Hormonal correlates of 'masculinization' in female spotted hyaenas (Crocuta crocuta). 2. Maternal and fetal steroids.

Concentrations of androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone), oestrogen and progesterone were measured in relation to pregnancy in the spotted hyaena (Crocuta crocuta). The gestation period was estimated to be about 110 days. There was a marked progressive rise in all the steroids starting in the first third of gestation. Chromatographic separation of plasma showed that much of the oestrogen is not oestradiol (only 12% of total measured) and that a significant fraction of the 'testosterone' may be dihydrotestosterone. In the final third of pregnancy, concentrations of androgen (especially testosterone plus dihydrotestosterone) in the female circulation reached the maximal values of adult males; the percentage of dihydrotestosterone relative to total testosterone plus dihydrotestosterone was higher in females (44 +/- 3.9%, n = 20) than in males (29.5 +/- 3.5%, n = 17). Plasma androstenedione was also significantly higher in females, but the increment was less than for oestrogen, testosterone and progesterone, and the temporal pattern was less clear. Samples from the maternal uterine and ovarian circulation showed that androstenedione is largely of ovarian origin and metabolized by the placenta, while testosterone, progesterone and oestrogen are primarily of placental or uterine origin. Fetal samples were taken from two mixed-sex sets of twins and one male singleton. Gradients across the placenta measured in the fetal circulation confirmed that the placenta metabolizes androstenedione and is a source of testosterone for the female fetus; there were no consistent differences in androgens between male and female fetuses. It is suggested that the conspicuous masculinization of the female spotted hyaena, especially evident in the external genitalia at birth, is a result, at least in part, of high placental production of testosterone or dihydrotestosterone derived from the metabolism of high maternal androstenedione.     

Temporal constraints on androgen directed laryngeal masculinization in Xenopus laevis

Temporal constraints on androgen regulated masculinization of three sexually dimorphic laryngeal properties--tension, fiber type, and fiber recruitment--were examined in Xenopus laevis frogs. Endocrine state was manipulated at PM0 when the larynx is similar in males and females, at PM2 when the larynx begins sexual differentiation, and at PM6 when sexual differentiation is complete. Removing the testes in developing males (PM0 or PM2) completely arrests laryngeal masculinization. Masculinization resumes when testosterone is replaced later in development (PM2 or PM6, respectively). Thus, testicular secretions, in particular androgens, are required for laryngeal masculinization. The ability of androgens to masculinize tension, fiber type, and fiber recruitment in developing and adult larynges was also determined. Five weeks of testosterone treatment in PM0 or PM2 males and females completely masculinizes laryngeal tension and fiber type, but only partially masculinizes fiber recruitment. However, fiber recruitment can be fully masculinized in PM6 males castrated at PM2. We conclude that androgen induced masculinization of tension and fiber type are not temporally constrained but that androgen induced masculinization of fiber recruitment is. Prolonged androgen treatment can override the temporal constraints on masculinization of the larynx. Testosterone treatment for more than 6 months fully masculinizes fiber recruitment in developing (PM0 or PM2) females. In addition, prolonged treatment (greater than 9 months) completely masculinizes tension, fiber type, and fiber recruitment in adult females; these properties were not fully masculinized by shorter (1-3 months) treatments in adult females. Testosterone induced masculinization in females is maintained for up to 8 months following testosterone removal; thus androgen effects are long lasting and possibly permanent.     

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.     

Androgens and male mating behavior in rough-skinned newts, Taricha granulosa

Cyproterone acetate was administered either orally or intraperitoneally to intact, adult male newts, Taricha granulosa. The number of males that exhibited the courtship behavior of clasping when tested with nuptial females was not altered by the antiandrogen treatments. In males which were unresponsive to nuptial females, the occurrence of clasping was not evoked by injections for 4 days of testosterone, dihydrotestosterone, or 11-ketotestosterone. Further, the incidence of clasping was not significantly elevated by injections of prolactin and/or testosterone for 30 days. The effect of sexual activity on testosterone and dihydrotestosterone levels in male newts was determined by radioimmunoassay of plasma collected from males which were: (1) isolated from females; (2) allowed to clasp a female for 2 min; or (3) allowed to clasp a female for 1 hr. The testosterone and dihydrotestosterone levels were unchanged during this period of clasping. In February and again in June, plasma androgen concentrations were measured in males which differed in their propensity to initiate courtship when paired with females. Androgen levels were similar for males that clasped a female and males that never attempted to clasp a female. Plasma androgen levels in the male newt are apparently not correlated with sexual responsiveness.     

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.     

