Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation.

Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.

Investigation of sexual dimorphisms through mouse models and hormone/hormone-disruptor treatments.

Sexual dimorphism in mouse reproductive tissues is observable in adult, post-natal, and embryonic stages. The development of sexually dimorphic tissues starts with an ambisexual structure. It is followed by sex-specific organogenesis as guided by different signaling pathways that occur from late embryonic stages. The measurement of the anogenital distance (AGD), and the observation of the external genitalia are practical ways to distinguish male and female pups at birth and thereafter. Careful observation of the morphological or histological features and the molecular signatures of the external genitalia and perineum enable identification of sex or feminization/masculinization of embryos. Aberrations in hormone signaling via castration or treatment with hormones or hormone disruptors result in dysmorphogenesis of reproductive tissues. Several hormone disruptors have been used to modulate different aspects of hormone action through competitive inhibition and exogenous hormone treatment. Concomitantly, the vast advancement of conditional mutant mouse analysis leads to the frequent utilization of Cre recombination technology in the study of reproductive/urogenital tissue development. Mouse Cre-lines that are tissue-specific and cell-specific are also effective tools in identifying the molecular mechanisms during sexually dimorphic development. Cre-lines applicable to different cell populations in the prostate, seminal vesicles, testis and ovaries, and mammary glands are currently being utilized. In the external genitalia and perineum, Cre lines that examine the signaling pathways of cells of endodermal, ectodermal, and mesenchymal origin reveal the roles of these tissues in the development of the external genitalia. The interaction of hormones and growth factors can be examined further through a variety of techniques available for researchers. Such cumulative information about various technologies is summarized.

Development of the external genitalia and their sexual dimorphic regulation in mice.

The study of the external genitalia is divided into 2 developmental stages: the formation and growth of a bipotential genital tubercle (GT) and the sexual differentiation of the male and female GT. The sexually dimorphic processes, which occur during the second part of GT differentiation, are suggested to be governed by androgen signaling and more recently crosstalk with other signaling factors. The process of elucidating the regulatory mechanisms of hormone signaling towards other signaling networks in the GT is still in its early stages. Nevertheless, it is becoming a productive area of research. This review summarizes various studies on the development of the murine GT and the defining characteristics of a masculinized GT and presents the different signaling pathways possibly involved during masculinization.

Nonmyocytic androgen receptor regulates the sexually dimorphic development of the embryonic bulbocavernosus muscle.

The bulbocavernosus (BC) is a sexually dimorphic muscle observed only in males. Androgen receptor knockout mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at embryonic day (E) 16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signaling in this process, muscle-specific androgen receptor (AR) mutation was introduced, which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as Myogenic differentiation 1, Myogenin, and paired box transcription factor 7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the nonmyocytic cells using spalt-like transcription factor 1 (Sall1) Cre driver mouse was performed, which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the Sall1 Cre:AR(L-/Y) mutant embryos, and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by cyclin-dependent kinase inhibitors. An increased expression of p21 was observed in the BC myoblast of the Sall1 Cre:AR(L-/Y) mutant and wild-type female. Altogether this study suggests that the nonmyocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.