The estrogenic arousal of aggressive behavior in female mice

Experiments were conducted to determine the conditions under which estrogen would promote male-like aggressive behavior in female mice. The results of the first experiment showed that most females chronically exposed to testosterone propionate (TP) in adulthood fought, whereas females similarly treated with estradiol benzoate (EB) did not display aggression. Another experiment found that, when either TP or EB was administered on the day of birth, adult females displayed aggression in response to daily EB injections during adult life. Also, the potentiating effect of neonatal hormone exposure declined over the first 12 days postpartum, as 100% of the Day 0, 75% of the Day 6, and 0% of the Day 12 and 18 TP-treated females fought in response to daily injections of 40 μg of EB in adulthood. The final study showed that, under the test conditions employed, the failure of a chronic adult EB regimen to promote aggression was not due to a competing tendency to display female sexual behavior.     

Prenatal exposure to androgen influences morphology and aggressive behavior of male and female mice

To assess the effects of prenatal exposure to androgen on adult aggressiveness in mice, pregnant mice were given injections of 1.5 mg testosterone propionate (TP) or oil from Days 12 to 16 of pregnancy. All offspring were gonadectomized on the day of birth. Neonatal treatment occurred on the day following birth and consisted of one-half of the animals from each prenatal treatment group being injected with 100 μg TP while the other half were injected with oil, yielding four Prenatal/Neonatal treatment groups for each sex. On postnatal Day 60, all offspring were given subcutaneous implants of encapsulated testosterone (T) and tested for 10 min every other day against a male opponent until aggression was observed. Female offspring of TP-treated mothers were indistinguishable from males on external examination at birth. The duration of exposure to T required to induce aggression provides an index of the sensitivity of the neural substrate to T. When arranged from the most sensitive to the least sensitive to the aggression inducing action of T, the four Prenatal/Neonatal treatment groups of females were significantly different from each other: Group TP/TP > Group OIL/TP > Group TP/OIL > Group OIL/OIL. A similar pattern was observed for the male offspring. There were no differences in the proportion of animals per group that exhibited aggression (virtually all animals fought) or the intensity of aggression once exhibited. The results demonstrate that morphological and behavioral masculinization can occur in response to exposure to androgen during prenatal as well as neonatal life in mice.     

Differential effects of testosterone metabolites in the neonatal period on open-field behavior and lordosis in the rat

Two experiments were done to compare the effects of neonatal exposure to testosterone and its major metabolites, dihydrotestosterone (DHT) and estradiol (E₂), on the development of sex differences in open-field behavior in the rat. In Experiment 1 female rats administered either testosterone propionate (TP), DHT, or estradiol benzoate (EB) were found as adults to have low activity scores, more typical of adult males, when compared to the high scores of oil-treated females. In Experiment 2 the adult open-field behavior of female rats treated neonatally with testosterone or the metabolites was compared to that of male rats treated from Day 1 to 10 of life with the aromatizing enzyme inhibitor, androst-1,4,6-triene-3,17-dione (ATD). These same animals were later tested for lordotic behavior after gonadectomy and priming with EB and progesterone. All male animals and female animals exposed neonatally to testosterone or to either of the metabolites had suppressed open-field activity scores compared to oil-treated females. However, the lordotic behavior of females exposed to DHT and of males exposed to ATD was not defeminized and was comparable to that of oil-treated females. These observations were discussed in terms of a role for the androgenic actions of testosterone in establishing sex differences in nonreproductive behavior in the rat.     

Mounting behavior and lordosis behavior in castrated male rats treated with testosterone propionate, or with estradiol benzoate or dihydrotestosterone in combination with testosterone propinonate.

