Evaluating the twin testosterone transfer hypothesis: A review of the empirical evidence

In this paper we review the evidence that fetuses gestated with a male co-twin are masculinized in development, perhaps due to the influence of prenatal androgens: the so-called twin testosterone transfer (TTT) hypothesis. Evidence from studies of behavioral, perceptual, cognitive, morphological and physiological traits in same- and opposite-sex human twins is considered. Apart from two studies reporting increases in aspects of sensation-seeking for females with a male rather than a female co-twin, there is sparse evidence supporting the TTT hypothesis in behavioral studies. Outcomes from studies of perception (in particular otoacoustic emissions) and cognition (in particular vocabulary acquisition and visuo-spatial ability) provide more consistent evidence in support of masculinized performance in twins with a male co-twin compared to twins with a female co-twin. The outcomes favorable to the TTT hypothesis for otoacoustic emissions and visuo-spatial ability are restricted to females. Studies of physiology and morphology (e.g., brain volume, tooth size and 2D:4D ratio) also show some influence of co-twin sex, but again these effects are often restricted to female twins. Because females produce little endogenous testosterone, the effects of gestation with a male co-twin may be more pronounced in females than males. Thus, while uneven, the evidence for the TTT hypothesis is sufficient to warrant further investigation, ideally using large samples of same- and opposite-sex twins, along with control groups of same- and opposite-sex siblings when the characteristics assessed are potentially open to social influences.     

Finger-length ratios show evidence of prenatal hormone-transfer between opposite-sex twins

Finger-length ratio (second to fourth finger; 2D:4D) has been associated with various measures thought to be related to prenatal androgens. In addition, hormone-transfer theory posits that hormones can transfer between twins. We examined 2D:4D in same-sex (SS) and opposite-sex (OS) dizygotic twins to test both propositions. Results show that 2D:4D is masculinized in OS females compared to SS females. This provides strong evidence that 2D:4D is laid down prenatally, and that hormones (likely androgens) can transfer from male to female fetuses. Implications for developmental timeframes for both hormone-transfer and 2D:4D are discussed.     

Sexual conflict in twins: male co-twins reduce fitness of female Soay sheep

Males and females often have different requirements during early development, leading to sex-specific interactions between developing offspring. In polytocous mammals, competition for limited resources in utero may be asymmetrical between the sexes, and androgens produced by male foetuses could have adverse effects on the development of females, with potentially long-lasting consequences. We show here, in an unmanaged population of Soay sheep, that female lambs with a male co-twin have reduced birth weight relative to those with a female co-twin, while there was no such effect in male twins. In addition, females with a male co-twin had lower lifetime breeding success, which appeared to be mainly driven by differences in first-year survival. These results show that sex-specific sibling interactions can have long-term consequences for survival and reproduction, with potentially important implications for optimal sex allocation.     

Effect of prenatal androgens on click-evoked otoacoustic emissions in male and female sheep (Ovis aries)

Otoacoustic emissions (OAEs) were measured in male and female Suffolk sheep (Ovis aries). Some sheep had been administered androgens or estrogens during prenatal development, some were gonadectomized after birth, and some were allowed to develop normally. As previously reported for spotted hyenas, gonadectomy did not alter the OAEs for either sex; accordingly, the untreated/intact and the untreated/gonadectomized animals were pooled to form the control groups. The click-evoked otoacoustic emissions (CEOAEs) exhibited by the female control group (N = 12) were slightly stronger (effect size = 0.42) than those in the male control group (N = 15), which is the same direction of effect reported for humans and rhesus monkeys. Females administered testosterone prenatally (N = 16) had substantially weaker (masculinized) CEOAEs than control females (effect size = 1.15). Both of these outcomes are in accord with the idea that prenatal exposure to androgens weakens the cochlear mechanisms that underlie the production of OAEs. The CEOAEs of males administered testosterone prenatally (N = 5) were not different from those of control males, an outcome also seen in similarly treated rhesus monkeys. Males administered dihydrotestosterone (DHT) prenatally (N = 3) had slightly stronger (hypo-masculinized) CEOAEs than control males. No spontaneous otoacoustic emissions (SOAEs) were found in any ears, a common finding in non-human species. To our knowledge, this is the first ruminant species measured for OAEs.     

