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Review
. Jan-Feb 2014;87(1-2):4-22.
doi: 10.1016/j.diff.2013.12.003. Epub 2014 Feb 28.

Development of the External Genitalia: Perspectives From the Spotted Hyena (Crocuta Crocuta)

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Review

Development of the External Genitalia: Perspectives From the Spotted Hyena (Crocuta Crocuta)

Gerald R Cunha et al. Differentiation. .
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Abstract

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.

Keywords: Androgens; Clitoris; Estrogens; Hyena; Penis; Urogenital sinus.

Figures

Figure 1
Figure 1
Drawing of a pregnant female spotted hyena with a fetus in a uterine horn. A segment of the reproductive tract caudal to the uterus exhibits vagina histology. The urethra joins the caudal end of the vaginal segment, and the common urogenital sinus (UGS) thus formed extends through the pelvic outlet and makes a ∼180-degree turn to traverse to the exterior through the penis-like clitoris. Note the absence of external vaginal orifice. (Adapted from Drea with permission).
Figure 2
Figure 2
(A) Drawing of the adult spotted hyena clitoris by Gordon with permission (Funk, 2012). (B) Photo of the adult spotted hyena clitoris. (C) Photo of the adult spotted hyena penis.
Figure 3
Figure 3
The adult erect hyena penis (A) and clitoris (B). Note the distinctive shapes of the glans. (A) and (B) adapted from (Place and Glickman, 2004) with permission). (C) Three infant spotted hyenas (3-4 months old) in full erection (From Glickman et al 2005) with permission. Based upon glans shape, these infants can be accurately sexed as indicated.
Figure 4
Figure 4
Three-dimensional reconstructions (3DR) (B-E) as well as sections (A, F-H) of the penis of an untreated adult male hyena. (A) Distal section, note urethral orifice (black arrowheads) opening onto the dorsal penile surface. Orange arrows pointing to 3DRs indicate positions of sections (A, F-H)). (B) 3DR, ¾ view of the distal end of the adult hyena penis. Skin is flesh color, urethral orifice (yellow) opens on the dorsal surface of the penis. (C) 3DR, side view with skin rendered semi-transparent to reveal the GEB (green) surrounding the urethra (yellow) and the corporal body (red). (D) 3DR of the mid-shaft of the penis. Note slit-like urethra (yellow arrow) surrounded by the purple corpus spongiosum. The tunica albuginea (white) surrounds both the corporal body (red), urethra and corpus spongiosum. Retractor muscles (blue) lie ventral to the penile urethra. (E) 3DR, side view with penile skin rendered invisible and the tunica (white) rendered semi-transparent to reveal internal structures. (F) Proximal section showing corporal body (CB), and corpus spongiosum (CS) surrounding the slit-like urethra (Ur). Retractor muscles (dotted lines) are ventral to the urethra. Orange arrow indicates the position in 3DR (E) from which section (F) was taken. (G) Section through to the glanular erectile body (GEB); the orange arrow indicates the position in 3DR (D) from which section (G) was taken. The tunica albuginea surrounds both the corporal body (CB) and urethra (Ur). (H) Section just proximal to the glanular erectile body (GEB); the orange arrow indicates the position in 3DR (E) from which section (H) was taken. The tunica surrounds the corporal body (CB), slit-like urethra (Ur) and corpus spongiosum (CS).
Figure 5
Figure 5
Three-dimensional reconstructions (3DR) of the clitoris (B-C) as well as sections (A, D, E) of an untreated adult female hyena. (A) Distal section, note corporal body (CB), glanular erectile body (black arrowheads), and UGS (white arrowheads) opening ventrally. Large white arrow pointing to 3DR (B) indicates the position of section (A) in the clitoris. (B) 3DR, ¾ view of the distal end of the clitoris. White arrow indicates urethral orifice on the ventral surface of the clitoris. (C) 3DR, side view with skin semi-transparent to reveal internal structures. Note color-coding of labels and that the retractor muscles are displayed in two different blue hues. (D) Section through the glanular erectile body (dotted lines) as indicated by the large white arrow pointing to a position in 3DR (C). Black arrowheads demarcate the tunica, and dotted lines denote the GEB. Note pleated UGS in ventral position. (E) Proximal section showing corporal body (CB), pleated UGS, tunica (arrowheads) surrounding corporal body only, and retractor muscles (dotted lines) dorsal to the UGS.
Figure 6
Figure 6
