Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. IFT172 is a component of the IFT complex. Global disruption of mouse Ift172 gene caused typical phenotypes of ciliopathy. Mouse Ift172 gene appears to translate two major proteins; the full-length protein is highly expressed in the tissues enriched in cilia and the smaller 130 kDa one is only abundant in the testis. In male germ cells, IFT172 is highly expressed in the manchette of elongating spermatids. A germ cell-specific Ift172 mutant mice were generated, and the mutant mice did not show gross abnormalities. There was no difference in testis/body weight between the control and mutant mice, but more than half of the adult homozygous mutant males were infertile and associated with abnormally developed germ cells in the spermiogenesis phase. The cauda epididymides in mutant mice contained less developed sperm that showed significantly reduced motility, and these sperm had multiple defects in ultrastructure and bent tails. In the mutant mice, testicular expression levels of some IFT components, including IFT20, IFT27, IFT74, IFT81 and IFT140, and a central apparatus protein SPAG16L were not changed. However, expression levels of ODF2, a component of the outer dense fiber, and AKAP4, a component of fibrous sheath, and two IFT components IFT25 and IFT57 were dramatically reduced. Our findings demonstrate that IFT172 is essential for normal male fertility and spermiogenesis in mice, probably by modulating specific IFT proteins and transporting/assembling unique accessory structural proteins into spermatozoa.
Figure S1. Examination of IFT172 testicular expression by Western blot analysis using the anti-IFt172 antibody from Harvard University. Notice that two major proteins were detected, a full length IFT172 protein (170 kDa) and a smaller 130 kDa protein.
Figure S2. Genotype identification of Ift172flox/+ and Ift172-/- mice by PCR. Representative PCR results showing mice with different genotypes.
Figure S3. Examination of Ift172 cKO effect by RT-PCR and Real-time PCR. (A) Schematic representation of the mouse Ift172 gene structure and locations of primers used in the study; (B) Representative RT-PCR results using two primer sets that amplify exons 1 to 3 (primers P1 and P2) and exons 1 to 4 (primers P1 and P3) (upper panel). Expression of 18s rRNA was also examined as a control (lower panel). Notice that very weak PCR product was observed in the cKO mice when P1/P2 primer set was used; a smaller PCR product (arrow) was amplified when the P1/P3 primer set was used. This primer set also amplified a weak band that has the same size as in the control, indicating non-complete deletion. (C) Real-time PCR results using two different primer sets (left: primers P4 and P5; right: primers P1 and P2). Compared to the control, Ift172 mRNA expression level was significantly reduced in the cKO mice.
Figure S4. Examination of IFT172 expression in testicular sections of control (A) and conditional Ift172 KO mice (B) by immunofluorescence staining Notice that specific IFT172 signal (red) was only detected in the manchette of elongating spermatids (inserts) of control mice. In the conditional Ift172 KO mice, the staining was absent in the elongating spermatids. The manchette was stained using an anti-tubulin antibody (green).
Figure S5. Morphological examination of epididymal sperm by light microscopy at low magnification. Sperm density of the control mice is significantly higher than those observed in the Ift172 cKO mice under the same dilution.
Figure S6. Reduced ODF2 and AKAP4 levels in sperm of the Ift172 knockout mice. The sperm were double stained with an anti-α-tubulin antibody. Notice that even if ODF2 and AKAP4 signals were reduced in the cKO mice, α-tubulin signal appeared to be the same.