Epac activation induces an extracellular Ca²⁺‐independent Ca²⁺ wave that triggers acrosome reaction in human spermatozoa

Abstract

Background:The signaling pathways of the intracellular second messengers cAMP and Ca 2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca 2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca 2+ influx but also by a release from intracellular Ca 2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca 2+ ] i and in the generation of complex Ca 2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca 2+ release involved in the AR. Objective: To identify the molecular entities involved in the Ca 2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. Materials and Methods:In capacitated human spermatozoa, we monitored Ca 2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca 2+ -free medium.Results: Epac activation by 2'-O-Me-cAMP induced a Ca 2+ wave that started in the midpiece and propagated to the acrosome region. This Ca 2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na + /Ca 2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca 2+ -ATPases).Discussion: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca 2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. Conclusion:The Ca 2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

3D structure and in situ arrangements of CatSper channel in the sperm flagellum

“…4 ). The CatSper-mediated increase in intracellular Ca 2+ is important for triggering hyperactivated motility during capacitation 34 – 36 . During this process, wild type mammalian sperm dramatically increased the xy -displacement of their flagellum, i.e., in the out-of-plane beating direction (Fig.…”

“…5c ). In contrast, disruption of the CatSper zigzag-rows and misalignment of short CatSper clusters from the longitudinal axis would dysregulate this domino-effect of Ca 2+ entry, and thus would impair maintenance of homeostasis and an efficient propagation of the intracellular Ca 2+ wave 35 , 36 , 38 , resulting in a proximally stiff flagellum and altered sperm motility (Supplementary Fig. 5c ).…”

Two‐pore channel 1 and Ca2+ release‐activated Ca2+ channels contribute to the acrosomal pH‐dependent intracellular Ca2+ increase in mouse sperm

“…To identify the molecular entities, cells were incubated with Ca 2+ channel inhibitors for 5 min prior to the addition of 30 μM Mib or 10 μM NNC. The contribution of inositol 1,4,5‐trisphosphate receptors (IP 3 Rs) was evaluated by pretreatment with membrane‐permeable IP 3 R inhibitors, Xc (2 μM, selective inhibitor) (Mata‐Martinez et al., 2021) and 2‐APB (100 μM, non‐selective inhibitor) (Gambardella et al., 2021) using a low extracellular (100 nM) Ca 2+ recording medium. To address the role of TPC1 channels, sperm were treated with the membrane‐permeant TPC inhibitor t Ned‐19 (100 μM, selective inhibitor) (Arndt et al., 2014) using a recording medium with 100 nM Ca 2+ .…”

Common methods in mitochondrial research (Review)

“…The fluorescence intensity of Fluo-3 combined with Ca 2+ is ~40 times higher than that of free cells, thus avoiding the fluorescence interference of the cells themselves (185,187). As a long-wavelength indicator, Fluo-3 can be used in confocal laser imaging studies that can analyze the distribution of ca 2+ in individual intact living cells and distinguish mitochondrial ca 2+ from ca 2+ in other organelles within the cell; this method is suitable for mitochondrial ca 2+ in various living cells and is easy to operate, stable in performance and highly specific (155,187). However, the current mitochondrial Ca 2+ fluorescent probes cannot distinguish mitochondrial Ca 2+ from different cells.…”