Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50% of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile. Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm head and disorganization of flagellar structures, including loss of the central pair of microtubules and disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.Fertilization is the process whereby sperm and eggs interact reciprocally to begin development. To initiate fertilization, mammalian sperm cells rely on the propulsive forces generated by their flagella to reach the site of fertilization in the oviduct and to penetrate the investments of the egg (8). All flagella contain an axoneme composed of structural elements and motor proteins that work in a coordinated and regulated fashion to produce wave forms that produce progressive movement (3,4,6,8,15,21). The axoneme consists of a central pair of microtubules (central apparatus) surrounded by nine doublets of microtubules with the associated force-generating dynein arms. The basic axonemal structure among cilia and flagella is conserved across species, and much of our understanding of the structure and function of the axoneme has been derived from the study of model organisms. Genetic studies on the green alga, Chlamydomonas, have revealed the importance of several genes for flagellar assembly, stability of specific axonemal structures, and motility (2-6, 15, 21). Inactivation of PF16, one of these Chlamydomonas genes, results in flagellar paralysis (2,20,21). Moreover, when the flagella from the pf16 mutant are demembranated to produce axonemes, the C1 microtubule is destabilized and C1-associated polypeptides are lost. We cloned the human and murine orthologues of PF16, named sperm-associated antigen 6 (Spag6), and found that the amino acid sequences of the mammalian and algal proteins were highly conserved, including the eight armadillo repeats required for the assembly of PF16 onto the C1 microtubule and for flagellar function (11,16,20,21). To determine if Spag6 plays a critical role in the function of the mammalian axoneme, we inactivated mouse Spag6. Males lacking Spag6 were infertile because their sperm had striking motility defects and were frequently decapitated and had disorganized flagellar structures. Approximately 50% of nullizygous males and females have enlarged heads and smaller bodies and die prematurely with hydrocephalus, presumably reflecting a...
The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.
Mammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer-assisted motion-analysis system was used to describe sperm translational movements. To determine which movements were characteristic of HA, Ca-dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca- sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca-dependent motility subgroups were apparent: 1) a "slow-speed" subgroup with a curvilinear velocity (Vc) less than 169 microns/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc greater than 169 microns/sec) and wider-amplitude head movements as measured by curvilinear progressiveness ratio (PRc less than 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.
The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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