Evidence for extended action of gonadal hormones on the organization of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica).

Male and female gray short-tailed opossums were gonadectomized (GDX), or treated with the estrogen receptor antagonist tamoxifen citrate (TX), or corn oil (OIL) (control) during the 5th postnatal week, a time period equivalent to the 3rd postnatal week in rats and associated with high levels of circulating gonadal hormones and neural aromatase activity in this marsupial species. In adulthood following gonadectomy (for animals not previously gonadectomized) and replacement therapy with estradiol or testosterone, GDX males showed less male-typical scent marking and had shorter phalluses than OIL and TX males. Following replacement therapy with estradiol, GDX females were more likely to fight with and less likely to mate with stimulus males than TX females; OIL females were intermediate in these measures. Along with previous findings, these results suggest that gonadal hormones act over an extended postnatal period to organize sexually dimorphic behavior and morphology in male gray opossums and may have some effect on the organization of aggressive behavior in females of this species.

Partner preference of intact and ovariectomized female gray short-tailed opossums (Monodelphis domestica).

Intact, ovariectomized and ovariectomized estradiol (E)-treated female gray short-tailed opossums were placed in a test situation in which they could choose between an intact and a castrated male. Intact females chose to visit intact males first and visited them more frequently and spent more time with intact than with castrated males. Ovariectomized (OVX) females did not show this preference for visiting intact males over castrates. When compared to OVX females with blank implants, OVX females with E implants spent less time with castrated males. Like intact females, OVX and OVX-E-treated females preferred to stay in close proximity to but not actually in the cage of intact rather than castrated males. To our knowledge, this is the first experimental study of partner preference and its relationship to hormonal condition in a female marsupial.

Perinatal exposure to estradiol masculinizes aspects of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica).

The effects on adult sexually dimorphic behavior of perinatal exposure to estrogen were examined by treating male and female gray opossums with estradiol (EST), an estrogen receptor antagonist (tamoxifen:TX) or oil control (OIL) during the first week of life, a time period corresponding in this marsupial to late gestation in rodent species. Following gonadectomy and replacement therapy with testosterone in adulthood, males showed more scent-marking behavior than females and EST animals showed more scent marking than TX or OIL animals. Also, phalluses were longer and body weight was higher in males than in females and in EST-treated animals than in TX-treated animals; OIL animals were intermediate in these morphological measures. EST animals of both sexes showed less female-typical screeching threat behavior than OIL or TX animals. Because these hormone manipulations were conducted on the "fetus" directly in this marsupial (rather than via the maternal circulation as in previously studied eutherian species), these findings provide unique confirming evidence for masculinization of aspects of behavior and morphology by early exposure to estradiol in mammals.

The hormonal control of birth behavior in the gray short-tailed opossum (Monodelphis domestica).

In all major groups of Australian marsupials, prostaglandin F2alpha (PGF) or oxytocin injection initiates birth behavior in adult females, adult males and pouch young. Because inhibitors of PGF synthesis block this initiation, oxytocin may activate birth behavior via the stimulation of PGF synthesis. In this study, the role of PGF and oxytocin in the activation of birth behavior was examined in an American marsupial, the gray short-tailed opossum (Monodelphis domestica). Adult male and female gray opossums were given PGF, oxytocin, or saline (control) before behavioral observation. On the next day, the animals in the oxytocin group were injected with the PGF inhibitor flunixin meglumide (Finadyne, Schering Corp., U.S.A.) before oxytocin reinjection and behavioral observation. Both males and females showed birth behavior in response to PGF but only females responded to oxytocin. There was no significant difference in the latency of response of females to oxytocin alone versus response to oxytocin after receipt of the PGF inhibitor. These results suggest that, in contrast to Australian species, in this American marsupial, oxytocin initiates birth behavior only in females and does not operate via stimulation of prostaglandin secretion.

Male gray short-tailed opossums (Monodelphis domestica) receive penile intromissions when treated with estrogen and progesterone in adulthood.