Three-dimensional reconstructions (3DR) (A, E & F) as well as sections (BD) of an adult male hyena penis exposed prenatally to flutamide (25mg/kg/day) from 32 to 110 days of gestation. (A) Side view with the skin (tan) semi-transparent to reveal internal structures. Note color-coding of labels throughout the figure. Prenatal flutamide has elicited the female pattern of the urethral orifice opening onto the surface ventral to the tip of the penis. (B) Distal section demonstrating the urethra (black arrowheads) opening ventrally. Large white arrow pointing to 3DR (B) indicates the position of section (A). (C) Section through the glanular erectile body (green in A & E) as indicated by the tip of the large white arrow pointing to 3DR (A). Note glanular erectile body (dotted lines), tunica (black arrowheads) only surrounding the corporal body (CB). The urethra (Ur) is large, pleated and ventrally positioned. (D) Proximal section through the retractor muscles (blue in A & F) as indicated by the tip of the large white arrow pointing to 3DR (A). Retractor muscles (dotted lines) are dorsal to the urethra (UR). The tunica (arrowheads) surrounds the corporal body (CB) only. (E) ¾ side view of proximal end of penis of a prenatally flutamide-treated adult male hyena. The arrangement of structures of this male specimen exhibits the female pattern (compare with Fig. 5C).
Figure 7
Figure 7
3DRs of the urethra/UGS (yellow) and glanular erectile body (green) of adult phalluses of (A) an untreated male, (B) an untreated female, and (C) a male treated with flutamide (25mg/kg/day) from 32 to 110 days of gestation. Side views (left) and proximal to distal views (right). Note male/female sexual dimorphism and that the prenatally flutamide/finasteride-treated male exhibits the adult female pattern.
Figure 8
Figure 8
Spotted hyena embryo at 27 days of gestation. The abdominal wall and abdominal viscera have been removed to reveal the urogenital ridges (UGR). The genital tubercle (GT) is already a prominent projection from the ventral body flanked by the hind limb buds.
Figure 9
Figure 9
(A and B) Male and female genital tubercles (GT) at 30 days of gestation. Note similarity in size of male and females GTs. (C) Genital tubercle dissected from a 30-day-old male spotted hyena fetus and photographed with oblique lighting. Note the whitish midline structure (arrow), which represents the solid urethral plate. Adapted from (Cunha et al., 2005) with permission.
Figure 10
Figure 10
(A) Histological section of an undifferentiated gonad from a 31-day-old fetus sexed by PCR as male. Note absence of seminiferous cords/tubules. (B & C) Wholemount images of spotted hyena genital tracts. (B) Wholemount of a 48-day-old female specimen. Note presence of Mullerian (MD) and Wolffian (WD) ducts and mesonephric tubules (small white arrow). (C) Wholemount of a 50-day-old male specimen. Note presence of Mullerian (MD) and Wolffian (WD) ducts and mesonephric tubules (small black arrow). Both of these specimens exhibit the ambisexual stage based upon differentiation status of the gonads, Wolffian and Mullerian ducts. Subtle texture at the periphery of the testis is indicative of seminiferous tubules, which was confirmed in tissue sections (not illustrated). Adapted from (Cunha et al., 2005) with permission.
Figure 11
Figure 11
Sections though mid-phallus of (A) untreated male 95 days of gestation, (B) newborn untreated female, (C) a stillborn male spotted hyena whose mother was treated from day 21 to 94 of gestation with flutamide (50mg/kg/day) plus finasteride (2 mg/kg/day) (Flut + Fin), and (D) a newborn female spotted hyena whose mother was treated with mibolerone from 37-114 days of gestation (6.6/μg/kg/day). These four specimens are of roughly the same age (94 to 114 days of gestation). Note that sexual dimorphism (A & B) at this age corresponds exactly to that seen in adulthood (compare with Figures 4 & 5). Note that the pattern seen in the flutamide/finasteride-treated male (C) is virtually identical to that of the untreated female (B) in regard to (a) position of the retractor muscles, (b) size and shape of the urethra (Ur)/urogenital sinus (UGS), (c) presence or absence of the corpus spongiosum, and (d) extent of the tunica. (D) The mibolerone-treated female exhibits the male pattern. Black arrows in (A-D) denote the retractor muscles. Adapted from (Cunha et al., 2005) with permission. Intact male= ♂, Castrated male= formula image, Intact female= ♀, Ovariectomized female= formula image
Figure 12
Figure 12
Phallic length in millimeters of adult (≥ 30 months) intact males and females versus adult males and females gonadectomized prepubertally. Analysis of variance revealed that there were highly significant differences among the four groups (F = 21.74, df = 3, p<.0001). Post-hoc tests indicated that there was a significant sex difference between intact males and intact females in phallic length (Fisher PLSD, p<.0001), and between Intact Females and Ovariectomized Females in clitoral length (Fisher PLSD, p<.01). Prepubertal castration had no significant effect on penile length (Fisher PLSD, p=.36) Numbers indicate numbers of specimens per group. ♂ = Male ♀= Female
