Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

Abstract

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.

FIG 1

Immunoblots demonstrating that sperm from CATSPER2+/− males contain the same levels of CatSper2 protein as sperm from wild-type (CATSPER2+/+) males. Upper blot, sperm extracts labeled with CatSper2 antibody. Lower blot, after removing CatSper2 antibody, blots were probed with anti-tubulin antibody to indicate numbers of sperm loaded per lane.

FIG 2

Flagellar curvature generated by thimerosal. Activated sperm swimming in medium alone (A). Prolonged exposure to thimerosal caused sperm to arrest with the flagellum curved in the direction opposite of the curve of the head (B). Scale bar = 10 μm.

FIG 3

Ca²⁺ imaging of fluo3-loaded sperm. Images of wild-type sperm were captured before (A, C) and 25 sec after (B, D) application of medium alone (B) or containing thimerosal (D). Warmer colors indicate higher fluorescent intensities and increased intracellular Ca²⁺. Scale bar = 10 μm.

FIG 4

Flagellar beating patterns of CATSPER1^−/− and CATSPER2^−/− sperm. CATSPER1^+/− (A) and CATSPER2^+/− (B) sperm display normal activated motility consisting of moderate amplitude flagellar bends and nearly symmetrical beating. CATSPER1^−/− (C) and CATSPER2^−/− (D) sperm display low bending amplitude and slightly asymmetrical flagellar beating (D). Black and gray indicate the principal and the reverse flagellar bends, respectively.

FIG 5

Ni²⁺ reduced thimerosal-induced hyperactivation. CATSPER2^+/− or CATSPER2^−/−sperm were treated with thimerosal after 5 min pre-incubation in 1 mM Ni²⁺. Bars labeled with different letters indicate significant differences between treatments (P < 0.05).