The counterbend response of mouse sperm: Evidence that the nexin links have an elastic limit.

 

Kathleen A. Lesich and Charles B. Lindemann

 

Mammalian sperm demembranated with Triton X-100 and inhibited with 50 M NaVO3 in the presence of 0.1 mM ATP are immotile and flaccid. When the proximal portion of the flagellum of these sperm is manipulated into a bend with a glass microprobe, the distal flagellum develops a bend in the direction opposite the imposed bend. This is the counterbend response. An analysis of this phenomenon in rat sperm (Lindemann et al., 2005, Biophys. J. Vol. 89:1-10) concluded that the counterbend can best be explained by the presence of inter-doublet elastic elements in the axoneme, most likely the nexin links. Mouse sperm also exhibit the counterbend response. In addition, mouse sperm are unique in their exaggerated response to calcium. Reactivated mouse sperm respond to the addition of 1 mM CaCl2 by going into a coiled shape best described as a curlicue. The curlicue shape can generate a shear angle at the flagellar tip as large as 16 radians. This amount of shear will produce an interdoublet sliding of 800 nm between doublets pairs 2-3, 3-4, 7, 8 and 8-9 in the distal flagellum. We hypothesized that stretching the interdoublet linkages this much would likely exceed the elastic limit and result in breakage. When reactivated mouse sperm were induced into the curlicue configuration with Ca2+, we subsequently found that they would not show a normal counterbend response after NaVO3 treatment. This confirmed the hypothesis that the interdoublet elastic linkages have an elastic limit and will break if this limit is exceeded. Supported by NSF grant MCB-0110024.

 

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Evidence that Ca2+ selectively affects dyneins on certain microtubule pairs in the mouse sperm axoneme.

 

Kristen Ponichter, Kathleen A. Lesich and Charles B. Lindemann

 

Mouse sperm were induced to undergo sliding disintegration of the axoneme by a two step method. First, epididymal sperm were detergent extracted with 0.1% Triton X-100 in a potassium glutamate based reactivation buffer in the presence of 0.3mM Mg-ATP at pH 7.8 as described in Lindemann and Goltz, 1988 (Cell Motil. Cytoskeleton, 10:420-431). A high percentage (~90%) of sperm exhibited a coiled curlicue shape if 1mM Ca2+ was also added to the mixture. Second, the cells were stripped of their mitochondrial sheath by transferring 0.5 ml of the reactivated cells to 4.5 ml of an extraction buffer that contained 0.1 M potassium glutamate, 2mM DTT, 0.02M Tris-HCl, 5mM MgCl2, 0.5mM EGTA, 1mMATP and 0.5% Triton X-100 at pH 9.5 and incubating at 34C for 5 min. After incubation in the extraction buffer, phase microscopy revealed disintegrated axonemes with extruded outer dense fibers and microtubules. The polarity of the disintegration patterns was analyzed with respect to the reference provided by the pointed asymmetric sperm head. In the absence of Ca2+ most cells had emergent fibers that came from the side of the axoneme opposite to the head point (47% vs. 14%). This ratio was shifted to almost equal numbers in the samples treated with Ca2+ (28% vs. 27%). There were also significantly more cells showing fibers ejected from both sides of the axoneme in the Ca2+ treated samples (6% control; 12% with Ca2+; p < 0.05). These results suggest that the dyneins on doublets #1 and #2 are more likely to actively initiate sliding in the presence of Ca2+. Supported by NSF grant MCB-0110024.