The flagellar protein Enkurin is required for mouse sperm motility and for transport through the female reproductive tract

Abstract

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.

Figure 1.

Enkur ^tm/tm mice have decreased male fertility. (A) Northern blots reveal an Enkur RNA in testis of wild-type mice (+/+) that cannot be detected in mice that are homozygous for the mutated Enkur allele (tm/tm). Lower panel: β-actin loading control. (B) Immunoblot of sperm proteins. A ∼35 kDa band is present in the insoluble fraction (pel) of extracts of wild-type sperm (+/+) but is absent in sperm from mice that are homozygous for the mutant Enkur allele (tm/tm). (C) Size of litters sired by Enkur^+/+ (+/+) and Enkur^tm/tm (tm/tm) males during mating with Enkur^+/+ females. Average litter size is indicated (grey horizontal line) and data points represent individual litters. Total number of mating trials is shown above the scatter graph.

Figure 2.

Enkur is critical for sperm transport in the female reproductive tract and fertilization in vitro. Data represents the mean ± SD. (A) Numbers of Enkur^+/+ (+/+) and Enkur^tm/tm (tm/tm) sperm found in the excised oviducts following natural mating (n = 5 independent experiments). (B) Fertilization of Enkur^+/+ oocytes (% of total oocytes) in vitro by Enkur^+/+ (+/+) or by Enkur^tm/tm (tm/tm). Data represents mean ± SD (n = 6 independent experiments). (A, B) Horizontal line above bars represents statistical differences by two tailed t-test.

Figure 3.

Kinematic effects of Enkur on sperm motility. (A) Immunoblot of isolated sperm heads and tails probed with anti-Enkur antibody. Enkur is detected only in the tail fraction. (B) Representative CASA traces of Enkur^+/+ and Enkur^tm/tm sperm following swim out from cauda epididymis. Genotype is indicated above the panels: Ba, Enkur^+/+; Bb, Enkur^tm/tm. Blue traces represent cases where sperm were tracked continuously during the 20-s acquisition period. This population was used for kinematic analysis. Other colors represent cells that were not tracked continuously throughout the acquisition window and were not analyzed further. (C) Kinematic properties of Enkur^+/+ (five independent experiments, 673 total sperm) and Enkur^tm/tm sperm (four independent experiments, 357 total sperm). Fraction of sperm that exhibited hyperactivation-like motility was calculated as described [42]. Data represent means ± SD (horizontal line above bars represents statistical differences by two tailed t-test). (Please see the online version for the color figure).

Figure 4.

Effects of Enkur genotype on flagellar bending. Flagellar motility of uncapacitated cauda epididymal sperm from Enkur^+/+ (+/+, uncap) and Enkur^tm/tm (tm/tm, uncap) mice were examined and compared with Enkur^+/+ following incubation under capacitating conditions in vitro (+/+, cap). (A) A representative montage of flagellar bends (+/+uncap, a1-j1; tm/tm uncap, a2-j2; +/+ cap, a3-j3) with about 33 ms elapsed between frames. (B) Traces obtained from montage shown in Panel A. Large circles mark the point where the flagellum contacts the base of the head. (C) Primary bend amplitudes of cauda epididymal sperm from Enkur^+/+ (+/+) and Enkur^tm/tm (tm/tm) mice at the beginning of incubation under capacitating conditions and after 90-min incubation. Data represent means ± SD (horizontal line above bars represents statistical differences by two tailed t-test).

Figure 5.

The response of Triton X-100 extracted sperm to Ca²⁺. (A) Representative fields of Enkur^+/+ (left panel) and Enkur^tm/tm sperm (right panel) following addition of 1 mM CaCl₂. Flagella of Enkur^+/+ sperm adopt a highly coiled anti-hook form while those of Enkur^tm/tm exhibit little or no curvature. (B) Curvature is expressed as the straight line distance between the head-flagellum junction and a point on the flagellum 20 μm from the head junction. Flagella of both genotypes were relatively straight in the absence of added Ca²⁺ medium. Following addition of 1 mM Ca²⁺ flagella of Enkur^+/+ sperm become coiled, but there is no observed effect on the flagella of Enkur^tm/tm sperm. Data represents mean ± SD of five animals for each genotype (>20 sperm were assessed for each animal; horizontal line above bars represents statistical differences by two tailed t-test).