Acute effects of sex-specific sex hormones on heat shock proteins in fast muscle of male and female rats

Heat shock protein (HSP) expression and sex hormone levels have been shown to influence several aspects of skeletal muscle physiology (e.g., hypertrophy, resistance to oxidative stress), suggesting that sex hormone levels can effect HSP expression. This study evaluated the effects of differing levels of sex-specific sex hormones (i.e., testosterone in males and estrogen in females) on the expression of 4: HSP70, HSC70, HSP25, and αB-crystallin in the quadriceps muscles of male and female rats. Animals were assigned to 1 of 3 groups (n = 5 M and F/group). The first group (Ctl) consisted of typically cage-housed animals that served as controls. The second group (H) was gonadectomized and received either testosterone (males) or estradiol (females) via injection for 12 consecutive days. The third group (Gx) was gonadectomized and injected as above, but with vehicle only, rather than hormones. Significant sex by condition interactions (P < 0.05 by two-way MANOVA) were found for all 4 proteins studied, except for HSP70, which exhibited a significant effect of condition only. The expression of all HSPs was greater (1.9–2.5-fold) in males vs. females in the Ctl group, except for HSP70, which was no different. Generally, gonadectomy appeared to have greater effects in males than females, but administration of the exogenous sex hormones tended to produce more robust relative changes in females than males. There were no differences in myosin composition in any of the groups, suggesting that changes in fiber type were not a factor in the differential protein expression. These data may have implications for sex-related differences in muscular responses to exercise, disuse, and injury.     

Sex-related differences in spatial divided attention and motor impulsivity in rats

The acquisition and performance of a self-paced test of spatial divided attention linked with frontal cortex function were assessed in postpubertal (> 60 days) normal or gonadectomized male and female rats. Males were more accurate at detecting relatively brief visual stimuli than females, but this difference was eliminated by increasing the target stimulus duration, indicating an attentional basis for this effect. Premature errors were, however, greater in males than in females, suggesting greater impulsivity in males. Subsequent experiments in gonadectomized rats suggest that circulating hormones influence attention and impulsivity, but not necessarily sex differences. These results demonstrate a double dissociation between components of impulse control and divided attention in male and female rats and may have implications for sex differences in disorders of attention and cognition.     

Sexual differentiation of play behavior in the ferret

Three experiments were performed to elucidate the endocrine mechanisms responsible for sex differences in the prepubertal play behavior of ferrets. In Experiment 1, gonadally intact adolescent males exhibited higher levels of "stand-over" behavior than females did in tests between 63 and 123 days of age with gonadally intact female partners of the same age. In Experiment 2, ferrets exposed to androgen or to ovarian steroids over Days 5-20 of postnatal life subsequently exhibited significantly higher levels of stand-over behavior in tests with female partners than did control females gonadectomized on Day 5 and not subsequently given any steroids. However, none of the subjects in Experiment 2 exhibited levels of stand-over behavior comparable to those of the gonadally intact males in Experiment 1. In Experiment 3, males gonadectomized and implanted subcutaneously with testosterone capsules on Day 70 and tested with female partners at 84-96 days of age exhibited levels of stand-over behavior comparable to those observed in Experiment 1 in gonadally intact males of the same age (Weeks 12-14). Males gonadectomized on Day 70 and given no hormone at the time of testing exhibited significantly lower levels of this behavior. Significantly lower levels of this behavior were also exhibited by males gonadectomized on Day 35 and females gonadectomized on Day 70 regardless of whether they were tested with testosterone present after Day 70. Sex differences in the expression of prepubertal play behavior of ferrets apparently result from differential exposure of males and females to androgen over an extended postnatal period.     

Sex differences in the activational effects of gonadal hormones on food intake and body weight

Estrogens have been shown to decrease, and androgens to increase body weight (BWt) of guinea pigs (GPs). The magnitude of the BWt sex difference shown by intact adult GPs is due primarily to these concurrent, or activational, effects of gonadal steroids. However, a small but significant sex difference in BWt persists in animals gonadectomized at birth, indicating that early hormonal exposure may permanently influence certain steroid sensitive weight regulatory mechanisms in the two sexes. Three experiments were therefore designed to investigate the short term effects of estradiol and testosterone on food intake (FI) and BWt of gonadectomized adult male and female GPs. In the first experiment, GPs gonadectomized in adulthood were given a single injection of 6 micrograms estradiol benzoate (EB). Although EB treatment reduced FI and BWt of both females and males, significantly larger reductions occurred in females. In the second experiment, GPs gonadectomized at birth received treatments of oil or 2 micrograms EB for 7 days. EB treatment also produced significantly larger effects on FI and BWt in the neonatally gonadectomized females. The third experiment involved GPs gonadectomized as adults who were injected with either oil or 1 mg/day testosterone propionate in oil (TP) for 32 days. Compared to changes in oil injected controls, TP produced significantly larger increases in male BWt than female BWt. Therefore, although GPs show only minor sex differences in BWt which might relate to prenatal gonadal hormonal exposure, significant sex differences remain in their responsiveness to the activational effects of gonadal steroids on FI and BWt in adulthood.     

