Figure 4

Confocal images of mRHBDD1 knockdown and control mouse testis sections. A. Autologous control tubules from the right epididymis of one mouse. pRNAT-H1.1/Hygro-negative control (encoding GFP) stably-expressing GC-1 cells were transplanted into the seminiferous tubules of the right testis. These cells differentiated into spermatids with GFP in the epididymal tubules. They had acrosomes that were stained with PNA-linked Rhodamine. I. PNA-stained acrosomes. II. Hoechst 33258-stained nuclei. III. GFP-labelled spermatids. IV. Merge. B. Heterologous control (seminiferous tubule). Stable pRNAT-H1.1/Hygro-negative control (encoding GFP) GC-1 cells were transplanted into the seminiferous tubules of testes from another animal, capable of producing spermatids in the seminiferous tubules, and were tagged with GFP. They contained acrosome structures stained with PNA-linked Rhodamine. I. PNA-stained acrosomes. II. Hoechst 33258-stained nuclei. III. GFP-labelled spermatids. IV. Merge. C. Test group tubules from the left epididymis of the same animal as in A. pRNAT-H1.1/Hygro-6# containing an effective RNAi plasmid against stable mRHBDD1-expressing GC-1 cells (cell line 2#) was transplanted into the seminiferous tubules of the left testis. The transplanted GC-1 cells and spermatids were not found in the seminiferous or epididymal tubules, indicating that these stable germ cells had died or lacked the ability to differentiate. I. PNA-stained acrosomes. II. GFP-labelled spermatids. III. Hoechst 33258-stained nuclei. D. Residual endogenous germ cells that had resisted busulfan treatment in mouse testes differentiated into sperm cells in the epididymal tubules of the left testis, indicating that these busulfan-treated seminiferous tubules could still support the generation of spermatids. Note: PNA specifically stains the acrosomes of spermatids. I. PNA-stained acrosomes. II. Hoechst 33258-stained nuclei. III. Merge. White triangles are PNA-stained acrosomes (AI, BI and DI). Open triangles are spermatids with GFP tag derived from stable GC-1 cells (AIII, BIII).