FIG. 2.

Differential distribution of PPP1CC isoforms in testis. a) Western blot analysis showing expression of all four PP1 isoforms in purified rat Sertoli cells isolated from Day 20 neonatal testis. About 20 μl per lane of Sertoli cell extracts representing ∼1 × 10⁶ cells were separated by SDS-PAGE. Western blotting was done using specific antibodies against each PP1 isoform as mentioned in the Materials and Methods section. Lane 1: among the four isoforms, PPP1CC1, PPP1CA, and PPP1CB could be clearly detected in Sertoli cells, while PPP1CC2 was absent in this cell type; lane 2: testis extracts were used as positive control for the presence of all four isoforms; lane 3: COS (kidney cell derived from African green monkey) cell extracts were used as a control for the absence of the PPP1CC2 isoform. Vimentin was used as a Sertoli cell marker. b) Immunoblot showing the expression of all four PP1 isoforms in mouse primary cultured SSCs derived from neonates (lane 3) and in 15P1 cells (lane 2). It is to be noted that PPP1CC2 is the only isoform absent in these cell types. GATA4 was used as a marker for Sertoli cells, while PLZF and VASA were used as control for the SSC marker. β-Actin was used as loading control for the lanes. c–f) Immunofluorescence detection of the PPP1CC1 isoform in paraffin-embedded testis sections of adult mice. c) At 20× magnification, specific Sertoli cell staining could be seen as radial spokes within the testis sections; the small, boxed region (white) is magnified and shown as an inset (large white box on bottom left corner) to show a PPP1CC1 presence in premeiotic cells, i.e., spermatogonia and preleptotene spermatocytes (yellow arrows). d) The corresponding DIC image. e) At 60× magnification, the radial spokes are clearly seen as Sertoli cell processes (white arrows). f) The corresponding superimposed DAPI counterstained image. The small, boxed region (white) was further zoomed and shown as an inset (bottom left corner) to clearly view the Sertoli cells and the adjacent germ cells. Sc, Sertoli cells. For immunodetection, the aforementioned PPP1CC1-specific antibody was used. All images are representative of observations from multiple sections in different animals.