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1 Department of Biomedical Sciences, College of Veterinary Medicine, Nursing and Allied Health and 2 Department of Biology and CBR/RCMI, Tuskegee University, Tuskegee, Alabama 36088, USA and 3 Department of Physiology and Pharmacology, Auburn University, Auburn, Alabama, USA
Correspondence should be addressed to H O Goyal; Email: goyalho{at}tuskegee.edu
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Abstract |
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 Top Abstract IntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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(ER). The resistance of ER knockout mice to develop these abnormalities implies an unequivocal role for ER in mediating maldevelopment of the penis. Additionally, the prevention of estrogen-inducible penile abnormalities by ER antagonist ICI 182 780 implies that a functional ER-mediated pathway is essential for inducing penile abnormalities. Likewise, the ability of testosterone or dihydrotestosterone to negate these abnormalities suggests a role for an androgen receptor (AR)-mediated pathway. Taken together, these observations led us to hypothesize that neonatal estrogen exposure, via an ER-mediated pathway (direct action) or an AR-mediated pathway (indirect action through decreased testosterone) or both pathways, up-regulates ER expression in stromal cells of the penis, which are then reprogrammed such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated. |
Introduction |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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reduced testosterone metabolite, is critical in development of organs derived from the urogenital sinus, genital tubercle, and genital swelling, including the scrotum, prostate, and penis (Anderson & Clark 1990, George & Wilson 1994, Wilson & Davies 2007). Alterations in androgenic activity during differentiation can cause maldevelopment of internal and external male genitalia, including hypospadias and shorter penis (Gray et al. 2001, Sultan et al. 2001, Kim et al. 2002, Foster & Harris 2005). However, unlike androgens, the role of estrogen in development of male reproductive organs, especially in the penis, remains largely unknown; although both estrogen receptors (ERs) and/or aromatase enzyme have been identified in the developing penis of a number of species, including humans (Crescioli et al. 2003, Schultheiss et al. 2003, Dietrich et al. 2004), rodents (Jesmin et al. 2002, 2004), and rabbits (Srilatha & Adaikan 2004). Epidemiological studies have shown links between inappropriate estrogen exposure and higher frequency of reproductive abnormalities in men and wild animals (Toppari et al. 1996, McLachlan et al. 2001, Safe et al. 2001, Mosconi et al. 2002, Fisher 2004, Vidaeff & Sever 2005, Storgaard et al. 2006, reviews). Male offspring of women exposed to diethylstilbestrol (DES) during pregnancy have higher incidence of epididymal cysts, cryptorchidism, hypospadias, and smaller penis (Gill et al. 1979, Swan 2000, Klip et al. 2002). Laboratory animals exposed neonatally to estrogen develop hypospadias (McLachlan et al. 1975, Kim et al. 2004, Newbold 2004). Neonatal exposure to estrogen at low doses enlarges the prostate gland, while higher doses have the opposite effect (vom Saal et al. 1997, Gupta 2000, Putz et al. 2001, vom Saal & Hughes 2005). Taken together, the above studies suggest that prenatal and/or neonatal exposure to estrogens can have permanent deleterious effects on the development of male reproductive organs; however, the mechanism underlying estrogen-inducible abnormal phenotypes in the male reproductive tract, as well as in the penis, remains unknown.
Recently, we reported permanent dysmorphogenesis of the penis and loss of fertility in adult rats treated neonatally with DES or EV (Goyal et al. 2004a, 2004b, 2005a, 2005b). Importantly, these studies showed replacement of smooth muscle cells and cavernous spaces by fat cells in the corpora cavernosa penis. Additionally, these effects were dose dependent, required a critical window of exposure, and were associated with decreased plasma testosterone and up-regulation of ER expression in the body of the penis. The objective of this review is to present the above data in a comprehensive manner and discuss the mechanism by which neonatal exposure to estrogen stimulates adipogenesis and inhibits smooth muscle cell differentiation during a critical period of penile development.
Hence, in this review, we will first present a brief description of the rat penis morphology at the gross and histological levels for the purpose of orientation. This will be followed by data that neonatal estrogen exposure results in permanent penile dysmorphogenesis characterized by replacement of cavernous spaces and smooth muscle cells by fat cells in the body of the penis (Goyal et al. 2004a); estrogen-induced penile disorders are dose-dependent (Goyal et al. 2005a), as well as are dependent upon estrogen exposure during a critical period of penile development (Goyal et al. 2005b); neonatal estrogen exposure results in up-regulation of ER, but without any alteration in ERß or androgen receptor (AR) expression, in the body of the penis (Goyal et al. 2004b); ER knockout (ERKO) mice are resistant to estrogen-inducible penile abnormalities (Goyal et al. 2007); neonatal estrogen exposure lowers testosterone secretion at puberty or adulthood (Goyal et al. 2004b) and suppresses neonatal testosterone surge (Goyal et al. 2005b); and the coadministration of ER antagonist or androgen with estrogen mitigates estrogen-inducible developmental penile abnormalities. Finally, we will discuss possible mechanisms by which neonatal estrogen exposure results in penile dysmorphogenesis.
