Histological analysis of wild-type and Tssk1/2 knockout testes and epididymal spermatozoa. (A,B) PAS staining of adult mouse testes, with a tubule cross-section at stage VIII of the spermatogenic cycle at the centre, showing the absence of conspicuous abnormalities in the testes from knockout mice (−/−) as compared with wild type (+/+). (C,D) Higher magnification of PAS-stained sections, showing stage IX of the cycle, with step 9 spermatids (arrowheads). There is a slight spermiation defect in the knockout, with several step 16 condensed spermatids (arrows) remaining present at stage IX. (E,F) Using an antibody targeting ERGIC53/p58, some dysregulation of the cytoplasmic reorganization of elongating spermatids in the knockout is detected, leading to enlarged cytoplasm associated with late spermatids at stage VIII of the cycle (arrowheads point to brown immunostaining of the cytoplasm of elongating spermatids). (G,H) HE staining of spermatozoa from cauda epididymis, showing marked morphological abnormalities of the knockout cells. Scale bars: 200 μm (A,B), 20 μm (C-H).

Cellular localization of TSSK1, TSSK2 and TSKS. (A-C) Immunofluorescent staining with anti-TSSK1 (A), anti-TSSK2 (B) and anti-TSKS (C) of adult wild-type testis (green). All three proteins show cytoplasmic localization in elongating spermatids near the luminal center of the cross-sections of the testicular tubules, with an accumulation in dots. Blue signal is the DAPI nuclear staining. In the Tssk1/2 knockout (insets in A-C), only the TSKS signal is maintained, but without the marked dots. (D,E) Anti-TSKS staining of wild-type testis at a higher magnification shows a ring (open arrowhead) and a satellite (closed arrowhead) near the nucleus in early elongating spermatids (D). At a later step of spermatid elongation (E) the ring has moved down the flagellum, where it is found at the distal end of the newly formed mitochondrial sheath (the double-arrow points to nucleus and ring within one cell). (F) The anti-TSKS signal (green) does not colocalize with the centrioles marked with anti-γ-tubulin antibody (red), in wild-type early elongating spermatids. (G) The ring and satellite marked with anti-TSKS antibody (green) do not colocalize with the Golgi remnant marked with anti-GM130 antibody (red), in early elongating spermatids. +/+, wild type; −/−, knockout. Scale bars: 40 μm (A-C), 20 μm (D,E), 5 μm (F,G).

Schematic presentation of CB ring and satellite. (A) In round spermatids, the CB contains MIWI and is found bouncing around the nucleus. The Golgi is associated with the growing acrosome. (B) In early elongating spermatids, the CB has lost MIWI but gained TSSK1, TSSK2 and TSKS, forming a ring around the tail, next to the annulus and close to the centriole. Because the satellite also contains TSSK1, TSSK2 and TSKS, this indicates a common origin of ring and satellite. The Golgi becomes disengaged from the fully developed acrosome. The mitochondria are scattered throughout the cytoplasm. (C) At later steps of spermatid elongation, the ring has become smaller and has moved down the tail with the annulus. In its slipstream, the mitochondria become associated with the axoneme. The centriole degrades, and the satellite and Golgi remnant also become less prominent. This step of spermatid elongation is followed by reduction of the cytoplasmic volume and formation of the residual body (not illustrated).

The chromatoid body in round and elongating spermatids. (A-D) Immunofluorescent staining of MIWI in CBs of round spermatids at step 8 (green dots) disappears when the spermatids reach step 9, both in the wild type (A,C) and in Tssk1/2 knockout testis (B,D). Cytoplasmic staining of MIWI is observed in spermatocytes. (E-H) Cross-section of the CB ring (open arrowhead) associated with the annulus (closed arrowhead) in wild-type early elongating spermatids (E). This CB ring material is absent in Tssk1/2 knockout spermatids (F). At later steps of elongation, the annulus is present at the border of the middle piece and principal piece, both in the wild type (G) and knockout (H). The insets in G,H show that some electron-dense material remains associated with the annulus in wild-type late spermatids, but not in the Tssk1/2 knockout. +/+, wild type; −/−, knockout. Scale bars: 20 μm (A-D), 500 nm (E,F), 1000 nm (G,H).