Ovarian influences on the development of sexual behavior in neonatally androgenized rats

Female sexual behavior was studied in male and female rats. Males were castrated on the day of birth (Day 1); some received ovarian implants at that time; others were injected on Day 3 with oil, 5 μg testosterone propionate (TP), or 50 μg TP. Females were ovariectomized at birth, 20, or 60 days of age; on Day 3 all were injected with oil, 5 μg, TP, or 50 μg TP. Prepuberal ovarian tenancy in females tended to counteract the effects on sexual receptivity of TP administered during neonatal life. In males ovarian implants facilitated female sexual behavior at adulthood in oil-injected animals, but did not significantly influence the effect on neonatally injected TP.     

Testicular hormones during the first few hours after birth augment the tendency of adult male rats to mount receptive females

In the male rat, a dramatic increase in serum testosterone of testicular origin occurs during the first two hours of postnatal life. This experiment was designed to determine whether this increase contributes to the development of the propensity for adult male rats to mount sexually receptive females. Male rats were castrated at either 0-hours (virtually at the moment of birth), or at 6 hours, or at 24 hours after birth. Some males castrated at 0-hours were injected with 1 microgram of testosterone after surgery. Control males were sham-operated at birth, and castrated in adulthood. At about 90 days of age, each male was given testosterone replacement therapy and tested over a period of 6 weeks for mounting. Castration of newborn males retards the development of mounting in the sense that males castrated at 0-hours require a longer period of hormone stimulation before beginning to mount, a greater percentage of males castrated at 0-hours fail to mount altogether, and even those who do mount do so at a frequency significantly lower than that for males castrated later in life. These effects are not seen when males are given an injection of testosterone immediately after castration at 0-hours, or when castration occurs at 6 hours after birth or later. We conclude that gonadal hormonal stimulation during the first several hours after birth contributes to, although is perhaps not essential for, the development of mounting.     

Effects of gonadal hormones on urinary behavior in dogs

The effects of testicular and ovarian hormones on urinary frequency and posture were studied in 8 groups of dogs. Three of the 4 female groups had been exposed to androgenic stimulation in utero, neonatally, or both in utero and in infancy. The fourth group of females consisted of ovariectomized controls. Males were intact or castrated as adults, as juveniles, or as neonates. Frequency of urination was increased in all groups by estrogen and by testosterone. Urinary posture was unaffected in males by castration in the adult or juvenile stages. Neonatal castration of males resulted in periodic regression from the adult male posture to the immature male posture. Control and prenatally androgenized females urinated in the feminine position. Females treated with large amounts of testosterone in infancy showed a limited degree of masculinization of urinary posture. Females exposed to testosterone before and immediately after birth urinated as females about 50% of the time and as adult males for approximately half of their urinations. Exogenous estrogen or androgen administered in adulthood had no effect on posture assumed for urination.     

Variation in infanticide and parental behavior in male mice due to prior intrauterine proximity to female fetuses: Elimination by prenatal stress

When sexually-naive male mice are placed together with newborn young, some males will commit infanticide (kill the young) while others will behave parentally (retrieve the young to a nest and keep them warm). The intrauterine position of male mouse fetuses, which is recorded at Cesarean delivery, has previously been found to influence the titers of estradiol that male fetuses are exposed to in utero. In adulthood, most male mice that developed in utero between male fetuses (2 M males) behaved parentally toward young, while most males that developed in utero between female fetuses (0 M males) committed infanticide. When 0 M and 2 M males were castrated at birth and tested with young in adulthood, few males committed infanticide. But, when these same males were tested with young after 25 days of treatment with testosterone, more 0 M than 2 M males committed infanticide and more 2 M than 0 M males behaved parentally. In contrast to the above findings, more 2 M than 0 M males that were castrated when 28 days old behaved parentally without treatment with testosterone; few of the non-parental males committed infanticide, but, instead, ignored the young. Finally, stressing pregnant mice by placing them under bright lights during the last third of pregnancy eliminated the effect of developing next to female fetuses in the male offspring, and all prenatally-stressed males resembled 2 M males in their behavior toward young: most prenatally-stressed males were parental rather than infanticidal when tested with young. The significance of these findings to models of hormonal effects on sexual differentiation is discussed.     

