Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays

Social behavior involves both the recognition and production of social cues. Mice with selective deletion (knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -α or -β display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knockouts (KO: OTKO, αERKO, βERKO) from the odors of their wildtype littermates (WT: OTWT, αERWT, βERWT). Females showed marked initial choices of the urine odors of OTWT and βERWT males over those of OTKO and βERKO males, and αERKO males over αERWT males. The odors of OTKO and βERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the αERWT and αERKO males induced aversive, analgesic responses, with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females displaying significantly greater responses to the odors of stressed OTKO and βERKO males than their WTs, and significantly lower analgesia to the odors of stressed αERKO than αERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners' modified responses to their odors.     

Reproductive Functions Illustrating Direct and Indirect Effects of Genes on Behavior

Effects of gene products on reproductive behavior which are relatively direct include those of the estrogen receptor and progesterone receptor. For example, work with estrogen receptor-deficient (ERKO) female mice has extended previous evidence contributing to the neurochemical analysis of lordosis behavior. On the other hand, sex differences in behavior present a classic example of indirect effects of genes on behavior. Work with ERKO male mice shows the necessity of ER gene expression for normal masculinization of the brain. In particular, behavioral assay results distinguish apparent motivational performance of ERKO males from male mating reflexes: the former is similar to that of wild-type males in important respects, while the latter are deficient in ERKO males. The present paper first reviews a small number of clear genetic contributions to reproductive behaviors, and then reports one experiment pertinent to the interpretation of the behavioral status of ERKO male mice.     

Evidence for Sex-Biased Behavioral Maternal Investment in the Gray Mouse Lemur (Microcebus murinus)

To study sex-differential allocation of maternal behavior in Microcebus murinus, I recorded behavioral patterns on 21 litters from parturition to the weaning period. After a pregnancy of 61.5 ± 0.9 days, females may produce from one to four young per litter. Litters were weaned in 40 days by mothers. Behavioral observations at the beginning of the nocturnal activity period demonstrated that close contacts between mothers and infants were more frequent in multiparous mothers than in primiparous ones (p < .01). Time in close contact with offspring aged >15 days old compared to contacts with neonates is significantly lower only in single-sex litters and is more marked for all-female litters (p < .01). Mother's approaches toward mixed litters or all-male litters were always significantly greater than approaches toward all-female litters (p = .04). However, mother's approaches within mixed sex litters were not biased toward either sex (p = .7). Males in a litter may be interpreted as a stimulator of maternal behavior. Similarly, using retrieving tests of 15-min duration, a significant maternal preference for male neonates is evident. Latency to first retrieval is significantly shorter in multiparous females than in primiparous ones (p < .05) independent of size and sex ratio of the litter. For multiparous females only, male neonates were chosen first for retrieval more often than females (p < .05). Finally, the calls of young played a stimulator effect on maternal retrieving (p < .001). Accordingly multiparous mothers exhibit more interest in their young, which appears to be biased toward male neonates.     

Circadian parameters are altered in two strains of mice with transgenic modifications of estrogen receptor subtype 1

There are sex differences in free‐running rhythms, activity level and activity distribution that are attributed, in part, to the action of gonadal hormones. We tested the hypothesis that non‐classical estrogenic signaling pathways at estrogen receptor subtype 1 (ESR1) modify the amplitude and phase of activity. We used ESR1 knock‐out mice (ERKO) and non‐classical estrogen receptor knock‐in mice (NERKI). ERKO animals are unable to respond to estrogen at the ESR1 and NERKI animals lack the ability to respond to estrogens via the estrogen response element‐mediated pathway, but can still respond via non‐classical mechanisms. We compared intact male and female ERKO, NERKI and wildtype (WT) mice with respect to total wheel‐running activity, activity distribution across the 24‐h day, phase angle of activity onset and free‐running period (τ) and the duration of activity in constant conditions. WT females had significantly greater activity than WT males, and this activity was more consolidated to the dark phase of the light:dark cycle. These sex differences were absent in the NERKI and ERKO animals. Among females, NERKI and ERKO animals had greater activity during the light phase than WT counterparts. Additionally, we have identified a novel contribution of non‐classical estrogen signaling pathways on the distribution of activity. Our data suggest that total activity is ESR1‐dependent and daily activity patterns depend on both classical and non‐classical actions of estrogens. These data will aid in identifying the mechanisms underlying sex differences in sleep–wake cycles and the influence of steroid hormones on circadian patterns.     

