SPNR was localized by immunocytochemistry (Fig. 1) and by immunofluorescence (Fig. 3) to Carnoy’s-fixed sections of testis from azh/azh males. Confocal microscopy of azh/azh sections labeled with anti-SPNR (Fig. 3, A and D) and anti-tubulin antibodies (Fig. 3, B and E) showed that as in the wild-type testis (Fig. 2), SPNR is coincident with tubulin. SPNR was first detected on the forming manchette in step 9 spermatids from azh/azh males (Fig. 3A), just as in wild type. Immunostaining followed the formation of the abnormal manchette up until its disassembly in step 15 spermatids. As the spermatids elongated, SPNr remained attached to mutant manchettes that had detached from the end of the nucleus (arrow in Azh panel, Fig. 1) and was also localized to large ectopic microtubule aggregates (Fig. 3F, arrow). As in wild type, SPNR immunostaining disappeared in step 15 spermatids coincident with the disassembly of the manchette, leaving the distorted head shape as the only recognizable abnormality in the mature sperm. buy diabetes drugs
Male mice homozygous for the hop mutation are infertile. As previously described, seminiferous tubules in hop/hop males have few elongated spermatids, and these cells have abnormal flagella. Manchette microtubules form at the appropriate time, but they overproliferate and begin to impinge on the integrity of the nucleus. The elongating spermatids appear to degenerate, forming large round cells that extend into the lumen of the seminiferous tubules. The presence of these cells in the lumen suggests that the elongating spermatids may be degenerating as they are being released from their Sertoli cell connection upon spermiation in stage VIII. No mature sperm are found in the epididymides of hop homozygotes.
FIG. 3. Confocal m i crographs of SPNR and tubul i n i mmunolocal i zati on i n azh (A-F) and hop (G-L) testes. Testi s secti ons were treated w i th rabb i t polyclonal affinity-purified anti-SPNR and/or mouse monoclonal anti-a-tubulin primary antibodies. Sections were then treated with an anti-rabbit FITC-conjugated and/or anti-mouse Texas red-conjugated secondary antibody. A, D, G, J) SPNR; B, E, H, K) tubulin; C, F, I, L) merged SPNR and tubulin staining. F) Arrow marks a microtubule aggregate. I) Arrow is pointing to degenerating spermatid; arrowhead highlights an abnormal elongated manchette. L) Arrowhead points to ”caplike” staining pattern.