Intrauterine position influences anatomy and behavior in domestic rabbits

In several rodent species, the sexual differentiation of a female offspring is known to be affected in utero by the testosterone produced in adjacent male littermates. The aim of this study was to investigate the effect of male neighbors on the sexual differentiation in domestic rabbits. For this, the intrauterine position (IUP) of a female offspring from unilaterally ovariectomized, multiparous mothers was determined by their birth order. Depending on the sex of the adjacent fetuses, pups were divided into 4 groups: 1. Males. 2. 2 M females (females with 2 adjacent males), 1 M females (females with 1 male neighbor), and 0 M females (females with zero adjacent male). Pups' anogenital distance (AGD) was measured at birth and on Day 180 postpartum, when spontaneous chin marking activity was also measured. Our results revealed that AGD was a reliable indicator of sex as male pups had larger AGD than females, both at birth and later on. Adjacent male fetuses had significant effect: the more adjacent male fetuses females have had the longer AGD they possessed. AGD at birth was a good predictor of AGD and behavior of adults, as 2 M does showed the longest AGD and the highest chin marking activity among females. We concluded that, similarly to rodents, proximity to males in utero affects both anatomy and behavior in rabbits.     

Studies on the effects of dehydroepiandrosterone and its metabolites on attack by castrated mice on lactating intruders

Triads of castrated male mice (residents) were injected each day for 2 weeks with oil vehicle or 280 nmol of dehydroepiandrosterone (D). They were tested for their attack on a lactating female (intruder) introduced in their home-cage 2,24 or 48 hr after their last injection. D significantly reduced male aggressive behavior for at least 24 hr. Other groups of castrated males were injected each day with vehicle or 280 nmol of D, dehydroepiandrosterone sulfate (DS) or androstenediol (ADIOL). D, but neither DS nor ADIOL, significantly reduced their aggressive responses to intruders. Neural levels of D, DS and testosterone (T) were measured in intact males injected with vehicle or D. The concentration of D in brain was unchanged by castration and was increased about 20 fold in D injected males. The concentration of neural T was 10 nmol/g of tissue in intact males, was almost undetectable in castrated males, and was slightly increased in D injected males. Further work will be needed to identify the steroid molecule(s) responsible for this behavioral effect of D.     

Initiation of mating behavior in developing male rats following peripheral electric shock

When shocked on the flanks in the presence of estrous females, juvenile male rats displayed their first series of intromissions and ejaculation at a significantly younger age than non-shocked males or males shocked 24 hr before sex tests. A premature release of testosterone was not responsible for the phenomenon, as judged by genital organ weights and blood testosterone determinations made 24 hr following the initial ejaculation. Males induced to copulate with shock at early ages continued to display intromissions and ejaculation on subsequent tests without further peripheral electrical stimulation. However, in prepuberally castrated male rats, peripheral shock in conjunction with injections of a high dose of testosterone propionate (TP) was less successful than TP alone in facilitating the initial intromission. The results are discussed in terms of a three-part hypothesis concerning the initial activation of adult sexual behavior in the male rat.     

Influence of neonatal androgen on the display of territorial marking behavior in the gerbil.

The effect of the presence or absence of androgen during the neonatal period on territorial marking behavior in the Mongolian gerbil was studied. Scent marking frequency was 20-40 fold greater in males than in females. Gonadectomy depressed marking in males but not in females. Testosterone propionate (TP) therapy completely restored marking in male but increased marking in intact and ovariectomized females to only one fourth that in males. Genetic males castrated within 2 days postpartum did not mark more frequently than TP-treated females after TP treatment in adulthood. Genetic females given a single TP injection within 6 days postpartum marked at male levels after TP treatment in adulthood. Males castrated after Day 2 and females given TP after Day 6 displayed marking frequencies intermediate between normal male and female levels after TP treatment in adulthood. This study suggests that sexual dimorphism in territorial marking behavior is due to a sex difference in the competency to respond to androgen, and it appears that development of this competency occurs during the neonatal period and is regulated, at least in part, by androgen. The onset of this differentiation process occurs earlier in the male than in the female.     

Sex differences in body growth in the rat.

Adult male rats are larger than females, due to a persistent difference in the growth rate from puberty onward. Gonadectomy at birth abolished, whereas gonadectomy on Day 21 caused a diminution of the sex differences. There were no differences in growth pattern between females spayed at birth and females spayed on Day 21. In male rats this was different: males castrated at birth became lighter and smaller than males castrated on Day 21. Thus males castrated at birth and females spayed at either age grew at comparable rates which were below the growth rate of males castrated at 21 days. This demonstrated the significant role of the neonatal testes on subsequent growth; prepuberal ovaries did not seem to play an important role. The administration of testosterone propionate (TP) to female rats prenatally suppressed growth of intact, but not of females spayed at birth. This TP effect is ovary-dependent. TP given neonatally promoted growth independently of the ovaries. It is concluded that neonatal androgens organize mechanisms which regulate subsequent body growth in the male rat, and that from puberty on ovarian secretions suppress the growth rate. These opposite actions of the gonads cause the sex differences in body growth of the rat.     