Chd8 haploinsufficiency impacts rearing experience in C57BL/6 mice

Mutations in CHD8 are one of the highest genetic risk factors for autism spectrum disorder. Studies in mice that investigate underlying mechanisms have shown Chd8 haploinsufficient mice display some trait disruptions that mimic clinical phenotypes, although inconsistencies have been reported in some traits across different models on the same strain background. One source of variation across studies may be the impact of Chd8 haploinsufficiency on maternal-offspring interactions. While differences in maternal care as a function of Chd8 genotype have not been studied directly, a previous study showed that pup survival was reduced when reared by Chd8 heterozygous dams compared with wild-type (WT) dams, suggesting altered maternal care as a function of Chd8 genotype. Through systematic observation of the C57BL/6 strain, we first determined the impact of Chd8 haploinsufficiency in the offspring on WT maternal care frequencies across preweaning development. We next determined the impact of maternal Chd8 haploinsufficiency on pup care. Compared with litters with all WT offspring, WT dams exhibited less frequent maternal behaviors toward litters consisting of offspring with mixed Chd8 genotypes, particularly during postnatal week 1. Dam Chd8 haploinsufficiency decreased litter survival and increased active maternal care also during postnatal week 1. Determining the impact of Chd8 haploinsufficiency on early life experiences provides an important foundation for interpreting offspring outcomes and determining mechanisms that underlie heterogeneous phenotypes.     

Nature Needs Nurture: The Interaction of Hormonal and Social Influences on the Development of Behavioral Sex Differences in Rhesus Monkeys

Thirty years of research on early social and hormonal environments and their relationship to the expression of behavioral sex differences in rhesus monkeys are reviewed. These studies demonstrate that whether aggressive and submissive behaviors are sexually dimorphic depends primarily on the social and not the hormonal environment. Early rearing environments without mothers or allowing brief periods of peer interaction produced higher levels of male aggression and female submission. Presenting behavior was expressed more by females than males in environments with high male aggressivity and female submissiveness. No sex differences in presenting occurred in low aggressivity environments, unless monkeys were reared isosexually, when males presented more than females. Rough and tumble play and foot-clasp mounting were consistently exhibited more by males than females across all rearing environments studied, but rearing environment affected the degree of the sex difference. When reared isosexually males displayed less, and females more, foot-clasp mounting than when heterosexually reared. No social environment increased the low frequency of female rough and tumble play. Suppressing neonatal androgen in males did not effect any sexually dimorphic behavior. Prenatal androgen administration to genetic females masculinized many aspects of their juvenile behavior, consistently increasing rough and tumble play and foot-clasp mounting across different social environments. Thus the sexually dimorphic behaviors which showed the smallest variability across social contexts were the most profoundly affected by the prenatal hormonal environment. These studies demonstrate that the expression of consistent juvenile behavioral sex differences results from hormonally induced predispositions to engage in specific patterns of juvenile behavior whose expression is shaped by the specific social environment experienced by the developing monkey.     

Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations.

