Development of the external genitalia: perspectives from the spotted hyena (Crocuta crocuta).

This review/research paper summarizes data on development of the external genitalia of the spotted hyena, a fascinating mammal noted for extreme masculinization of the female external genitalia. The female spotted hyena is the only extant mammal that mates and gives birth through a pendulous penis-like clitoris. Our studies indicate that early formation of the phallus in both males and females is independent of androgens; indeed the phallus forms before the fetal testes or ovaries are capable of synthesizing androgens. Likewise, pre- and postnatal growth in length of the penis and clitoris is minimally affected by "androgen status". Nonetheless, several internal morphologies, as well as external surface features of the phallus, are androgen-dependent and thus account for dimorphism between the penis and clitoris. Finally, estrogens play a critical role in penile and clitoral development, specifying the position of the urethral orifice, determining elasticity of the urethral meatus, and facilitating epithelial-epithelial fusion events required for proper formation of the distal urethra/urogenital sinus and prepuce. Accordingly, prenatal inhibition of estrogen synthesis via administration of letrozole (an aromatase inhibitor) leads to malformations of the glans as well as the prepuce (hypospadias). The effects of prenatal androgens, anti-androgens and impaired estrogen synthesis correlated with the tissue expression of androgen and estrogen receptors.

Analysis of the effect of estrogen/androgen perturbation on penile development in transgenic and diethylstilbestrol-treated mice.

Because both androgens and estrogens have been implicated in penile morphogenesis, we evaluated penile morphology in transgenic mice with known imbalance of androgen and estrogen signaling using scanning electron microscopy (SEM), histology, and immunohistochemistry of androgen and estrogen receptors α/ß. Penises of adult wild-type, estrogen receptor-α knockout (αERKO), estrogen receptor-ß knockout (ßERKO), aromatase knockout (Arom-KO), and aromatase overexpression (Arom+) mice were evaluated, as well as adult mice treated with diethylstilbestrol (DES) from birth to day 10. Adult penises were examined because the adult pattern is the endpoint of development. The urethral orifice is formed by fusion of the MUMP (male urogenital mating protuberance) with the MUMP ridge, which consists of several processes fused to each other and to the MUMP. Similarly, the internal prepuce is completed ventrally by fusion of a ventral cleft. In adult murine penises the stromal processes that form the MUMP ridge are separated from their neighbors by clefts. αERKO, ßERKO, and Arom-KO mice have penises with a MUMP ridge clefting pattern similar to that of wild-type mice. In contrast, Arom+ mice and neonatally DES-treated mice exhibit profound malformations of the MUMP, MUMP ridge clefting pattern, and internal prepuce. Abnormalities observed in Arom+ and neonatally DES-treated mice correlate with the expression of estrogen receptor-beta (ERß) in the affected structures. This study demonstrates that formation of the urethal orifice and internal prepuce is due to fusion of separate epithelial-surfaced mesenchymal elements, a process dependent upon both androgen and estrogen signaling, in which ERß signaling is strongly implicated.

New insights on the morphology of adult mouse penis.

The adult mouse penis represents the end point of masculine sex differentiation of the embryonic genital tubercle and contains bone, cartilage, the urethra, erectile bodies, several types of epithelium, and many individual cell types arrayed into specific anatomical structures. Using contemporary high-resolution imaging techniques, we sought to provide new insights to the current description of adult mouse penile morphology to enable understanding of penile abnormalities, including hypospadias. Examination of serial transverse and longitudinal sections, scanning electron microscopy, and three-dimensional (3D) reconstruction provided a new appreciation of the individual structures in the adult mouse penis and their 3D interrelationships. In so doing, we discovered novel paired erectile bodies, the male urogenital mating protuberance (MUMP), and more accurately described the urethral meatus. These morphological observations were quantified by morphometric analysis and now provide accurate morphological end points of sex differentiation of mouse penis that will be the foundation of future studies to identify normal and abnormal penile development.

The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben).

Studies were conducted to elucidate the importance of androgen-mediated induction of the extreme masculinization of the external genitalia in female spotted hyenas. Phallic size and shape; androgen receptor (AR) and alpha-actin expression; and sex-specific differences in phallic retractor musculature, erectile tissue, tunica albuginea, and urethra/urogenital sinus were examined in male and female fetuses from Day 30 of gestation to term. Similar outcomes were assessed in fetuses from dams treated with an AR blocker and a 5alpha-reductase inhibitor (antiandrogen treatment). Clitoral and penile development were already advanced at Day 30 of gestation and grossly indistinguishable between male and female fetuses throughout pregnancy. Sex-specific differences in internal phallic organization were evident at Gestational Day 45, coincident with AR expression and testicular differentiation. Antiandrogen treatment inhibited prostatic development in males and effectively feminized internal penile anatomy. We conclude that gross masculinization of phallic size and shape of male and female fetuses is androgen-independent, but that sexual dimorphism of internal phallic structure is dependent on fetal testicular androgens acting via AR in the relevant cells/tissues. Androgens secreted by the maternal ovaries and metabolized by the placenta do not appear to be involved in gross masculinization or in most of the sex differences in internal phallic structure.