Treatment of prepuberally castrated male rats with testosterone propionate (TP, 50, 200, 500, or 1000 μg for 30 days) in adulthood stimulated the display of both mounting behavior and lordosis behavior. No correlation between mounting and lordosis behavior could be detected at any TP dose level. Treatment of prepuberally castrated male rats with either 1 μg estradiol benzoate (EB) or 500 μg dihydrotestosterone (DHT) for 60 days stimulated the display of mounting behavior in three of eight and four of eight rats, respectively. Treatment with 200 μg TP for the last 30 days of rats receiving either EB or DHT for 60 days resulted in an abrupt onset on mounting behavior as compared to rats treated with oil for 60 days. These results show additive effects of EB or DHT and TP upon mounting behavior by male rats and are interpreted as a support for the suggestion that testosterone to estrogen as well as testosterone to DHT conversion may be involved in the mechanism whereby testosterone activates the mounting behavior of castrated rats.     

The influence of caging conditions and hormone treatments on fighting in male and female hamsters

After gonadectomy, more individually caged female hamsters fought prior to the initiation of hormone treatments than did group-caged females. Daily injections of testosterone propionate (TP), estradiol benzoate (EB), or progesterone (Prog) had no influence on the number of individually caged females that fought. However, TP and EB were effective in increasing the number of group-caged females that fought. In contrast to females, both individually and group-caged males fought infrequently after castration. Daily injections of TP, EB, or Prog were effective in increasing the number of individually caged males that fought, while only TP and EB were effective in group caged males. Prog failed to increase the number of group-caged hamsters of either sex that fought.     

Influence of prolactin, androgens and bromocriptine on seminal vesicular and prostatic adenosine triphosphatases in castrated adult monkeys Macaca radiata

E ects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combinations of these androgens with PRL/Br on specific activities of adenosine triphosphatases (ATPases) of seminal vesicles and cranial and caudal prostates were studied in castrated adult bonnet monkeys. Castration decreased all ATPases (sodium/potassium, magnesium and calcium dependent) of seminal vesicles and both the lobes of prostate. PRL restored the normal activities of all ATPases in both the organs. Br given alone decreased all ATPases of prostate but caused no significant alteration, particularly calcium dependent ATPases of seminal vesicles and caudal prostate. TP/DHT replacement restored all ATPases of both the organs to the normal levels. PRL + TP/DHT further enhanced all the ATPases activities of all the regions studied. Br + TP/DHT decreased all ATPases but it did not produce any alteration in the calcium ATPases of seminal vesicles. The results suggest that prolactin facilitates membrane transport enzymes in the cranial and caudal prostate and seminal vesicles of adult castrated bonnet monkeys.     

Differential responsiveness to replacement therapy of pre- and postpubertally castrated mice with respect to intermale aggression

Male mice castrated before Day 6 of postnatal life differ from adult castrates in that they do not exhibit normal patterns of intermale aggression following replacement therapy as adults. Two experiments sought to determine the effect castration after Day 6, but before puberty, would have on this response to adult replacement therapy. It was found that adult castrates showed some increases in fighting as early as six hr after a subcutaneous (s.c.) injection of 2 mg testosterone propionate (TP). At 30 hr after treatment 14/15 mice fought, while at 72 hr 15/15 fought and the number of fights in 10 min was significantly higher than at 30 hr. In the second experiment, the response to adult treatment with 2 mg TP (s.c.) in mice castrated in Day 10 or Day 50 was compared. Different groups were tested at 16, 40, 64, and 88 hr after TP treatment. The latency to respond to TP was significantly less and the level of fighting obtained was significantly greater in Day 50 castrates than in Day 10 castrates at each time of testing. The proportion of Day 10 (14/15) and Day 50 (15/15) castrates fighting was equal by 88 hr, while the mean fighting frequency was significantly lower in Day 10 castrates (M = 4.93 ± 4.78) than in Day 50 castrates (M = 8.06 ± 1.63). Day 50 castrates fought significantly more often than controls at 40 hr, whereas the level of fighting attained by Day 10 castrates was not significant until 64 hr. These results suggest that even after the organizational period for aggression, testosterone is necessary for maintenance and/or preparation of the substrate essential to the elicitation of aggression.     