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Morphology of the rat penis |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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). Microscopically, the body consists of a pair of corpora cavernosa, which are located dorsolateral to the urethra and are partially separated by a septum bringing blood vessels and nerves to the penis, and a corpus spongiosus that surrounds the urethra (Fig. 1B). The corpora cavernosa are surrounded by a thick connective tissue capsule called tunica albuginea, which consists of an inner cellular layer and an outer fibrous layer, and contain endothelial-lined cavernous spaces (also called sinusoids or lacunae), smooth muscle cells, and collagen fibers (Fig. 1C). Additionally, they also contain a few fat cells that are more clearly identified in paraffin sections stained with osmium tetroxide (Fig. 1D). The corpus spongiosus is structurally similar to the corpora cavernosa, but cavernous spaces and smooth muscle cells are less developed (Fig. 1E).
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Neonatal exposure to estrogen induces permanent penile dysmorphogenesis |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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), the os penis was under-developed and the thickness of the corpora cavernosa, but not that of the corpus spongiosus, was dramatically reduced (Fig. 1G). The cavernous spaces and smooth muscle cells in the corpora cavernosa were replaced by empty appearing cells, which were confirmed as adipocytes in paraffin sections stained with osmium tetroxide (Fig. 1H and I). Conversely, the morphology of the corpus spongiosus was unaltered, as a result of DES treatment (Fig. 1J). While all females mated with control males (n=7) had 3–5 copulatory plugs each and delivered pups (fertility index: 100%), none of the females mated with treated males (n=14) had pups (fertility index: 0%) and only one female had a small copulatory plug. In conclusion, adult rats treated neonatally with estrogen develop permanent dysmorphogenesis of the penis and loss of fertility, which probably results from erectile dysfunction as indicated by treated males’ inability to deposit vaginal plugs. |
Penile dysmorphogenesis is dose- and time-dependent |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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); the weight and length of the penis were decreased in a dose-dependent manner (Fig. 2A); and smooth muscle cells and cavernous spaces were replaced by adipocytes (not shown because effects were similar to those shown in Fig. 1H and I). Conversely, penile morphology and fertility were similar to controls in the lower dose groups, except four out of seven males in the 100 ng DES group, which did not sire pups. The loss of fertility in all these four males was associated with penile abnormalities similar to those observed in the higher dose groups. In conclusion, neonatal exposure to DES or EV at a dose of 1 µg (0.1 mg/kg) or higher per day on alternate days from postnatal days 2–12 results in abnormal development of the penis and loss of fertility in 100% of the treated rats, although a lower dose (0.01 mg/kg) also causes infertility, but at a reduced percentage.
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) and varying degrees of abnormal accumulation of adipocytes, which was maximal in the 1–6-day group (Fig. 2C). In conclusion, estrogen exposure prior to 12 days of age, as short as 1–3 postnatal days, results in permanent penile deformities and loss of fertility. |
Penile dysmorphogenesis is associated with ER up-regulation
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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, ERß, and AR expression in the body of the penis. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between controls and treated rats in any age group (Fig. 3A and B). Conversely, ER-positive cells (mostly stromal), while few and faintly stained in controls, were abundant and intensely stained in all age groups of treated rats, especially at 18 days (Fig. 3C and D). RT-PCR confirmed immunocytochemical data where ER expression was enhanced and AR expression was unaltered, as a result of neonatal DES exposure (Fig. 3E). Unlike ER, ERß expression was high in both control and treated groups (Fig. 3F). In conclusion, neonatal estrogen exposure results in ER up-regulation, but without an alteration in expression for ERß or AR.
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ER presence is essential in estrogen-inducible penile dysmorphogenesis
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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null allele were crossed to generate ERKO and wild-type C57BL/6 mice in a continuous breeding scheme at the University of Illinois, Champaign/Urbana. Pups were treated with DES at a dose of 200 ng (0.1 mg/kg same as in the rat) in 25 µl olive oil, per pup, every other day, from postnatal days 2–12. Controls received oil only. Between 8 and 12 days of age, DNA was isolated from tail tissues using Direct PCR Lysis Reagent (Viagen Biotech Inc., Los Angeles, CA, USA) and used to determine genotype. Animals were shipped to Tuskegee University at adulthood for further study. Fertility was tested at 90–120 days of age (n=5–7 each group) and tissues were collected at 96–153 days of age.
