Reduced Inter-doublet Sliding and the Response to Calcium in Mouse Sperm Lacking the Central Apparatus Protein, SPAG16L.

K. A. Lesich,1 Z. Zhang,2 J. F. Strauss,2 C. B. Lindemann1

 

1Biological Sciences, Oakland University, Rochester, MI, 2Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA

A targeted disruption of the PF20 gene in mice affected spermatogenesis and resulted in sperm cells with reduced flagellar motility (Zhang et al., 2002, Mol.Cell.Biol. 22: 7993-8004). The same study determined that the defective protein associated with this mutation, sperm-associated antigen 16 (SPAG16L), is localized to the central apparatus of the flagellar axoneme. To assess the potential role of the SPAG16L protein in sperm motility we compared the primary motor function of sperm from mice carrying this defect to sperm from age and strain-matched controls. Axonemal sliding disintegration was assayed by a method that utilized a 0.5% Triton X-100 / 2 mM DTT extraction buffer (pH 9.4) to remove the mitochondrial sheath. Sliding disintegration was initiated with 1mM ATP and the number of disintegrating cells vs. non-responders was counted and the pattern of disintegration recorded. Fewer SPAG16L sperm cells showed disintegration than control cells (27 % vs. 52%, p<0.01), and of the responding cells significantly fewer cells from the SPAG16L strain extruded doublets from both sides of the axoneme (6% vs. 14%, p<0.05). We evaluated the ability of demembranated sperm to respond to Ca2+, which is evident by a change in direction of flagellar curvature following exposure. Significantly less of the sperm from the mutant mice responded to Ca2+ (37%) compared to the control sperm (74%), p=0.006. Furthermore, control sperm responded to 1 mM Ca2+ by assuming a curlicue configuration (1.9 105 radians/meter) while the SPAG16L sperm cells showed a significantly reduced curvature development in the midpiece region (2.5 104 radians/m; p <0.001). This suggests a reduced ability of sperm cells from SPAG16L mice to develop bending torque. Taken together, our results implicate a central role for the SPAG16L protein in primary motor function of sperm flagella. Supported by N.S.F. grant #MCB- 0516181 and N.I.H grant # HD37416.

 

*************************************************************************************************************************

 

An Investigation of the Mechanical Properties of Passive Sea Urchin Sperm Flagella.

 

Dominic Pelle, Kathleen A. Lesich and Charles B. Lindemann

 

Biological Sciences, Oakland University, Rochester, MI

 

Recently, our lab reported that passive, vanadate-inhibited (50 M + 0.1 mM ATP), rat sperm flagella exhibit a phenomenon described as counter-bend formation (Lindemann et al. 2005 Biophys. J. 89:1165-1174). When the basal portion of the flagellum is bent with a glass microprobe, the portion of the flagellum distal to the probe contact point develops a bend in the opposite direction to the imposed bend. In this study, we confirm that a similar phenomenon is observed in sea urchin flagella. Therefore, the counter-bend phenomenon is integral to the basic 9 + 2 microtubular axoneme and is not derived from the special features of mammalian sperm morphology. We further determined that removing the basal end of the flagellum by micro-dissection eliminates the response, as does mechanically splitting the flagellum longitudinally. If the axoneme is immobilized in the middle of the flagellum, bends imposed on the basal section result in a counterbend in the distal section, but manipulation of the distal portion does not produce a counterbend in the basal portion. All of these results confirm that the phenomenon depends on the generation of inter-doublet shear. Based on measurements under our experimental conditions the flagellum has a total stiffness of ~ 6.0 10-22 Nm2. We used this value to calculate the torque produced in the counterbend and find a shear resistance of ~ 7.4 pN/nm for the flagellar microtubules. On this basis, the shear resistance of the sea urchin flagellum would account for ~ of the total stiffness of the passive axoneme. Supported by N. S. F. grant #MCB-0516181.