Effect of flutamide (an antiandrogen) and diethylstilbestrol on the reproductive behavior of Japanese quail

Three experiments were conducted in order to investigate the role of brain androgen and estrogen receptors in sex hormone activated male reproductive behavior in Japanese quail. In Experiment 1, castrated male quail were injected with oil, testosterone propionate (TP), flutamide (FLUT), an androgen antagonist, or TP+FLUT. Males given TP+FLUT, compared with birds receiving TP alone, strutted much less and had smaller proctodeal (foam) glands. Copulation was reduced by FLUT only on the last test day and only on one measure (number of head grabs + mounts). These results suggest that binding of testosterone or one of its metabolites to an androgen receptor is part of the mechanism of TP activated strutting, and therefore that central nervous system androgen receptors are involved in a male reproductive behavior pattern. In Experiment 2, castrated male quail were injected with oil, with 50 micrograms estradiol benzoate (EB), or with 25, 50 or 100 micrograms diethylstilbestrol (DES), a synthetic estrogen that does not bind to androgen receptors. EB but not DES activated copulation to a significant extent. In Experiment 3 male and female quail with photically regressed gonads were given intraperitoneal Silastic implants of DES, estradiol (E) or cholesterol. DES was highly effective at activating male-typical copulation in males and receptivity in both sexes. Thus hormonal interaction with estrogen receptors alone is sufficient for the activation of male-typical as well as female-typical copulatory behavior in this species.     

Hormonal factors controlling the development of sexual behavior in the male golden hamster

Masculine and feminine responses of male hamsters castrated on the day of birth (Day 1) and treated with androgen, estrogen or control substances on Days 2–4 were investigated. Male hamsters treated with testosterone, testosterone propionate, estradiol, estradiol benzoate, or diethylstilbestrol displayed mounting behavior after TP treatment in adulthood. Androsterone, dihydrotestosterone or control substances given neonatally failed to induce the potential for masculine sexual behavior. These data support the hypothesis that the development of masculine sexual behavior is dependent upon the presence of estrogen or androgens which can be converted to estrogen during sexual differentiation. Sexual receptivity in response to adult ovarian hormone treatment was decreased in Day 1 castrated males which received testosterone propionate or estrogens neonatally. Receptivity measures of males treated with testosterone, androsterone or dihydrotestosterone were not different from controls. Treatment of the neonatally castrated male with androgens early in life resulted in significant development of the penile bone cartilage, whereas the peripheral morphology of males treated neonatally with estrogen was not different from control males. These results indicate that alterations of peripheral morphology and mechanisms involved in the differentiation of sexual behavior may be hormone specific.     

Maternal behavior in male rats: Critical times for the suppressive action of androgens

To determine whether the first day of life is the only period during infancy when removal of the testes permanently alters maternal behavior in rats, male Sprague-Dawley rats were castrated on either Day 1, 5, 10, 25 or 70 of life and were tested as adults for maternal behavior following testosterone replacement. Males castrated on Day 1, 5 or 10 responded significantly sooner to pups and spent longer times exhibiting maternal behavior than did males castrated on Day 25 or later. However removal of the testes on the first day of life was maximally effective. Treatment with testosterone neither inhibited nor facilitated maternal behavior. Thus there is no one critical period in infancy for differentiation of the neural substrate for maternal behavior, although removal of androgen at birth is maximally effective for the onset of maternal behavior in the adult.     

Neonatal bonadal hormones: effect on maternal and sexual behavior in the male rat

Neonatal exposure of rats to androgen results in alteration of adult sex behavior and gonadotropin release. Other sexually dimorphic adult behaviors have also been shown to be dependent, either in part or in full, upon exposure to androgen neonatally. The present study was conducted to determine the effect of neonatal androgens in organizing the brain of the male rat (Long-Evans strain) with regard to maternal behavior. The results indicate that males neonatally exposed to androgen exhibit poor maternal behavior as adults when compared to males castrated at birth and males receiving gonadotropin antiserum in infancy. The males castrated at birth and males receiving gonadotropin antiserum in infancy, when primed with estrogen and progesterone, showed high levels of female sexual behavior when compared to controls. In terms of male sex behavior, the control groups performed slightly better than the males castrated at birth and males receiving antisera in infancy. The results suggest that the neonatal pituitary gland has an indirect role in the process of sexual differentiation.     