Estrogens affect the development, maturation, and function of multiple organ systems, including the immune system. One of the main targets of estrogens in the immune system is the thymus, which undergoes atrophy and phenotypic alterations when exposed to elevated levels of estrogen. To determine how estrogens influence the thymus and affect T cell development, estrogen receptor alpha (ERalpha) knockout (ERKO) mice were examined. ERKO mice have significantly smaller thymi than their wild-type (WT) littermates. Construction of ER radiation bone marrow chimeras indicated that the smaller thymi were due to a lack of ERalpha in radiation-resistant tissues rather than hemopoietic elements. ERKO mice were also susceptible to estradiol-induced thymic atrophy, but the extent of their atrophy was less than what was seen in WT mice. The estradiol-treated ERKO mice failed, however, to manifest alterations in their thymic CD4/CD8 phenotypes compared with WT mice. Therefore, ERalpha is essential in nonhemopoietic cells to obtain a full-sized thymus, and ERalpha also mediates some of the response of the thymus to elevated estrogen levels. Finally, these results suggest that in addition to ERalpha, another receptor pathway is involved in estradiol-induced thymic atrophy.     

Lack of functional estrogen receptor beta gene disrupts pubertal male sexual behavior

The estrogen receptor-beta (ERbeta) mediates estrogen action in the female gonads, reproductive tract, and central nervous system. In addition, in rats and mice, gonadotropin-releasing hormone (GnRH-I) neurons coexpress ERbeta. Here we asked if ERbeta plays a role in the onset of puberty and in hypothalamic-pituitary-gonadal (HPG) axis function in male mice. We examined mating behavior, testosterone concentrations, steroid negative feedback on gonadotropins, and GnRH-I function in male ERbeta knockout (ERbetaKO) and wild-type (WT) mice. Peripubertal ERbetaKO males displayed their first ejaculation at a significantly older age than WT littermates. Castrated, adult ERbetaKO mice had significantly higher plasma luteinizing hormone (LH) than WT counterparts. Estradiol (E2) treatment reduced LH and follicle stimulating hormone (FSH) concentrations to an equivalent degree in castrates of both genotypes. In three different measures of the adult GnRH-I system, no genotypic differences were observed. These data show that ERbeta plays an important role in the timing of male sexual behavior at puberty, but does not appear to be involved in adult HPG axis functioning. Furthermore, our data suggest that a primary role of ERbeta may be to regulate ejaculatory behavior.     

Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase

Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.     

Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules

Previous studies of the estrogen receptor-alpha knockout (alpha ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the alpha ERKO mouse lacks a functional estrogen receptor alpha (ER alpha) throughout development, it was not known whether the morphological and physiological abnormalities observed in the alpha ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER alpha in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in alpha ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent alpha ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in alpha ERKO siblings of the same age. Based on this study, we conclude that ER alpha has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the alpha ERKO mouse is likely the result of a concurrent response to the lack of functional ER alpha, and not solely due to the lack of ER alpha during early developmental times.     

Sensorimotor development in neonatal progesterone receptor knockout mice

Early exposure to steroid hormones can permanently and dramatically alter neural development. This is best understood in the organizational effects of hormones during development of brain regions involved in reproductive behaviors or neuroendocrine function. However, recent evidence strongly suggests that steroid hormones play a vital role in shaping brain regions involved in cognitive behavior such as the cerebral cortex. The most abundantly expressed steroid hormone receptor in the developing rodent cortex is the progesterone receptor (PR). In the rat, PR is initially expressed in the developmentally‐critical subplate at E18, and subsequently in laminas V and II/III through the first three postnatal weeks (Quadros et al. [2007] J Comp Neurol 504:42–56; Lopez & Wagner [2009]: J Comp Neurol 512:124–139), coinciding with significant periods of dendritic maturation, the arrival of afferents and synaptogenesis. In the present study, we investigated PR expression in the neonatal mouse somatosensory cortex. Additionally, to investigate the potential role of PR in developing cortex, we examined sensorimotor function in the first two postnatal weeks in PR knockout mice and their wildtype (WT) and heterozygous (HZ) counterparts. While the three genotypes were similar in most regards, PRKO and HZ mice lost the rooting reflex 2–3 days earlier than WT mice. These studies represent the first developmental behavioral assessment of PRKO mice and suggest PR expression may play an important role in the maturation of cortical connectivity and sensorimotor integration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 16–24, 2014     

Hepatic lipase deficiency attenuates mouse ovarian progesterone production leading to decreased ovulation and reduced litter size

