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Biol Reprod. 2009 September; 81(3): 507–516.
Published online 2009 May 6. doi:  10.1095/biolreprod.108.071951
PMCID: PMC2731982

Estrogen-Induced Developmental Disorders of the Rat Penis Involve Both Estrogen Receptor (ESR)- and Androgen Receptor (AR)-Mediated Pathways1

Abstract

This study tested the hypothesis that the estrogen receptor (ESR) pathway, androgen receptor (AR) pathway, or both mediate estrogen-induced developmental penile disorders. Rat pups received diethylstilbestrol (DES), with or without the ESR antagonist ICI 182,780 (ICI) or the AR agonist dihydrotestosterone (DHT) or testosterone (T), from Postnatal Days 1 to 6. Testicular T concentration, penile morphology and morphometry, and/or fertility was determined at age 7, 28, or 150 days. DES treatment alone caused 90% reduction in the neonatal intratesticular T surge; this reduction was prevented by ICI coadministration, but not by DHT or T coadministration. Unlike the T surge, coadministration of ICI and coadministration of DHT or T mitigated penile deformities and loss of fertility. Generally, ICI, DHT, or T treatment alone did not alter penile morphology; however, fertility was 20% that of controls in ICI-treated rats vs. 70%–90% in DHT- or T-treated rats. The lower fertility in the rats treated with ICI alone could be due to altered sexual behavior, as these males did not deposit vaginal plugs. In conclusion, observations that both an ESR antagonist and AR agonists prevent penile deformities and infertility suggest that both pathways are involved in estrogen-induced penile disorders. Observations that coadministration of ICI, but not DHT or T, prevents the DES-induced reduction in the neonatal T surge suggest that, although ICI exerts its mitigating effect both at the level of Leydig cells and penile stromal cells, DHT and T do so only at the level of stromal cells.

Keywords: androgen receptor, erectile dysfunction, estradiol, estradiol receptor, estrogen, estrogen receptor, penis, testosterone

INTRODUCTION

According to a 2001 World Health Organization report, at least 80 million people worldwide are affected by infertility, and at least 40% of them are males [1]. Although causes of infertility in most men are multifactorial, exposure to environmental estrogens during development has been linked with higher incidence of reproductive disorders and cancers [2, 3]. More than 2 million male offspring of women treated worldwide with diethylstilbestrol (DES) to prevent miscarriages between 1950 and 1970 have higher incidences of testicular cancer, cryptorchidism, hypospadias, and smaller penis [4, 5]. Pregnant mothers with high intake of phytoestrogens are more likely to give birth to boys with hypospadias [6]. A drop in the ratio of boys to girls in the United States and Japan since 1970 is suspected from prenatal exposure to estrogens [7]. Laboratory animals exposed during development to estrogens develop hypospadias [8] and are predisposed to a precancerous growth of the prostate in adulthood by an epigenetic mechanism [9]. Alligators from Lake Apopka, FL, which is contaminated with industrial estrogenic chemicals, have smaller phalluses [10, 11]. Turtles from Moody Pond, MA, exposed to xenobiotic contaminants have impaired reproductive functions [12]. Thus, perinatal exposure to estrogens has permanent, even transgenerational, deleterious effects on the development of reproductive organs in both humans and wildlife; however, mechanisms underlying these reproductive disorders are not well understood.

Our long-range goal is to understand cellular and molecular mechanisms of estrogen-inducible developmental reproductive disorders in males. In this pursuit, we previously reported a rat model of estrogen-inducible permanently maldeveloped penis characterized by accumulation of fat cells and loss of smooth muscle cells and cavernous spaces in the corpus cavernosum, but not in the corpus spongiosum, part of the body of the penis [13]. These region-specific effects of neonatal estrogen exposure in the body of the penis are dose dependent [14], require a critical window of exposure [15], and are associated with suppressed neonatal testosterone (T) surge from ages 5 to 8 days [15] and with up-regulation of estrogen receptor alpha (ESR1) in penile stromal cells [16]. In addition, observations that Esr1-knockout (Esr1−/−) mice are resistant to estrogen-inducible penile abnormalities present in the wild-type littermates imply an unequivocal role for ESR1 in mediating maldevelopment of the penis [17].