Fetal, hormonal and experiential factors influencing the mating-induced regulation of infanticide in male house mice

When a male house mouse encounters a neonate he either attempts to kill it or he does not harm it. An unusual aspect of his response is that adaptive, time-dependent changes in behavior toward pups result from a unique stimulus-response system triggered specifically by ejaculation. In virgin male CF-1 mice, about 50% of all males are spontaneously infanticidal when they encounter a pup while the other 50% are typically "parental." The stimulus of ejaculation causes virtually all males to kill pups; however, by the time offspring are born three weeks after mating, infanticide is inhibited and almost all males now behave parentally toward pups. Our experiments examine fetal, experiential, and hormonal factors influencing the changes in infanticidal and parental behavior that occur in male mice as a result of mating. Males who developed in utero between two female fetuses, and were thus exposed to relatively low testosterone concentrations during fetal development, were significantly more likely to exhibit infanticide--both before and after mating--than were males who developed between two male fetuses. Concurrent exposure to testosterone appears required in order for naive males to exhibit infanticide and for spontaneously parental males to become infanticidal after ejaculation. In contrast, neither testosterone nor pituitary hormones appeared responsible for the timed inhibition of infanticide occurring by three weeks after mating, since castrated and hypophysectomized males showed a response pattern similar to intact males. The mating-induced inhibition phenomenon appears to be a neurally timed and mediated response that operates independently from pituitary hormone secretions or changes in gonadal hormones resulting from mating.     

Comparison of the effects of testosterone and dihydrotestosterone on the behavior of male cynomolgus monkeys (Macaca fascicularis)

To compare the behavioral effects of testosterone propionate (TP) and diyhdrotestosterone propionate (DHTP) at doses producing plasma levels of androgens within the physiological rage, observations were made on 4 castrated male cynomolgus monkeys during successive 4-week treatment periods while they received 25, 50, 100, 200, 400 and 800 micrograms of either TP or DHTP SC/day in counterbalanced order. Males were tested with each of the same 4 female partners (16 pairs, 1024 1-hr behavior tests). Males were injected at 1600 hr and blood samples were obtained at 0800 hr (256 samples, 456 hormone determinations). Physiological plasma levels of T resulted from the 200 micrograms and 400 micrograms TP treatments, and were associated with significantly increased ejaculatory behavior. Physiological plasma levels of DHT resulted from the 50 micrograms and 100 micrograms DHTP treatments, but there were no changes in ejaculatory behavior over the entire DHTP dose range used. This difference in the behavioral effects of TP and DHTP, not previously reported for a primate, could not be accounted for by the effects of treatment order, season, long-term behavioral testing, female sexual motivation or behavior reflecting the peripheral action of androgens.     

Effect of prenatal uterine position on male and female rats sexual behavior.

It is well known that female rats developing close to a male in utero show a higher frequency of heterotypical or male-like behavior in adulthood, and have longer anogenital distances. The present investigation was designed to evaluate the in utero masculine influence on the homotypical sexual behavior of male and female rats. Also explored was the influence on body weight on Gestation Day 21 (day of cesarean delivery) and 21, 40. and 120 days after birth, testicle weight following the gonadectomy of males on Day 40, and serum testosterone in males and females on the day of delivery. The presence of a contiguous male fetus located caudally with respect to uterine blood flow led to the masculinization of male-like behavior in male rats, the defeminization of female like behavior in females and to increased body weights on Day 21 of gestation in both sexes. No significant differences were detected in the remaining parameters. Findings indicated a gradation in the intensity of expression of male and female sexual behavior in adulthood related to the intrauterine position resulting in interindividual variability. The possible implication of this physiological phenomenon in the structure of rodent populations is discussed.     