The lipolytic enzyme hepatic lipase (HL) may facilitate mobilization of cholesterol substrate for ovarian steroidogenesis. We investigated whether HL was necessary for optimum reproduction in the female mouse by analyzing breeding performance and ovarian responses to gonadotropins in HL-/- mice. HL-/- female mice bred with HL-/- males had the same pregnancy success rate and pup survival rate as did wild-type (WT) mice but had significantly smaller litters, producing 1.7 fewer pups per litter. Mice were primed with eCG/hCG, and at 6 h post-hCG the HL-/- mice had smaller ovaries than did the WT mice. HL deficiency specifically affected ovarian weight; adrenal gland weights did not differ between WT and HL-/- mice. HL-/- mice weighed more than age-matched WT mice. Between the two mouse genotypes, uterine weights were the same, indicating that estrogen production was equivalent. However, the HL-/- ovaries produced significantly less progesterone than did the WT ovaries within 6 h of hCG stimulation. HL-/- ovaries had the same number of large antral follicles as did the WT ovaries but had fewer hemorrhagic sites, which represent ovulations, fewer corpora lutea, and more oocytes trapped in corpora lutea. We suggest that reduced progesterone synthesis following hCG stimulation attenuated the final maturation of preovulatory follicles, resulting in smaller ovaries. Furthermore, reduced progesterone production limited the expression of proteolytic enzymes needed for tissue remodeling, resulting in fewer ovulations with a corresponding increase in trapped or unovulated oocytes and providing a possible explanation for the smaller litter size observed in spontaneously ovulating HL-/- mice.     

Relaxin-3 null mutation mice display a circadian hypoactivity phenotype

Characterizing the neurocircuits and neurotransmitters that underlie arousal and circadian sleep/wake patterns is an important goal of neuroscience research, with potential implications for understanding human mental illnesses, such as major depression. Recent anatomical and functional studies suggest that relaxin-3 neurons and their ascending projections contribute to these functions via actions on key cortical, limbic and hypothalamic circuits. This study reports the behavioral phenotype of C57BL/6J backcrossed relaxin-3 knockout (KO) mice. Cohorts of adult, male and female relaxin-3 KO and wild-type (WT) littermate mice were subjected to a battery of behavioral tests to assess sensorimotor function and complex behavior. No overt deficits were detected in motor-coordination, spatial memory, sensorimotor gating, anxiety-like behavior or locomotor behavior in novel environments; and no marked genotype differences were observed in response to a chronic stress protocol. Notably however, compared to WT mice, relaxin-3 KO mice displayed robust hypoactivity during the dark/active phase when provided with free home-cage access to voluntary running wheels. This circadian hypoactivity was reflected by reduced time spent and distance traveled on running wheels, coupled with an increase in the time spent immobile, possibly reflecting increased sleeping. Overall, these studies support a role for relaxin-3 signaling in the control of arousal and sleep/wakefulness, and identify the relaxin-3 KO mouse as a useful model to study this role further.     

Masculine Sexual Behavior Is Disrupted in Male and Female Mice Lacking a Functional Estrogen Receptor α Gene

Masculine sexual behavior is regulated by testosterone (T). However, T can be metabolized to form estrogens or other androgens, which then activate their own receptors. We used knockout mice lacking a functional estrogen receptor α (ERα) gene to test the hypothesis that, following aromatization, T acts via the ERα to activate normal masculine sexual behavior. After gonadectomy and T replacement, wild-type (WT) male and female mice displayed masculine behavior. However, given the same T treatment, little masculine behavior was displayed by mice of either sex that lack a normal copy of the ERα gene. In particular, the latency to display masculine sex behavior and the number of mount attempts per trial were significantly reduced in the ERα⁻mice compared to WT littermates (P< 0.05). In addition, we found that in both sexes, ERα⁻mice have a smaller cluster of androgen receptor immunoreactivity in the bed nucleus of the stria terminalis. Using adult ERα⁻mice we were unable to determine whether these genotypic differences are due to organizational or activational effects. However, it is clear that the ERα plays a key role in the expression of masculine sexual behavior and in the regulation of androgen receptors in a neuronal cell population involved in the display of motivated behaviors.     