Serum estradiol, testosterone and dihydrotestosterone in male monkeys treated with testosterone propionate.

Serum estradiol (E2), testosterone (T) and dihydrotestosterone (DHT) were measured in juvenile (pre-pubertal) male rhesus monkeys injected with either 8 mg or 80 mg of testosterone propionate (TP). After one week, the three steroids were elevated and remained essentially unchanged for the duration of the study. There was little difference in serum E2 or DHT when comparing the two groups of steroid-treated monkeys. In contrast, T levels were consistently greater in the animals given the high dosage of TP.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

The effects of testosterone administered neonatally on the incorporation of 3H-leucine into protein by thyroid gland of rat: a neonatal priming.

Rats were castrated on day 2 after birth, given one injection of testosterone pronate (TP: 2.5 mg) or oil just after operation and then received TP or oil when adult. 3H-Leucine was injected intravenously 2.5 hours before killing. The incorporation of labelled amino acid into protein was investigated in the thyroid glands. Males receiving TP both neonatally and when adult incorporate twice as much labelled leucine into protein as compared to their counterparts. Grain density was highest in the thyroid gland of early hormone treated rats when challanged with testosterone in adulthood. Thyroid gland priming by early hormone treatment has been discussed.     

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Neuroendocrine consequences of prenatal androgen exposure in the female rat: absence of luteinizing hormone surges, suppression of progesterone receptor gene expression, and acceleration of the gonadotropin-releasing hormone pulse generator

Preovulatory GnRH and LH surges depend on activation of estrogen (E2)-inducible progesterone receptors (PGRs) in the preoptic area (POA). Surges do not occur in males, or in perinatally androgenized females. We sought to determine whether prenatal androgen exposure suppresses basal or E2-induced Pgr mRNA expression or E2-induced LH surges (or both) in adulthood, and whether any such effects may be mediated by androgen receptor activation. We also assessed whether prenatal androgens alter subsequent GnRH pulsatility. Pregnant rats received testosterone or vehicle daily on Embryonic Days 16-19. POA-hypothalamic tissues were obtained in adulthood for PgrA and PgrB (PgrA+B) mRNA analysis. Females that had prenatal exposure to testosterone (pT) displayed reduced PgrA+B mRNA levels (P < 0.01) compared with those that had prenatal exposure to vehicle (pV). Additional pregnant animals were treated with vehicle or testosterone, or with 5alpha-dihydrotestosterone (DHT). In adult ovariectomized offspring, estradiol benzoate produced a 2-fold increase (P < 0.05) in PgrA+B expression in the POA of pV females, but not in pT females or those that had prenatal exposure to DHT (pDHT). Prenatal testosterone and DHT exposure also prevented estradiol benzoate-induced LH surges observed in pV rats. Blood sampling of ovariectomized rats revealed increased LH pulse frequency in pDHT versus pV females (P < 0.05). Our findings support the hypothesis that prenatal androgen receptor activation can contribute to the permanent defeminization of the GnRH neurosecretory system, rendering it incapable of initiating GnRH surges, while accelerating basal GnRH pulse generator activity in adulthood. We propose that the effects of prenatal androgen receptor activation on GnRH neurosecretion are mediated in part via permanent impairment of E2-induced PgrA+B gene expression in the POA.     

Effects of Androgens and Oestrogens on the Behaviour of Chicks in an Imprinting Situation

The behavioural effects of testosterone propionate (TP), 5α-dihydrotestosterone (DHT) and oestradiol benzoate (OB) were investigated in day-old chicks during imprinting sessions to a duck model. TP increased the duration of peeping while inhibiting the following reaction and the twitters. DHT had more or less the same effects while OB induces the reverse behavioural changes. The behavioural effects of hormone injections agree with behavioural sex differences observed in non-injected animals: ♂♂ peep more than ♀♀ which on the other hand produce more twitters. This could be related to sex differences in the hormonal status of the birds at hatching, as it is known that during incubation male chick embryos have higher plasma testosterone levels than ♀♀ of corresponding ages.     

