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In JoVE (1)
Other Publications (8)
Articles by Jackson C. Kirkman-Brown in JoVE
Techniques for Imaging Ca2+ Signaling in Human Sperm
Katherine Nash1, Linda Lefievre2, Ruben Peralta-Arias1, Jennifer Morris1, Aduen Morales-Garcia1, Tom Connolly2, Sarah Costello1, Jackson C. Kirkman-Brown3, Stephen J. Publicover1
1School of Biosciences, University of Birmingham, 2School of Medicine, University of Birmingham, 3Centre for Human Reproductive Science, Birmingham Women’s Hospital
Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.
Other articles by Jackson C. Kirkman-Brown on PubMed
Journal of Andrology. May-Jun, 2002 | Pubmed ID: 12002428
The Biochemical Journal. Jun, 2003 | Pubmed ID: 12614198
Progesterone induces a biphasic Ca(2+) influx and consequent acrosome reaction in human spermatozoa. We have used two procedures to vary the stimulus (dosage and prior receptor desensitization) to investigate the encoding of stimulus strength by intracellular [Ca(2+)] ([Ca(2+)](i)). Acrosome reaction and amplitude (but not kinetics) of the transient [Ca(2+)](i) response (population measurement) showed sigmoidal dose sensitivity over the range 0.3 nM-3 microM, saturating at approximately 300 nM (ED(50) approximately 30 nM). The amplitude of the sustained response saturated at 3 microM. Single-cell imaging showed that the amplitudes of both transient and sustained [Ca(2+)](i) responses were highly dose-dependent, but that their frequency of occurrence and kinetics were largely dose-independent. Fluorimetric measurements confirmed that progesterone-induced [Ca(2+)](i) influx was subject to desensitization, with second and subsequent applications of 3 microM progesterone being ineffective. However, sequential additions of 3 nM, 30 nM and 3 microM progesterone generated transient [Ca(2+)](i) responses at each concentration, the amplitude and duration of the response to 3 microM progesterone being reduced compared with non-pretreated cells. Single-cell imaging revealed that pretreatment had no effect on the proportion of responsive cells, but single-cell responses, similarly to population responses, were smaller and markedly reduced in duration, consistent with an effect of desensitization on a late component of the [Ca(2+)](i) transient. We conclude that the strength of the progesterone stimulus, when varied by dosage or by desensitization, is encoded by an analogue [Ca(2+)](i) signal. Dose dependency of the acrosome reaction is therefore determined not by the number of progesterone-responsive cells but by variation in the probability of exocytosis in a 'constant' responsive population.
The Biochemical Journal. Mar, 2004 | Pubmed ID: 14606954
We have used single-cell imaging to investigate intracellular Ca2+ signalling in human spermatozoa stimulated with progesterone (3 microM). In approx. 9% of cells progesterone caused the activation of slow repetitive [Ca2+]i (intracellular Ca2+ concentration) oscillations, with a period of 1-4 min, which persisted for the duration of recording (20-30 min). Pretreatment with nifedipine, which blocks T- and L-type voltage-operated Ca2+ channels in spermatogenic cells, did not modify the characteristics of the oscillations, but reduced the proportion of cells in which they were observed. Stimulation with Bay K 8644 or FPL64176 induced [Ca2+]i oscillations in 5-10% of cells, but their frequency was low (period, 4-5 min). Application of valinomycin (1 microM) to clamp membrane potential at E(K) (equilibrium potential for potassium) did not modify activity in oscillating cells, showing that plasma membrane potential and activation of voltage-operated conductances are not involved in the mechanism by which sperm [Ca2+]i oscillations are generated.
Kinetics of the Progesterone-induced Acrosome Reaction and Its Relation to Intracellular Calcium Responses in Individual Human Spermatozoa
Biology of Reproduction. Dec, 2006 | Pubmed ID: 16957023
Progesterone at 3 microM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca(2+)](i)) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca(2+)](i) signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca(2+)](i) signal is not the critical determinant of AR. Chelation of extracellular calcium to arrest AR at varying times after progesterone stimulation revealed that maximal AR occurred immediately following progesterone stimulation, during the initial transient calcium influx rather than during the sustained calcium response. Attempts to follow acrosomal dispersal in real-time by staining with the acidic organelle probes LysoTracker DND-99 and dapoxyl (2-aminoethyl) sulphonamide (DAES) proved inconclusive due to heterogeneous labeling of the cell population. Surprisingly, the dye was often not confined to the acrosome but stained the whole sperm head, which suggests that only a subpopulation of human sperm cells contains a sufficiently acidic acrosome.
Seminars in Reproductive Medicine. May, 2007 | Pubmed ID: 17447210
As sperm traverse the female tract from vagina to oocyte, they experience a steroid milieu, which due to transcriptional inactivity, they can only respond to via non-genomic signaling. This environment mediates events including capacitation, changes in motility patterns, chemotaxis, and acrosome reaction. Current knowledge of the events, calcium signaling pathways, and potential identity of receptors involved is reviewed in light of recent data, with a context for further work in the field, and emphasizing the importance of steroids as a mixed stimulant. Progesterone receptor candidates are considered in light of recent findings, including novel classes of receptors such as a progesterone membrane receptor component-1 or -2 complex with serpine-1 mRNA binding protein, the best candidate so far for progesterone activity in human sperm. Given the number of other alternative candidates and the apparent diversity of the signaling pathways activated, the presence of multiple species of progesterone receptors should not be excluded. Given that sperm dysfunction is the most common defined cause of infertility, advances in our currently limited knowledge of these pathways and events are crucial to not only create better therapies but also improve rational diagnosis.
Reproduction (Cambridge, England). Apr, 2007 | Pubmed ID: 17504912
Although sperm dysfunction is the single most common cause of infertility, we have poor methods of diagnosis and surprisingly no effective treatment (excluding assisted reproductive technology). In this review, we challenge the usefulness of a basic semen analysis and argue that a new paradigm is required immediately. We discuss the use of at-home screening to potentially improve the diagnosis of the male and to streamline the management of the sub-fertile couple. Additionally, we outline the recent progress in the field, for example, in proteomics, which will allow the development of new biomarkers of sperm function. This new knowledge will transform our understanding of the spermatozoon as a machine and is likely to lead to non-ART treatments for men with sperm dysfunction.
Molecular Human Reproduction. Aug, 2011 | Pubmed ID: 21653751
The success of internal fertilization is reliant upon successful sperm migration through the female tract. Timely location of the oocyte in what is a complex three-dimensional, highly invaginated series of moist opposed surfaces is a challenge at which only tens of sperm ever succeed. In part this could be due to the differences in scale, with a 50 µm long cell facing a probable migration of well over 20 cm due to the complex architecture. Many groups have focused upon the role for a chemotactic 'attractive egg' effect in guiding sperm to increase numbers at the fertilization site. What most research has neglected to consider is the role that the viscosity of the mucous layers, which coat the entire tract and through which sperm must swim, plays in both sperm selection and ongoing modulation of their behaviour. From allowing sperm to enter through the cervix during the ovulation phase, to denying them entrance through action of the female contraceptive pill, viscous effects are fundamental in controlling the migrating sperm population throughout the tract. The physiological effects of viscosity are also crucial to consider when designing and extrapolating data from in vitro experiments to the in vivo situation.