Following treatment with estradiol and progesterone, gonadectomized male as well as female gray opossums received penile intromissions from intact stimulus males. Intromission was possible in male gray opossums because, like marsupials of both sexes, they possess a single cloaca-like anogenital opening. All subjects that allowed intromission showed anogenital dragging just prior to intromission. While intromission latency was similar in tests involving male and female subjects, total intromission duration was longer in tests involving male subjects than in tests involving female subjects, and sex locks were seen only in tests involving female subjects. These findings are discussed with respect to the potential usefulness of gray opossums for studying the effects of peripheral vs central factors on the display of sex differences in behavior.

Sex differences and the development of social behavior in a marsupial, the gray short-tailed opossum (Monodelphis domestica).

The effects of sex and age on social behavior were examined in gray short-tailed opossums (Monodelphis domestica), small didelphid marsupials. Each animal received five behavior test batteries spanning prepubertal to postpubertal ages. Each test battery consisted of two tests with animals of the same age, one with a male and one with a female. Precopulatory behavior toward females, intermale fighting requiring test interruptions as well as scent marking behavior were seen at higher levels in males than in females and were seen more frequently around and after puberty than before puberty. Females showed more threat behavior than males in mixed-sex and in same-sex interactions. This sex difference was apparent after puberty in tests with male partners and prior to as well as around puberty in tests with female partners. Because climbing over and boxing with another animal were seen more frequently prior to than after puberty, these behaviors may be elements of play fighting (i.e. attack and defense without submission and threat). These findings are discussed with respect to the role of gonadal hormones in the organization and activation of behavior and with reference to their comparative significance in mammals.

The hormonal induction of mating behavior in female gray short-tailed opossums (Monodelphis domestica).

The activating effects of estradiol alone and of estradiol plus progesterone on copulation and related behavior were examined in ovariectomized female gray short-tailed opossums. Following receipt of subcutaneous estradiol (E) or cholesterol (C) implants, E-females showed less screeching threat behavior toward intact males than C-females. Intact males showed more precopulatory behavior toward E- than toward C-females, and 20% of E-females and no C-females copulated with males. In a second experiment, females that had previously received subcutaneous E implants received injections of either corn oil alone (E + OIL) or 500 micrograms progesterone in corn oil (E + P) 4 hr before behavioral testing. Of E + P-females, 88% copulated with males, compared to 38% of E + OIL-females. All females that copulated in both experiments showed anogenital dragging just prior to copulation. Hormonal induction of estrous behavior and copulation has not been reported previously in a female marsupial.

The effects of neonatal treatment with tamoxifen on sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica).

The effects of postnatal exposure to the antiestrogen tamoxifen on sexually dimorphic behavior and morphology in adult male and female gray short-tailed opossums were examined. Following gonadecomy and treatment with estradiol in adulthood, neonatally tamoxifen-treated (TX) females showed less female-typical screeching threat behavior and TX males showed less male-typical precopulatory behavior than same-sex controls. While testes failed to descend and phalluses were shorter in juvenile TX males than in juvenile control males, behavior did not differ between TX and control animals following treatment with testosterone in adulthood. The findings that activity level following treatment with estradiol in adulthood and body weight in juveniles and adults were significantly higher in TX than in control animals of both sexes suggest that perinatal estrogens are involved also in the organization of these characteristics. The significance of these findings is discussed with respect to the development of sex differences in behavior and morphology in eutherian mammals.

Sex differences and the role of aromatization in the control of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica).

Sex differences in the activating effects of an aromatizable (testosterone, T) and a nonaromatizable (dihydrotestosterone, DHT) androgen on sexually dimorphic scent marking and aggressive behavior were examined in gonadectomized gray short-tailed opossums. When compared with males, females showed less chest, head, flank, and hip marking and more fighting behavior in tests with stimulus females and threat behavior in tests with stimulus males following receipt of subcutaneous T, DHT, or blank (B) implants. Testosterone but not DHT activated hip and head marking while both T and DHT activated flank marking. In tests with stimulus females, only males showed clicking vocalizations and T but not DHT or B stimulated clicking. Animals that received T had significantly larger suprasternal scent glands than those that received DHT or B. The significance of these findings is discussed with respect to the development of sex differences in behavior in eutherian mammals.

Effects of postnatal exposure to alcohol on reproductive physiology and sexually dimorphic behavior in a marsupial, the gray short-tailed opossum (Monodelphis domestica).