The objective of this study was to test the hypothesis that estrogen-inducible developmental penile deformities are mediated via the ESR pathway and/or the androgen receptor (AR) pathway. The working model is that estrogen exposure down-regulates neonatal T surge, which in turn reprograms stromal cell differentiation toward increased adipogenesis in the body of the penis. In other words, the perinatal T surge, typical for rodents from late gestation to the first week of life [18, 19], may be a natural mechanism of safeguarding normal differentiation of stromal cells into smooth muscle cells and cavernous spaces at a critical period of penile development. Hence, we aimed to determine the effect of blocking the ESR pathway or activating the AR pathway on the perinatal T surge, penile development, and fertility by treating neonatal rat pups with DES, with or without coadministration of the ESR antagonist ICI 182,780 (ICI) or the AR agonist dihydrotestosterone (DHT) or T.

MATERIALS AND METHODS

Animals and Treatments

Neonatal and/or adult Sprague-Dawley male and female rats (Harlan Sprague Dawley, Indianapolis, IN) were maintained at 22–23°C ambient temperature, 55%–60% relative humidity, and 12L:12D cycle and had free access to food (Rodent Chow 5001; Purina Mills, St. Louis, MO) and water for 24 h. Animals were handled in accord with the guidelines stipulated in the Guide for the Care and Use of Laboratory Animals (Washington, DC: Institute of Laboratory Animal Resources, National Research Council, National Academy Press; 1996). The Tuskegee University Institutional Animal Care and Use Committee approved all animal procedures.

Timed-pregnant female rats were housed individually. Within 24 h of delivery, seven to eight male pups from different litters (one pup from each litter to avoid any litter effect) were assigned to each group, and the number of pups per group was adjusted to eight with an extra female, where appropriate. Pups received daily s.c. injections of 25 μl of olive oil containing DES, with or without the ESR antagonist ICI or the AR agonist DHT or T. Daily doses per pup used for each of these compounds were as follows: DES (1 μg [0.1 mg/kg]), DHT (200 μg [20 mg/kg]), T (200 μg [20 mg/kg]), and ICI (250 μg [25 mg/kg]). The dosage in milligrams per kilograms of weight was calculated by averaging the mean weight of pups on Postnatal Day 1 (6.5 g) and on Postnatal Day 6 (13.5 g), as most pups received treatments from Postnatal Days 1 to 6 (with Postnatal Day 0 being the day of birth). Controls received oil only. The dose and duration of DES treatment were selected based on data from our previous studies [14, 15] in which a similar treatment regimen resulted in permanent penile maldevelopment and infertility in 100% of the treated rats. Likewise, these doses of ICI [20], DHT, and T [21] have been shown to block estrogenic effects in the male reproductive organs. Animals were killed by asphyxiation with CO2 in a closed chamber. At different developmental periods, animals were examined, and tissues were collected as described herein.

Neonatal Animals

The objective of this study was to determine the extent to which DES treatment suppresses the neonatal (age 4–7 days) intratesticular T surge and the extent to which this suppression can be prevented by the ESR antagonist ICI and the AR agonists DHT and T. In experiment 1, pups received daily injections of DES or oil from Postnatal Days 1 to 3, 1 to 4, or 1 to 5, and testes were collected at ages 4, 5, and 6 days, respectively. In experiment 2, pups received daily injections of DES, DES + ICI, DES + DHT, DES + T, ICI, DHT, T, or oil from Postnatal Days 1 to 6, and testes were collected at age 7 days. Testes from both experiments were frozen at −20°C until assayed for intratesticular T concentration using a COAT-A-COUNT T radioimmunoassay (Siemens Corp., New York, NY) as described previously by our laboratory [15]. Briefly, 10–30 mg of testicular tissue was homogenized in PBS. Eight volumes of ethyl ester were added to the homogenate and vortexed vigorously. The aqueous phase was snap frozen, and the organic supernatant was transferred to a secondary tube and air dried. Just before assay, samples were resuspended in PBS. The sensitivity of the assay was 0.2 ng/ml. All samples were quantified in a single assay, and the intraassay coefficient of variation was 7%.

Prepubertal Animals

The objective of this study was to determine the extent to which the coadministration of ICI, DHT, or T with DES mitigates DES-induced effects on the prepubertal (age 28 days) penis and other related parameters. Neonatal pups received daily injections of DES, DES + ICI, DES + DHT, DES + T, ICI, DHT, T, or oil from Postnatal Days 1 to 6. Various tissues, including the penis, penile skeletal muscles, testes, and blood, were collected at age 28 days and examined for different parameters as described in Adult Animals.

