Ned J. Place

Morphology of Mouse External Genitalia: Implications for a Role of Estrogen in Sexual Dimorphism of the Mouse Genital Tubercle

PURPOSE We examined the role of androgens and estrogens in mammalian sexual differentiation by morphological characterization of adult wt and mutant mouse external genitalia. We tested the hypothesis that external genitalia development depends on androgen and estrogen action. MATERIALS AND METHODS We studied serial sections of the external genitalia of the CD-1 and C57BL6 wt strains of adult mice (Charles River Laboratories, Wilmington, Massachusetts). We recorded linear measurements of key structures in each specimen, including the urethra, erectile tissue, bone and cartilage. We used similar methodology to analyze mice mutant for estrogen receptor α (αERKO) and androgen receptor (X(Tfm)/Y) (Jackson Laboratory, Bar Harbor, Maine). RESULTS Morphology in X(Tfm)/Y adult murine external genitalia was remarkably similar to that in wt females. Bone and clitoral length was similar in wt females and X(Tfm)/Y mice. Conversely the αERKO clitoris was 59% longer and bone length in αERKO females was many-fold longer than that in female wt mice or X(Tfm)/Y mutants. The αERKO clitoris contained cartilage, which is typical of the wt penis but never observed in the wt clitoris. Serum testosterone was not increased in female αERKO mice 10 days postnatally when sex differentiation occurs, suggesting that masculinization of the αERKO clitoris is not a function of androgen. CONCLUSIONS Masculinization of the αERKO clitoris suggests a role for estrogen in the development of female external genitalia. We propose that normal external genital development requires androgen and estrogen action.

Exposure to naturally circulating androgens during foetal life incurs direct reproductive costs in female spotted hyenas, but is prerequisite for male mating

Among all extant mammals, only the female spotted hyena (Crocuta crocuta) mates and gives birth through the tip of a peniform clitoris. Clitoral morphology is modulated by foetal exposure to endogenous, maternal androgens. First births through this organ are prolonged and remarkably difficult, often causing death in neonates. Additionally, mating poses a mechanical challenge for males, as they must reach an anterior position on the females abdomen and then achieve entry at the site of the retracted clitoris. Here, we report that interfering with the actions of androgens prenatally permanently modifies hyena urogenital anatomy, facilitating subsequent parturition in nulliparous females who, thereby, produce live cubs. By contrast, comparable, permanent anatomical changes in males probably preclude reproduction, as exposure to prenatal anti–androgens produces a penis that is too short and has the wrong shape necessary for insertion during copulation. These data demonstrate that the reproductive costs of clitoral delivery result from exposure of the female foetus to naturally circulating androgens. Moreover, the same androgens that render an extremely unusual and laborious process even more reproductively costly in the female are apparently essential to the males physical ability to reproduce with a normally masculinized female.

The Ontogeny of the Urogenital System of the Spotted Hyena (Crocuta crocuta Erxleben)

Abstract 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 α-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 5α-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.

Short Day Lengths Delay Reproductive Aging

Abstract Caloric restriction and hormone treatment delay reproductive senescence in female mammals, but a natural model of decelerated reproductive aging does not presently exist. In addition to describing such a model, this study shows that an abiotic signal (photoperiod) can induce physiological changes that slow senescence. Relative to animals born in April, rodents born in September delay their first reproductive effort by up to 7 mo, at which age reduced fertility is expected. We tested the hypothesis that the shorter day lengths experienced by late-born Siberian hamsters ameliorate the reproductive decline associated with advancing age. Short-day females (10L:14D) achieved puberty at a much later age than long-day animals (14L:10D) and had twice as many ovarian primordial follicles. At 10 mo of age, 86% of females previously maintained in short day lengths produced litters, compared with 58% of their long day counterparts. Changes in pineal gland production of melatonin appear to mediate the effects of day length on reproductive aging; only 30% of pinealectomized females housed in short days produced litters. Exposure to short days induces substantial decreases in voluntary food intake and body mass, reduced ovarian estradiol secretion, and enhanced production of melatonin. One or more of these changes may account for the protective effect of short day lengths on female reproduction. In delaying reproductive senescence, the decrease in day length after the summer solstice is of presumed adaptive significance for offspring born late in the breeding season that first breed at an advanced chronological age.

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. J. Comp. Neurol. 498:80–92, 2006.

Ultrasonic measurements of second and third trimester fetuses to predict gestational age and date of parturition in captive and wild spotted hyenas Crocuta crocuta

Parturition in spotted hyenas (Crocuta crocuta) is a fascinating event to witness, as females of this species are highly masculinized and give birth through a penis-like clitoris. Furthermore, shortly after birth, a high rate of aggression occurs between littermates that can sometimes end in siblicide. To study these events thoroughly, an accurate estimate of the date of parturition is necessary. To this end, we performed transabdominal ultrasounds every 20-30 days in five captive spotted hyenas of known gestational age, beginning approximately 30 days after mating. We measured the femur length (FL), abdominal circumference (AC), and biparietal diameter (BPD) of eight fetuses from Days 42 to 100 of their 110 days of gestation. FL proved to be the most effective measurement, as it correlated well with gestational age and was easy to obtain consistently. The relationship between estimated gestational age (EGA) and FL is described by the equation: [EGA = 37.3 + (14.0 x FL)]. AC also correlated well with EGA, but was more difficult to measure than FL. Measuring BPD became increasingly difficult as pregnancies advanced beyond 70 days of gestation. Because gestational age is often not known in captive and free-ranging spotted hyenas, measuring fetal FL ultrasonographically is a rapid and reliable way to determine an approximate date of parturition. This technique proved invaluable when used to track and monitor a free-ranging spotted hyena during the days just before and after parturition.

Effects of Prenatal Treatment with Antiandrogens on Luteinizing Hormone Secretion and Sex Steroid Concentrations in Adult Spotted Hyenas, Crocuta crocuta

Abstract 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.

Effects of experimentally elevated testosterone on plasma glucocorticoids, body mass, and recapture rates in yellow-pine chipmunks, Tamias amoenus.

In male yellow-pine chipmunks plasma levels of glucocorticoids (GCs) are low while plasma testosterone (T) levels peak during the mating season, suggesting that T suppresses GC levels. To test this hypothesis, free-living, post-reproductive males were implanted during summer with either a T-filled (T-males) or an empty silastic implant (controls or C-males). Body mass and plasma levels of corticosterone, cortisol, and T were measured immediately before and 1 month after implantation. Exogenous testosterone increased T to high physiological levels typical of reproductively active males. By 1 month after implantation, T-males decreased their mean body mass and plasma GC levels, while C-males maintained their mean body mass and GC levels. Even though T-males lost mass, recapture success 1 month after implantation for T-males (71%) was equal to that of C-males (71%). However, the overwinter recapture rate of C-males (83%) was significantly greater than that of T-males (20%). The results support the hypothesis that high plasma T of males during mating has a suppressive effect on plasma GC levels. Additionally, experimentally elevated T significantly reduced the rate of recapture during the following spring, and this may reflect a reduction in local overwinter survival. The suppression of adrenocortical activity by T may contribute to the reductions in prehibernation body mass and post-emergence recapture success. J. Exp. Zool. 287:378-383, 2000.