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.     

Intersexuality in the mysid Neomysis integer

Intersexuality is described in Neomysis integer (Leach), apparently the first occurrence among the Mysidacea. Seven out of 13,700 specimens examined were masculinized females possessing elongated fourth pleopods, a male secondary sexual characteristic. Each masculinized female appeared to have reproduced as a normal female; there was no evidence of testicular tissue.     

Freemartins in beef cattle twins induced by embryo transfer

The incidence of freemartinism in heterosexual twins (male-female) resulting from embryo transfer was studied by determining sex chromosome chimerism in lymphocytes and masculinization of female reproductive tracts at slaughter. In one group of calves, ten of 11 heifers born co-twin to full sib, paternal half sib, or unrelated bull calves exhibited sex chromosome chimerism, a proportion in close agreement with that observed in naturally occurring twins. The ten calves with sex chromosome chimerism also had masculinized tracts whereas the other had an apparently normal female tract. Bull calves had a percentage of XY cells similar to their female co-twins, except for the twin set from which the “normal” female was obtained. The bull calf from this set had 5.6% XX cells although no XY cells were observed in the heifer in 66 metaphase spreads. No association was observed between the degree of sex chromosome chimerism and abnormalities of the female tract. Reproductive tracts from all female-female twin sets were normal. In another group of calves, all 20 heifers from heterosexual twin sets had masculinized reproductive tracts. It is concluded that the induction of twins by embryo transfer results in normal expression of freemartinism even though calves may be unrelated and are known to develop in separate uterine horns.     

Freemartinism in red deer (Cervus elaphus L.)

Several mixed-sex twins were produced by red deer treated with progesterone and pregnant mare's serum gonadotrophin. Of seven females karyotyped, two were 68,XX/68,XY chimeras. One male had the same chimeric karyotype as its female co-twin. These are the first reported cases of freemartinism in deer.     

Altruistic Behavior among Twins

According to kin selection theory, indirect reproductive advantages may induce individuals to care for others with whom they share genes by common descent, and the amount of care, including self-sacrifice, will increase with the proportion of genes shared. Twins represent a natural situation in which this hypothesis can be tested. Twin pairs experience the same early environment because they were born and raised at the same time and in the same family but their genetic relatedness differs depending on zygosity. We compared the degree of willingness to fight and sacrifice for the co-twin among monozygotic (MZ) and dizygotic (DZ) pairs in a sample of 1443 same-sex and opposite-sex twins. We also analyzed the effect of the subject’s gender and that of the co-twin on those altruistic behaviors. Results partly supported the postulated explanation. MZ twins (who share nearly their entire genome) were significantly more likely than DZ twins (who on average share half of their segregating genes) to self-sacrifice for their co-twins, but zygosity did not affect willingness to fight for him/her. The genders of the subject and of the co-twin, not genetic relatedness, were the best predictors of aggressive altruistic intentions.     

Gonadal hormones modulate sex differences in judgments of relative numerousness in meadow voles, Microtus pennsylvanicus

Animals in a variety of taxa discriminate between a greater quantity and a lesser quantity of the same object, an ability that is referred to as relative numerousness judgment. For example, meadow voles can distinguish between areas containing more over-marks by one opposite-sex scent donor and fewer over-marks by another opposite-sex scent donor. Females appear to be able to make better discriminations between more or less over-marks than do males. In that gonadal hormones have been implicated in modulating cognitive function associated with spatial tasks, we tested the hypothesis that high titers of testosterone and estradiol are necessary for male and female voles, respectively, to distinguish between the top- and bottom-scent donors in an area containing mixed over-marks. We gonadectomized voles, giving them either gonadal hormone replacement (testosterone for males and estradiol for females) or no hormone replacement, and tested their spontaneous judgments of distinguishing between the top- and bottom-scent donors in an area containing mixed over-marks; a task involving judgments of relative numerousness. Female voles given replacement estradiol performed better than did female voles not given replacement estradiol in determining the top-scent and bottom-scent males in areas containing mixed over-marks. In contrast, males not treated with replacement testosterone performed better than did males treated with testosterone in determining the top-scent and bottom-scent males in areas containing mixed over-marks. Thus, high titers of estradiol and low titers of testosterone are associated with better performance on tasks involving relative numerousness in female and male voles, respectively. The results of this task on relative numerousness judgments are discussed in relation to the effects of gonadal steroid hormone on spatial ability, a closely related cognitive domain, and the social biology of meadow voles.     