Adult Animals

The objective of this study was to determine the extent to which the coadministration of ICI, DHT, or T with DES mitigates DES-induced effects on the adult (age 130–150 days) penis and other related parameters, as well as on fertility. To prevent overcrowding of animals at a given time and to include sufficient numbers of animals in a treatment, the study was completed in two replicates. In the first replicate, neonatal pups received daily injections of DES, DES + ICI, DES + DHT, ICI, DHT, or oil from Postnatal Days 1 to 6. In addition, two groups received daily injections of DES from Postnatal Days 1 to 6, followed by daily injections of DHT from Postnatal Days 7 to 12 in the first group and from Postnatal Days 13 to 18 in the second group. The rationale was to determine whether DHT treatment outside of the time of DES exposure mitigates DES-induced penile and other related developmental abnormalities. In the second replicate, treatments were the same, except that T was used instead of DHT and treatments were given from Postnatal Days 1 to 6 only. During the course of the study, animals were examined for testicular descent (age 22–38 days), preputial sheath release (age 38–70 days), and fertility (age 90–120 days). Animals were killed at age 130–150 days, and tissues were collected and processed for various parameters as described herein. Animals from both trials were combined for statistical analysis, as there was no significant (P < 0.05) replicate effect on the weight of the testis, penis, or bulbospongiosus muscle or on the length of the penis.

Descent of Testes

Testicular descent was observed every other day from ages 22 to 38 days. Testes were characterized as fully descended when they were palpated in the scrotum while holding the animal in a supine position. The formation of a scrotal bulge marked the descent of testes.

Release of Preputial Sheath

The preputial sheath was examined every third day from ages 38 to 70 days. While holding the animal in a supine position, the prepuce was gently pushed proximally and was characterized as fully released when it completely retracted from the glans penis.

Body and Testes Weights

Animals were weighed just before necropsy. Testes were freed of the epididymis and adjoining fat before recording their weights. After weighing, the left testis and epididymis were frozen at −20°C until thawed for sperm count. The right testis was used for T assay as described herein.

Penis and Penile Skeletal Muscles

The penis was measured for length and weight and was processed for histopathology and histochemistry as described previously by our laboratory [14, 15]. Briefly, the penis was exposed up to the ischial arch, and its stretched length was measured from the tip of the glans penis to the midpoint of the ischial arch. For histopatholgy, tissues were fixed in 10% formaldehyde for 2–3 days, and 5-μm-thick paraffin sections were stained with hematoxylin-eosin. For histochemical demonstration of fat, formaldehyde-fixed tissues were stained en bloc for 8 h with 1% osmium tetroxide dissolved in 2.5% potassium dichromate solution and then processed for paraffin embedding. In addition, the os penis was radiographed (one animal per group), and the bulbospongiosus and levator ani muscles were isolated and weighed [14, 15]. Montage figures were assembled using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).

Daily Sperm Production, Epididymal Sperm Reserves, and Sperm Morphology

The daily sperm production and sperm reserves in the tail of the epididymis (measures of efficiency of spermatogenesis), as well as sperm morphology (a measure of normal spermatogenesis), were examined as described previously [15]. For sperm morphology, sperm were collected from the right tail of the epididymis, near its junction with the ductus deferens, in 2 ml of PBS containing 10 μl of 10% formalin. A 10-μl drop of the diluted sample was placed on a glass slide, covered with a coverslip, and examined using a phase-contrast microscope (400× total magnification). Two hundred sperm from each animal were evaluated and classified as normal, head defect, midpiece defect, principal piece defect, proximal droplet, distal droplet, or detached head [22].

Fertility

Six to 12 adult male rats for each group from two replicates were transferred to mating cages floored with a mesh grid and were cohabitated with untreated 70-day-old females (1:1) for 12 days. Cages were checked twice daily for the presence of copulatory plugs. The plug-positive females were separated and evaluated for the presence of sperm in vaginal washings. Females were allowed to deliver, and the number of pups per litter was recorded.

Plasma and Intratesticular T

For plasma T, one blood sample was collected from the heart of each animal just before complete asphyxiation with CO2; for intratesticular T, a part of the right testis was collected from each animal at the time of necropsy. Both plasma parenchyma and testicular parenchyma were frozen at −20°C until assayed as described in Neonatal Animals, except that approximately 100 mg of testicular tissue was homogenized for intratesticular T.

