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. Author manuscript; available in PMC: 2013 May 14.
Published in final edited form as: J Androl. 2010 Oct 7;32(3):282–294. doi: 10.2164/jandrol.110.010769

Genetically-induced Estrogen Receptor Alpha mRNA (Esr1) Overexpression Does Not Adversely Affect Fertility or Penile Development in Male Mice

John Heath *, Yazeed Abdelmageed *, Tim D Braden , Carol S Williams *, John W Williams **, Tessie Paulose §, Isabel Hernandez-Ochoa §, Rupesh Gupta §, Jodi A Flaws §, Hari O Goyal *
PMCID: PMC3653629  NIHMSID: NIHMS302412  PMID: 20930192

Abstract

Previously, we reported that estrogen receptor alpha mRNA (Esr1) or protein (ESR1) overexpression resulting from neonatal exposure to estrogens in rats was associated with infertility and mal-developed penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing Esr1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls, as a result of Esr1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P < 0.05) lower as compared to controls; however, the weight of the testis, the morphology of the testis and epididymis, and the plasma and testicular testosterone concentration were not different from controls. Hence, the genetically-induced Esr1 overexpression alone, without an exogenous estrogen exposure during the neonatal period, is unable to adversely affect the development of the penis as well as other male reproductive organs, except limited, but significant, reductions in weights of the seminal vesicles and BS/LA muscles.

Keywords: Esr1 overexpression, mutant mice, fertility, penis, testis, seminal vesicles

INTRODUCTION

Infertility affects at least 80 million people world-wide, and nearly 40% of them are males (2001 WHO Report, Barrett, 2006). Epidemiological studies have shown links between exposure to environmental estrogens, also referred to as endocrine disruptors, and increasing frequency of reproductive abnormalities in humans and wildlife (McLachlan et al, 2001; Toppari et al, 1996). More than two million male offspring of women exposed to diethylstilbestrol (DES) during pregnancy have higher incidence of testicular cancer, cryptorchidism, epididymal cysts, and smaller penis (Gill et al, 1979; Swan, 2000). Laboratory animals exposed perinatally to estrogens develop male reproductive tract abnormalities, including hypospadias (McLachlan et al, 1975; Newbold, 2004; Martin et al, 2008), and are predisposed to a pre-cancerous growth of the prostate at adulthood by an epigenetic mechanism (Prins et al, 2007). Alligators from Lake Apopka (FL) contaminated with industrial estrogenic chemicals have smaller phallus (Guillette et al, 1996; Milnes et al, 2005). Thus, perinatal exposure to estrogens has permanent deleterious effects on the development of reproductive organs in both humans and wildlife; however, mechanisms underlying reproductive disorders are not clearly understood.

Estrogens, as well as estrogenic compounds such as methoxychlor and bisphenol A, mediate their effects by binding with estrogen receptors ESR1 and ESR2. Although both receptors are present in the male reproductive tract (Couse et al, 1997; Hess et al, 1997a; Kuiper et al, 1997; Atanassova et al, 2001), including the penis (Jesmin et al, 2002; Goyal et al, 2004a; Mowa et al, 2006), ESR1 plays a critical role in reproductive physiology, as well as pathology. For example, whereas Esr1-knockout (Esr1−1−) male mice are infertile (Eddy et al, 1996) and are resistant to estrogen-inducible reproductive abnormalities observed in the wild-type mice (Prins et al, 2001), Esr2-knockout (Esr2−2−) male mice are fertile (Krege et al, 1998) and develop estrogen-inducible abnormalities similar to those present in the wild-type mice (Prins et al, 2001). Additionally, estrogen-inducible reproductive tract abnormalities are associated with ESR1 or Esr1 up-regulation not only in males (Goyal et al, 2004a; Mansour et al, 2008; Prins and Birch, 1997; Sato et al, 1994; Williams et al, 2001) but also in females (Yamashita et al, 1990; Markey et al, 2005; Tekmal et al, 2005).

Our long-range goal is to understand the mechanisms of estrogen-inducible reproductive disorders, especially affecting the penis in rats. In this regard, we reported previously that neonatal estrogen exposure prior to 12 days of age, especially from 1–6 days of age, resulted in permanent loss of fertility and mal-developed penis, which had an abnormal accumulation of fat cells and loss of smooth muscle cells and cavernous spaces (blood vessels) (Goyal et al, 2005a). Additionally, the mal-development of the penis was associated with ESR1 (Goyal et al, 2004a) and Esr1 up-regulation (Mansour et al, 2008). Both penile mal-development and Esr1 up-regulation were mitigated by ER antagonist ICI 182,780 (Mansour et al, 2008; Goyal et al, 2009). Esr1−1− mice were resistant to estrogen-inducible penile deformities present in the wild-type mice (Goyal et al, 2007). Collectively, the above observations provide evidence that an ESR1-mediated pathway, possibly via Esr1 up-regulation, is critical in inducing the harmful effects of neonatal estrogen exposure in the developing penis.