Regulation of Noncontact Erection in Rats by Gonadal Steroids

Male rats exhibit erections in the presence of inaccessible estrous females, and we investigated which gonadal steroids regulate these noncontact erections (NCEs). Sexually experienced Wistar males (n >/= 8/group) were tested for NCE four times (every 3 days) before castration, after castration, and after receiving subcutaneous implants of 10-mm Silastic capsules that were empty or filled with crystalline testosterone propionate (TP), dihydrotestosterone (DHT), estradiol benzoate (EB), or DHT + EB (10 mm each). Before castration, males responded with NCE in approximately 50% of tests. No males had NCEs after castration, beginning 3 days after surgery. Also, no males responded after treatment with EB or empty capsules. After receiving implants of TP, DHT, or DHT + EB, 50% of males had NCEs, beginning with the first test 3 days after treatment. On every measure of NCE, males treated with DHT or DHT + EB were indistinguishable from each other and from TP-treated males. Among the sexual responses of male rats, NCE appears to be more sensitive than other behaviors to changes in gonadal condition. In its profile of response to gonadal steroids (testosterone+, dihydrotestosterone+, estradiol-), NCE is similar to reflexive erection, for which spinal systems are sufficient, and unlike copulation (T+, DHT-, E+), which depends on discrete areas of the brain. We nonetheless conclude that NCE depends on androgen-sensitive systems in the brain, but androgen-sensitive neurons in the lumbosacral spinal cord may also play a role.     

Influences of pre- and postnatal testosterone treatment on defeminization of sexual receptivity in pigs

Sexual receptivity was evaluated in female and male pigs that had experienced varying periods of exposure to testosterone pre- and postnatally. For prenatal exposure, pregnant sows were treated with testosterone propionate (TP) from Day 29-35 or Day 39-45 of gestation at a dosage that caused virilization of the external genitalia of their female offspring. Eighty-three percent of the females that received TP prenatally had regular estrous cycles, but reached puberty later than control females. Only 26% of the females that received TP both pre- and postnatally (4-6 mo of age) were observed in estrus by 10 mo of age. After ovariectomy and acute treatment with estradiol benzoate (EB), the proportion of females that showed the immobilization response (receptivity) was similar for all groups of females independent of pre- or postnatal TP treatment. Females treated prenatally from Day 39-45 showed the immobilization response for fewer days after treatment with a high dosage of EB than did controls. On the basis of these observations, we conclude that receptivity in female pigs is not affected greatly by testosterone treatment at the stages of development that were investigated. Males castrated at birth and treated with a single injection of EB after 9.5 mo showed the immobilization response. In contrast, few males castrated at 8 mo or castrated at birth and treated with TP from 3 to 6 mo showed the immobilization response after EB treatment. These observations provide direct evidence for a postnatal component of testosterone-dependent defeminization of receptivity in male pigs.     

Sexual behavior in male rats treated with estrogen in combination with dihydrotestosterone

Male rats castrated at 30 days of age were treated with estradiol benzoate (dose range: 0.05–50 μg EB for 26 days) and dihydrotestosterone (1 mg DHT for 36 days) as adults. The combined EB and DHT treatments resulted in display of male sexual behavior which did not differ from the behavior shown by intact untreated males or castrated, testosterone propionate (1 mg TP for 26 days) treated males. EB alone or DHT alone were relatively ineffective in activating male behavior in castrated males.     

