Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction

Abstract

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

Unexplained infertility is frequently caused by defective CatSper function preventing sperm from penetrating the egg coat

“…8B), confirming that it is not per se affected by loss of CatSper function (Luo et al ., 2019); see however (Wang et al ., 2021b). To elucidate the role of Ca 2+ influx via CatSper, we studied the action of progesterone, which in most (Harper et al ., 2006; Krausz et al ., 1996; Luo et al ., 2019; Rahban et al ., 2021; Rehfeld et al ., 2020; Rennhack et al ., 2018; Schaefer et al ., 1998; Tamburrino et al ., 2014) but not all (Prajapati et al ., 2022; Uhler et al ., 1992) previous studies sufficed as a physiological stimulus for acrosomal exocytosis and was abolished in sperm lacking functional CatSper (Luo et al ., 2019). Under the experimental conditions used here, the fraction of acrosome reacted control sperm was not significantly enhanced by progesterone (Fig.…”

“…Thus, studies mimicking in vitro the complex chemical, topographical, and hydrodynamic landscapes of the female genital tract (Kirkman- Brown & Smith, 2011;Suarez, 2008;Suarez & Pacey, 2006) are required to gain further insights into the role of CatSper for sperm migration and navigation in vivo. Future studies are also required to understand the role of Ca 2+ influx via CatSper for acrosomal exocytosis using, e.g., physiological combinations of ligands and/or intracellular alkalization, which is more efficacious than progesterone alone in stimulating Ca 2+ influx via CatSper (Brenker et al, 2018a;Prajapati et al, 2022) and acrosomal exocytosis (Prajapati et al, 2022;Schaefer et al, 1998). Finally, whether impaired CatSper function like in patient C9 also affects sperm function and male fertility remains to be determined.…”

Sperm Toolbox – A selection of small molecules to study human spermatozoa

“…Both modes are capable of measuring induction of the acrosome reaction. However, in the acrosome 2 assay, a physiological agonist mix of the acrosome reaction is used thus we also also measures inhibition of acrosome reaction (Prajapati et al, 2022).…”

“…For experiments in capacitating conditions, we used a buffer system slightly modified from the HTF from (Prajapati et al, 2022) to a final composition of 97.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 0.33 mM Na-pyruvate, 12.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, 25 mM NaHCO3 and 3 mg/mL BSA. All buffer components were supplied by Sigma-Aldrich unless otherwise stated and all buffer systems were adjusted to pH 7.4.…”

SKF96365 modulates activity of CatSper channels in human sperm

“…Intracellular Ca 2+ concentration ([Ca 2+ ] i ) signalling is crucial to sperm function, being a key regulator of motility, acrosome reaction, and capacitation ( Darszon et al , 2005 ; Publicover et al , 2008 ; Prajapati et al , 2022 ). A number of stimuli, including zona pellucida (ZP), are believed to regulate human sperm function by this pathway, the best characterized being the steroid progesterone (P4), which is secreted by the cells of the cumulus and is present throughout the female tract ( Correia et al , 2007 ).…”