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)-sensitive Ca2+-influx through CatSper channels. WHAT IS KNOWN ALREADY A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although 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 1000 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. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight 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 the 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 in 36% and 96% of oscillating cells, respectively (P < 10−10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations. LARGE SCALE DATA N/A LIMITATIONS, 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 regulation and generation (or failure) of these oscillations. STUDY FUNDING/COMPETING INTEREST(S) E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest.

Mitochondrial permeability transition pore (MPTP) is a protein complex whose opening promotes an abrupt increase in mitochondrial inner membrane permeability. Calcium signaling pathways are described in gametes and are involved in the fertilization process. Although mitochondria may act as Ca(2+) store and have a fast calcium-releasing mechanism through MPTP, its contribution to fertilization remains unclear. The work aimed to investigate the MPTP phenomenon in sea urchin spermatozoa and its role on the fertilization. Several pharmacological tools were used to evaluate the MPTP's physiology. Our results demonstrated that MPTP occurs in male gametes in a Ca(2+) - and voltage-dependent manner and it is sensitive to cyclosporine A. Additionally, our data show that MPTP opening does not alter ROS generation in sperm cells. Inhibition of MPTP in spermatozoa strongly improved the fertilization rate, which may involve mechanisms that increase the spermatozoa lifespan. The present work is the first report of the presence of a voltage- and Ca(2+) -dependent MPTP in gametes of invertebrates and indicates MPTP opening as another evolutionary feature shared by sea urchins and mammals. Studies about MPTP in sea urchin male gametes may contribute to the elucidation of several mechanisms involved in sperm infertility.

Mitochondrial permeability transition pore (MPTP) has been associated to calcium homeostasis and reactive oxygen species (ROS) generation in several cell types. While extensively investigated in somatic cells, there are few data regarding MPTP phenomenon in gametes. The aim of the present work was to investigate MPTP occurrence in sea urchin female gametes. The protonophores CCCP and FCCP, and the Ca ionophore ionomycin, were used as pore inductors. Pore opening was monitored by mitochondrial potential sensitive probes and cobalt-quenched calcein assay. The pore desensitizer cyclosporin A (CsA) prevented the loss of mitochondrial inner membrane potential (ΔΨ) and pore opening induced by MPTP activators. The disruption of ΔΨ led to an increase in ROS generation, which was completely prevented by CsA. Our data also demonstrated that the increase in ROS production induced by MPTP opening requires extracellular Ca. In summary, the current study provides evidence about the occurrence of MPTP in sea urchin eggs in a similar manner as described in vertebrate somatic cells - CsA-sensitive, voltage- and Ca-triggered - and shows MPTP as a highly conserved physiological event through the evolution.

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 > 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 > 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 emphasises that the drug does not fully mimic the actions of P4.

Repetitive [Ca2+]i transients (oscillations), observed in progesterone (P4)-stimulated immobilised human sperm, reversibly modifiy flagellar beating. Here we report that such oscillations also occur in P4-stimulated free-swimming sperm and regulate (or contribute to) behavioural switching.