Figure 13
Figure 13
Transverse sections through the distal aspect of the phallus of an untreated 45-day male (A), an untreated female (B) and a hyena fetus treated from 25 to 45 days with letrozole (12.5mg/day to the pregnant mother). Note the attachment of the urethral plate, which is dorsal in the male (A), ventral in the female (B), and ventral in the letrozole-treated male (C). (D) Three-dimensional reconstructions of the three specimens above showing additional sexual dimorphism in regard to the relationship between the corporal body (CB, red), tubular urethra (Ur, blue) and solid urethral plate (UP, yellow). The brown skin has been rendered semi-transparent.
Figure 14
Figure 14
External genitalia of untreated (A and E) and letrozole-treated (B-D, F, G) hyenas. (A) ExG of a 68-day untreated male hyena fetus. Note sharply defined ventral raphe (arrows). (B) ExG of a 67-day male hyena fetus treated from 23 to 67 with letrozole (12.5mg/day to the pregnant mother). Note the deeply furrowed ventral raphe (white arrows). (C) ExG of a 68-day male hyena fetus treated from 23 to 68 with letrozole (12.5mg/day to the pregnant mother). Note the deeply furrowed ventral raphe and bifid scrotum. (D) Flaccid adult clitoris of a female hyena treated from 58 to 108 days of gestation with letrozole. Note prominent preputial hypospadias. (E) Flaccid adult clitoris of an untreated adult female hyena. (F) Dorsal view of the penis of an adult male hyena treated from days of gestation 82 to 109 with letrozole (12.5mg/day to the pregnant mother). Note that the tip of the penis (yellow double arrow) extends over 1cm beyond the distal edge of the prepuce (white arrowheads). Normally the prepuce completely covers the glans penis. (G) Ventral view of the letrozole-treated penis in (F). Note the inordinately large urethral orifice (hypospadias, white arrowheads) situated on the ventral aspect of the glans penis. Normally the urethra opens on the dorsal aspect of the glans penis. (D and E) from (Frank et al., 1990) with permission.
Figure 15
Figure 15
Drawings of morphogenesis of the human penile urethra. The solid urethral plate canalizes to form the urethral groove, whose edges fuse in the midline to form the tubular penile urethra. Small opposing arrows indicate future fusion of adjacent epithelial surfaces. This developmental process has been observed in all species examined to date and appears to also apply to the spotted hyena. (adapted from Yamada, Satoh, Baskin, L. and Cunha, 2003 with permission)
Fig. 16
Fig. 16
Androgen receptor (AR) (A, B, E), estrogen receptor beta (ERβ) (C and F) and estrogen receptor alpha (ERD) (D and G) immunohistochemistry of 62-day hyena clitoris. In (B) the preputial lamina circumscribes the clitoris, which contains a corporal body dorsally, and a UGS ventrally. The preputial lamina (PPL) is AR-positive (A), ERβ-positive (C), and ERα-negative (D). AR, ERα, and ERα were detected in cells of the corporal body (E-G). The tunica albuginea, which is apparent by virtue of its blue color in (B) and indicated by arrowheads in (E and G), exhibits reduced or negative staining for AR and ERα.
Figure 17
Figure 17
Androgen receptor (AR) (A, D, E), estrogen receptor beta (ERβ) (B, F, G) and estrogen receptor alpha (ERα) immunohistochemistry (C, E) of a 62-day hyena clitoris. AR was detected in the preputial lamina (PPL), preputial skin and mucosa (PP), and mesenchyme surrounding the UGS (D), but was undetectable in the epithelium of the UGS (F). ERβ was detected in the epithelium of the UGS (F) and preputial skin and mucosa (PP). ERα was detected in the mesenchyme surrounding the UGS (E), but was undetectable in the epithelia of the UGS, preputial lamina, and preputial skin and mucosa (PP).
Fig. 18
Fig. 18
Androgen receptor (AR) immunohistochemistry of 68-day hyena penis. (A) Low magnification overview showing preputial lamina, tunica, urethra (Ur), corporal body (CB), and glanular erectile body (dotted lines). (B) Higher magnification of left box. Almost all cells within the corporal body (CB) are AR-positive. The tunica (between the two opposing arrows) consists of a mixture of AR-positive and AR-negative cells. (C) Higher magnification of right box in (A). The glanular erectile body contains a high percentage of AR-positive cells. (D) The urethral epithelium is mostly AR-negative, but is surrounded by AR-positive mesenchymal cells.

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