The objective of the present study is to determine whether genetically-induced Esr1 up-regulation, in the absence of an exogenous estrogen exposure, results in loss of fertility and penile mal-development. To fulfill this objective, we have used, from our collaborators’ laboratory, transgenic mice in which Esr1 is up-regulated throughout the prenatal and postnatal developmental periods in male and female reproductive organs (Tomic et al, 2006, 2007). Specifically, the present study has focused on effects of genetically-induced Esr1 overexpression on fertility-related parameters in males, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, and penile development.

MATERIALS AND METHODS

Animals

Transgenic mice overexpressing Esr1 and control mice were generated and screened/genotyped as described below at the University of Illinois at Urbana-Champaign. Animals were shipped to Tuskegee University at adulthood for further study. Animals at both universities 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 accordance with the guidelines for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy Press, Washington, DC, 1996). All animal procedures were approved by the Institutional Animal Care and Use Committees of the University of Illinois at Urbana-Champaign and Tuskegee University. The fertility study was conducted at 125–140 days of age and tissues were collected following asphyxiation with CO2 at 150–170 days of age.

Generation of Esr1 Overexpressing and Control Mice

The Esr1 overexpressing and control mice used in this study were generated using C57BL6 and FVB mice as previously described (Tomic et al, 2006, 2007). Briefly, transgenic mice carrying a transgene composed of the coding sequence for murine Esr1 placed under the regulatory control of a tet-op promoter (tet-op-Esr1 mice) (Hruska et al, 2002) were mated to tet-op-tTA/tet-op-luciferase mice (Shockett et al, 1995) to produce triple-transgenic mice, tet-op-tTA/tet-op-luciferase/tet-op-Esr1. The transcription of Esr1 transgene is only achieved in the presence of a tetracycline-responsive transactivator (tTA) protein (Fig. 1). In the triple-transgenic mice that were produced, the initial transcription of tTA protein occurs through a leaky transcription from the tet-op promoter, producing only small amounts of tTA protein. However, these small amounts then positively feedback on the tet-op promoter and further increase tTA protein production (Shockett et al, 1995). The tTA protein binds to the tet-op promoter linked to the Esr1 transgene as well, driving the expression of transgenic Esr1 and luciferase. Tet-op-tTA/tet-op-luciferase mice (Shockett et al, 1995) were used as control mice in this study.

Figure 1.

Figure 1

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Transcription of tTA, luciferase, and Esr1 in tet-op-tTA/tet-op-luciferase/tet-op- Esr1 (Esr1 overexpressing) triple-transgenic mice. The transgene Esr1 is subcloned downstream of a tetracycline-responsive promoter (tet-op). Transcriptional activation of this construct is achieved in the presence of a tTA protein. Specifically, tTA protein binds to the tet-op promoter and increases the production of tTA protein through a positive feedback mechanism. In addition, tTA protein binds to the tet-op promoter (in the tet-op-Esr1 transgene and in the tet-op-luciferase) driving the expression of transgenic Esr1 and luciferase.

Screening/genotyping Esr1 Overexpressing and Control Mice

Mice were genotyped using polymerase chain reaction (PCR)–based assays. Briefly, ear punch tissues from pups were lysed in proteinase K buffer (50mM Tris-HCl, 20mM NaCl, 1mM EDTA, and 1% SDS, pH 8.0) containing 1 μl of 20 mg/ml proteinase K (Qiagen Inc., Valencia, CA). Digestion was carried out at 100°C for 3 min. The lysate was then subjected to PCR using primers: (1) 5′-CGAGCTCGGTACCCGGGTCG-3′ and (2) 5′-GAACACAGTGGGCTTGCTGTTG-3′ for tet-op-Esr1 and primers (1) 5′-CGAGCTCGGTACCCGGGTCG-3′ and (2) 5′-GCAAAAGTGAGTATGGTGCC- 3′ for tet-op-tTA. The conditions for tet-op-tTA PCR were 94°C for 3 min of initial denaturation followed by 35 cycles at 94°C for 60 s, 61°C for 60 s, 72°C for 180 s, and final extension at 72°C for 10 min. The conditions for tet-op-Esr1 were 94°C for 3 min of initial denaturation followed by 35 cycles at 94°C for 60 s, 57°C for 90 s, and 72°C for 120 s. PCR products were then subjected to agarose gel electrophoresis. The presence of a 372-bp fragment indicated that the mice were controls (tet-op-tTA/tet-op-luciferase) and the presence of both 372- and 369-bp bands indicated that the mice were Esr1 overexpressors (tet-op-tTA/tet-op-luciferase/tet-op-Esr1).