The effects of postnatal exposure to alcohol on reproductive physiology and sexually dimorphic behavior and anatomy in adult male and female gray short-tailed opossums were examined. Female responsiveness to male pheromones and fertility in both sexes were essentially normal in postnatally alcohol-treated animals. However, aspects of sexually dimorphic behavior were masculinized and defeminized in females and demasculinized in males following gonadectomy in adulthood and treatment with male (testosterone) or female (estradiol) hormones. The possible role of alterations in neural aromatase activity by perinatal alcohol exposure in mediating these behavioral effects and the potential use of this marsupial species in perinatal alcohol studies are discussed.

Neural aromatase activity in a marsupial, the gray short-tailed opossum (Monodelphis domestica): ontogeny during postnatal development and androgen regulation in adulthood.

Neural aromatase activity (AA) was measured in gray short-tailed opossums (Monodelphis domestica) on the day of birth and at selected ages through adulthood. In adulthood, regulation by testicular androgens was examined. In mixed-sex samples of whole brain, AA was present at birth and increased until postnatal day (PD) 16. In hypothalamus-preoptic area (HPOA), significantly higher levels of AA were seen in animals on PD 16 than on PD 30 and PD 30 animals had higher levels of AA than all older ages including adults. Significant sex differences in HPOA AA with male levels higher, were seen only on PD 16 and in adulthood. While lower overall than in HPOA, AA was present also in cerebral cortex (CX). In CX, AA was higher on PDs 16 and 30 than at older ages. Significant sex differences in CX AA were observed only in adulthood. One week following castration in adulthood, AA dropped significantly in CX but not in HPOA. These findings are compared with those obtained from other marsupial and eutherian mammals with reference to the possible significance of AA in sexual differentiation of the gray opossum brain.

Estrogen in peripheral plasma during postnatal development in gray short-tailed opossums.

Plasma samples obtained from gray short-tailed opossums (Monodelphis domestica) at selected ages through adulthood were assayed for estrogen (E). Levels of E in one mixed-sex plasma pool of animals aged postnatal day (pd) 4 and one of two mixed-sex plasma pools of animals aged pd 8 were over 300 pg/ml. On pd 16, E levels in males and females averaged 30 and 47 pg/ml, respectively. While no significant sex differences in E levels were seen on pd 30 or pd 60, mean E levels for animals on pd 30 were significantly higher (275 pg/ml in males and 181 pg/ml in females) than on pd 60 (78 pg/ml in males and 85 pg/ml in females) or pd 145 (adults). In adult animals, estrogen levels in females averaged 54 pg/ml; all adult male E levels were below the limit of sensitivity of the assay. Maternal E levels, which did not vary significantly by age of litter, averaged 10 pg/ml overall. These findings are discussed with respect to possible significance of high E levels in developing marsupials for sexual differentiation and general brain development.

Evidence for high levels of androgen in peripheral plasma during postnatal development in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

Plasma samples were assayed for androgen in gray short-tailed opossums (Monodelphis domestica) on the day of birth and at selected ages through adulthood. Levels of androgen in mixed-sex plasma pools of animals 4 and 8 days of age were higher than in either sex at all other ages examined. At postnatal Days 16, 30, and 60 (weaning), levels of androgen were equivalent in males and females and as high as in adult males. In both sexes, androgen levels were lower at postnatal Day 84 (juveniles) than at younger ages; after puberty, levels were significantly higher in males than in females. These findings are discussed with respect to similarities and differences between marsupials and eutherians in hormonal environment during the perinatal period and with respect to the possible role of androgens in sexual differentiation of the gray opossum brain.

Early differentiation of the gonads in the gray short-tailed opossum (Monodelphis domestica).

The time course for gonadal development in gray short-tailed opossums was examined in this study. It was found that the gonads were not differentiated on day 1 of postnatal life (the day of birth). While testis development was seen by postnatal day 4, ovarian development did not occur until after postnatal day 16. In both sexes, primordial germ cells were not identified until after postnatal day 1. These findings are discussed with respect to gonadal differentiation in other marsupial species.

The effects of gonadal hormones on scent marking and related behavior and morphology in female gray short-tailed opossums (Monodelphis domestica).