Statistical Analysis

Statistical analyses were performed using ProStat statistical software (Polysoftware International, Pearl River, NY). ANOVA was performed on all parameters, except fertility data, which were analyzed by Fisher exact test (see Table 4). Treatment groups with means significantly different (P < 0.05) from those of controls were identified using Duncan test or Student t-test. When data were not distributed normally or heterogeneity of variance was identified, analyses were performed on transformed data or ranked data. Data are expressed as the mean ± SEM throughout the text.

TABLE 4.
Effect of neonatal DES exposure, with or without ESR antagonist ICI 182,780 or AR agonist dihydrotestosterone (DHT) or testosterone (T), on fertility in male rats.a

RESULTS

Neonatal Intratesticular T Surge

The mean concentrations of neonatal intratesticular T surge in control rats were 1474 ± 403 ng/g at age 4 days, 952 ± 112 at 5 days, and 633 ± 195 at 6 days (Fig. 1A). DES treatment, regardless of whether the treatment was from Postnatal Days 1 to 3, 1 to 4, or 1 to 5, reduced the surge by more than 90%; this level of decline had already occurred by age 4 days (Fig. 1A). DES treatment from Postnatal Days 1 to 6 caused a similar percentage of decrease in the surge at age 7 days in terms of both testicular T per gram of tissue (Fig. 1B) and total testicular T per testis (Fig. 1C). The coadministration of ICI with DES restored the surge to the control level, but the coadministration of DHT or T with DES had virtually no restorative effect. While ICI treatment alone had no diminutive effect on the surge, DHT or T treatment alone (similar to DES treatment) reduced the surge by more than 90%.

FIG. 1.
A) Testicular T concentration (ng/g) in rat pups at ages 4, 5, and 6 days treated, respectively, with DES from Postnatal Days 1 to 3, 1 to 4, and 1 to 5. B and C) Testicular T concentration (ng/g and ng/testis) in rat pups at age 7 days treated with DES, ...

Plasma and Intratesticular T at Prepuberty and Adulthood

The mean plasma and intratesticular T concentrations in control rats were 0.16 ± 0.04 ng/ml and 16.00 ± 1.63 ng/g at age 28 days and 1.20 ± 0.11 ng/ml and 140.00 ± 22.47 ng/g at adulthood, respectively. Neither plasma nor intratesticular concentration at age 28 days or at adulthood was significantly (P < 0.05) different from that of controls in any of the treatment groups regardless of whether the treatment included DES (with or without ICI, DHT, or T) or the treatment included ICI, DHT, or T alone (data not shown).

Body Weight

The mean body weight at age 28 days in control rats was 77.1 ± 3.1 g, which was not significantly (P < 0.05) altered in any of the treatment groups (data not shown). Similarly, the mean body weight at adulthood was similar between controls (467.2 ± 11.6 g) and all treated groups (Table 1), except in the group that received DES (Postnatal Days 1–6) + DHT (Postnatal Days 13–18), in which it was significantly (P < 0.05) increased to 528.0 ± 15.9 g.

TABLE 1.
Effect of neonatal DES exposure, with or without ESR antagonist ICI 182,780 or AR agonists dihydrotestosterone (DHT) or testosterone (T), on mean (± SEM) body weight and relative organ weight (per 100 g body weight) of the paired testes, bulbospongiosus ...

Testis Weight

The mean weight of both testes at age 7 days in control pups was 27.5 ± 1.0 mg, which was decreased by 13%–16% in all treated groups, except in the groups treated with DES or T alone, in which the decrease was almost 30% (Fig. 2A). The mean weight of both testes at age 28 days in control rats was 0.630 ± 0.030 g, which was decreased by almost 50% in pups that received DES alone; this decrease was completely negated by ICI coadministration with DES but was not affected by DHT or T coadministration (Fig. 2B). While ICI treatment alone had no diminutive effect on the testis weight at Day 28, DHT or T treatment alone reduced it by 20%–30%. Similar to the 28-day-old group, the mean testis weight at adulthood was decreased by almost 30% as a result of DES treatment (Fig. 2C and Table 1). ICI coadministration with DES restored it to the control level. Neither T nor DHT coadministration with DES, regardless of the postnatal period of DHT coadministration (age 1–6, 7–12, or 13–18 days), had any restorative effect. While ICI or T treatment alone had no significant diminutive effect on the testis weight, DHT treatment alone reduced it significantly (P < 0.05) by almost 15%.

