Are there intracellular Ca2+ oscillations correlated with flagellar beating in human sperm? A three vs. two-dimensional analysis

Abstract

Study question: Are there intracellular Ca2+ ([Ca2+]i) oscillations correlated with flagellar beating in human sperm?

Summary answer: The results reveal statistically significant [Ca2+]i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D).

What is known already: Fast [Ca2+]i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca2+]i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella.

Study design, size, duration: Healthy donors whose semen samples fulfill the WHO criteria between the age of 18-28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed.

Participants/materials, setting, methods: Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca2+ sensitive dye) or with Calcein (Ca2+ insensitive dye). Ni2+ was used as a non-specific plasma membrane Ca2+ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D.

Main results and the role of chance: We have implemented a new system that allows [Ca2+]i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca2+]i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca2+ transporters, as they were insensitive to external Ni2+, a non-specific plasma membrane Ca2+ channel blocker.

Large scale data: N/A.

Limitations reasons for caution: Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed.

Wider implications of the findings: The possibility of recording flagellar [Ca2+]i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node.

Study funding and competing interest(s): This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.

Keywords: 3D microscopy; 3D sperm flagella; fertilization; human sperm; image analysis; intracellular calcium.