Techniques for imaging Ca2+ signaling in human sperm.

Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca(2+)-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca(2+) response as % change in fluorescence is obtained.

Molecular mechanism for human sperm chemotaxis mediated by progesterone.

Sperm chemotaxis is a chemical guiding mechanism that may orient spermatozoa to the egg surface. A picomolar concentration gradient of Progesterone (P), the main steroidal component secreted by the cumulus cells that surround the egg, attracts human spermatozoa. In order to elucidate the molecular mechanism of sperm chemotaxis mediated by P, we combine the application of different strategies: pharmacological inhibition of signaling molecules, measurements of the concentrations of second messengers and activation of the chemotactic signaling. Our data implicate a number of classic signal transduction pathways in the response and provide a model for the sequence of events, where the tmAC-cAMP-PKA pathway is activated first, followed by protein tyrosine phosphorylation (equatorial band and flagellum) and calcium mobilization (through IP(3)R and SOC channels), whereas the sGC-cGMP-PKG cascade, is activated later. These events lead to sperm orientation towards the source of the chemoattractant. The finding proposes a molecular mechanism which contributes to the understanding of the signal transduction pathway that takes place in a physiological process as chemotaxis.

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract.

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.