Transient masculinization in the fossa, Cryptoprocta ferox (Carnivora, Viverridae)

In at least 9 mammalian species, females are masculinized throughout life, but the benefits of this remain unclear despite decades of thorough study, in particular of the spotted hyaena (Crocuta crocuta) in which the phenomenon has been associated with a high fitness cost. Through examination of wild and captive fossas (Cryptoprocta ferox, Viverridae), androgen assays, and DNA typing for confirmation of gender, we made the first discovery of transient masculinization of a female mammal. Juvenile female fossas exhibited an enlarged, spinescent clitoris supported by an os clitoridis and a pigmented secretion on the underpart fur that in adults was confined to males. These features appeared to diminish with age. The majority of adult females lacked them, and os clitoridis length was inversely related to head-body length. No evidence was found to link this masculinization to elevated female androgen levels. Circulating concentrations of testosterone and androstenedione, but not dihydrotestosterone, were significantly lower in females than in males. No significant differences in testosterone, androstenedione, or dihydrotestosterone levels were found between juvenile (masculinized) and adult (nonmasculinized) females. There are several possible physiological mechanisms for this masculinization. None of the hypotheses so far proposed to explain the evolutionary basis of female masculinization in mammals are applicable to our findings. We present 2 new hypotheses for testing and development.     

Sex-specific effects of gonadal steroids on conspecific odor preference in the rat

Effects of gonadal steroids on conspecific odor preference for either (1) sexually active male or active female, (2) sexually active or gonadectomized (gdx) males, (3) sexually active or gdx females, and (4) gdx males or gdx females were determined in male and female rats in a three-chamber apparatus. For the first test, gdx females were made sexually active by treatments with estradiol benzoate (EB) and progesterone (P), and sexually active males were selected by prior screening. Sexually active males and females preferred opposite-sex odor over same-sex odor. Odor of sexually active opposite-sex conspecifics was preferred over that of inactive ones. Immediately after the completion of the first test, sexually active males were gdx and females were left without hormonal treatment. Second and third tests were carried out 2 and 5 weeks after the first test. In the second test, gdx males preferred odor of sexually active males rather than that of receptive females (male-directed preference); in the third test, both males and females showed no preference when tested with four stimulus pairs. The final tests were carried out in gdx males with EB and P, and gdx females with 2-week exposure to testosterone (T). Males with EB and P showed a male-directed preference again, whereas T-treated females kept their own female preference. Injection of EB alone to gdx males did not induce any preference. The present study clearly demonstrated sex difference in conspecific odor preference. Although both male and female preferences depend on their circulating sex steroids, the direction of male preference is more susceptible to their hormonal states, compared to that of females.     

Sexual experience interacts with steroid exposure to shape the partner preferences of rats

A three-phase experiment manipulated sexual experience and hormone exposure (perinatally and in adulthood) in female rats housed individually from weaning so as to limit peripubertal social and sexual experience. Noncontact partner preference for a male or estrous female rat was measured both before and after sexual experience, first while rats were under the influence of circulating testosterone propionate (TP) and later after priming them with ovarian hormones (estradiol benzoate and progesterone; EB & P). When implanted with TP capsules and tested while sexually naive, all groups of female rats preferred females to males without differing statistically. However, following three sexual experience sessions with estrous females, differences emerged between the masculinized and control groups in the magnitude of their female-directed preference, with masculinized females demonstrating a significantly greater preference for estrous females. Sexual experience with male rats under EB & P did not result in a significant shift in preference in any group. Histological assessment indicated that the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was increased by exposure to TP postnatally, and SDN-POA volume correlated positively with partner preference scores but only when rats were both sexually experienced and exposed to circulating TP in adulthood. These results suggest that sexual experience interacts with steroid exposure to shape partner preference.     

