Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(6)

The change in development of cells rich in H+-ATPase induced by neonatal administration of steroids emphasizes the importance of the steroid hormone environment for maintaining the normal timing and intensity of expression of genes such as H+-ATPase and that altering the steroid environment can repress or retard their expression.

In summary, this study examined whether androgens, estrogens, or both are involved in the development of cells that are rich in H+-ATPase.

Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(5)


Recent evidence suggests that 3a-diol is a ligand for ERs (preferentially ERp) and that it acts to regulate AR levels in the rat prostate. Whether or not 3a-diol regulates AR levels in any other regions of the male reproductive tract has not been investigated, but because one of the major effects of neonatal DES administration is the virtual abolition of AR protein expression in the epididymis, the potential involvement of 3a-diol is a possibility. Studies of the male rat anterior pituitary have shown that the conversion of DHT to 3a-diol is reversible and that 3a-hy-droxysteroid dehydrogenase (3a-HSD) can convert 3a-diol back into DHT.

Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(4)

We have shown previously that some other morphological effects on the reproductive tract that were observed after neonatal DES administration, such as rete testis distension, loss of aquaporin-1 expression in the efferent ducts, and malformation of the epididymis/vas deferens, were not altered after GnRHa administration, suggesting an estrogenic action of DES in addition to androgen suppression. In addition, previous reports have suggested that estrogens might be involved in the maturation of some epididymal cell types that express H+-ATPase.

Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(3)


The comparable levels of the reduction in the number of H+-ATPase-rich cells observed in animals treated with DES and GnRHa are suggestive of a common mechanism in altering the development of this cell phenotype. This most likely centers on interference with androgen production, action, or both, because both GnRHa and DES treatments suppress blood levels of testosterone, and DES (but not GnRHa) treatment causes a major reduction in expression of the androgen receptor protein in the epididymis and vas deferens.

Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(2)

Previous data in the literature suggested that narrow and clear cells (also called apical mitochondria-rich cells) were under the control of estrogens and not androgens. Another study showed that lumenal acidification in the rat epididymis is under androgen control. The current study was, therefore, designed to test whether neonatal manipulation of sex steroid levels affected the development of ep-ididymal H+-ATPase-rich cells.

Modulation of the Onset of Postnatal Development of H+-ATPase: DISCUSSION(1)


Vacuolar H+-ATPase is involved in the process of lu-menal acidification in the male reproductive tract of both rats and humans. The acidic lumenal environment is believed to play a role in maintaining and storing sperm in an immotile state prior to ejaculation. We have previously shown that H+-ATPase is expressed in a subpopulation of epididymal epithelial cells, the narrow cells in the initial segments, and clear cells in the remainder of the epididymis, and that these cells are present as early as 2 wk after birth. At this time point, a large number of H+ATPase-rich cells were observed in the vas deferens, whereas only a few positive cells were detected in the epididymis, suggesting that the ability to secrete protons may begin in the vas deferens and progress back toward the epididymis during postnatal development.

Modulation of the Onset of Postnatal Development of H+-ATPase: RESULTS(5)

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.

Modulation of the Onset of Postnatal Development of H+-ATPase: RESULTS(4)


The epididymides of animals treated with GnRHa and DES showed retarded development, as evidenced by smaller epididymal lumens when compared with control rats or those treated with DES + TE (Fig. 4, A and D, respectively). Coadministration of DES + TE resulted in the maintenance of epididymal epithelial H+-ATPase immunoexpression at levels similar to controls. Morphologically, the epididymal epithelium of the animals treated with DES + TE appeared broadly similar to that in controls. However, despite the maintenance of H+-ATPase levels, the testes weight of these animals were similar to those measured in rats treated with DES alone or with GnRHa (Table 1).

Modulation of the Onset of Postnatal Development of H+-ATPase: RESULTS(3)

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.

Modulation of the Onset of Postnatal Development of H+-ATPase: RESULTS(2)


Study 2

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.