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep: a sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 mum intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis responses to stress.     

Effects of septal lesions on shock thresholds in weanling male and female rats

Twenty-four-day old weanling male and female rats were either lesioned in the septal area, gonadectomized, lesioned and gonadectomized, or untreated. Tests for reactivity to electric shock were conducted at 26 and at 40 days of age. Neither males nor females showed any effect of the lesions are measured by flinch and jump thresholds to electric shock immediately following surgery; both male and female lesioned groups showed a significant lowering of thresholds when tested at 40 days of age. The effects of septal lesions on response to electric shock were dissociated from the effects on hyperemotionality measured over the same period of time after the lesions, and appeared to have their effect independent of the sex or hormonal condition of the animals.     

Impact of testosterone and oestradiol on region specificity of skeletal muscle-ATP, creatine phosphokinase and myokinase in male and female Wistar rats.

The main aim of the present study was to test the hypothesis that skeletal muscle ATP concentration, creatine phosphokinase and myokinase enzyme activities are stimulated by the sex steroids in both male and female rats (animals were not subjected to any kind of exercise or any training). To test the hypothesis healthy mature (90-120 days old, weighing about 160-180 g) male and female rats were gonadectomized. Gonadectomized male and female rats were administered with testosterone (Sigma Chemical, St Louis, MO, USA) at a dose of 100 microg (100 g body weight)-1 day-1 for males and 5 microg (100 g body weight)-1 day-1 for females for 30 days from day 31 post-castration onwards; and oestradiol at a dose of 5 microg (100 g body weight)-1 day-1 for 30 days from day 31 post-castration onwards for both males and females (17beta oestradiol, Sigma Chemical Company, St Louis, MO, USA). The ATP content, creatine phosphokinase and myokinase enzyme activities of skeletal muscles were significantly higher than that of skeletal muscles of female control rats. Gonadectomy resulted in a significant decrease in ATP content and creatine phosphokinase myokinase enzyme activities in both male and female rats. Testosterone treatment to gonadectomized male rats brought back the parameters to normalcy whereas the same to the female rats enhanced the enzyme activities and ATP contents to the level of control male rats. Oestradiol treatment to castrated male rats did not bring about any significant alterations whereas the same in gonadectomized female rats brought them back to normalcy. Therefore from the present study it is concluded that testosterone is effective in both males and females whereas oestradiol was effective only in the females in enhancing skeletal muscle energy metabolism.     

Impact of testosterone and oestradiol on region specificity of skeletal muscle-ATP,creatine phosphokinase and myokinase in male and female Wistar rats

The main aim of the present study was to test the hypothesis that skeletal muscle ATP concentration, creatine phosphokinase and myokinase enzyme activities are stimulated by the sex steroids in both male and female rats (animals were not subjected to any kind of exercise or any training). To test the hypothesis healthy mature (90–120 days old, weighing about 160–180 g) male and female rats were gonadectomized. Gonadectomized male and female rats were administered with testosterone (Sigma Chemical, St Louis, MO, USA) at a dose of 100 μg (100 g body weight)^–1 day^–1 for males and 5 μg (100 g body weight)^–1day^–1 for females for 30 days from day 31 post-castration onwards; and oestradiol at a dose of 5 μg (100 g body weight)^–1day^–1 for 30 days from day 31 post-castration onwards for both males and females (17β oestradiol, Sigma Chemical Company, St Louis, MO, USA). The ATP content, creatine phosphokinase and myokinase enzyme activities of skeletal muscles were significantly higher than that of skeletal muscles of female control rats. Gonadectomy resulted in a significant decrease in ATP content and creatine phosphokinase myokinase enzyme activities in both male and female rats. Testosterone treatment to gonadectomized male rats brought back the parameters to normalcy whereas the same to the female rats enhanced the enzyme activities and ATP contents to the level of control male rats. Oestradiol treatment to castrated male rats did not bring about any significant alterations whereas the same in gonadectomized female rats brought them back to normalcy. Therefore from the present study it is concluded that testosterone is effective in both males and females whereas oestradiol was effective only in the females in enhancing skeletal muscle energy metabolism.     

