Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes.

Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.