FIG. 2.
The absolute paired testis weight at ages 7 (A), 28 (B), and 130–150 (C) days in rats treated with DES, with or without coadministration of ICI, DHT, or T, from Postnatal Days 1 to 6, except in the case of DHT, which was also coadministered from ...

Descent of Testes

Testes descended at age 24–25 days in controls, but the descent was delayed by 8–12 days in the DES-treated group (Table 2). The delay was partially mitigated in rats that received DES + ICI (age 25–29 days) or DES + DHT (age 29–33 days), regardless of the postnatal period of DHT administration. The descent time in the rats treated with ICI alone was similar to that of controls (age 25 days), but it was delayed by 4–6 days in rats treated with DHT alone (age 29–31 days).

TABLE 2.
Effect of neonatal DES exposure, with or without ESR antagonist ICI 182,780 or AR agonist dihydrotestosterone (DHT), on testicular descent and preputial sheath release.a

Release of Preputial Sheath

The preputial sheath was separate from the glans penis at age 47–50 days in the control rats, in contrast to at least age 70 days in the rats treated with DES alone (Table 2). The release time was essentially similar between controls and the DES + ICI-treated rats (age 49–52 days), but it was delayed by 4–11 days in rats treated with DES + DHT on Postnatal Days 1–6 and by 13–19 days in rats treated with DES + DHT on Postnatal Days 7–12. It was still attached at age 70 days in rats treated with DES + DHT on Postnatal Days 13–18. ICI treatment alone had no delaying effect (age 49–52 days), but DHT treatment alone delayed the release by 2–6 days (age 52–56 days).

Penile Measurements

The mean weight and length of the penis at age 28 days in the rats treated with DES alone were reduced, respectively, to almost 55% and 85% those of controls (55 ± 2 mg and 19.4 ± 0.2 mm in controls) (Fig. 3A). Both parameters were increased to 100% those of controls by ICI coadministration, in contrast to 80%–90% those of controls by DHT or T coadministration with DES. Neither parameter was different from that of controls in animals that received ICI, DHT, or T alone. At adulthood, both parameters in the rats treated with DES alone were significantly (P < 0.05) decreased at percentage levels similar to those observed at age 28 days (Fig. 3B and Table 1), and both were restored to almost the control level by ICI, T, or DHT coadministration with DES on Postnatal Days 1–6. Coadministration with DHT on Postnatal Days 7–12 increased both parameters significantly (P < 0.05) compared with the group treated with DES alone (Table 3); however, they were still significantly lower than those of controls. Conversely, DHT coadministration on Postnatal Days 13–18 further exacerbated DES-induced decreases in the penile weight (P < 0.01) (Table 3) and length (P < 0.001). Neither parameter was different from that in controls in animals that received ICI, DHT, or T alone.

FIG. 3.
The absolute penis weight and length at ages 28 (A) and 130–150 (B) days in rats treated with DES, with or without coadministration of ICI, DHT, or T, from Postnatal Days 1 to 6, except in the case of DHT, which was also coadministered from Postnatal ...
TABLE 3.
Comparison of mean (±SEM) measurements of the length and weight of the penis and weight of the bulbosponsiosus muscle between and among the DES, DES+DHT (1–6d), DES+DHT (7–12d), DES +DHT (13–18d) groups.

Penile Skeletal Muscles

At age 28 days, the mean weight of the bulbospongiosus was reduced to 60% that of controls (26 ± 1 mg vs. 41 ± 3 mg in controls) as a result of DES treatment. The coadministration of ICI, DHT, or T increased it to almost the control level, and ICI, DHT, or T treatment alone had no diminutive effect (data not shown). Similar to the 28-day-old group, the bulbospongiosus muscle at adulthood exhibited an almost 40% decrease in weight as a result of DES treatment (592 ± 30 mg vs. 908 ± 20 mg in controls), exhibited mitigation of this decrease by 80%–100% as a result of ICI, T, DHT coadministration on Postnatal Days 1–6, and exhibited no significant diminutive effect as a result of ICI, DHT, or T treatment alone (Fig. 4 and Table 1). Conversely, DHT coadministration on Postnatal Days 7–12 or 13–18 further exacerbated DES-induced weight loss, although significantly so (P < 0.05) only in the case of the group treated on Postnatal Days 13–18 (447 ± 20 mg vs. 592 ± 30 mg in the DES group) (Table 3). Generally, the mean weight of the levator ani did not differ between controls and any of the treated groups at age 28 days (20 ± 1 mg in controls [other data not shown]) or at adulthood (258 ± 9 mg in controls [other data not shown]), except in the DHT group treated on Postnatal Days 13–18, in which it was significantly reduced to 202 ± 11 mg.

