The present study demonstrates that the mouse ICI 182,780 treatment model, when extended from 4 days to 125 days post treatment, resembles the αERKO model in nearly every feature, except for the transient increase in testis weight and testicular atrophy. The decrease in sperm concentration in cauda epididymis and luminal dilation of the rete testis and efferent ductules following treatment clearly support the overall hypothesis that a functional ERα is required for normal fluid reabsorption in the efferent ductules, as concluded from prior studies of the αERKO male [4, 7].
In the present model, treatment was started at 30 days of age. This age represents early puberty and the males were still growing, as evidenced by the increase in body and testis weights in controls and treated animals over the next 60 days. Except for an unexplained difference in testis weight on day 16 post treatment, there was no effect on testis weight out to day 25, similar to a previous short-term study . However, on day 59 of treatment testis weight differences began to appear, with a decrease in the treated animals. Thus, with a longer treatment period the mouse showed limited atrophy of seminiferous tubules, but not total atrophy as seen in the αERKO males .
At the beginning of puberty, spermatogenesis appears normal in the αERKO testis, but as the male ages seminiferous tubules begin to degenerate . The αERKO testis weight nearly doubles in approximately 40 days, starting at puberty; then testis weight declines and eventually total testicular atrophy occurs at 185 days of age . ICI 182,780 treatment in the rat induces a similar pattern of effect, with both testicular swelling and long-term atrophy of the testis, nearly identical to the αERKO male [14, 15]. Why the mouse testis did not respond with a similar increase in testis weight and then atrophy remains a major question. However, the rete testis was dilated in the mouse, as in αERKO and ICI-treated rat males, suggesting that ability of the mouse testis to handle fluid accumulation is dependent upon developmental factors that are lacking in the αERKO male. One possibility is that the mouse epididymis is capable of compensatory reabsorption of the excess fluid. Future studies are required to understand these differences in response.
The effects observed on spermatogenesis did not appear to be due to fluid accumulation because there was no dilation of the seminiferous tubular lumen, in contrast to what was discovered in the αERKO testis . Furthermore, abnormal spermatogenesis did not occur until day 59, suggesting that the degenerative process required long-term inhibition of ER. Because ICI 182,780 blocks both ERα and ERβ , the delayed testicular response may be due to ICI's inhibition of ERβ and not ERα. ERα is not expressed in the seminiferous epithelium, but ERβ is abundant in Sertoli and germ cells [16, 21–25]. Therefore, ICI's effects on the epithelium may be through long-term inhibition of ERβ. It is interesting that the aromatase knockout mouse also shows a delayed disruption of spermatogenesis [26, 27], similar to the effects of ICI 182,780 in the mouse testis. Although the ERβ knockout mouse shows no apparent phenotype [3, 10, 28], in light of the current findings, it would be worthwhile to examine the βERKO male for specific aging effects on the testis.
Dilation of the rete testis lumen was less extensive following ICI 182,780 treatment, compared to the αERKO mouse . The present study demonstrated that this effect begins sooner than previously thought, with dilation being observed on day 10. On the other hand, the rete epithelium was reduced in height as early as day 8. It is assumed that the decrease in epithelial height is related to the accumulation of luminal fluid; however, it may also be due to overgrowth of the rete epithelium because neonatal exposure to environmental estrogens has produced a similar dilation and overgrowth of the rete testis [29, 30].
A prior study did not find these early effects of ICI 182,780 treatment . However, here we report effects as early as day 4, at which time proximal regions of the efferent ductules were dilated. This observation is important because the dilatation did not induce a reduction in epithelial cell height, suggesting that the two effects are not directly linked. Others have suggested that the decrease in epithelial height is due to alterations in ion transporter expression , but the present study does not support this hypothesis, as fluid reabsorption detected by luminal dilation occurred without a decrease in epithelial height. Other early effects were subsequent reduction in efferent ductule epithelial height (day 8) and protrusion of the narrow cells in the epididymis. Effects on the testis were not observed until much later. These early morphological effects were maintained throughout the 125-day treatment period and are in agreement with our previous studies of αERKO mouse [4, 5] and ICI 182,780 treatment in mice . Thus, it may be possible to use the mouse antiestrogen treatment model during shorter periods of treatment for the study of fluid dynamics within the efferent ductules, but also to study testicular effects that require more than 50 days of exposure.
Overall effects of ICI 182,780 on the mouse efferent ductules were identical to those observed in the αERKO male, including the loss of NHE3 staining and decrease in staining for AQP-1 and CAII . These data further support the hypothesis that estrogen's regulation of sodium transport by this epithelium is essential for maintenance of fluid reabsorption. Antiestrogen treatment also caused a decrease in ERα staining, which has been reported in female tissues , but the compound had no effect on ERβ.
The effects noted in epididymis could be due to direct action of ICI on the epididymal epithelium, as recent studies have shown that ERα is expressed more extensively in the epididymis of the mouse , than in the rat . Cell types that responded to treatment, the narrow and apical cells of initial segment, apical cells of the caput, and clear cells of the corpus and cauda, are all strongly ERα-positive . In the ICI treated group, apical vesicles in the narrow cells and vesicles in clear and principal cells in corpus and distal caudal epididymis were increased in size and number. An accumulation of large PAS-positive granules was also observed in the apical cells of the caput. These findings are nearly identical to epididymal changes seen in αERKO  and may indicate an attempt to increase reabsorption of fluid by the epididymis, to compensate for reduced reabsorption found in the proximal region of the efferent ductules [4, 7].
In the control animals, epididymal sperm concentrations reached their peak on day 59, showing a gradual increase starting at 38 days of age. However, the ICI treated group did not experience this normal increase in sperm concentration and beginning on day 16 showed a divergence from controls so that on days 59 and 125 post treatment there were decreases of 30 and 41%, respectively. These differences are considerably more than the 22% decline seen in αERKO males . On day 25 (data not shown), there was a 12.4% decline in sperm concentration, which was similar to our previous experiment, which showed an 11% decrease after 35 days of antiestrogen treatment . Thus, the overall trend in the mouse antiestrogen treatment model is for increasing dilution of cauda sperm and accumulation of fluids in the efferent ductules and testis. It is noteworthy that the decrease in fertility did not occur until day 125 post treatment, the time period that coincided with the maximum decrease in sperm concentration. In the rat, decreased fertility coincided with the onset of testicular atrophy [14, 15], therefore it was questioned whether infertility and subfertility was due to abnormal sperm maturation due to dilution in the epididymis or if atrophy of the seminiferous epithelium was required before sperm concentrations could reach levels that would produce infertility. The current study suggests that subfertility in the mouse antiestrogen treatment model is related to the sperm dilution effects that occur in the epididymis, which appears to induce abnormal maturation of the cauda sperm. Even in the αERKO male, testicular sperm are fertile when transplanted into germ cell depleted normal testes [12, 13], which suggests that the epididymal environment is one of the more important factors leading to infertility in the absence of ERα expression in the male.