Female masculinization and reproductive life history in the western mosquitofish (<Emphasis Type="Italic">Gambusia affinis</Emphasis>)

In this study, we explored the relationship between female masculinization and various morphological and reproductive parameters of male and female western mosquitofish (Gambusia affinis), including body size, anal fin length, testes mass and female fecundity. We detected high levels of female masculinization in the Brazos and Little Brazos Rivers (Falls County, Texas) in March 2008, but not 2009. In both populations, masculinized females were smaller, possessed shorter anal fins, and had significantly lower total fecundity (collective number of eggs and embryos) compared to non-masculinized females. In addition, 2008 non-masculinized females were smaller and less fecund than 2009 non-masculinized females in both populations. Moreover, 2008 males were smaller, had shorter gonopodia, and smaller testes than 2009 males. Our results support the hypothesis that female masculinization can have a strong influence on reproductive life history and population dynamics of livebearing fishes.     

Sex and seasonal differences in aggression and steroid secretion in Lemur catta: are socially dominant females hormonally 'masculinized'?

Female social dominance characterizes many strepsirrhine primates endemic to Madagascar, but currently there is no comprehensive explanation for how or why female lemurs routinely dominate males. Reconstructing the evolutionary pressures that may have shaped female dominance depends on better understanding the mechanism of inheritance, variation in trait expression, and correlating variables. Indeed, relative to males, many female lemurs also display delayed puberty, size monomorphism, and 'masculinized' external genitalia. As in the spotted hyena (Crocuta crocuta), a species characterized by extreme masculinization of the female, this array of traits focuses attention on the role of androgens in female development. Consequently, I examined endocrine profiles and social interaction in the ringtailed lemur (Lemur catta) to search for a potential source of circulating androgen in adult females and an endocrine correlate of female dominance or its proxy, aggression. I measured serum androstenedione (A(4)), testosterone (T), and estradiol (E(2)) in reproductively intact, adult lemurs (10 females; 12 males) over four annual cycles. Whereas T concentrations in males far exceeded those in females, A(4) concentrations were only slightly greater in males than in females. In both sexes, A(4) and T were positively correlated, implicating the Delta(4)-biosynthetic pathway. Moreover, seasonal changes in reproductive function in both sexes coincided with seasonal changes in behavior, with A(4) and T in males versus A(4) and E(2) in females increasing during periods marked by heightened aggression. Therefore, A(4) and/or E(2) may be potentially important steroidal sources in female lemurs that could modulate aggression and underlie a suite of masculinized features.     

Defense of females, but not social status, predicts plasma androgen levels in male spotted hyenas

A positive correlation between male social status and testosterone levels is expected and often found in social species with high rates of agonistic interactions or when social relationships among males are unstable. In contrast, in species with low rates of agonistic interactions or when social relationships are stable, testosterone levels should not correlate with social status. The "challenge hypothesis" predicts that androgen levels should rise during periods of courtship or mate guarding. We addressed these questions in free-ranging spotted hyenas, a species with low rates and low intensities of aggression among males but where males spend extensive effort to court females. In males, we measured testosterone, its precursor androstenedione, and its metabolite 5alpha-dihydrotestosterone. As predicted, testosterone levels were significantly higher and androstenedione levels tended to be higher in males that, at the time of sampling, defended a female, compared with males that did not defend a female. Also, as predicted, there was no correlation between social status and androgen levels in male spotted hyenas.     

Plasma androgens in spotted hyaenas (Crocuta crocuta): influence of social and reproductive development

Differences in plasma testosterone and androstenedione concentrations in male spotted hyaenas belonging to various reproductive and social categories 4 clans resident in South Africa and Botswana suggest that central-immigrant males have the highest concentrations of androgens, reflecting their roles as mating males. Social inhibition of reproductive function may occur in other males. A reversal in the ratio of testosterone: androstenedione occurs at puberty in most individuals, testosterone becoming the dominant hormone, especially in males that have procured mating rites. Cubs of either sex had low testosterone concentrations, except for a 4-day-old male that displayed adult concentrations, which were accompanied by a temporary testicular descent. Plasma testosterone concentrations in females largely reflected ovarian activity and showed no correlation with androstenedione concentrations. Female cubs had androstenedione concentrations significantly higher than those in all other social categories except for the central-immigrant males. The androgen profiles presented here suggest that the key to the behavioural dominance of female spotted hyaenas over males may lie with the neonatal developmental stages, rather than with the androgen patterns of adult animals.     