Ethanol self‐administration in serotonin transporter knockout mice: unconstrained demand and elasticity

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self‐administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self‐administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self‐administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol .     

Do Laboratory Mouse Females that Lose Their Litters Behave Differently around Parturition?

Efficiency in laboratory mouse breeding is hampered by poor reproductive performance, including the loss of entire litters shortly after birth. However, the underlying mechanisms are not yet fully understood and establishing the cause of death in laboratory mouse pups can be complicated. Newborn mouse pups are generally hidden in nests, dead pups are often eaten by the female, and the widespread practice of leaving periparturient females undisturbed complicates inspection, which may delay the discovery of pup loss. In order to efficiently prevent problems with litter loss, it is important to find key factors for survival. We investigated differences in periparturient behavior between female laboratory mice whose pups survived until weaning and females whose entire litters were lost. Video recordings of 82 primiparous females of the C57BL/6 strain or knockouts with C57BL/6 background were used. The mice were observed from 24 h before until 24 h after parturition and female behaviors coded using a pre-established ethogram. The relationship between behavior and survival was analyzed using logistic models, where litter survival was regressed on the proportion of 30-s observations with at least one occurrence of the behavior. We found that females with surviving litters performed more nest building behavior during the last 24 h before parturition (p = 0.004) and spent less time outside the nest during the entire observation period (p = 0.001). Increased litter survival was also associated with more passive maternal behaviors and the female ignoring still pups less. Females that lost their litters performed more parturition-related behaviors, suggesting prolonged labor. The results indicate that maternal behavior plays a significant role in laboratory mouse pup survival. Complications at parturition also contribute to litter mortality.     

Environmental maternal influences on body composition in mice selected for body weight

Summary The effect of the postnatal maternal environment, simulated by rearing mice in litters of three, six or nine, on body weight and body composition was investigated in three lines of mice differing widely in growth rate. The lines were selected for high (H₆) and low (L₆) 6-week body weight while the control line was maintained by random selection. Body weight and weights and percentages of ether extract, water, ash and protein at 21, 42, 63 and 84 days were recorded. With few exceptions, there were positive correlated responses to selection in body weight and in weights of body components. At 21 and 42 days the correlated responses were larger in L₆ mice than in H₆ mice. Body weight and weights of body components were larger for mice reared in litters of three than for those reared in litters of nine. Also, mice reared in litters of six were intermediate in body weight and weights of some of the body components between those reared in litters of three and nine. Differences in body weight and weights of body components due to postnatal maternal environment were small by comparison with differences due to genetic line. There were significant line by maternal environment interactions in body weight at 21 days and in ether extract weight at 21 and 63 days. Line and maternal environment differences in percentages of body components did not follow any consistent trend. The results for percentages of body components were further complicated by line x maternal environment interactions. In general, both line and postnatal maternal environmental differences in percentages of body components diminished with age.Paper No. 5670 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27650. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned     

The effects of parity, litter size, season, and breeding protocol on the number of DBA/2J Ten mice available for weaning.

Data on the reproductive behavior of DBA/2J Ten mice, collected from October 1970 through March 1973 and utilizing a 2-female, 1-male breeding system, with the mice remaining together throughout their reproductive life indicates: a) Litter size tends to increase through the fourth litter, the first litter being much smaller than successive litters; b) Survival rates of litters with 2--4 pups increase with successive deliveries; c) Survival rates of litters with 5--9 pups are approximately the same regardless of parity; d) There is a circannual rhythm in the number of pups available for weaning da 21, which is attributable to changes in postnatal viability. Data collected using a breeding pen system, with isolation of females pre- and post-delivery (March 1973 to March 1974) indicates: a) There is a reduction in live litter sizes in later litters with this system; b) There is a large reduction in the proportion of pups surviving to weaning with this system when compared with the other breeding system.     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.