Enhancement of female receptivity in neonatally castrated males by prepuberal ovarian transplants

Male rats castrated within 6 hr after birth had ovaries transplanted from littermates at 5, 15, or 25 days of age. All grafts were removed at 40 days of age and mating tests were initiated at either 50 or 90 days of age. Males developing with ovaries were more responsive to exogenous estrogen and progesterone at early and at later ages than nonimplanted castrated control animals. No differences in receptivity were associated with age at implantation. All groups tested initially at 50 days were more responsive on the first 2–3 mating experiences than those first tested at 90 days of age. By 584 days of age, ovarian-implanted but not castrated control males responded to replacement hormones with moderate levels of receptivity. After puberty and before initiation of testing and hormone injections, males developing with ovarian tissue had lighter body weights than the nonimplanted controls.     

Effects of neonatal testosterone treatment on sex differences in formalin-induced nociceptive behavior in rats

There are sex differences in nociceptive behavior induced by formalin in rats. To determine whether these sex differences are the result of the sexual differentiation of the brain, that is masculinization and defeminization [A.P. Arnold, R.A. Gorski, Gonadal steroid induction of structural sex differences in the central nervous system, Annu. Rev. Neurosci. 7 (1984) 413–442; M.M. McCarthy, A.T.M. Konkle, When is a sex difference not a sex difference? Front Neuroendocrinol. 26 (2005) 85–102], some female rats were injected with testosterone propionate (TP, 100 μg/25 μl/rat) on the day of birth and on the following day. As controls, other female rats and all male rats were injected with the same volume of sesame oil. They were castrated at the age of 8 weeks, and implanted with a silicon tube containing 20% of 17β-estradiol or cholesterol. Two weeks after the implantation, rats were injected with 50 μl of 2% formalin in the right hind paw and their behavioral changes were observed for 1 h. In cholesterol-implanted rats, all rats exhibited three typical phases of pain response and there were no significant differences in the scores of nociceptive behavior. In 17β-estradiol implanted rats, female and TP-treated female rats had a significantly higher score of nociceptive behavior than male rats. These results indicate that estrogen produces sex differences in nociceptive behavior induced by formalin, and suggest that these differences are not due to the sexual differentiation of the brain, since the dose and the timing of the TP treatment effectively defeminize and masculinize female rats. Alternatively, sexual differentiation of the brain response to formalin-induced nociceptive behavior may be different from ordinary sexual differentiation.     

Urinary testosterone levels in the male blind mole rat (Spalax ehrenbergi) affect female preference

This study investigated the sexual attraction of female blind mole rats to four groups of male mole rats: (a) intact males raised in captivity; (b) intact males trapped in the field; (c) captive males injected with testosterone; (d) captive castrated males. In the first part we measured blood testosterone, androstenedione, and dihydrotestosterone (DHT) levels, by radioimmunoassay; and urine testosterone levels, measured by GC-MS. The second part examined the relationship between urine testosterone levels in males and their attractiveness to females. Higher blood and urine testosterone levels were found in the field animals and in those injected with testosterone compared to captive intact or castrated animals: urine testosterone levels in the two other groups were not detectable. Blood androstenedione levels were also higher in the field animals and in those injected with testosterone compared to captive intact or castrated mole rats. Blood dihydrotestosterone levels were not detectable in all four experimental groups. Female mole rats chose to spend a longer period of time next to males with high blood and urine testosterone levels and high blood androstenedione levels than next to those with lower levels of these hormones. Because courtship and sexual behavior are influenced both by high levels of blood and urine testosterone and high levels of blood androstenedione, we suggest that the low levels of courtship and other sexual behavior in captive mole rats may be related to the lack of female attraction to these males, which display low levels of all three parameters.     

Estradiol benzoate facilitates lordosis and ear wiggling of 4- to 6-day-old rats

The effects of exogenous and endogenous steroids on components of female sexual behavior of neonatal male and female rats were investigated. In Experiment 1, 4-day-old rats were treated with 0, 0.1, 1.0, 10, or 100 micrograms/10 g body weight estradiol benzoate (EB) and were tested 44 hr later. In Experiment 2, male rats castrated within 24 to 48 hr of birth were compared with sham operated controls and castrates given steroid replacement. The results indicated that most 6-day-old pups will display lordosis and ear wiggling; therefore, the display of these responses is not dependent upon exogenous steroids. However, a fine-grained behavioral analysis revealed that EB treatment increased the frequency, duration, and intensity of lordosis and the frequency of ear wiggling in infant females, and it increased lordosis duration in males. Castration of infant males decreased the likelihood that male infants would display lordosis, whereas testosterone replacement restored behavior to control levels. These data question the concept that organizational and activational actions of estrogens occur during completely separable times in development and should provide new insights into the development of estrogen receptor function and the process of sexual differentiation of brain and behavior.