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.

The anti-androgen combination, flutamide plus finasteride, paradoxically suppressed LH and androgen concentrations in pregnant spotted hyenas, but not in males.

The androgen receptor blocker flutamide and the 5α-reductase inhibitor finasteride have been used in a variety of species to investigate the ontogeny of sexual dimorphisms by treating pregnant females or neonates at critical periods of sexual differentiation. Likewise, we have used these drugs to study the profound masculinization of the external genitalia in female spotted hyenas. However, a potential pitfall of administering flutamide, either alone or in combination with finasteride, is that it maintains or even raises plasma concentrations of luteinizing hormone (LH) and testosterone (T), because negative feedback of the hypothalamic-pituitary-gonadal axis is disrupted. Contrary to expectations, when pregnant spotted hyenas were treated with flutamide and finasteride (F&F), the concentrations of T during late gestation were suppressed relative to values in untreated dams. Herein, we further investigate the paradoxical effects of F&F treatment on a battery of sex hormones in spotted hyenas. Beyond the effects on T, we found plasma concentrations of LH, estradiol, progesterone and androstenedione (A4) were also significantly lower in F&F-treated pregnant hyenas than in controls. Flutamide and finasteride did not have similar effects on LH, T, and A4 concentrations in male hyenas. The paradoxical effect of F&F treatment on LH and T concentrations in the maternal circulation suggests that negative feedback control of gonadotropin and androgen secretion may be modified in spotted hyenas during pregnancy.

Transient genital abnormalities in striped hyenas (Hyaena hyaena).

The highly masculinized genitalia of female spotted hyenas Crocuta crocuta is unique among mammals: Crocuta have no external vagina so urination, penile intromission and parturition take place through the clitoris, which mimics a fully erectile male penis. Among hyenids, virilization of external female genitalia has previously been observed only in Crocuta, so functional explanations of masculinization have focused on aspects of social ecology unique to the species. Here we first show that the striped hyena Hyaena hyaena exhibits both unusual similarity in male and female androgen concentrations and transient genital anomalies characterized by a convergence in genital appearance among young males and females. We then evaluate hypotheses regarding the evolution of genital masculinization in the Hyaenidae and other taxa. Hyaena are behaviorally solitary, so discovery of unusual genital development patterns in this species does not support any current evolutionary models for masculinization in Crocuta, which all rely on the trait originating within a highly social species. Some hypotheses can be modified so that masculinization in Crocuta represents an extreme elaboration of a preexisting trait, shared as a homology with Hyaena.

Distribution of vasopressin in the forebrain of spotted hyenas.

The extreme virilization of the female spotted hyena raises interesting questions with respect to sexual differentiation of the brain and behavior. Females are larger and more aggressive than adult, non-natal males and dominate them in social encounters; their external genitalia also are highly masculinized. In many vertebrates, the arginine vasopressin (VP) innervation of the forebrain, particularly that of the lateral septum, is associated with social behaviors such as aggression and dominance. Here, we used immunohistochemistry to examine the distribution of VP cells and fibers in the forebrains of adult spotted hyenas. We find the expected densely staining VP immunoreactive (VP-ir) neurons in the paraventricular and supraoptic nuclei, as well as an unusually extensive distribution of magnocelluar VP-ir neurons in accessory regions. A small number of VP-ir cell bodies are present in the suprachiasmatic nucleus and bed nucleus of the stria terminalis; however, there are extensive VP-ir fiber networks in presumed projection areas of these nuclei, for example, the subparaventricular zone and lateral septum, respectively. No significant sex differences were detected in the density of VP-ir fibers in any area examined. In the lateral septum, however, marked variability was observed. Intact females exhibited a dense fiber network, as did two of the four males examined; the two other males had almost no VP-ir septal fibers. This contrasts with findings in many other vertebrate species, in which VP innervation of the lateral septum is consistently greater in males than in females.

Effects of prenatal treatment with antiandrogens on luteinizing hormone secretion and sex steroid concentrations in adult spotted hyenas, Crocuta crocuta.

Prenatal androgen treatment can alter LH secretion in female offspring, often with adverse effects on ovulatory function. However, female spotted hyenas (Crocuta crocuta), renowned for their highly masculinized genitalia, are naturally exposed to high androgen levels in utero. To determine whether LH secretion in spotted hyenas is affected by prenatal androgens, we treated pregnant hyenas with antiandrogens (flutamide and finasteride). Later, adult offspring of the antiandrogen-treated (AA) mothers underwent a GnRH challenge to identify sex differences in the LH response and to assess the effects of prenatal antiandrogen treatment. We further considered the effects of blocking prenatal androgens on plasma sex steroid concentrations. To account for potential differences in the reproductive state of females, we suppressed endogenous hormone levels with a long-acting GnRH agonist (GnRHa) and then measured plasma androgens after an hCG challenge. Plasma concentrations of LH were sexually dimorphic in spotted hyenas, with females displaying higher levels than males. Prenatal antiandrogen treatment also significantly altered the LH response to GnRH. Plasma estradiol concentration was higher in AA-females, whereas testosterone and androstenedione levels tended to be lower. This trend toward lower androgen levels disappeared after GnRHa suppression and hCG challenge. In males, prenatal antiandrogen treatment had long-lasting effects on circulating androgens: AA-males had lower T levels than control males. The sex differences and effects of prenatal antiandrogens on LH secretion suggest that the anterior pituitary gland of the female spotted hyena is partially masculinized by the high androgen levels that normally occur during development, without adverse effects on ovulatory function.