Effects of early sex steroid hormone treatment on courtship behavior and sexual attractivity in the red-sided garter snake, Thamnophis sirtalis parietalis

Previous research has shown that exogenous androgen fails to elicit courtship (chin-rubbing) behavior in adult male or female red-sided garter snakes (Thamnophis sirtalis parietalis). However, gonadectomized and intact newborn male and female red-sided garter snakes given silastic capsules containing testosterone exhibit chin-rubbing behavior; gonadectomized and untreated animals do not show this behavior. Both males and females also exhibit chin-rubbing behavior when treated with testosterone as yearlings. Hibernation stimulates chin-rubbing behavior only in males that have received androgen treatment as a neonate or as a yearling. Previous research has also shown that adult females, but not adult males, are courted if given estrogen treatment. Both newborn males and females will elicit chin-rubbing behavior from adult males if given estrogen treatment, indicating production and release of an attractiveness pheromone characteristic of adult females. Male red-sided garter snake breed for the first time on emergence from their second hibernation, whereas females probably do not breed until their third year of life. These data suggest that in the red-sided garter snake, a species that exhibits a dissociated reproductive tactic, sex steroid hormones act to organize central nervous system mechanisms subserving courtship behavior such that temperature, and not hormonal, fluctuations activate sexual behavior in the adult organism.     

The emission and elicitation of mouse ultrasonic vocalizations: the effects of age, sex and gonadal status

Subject' mice of varying gonadal status (castrate males; intact or neonatally gonadectomized females and males) were paired for 3-min with intact 'stimulus' females and ultrasonic vocalizations were monitored. Vocalization patterns from home cages differed from the test pairings. The results suggested that the age, experience and gonadal status of the subject influenced the vocalizations from the pair. As the source of ultrasonic calls from these vocally intact pairs could not be individually identified, the 'subjects' were paired with a range of ultrasonically silent (inferior laryngeal nerve-transected) stimulus animals. Vocalizations were detected from all combinations of animals. Gonadally intact females were most effective in eliciting ultrasonic vocalizations from the subjects and gonadally intact males were least effective. The responses of castrate males were lower than from intact males. Anesthetized adults of either sex elicited only poor vocalization responses from other adults. Ultrasonic calls have often previously been studied using vocally intact 'subject' and 'stimulus' animals: the present results confirm the difficulty of establishing who is who in such situations.     

Pup-killing in mice: the effects of gonadectomy and testosterone administration.

The administration of testosterone to intact and to gonadectomized adult female mice induced pup-killing whereas such treatment failed to induce killing in intact males and in males gonadectomized in adulthood. Testosterone treatment was effective in inducing killing when given to adult, neonatally gonadectomized, males. Also, gonadectomy of males performed prior to experience with young reduced the number of animals that subsequently killed. Gonadectomy performed after animals had killed pups did not eliminate killing behavior. Finally, practically all females that exhibited pup-killing in response to the administration of testosterone also displayed intraspecific aggression than killed pups.     

Neonatal hormone experience and adult lordosis and fighting in the golden hamster.

Androgenization by testicular secretions or exogenous testosterone propionate (TP treatments administered 24-48 hr post partum) suppressed sexual receptivity in the golden hamster. In response to prolonged adult estradiol benzoate (EB) treatment, gonadectomized normal females and neonatally castrated males exhibited significantly longer total lordosis durations than normal male or neonatally TP-treated (20 mug or 200 mug) females. These results suggest that one aspect of androgen-induced masculinization in the hamster involves reduced estrogen sensitivity. Responses to sequential EB followed by progesterone treatment were also lower in the androgenized groups. Neonatally castrated males did not differ significantly from normal females in their lordosis behavior. Irrespective of adult hormone treatment, androgenized animals fought more than normal females or neonatally castrated males. A genital mask was used to reduce sex differences in peripheral stimulation during testing.     

Sex,age and shock-intensity as factors in passive avoidance

Sex, age and shock-intensity were studied in a step-through passive avoidance test. In adult rats (140 days) males showed more inhibition of a previously punished approach response. No sex differences were observed in young adult animals (60 days). In prepuberal animals (30 days), on the other hand, it was the females which showed more response inhibition. In prepuberty females showed far more response inhibition than in adulthood. Even when using 1.6 mA as a reinforcer, only 17% of the adult females showed inhibition after punishment of the entering response.     