Embryonic and posthatching treatments with sex steroids demasculinize the motivational aspects of crowing behavior in male Japanese quail

Demasculinizing action of embryonic estrogen on crowing behavior in male Japanese quails was examined. Eggs were treated with either 20 μg of estradiol benzoate (EB) or vehicle on the 10th day of incubation. Chicks hatched from both groups of eggs were injected daily with either testosterone propionate (TP; 10 μg/g b.w.), 5α-dihydrotestosterone (DHT, a non-aromatizable androgen; 10 μg/g b.w.), or vehicle from 11 to 50 days after hatching, and during this period their calling behaviors were observed. Irrespective of embryonic treatments, all birds received posthatching treatment with either TP or DHT, but not with vehicle, emitted crows in place of distress calls in a stress (non-sexual) context of being isolated in a recording chamber. The posthatching TP, but not posthatching DHT, induced crowing in a sexual context (crowing in their home-cages) from much earlier age than posthatching vehicle in the birds received control embryonic treatment with vehicle. The same TP treatment, however, completely eliminated the crowing in a sexual context in the birds received EB during their embryonic life. In the birds treated with either posthatching DHT or posthatching vehicle, the crowing in a sexual context was only slightly decreased by embryonic EB treatment. These data suggest that posthatching estrogen, derived from testosterone aromatization, enhances the demasculinizing action of embryonic estrogen, and thus strongly reduces the sexual motivation for crowing behavior. This demasculinizing action, however, would not influence vocal control system which generates acoustic pattern of crowing in the presence of androgens allowing the birds to crow in a non-sexual context.     

Induction of male mating behavior in androgen-insensitive (tfm) and Normal (King-Holtzman) male rats: effect of testosterone propionate, estradiol benzoate, and dihydrotestosterone

Castrated androgen-insensitive rats exhibited mounting and intromission patterns in response to testosterone propionate (TP), estradiol benzoate (EB), or EB combined with dihydrotestosterone (DHT) treatment in adulthood. Treatment with DHT alone was ineffective in stimulating male mating behavior in the mutant rats. Since androgen-insensitive rats, like normal males, have the potential to show mounting behavior following hormone treatment in adulthood, the neural substrate underlying this behavior must be masculinized during development. The effectiveness of gonadal hormones in activating the entire copulatory sequence in castrated littermate males (King-Holtzman) was also examined. TP treatment induced mating behavior in the control rats. DHT also stimulated the complete copulatory pattern, although it was not as effective as TP. The administration of EB, however, did not induce ejaculation in control rats. These results do not support the hypothesis that the activation of male mating behavior by testosterone requires its metabolite estrogen (aromatization hypothesis).     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Parental responsiveness is feminized after neonatal castration in virgin male prairie voles, but is not masculinized by perinatal testosterone in virgin females

We previously found a large sex difference in the parental responsiveness of adult virgin prairie voles (Microtus ochrogaster) such that most males are spontaneously parental, whereas most females are not. Because this sex difference is independent of the gonadal hormones normally circulating in adult virgin voles, the present study examined whether perinatal hormones influence the development of this sex difference. Males were treated prenatally (via their pregnant dam) with both the androgen receptor blocker flutamide (5 mg/day/dam) and the aromatase inhibitor ATD (1 mg/day/dam), or oil, for the last 2 weeks of gestation. Half of the subjects from each group were castrated on the day of birth and the other half received a sham surgery. As adults, intact males were castrated and all males received a silastic capsule filled with testosterone. Prenatal treatment with flutamide and ATD had no effect on males' behavior toward pups, but neonatal castration significantly reduced the percentage of males acting parentally. In a second experiment, females were exposed to testosterone propionate (TP; 50 microg/day/dam) or oil via their dam during the last 2 weeks of gestation. For the first neonatal week, half of the females from each group were injected with TP (1 mg/day) and the other half oil. As adults, females were ovariectomized and half from each group received a testosterone-filled capsule and the other half received an empty capsule. None of the perinatal TP treatments increased females' parental responsiveness, although females from all groups that received testosterone capsules as adults were highly parental. Therefore, although postnatal testicular hormones are necessary for high parental responsiveness in males, the behavior of females is not influenced by perinatal exposure to testosterone.