Genetically induced estrogen receptor α mRNA (Esr1) overexpression does not adversely affect fertility or penile development in male mice

Abstract

Previously, we reported that estrogen receptor α mRNA (Esr1) or protein (ESR1) overexpression resulting from neonatal exposure to estrogens in rats was associated with infertility and maldeveloped 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 < .05) lower as compared with 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, 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 for limited, but significant, reductions in weights of the seminal vesicles and BS/LA muscles.

Figure 1

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.

Figure 2

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).

Figure 3

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).

Figure 4

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.

Figure 5

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.

Figure 6

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