Sex differences in self-grooming of rats: effects of gonadal hormones and context

When prepubescent males and females were placed alone in a novel cage, males were found to groom their genitals, but not other body regions, more than females. This sex difference was present in untreated and in testosterone-treated gonadectomized rats as well as in intact rats. Neither the presence of gonads nor testosterone treatment affected grooming in this context. However, when similarly treated rats of the same age were observed in groups in the home cage, no sex difference in genital grooming of intact or gonadectomized rats was found. Testosterone significantly increased nongenital grooming in both sexes and genital grooming in males. Thus, both the presence of sex differences and the effects of testosterone on self-grooming depend on the behavioral context.     

Differences in reserpine-induced striatal dopamine output and content between female and male mice: implications for sex differences in vesicular monoamine transporter 2 function

In this report a series of six in vitro experiments in which reserpine-evoked dopamine output and two in vivo experiments in which the effects of reserpine injections upon dopamine content from striatal tissue of female and male mice were performed as a means to assess possible sex differences in vesicular monoamine transporter 2 (VMAT2) function. Significantly greater amounts of dopamine were obtained from striatal tissue of female mice in response to either a brief (experiment 1) or continuous (experiment 2) infusion of reserpine. Similarly, reserpine-evoked dopamine output from striatal tissue of gonadectomized females was significantly greater that that of gonadectomized males (experiment 3). When reserpine-evoked dopamine responses were compared directly between intact versus gonadectomized females (experiment 4) or males (experiment 5) no statistically significant differences were obtained. Finally, comparisons of gonadectomized females treated or not with estrogen revealed no statistically significant differences in reserpine-evoked dopamine output (experiment 6). Injections of reserpine produced significantly greater depletions of striatal dopamine content within intact female versus male mice (experiment 7). Dopamine contents of gonadectomized females treated or not with estrogen did not differ following treatment with reserpine, but were significantly greater than that of gonadectomized males (experiment 8). Taken together, these results show that female striatal tissue is more responsive to reserpine-evoked dopamine output, and this sex difference appears to be estrogen independent. Similarly, the dopamine depleting effects of reserpine are greater in intact female mice, however, gonadectomy reverses this effect in an estrogen independent manner. The data suggest that female mice may have a greater amount/activity of VMAT2 function as revealed by the increased responsiveness to the VMAT2 blocking drug, reserpine. Such differences in VMAT2 function may be related to the gender differences observed in conditions like Parkinson's disease and drug addiction.     

Ontogeny of sex differences in open-field ambulation in the rat

The effects of age and gonads were studied in rats subjected to open-field tests, during which ambulation behavior was recorded. Subjects were three groups of male and female rats: sham-operation on day 1 and day 21; gonadectomy on day 1 and sham-operation on day 21; and sham-operation on day 1 and gonadectomy on day 21. Half of each group were tested in a circular open field (3 min/day, 3 consecutive days) on days 28-30; the others were tested on days 47-49. Representatives of both batches were tested again in a square open field on days 76-78. There was a sex difference in ambulation at 77 days, but not at earlier ages. In animals gonadectomized on day 1 or day 21 the sex difference in adulthood failed to occur, because castration caused the males to ambulate as much as sham-operated and ovariectomized females. On the basis of our results and reports in the literature it is suggested that testicular secretions around puberty have an organizing effect on ambulation behavior. The intact adult male rat ambulates less than the adult female and this difference persists after castration in adulthood. Castration well before puberty prevents the development of the adult sex difference.     

Effects of adrenalectomy and gonadectomy on sex and stress-induced differences in bicuculline-induced convulsions

The effects of adrenalectomy, gonadectomy and combined adrenalectomy plus gonadectomy on the previously described sex-dependent anticonvulsive effect of swim stress were studied in rats. The convulsive signs (myoclonic twitch, generalized convulsions, tonic hindlimb extension) were produced by constant i.v. infusion of γ-aminobutyric acid_A (GABA_A) antagonist bicuculline, which started 15 min after termination of swim stress (10-min swim at 18–19°C). Adrenalectomy decreased the threshold doses of bicuculline producing the first myoclonic twitch and the onset of generalized convulsions only in females. In adrenalectomized females, but not in males, swim stress enhanced the threshold dose of bicuculline producing generalized convulsions, but, unlike in adrenal-intact animals, it failed to enhance the dose of bicuculline producing tonic hindlimb extension. In gonadectomized stressed and unstressed animals all sex differences disappeared, and swim stress enhanced in both sexes only the threshold doses of bicuculline producing tonic hindlimb extension. Adrenalectomized plus gonadectomized animals displayed clear sex differences in doses of bicuculline necessary to produce all the convulsive signs. In the same animals swim stress postponed, especially in females, the onset of the first myoclonic twitch and generalized convulsions, but not the onset of tonic hindlimb extension. In summary, our results suggest that hormones of the adrenal and gonadal glands are only partly responsible for decreased susceptibility, especially of female rats, to the GABA_A antagonist bicuculline. Moreover, they have demonstrated that stress produces a gender-specific anticonvulsive effect even in the animals completely deprived of steroid hormones of peripheral origin. Received: 4 June 1998 / Accepted: 11 June 1999     