Adaptive attunement to the sex of individuals at a competition: the ratio of opposite- to same-sex individuals correlates with changes in competitors' testosterone levels

Evolutionary theories (e.g., the challenge hypothesis) suggest that testosterone plays an important role in intrasexual competition. In addition, those theories suggest that testosterone responses during competition should depend upon the presence of potential, immediate mating opportunities associated with the competition. The current research tested the hypothesis that the sex composition of individuals at a competition (ratio of opposite-sex, potential mates to same-sex individuals) would influence changes in competitors' testosterone levels. Consistent with our hypotheses, higher ratios of opposite- to same-sex individuals at an ultimate frisbee tournament were associated with greater increases in salivary testosterone among competitors. The relationship between sex ratio and increased salivary testosterone was observed for both male and female competitors and occurred regardless of whether competitors won or lost. Findings are consistent with the hypothesis that testosterone responses during competition are influenced by cues of potential, immediate mating opportunities.     

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.     

Testosterone increases singing and aggression but not male-typical sexual partner preference in early estrogen treated female zebra finches

Female zebra finches given estradiol benzoate (EB) as nestlings and testosterone propionate (TP) as adults show masculinized sexual partner preference, preferring females instead of males. This suggests an organizational effect of EB on sexual partner preference in a socially monogamous species that pairs for life. It is not known whether there is an activational hormone effect on sexual partner preference in this species, or whether adult testosterone treatment is necessary for masculinized preference to be expressed. In this experiment females were injected with EB daily for the first 2 weeks posthatching. As adults they were given TP filled or empty implants. Subjects were then given two-choice preference tests with male vs female stimuli, in which singing as well as proximity to the stimuli was recorded, followed by tests in a group aviary for social behavior and pairing preference. Females with TP implants sang more than females with empty implants and were more aggressive toward other females. They did not, however, differ from females with empty implants in any measure of sexual partner preference. Neither group showed a marked preference for males; instead both groups were equally interested in males and females. Thus adult testosterone treatment is not necessary for early estrogen treated females to show a shift in sexual partner preference in the male-typical direction.     

Genetic influence on human lifespan and longevity

There is an intense search for longevity genes in both animal models and humans. Human family studies have indicated that a modest amount of the overall variation in adult lifespan (approximately 20–30%) is accounted for by genetic factors. But it is not known if genetic factors become increasingly important for survival at the oldest ages. We study the genetic influence on human lifespan and how it varies with age using the almost extinct cohorts of Danish, Finnish and Swedish twins born between 1870 and 1910 comprising 20,502 individuals followed until 2003–2004. We first estimate mean lifespan of twins by lifespan of co-twin and then turn to the relative recurrence risk of surviving to a given age. Mean lifespan for male monozygotic (MZ) twins increases 0.39 [95% CI (0.28, 0.50)] years for every year his co-twin survives past age 60 years. This rate is significantly greater than the rate of 0.21 (0.11, 0.30) for dizygotic (DZ) males. Females and males have similar rates and these are negligible before age 60 for both MZ and DZ pairs. We moreover find that having a co-twin surviving to old ages substantially and significantly increases the chance of reaching the same old age and this chance is higher for MZ than for DZ twins. The relative recurrence risk of reaching age 92 is 4.8 (2.2, 7.5) for MZ males, which is significantly greater than the 1.8 (0.10, 3.4) for DZ males. The patterns for females and males are very similar, but with a shift of the female pattern with age that corresponds to the better female survival. Similar results arise when considering only those Nordic twins that survived past 75 years of age. The present large population based study shows genetic influence on human lifespan. While the estimated overall strength of genetic influence is compatible with previous studies, we find that genetic influences on lifespan are minimal prior to age 60 but increase thereafter. These findings provide a support for the search for genes affecting longevity in humans, especially at advanced ages.     