Hormone effects on male sex behavior in adult South African clawed frogs, Xenopus laevis

Intact, adult male Xenopus laevis were injected with human chorionic gonadotropin (HCG) and tested with intact HCG-primed females. Under these conditions, males displayed high levels of sex behavior (clasping of females). By 2 weeks after castration, these males had ceased clasping. Testosterone and dihydrotestosterone reinstated clasping in male castrates. Following removal of testosterone or dihydrotestosterone pellets, castrated males ceased to clasp. No male was ever observed to clasp following estradiol implanted in pellets or in silastic capsules. In experiments on castrated, adult, female Xenopus laevis, both testosterone and testosterone propionate pellets reliably produced male sex behavior in the form of clasping. The clasping of testosterone-implanted female and male castrates was very similar in form and duration. The behavioral effectiveness of testosterone in both sexes and the ineffectiveness of estradiol in eliciting clasping is paralleled by autoradiographic localization of sex steroids in brain where the distribution of testosterone-concentrating, cells is the same for males and females, but different from the distribution of estradiol-concentrating cells.     

Stress hormone masculinizes female morphology and behaviour

Sex steroids play major roles in vertebrate sexual differentiation. Unexpectedly, we now find that exposure to elevated levels of the naturally occurring stress hormone cortisol can also masculinize sexually dimorphic morphological characters and behaviour in adult female mosquitofish (Gambusia affinis) in a dose-dependent manner. Females masculinized by cortisol developed elongated anal fins with distal tip features similar to those of mature males. Most masculinized females also attempted to copulate when placed with normal females. Although the mechanism of masculinization is currently unknown, we propose a role for an enzyme that both inactivates cortisol and catalyzes the final step in synthesis of a major teleost androgen. This mechanism may also help explain some previously reported effects of stress on sexual development across vertebrate taxa. Our findings underscore the need to understand the full range of chemicals, both naturally occurring hormones and human-produced endocrine disruptors, that can influence sexual differentiation and reproductive function.     

Treatment of pregnant rhesus macaques with testosterone propionate: observations on its fate in the fetus

Castrated male primates, unlike castrated male rodents, respond to exogenous estrogen by releasing gonadotropin. Although this disparity is unexplained, it may occur because the amount of testicular androgen secreted during a critical period for sexual differentiation is not sufficient to completely androgenize the anlagen of the central nervous system (CNS) in male primates. Therefore, male primates might be incompletely masculinized, and if fetal males were exposed to additional androgen during sexual development, they would no longer display the positive feedback to estrogen that usually characterizes females. Besides the development of mechanisms mediating the release of gonadotropins, questions about relationships between adult male sexual behaviors and the intensity of the androgen stimulus upon developing neural structures are of interest. We tested some of these possibilities by injecting androgen into 8 pregnant rhesus macaques from Days 40 through 50 of gestation, and we compared serum levels of testosterone (T), dihydrotestosterone (DHT), and androstenedione (delta 4) in the fetal circulation with that of 5 untreated control males. Fetal sera (both male and female) from treated pregnancies did not contain significantly greater quantities of T and DHT than sera of intact control males from untreated mothers. The maternal sera, however, contained large amounts of T (125.05 +/- 27.40 [SEM] ng/ml, n = 8) and significant elevations of DHT and delta 4 after treatment. The concentrations of delta 4 in the fetal circulation were significantly elevated (p less than 0.01) in treated fetuses compared to intact control males, probably due to the actions of the 17 beta-hydroxysteroid dehydrogenases in the placenta, the fetus, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of induced masculinization on reproductive and aggressive behaviors of the female mosquitofish,Gambusia affinis affinis

Synopsis FemaleGambusia affinis affinis were masculinized with the degraded products of 65% stigmastanol-30% B-sitosterol, a phytosterol. The masculinized females were paired with non-treated males, non-treated females, and other masculinized females. The pairs were analyzed for reproductive and aggressive behaviors exhibited. The behavioral patterns of these pairs were compared statistically to the behavior patterns of the following non-treated pairs: male with male, female with female, and male with female. The masculinized females behaved like males in that they followed, swung, and thrust their gonopodia at non-treated females and larger masculinized females. However, the male-like behavior of the masculinized females was not as intense as that of normal males. Also, the effect of masculinization on the behavior of the treated female was context-dependent as seen when placed with a male or a smaller masculinized female. Under these conditions the masculinized females behaved like typical females and exhibited no masculinized behaviors. The effects of anal spots and size differences are discussed as possible explanations for the variability in behaviors exhibited. The masculinized females displayed no change in aggressive behaviors.     