Evaluation of Fertility

In the fertility study, Esr1 overexpressor males at 125–140 days of age were cohabited with 70–80-day-old FVB wild-type females (1 male to 2 females per cage). At the end of 12 days of cohabitation, females were separated and allowed to deliver at term. The fertility-related parameters studied were: i) fertility index, ii) fecundity index, iii) numbers of pups per litter, and iv) numbers of days from the day of cohabitation to the day of delivery. The fertility index is defined as the ability of a male to sire pups and was calculated as follows: total number of males that sired pups/total number of males cohabited x 100. The fecundity index is defined as the prolific ability of a male to impregnate a number of females and was calculated as follows: total number of females being impregnated/total number of females cohabited x 100.

RNA Isolation and Real-Time PCR Analysis

Penile tissues excluding the os penis were snap frozen from adult Esr1 overexpressors and control mice and were subjected to quantitative real-time PCR for Esr1. Real-time PCR analysis was performed as previously described (Tomic et al, 2007). Total RNA was isolated from tissue using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. RNA was quantified spectrophotometrically at OD260 and the purity was assessed by measuring the OD260/OD280 ratio using Nanodrop ND-1000. Reverse-transcriptase generation of cDNA was performed with 1μg of total RNA using Omniscript RT kit (Qiagen Inc., Valencia, CA) with random primers according to the manufacturer’s protocols.

Real-time PCR was conducted using a MJ Research (OPTICON) real-time PCR machine and accompanying software according to the manufacturer’s instructions. The OPTICON quantifies the amount of PCR product generated by measuring the dye (Dynamo SYBR green, Finnzymes, New England Biolabs, Ipswich, MA) that fluoresces when bound to double-stranded DNA. A standard curve was generated from six serial dilutions including a blank, of purified PCR product. Primers specific for mouse β-actin were used as an internal control as previously described (Tomic et al, 2007). Primer sequences were: for Esr1 (forward) 5′-AATTCTGACAATCGACGCCAG-3′ and (reverse) 5′-GTGCTTCAACATTCTCCCTCCTC-3′ (Tomic et al, 2007) and for β-actin (forward) 5′-GGGCACAGTGTG-GGTGAC-3′ and (reverse) 5′-CTGGCACCACACCTTCTAC-3′ (Weihua et al, 2000). The specificity of the primers was verified by the lack of amplification from genomic DNA. An initial incubation of 95°C for10 min was followed by 40–50 cycles of 94°C for 10 s, 55°C for β-actin and 60°C for Esr1 for 10 s, and 72 °C for 10 s, with final extension at 72°C for 10 min.

Genomic equivalents (an arbitrary term generated by OPTICON) denote the relative gene expression level in the experimental samples. These values are calculated based on the standard curve and are read at the cycle at which the gene begins amplifying exponentially. This cycle number is 18 for β-actin and 20 for Esr1. For each primer, a melting curve was performed at 55–90°C to monitor the generation of a single product. Genomic equivalents from Esr1 overexpressor and control samples were normalized by obtaining the ratio Esr1/β-actin. Fold changes were calculated as a ratio of normalized values of Esr1 overexpressors and mean normalized values of controls. All experiments were performed in triplicate.

Western Blot Analysis

Penile tissue from control and ESR1 overexpressing mice was homogenized in T-Per-Tissue Protein Extraction Reagent (Thermo Fisher Scientific, IL) containing a protease inhibitor cocktail (Roche Diagnostics, Switzerland). The amount of protein in each homogenate was determined with a bicinchoninic acid protein assay kit. Protein lysates were subjected to Western blot analysis using antibodies against ESR1 (SC 542; Santa Cruz Biotechnology, Inc., CA) or androgen receptor (PG21–36; University of Illinois at Chicago). Immune complexes were visualized using an enhanced chemiluminescence detection kit (Cell Signaling Technologies Inc, Beverly, MA) with α-tubulin as a control for loading. Scanning densitometry (ImageJ software [NIH]) was used to quantify changes in protein levels and data normalized to α-tubulin.

