Acrosome Reaction and Ca2+ Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2+]i1

Abstract

The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²⁺ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²⁺ indicator. These AR and intracellular Ca²⁺ ([Ca²⁺]i) recordings revealed that the presence of [Ca²⁺]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²⁺]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²⁺]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²⁺]i.

Veratridine-sensitive Na + channels regulate human sperm fertilization capacity

“…A recent report has shown that stimulation of AR by progesterone in mouse sperm is absolutely dependent on a specific P4-induced transitory increase in [Ca 2+ ]i [37]. Moreover, alterations of the P4-induced [Ca 2+ ]i signal prevent the AR in human spermatozoa [38]. Thus, to investigate further the role of veratridine on AR, we evaluated its effect on the [Ca 2+ ]i response to P4.…”

“…In the presence of veratridine, the duration of the P4-induced [Ca 2+ ]i transient was increased, leading to a retard in the time to peak, and the phase of decay of the [Ca 2+ ]i transient was delayed. The broader [Ca 2+ ]i signal produced by P4 in the presence of veratridine might reflect a deregulation of the sperm Na + /Ca 2+ balance [39,40] and could avoid the presence of the second [Ca 2+ ]i peak to P4, necessary for the initiation of the AR, which is a tightly regulated mechanism [3,37,38].…”

Fertilization Failure is Not Associated with Sperm Motility Following Human In vitro Fertilization

“…Progesterone is also known to stimulate several sperm functions, including hyperactivation 5 and acrosome reaction. 6 Hyperactivated motility might assist the sperm in penetrating the ZP, which is a critical process for fertilization. 7 Thus, we conclude that sperm motility becomes more vigorous in TFF/PFF patients, and sperm hyperactivation induced by progesterone is unlikely to induce TFF/PFF.…”

Preparation and observation methods can produce misleading artefacts in human sperm ultrastructural morphology

“…With the application of scanning electron microscopy (SEM) in reproductive medicine, the evaluation of human sperm ultrastructural morphology (SUM) has greatly increased. The SEM enables to observe and interpret the surface morphology of a human spermatozoon more easily and completely than with conventional light microscopy (Chemes, ; Moretti, Pascarelli, Belmonte, Renieri, & Collodel, ; Nussdorfer, Cilenšek, Zorn, & Petrovič, ; Ricci et al., ; Sánchez‐Cárdenas et al., ). Thus, examination of SUM using SEM can provide a useful additional approach to identify the status of SUM and aid a better understanding for certain etiologies of male infertility or fertilising failure.…”