Summary
Intraflagellar transport (IFT) is an evolutionarily conserved mechanism for cilia formation. Defects in IFT/cilia
have been linked to cilia-related diseases. Although the roles of IFT in somatic tissues have been extensively
studied, little is known about its role in sperm flagella formation, which are specialized motile cilia with
accessory structures. Using conditional knockout (cKO) strategies, our laboratory analyzed male germ cell-
specific IFT mutant mice and discovered that all the analyzed IFTs are required for normal sperm
formation/function. Among the Ift genes, Ift25 and Ift27 hold particular interest. The two IFTs form a
heterodimer through their unique characterized structures. Although these two genes are not required for cilia
assembly in somatic cells, both are essential for sperm formation and function. Specific elimination of each of
these genes in male germ cells resulted in almost identical sterile phenotypes. Sperm from these mice were
immotile and had disorganized accessory structures, especially the fibrous sheath. Levels of testicular pro-
AKAP4, the precursor protein of AKAP4, an A-kinase anchor protein (AKAP) and significant component of the
sperm fibrous sheath, were increased; on the contrary, the mature AKAP4 was significantly reduced in
both Ift25 and Ift27 cKO mice. IFT25 associates with dynactin 4 (DCTN4), a dynein-associated protein. In
addition to IFT25, IFT27 also associates with signal peptide peptidases like 2a (SPPL2a), which functions as
a protease and is present in developing sperm flagella. The formation of mature AKAP4 was also affected in
the Sppl2a KO mice. Based on these observations, we propose the following central hypotheses: 1
) IFT25 and
IFT27 are dedicated to the movement and placement of accessory structure components critical for functional
sperm, and 2) The IFT25/IFT27 complex use specific domains to form IFT complex particles for sperm flagella
assembling. To test these hypotheses, we propose the following Specific Aims: 1. To characterize the
IFT25/IFT27 complex components in the testis essential for normal sperm morphogenesis, particularly the
formation of accessory structures; 2. To investigate sperm accessory structure defects of Ift25 cKO mice
dynamically and develop an in vivo system to track the IFT25 complex trafficking in live germ cells for sperm
flagella assembly; and 3. To explore functional consequences of IFT25/27 disruption in sperm signaling. We
propose that the IFT25/IFT27 heterodimer forms a transporting complex containing SPPL2a and DCTN4
through specific domains in male germ cells for normal sperm accessory structure assembly; we expect
defects in accessory structures in the Ift25 cKO mice will occur at specific developmental steps. The dynamic
trafficking process of the IFT25 complex in live male germ cells can be tracked. We hypothesize that SPPL2a
is involved in processing pro-AKAP4 to mature into AKAP4, resulting in normal PKA signaling in mature sperm.
The proposed research will elucidate the mechanisms of the two IFT proteins in the formation of functional
sperm and
build a platform to study the roles of other IFT components in male and female reproduction.
.