Differential effects of prenatal testosterone timing and duration on phenotypic and behavioral masculinization and defeminization of female sheep

The process of sexual differentiation leaves genetically female individuals at risk of being masculinized by exogenous androgens. Previous research with sheep indicates that exposure to excess testosterone from Gestational Day (GD) 30 to GD 90 of the 147-day gestation masculinizes and defeminizes behavior as well as genitalia. Lower doses and shorter durations produce animals with varying degrees of genital virilization and alterations of the hypothalamic-pituitary-gonadal axis, but to our knowledge, the effects on complex behavior and its prediction by the amount of external virilization have not been explored. Previous research in rodents has suggested that sexual differentiation of the central nervous system and the external genitalia can be dissociated. Therefore, we hypothesized that the extent of virilization of external genitalia would not be predictive of the lack of female-typical, or the presence of male-typical, mating behavior. To test this hypothesis, we compared control females, females exposed to exogenous testosterone from GD 30 to GD 90 (T60 females) that have virilized genitalia, and females exposed to testosterone from GD 60 to GD 90 (T30 females) that have female-typical genitalia. Both natural behavioral estrus in the flock and hormonally controlled behavioral tests were used to explore reproductive behavior. The T60 and T30 females exhibited more masculinized reproductive behavior than the controls; however, the T30 females also exhibited feminine behavior. Neither testosterone-treated group was receptive or was mounted at rates comparable to those of controls. These data illustrate that variation in the timing or duration of exposure to prenatal testosterone during a critical period for masculinization can have variable effects on defeminization and that the effects of testosterone on genitalia are not entirely predictive of behavior.     

Self-Grooming as a Tactic Used by Prairie Voles Microtus ochrogaster to Enhance Sexual Communication

Observations of numerous mammals suggest males self-groom more than females in response to the odours of opposite-sex conspecifics. Two experiments tested the hypothesis that self-grooming may be a tactic used by males to attract mates in prairie voles Microtus ochrogaster . In the first experiment, we measured the amounts of time voles self-groomed during exposure either to male-scented cotton bedding, female-scented cotton bedding, or clean cotton bedding. Results from this experiment support the hypothesis and also show that female prairie voles self-groom in response to odours of males. In addition, male prairie voles groom more in response to male odours than to female odours, suggesting that self-grooming also serves a role in male–male competition. In the second experiment, male and female voles spent more time investigating scent marks of opposite-sex conspecifics that recently self-groomed at a higher rate than those of opposite-sex conspecifics that self-groomed at a lower rate. Female, but not male prairie voles, spent more time investigating scent marks of opposite-sex conspecifics that self-groomed at a high rate than those of same-sex conspecifics that self-groomed at a high rate. For prairie voles, self-grooming may increase the detection of their scent marks by conspecifics. By self-grooming, prairie voles may be attempting to attract opposite-sex conspecifics, and males may also be attempting to deter encounters with male conspecifics.     

Experimental Addition of Green Plants to the Nest Increases Testosterone Levels in Female Spotless Starlings

Multiple male traits and displays may act in signalling sexually selected processes during courtship. Spotless starling males (Sturnus unicolor) carry green plants into their nests before egg laying, and recent studies have shown that this behaviour is related to female breeding decisions and the production of male‐biased broods. Although the functional implications of this effect on females are not yet clear, data suggest that it could be mediated by female circulating hormones. Additionally, females may show higher androgen levels as a consequence of the increased female–female competition generated by the increase in male attractiveness. We tested this hypothesis using the same manipulation of green nesting material that has been previously shown to result in an increase of male attractiveness in male spotless starlings. We found that females in experimental nests increased their circulating testosterone levels during the laying period. In addition, there was an increase of social interferences in the experimental nests because of the addition of green plants. We hypothesise that testosterone may allow females to maintain their mating status when competing with other females for the preferred males. Addition of green plants also increased the variance in the levels of circulating testosterone, suggesting plasticity between females in their response to the manipulation. We propose that there is a functional link between high testosterone levels, male‐biased sex ratios and female resource‐holding potential in intra‐sexual competition in this species.