Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm.
This mutation is highly disruptive to ciliary assembly in other organs.Ift88(-/-) mice are completely sterile.The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins.
Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605.
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Figure 3: Timing and location of IFT88 expression in wild-type testis. (A) Immunofluorescence micrographs of sections through (a) stage II-III and (b) stage VIII seminiferous tubules labeled with anti-IFT88 antibody (green), anti–α-tubulin (red), and the nuclear stain TOTO-3 (blue). Arrows point out pachytene cell nuclei, in which the chromatin is condensed into multiple distinct foci; in a, IFT88 is just beginning to appear in the cytoplasm surrounding the pachytene nuclei. The bright red tubulin labeling marks Sertoli cells, through which the developing spermatids pass. es, elongated spermatids; f, flagella; L, lumen; p, pachytene; rs, round spermatids. Scale bar, 20 μm. (B) Immunofluorescence micrographs of individual isolated spermatids labeled with anti-IFT88 (green), anti–α-tubulin (red), and TOTO-3 (blue). (a) The round head and short flagellum suggests that this is an early step 2-3 spermatid. (b) The round head and longer flagellum indicates that this is a step 2-3 or slightly later spermatid. (c) The nucleus has just begun to elongate, but the head is round and lacks a cytoplasmic lobe, indicating that this is a step 7 or possibly step 8 spermatid. (d) The more elongated shape of the head and nucleus and the distinct cytoplasmic lobe suggest that this is a step 10 spermatid. (e) The nucleus is highly condensed, and the head is more elongated than in d, suggesting that this is a step 11 spermatid. (f) The condensed nucleus, sharp narrow apex of the head, and presence of long narrow cytoplasmic lobe indicate that this is a later, probably step 15, spermatid. (g) The shape of the head (which is properly oriented to display the typical hook only in the spermatid on the right) and absence of cytoplasmic lobes suggest that these are either late step 16 spermatids that have already lost their cytoplasmic lobes or mature sperm from the tubule lumen. IFT88 labeling is present in the heads and tails of step 2-3 through 11 spermatids, limited largely to the cytoplasmic lobe in the step 15 spermatid, and no longer detected in the step 16 spermatids/mature sperm. Asterisks mark heads from other spermatogenic cells. Scale bar, 10 μm. (C) Immunofluorescence micrographs of epididymal sperm labeled with anti-acetylated α-tubulin (red), anti-IFT88 (green), and DAPI (blue). (a, b) Merged images; (a′, b′) just the green (IFT88) channel for the respective images. Tubulin labeling is readily apparent, but IFT88 is not detectable in the sperm head or tail. Although the distal part of the tail has doubled back on itself in the sperm shown in b and b′, the image shows what appears to be a cytoplasmic droplet (arrow) that serves as a positive control for the IFT88 labeling. Scale bar, 10 μm.
To determine when and where during spermatogenesis IFT88 is expressed in testis, we used immunofluorescence microscopy of sections through wild-type seminiferous tubules (Figure 3A). We found that IFT88 was first apparent in pachytene spermatocytes of stage II-III (Figure 3Aa) and was strongly labeled in pachytene cells at stage VIII (Figure 3Ab). It continued to be expressed as the spermatocytes underwent maturation, as shown by its presence in the tails of developing spermatids at both of these stages (Figure 3A, a, and b).