Induction of reproductive tract developmental abnormalities in the male rat by lowering androgen production or action in combination with a low dose of diethylstilbestrol: evidence for importance of the androgen-estrogen balance
2002 Study Abstract
This study tested the hypothesis that testis/reproductive tract abnormalities induced in the rat by neonatal treatment with diethylstilbestrol (DES) result from disturbance of the androgen-estrogen balance. Male rats were treated neonatally with a dose of DES (0.1 micro g) that induced either no or small effects on its own or with a dose (10 micro g) that induced major reproductive tract abnormalities.
To allow quantification, the abnormalities chosen for study were distension of the rete testis and efferent ducts and reduction in epithelial cell height in the efferent ducts and vas deferens.
To alter the androgen-estrogen balance, other rats were treated with DES (0.1 micro g) in combination with a treatment to suppress either androgen production [GnRH antagonist (GnRHa)] or androgen action (flutamide); other rats were treated with GnRHa or flutamide alone. Testosterone levels were measured to verify the effects of treatment.
Combined administration of DES (0.1 micro g) plus GnRHa or flutamide induced significantly greater distension/overgrowth of the rete testis and efferent ducts (ED) and a reduction in epithelial cell height of the ED than did DES (0.1 micro g) administered alone. Neither GnRHa nor flutamide affected rete or ED distension when administered alone, but both significantly reduced ED epithelial cell height. Neonatal treatment with bisphenol-A (100 micro g) with or without GnRHa had no significant effect on any of these parameters. In contrast to the ED, a reduction in cell height of the vas deferens was induced to an equal extent by DES (10 micro g), DES (0.1 micro g) with GnRHa, and GnRHa alone, suggesting greater sensitivity of this tissue to both androgen and estrogen action. The induction of major abnormalities in rats treated with DES (10 micro g) was coincident with loss of androgen receptor immunoexpression in affected tissues. Reduced androgen receptor immunoexpression was also induced by combined treatment with DES (0.1 micro g) plus GnRHa or flutamide, whereas treatment with any of these compounds alone had no or only minor effects.
These findings suggest that reduced androgen action sensitizes the reproductive tract to estrogens, demonstrating that the balance in action between androgens and estrogens, rather than their absolute levels, may be of fundamental importance in determining normal or abnormal development of some regions of the male reproductive tract.
Discussion
The primary aim of the present studies was to test the hypothesis that testis/reproductive tract abnormalities, which are induced in the neonatal rat by treatment with relatively high doses (10 μg) of DES, result from disturbance of the androgen-estrogen balance rather than from the estrogenic effect of DES alone (23, 24). This was tested by treating male rats neonatally with a dose of DES (0.1 μg) that induced either no or small effects on its own and combining this with a treatment that suppressed either androgen production (GnRHa) or androgen action (the androgen receptor antagonist flutamide). The results show unequivocally that either of these combined treatments was able to significantly induce greater abnormalities of the rete testis and efferent ducts than did 0.1 μg DES administered alone. Neither GnRHa nor flutamide induced any of the abnormalities when administered alone, whereas treatment with a high dose of DES (10 μg) induced major abnormalities coincident with loss of immunoexpression of the AR in affected tissues. Reduced immunoexpression of AR was also induced by combined treatment with 0.1 μg DES and GnRHa or flutamide, whereas treatment with any of the compounds alone had no or only minor effects on AR immunoexpression. These results together with our previous demonstration that induction of reproductive abnormalities by neonatal treatment with 10 μg DES can be blocked by cotreatment with testosterone (24) provide support for the suggestion that these abnormalities result from a disturbance of the normal androgen-estrogen balance rather than from a lowering of androgen action or an elevation of estrogen action per se. A slightly alternative view is that the level of androgen exposure determines the sensitivity of the developing male reproductive tract to estrogens, such that low androgen levels increase sensitivity to estrogens and vice versa. Either interpretation implies that normal or abnormal development of the male reproductive system may be governed by the relative levels (balance) of androgens and estrogens rather than by the absolute level of either hormone. This has several implications, as discussed below.
The finding of key importance from the present study is the demonstration that combined treatment with a relatively low dose (0.1 μg) of DES plus treatments to either lower androgen production (GnRHa) or block androgen action (flutamide) was able to induce significantly greater distension of the rete testis or efferent ducts, as measured by lumenal area, than any of the treatments administered alone; of the latter, only 0.1 μg DES had any (minor) effect on its own. Although these contrasting effects were clear-cut, the use of another end point, epithelial cell height in the efferent ducts or proximal vas deferens, resulted in a more complex picture, as all single treatments other than bisphenol-A exerted significant adverse effects. In the efferent ducts it was still clear that combined treatment with 0.1 μg DES plus either GnRHa or flutamide was able to reduce epithelial cell height to a significantly greater extent than any of these treatments individually; indeed, the combined treatments caused an effect of similar magnitude to that induced by treatment with 10 μg DES alone. In contrast, several of the individual treatments (10 or 0.1 μg DES plus GnRHa) induced very similar effects on epithelial cell height in the proximal vas deferens, and combined treatment with 0.1 μg DES and either GnRHa or flutamide was unable to exacerbate this effect compared with administration of 0.1 μg DES or GnRHa alone. This suggests that more posterior parts of the reproductive tract, such as the vas deferens, may be more sensitive to androgens and estrogens than the more anterior parts (rete and efferent ducts) such that all single treatments, other than flutamide and bisphenol-A, induced maximal effects. This makes it fundamentally difficult to test whether the androgen-estrogen balance is as important in the vas as it appears to be in the more anterior regions of the tract.
