A separate cohort of rats was treated with vehicle or the androgen receptor antagonist flutamide (Fig. 6, A and B, respectively). Similar to GnRHa, DES, and EE treatments, administration of flutamide induced a reduction in the number of H+-ATPase positive cells, although this effect was less marked than in animals treated with DES alone or with GnRHa. The tubule lumen was smaller in the treated group, indicating that flutamide treatment also retarded epididymal lumen development. These observations are in accordance with published data. Quantification analysis showed that the rats treated neonatally with flutamide exhibited a 34% reduction in the mean number of H+-ATPase positive cells per millimeter of epididymal basal membrane (Fig. 7) compared with their controls.
Western Blot Analysis of H+-ATPase Protein Levels
To confirm the changes in the expression of H+-ATPase that was detected by immunofluorescence after neonatal hormone treatments, Western blot analysis was performed (Fig. 8). In homogenates from control epididymides, the affinity-purified antibody revealed a strong band at around 31 kDa and an additional band at around 60 kDa, as we have described previously (Fig. 8, A and B).
Treatments with GnRHa, DES, or EE induced a marked reduction in the intensity of these bands, whereas coadministration of DES + TE prevented the reduction in H+-ATPase expression that occurred after DES treatment alone (Fig. 8A). Similarly, Western blot analysis confirmed that neonatal treatment with flutamide induced a reduction in H+-ATPase protein levels in comparison to control levels (Fig. 8B). To demonstrate equal protein loading in each sample, a Western blot against p-actin was also performed (Fig. 8, C and D).
FIG. 6. Immunoexpression of H+-ATPase in Day 25 rat cauda epididymis from a control rat (A) and from an animal treated neonatally with fluta-mide (B). Note the clear reduction in the number of H+-ATPase-rich cells in the latter group. Bars = 40 ^m.
FIG. 7. Quantification of the number of H+-ATPase-positive cells per millimeter of epididymal tubule basal membrane at Day 25 in control rats and in rats treated neonatally with flutamide. **P < 0.01 in comparison with control. Data are means ± SEM for n = 3.
FIG. 8. A and B) Western blot analysis of H+-ATPase protein levels in epididymides from 25-day control rats (lanes 1 and 6) and from rats treated neonatally with GnRHa (lane 2), DES (lane 3), EE (lane 4), DES + TE (lane 5), or flutamide (lane 7; this lane should be compared with the control in lane 6). A significant decrease in the H+-ATPase signal was observed after GnRHa, DES, EE, and flutamide treatments. Coadministration of TE with DES prevented the inhibition induced by DES treatment alone. C and D) Western blot analysis of p-actin protein levels in the same groups. No difference in signal was observed, suggesting that equal protein levels were added to each lane.