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

Abstract

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.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm

“…This is in accordance to recent observations by Torrezan-Nitao and coworkers. 52 Previous reports have shown that the presence of a [Ca 2+ ] i oscillatory pattern during P4 stimulation prevented the occurrence of AE in human. 41,53,54 Using the same strategy to monitor both AE and [Ca 2+ ] i , we also found that an oscillatory pattern during a [Ca 2+ ] i response to P4 inhibits AE.…”

The Action of Reproductive Fluids and Contained Steroids, Prostaglandins, and Zn2+ on CatSper Ca2+ Channels in Human Sperm

“…However, it needs to be systematically assessed whether the efficacy of steroids to increase [Ca 2+ ] i and their ability to evoke particular cellular responses indeed correlate. Furthermore, on a single-cell level, progesterone activation of CatSper triggers long-lasting oscillations of [Ca 2+ ] i ( Kirkman-Brown et al, 2004 ; Bedu-Addo et al, 2007 ; Brown et al, 2017 ; Kelly et al, 2018 ; Mata-Martinez et al, 2018 ; Torrezan-Nitao et al, 2021 ) that seem to involve Ca 2+ -induced Ca 2+ release and an interplay of CatSper with Slo3 ( Torrezan-Nitao et al, 2021 ). Whether steroids other than progesterone also trigger such [Ca 2+ ] i oscillations is unknown.…”

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm

“…A major focus of research on the cation channel of sperm (CatSper) has been to understand its impact on fertilization ( Quill et al, 2001 ; Ren et al, 2001 ). Activation of CatSper triggers calcium influx and release from internal stores leading to oscillations in intracellular calcium ([Ca 2+ ] i ) that originate in the flagellum and spread to the neck and head ( Torrezan-Nitao et al, 2021 ). Calcium entry drives hyperactivated motility (HAM) that is required for fertilization ( Quill et al, 2003 ; Qi et al, 2007 ).…”