Determination of oxytetracycline induced fluorescence and autofluorescence as an indicator of age and sex differences in rat femur

Summary Simple and rapid methods for the determination and detection of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic) were used to estimate age and sex related differences in the rat femur. The autofluorescence always had collagen characteristics; it increased with age and was not sex dependent. The intensity of OTC induced fluorescence decreased with age, being higher in males than in females. In the oldest animals, however a slight increase in the fluorescence intensity was observed, and sex differences disappeared.The spectrofluorometric measurements of intact femora indicate a higher OTC retention on the bone surface of young animals and adult males but not in adult females. The results indicate that the determination of OTC retention in the bone using our quantitative fluorometric method and the detection of OTC deposition at bone surface using a spectrofluorometric technique can be a sensitive and inexpensive tool for experimental studies of bone changes in physiological and pathological conditions.This study was supported in part by the Pliva, Pharmaceutical Works, Zagreb, Yugoslavia and US Department of Agriculture, Beltsville, Maryland     

Failure of ACTH to prolong extinction of a conditioned taste aversion in the absence of the testes

Both corticotropin (ACTH) and testosterone prolong the extinction of a conditioned taste aversion in water-deprived intact male rats. An investigation was made to determine whether ACTH affects extinction in the absence of the testes and also to determine the effect of ACTH on serum testosterone levels. Water-deprived intact males showed prolonged extinction after ACTH injections; water-deprived gonadectomized males and intact females did not. All three of these groups showed elevated testosterone levels after ACTH administration, but testosterone levels were higher in the intact males than in the gonadectomized males or intact females. These results clearly show that in the absence of the testes ACTH is unable to prolong extinction. It is proposed that the increased level of testosterone following ACTH injection in water-deprived intact males is responsible for the prolonged extinction of a conditioned taste aversion. Although testosterone levels may increase in females and castrated males following ACTH injection, the increase is not sufficient to prolong extinction in these water-deprived animals.     

Dose- and sex-dependent effects of the neurotoxin 6-hydroxydopamine on the nigrostriatal dopaminergic pathway of adult rats: differential actions of estrogen in males and females

Epidemiological and clinical studies provide growing evidence for marked sex differences in the incidence of certain neurological disorders that are largely attributed to the neuroprotective effects of estrogen. Thus there is a keen interest in the clinical potential of estrogen-related compounds to act as novel therapeutic agents in conditions of neuronal injury and neurodegeneration such as Parkinson's disease. Studies employing animal models of neurodegeneration in ovariectomised female rats treated with estrogen support this hypothesis, yet experimental evidence for sex differences in the CNS response to direct neurotoxic insult is limited and, as yet, few studies have addressed the role played by endogenously produced hormones in neuroprotection. Therefore, in this study we aimed to determine (1) whether the prevailing levels of sex steroid hormones in the intact rat provide a degree of protection against neuronal assault in females compared with males and (2) whether sex differences depend solely on male/female differences in circulating estrogen levels or whether androgens could also play a role. Using the selective, centrally administered neurotoxin 6-hydroxydopamine, which induces a lesion in the nigrostriatal dopaminergic pathway similar to that seen in Parkinson's disease, we have demonstrated a sexually dimorphic (male-dominant), dose-dependent susceptibility in rats. Furthermore, following gonadectomy, dopamine depletion resulting from a submaximal dose of 6-hydroxydopamine (1 microg) was reduced in male rats, whereas in females, ovariectomy enhanced dopamine depletion. Administration of the nonaromatizable androgen dihydrotestosterone to gonadectomized animals had no significant effect on 6-hydroxydopamine toxicity in either males or females, whereas treatment of gonadectomized males and females with physiological levels of estrogen restored the extent of striatal dopamine loss to that seen in intact rats, viz, estrogen therapy reduced lesion size in females but increased it in males. Taken together, our findings strongly suggest that there are sex differences in the mechanisms whereby nigrostriatal dopaminergic neurones respond to injury. They also reveal that the reported clinically beneficial effects of estrogen in females may not be universally adopted for males. While the reasons for this gender-determined difference in response to the activational action of estrogen are unknown, we hypothesize that they may well be related to the early organizational events mediated by sex steroid hormones, which ultimately result in the sexual differentiation of the brain.