Functional deficiencies and a reduced response to calcium in the flagellum of mouse sperm lacking SPAG16L

Abstract

The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action. In addition, the passive flagella, inhibited by sodium metavanadate to disable the dyneins, were examined for mechanical abnormalities. Live Spag16L-null sperm exhibited much less bending of the flagellum during the beat. The amount of microtubule sliding in the R-bend direction of the beat was selectively restricted, which suggests that there is limited activation of the dyneins on one side of the axoneme in the live cells. This is corroborated by the results on detergent-extracted sperm models. The flagellar response to calcium is greatly reduced. The calcium response requires the activation of the dyneins on outer doublets 1, 2, 3, and 4. These are the same dyneins required for R-bend formation. In axonemes prepared to disintegrate by microtubule sliding, we observed little or no extrusion of doublets 1 and 2, consistent with a reduced activity of their dyneins. This deficit in motor function, and an increased rigidity of the midpiece region which we detected in the passive flagella, together can explain the observed motility characteristics of the Spag16L-null sperm.

FIG. 1.

Shear angle development of WT and Spag16L^−/− mouse sperm during one beat cycle. Shear angles were measured on live, free-swimming mouse sperm in MKRB media at 24°C and are plotted as a function of flagellar length (in micrometers) from the head-tail junction. The WT sperm (A) exhibit a greater development of shear in both the principal and reverse directions of the beat compared with Spag16L^−/− mouse sperm (B). The largest difference between the two plots is in the shear excursion in the R-bend direction (downward deflection) of the Spag16L^−/− sperm. Tracings are at approximately 33-ms intervals. Principal bend (P) and reverse bend (R) directions of the beat are identified.

FIG. 2.

Response of Triton X-100-extracted mouse sperm reactivated at room temperature with 0.3 mM Mg-ATP, 0.5 mM EGTA, and 1 mM CaCl₂. A) Sperm from WT mice exhibit the typical curlicue response to CaCl₂ which is a characterized by a strong development of curvature over the entire length of the flagellum that can result in two, or even three complete 360° coils. B) Spag16L^−/− sperm do not exhibit the same strong curlicue response to 1 mM CaCl₂. Although curvature development of the flagellum is changed to the opposite direction of normal, untreated cells (which is a characteristic “C” shape in the same direction as the pointed sperm head), the response is drastically reduced compared with wild-type cells, with Spag16L^−/−cells developing a slight change of curvature. Circled areas were selected for the magnified inset views. Arrow indicates the commonly found kinking at the midpiece-principal piece junction. Bars = 30 μm; inset bars = 10 μm.

FIG. 3.

Phase-contrast imaging of MT disintegration patterns observed in mouse sperm after reactivation with 1 mM CaCl₂ and 0.3 mM Mg-ATP and subsequent removal of the mitochondrial sheath. After preparing the sperm sample as detailed in Materials and Methods, four basic patterns of MT extrusion from the flagellum are observed relative to the polarity defined by the sperm head. Some sperm remain intact and do not extrude MTs, as shown in A. Other sperm extrude only doublet fibers 9, 1, and 2, as in B (front), or 4, 5, 6, and 7, as in C (back), or from both sides of the axoneme, as in D. Bars = 10 μm.

FIG. 4.

The TEM examination of Ca²⁺-treated, demembranated, and disintegrated WT sperm samples. The images shown are representative of the types of MT extrusion patterns/examples that we counted for an ultrastructural analysis of disintegrated WT sperm and confirmation of our optical microscopy results. Intact axonemes are shown in A and B. An image of MT extrusion from both sides of the axoneme is shown in C. Images of the sliding of MTs 5 and 6 from the axoneme are shown in D and E. Individual 5–6 MTs and their associated ODFs completely extruded from the axoneme are shown in F–H. Wild-type sperm respond to Ca²⁺ by extruding a significantly greater number of MTs from the 9, 1, 2 side of the axoneme. Examples of free MTs 1 and 2 or the MT 1 alone (and corresponding ODFs) are shown in I–L. The standard numerical assignments of the ODFs associated with their MT doublets are provided. Bar = 500 nm.

FIG. 5.

The TEM examination of Ca²⁺-treated, demembranated, and disintegrated Spag16L-null sperm samples. Images shown represent the most common types of MT extrusion patterns found in electron micrographs of Spag16L-null sperm. Compared with wild-type sperm, null sperm remain intact more often, as shown in A–C, G, and H. Null sperm most frequently extrude fibers from only the side of the axoneme that corresponds to activation of doublets numbered 4, 5, 6, and 7. The reduced number of images where the doublet 1 and ODF were extruded implicates a reduced response to Ca²⁺. Examples of this pattern of disintegration are shown in D, I, and J. Examples of free 5–6 doublet pairs (and the corresponding ODFs) are found in F, J, L, and M. In a number of instances it was impossible to determine which side of the axoneme extruded MTs, even though sliding was evident, as in E and K. The extrusion of doublets 1 and 2 requires the action of the same dyneins that bend the flagellum in the direction of the Ca²⁺ response. An individual doublet 1 was found, a rare occurrence, as in J. It is notable that we could not find any comparable images of the 9, 1, 2 group as was found for WT. The standard numerical assignments of the ODFs associated with their MT doublets are provided. Bar = 500 nm.

FIG. 6.

A typical field-view micrograph of demembranated and disintegrated WT and Spag16L-null sperm treated with 1 mM CaCl₂. A) Wild-type sperm characteristically slide a larger number of individual fibers from the axoneme compared with Spag16L-null sperm, which is evident by the four individual 5–6 doublet pairs (and corresponding ODFs) observed. B) More intact axonemes are identified from Spag16L^−/− sperm after disintegration compared with WT sperm. Additionally, only a very small percentage of cells extrude doublet fibers from the 9, 1, 2 side of the axoneme. Bar = 500 nm.

FIG. 7.

The mean frequency (percent sliding of total sperm count) ± SEM of the disintegration patterns of WT and Spag16L^−/− mouse sperm treated with 1 mM CaCl_2. A) Eleven separate experiments were examined by light microscopy to determine the frequency of MT disintegration patterns (as shown in Fig. 3) after treatment with 1 mM CaCl₂. The mean percent frequency ± SEM is plotted as a function of the disintegration pattern observed for WT (black bars) and Spag16L^−/− (white bars) sperm cells. The frequency of sliding of the 9, 1, 2 group of MTs in WT mouse sperm is statistically significantly different from Spag16L^−/− mouse sperm (^aP = 0.003; ^bP = 0.007). B) Three of the MT sliding experiments were subsequently processed for TEM to confirm the results. The mean percent ± SEM of each sliding pattern observed from TEM processing of both sperm types is plotted in B. Although not enough experimental repeats were performed to establish statistical significance, the frequency of sliding in the 9, 1, 2 group of MTs from both WT and Spag16L^−/− mouse sperm follows the trend observed at the light-microscopy level.

FIG. 8.

Response to manipulation in vanadate-treated sperm cells. Wild-type (A and B) and Spag16L^−/− (C and D) Triton X-100-extracted sperm were treated with 50 μM vanadate and manipulated with a glass microprobe at room temperature. Wild-type cells exhibit a typical response, which is the smooth development of curvature in the midpiece region when bent in both directions. Spag16L^−/− cells do not display this same response; rather, they are quite stiff in the midpiece region, and smooth curvature development occurs only after the midpiece-principal junction (arrowheads). See Supplemental Movies S1 and S2 to view the manipulation of WT and Spag16L^−/− cells, respectively. Bar = 20 μm.