Figure 7. CRB3 knockdown in adult rat testes in vivo impairs spermatid and phagosome transport.

Adult rat testes were transfected with corresponding siRNA duplexes on day 0. On day 5, testes were collected for IF or histological analysis. (A) Frozen sections of testes were stained for CRB3 (red), illustrating considerable CRB3 KD in this early stage VIII tubule. Cell nuclei were visualized by DAPI (blue). Scale bar, 30 μm. IF data were summarized in this histogram with each bar a mean ± SD of n = 3 rats using ~100 randomly selected tubules, illustrating a KD efficiency >70%. **P < 0.01. (B) In control RNAi testes, elongated spermatids lined up at the tubule lumen in this stage VIII tubule, ready for spermiation. However, elongated spermatids remained entrapped in the epithelium at stage VIII in the CRB3 RNAi group (blue arrowheads in the micrographs). In control testes, phagosomes were transported to the base of the epithelium at stage IX (green arrowheads) as earlier reported⁵⁹. But CRB3 KD led to retention of phagosomes near the luminal edge (yellow arrowheads), illustrating defects in phagosome transport, with elongated spermatids remained entrapped in the epithelium in this early stage IX tubule (blue arrowheads). In Ctrl RNAi testes, only step 12 elongating spermatids were found in the epithelium of stage XII tubules; however, in CRB3 KD testes, step 19 spermatids were persisently found in stage XII tubules (blue arrowheads) and phagosomes also persisted in stage XII tubules (yellow arrowhead), illustrating CRB3 KD caused defects in spermatid and phagosome transport. Scale bar, 30 μm. (C) Steady-state mRNA level of CRB3 was estimated by qPCR, illustrating ~60% KD of CRB3. Each bar is a mean ± SD of n = 5 rats. **P < 0.01. (D) Percentage of stages VII and VIII tubules in the testes after CRB3 KD, but not IX tubules, was considerably altered after CRB3 KD. About 600 staged tubules from n = 3 rats were scored. *P < 0.05, **P < 0.01. E) Staged tubules that had defects in spermatid and/or phagosome transport as noted in (B) were scored with a total of ~300 tubules from n = 3 rats. **P < 0.01.