The treatments performed in study 1 were repeated and, in addition, a cohort of rats was also treated neonatally with DES + TE to determine whether the administration of testosterone could prevent the negative effects of DES observed in study 1. Similarly, to determine whether androgen action via the AR is important in establishing or maintaining cells that are rich in H+-ATPase, a cohort of rats was treated neonatally with the AR antagonist flutamide.
Administration of GnRHa, EE, DES, or DES + TE induced similar effects on testis weight at 25 days of age, causing >85% reduction in weight (Table 1). These findings demonstrate the effectiveness of the respective treatments in retarding testicular development. In contrast, neonatal administration of flutamide was without significant effect on testicular weight in this particular study.
Specificity of the H+-ATPase Antibody
To confirm specificity of the H+-ATPase antibody, immunofluorescence experiments were conducted using antibodies that had been preabsorbed with an excess of the immunizing peptide prior to the first incubation step. As shown in Figure 2, the immunoreactivity was completely abolished by preabsorption of the antibodies (compare Fig. 2, A and B), indicating specificity of the staining.
FIG. 2. Specificity of the H+-ATPase antibody. Epididymis sections were incubated with the H+-ATPase antibody (A) or with antibody that had been preabsorbed with the immunizing peptide (B). The staining is completely abolished in the presence of the peptide.