Measurement of the Body Weight and Organ Weights

All animals were weighed and terminated at 150–170 days of age. The testes and seminal vesicles were weighed as markers for effects of Esr1 overexpression on reproductive organs other than the penis since estrogens are known to have an inhibitory effect on both of these organs (Goyal et al, 2003, 2004a,b, 2005a,b).

Measurement of the Penis and Penile Skeletal Muscles

The penis was measured for length and diameter with a caliper (calibrations up to 0.1 mm) and weighed as described previously (Goyal et al, 2005b). 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, and the diameter was measured from the middle of the body of the penis. After removing the free loose connective tissue, the entire penis was weighed. The rodent penis is surrounded by three pairs of skeletal muscles: ischiocavernosus extends from the ischial arch to the middle of the dorsal surface of the body of the penis; bulbospongiosus surrounds the ventro-lateral surface of the bulb of the penis; and levator ani forms a sling-like band around the anus and is connected dorsally to the bulbospongiosus. The bulbospongious and levator ani muslces were isolated, freed from connective tissue and fat, and weighed together.

Radiography of the Penis

Two penises each from the control and Esr1 overexpressing mice were fixed in 10% formaldehyde and radiographed to determine effects of Esr1 overexpression on development of the os penis as described previously (Goyal et al, 2004a).

Histopathology of the Penis

After weighing, 2 to 3 mm-long sections from the middle of the body of the penis were processed for histopathology and histochemistry (n = 5–6 each group). For histopathology, tissues were fixed in 10% formalin for 24–48 h and processed for paraffin embedding using an automatic tissue processor. Five-μm-thick paraffin sections were stained with hematoxylin and eosin (H&E) and examined for routine histopathology using a light microscope. For fat demonstration, formaldehyde-fixed tissues were en block stained for 8 h with 1% osmium tetroxide dissolved in 2.5% potassium dichromate solution, and then processed for paraffin embedding. Five-μm-thick osmium-fixed, unstained, undeparaffinized sections of the body of the penis were examined using light microscopy, and the adjacent serial sections were stained with H&E to allow for examination of histopathology. Such examination of undeparaffinized and unstained sections is useful in viewing the full extent of fat infiltration in the penis because it rules out the possibility of fat droplets being washed out during H&E staining. In addition, 1- mm-thick pieces of tissues from the middle of the body of the penis were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy, as described previously from our laboratory (Goyal and Williams, 1991). One-μm-thick epoxy sections were stained with 1% toluidine blue in 1% borax. Digital images of paraffin and epoxy sections, as well as of the radiographs, were captured with a Leitz Orthoplan microscope (Vashaw Scientific, Inc, Norcross, GA) and the Kodak Microscopy Documentation System 290 (Eastman Kodak Company, Rochester, NY) and were assembled with the use of Adobe Photoshop CS2.

Histopathology of the Testis and Epididymis

After weighing, 2–3 mm thick pieces from the cranial end of testes (near the rete testis) were fixed in Bouin’s fluid for 24 h, and the head, body and tail of the epididymis were fixed in 10% formalin for 24–48 h. After fixation, tissues were processed, embedded, sectioned, stained, and examined for routine histopathology using a light microscope as described above for the penis.

Plasma and Intratesticular Testosterone

For plasma testosterone, one blood sample was collected by cardiac puncture from each animal just prior to necropsy; and for intratesticular testosterone, the middle part of the right testis was collected from each animal at the time of necropsy. Both plasma and testicular parenchyma were frozen at −20°C until assayed. Testes were processed in accordance with the protocol described by Park et al (2002). Briefly, 10–30 mg of testicular tissue was homogenized in phosphate-buffered saline. Eight volumes of ether 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 prior to assay, samples were re-suspended in phosphate-buffered saline. Testosterone in plasma and testicular tissue was measured using a COAT-A-COUNT testosterone radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA) according to manufacturer’s protocol. The sensitivity of the assay was 0.2 ng/ml. The intra-assay and inter-assay coefficients of variations were 6% and 12%, respectively.

Statistics

Statistical analyses were performed using ProStat statistical software (Polysoftware International, Pearl River, NY). Analysis of variance was performed on all parameters except those from the mating study. Treatment groups with means significantly different (P < 0.05) were identified using Duncan’s Multiple Range test. When data were not distributed normally, or heterogeneity of variance was identified, analyses were performed on transformed or ranked data. Data from the mating study were evaluated using Fisher’s Exact Test. Data are expressed as mean ± SE throughout the text.