Adrenal and gonadal contribution to circulating androgens in spotted hyaenas (Crocuta crocuta) as revealed by LHRH, hCG and ACTH stimulation

Single venous blood samples were collected from 52 hyaenas and serial samples via indwelling jugular catheters from 8 hyaenas. Social status of the hyaenas was unknown. Radioimmunoassay estimations were specific for testosterone, androstenedione, cortisol and LH. No significant differences could be found between the sexes for concentrations of testosterone (adult males 2.9 +/- 1.3 nmol/l; adult females (non-pregnant) 1.6 +/- 0.3 nmol/l). Androstenedione concentrations in sub-adult males were significantly lower than those in females (2.3 +/- 0.05 and 5.5 +/- 1.2 nmol/l). Testosterone and androstenedione concentrations were significantly higher in parous pregnant than in parous non-pregnant females (4.3 +/- 1.4 and 1.6 +/- 0.3; and 23.7 +/- 11.6 and 6.7 +/- 0.9 nmol/l respectively). Both LHRH and hCG elicited elevated androgen concentrations in females in different reproductive categories. Androgens are produced by the ovary in the absence of follicular or luteal structures, indicating androgen production by the interstitial tissue of the ovarian stroma.     

Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: IV. Masculinization of the spinal nucleus of the bulbocavernosus with testosterone metabolites

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. SNB motoneurons and their perineal target muscles are present in adult males but reduced or absent in females. This sexual dimorphism is due to the presence of androgen during development; females treated with testosterone (T) perinatally have a masculine SNB system. To assess whether masculinization of the SNB could involve the conversion of testosterone into its active metabolites, dihydrotestosterone (DHT) and estrogen, we examined the development of the SNB in females treated perinatally with estrogen alone or in combination with dihydrotestosterone. Counts of motoneurons in the developing SNB in all groups showed the typical prenatal increase followed by a differential postnatal decline; the incidence of degenerating cells reflected this decline. Motoneuron numbers and the frequency of degenerating cells in females treated with estrogen (E) alone did not differ from those of normal females, with both groups losing large numbers of motoneurons and having a high incidence of degenerating cells. In contrast, females treated with both estrogen and dihydrotestosterone did not show the female-typical decline in motoneuron number and had a low, masculine incidence of degenerating cells. By postnatal day 10, females treated with estrogen and dihydrotestosterone had a fully masculine SNB motoneuron number, suggesting that dihydrotestosterone alone or in conjunction with estrogen may be involved in the development of the sexually dimorphic SNB system.     

Masculinization of Female Crayfish,Procambarus Clarki (Girard)

Summary

Twenty-three immature female crayfish (Procambarus clarki (Girard); carapace length = 8.2–17.9 mm), implanted with androgenic gland implants obtained from P. clarki males, were observed for 326 days for evidence of masculinization. Number of molts completed ranged from three to eight. Of eleven surviving females, three were masculinized as evidenced by the partial transformation of their first pair of pleopods into male-type gonopods. No other evidence of masculinization, either internal or external was seen. Masculinization of the first pair of pleopods was unequal and first noted 290 days post-implantation. We conclude that androgenic gland implantation can induce masculinization of P. clarki females, albeit only with great difficulty. This is the first report of masculinization of female macruran decapods by androgenic gland implantation.     

Androgen levels in the Spotted hyaena Crocuta crocuta: the influence of social factors

The Spotted hyaena is characterized by a suite of unique morphological and behavioural adaptations in the female: the external genitalia are nearly indistinguishable from those of the male, and females dominate males in most behavioural interactions. In a previous study of southern African hyaenas, males and females were found to have similar levels of circulating androgen, and it was suggested that relatively high foetal and adult levels are associated with female genital masculinization and dominance over males, respectively. This study shows that females from an East African population have androgen levels significantly lower than males, and that these levels in both sexes are strongly affected by individual social status; alpha animals had the highest androgen levels in each sex. These data cast doubt on the earlier hypothesis that aspects of sexual monomorphism in the Spotted hyaena are related solely to elevated female androgen titres.     

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.     

Masculinized female yellow-bellied marmots initiate more social interactions

The presence of male siblings in utero influences female morphology and life-history traits because testosterone transferred among foetuses may masculinize females. Similarly, litter sex composition might alter the display of sexually dimorphic behaviour, such as play and allogrooming, since they are modulated by androgens. We explored whether masculinization alters the frequency of play and sociopositive behaviour in female yellow-bellied marmots (Marmota flaviventris). We found that masculinized juvenile females were more likely to initiate play and allogrooming, but yearling females exhibited higher levels of oestrogen-modulated sociopositive behaviours. Additionally, the more they interacted, the greater number of different partners they interacted with. Our results suggest that masculinization increases the rate of age-dependent social behaviour. This probably works by increasing exploration that predisposes individuals to higher encounter rates. Further support comes from previous findings showing that masculinized females were more likely to disperse. Our study stresses the importance of considering litter sex composition as a fitness modulator.