Plasma Testosterone Levels in Neonatally Treated Rats
Testosterone levels in 25-day-old rats were significantly reduced in all cohorts treated neonatally with GnRHa, DES, or EE as shown in Figure 3A. Plasma testosterone levels were assessed in 18-day-old rats after neonatal flutamide treatment (Fig. 3B). This graph illustrates the variable effect of flutamide treatment on testosterone levels, but overall, there was no significant increase in levels after flutamide treatment.
Quantification of H+-ATPase-Rich Cells in Control and Neonatally Treated Rats
The epididymides of control rats all exhibited a large subpopulation of H+-ATPase immunopositive cells. As demonstrated in study 1, the immunofluorescence was largely present in the apical pole (Fig. 4A). The morphology of the positive cells appeared similar throughout the epididymis, with tall columnar epithelial cells being evident in all regions. Panels B and C in Figure 4 illustrate cauda epididymides from Day 25 rats after neonatal treatment with GnRHa or DES, respectively (EE not shown), and demonstrate broadly similar reductions in the number of H+-ATPase-positive cells to those illustrated in Figure 1.
FIG. 3. The effect of neonatal treatment with vehicle, GnRHa (n = 8), DES (n = 8), or EE (n = 7) on plasma testosterone levels (ng/ml) at Day 25 (A) and the effect of neonatal treatment with flutamide (n = 5) or vehicle (n = 5) on plasma testosterone levels at Day 1 8 (B). ***P < 0.001, in comparison with the respective control group. Data are means ± SEM.
FIG. 4. Immunoexpression of H+-ATPase in Day 25 rat cauda epididymidis from a control rat (A) and rats treated neonatally with either GnRHa (B), DES (C), or DES + TE (D). Note the reduction in H+-ATPase immunostaining similar to that shown in Figure 1 after DES and GnRHa treatment, but also note the similarity in the intensity and number of immunopositive cells in control animals and those treated with DES + TE. Bar = 40 |xm.