Estrogen-induced maldevelopment of the penis involves down-regulation of myosin heavy chain 11 (MYH11) expression, a biomarker for smooth muscle cell differentiation

Abstract

Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%-80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect.

FIG. 1

Microarray analysis from the penis (A) and testis (B) at 7 days of age in rats treated from 1 to 6 days of age with DES. Compared to controls, note in the penis 60%–90% reduction in gene expression profiles of markers for smooth muscle cell or angiogenesis and, in the testis, 80%–95% reduction in gene expression profiles of steroidogenic enzymes.

FIG. 2

Quantitative real time-PCR of Myh11 expression at 7 (A) and 21 (B) days of age and a temporal change in Myh11 expression (C) from 7 to 21 days of age in rats treated from 1 to 6 days of age with DES, with or without coadministration of ICI or DHT. Note nearly 50%–70% reduction in Myh11 expression as a result of DES treatment and its reversal to the control level by ICI or DHT coadministration at 7 and/or 21 days. Note a temporal threefold increase in Myh11 expression in controls versus a nonsignificant onefold increase in the DES group. Temporal changes in other treatments are not shown. D) Note almost 80% reduction in MYH11 expression as a result of DES treatment. E) Western blot analysis of MYH11 protein expression at 10 days of age in control and DES-treated rats. Data are expressed as means ± SEM. Means with different lowercase letters are significantly different from each other (P < 0.05). Asterisks denote significant difference from controls at the level of *P < 0.05 or **P < 0.001. In A and B, numbers within bars indicate the number of animals in each experiment group.

FIG. 3

Immunohistochemical localization of smooth muscle alpha actin in the body of the penis at 7, 10, 21, days of age and at adulthood in control (A–D) and DES-treated (E–H) rats. Note cavernous spaces (arrowheads) as outlined by the surrounding alpha actin-positive cavernous smooth muscle cells in the corpora cavernosa (CC). Both structures are much less developed in DES-treated animals than controls. Note arterioles or metarterioles (arrows) in DES-treated animals. Other alpha actin-positive structures are blood vessels (BV) present in the intercrural septum located dorsally. Corpus spongiosum (CS) is located ventrally. The control section incubated with blocking serum in place of primary antibody did not stain smooth muscle cells surrounding the cavernous spaces or blood vessels in the intercrural septum and is not shown. All photomicrographs were obtained at the same magnification. Bar = 100 μm.

FIG. 4

Histochemical localization of fat cells in the body of the penis at 7, 10, and 21 days of age and at adulthood in control (A–D) and DES-treated (E–H) rats. Note accumulation of fat cells in DES-treated animals compared to that in controls. Also, note the absence of fat cells in the corpus spongiosum (CS) that is located ventrally and surrounds the urethra. En block staining with osmium tetroxide. CC, corpora cavernosa. Bar = 100 μm.

FIG. 5

Quantitative real time-PCR shows a relative fold change in Esr1, Esr2, Ar, and Pparγ expression levels at 7 (A) and 21 days (B) of age as a result of DES treatment and shows a temporal fold change in Esr1 (C), Esr2 (D), Ar (E), and Pparγ (F) expression levels from 7 to 21 days of age in controls and DES-treated rats. Note significant up-regulation in Esr1 expression at 7 and 21 days and in Pparγ expression at 21 days as a result of DES treatment. Asterisks denote significant differences from controls at the level of *P < 0.05 or **P < 0.001.

FIG. 6

Testicular testosterone (ng/g) at 7 days of age in rats treated from 1 to 6 days of age with DES with or without coadministration of ICI or DHT. Note nearly 90% reduction in the neonatal testosterone surge as a result of DES treatment and its reversal to the control level by ICI coadministration, but not by DHT coadministration. Data are expressed as means ± SEM. Means with different lowercase letters are significantly different from each other (P < 0.05). Numbers within bars indicate the number of animals in each experiment group.

FIG. 7

The penis weight at 7 days of age (A) and the penis weight and length at 21 days of age (B) in rats treated from 1 to 6 days of age with DES, with or without coadministration of ICI or DHT. Note that the coadministration of ICI or DHT mitigated DES-induced reductions in penile measurements in both age groups. Data are expressed as mean ± SEM. Means with different lowercase letters are significantly (P < 0.05) different from each another. Numbers within bars indicate the number of animals in each experiment group.