Penises from the wild-type treated mice only showed abnormalities, which were similar to those observed in the rat penis, including maldevelopment of the os penis (Fig. 3G) and reductions in the length and weight of the penis (Fig. 3H). While all wild-type control males sired pups, none did in the wild-type treated or knockout control or knockout treated males. In conclusion, the ERKO mice are resistant to estrogen-inducible penile abnormalities present in the wild-type littermates implying an unequivocal role for ER in mediating maldevelopment of the penis.
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Penile dysmorphogenesis is associated with lower plasma and intra-testicular testosterone |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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).
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0.05) different from that of controls (2.78 ng/ml), it was significantly reduced in the 100 ng and higher dose DES and EV groups (Fig. 4B).
Effect of the time and length of estrogen exposure
Male pups received EV at a dose of 1 µg per pup, each day on postnatal day (s) 1, 1–3, 4–6, 1–6, 7–12, 13–18, 19–24, or 25–30. Plasma samples were assayed at 140–150 days of age. The mean plasma testosterone concentration was significantly lower in the 1—6 and 4–6 day groups only (Fig. 4C).
Intra-testicular testosterone
The intra-testicular testosterone concentration was determined in rats treated with EV at a dose of 1 µg per pup, each day on 1–3 postnatal days. Testes were collected and frozen on postnatal days 5, 8, and 12. The rationale was to find out whether estrogen treatment results in suppressing the neonatal intra-testicular testosterone surge that is typical for rodents from late gestation (18.5–19.5 days) to the first week of life (Ward & Weisz 1984, El-Gehani et al. 1998). The reason for treating pups for 1–3 postnatal days was that, among various treatment groups, this was the earliest and the shortest developmental period in which estrogen exposure resulted in abnormal penis and loss of fertility (see above section C, Time of Exposure).
The mean intra-testicular testosterone concentration in control rats at 5 and 8 days of age was 160 and 98 ng/g respectively as opposed to <10% of the control values in the treated rats (Fig. 4D), implying that neonatal estrogen exposure suppresses the perinatal testosterone surge.
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ER antagonist or androgen coadministration with estrogen mitigates estrogen-induced penile dysmorphogenesis |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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); although, comparatively, the mitigation was somewhat lower in the case of DES and testosterone. In conclusion, the prevention of estrogen-inducible penile abnormalities by ICI treatment implies that a functional ER is essential for inducing penile abnormalities. Likewise, the ability of DHT or testosterone to negate these abnormalities, although at a lower level than with ICI, suggests a role for AR-mediated pathway, probably by negating estrogen-induced lower androgen action at the level of penile stromal cells. |
Mechanism of estrogen-induced penile dysmorphogenesis |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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Studies aimed at elucidation of mechanisms through which aberrant estrogen exposure affects restructuring of the corpora cavernosa will provide important insights into mechanisms governing early development of the penis. Based upon observations from other studies (Murakami 1986, 1987) that both smooth muscle cells and cavernous spaces were absent in the rat penis at birth and started to differentiate from stromal cells after 5–6 days of age, we hypothesized that the structural changes that we observed in the corpora cavernosa may have resulted from estrogenic effects limited to the early period of penile development. Indeed, this was confirmed in our developmental study in which rats treated prior to 12 days of age, but not after, developed permanent penile abnormalities and loss of fertility, implying postnatal days 1–12 as the period when penis was sensitive to estrogen exposure (Goyal et al. 2005b). Additional observations confirmed that the magnitude of these effects (for example, increase in the number, size and percent area of adipocytes) was higher in the 1–6 day group than in the 7–12 day group determined 1–6 postnatal days as the most sensitive period. It is noteworthy here that the rat penis during this estrogen-sensitive period contains only stromal cells in the corpora cavernosa (lacks smooth muscle cells and cavernous spaces) and thus is similar developmentally to the human penis in the first and second trimesters of pregnancy (Williams-Ashman & Reddi 1991, George & Wilson 1994, Klonisch et al. 2004).
Whether the observed effects of estrogen on penile dysmorphogenesis result from an ER-mediated pathway or an AR-mediated pathway, or both ways remains unclear. Support for an ER-mediated pathway comes from observations that both ER and ß are present in stromal cells in 1-day-old rat penis (Jesmin et al. 2002, Mowa et al. 2006), estrogens inhibit proliferation of smooth muscle of injured blood vessels (Goyal & Oparil 2001), and the estrogen metabolite, 2-methoxyestradiol, inhibits angiogenesis (Fotsis et al. 1994). Additionally, ER up-regulation is associated with abnormal development in rodents of the female reproductive tract (Yamashita et al. 1990, Markey et al. 2005), mammary gland (Tekmal et al. 2005), male reproductive tract (Sato et al. 1994), prostate gland (Prins & Birch 1997, Prins et al. 2001), and seminal vesicles (Williams et al. 2001). In addition, ER overexpression is shown to inhibit growth and angiogenic factors in the endometrial carcinoma cell line Ishikawa (Ali et al. 2004); and ER is the main regulator of estrogenic effect on adipose tissue, especially that an alteration in estrogen/ER signaling during development results in dramatic changes in adipocyte number (Cooke & Naaz 2004). Thus, evidence in our studies of association between estrogen-inducible penile abnormalities and enhanced ER expression, mitigation of estrogen-inducible penile deformities by ER antagonist ICI 182 780, and lack of estrogen-inducible abnormalities in the penis of ERKO mice indicates that a functional ER pathway is essential in induction of aberrant penile development.
