course. Upon rapid application of progesterone this response peaked within 5-20 s. In 35% of progesterone-treated spermatozoa a sustained elevation of [Ca(2+)](i) occurred, which became discernible during the falling phase of the transient response and persisted for at least 20 min. Both [Ca(2+)](i) responses were localised to the postacrosomal region. Averaging of large numbers of single cell responses generated traces similar to those seen in fluorimetric studies. Although the sustained response was strongly associated with the initial, transient response, a few spermatozoa generated sustained responses that were not preceded by a significant transient response (5% of cells). It is concluded that a genuine biphasic [Ca(2+)](i) signal is activated by progesterone and that the sustained response is a discrete signalling event with biological significance.