RESULTS

Fertility

Fertility-related parameters studied were fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per litter. None of these parameters was significantly (P < 0.05) different between the control and Esr1 overexpressors (Table 1). Considering that the gestation length in mice averages 19 days, male mice took, on average, 5 days from the day of cohabitation to impregnate, implying that most females got pregnant within the first estrous cycle.

Table 1.

Fertility Data from the Control and ERα Over-expressing (^) Male Mice Cohabited with Control Females from Two Trials.

Group Fertility Index Fecundity Index Days from the Day of Cohabitation to the Day of Delivery Pups/Female
Control 80% (8/10 M) 65% (13/20 F) 25±2 10.2±0.6
ERα(^) 100% (10/10 M) 100% (20/20 F) 24±1 9.6±0.4
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Data are expressed as mean ±SEM

Esr1 Expression in the Penis

The body of the penis collected from Esr1 overexpressing mice had an almost 28-fold increase in Esr1 levels as compared with that of controls (Fig. 2A), confirming that the transgenic mice over-expressed Esr1 in the penis.

Figure 2.

Figure 2

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Expression of Esr1 mRNA (A) and ESR1 and AR protein (B) in the body of the penis of control and Esr1 overexpressing mice. Esr1 expression was analyzed by real-time PCR using specific primers and quantified relative to β-actin (internal control). ESR1 and AR protein were analyzed by Western Blot (shown) and quantified relative to α-tubulin (internal control). Data are expressed as mean ± SEM. *Significant difference from controls (P<0.05).

ESR1 and Androgen Receptor (AR) Expression in the Penis

The level of ESR1 in the body of the penis was significantly higher than that of controls, but that of AR was not different from controls (Fig. 2B).

Body and Organ Weights

The mean body weight did not significantly (P < 0.05) differ between the control and Esr1 overexpressing mice and averaged 32.5 g and 31.0 g, respectively. Likewise, neither the absolute nor the relative weight (per 100 g body weight) of the paired testes significantly differed between groups (Fig. 3A, Table 2); however, there was a tendency for the testicular weight to be a bit lower in Esr1 overexpressing mice. For example, the paired absolute testicular weight was 0.190 g or higher in eight out of 10 mice in the control group versus only three out of 10 mice in the Esr1 overexpressor group. Unlike the testes, both absolute and relative weights of the paired seminal vesicles, including the coagulating glands, were significantly lower in the Esr1 overexpressor group and the decrease was almost 20%, as compared to controls (Fig. 3B, Table 2).

Figure 3.

Figure 3

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The absolute paired weight of the testis and seminal vesicle (SV), the absolute weight of the penis and bulbospongiosus and levator ani muscles (BS/LA), and the length and diameter of the penis in the control and Esr1 overexpressing adult mice. Note significant reductions in weights of the seminal vesicle and BS/LA muscles in the Esr1 overexpressing mice, as compared to controls. Data are expressed as mean ± SEM. *Significant difference from controls (P<0.05).

Table 2.

The Body Weight (BW), the Paired relative Weight (per 100 g BW) of the Testis (T), Seminal Vesicle (SV), Relative Weight of the Bulbospongiosus and Levator Ani (BS/LA) Muscles, and the Penis in the Control and ERα Over-expressing (^) Adult Male Mice (n=10/treatment group).

Group BW (g) Testis (g) SV (g) BS/LA (g) Penis (g)
Control 32.5 0.1898 A0.4010 A0.1109 0.0349
±1.6 ±0.0168 ±0.0291 ±0.0050 ±0.0016
ERα(^) 31.0 0.1811 B0.3322 B0.0987 0.0362
±0.6 ±0.0050 ±0.0139 ±0.0017 ±0.0006
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Data are expressed as mean ±SEM

A,B

superscripts within a column indicate significant differences (p<0.05)

Measurements of the Penis and Penile Skeletal Muscles

The penile measurements, including weight, length, and diameter, were not significantly (P < 0.05) different between the control and Esr1 overexpressing mice (Figs. 3C–E, Table 2). However, both absolute and relative weights of the combined bulbospongiosus and levator ani muscles were significantly lower in the Esr1 overexpressor group and the decrease was almost 10%, as compared to controls (Fig. 3F, Table 2).