FIG. 4.
The absolute bulbospongiosus muscle weight at age 130–150 days in rats treated with DES, with or without coadministration of ICI, DHT, or T, from Postnatal Days 1 to 6, except in the case of DHT, which was also coadministered from Postnatal Days ...

Penile Histopathology and Histochemistry

Grossly, the rat penis consists of a cylindrical body, a bulbous glans penis, a right angle between the body and the glans, and an os penis within the glans (Fig. 5). Histologically, the body of the penis consists of three erectile bodies: two corpora cavernosa that are located dorsolateral to the urethra and a corpus spongiosus that surrounds the urethra (Fig. 6). Both the corpora cavernosa and the corpus spongiosus contain smooth muscle cells and cavernous spaces (also called sinusoids or blood vessels), but both of these structures are more developed in the corpora cavernosa. Because treatment effects were similar in the 28-day-old and adult groups, they are described in the adult group only. Grossly, DES treatment caused malformation of the penis as characterized in radiographs by rarefaction of the body of the penis and underdevelopment and undercalcification of the os penis (Fig. 5). The coadministration of ICI, T, or DHT with DES on Postnatal Days 1–6 prevented these developmental gross anomalies; however, the level of prevention was marginal in rats coadministered DHT on Postnatal Days 7–12 or 13–18, especially in the latter (Fig. 5). Histopathologically, DES-induced changes were present only in the corpora cavernosa and included loss of smooth muscle cells and cavernous spaces, accumulation of fat cells, and reduction in the thickness of the tunica albuginea capsule surrounding the corpora cavernosa (Fig. 6). Generally, the coadministration of ICI, T, or DHT on Postnatal Days 1–6 mitigated histopathological abnormalities; however, the level of mitigation was somewhat limited in rats coadministered DHT on Postnatal Days 7–12 and was virtually nonexistent in rats coadministered DHT on Postnatal Days 13–18 (Fig. 6). None of the DES-induced gross or histopathological abnormalities were observed in rats that received ICI, DHT, or T alone.

FIG. 5.
Radiographs of the penis in adult rats treated neonatally with oil (control) or DES, with or without coadministration of ICI, DHT, or T, from Postnatal Days 1 to 6, except in the case of DHT, which was also coadministered from Postnatal Days 7 to 12 or ...
FIG. 6.
Micrographs of the body of the penis in adult rats treated neonatally with oil (control) or DES, with or without coadministration of ICI, DHT, or T, from Postnatal Days 1 to 6, except in the case of DHT, which was also given from Postnatal Days 7 to 12 ...

Daily Sperm Production, Epididymal Sperm Reserves, and Sperm Morphology

Compared with controls, both the daily sperm production per gram of testicular parenchyma and the total number of sperm per testis were 10%–20% lower in all treated groups, and the decrease was significant (P < 0.05) in most cases (Fig. 7). Conversely, the total number of sperm reserve in the tail of the epididymis was significantly (P < 0.05) lower only in the case of rats treated with DES alone (132. 2 ± 7 × 106 vs. 184.6 ± 14.8 × 106 in controls). The mean percentage of normal sperm (95% in controls), as well as the mean incidence of abnormalities affecting the head, midpiece, and principal piece of the tail, was similar between controls and all treated groups (data not shown).

FIG. 7.
Daily sperm production (DSP) per gram of testicular parenchyma and total numbers of sperm per testis at age 130–150 days in rats treated with DES, with or without coadministration of ICI or T, from Postnatal Days 1 to 6. Data are expressed as ...

Fertility

While 10 of 11 control males (91%) sired pups and deposited plugs and sperm in the vagina, none did so in males treated with DES alone or DES coadministered with DHT on Postnatal Days 7–12 or 13–18 (Table 4). Conversely, ICI, T, and DHT coadministration on Postnatal Days 1–6 restored fertility to 73%, 78%, and 47% of the control level, respectively. Fertility in male rats treated with T and DHT alone was 96% and 78% that of controls, respectively, but was only 20% that of controls in rats treated with ICI alone. In addition, only three to four of 11 males in the latter group deposited plugs and/or sperm in the vagina.