The hormonal control of scent marking and related behavior and morphology was examined in female gray short-tailed opossums. Females rarely scent marked when intact or following ovariectomy. Testosterone (T) but not estradiol (E) treatment stimulated chest marking while either hormone stimulated head marking in ovariectomized females tested alone. When the same females were tested with males, T-treated females showed little scent marking of any type; E-treated females showed hip marking in significantly more tests than females in the other treatment groups. Suprasternal scent glands (absent in intact females) and phalluses of females that received T were significantly larger than those of animals that received E or control animals. These findings are discussed with respect to similarities and differences between marsupial and eutherian females and between male and female gray opossums in the hormonal control of sexually dimorphic behavior and morphology.

The luteinizing hormone-releasing hormone (LH-RH) system in neonatally estradiol-feminized male gray short-tailed opossums (Monodelphis domestica).

In this study, the luteinizing hormone-releasing hormone (LH-RH) system was examined in adult gray short-tailed opossums that had been treated with estradiol benzoate (EB) on days 1 and 3 of postnatal life, a treatment which results in complete block of testicular development. The finding that the organization of the LH-RH system in neonatally EB-treated males did not differ from that of neonatally untreated animals indicates that the LH-RH system can develop normally despite the absence of gonads throughout perinatal life.

The hormonal control of scent marking and precopulatory behavior in male gray short-tailed opossums (Monodelphis domestica).

Little is known about the hormonal control of behavior in marsupials. In the present study, the effects of castration and of testosterone or estradiol replacement therapy on scent marking and precopulatory behavior in male gray short-tailed oppossums (Monodelphis domestica) were examined. It was found that castration resulted in decreases in chest and flank/hip marking displayed by male gray opossums. Testosterone but not estradiol stimulated chest marking in castrates. Males treated with either estradiol or testosterone displayed more flank/hip marking than control males. Highest levels of female aggression toward males were seen when the males had received testosterone treatment. These findings are discussed with respect to similarities and differences between marsupials and eutherians in the neural metabolism of testosterone and the hormonal control of scent marking behavior.

Ontogeny of estrogen binding sites in the brain of gray short-tailed opossums (Monodelphis domestica).

This study evaluated the binding of [3H]estradiol to brain cytosols from gray short-tailed opossums ranging in age from newborn to 63 days postnatal. Estrogen binding was undetectable in whole-brain cytosol of newborn opossums. By postnatal day 4, high affinity (Kd = 0.2 nM), saturable (Bmax = 3 fmol/mg protein) estrogen binding sites were present, and estrogen binding in whole-brain remained low but detectable (2-5 fmol/mg) through day 63. In contrast, estrogen binding sites in the hypothalamus-preoptic area increased substantially from 3.4 fmol/mg on day 16 to 14 fmol/mg by day 63.

The effects of pheromonal stimuli on estrus and peripheral plasma estradiol in female gray short-tailed opossums (Monodelphis domestica).

Exposure to male pheromones is associated with the activation of vaginal estrus in gray short-tailed opossums. The effects of such exposure on peripheral plasma estradiol-17 beta (E) levels in this marsupial species were examined in this study. Mean E levels of 27.8 pg/ml +/- 4.4 in diestrous females living in a room containing only females were similar to those seen in other marsupials. Direct naso/oral exposure to pheromonal cues provided by males resulted in vaginal estrus in 75% of these females within 4 11 days. None of the females exposed to clean cages came into vaginal estrus. Animals that were in estrus at the time of blood sampling or came into estrus over the experimental period had significantly higher E levels (58.1 +/- 12.6 pg/ml) than females in the pheromone-exposed and control groups that did not come into estrus (23.3 +/- 8.2 pg/ml). These findings are discussed with respect to other marsupial and eutherian species.

Limb defects in gray short-tailed opossums (Monodelphis domestica) following postnatal injection with ethanol or saline.

Because the hindlimbs of marsupials at birth are in an embryonic stage of development, this group can prove useful for studies of limb teratology. In this study, injection of neonatal gray opossums with 2 mg ethanol (ETOH) or saline (SAL) in the right or left hindquarter resulted in defects of the associated limb in 44% of ETOH animals and 16% of SAL animals. Affected SAL animals showed gait abnormalities, foot clubbing, and moderately reduced limb size. Affected ETOH animals showed these abnormalities as well as fused digits, missing digits, and, in one case, a partially missing limb. Thus, while injection itself may have resulted in altered limb development, local infusion of alcohol had a further teratogenic effect on such development.