Radiography of the Os Penis

The penis was radiographed to determine whether Esr1 overexpression altered development of the os penis, which extends from the distal end of the body of the penis to the tip of the glans penis. Its proximal end (near the body) is a bit wider and has a marrow cavity. The os penis was evaluated for length, thickness and level of calcification. None of these parameters was affected by Esr1 overexpression, as compared to controls (Figs. 4A, 4G).

Figure 4.

Figure 4

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Radiographs of the penis and micrographs of the penis in the control (A–F) and Esr1 overexpressing adult mice (G–L). A, G): Radiographs of the penis. Note similar development of the os penis in both groups of mice. B, H): Paraffin sections of the body of the penis stained with hematoxylin and eosin (H&E). Note that different parts of the body, including the intercrural septum containing blood vessels (bv) and nerve fibers (n), the paired corpora cavernosa (cc), and the corpus spongiosus (cs) are similarly developed in both groups. C, I): Paraffin sections of the corpus cavernosus stained with H&E. Note similar development in both groups of different components of the corpus cavernosus: cavernous spaces (cs), smooth muscles underlying the cavernous spaces (not clearly identified with H&E stain), and wide connective tissue septa (ct) between cavernous spaces. D, J): Epoxy sections of the corpus cavernosus stained with toluidine blue. Darkly stained smooth muscles (arrows) surrounding the cavernous spaces (cs) are clearly seen in epoxy sections of both groups. E, K): Paraffin sections of the corpus spongiosus stained with H&E. Note both groups have similar development of different components of the corpus spongiosus: urethra (u) lined by stratified cuboidal to columnar epithelium, cavernous spaces (arrows), smooth muscles (sm), and the connective tissue capsule called tunica albuginea (ta). F, L): Undeparaffinized sections of the corpora cavernosa. Note the presence of a few, small, black-staining fat cells (circles) in both groups.

Histopathology of the Penis

The body of the penis was examined using paraffin sections stained with H&E and epoxy sections stained with toluidine blue for morphology and using undeparaffinized sections fixed with formaldehyde and osmium tetroxide for fat infiltration. The penis body consists of paired corpora cavernosa that are located dorsolateral to the urethra and a corpus spongiousus that is located ventrally and surrounds the urethra (Fig. 4B). The connective tissue septum separating the two cavernosa is incomplete and contains branches of the dorsal artery and vein of the penis and nerve bundles. Histologically, both corpus cavernosus and corpus spongiosus contain endothelial-lined cavernous spaces, which are underlain by smooth muscle cells and bundles of dense collagen fibers (Figs. 4C–E). A dense connective tissue capsule, tunica albuginea, surrounds both the corpora cavernosa and corpus spongiosus. All of the histological structures in both corpus cavernous and corpus spongiosus, including cavernous spaces, smooth muscle cells, dense collagen bundles and tunica albuginea, were morphologically similar in the control and Esr1 overexpressing mice (compare Figs. 4C–E with Figs. 4H–K). Likewise, a few fat cells were invariably found in the corpus cavernosus, but not in the corpus spongiosus, of both groups (Figs. 4F, 4L). There was no evidence of fat infiltration, as a result of Esr1 overexpression.

Histopathology of the Testis and Epididymis

Both the testis and epididymis were examined to determine if Esr1 overexpression resulted in cryptorchidism or an abscess in the tail of the epididymis at the gross level and resulted in abnormal lymphocytic infiltration in the epididymis at the histopathological level since these were the most common abnormalities associated with neonatal exposure to estrogens (Goyal et al, 2009). For the sake of consistency and reproducibility, stage VII-VIII seminiferous tubules, which are androgen-dependent (Sharpe et al, 1992) and are characterized by a wide lumen and elongated spermatids lining the lumen or free in the lumen (Fig. 5A), were examined. Likewise, among different segments of the epididymis, the initial segment and the tail of the epididymis were examined. The initial segment has the tallest epithelium with long stereocilia and has the least numbers of sperm in the lumen, which is usually stellate shape (Fig. 5B). Conversely, the tail of the epididymis has the shortest epithelium with dense and short microvilli and has the widest lumen with the largest numbers of sperm (Fig. 5C). In addition, the epithelium is interspersed with clear cells, which are characterized by lightly stained, clear cytoplasm (Fig. 5C).

Figure 5.