DISCUSSION

Data from the present study showing that DES-inducible developmental abnormalities in the penis (including delayed preputial sheath separation, malformation of the os penis, reductions in the length and weight of the penis, and accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis) are mitigated completely by the ESR antagonist ICI and almost completely by the AR agonists DHT and T provide evidence that both ESR and AR pathways mediate maldevelopment of the penis. The role of the ESR pathway in mediating penile maldevelopment should not be surprising, as both ESR1 and ESR2 are present in the neonatal rat penis [16, 20] and Esr1-knockout (Esr1−/−) mice are resistant to estrogen-inducible abnormalities observed in the wild-type mice [17]. Estrogen-induced histopathological effects have been reported in a rabbit model in which bisphenol A [23] or tetrachlorodibenzodioxin [24] treatment at puberty has resulted in deposition of fat and reduction in cavernous spaces in the body of the penis. An alteration in estrogen/ESR1 signaling during development results in dramatic changes in adipocyte number [25]. Laboratory animals exposed neonatally to estrogens develop a smaller penis [8]. Alligators have smaller phalluses in Florida's Lake Apopka, which is contaminated with industrial estrogenic chemicals [10, 11]. Male offspring of 2–3 million women worldwide exposed to DES during pregnancy from 1950 to 1970 have higher incidences of penile abnormalities, as well as a smaller penis [4, 5, 26].

Similarly, the role of the AR pathway in mediating penile maldevelopment should be expected, as ARs are present at peak concentration in the prepubertal rat penis [27, 28] and castration in rats at birth produces penile deformities, which are partially corrected with androgen substitution at the time of castration [29]. Rodents treated perinatally with the AR antagonist vinclozolin [30] or flutamide [31], the T synthesis inhibitor phthalates [32], or the 5α-reductase type II inhibitor finasteride [33] develop male reproductive tract abnormalities, including a malformed penis. Development of the os penis is androgen dependent in both rats and mice [34, 35], antiandrogen treatment with flutamide results in underdevelopment and undercalcification of the os penis [36], and castration induces fat deposition and loss of smooth muscle cells in the corpora cavernosa of the rabbit penis [37].

Our observations that both the ESR antagonist ICI and the AR agonists DHT and T mitigate DES-induced penile developmental abnormalities but that only ICI mitigates DES-induced suppression in the neonatal T surge suggest an indirect mode of action. This also means that normal development of the rat penis during Postnatal Days 1–6 is dependent on activation of ARs, which is initiated by the neonatal T surge in the case of control rats and by exogenous DHT or T in the case of DES-treated rats. Additional data that rats treated with DHT or T alone have a normal penis but a suppressed neonatal T surge, as well as that the DES-treated rats receiving DHT on Postnatal Days 1–6 (but not on Postnatal Days 7–12 or 13–18) develop a normal penis, provide further support to the concept that AR activation during Postnatal Days 1–6 is critical for normal development of the rat penis. In terms of the development of reproductive organs in humans, this period roughly corresponds to the following: the end of the first trimester and the beginning of the second semester of pregnancy [38, 39], the fetal T surge [39], and when a cohort of daughters were exposed to large doses of DES [26].

It is generally accepted that the perinatal T surge in rodents extends from late gestation (Days 18–19) to the first week of life, with the highest level of T secretion from 18 to 19 days of gestation [18, 19]. The present data support these observations and provide evidence for a day-to-day gradual decline in the neonatal T surge from ages 4 to 7 days (1474 ± 403 ng/g at 4 days to 280 ± 70 ng/g at 7 days), which reaches almost a nadir level between ages 8 and 12 days [15]. In addition, it is reported that the serum level of total androgens is 3-fold to 4-fold higher than that of T alone in neonatal male rats, especially on Postnatal Days 1–2 [40], and that the basal level of total androgens secreted by the progenitor Leydig cells is 14 times higher than that of T alone at age 21 days [41]. These observations imply that other androgens, in addition to T, are important in the masculinization and development of the neonatal male reproductive tract.