Alternatively, ARs are present throughout the body of the rat penis (Goyal et al. 2004b); AR concentration reaches a peak level at or prior to puberty when stromal cells are differentiating into smooth muscle cells and cavernous spaces (Rajfer et al. 1980, Takane et al. 1990); and neonatal estrogen exposure reduces AR expression in male reproductive organs (Prins & Birch 1995, McKinnell et al. 2001, Williams et al. 2001, Woodham et al. 2003) and lowers plasma testosterone (Sharpe et al. 1998, Atanassova et al. 2000). Additionally, the coadministration of testosterone with DES prevents most of the histopathological abnormalities affecting the male reproductive tract in rats (Rivas et al. 2003). Low androgen in vitro inhibits smooth muscle differentiation and promotes adipocyte differentiation in pluripotent mesenchymal cells (Bhasin et al. 2003, Singh et al. 2003); and castration induces fat deposition and loss of smooth muscle cells in the corpora cavernosa of the rabbit penis (Traish et al. 2005).
Collectively, the above observations suggest a lower testosterone and/or decreased AR activation as factors contributing indirectly to development of estrogen-inducible penile abnormalities. Our observations that coadministration of testosterone or DHT negates estrogen-inducible penile abnormalities and that estrogen exposure for 1–3 postnatal days reduces the perinatal testosterone surge by almost 90% at postnatal day 5 or 8 (Goyal et al. 2005b), the developmental period when stromal cells start differentiation into smooth muscle cells in the rat penis (Murakami 1986, 1987, provide credence to the indirect effect hypothesis. In other words, the perinatal testosterone surge, typical for rodents from late gestation to first week (Ward & Weisz 1984, El-Gehani et al. 1998), may be a natural mechanism of dampening ER expression in stromal cells and, thereby, safeguarding their normal differentiation into smooth muscle cells and endothelial-lined cavernous spaces at a critical period of penile development.
Hence, our working hypothesis of estrogen-inducible abnormal development of the penis in our rat model is as follows (Fig. 5): neonatal estrogen exposure inhibits the perinatal testosterone surge by the fetal Leydig cells; the resulting lower androgen action up-regulates ER in penile stromal cells; ER overexpressing stromal cells, when exposed to sufficient estrogen, are reprogrammed toward increased adipogenesis (in other words, a mere ER overexpression may not be adequate for stromal cell reprogramming).
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in stromal cells, normal stromal cell differentiation); testosterone coadministration will reverse DES effects by acting primarily at the stromal cells (lower testosterone surge, low ER in stromal cells, and normal stromal cell differentiation); and anti-androgenic action alone, resulting from gonadotrophin-releasing hormone antagonist (GnRH-A) and/or flutamide treatments, will up-regulate ER in stromal cells, but they may or may not be reprogrammed toward increased adipogenesis because of insufficient estrogen exposure.
In summary, we describe a rat model of permanently abnormally developed penis resulting from neonatal DES exposure for a very short period (as short as for 1–3 postnatal days) and at a moderate dose (0.1 mg/kg body weight; note this dose is miniscule in comparison to a total median dose of 200 mg/kg given to a cohort of women at the Boston Lying-in hospital in 1950–1970, Heinonen 1973). Additional experiments using pharmacological approaches (ER antagonist ICI 182 780, AR agonist DHT, centrally acting anti-androgen GnRH-A, and peripherally acting anti-androgen flutamide) are warranted to further understand mechanisms whereby increased ER activity or lower AR activity or both reprogram stromal cells such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated. These findings will be significant because humans and animals are continuously exposed to environmental estrogens (referred to as endocrine disruptors) and anti-androgens (vinclozolin and phthalates) and thus may be more prone to higher incidence of reproductive disorders.
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Acknowledgements |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMorphology of the rat...Neonatal exposure to estrogen...Penile dysmorphogenesis is dose-...Penile dysmorphogenesis is...ER{alpha} presence is essential...Penile dysmorphogenesis is...ER antagonist or androgen...Mechanism of estrogen-induced...AcknowledgementsReferences |
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