Figure 5

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Micrographs of the testis, initial segment of the epididymis, and tail of the epididymis in the control (A–C) and Esr1 overexpressing (D–F) adult mice. A, D): Paraffin sections of the testis stained with H&E showing stage VIII seminiferous tubules with elongated spermatids lining the lumen. Note similar spermatogenesis in both groups. B, E): Paraffin sections of the initial segment of the epididymis stained with H&E. Note the irregular lumen with barely any sperm and pseudostratified columnar epithelium with long stereocilia in both groups. C, F): Paraffin sections of the tail of the epididymis stained with H&E. As compared to the initial segment, note here the larger lumen filled with sperm and shorter epithelial height in both groups. Arrows indicate clear cells in the epithelium.

Neither cryptorchidism nor abscessed epididymis was observed in any of the control or Esr1 overexpressing mice. The stage VII-VIII seminiferous tubules in both groups were morphologically similar and were lined, beginning from the basal lamina to the lumen, with spermatogonia, spermatocytes, spermatids, and elongated spermatids with residual cytoplasmic droplets (compare Fig. 5A with Fig. 5D). In no circumstance, did we encounter any evidence of infiltration of lymphocytes, degeneration of germ cells, or retention of sperm in the seminiferous epithelium in the control or Esr1 overexpressing mice. Similarly, morphology of the initial segment and the tail of the epididymis was similar in the control and Esr1 overexpressor groups in that mice in both groups had the tallest epithelium and the least numbers of sperm in the initial segment, the shortest epithelium and the largest numbers of sperm in the tail of the epididymis, and contained a few clear cells in the epithelium of the tail of the epididymis (compare Figs. 5B & C with Figs. 5G & H)

Plasma and Intratesticular Testosterone

Both the plasma and intratesticular testosterone levels were similar between the control and Esr1 overexpressing mice (Figure 6)

Figure 6.

Figure 6

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Note similar concentration of plasma and intratesticular testosterone in the control and Esr1 overexpressing adult mice. Data are expressed as mean ± SEM.

DISCUSSION

The objective of this study was to determine whether genetically-induced Esr1 overexpression adversely affects male reproductive functions, especially fertility and development of the penis. Results revealed that fertility-related parameters, including fertility index, fecundity index, numbers of days between the first day of cohabitation and the day of impregnation, and numbers of pups per litter, were similar between controls and Esr1 overexpressors. Likewise, penile parameters, including os penis development, penile measurements, and penile morphology, were also similar between controls and Esr1 overexpressors. Hence, based on these results, it is reasonable to conclude that genetically-induced Esr1 overexpression is unable to adversely affect male fertility and penile development.

To our knowledge, this is the first in-depth study reporting effects of Esr1 overexpression on male fertility. Our data showing no statistical difference in numbers of pups sired per litter between controls and Esr1 overexpressor males are in agreement with those of a previous study from our coauthors’ laboratory in which control C57BL6 female mice were mated with Esr1 overexpressing males (Tomic et al, 2007). However, the latter study did not report on any of the other fertility-related parameters described in the present study. Unlike in males, Esr1 overexpressing females had a lower fertility, especially when they were mated with Esr1 overexpressing males (Tomic et al, 2007). According to these authors, the lower fertility was attributed, in part, to decreased numbers of implantation sites in the uterus and increased numbers of apoptotic endometrial epithelial cells (Tomic et al, 2007). Similarly, in another study, transgenic female mice overexpressing Esr1 had reduced fertility, which was associated with prolonged gestation length and dystocia (Davis et al, 1994).

Furthermore, our study showed that Esr1 overexpression did not adversely affect testicular or epididymal functions, as the seminiferous tubules, initial segment of the epididymis, and tail of the epididymis were morphologically similar between controls and Esr1 overexpressors. Additionally, the pituitary-testis axis was also unaffected, as the plasma and intratesticular levels of testosterone were similar between controls and Esr1 overexpressors. Similarly, Esr1 overexpressing and control female mice had similar age of vaginal opening, length of estrous cycle, numbers of primordial and primary follicles per ovary, height of uterine epithelium, and serum levels of estradiol, FSH and LH (Tomic et al, 2007). However, while numbers of preantral and antral follicles per ovary were similar on postnatal days 7 and 28 between controls and Esr1 overexpressors, they were significantly higher on day 90 in Esr1 overexpressors (Tomic et al, 2007), suggesting, according to authors, an enhanced follicular growth. In another study, exposure to methoxychlor, a pesticide that binds to ESR1, increased the percentage of atretic follicles in Esr1 overexpressing mice compared to controls, suggesting heightened sensitivity to the pesticide (Tomic et al, 2006).