An important question that needs to be answered is how does DES-induced suppression in the neonatal T surge divert penile differentiation toward increased adipogenesis? In this connection, it is relevant to know that differentiation of stromal cells into cavernous spaces and smooth muscle cells in the body of the rat penis does not begin until age 5–6 days [34, 35] and that these stromal cells express higher ESR1 in response to neonatal estrogen exposure [16]. In light of this information, it is hypothesized that the lower androgenic action resulting from estrogen-induced inhibition of the neonatal T surge up-regulates ESR1 expression in penile stromal cells, which are then reprogrammed toward enhanced adipogenesis. Supporting this hypothesis are our recent findings that the expression of peroxidase proliferator-activated receptor, a marker for differentiation of preadipocyte to adipocyte [42], is more than 6-fold higher in the rat penis treated neonatally with DES than in controls, as well as its restoration to the control level by the ESR antagonist ICI [43].

In agreement with previous studies on the effects of estrogen exposure on the testis [4447] and penile skeletal muscles [14, 15], weights of both organs were significantly reduced at age 28 days and at age 130–150 days. However, an important finding of the present study was a differential response of the testis to ICI, DHT, and T exposures. While the testis weight at age 28 days and at age 130–150 days was not different from that of controls in rats treated with ICI alone, it was significantly lower at age 28 days in rats treated with DHT or T alone and was significantly lower at age 130–150 days in rats treated with DHT alone. Likewise, although the coadministration of ICI restored DES-induced testicular weight loss to the control level in both age groups, the coadministration of DHT or T had minimal to no restorative effect in either age group. These differences may be attributed to various effects of these compounds on the neonatal T surge, which is reduced to 10% that of controls in the case of DHT or T exposure but is not altered from that of controls in the case of ICI. Alternately, differences in susceptibility to estrogens and androgens may have some role, as the rat testis contains both ESRs [48, 49] and ARs [50, 51].

A noteworthy effect of neonatal estrogen exposure that, to our knowledge, is reported for the first time is an exaggeration of DES-induced significant decline in the weight of the bulbospongiosus muscle and in the weight and length of the penis in rats receiving DHT on Postnatal Days 13–18. These observations imply that DHT exposure on Postnatal Days 13–18 is detrimental for the later development of the bulbospongiosus muscle and penis. These findings are clinically significant in light of the controversy as to whether androgen therapy for treating micropenis should be started before or after puberty, as well as in light of the information that androgen administration to prepubertal, but not postpubertal, hypogonadotropic hypogonadal rats resulted in a shorter penis at adulthood [52].

Another notable, but unexpected, finding of the study is the inability of 70%–80% of the rats treated with ICI alone to sire pups or to deposit plugs or sperm, in contrast to only 10%–20% of the males treated with DHT or T alone. This was the case in spite of the fact that all three groups of animals had a normal and similarly developed penis, penile skeletal muscles, and testis. In addition, the daily sperm production, total numbers of sperm per testis, caudal sperm reserve, and percentage of normal sperm were also similar. Based on these similarities, the lower fertility in the rats treated with ICI alone is difficult to explain but could be attributed to altered sexual behavior, as they were unable to deposit plugs or sperm. Similar to our results, 75% of male rats treated with ICI at a dose of 10 mg per rat per week from ages 30 to 150 days were infertile; however, unlike our results, the infertility was attributed to faulty spermatogenesis, probably stemming from a buildup of testicular fluid in the seminiferous tubules, rete testis, and efferent ductules [53]. Differences between our results and those of the latter authors may be attributed to differences in the time of treatment (neonatal vs. pubertal to adulthood) and/or the dose of ICI (0.25 mg vs. 10 mg).

In conclusion, adult male rats treated neonatally with DES develop penile deformities, including delayed preputial sheath separation, reductions in penile measurements, and accumulation of fat cells and loss of smooth muscle cells and cavernous spaces in the corpus cavernosum penis; however, all of these developmental malformations are mitigated by coadministration of the ESR antagonist ICI or the AR agonist DHT or T with DES, implying that both ESR and AR pathways mediate maldevelopment of the penis. Furthermore, results support our working model that exposure of neonatal male rats to estrogen suppresses the neonatal T surge; the resulting lower androgenic action during a critical period of development (age 1–6 days) reprograms penile stromal cells toward increased adipogenesis and leads to a permanently maldeveloped penis.

Acknowledgments

We acknowledge the assistance of graduate student Ensa Mathews and of Drs. Prasad Dalvi, Yazeed Abdelmageed, and John Heath. We thank Dr. Tsegaye Habtemariam, dean, for his encouragement and support.

Footnotes

1Supported by NIH grants MBRS-5-S06-GM-08091 (to H.O.G.) and RCMI-5-G128803059 and by USDA grant CSR-EES-ALX-TU-CTIF.

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