Progesterone-induced [ca2+]i oscillations and regulation of human sperm behaviour

Ca2+ signalling is crucial to modulate sperm motility and changes in the intracellular calcium concentration [Ca2+]i may underlie ‘switching’ of sperm swimming behaviour in human spermatozoa, which is important for sperm progression in the female tract. I investigated the mechanism by which progesterone induces [Ca2+]i oscillations and also investigated the role of [Ca2+]i oscillations for sperm swimming behaviour and penetration ability into mucus. Sperm were loaded with Ca2+-indicator fluo4/AM, treated with different agonists and analysed in single live-cell imaging and functional assays were also performed in free-swimming sperm. Approximately 25% of the human sperm population exhibit [Ca2+]i oscillations, independently of sperm capacitation. [Ca2+]i rise first in flagellum then spread actively to the head, apparently triggering CICR. Membrane potential (Vm), Slo3 channels and CatSper channels contribute to [Ca2+]i oscillations generation. [Ca2+]i oscillations occur in free-swimming spermatozoa and are associated with switching of sperm swimming behaviour in both low and high viscosity environments: drives velocity, sperm turning, hyperactivated motility and regulates attachment/detachment from substrate. Consistent with the involvement of CatSper in generation of [Ca2+]i oscillations, regulation of sperm swimming velocity and cell progression are dependent on CatSper channels. Additionally, sperm ability to penetrate into mucus environment is also dependent on CatSper activity

Behavioural switching during oscillations of intracellular Ca²⁺ concentration in free-swimming human sperm

A human sperm must swim to the egg to fertilise it. To do this the sperm uses different types of swimming (behaviours) as they are needed. When we watch sperm swimming we see that they regularly change behaviour, sometimes repeatedly switching between two different types. Calcium ions inside cells are crucial in controlling many cell functions and in sperm they play a key role in regulating their behaviour. Here we have measured the concentration of calcium ions inside swimming human sperm. We found that in 12/35 (34%) of the cells we assessed, the concentration of calcium changed repeatedly, averaging more than one cycle of rise and fall per minute. These changes in the concentration of calcium ions occurred as the sperm switched swimming stroke, suggesting that oscillation of calcium concentration is involved in controlling the switching of sperm behaviour. Impaired sperm motility is an important cause of subfertility in men. Understanding how sperm behaviour is controlled will allow the development of treatments that can rescue the fertility of sperm with impaired motility.Fil: Torrezan Nitao, Elis. University of Birmingham; Reino UnidoFil: Guidobaldi, Héctor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Giojalas, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Barratt, Christopher. University of Dundee; Reino UnidoFil: Publicover, Stephen. University of Birmingham; Reino Unid

[Ca2+]_i oscillations in human sperm are triggered in the flagellum by membrane potential- sensitive activity of CatSper

Study question: How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated?Summary answer: P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane-potential (Vm)-dependent Ca2+-influx through CatSper channels, which then induce secondary Ca2+ mobilisation at the sperm head/neck region.What is known already: A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Though pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required.Study design, size, duration: This was a laboratory study, that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1,000 individual cells over a period of 2 years.Participants/materials, setting, methods: Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp.Main results and the role of chance: P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1-2 s). K+-ionophore valinomycin (1 μM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly ‘reset’ upon manipulation of [K+]o. Pretreatment of sperm with valinomycin ([K+]o=5.4 mM) had no effect on the P4-induced [Ca2+] transient (P=0.95; 8 experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n=257; P=5*10-55 compared to control) and significantly reduced both amplitude and frequency of persisting oscillations (p=0.0001). Upon valinomycin washout oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+] the inhibitory effect of valinomycin was reduced and was dependent on EK (P=10-25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P<0.01). The CatSper inhibitor RU1968 (4.8 and 11 μM) caused immediate and reversible arrest of activity in36% and 96% of oscillating cells respectively (P<10-10). 300 μM quinidine which blocks the sperm K+ current (Ksper) completely inhibited [Ca2+]i oscillations.Large scale data: n/aLimitations, reasons for caution: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm.Wider implications of the findings: [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the generation (or failure) and regulation of these oscillations.Study funding/competing interest(s): ET was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). The authors have no conflicts of interest

SKF96365 modulates activity of CatSper channels in human sperm

Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in &gt;80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in &gt;95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.</p

ATP synthase: evolution, energetics, and membrane interactions

The synthesis of ATP, life's 'universal energy currency', is the most prevalent chemical reaction in biological systems, and is responsible for fueling nearly all cellular processes, from nerve impulse propagation to DNA synthesis. ATP synthases, the family of enzymes that carry out this endless task, are nearly as ubiquitous as the energy-laden molecule they are responsible for making. The F-type ATP synthase (F-ATPase) is found in every domain of life, and is believed to predate the divergence of these lineages over 1.5 billion years ago. These enzymes have therefore facilitated the survival of organisms in a wide range of habitats, ranging from the deep-sea thermal vents to the human intestine. In this review, we present an overview of the current knowledge of the structure and function of F-type ATPases, highlighting several adaptations that have been characterized across taxa. We emphasize the importance of studying these features within the context of the enzyme's particular lipid environment: Just as the interactions between an organism and its physical environment shape its evolutionary trajectory, ATPases are impacted by the membranes within which they reside. We argue that a comprehensive understanding of the structure, function, and evolution of membrane proteins -- including ATP synthase -- requires such an integrative approach.Comment: Review article; 29 pages, 6 figures/1 tabl