Although the different responses of epithelial cells from the efferent ducts and vas deferens to altered androgens and estrogens remain to be explained, the fact that normal development of the rete and efferent ducts appear particularly dependent on a normal androgen-estrogen balance may have relevance to findings in ERα knockout (ERKO) mice. The latter exhibit distension of both the rete and efferent ducts in the face of supranormal testosterone levels and the absence of normal expression of ERα in the epithelium of both the rete and efferent ducts. This raises the possibility that the abnormalities reported in ERKO males do not result only from the loss of ERα-mediated estrogen action, but might also be affected by disturbance of the androgen-estrogen balance, although in the opposite direction (supranormal androgen plus subnormal estrogen action) to that induced in the present studies. However, this would fail to explain why aromatase knockout mice, which also have elevated testosterone levels, do not show the same structural abnormalities as ERKO mice. Nevertheless, the present findings caution that whenever androgen and/or estrogen levels are altered substantially from normal in the developing male, changes resulting from disruption of the androgen-estrogen balance should be kept in mind.
There are examples in the literature indicating a role for the androgen-estrogen balance. For example, gynecomastia in men can be induced either by raising estrogen levels or by lowering androgen levels, such that in either situation the androgen-estrogen balance is altered in favor of estrogens. Another example is clover disease, in which castrated rams that fed on Mediterranean clover containing weakly estrogenic phytoestrogens died from urinary retention due to overgrowth of the bulbo-urethral glands, which are both an estrogen and an androgen target. Intact rams or castrated rams treated with androgens and fed on the same clover showed no overgrowth or ill effects. Thus, in castrated rams with low androgen levels, phtyoestrogens induced a catastrophic effect, whereas in intact rams with high androgen levels or in castrated rams supplemented with androgens, no effect occurred despite exposure to the same “estrogen” level. In many respects this example arises from a situation in which hormonal status would be comparable to that induced in the present studies by combined treatment with 0.1 μg DES plus GnRHa. There may be other examples in which a role for altered androgen-estrogen balance could be important. For example, abnormal prostatic structure and growth induced by neonatal estrogen treatment are known to involve both altered androgen and estrogen action, and this may apply also to reproductive tissues in the female.
If the relative, rather than absolute, levels of androgens and estrogens are important for normal development and/or function of the testis and reproductive tract, as our findings suggest, how would this work at the cellular level, given that the present understanding is that androgens and estrogens act via separate, if related, signaling systems? There are various possibilities based on published data, although none has been shown to operate physiologically. For example, estrogens can trans-activate the AR/ARA70 complex at high concentrations and thus activate the transcription of androgen-dependent genes, although DES could not exert this effect. Interaction between the C-terminal domain of ERα and the AR has been demonstrated using two-hybrid systems, and cotransfection of the two receptors into CV-1 cells has demonstrated a mutual ability of each receptor to antagonize trans-activation mediated by ligand binding to the other receptor. An alternative explanation might be activation of the Src-Raf1/Shc-Erk2 pathway, in which androgens and estrogens may induce assembly of a novel ternary complex comprising the AR, ER (either ERα or ERβ), and Src. This complex triggers activation of the protein kinase domain of Src and downstream effects, such as cell proliferation or inhibition of apoptosis. The androgen-AR and estrogen-ER complexes bind to separate domains on the Src protein, and antagonists of either the AR or ER can block activation of this pathway by either androgens or estrogens. Our recent findings that expression of classical androgen-regulated genes in the prostate can also be regulated by estrogens and that antiestrogens can block androgen activation of these genes in vivo are consistent with the activation of such a pathway.
Regardless of the pathway involved, the present findings have implications for issues such as endocrine disruptors, in which considerations of risk are focused largely on the absolute dose/level of exposure as opposed to the relative levels of androgen and estrogens. The present study has shown that combined treatment with a weak environmental estrogen, bisphenol-A, plus a GnRHa was unable to induce any of the abnormalities induced by 0.1 μg DES plus GnRHa, suggesting that in this situation the estrogenicity of the bisphenol-A, when injected in moderately high amounts (100 μg/injection), was still insufficient to perturb the androgen-estrogen balance. Whether this balance can be disturbed by higher doses of this or other environmental estrogens or in combination with environmental antiandrogens are obvious questions that need to be addressed.
In summary, the present findings add to the growing evidence of a close interrelationship between the actions of androgens and estrogens in regulating normal and abnormal development of the male reproductive system. Our findings suggest that the balance in action between androgens and estrogens, rather than the absolute levels of either hormone, may be of fundamental importance at least for some regions of the reproductive tract. From a physiological perspective, local regulation of relative levels of androgens and estrogens, for example by differential expression of aromatase or 5α-reductase, may be critical factors that ensure an appropriate steroid milieu for specific regions of the reproductive system
References
- Full study (free access) : Induction of reproductive tract developmental abnormalities in the male rat by lowering androgen production or action in combination with a low dose of diethylstilbestrol: evidence for importance of the androgen-estrogen balance, Endocrinology, Volume 143, Issue 12, Pages 4797–4808, doi.org/10.1210/en.2002-220531, 01 December 2002.
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DES DIETHYLSTILBESTROL RESOURCES
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