Intraflagellar transport protein 74 is essential for spermatogenesis and male fertility in mice†

Abstract

Intraflagellar transport protein 74 (IFT74) is a component of the core intraflagellar transport complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axonemes in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 and sperm-associated antigen 16L proteins were also not changed. However, the processed A-Kinase anchor protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.

Keywords: intraflagellar transport protein 74; male fertility; microtubules; spermatogenesis.

Figure 1.

Mouse IFT74 protein is highly expressed in the testis and developmentally regulated during spermatogenesis. (A) Western blot analysis of mouse IFT74 protein, using a high sensitive Femto system. Note that IFT74 is highly expressed in the testis and is also present in the tissues bearing motile and primary cilia, including brain, lung and kidney. (B) IFT74 expression during the first wave of spermatogenesis. Western blot result shows that its expression is significantly increased at day 20 after birth.

Figure 2.

Localization of IFT74 in male germ cells. (A) Immunofluorescence staining of IFT74 in germ cells of wild-type mice. The top row shows the germ cells with phase contrast microscopy. No specific signal was detected in the negative control using preimmune serum (a). It is present as vesicles in spermatocytes (arrow and inset in b) and round spermatids (dashed arrow and inset in c). It appears to be present in the acrosome and centrosome of elongating spermatid (d), and developing tail (inset in e). (B) The cells were double stained with a lectin acrosome marker, peanut agglutinin (PNA). IFT74 was colocalized with PNA. In addition, it was also present as a dot at the opposite region of the acrosome (white arrow), presumably the centriole.

Figure 3.

Significant reduction in sperm numbers and motility in the conditional Ift74 KO mice. (A) Western blot analysis of testicular IFT74 protein expression in control and conditional Ift74 KO mice. Note that IFT74 protein was missing in the conditional KO mice. Epididymal sperm were collected and physiologic parameters were compared between the control and conditional Ift74 KO mice. In the conditional Ift74 KO mice, there are significant reductions in sperm count (B), percentage of motile sperm (C), and sperm motility (D). Data are expressed as the Means ± SD (n = 4). *P < 0.05 compared with the control mice.

Figure 4.

Abnormal epididymal sperm in the conditional Ift74 KO mice. Examination of epididymal sperm by SEM. (a) Representative image of epididymis sperm with normal morphology from a control mouse. The sperm has a normally shaped head (white arrow) and a long, smooth tail (white dashed arrow). (b–d) Representative images of epididymal sperm from a conditional Ift74 KO mice. All sperm have short tails (black dashed arrow) and most have grossly abnormal heads (black arrow). Panels c to f show sperm with both abnormal heads (black arrow) and short abnormal tails (black dashed arrow).

Figure 5.

Abnormal spermiogenesis in the conditional Ift74 KO mice (HE, ×40). (A) Testis histology from control and the conditional Ift74 KO mice showing cross-sections of seminiferous tubules. Bar = 20 μm. (a) Control seminiferous tubule Stage VIII-IX, showing steps 8 and 9 round spermatids, flagella (F) of sperm being released into the lumen and residual bodies (Rb) of germ cell cytoplasm being phagocytized by Sertoli cells. P, pachytene spermatocyte. (b) The conditional Ift74 KO seminiferous tubule in Stage VIII, showing normal step 8 round spermatids and pachytene spermatocytes (P). Abnormal step 16 spermatids (Ab16) are seen being phagocytized and failing to spermiation. Residual bodies are not forming but small pieces of germ cell cytoplasm (Cy) are retained at the luminal border. (c) The conditional Ift74 KO Stage XI with abnormal step 11 spermatids (Ab11) with abnormally shaped heads and absence of tails. P, pachytene spermatocyte. (d) The conditional Ift74 KO tubule showing normal round spermatids but abnormal step 13 elongating spermatids (Ab13) that are lacking tails. Excess cytoplasm (Cy) of the elongating spermatids appears to be sloughed into the lumen. (B) Cauda epididymis from control and the conditional Ift74 KO mice. Bar = 20 μm. (a) Control epididymis showing an epithelium (Ep) lining the lumen that is filled with normal sperm aligned with their heads (Hd) and tails (T). (b) The conditional Ift74 KO epididymis showing a lumen filled with numerous, large cytoplasmic bodies that are likely residual bodies (Rb) and sperm with abnormal heads (Ab) and short or absent tails. Gc, sloughed round spermatid.

Figure 6.

Ultrastructural changes in the testis of conditional Ift74 KO mice. Testicular ultrastructural was analyzed in the conditional Ift74 KO mice. (a) Control mouse TEM image. The arrows in “a” point to the axoneme/tail of sperm, and dashed arrows point to the well-formed heads. Numerous axonemes of sperm tails are seen. (b–h) Ift74 mutant mouse testis. “b” shows the lumen area with the absence of normal axonemal structures. The arrows in (c) points to disorganized microtubules; the inset in “d” shows clusters of microtubules (arrows) and mitochondria (dashed arrows) without a core axoneme structure; the inset in “e” shows an abnormally formed axoneme without the central microtubules; the inset in “f” shows an abnormally formed elongating spermatid; “g” shows sloughed residual bodies and abnormal spermatids; the inset in “g” shows an abnormal spermatid. The developing acrosome appears to be normal (h). The arrows in “f” and “h” point to abnormal spermatid heads.

Figure 7.

IFT74 regulates expression levels of some IFT and flagellar proteins. (A) Examination of selective IFT protein expression levels in the control and conditional Ift74 KO mice by western blot. Compared to the controls, the expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140, but not IFT20 and IFT25 are reduced in the conditional Ift74 KO mice. (a) Representative western blot result; (b) quantitative analysis of selective IFT protein expression. (B) Examination of testicular expression levels of ODF2, a component of sperm tail outer dense fibers, AKAP4, a major component of fibrous sheath, and SPAG16L, a component of axonemal central apparatus protein in the control and conditional Ift74 KO mice. There was no difference in ODF2 and SPAG16L expression. However, the processed AKAP4 was significantly reduced in the conditional Ift74 KO mice. β-ACTIN was used as controls. (a) Representative western blot result; (b) quantitative analysis of ODF2 and SPAG16L; (c) quantitative analysis of AKAP4. Data are expressed as means ± SD (n = 3). *P < 0.05 compared with the control mice.