A noteworthy finding of the present study was a significant (P < 0.05) decline in the weight of seminal vesicles and bulbospongiosus/levator ani muscles by almost 20% and 10%, respectively, in Esr1 overexpressors compared to controls. The mean weight of the testes also decreased by almost 5%, but the decrease was not significant. Reasons for weight decline, and more importantly, differential weight decline, in these reproductive organs of Esr1 overexpressing mice are difficult to explain with the limited data available on this topic in the literature at this time. However, it is noteworthy that previous studies have shown that, among male reproductive organs, seminal vesicles are most sensitive, and testes the least sensitive, to estrogen exposure. For example, following DES exposure to neonatal male rat pups at a dose of 0.1 mg/kg, daily, from postnatal days 1–6, the weight reduction at adulthood was almost 70% in seminal vesicles vs 50% in bulbospongiosus/levator ani muscles vs 30% in testes, as compared to controls (Goyal et al, 2005b). Similarly, adult rats treated neonatally with estradiol benzoate had 80% reduction in the weight of seminal vesicles, in contrast to 20% in the testes (Putz et al, 2001). Reasons for differential response in estrogen-treated rats may be attributed, in part, to differences in susceptibility among these reproductive organs to estrogens since all of them contain estrogen receptors (Hess et al, 1997a; Pelletier et al, 2000). The secretion of seminal vesicles forms a significant part of the ejaculate and its fructose content provides nutrients to sperm in the female genital tract (Luke and Coffey, 1994), and the contraction of penile skeletal muscles aids in erection and ejaculation (Sachs, 1982). However, it is worth noting that the decline in weights of the seminal vesicles and bulbourethral/levator ani muscles was not sufficient to adversely affect the fertility of Esr1 overexpressors.

The role of estrogen receptors, especially ESR1, in the male reproductive tract is well established. Both ESR1 and ESR2 have been localized in the male reproductive tract of most mammals studied previously (Danzo and Eller 1979; West and Brenner 1990; Iguchi et al, 1991; Goyal et al, 1997; Hess et al, 1997a), with the highest concentration of ESR1 in the efferent ductules, whose major function is to absorb almost 90% of the fluid leaving the testes (Ilio and Hess, 1994). Mutant mice lacking Esr1 are infertile (Eddy et al, 1996), and the infertility is attributed to the inability of efferent ductules to absorb testicular fluid, thus leading to a fluid build-up in the seminiferous tubules, followed by degeneration of germ cells (Hess et al, 1997b). Our own observations, that Esr1−1− mice are resistant to estrogen-inducible developmental penile abnormalities observed in wild-type mice (Goyal et al, 2007) and that the co-administration of ER antagonist ICI 182780 with DES mitigates DES-induced maldevelopment of the rat penis (Goyal et al, 2009), support the role for ESR1 in mediating harmful effects of estrogen exposure. Additionally, ESR1 or Esr1 up-regulation is associated with abnormal development of the male reproductive tract (Sato et al, 1994; Atanassova et al, 2000; Goyal et al, 2004a), prostate gland (Prins and Birch 1997; Prins et al, 2001) and seminal vesicles (Williams et al, 2001). Taken together, the above studies provide unequivocal evidence of an obligatory role for ESR1 in mediating male reproductive physiology and pathogenesis.

Therefore, our observations of normal fertility and penile development in Esr1 overexpressing mice suggest that Esr1 overexpression alone, without an exogenous estrogen exposure during the neonatal period, is unable to induce mal-development of the penis and infertility. ER alpha up-regulation in penile stromal cells can result directly from an excessive estrogen exposure to neonatal pups or indirectly from an estrogen-induced down-regulation of the neonatal testosterone surge. Our previous observations that estrogen-induced down-regulation of the neonatal testosterone surge (Goyal et al, 2009) and up-regulation of peroxidase proliferator-activated receptor, a marker for differentiation of adipocytes (Tontonoz et al, 1994), are mitigated by the ESR antagonist ICI (Mansour et al, 2008) provide support to the indirect mechanism.

In conclusion, the genetically-induced Esr1 overexpression alone, without an exogenous estrogen exposure during neonatal period, is unable to adversely affect the development of the penis as well as other male reproductive organs, except limited, but significant, reductions in weights of the seminal vesicles and bulbospongiosus/levator ani muscles.

Acknowledgments

Authors thank Dr. Abdalla Eljack, Department Head, and Dr. Tsegaye Habtemariam, Dean, for their encouragement and support. There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. This work was supported by National Institute of Health grants NIH/NIEHS 1SC1ES019355-01 (to H.G.) and RCMI-5-G12RR03059.

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