The results of study 1 are presented first, followed by those obtained in study 2. Study 1 examined the effect of neonatal treatment with DES, ethinyl estradiol (EE), or GnRHa on the number of cells in the epididymis that were rich with H+-ATP ase at 25 days of age. The number of cells positive for H+-ATPase was then quantified and calculated as the number of positive cells per millimeter of epididymal tubular basal membrane.
Representative images illustrating the number of H+-ATPase-rich cells in the cauda epididymis of control and treated animals are shown in Figure 1. Panels A and B illustrate H+-ATPase immunostaining within control epididymides. Both images demonstrate that H+-ATPase is principally expressed in the apical pole of a subpopulation of epithelial cells. In control animals, a large number of H+-ATPase-positive cells were detected at Postnatal Day 25. However, after neonatal administration of either GnRHa, DES (Fig. 1, C and D, respectively), or EE (data not shown), the number of cells expressing H+-ATPase was much smaller.
These results were confirmed by quantifying the number of H+-ATPase immunopositive cells for each animal. Epididymides from control animals had a mean of 27 ± 4.5 H+-ATPase positive cells per millimeter, whereas treatment with either DES or EE significantly reduced (P < 0.001) this to 2.44 ± 0.92 and 5.86 ± 4.7, respectively. Neonatal treatment with GnRHa also reduced the number of H+-ATPase positive cells per millimeter (14.71 ± 7.7),but this decrease failed to reach statistical significance. This group had three animals, two of which showed a major decrease, and a third that showed little difference from control values. This variation in GnRHa response was one reason why a larger follow-up study was performed.
FIG. 1. Immunoexpression of H+-ATPase in the 25-day rat epididymis visualized by fluorescence (bright green staining). Tissue sections from a control rat cauda epidi-dymidis are shown at low (A) and high power magnification (B) to demonstrate the location of H+-ATPase in the apical membrane. Comparable low power sections from rats treated neonatally with GnRHa (C), or DES (D) are shown to demonstrate the reduced frequency of immu-nopositive cells. Bars = 40 ^m in A, C, and D, and 20 |xm in B.