Calcium Signaling in Mammalian Fertilization

During mammalian fertilization, a sperm cell has to perform a long list of physiological tasks to achieve its final goal: to fertilize an egg. This list includes capacitation, a process by which a sperm acquires hyperactivated motility essential for its long journey of migration to reach the site of fertilization. Within short range of an egg, the sperm is attracted by chemicals (progesterone in humans) secreted by the egg. Upon contact with the egg, the sperm is further activated by the egg coat proteins (zona pellucida, ZP) and releases its stored acrosomal enzymes via the acrosome reaction. The combination of hyperactivated motility and the action of the released enzymes enable the sperm to penetrate the thick ZP layer surrounding the egg and to achieve the final sperm-egg fusion to generate a new life.

It was known that, in each of the steps, calcium ions enter sperm cells. For example, BSA, a component used for sperm capacitation, progesterone, and ZP proteins were all known to induce Ca2+ entry to sperm. However, it was not clear how Ca2+ enters the sperm. There was also no genetic evidence supporting that Ca2+ entry is indeed required for fertilization. It was thought for several decades that Ca2+ enters sperm through the head region via voltage-gated Ca2+ channels CaVs that are also used by neurons and muscles.

We directly recorded whole-cell ion channel activities using patch clamping recording from mouse sperm and their precursor cells spermatocytes. We surprisingly found that, although CaV currents are present in spermatocytes, mature sperm do not have functional CaV  (Xia & Ren, 2009). Instead, sperm have currents from CatSper, a unique Ca2+ channel localized in sperm tail (discovered in the Clapham lab). We also purified the CatSper complex and discovered two major auxiliary subunits CatSperβ and CatSperγ. Our findings suggest that during the maturation process, the germ cells acquires a unique Ca2+ channel instead of the “conventional” CaVs.

Ion channel currents recorded from sperm and their precursors spermatocytes. Sperm have CatSper currents, but not CaV current. Spermatocytes have CaV currents.

Using Ca2+ imaging, we found that Ca2+ enters from the end segment of sperm flagellum (the principal piece), not the head, via CatSper. We also found that most of the known physiologically important sperm “activators” such as egg coats, cyclic nucleotides, BSA and intracellular alkalinization all trigger Ca2+ influx through CatSper in the tail, and a Ca2+ signal propagation from tail to head within a few seconds. Other investigators found that, in human, CatSper also mediates the progesterone-induced Ca2+ entry. Those findings suggest that CatSper is perhaps the most important Ca2+ channel in sperm function. Men and mice lacking CatSper have sperm lacking hyperactivated motility, and are infertile but with no other obvious phenotype, suggesting that nature has evolved a unique Ca2+ signaling pathway for the important function of sperm cells.

Recording sperm electrical activities with patch clamp recording and Ca2+ dynamics with imaging. Electrical activities of a sperm are recorded with a glass pipette from the droplet of a sperm cell. Intracellular Ca2+ concentrations (represented as fluorescence change ΔF/F0) are imaged from sperm cells loaded with Fluo-4 Ca2+ indicator. Upon application of egg coat proteins zona pellucida (time point zero), [Ca2+]i increases starts in the sperm tail (principal piece, PP), propagates through the mid-piece (MD), and finally reaches the head region in several seconds (imaged at room temperature). In sperm lacking CatSper channels, there is no [Ca2+]i rise.

Sperm Ca2+ imaging. A sperm cell was loaded with a Ca2+-sensitive dye Fluo-4 and imaged. After egg coat proteins zona pellucida were added, [Ca2+]i rises started in the tail and propagated to the head.


Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S., Shi, Q., Tilly, J.L. and Clapham, D.E. (2001) A sperm ion channel required for sperm motility and male fertility. Nature 413: 603-609

Liu, J., Xia, J., Cho, K-H., Clapham, D.E. and Ren, D. (2007) CatSperβ, a novel transmembrane protein in the CatSper channel complex. J. Biol. Chem. 282: 18945-18952.

Xia, J. and Ren, D. (2009) Egg coat proteins activate calcium entry into mouse sperm via CATSPer channels. Biol. Reprod. 80: 1092-1098

Wang, H., Liu, J., Cho, K-H. and Ren, D. (2009) A novel, single transmembrane protein CATSPERG is associated with CATSPER1 channel protein. Biol. Reprod. 81: 539-544

Ren, D. and Xia, J. (2010) Calcium signaling through CatSper channels in mammalian fertilization. Physiology 25: 165-75

Lishko, P., Kirichok, Y., Ren, D., Navarro, B., Chung, J.-J., and Clapham, D.E. (2011) The control of male fertility by spermatozoan ion channels. Annu. Rev. Physiol. 74: 453-475