Involvement of postnatal apoptosis on sex difference in number of cells generated during late fetal period in the sexually dimorphic nucleus of the preoptic area in rats

Postnatal apoptosis is involved in formation of the sex difference in neuron number of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. In this study, we examined the origin of neurons that die with apoptosis on the postnatal period to exhibit the sex difference in neuron number of the SDN-POA. First, we measured the number of cells that were labeled with 5-bromo-2'-deoxyuridine (BrdU) on embryonic day (ED) 17, ED18, and ED19 in the SDN-POA of rats on postnatal day (PD) 4 and PD8. The SDN-POA had many more cells labeled with BrdU on ED17 and ED18 than those on ED19. Significantly fewer cells labeled with BrdU on ED18 in the female SDN-POA from PD4 to PD8 resulted in a significant sex difference in the number at PD8. Next, combination analyses of BrdU-labeling and immunohistochemistry for single-stranded DNA (ssDNA), an apoptotic marker, were succeeded to investigate whether SDN-POA neurons generated during ED17-18 were removed by apoptosis. Many more ssDNA-immunoreactive cells that had been labeled with BrdU during ED17-18 were found in the SDN-POA of PD8 females, but few in the SDN-POA of PD8 males and PD4 females and males. These results suggest that the sex difference in the number of SDN-POA neurons generated during the late fetal period was caused by postnatal apoptosis.     

Neonatal manipulation of oxytocin affects expression of estrogen receptor alpha

In adult females many of the effects of the neuropeptide oxytocin are steroid, and especially estrogen dependent. Here we demonstrate for the first time that neonatal manipulation of oxytocin can affect the expression of estrogen receptor alpha. On the first day of postnatal life male and female prairie voles (Microtus ochrogaster) were randomly assigned to receive one of four treatments; (a) 50 microl i.p. injection of 3 microg oxytocin (approximately 1 microg/g), (b) 0.3 microg of an oxytocin antagonist (approximately 0.1 microg/g), or (c) isotonic saline. A fourth group was handled, but not injected. On postnatal day 8 or 21, brain tissue was collected, fixed and sectioned. Free-floating sections were stained for estrogen receptor alpha using immunocytochemistry, and estrogen receptor alpha immunoreactive neurons were compared by age, treatment, and sex. To compare the temporal expression of estrogen receptor alpha an additional set of brains was collected from untreated males and females on the day of birth. The effects of oxytocin manipulations were age dependent, sexually dimorphic, and site-specific. While there were no significant treatment effects on postnatal day 8, by postnatal day 21 females that received oxytocin showed a significant increase in the number of cells expressing estrogen receptor alpha-immunoreactivity in the ventromedial nucleus of the hypothalamus. Treatment with oxytocin antagonist resulted in a significant decrease in estrogen receptor alpha-immunoreactivity in the medial preoptic area in postnatal day 21 females. While there were no significant effects in males, males treated with oxytocin antagonist trended toward a reduction in estrogen receptor alpha-immunoreactivity in the medial amygdala. The results indicate that oxytocin can have organizational effects on the expression of estrogen receptor alpha, that these effects are sexually dimorphic, and finally that during the preweaning period the development of estrogen receptor alpha is sexually dimorphic.     

Reduction of the volume of the sexually dimorphic nucleus of the preoptic area by in utero ethanol exposure in male rats

Ethanol consumption during pregnancy has been demonstrated to result in alterations in central nervous system structure and function. Albino female rats were given either a liquid diet containing ethanol (5% w/v), a liquid diet which contained maltose-dextrin substituted isocalorically for the alcohol, or laboratory chow and water throughout pregnancy and for 14 days following birth. The volumes of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the nucleus accumbens septi (ACN) were measured in adult male rats exposed in utero and postnatally to these diets. Adult male rats exposed to ethanol in utero exhibit significantly smaller SDN-POA volumes (P less than 0.02) when compared to those of animals which received the control diets during the gestational period and postnatally. Ethanol treatment did not significantly influence the volume of the ACN. The results indicate that maternal ethanol consumption during pregnancy retards or inhibits SDN-POA development in fetal ethanol exposed male rats when compared to the nuclear volume in rats whose mothers were not exposed to ethanol during pregnancy.     

Prenatal ethanol and the prepubertal sexually dimorphic nucleus of the preoptic area

The volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus (SDN-POA) was determined in 14-31-day-old male and female rats whose mothers received a liquid diet containing 5% w/v ethanol from day 8 of gestation to parturition. Pair-fed dams received as a nutritional control an equal volume of an isocaloric liquid diet with maltose-dextrin in place of ethanol. Normal controls had laboratory rat chow and water available ad lib. The SDN-POA volume of ethanol-exposed males was significantly reduced compared to the pair-fed and normal males, and became indistinguishable from the SDN-POA volumes of the pair-fed and normal females. Ethanol-treated females also had a markedly reduced SDN-POA volume compared to the pair-fed and normal females. Our findings indicate that the SDN-POA of prepubertal rats of both sexes is sensitive to the effects of in utero ethanol exposure. While plasma testosterone, progesterone and estradiol titers, which we measured in fetuses on gestation day 22, were differentially affected by maternal ethanol consumption, the alterations by themselves cannot adequately explain the effects of prenatal ethanol exposure on the developing SDN-POA.     

Activational and organizational actions of gonadal hormones and the sex-specific effects of prolactin on food intake by rats.

Results of previous studies indicate that there is a sex-specific feeding response to prolactin (PRL) by rats: Only female rats significantly increase their food intake. The possible roles of the activational and/or organizational actions of the gonadal hormones in this sex difference were explored. In the first experiment, activational hormone exposure was manipulated in adult male and female rats. The results suggest that the activational actions of estrogen are not necessary for, and that testosterone does not block, PRL-induced increases in food intake by female rats. In a second experiment, organizational hormone exposure was manipulated in male and female rats during the early postnatal period. Genetic male rats organized as females by postnatal Day-1 castration significantly increased their food intake, while genetic female rats organized as males by postnatal androgen treatment maintained baseline levels of food intake. Overall, these experiments suggest that organizational, but not activational, gonadal hormone exposure plays a critical role in the development of this sex-specific response to PRL.     

Sex differences in estradiol-induced progestin receptor immunoreactivity in the guinea pig preoptic area and hypothalamus

Progesterone exerts on the central nervous system a number of effects, some of which are estrogen dependent mostly in the preoptic area and the mediobasal hypothalamus. In these regions, an immunocytochemical study was used to evaluate differences in progesterone receptor (PR) immunoreactivity between the male and the female guinea pig in response to 10 μg/day estradiol benzoate (EB) for 5 consecutive days. Compared to EB-treated females, EB-treated males showed a slightly lower number of PR-immunoreactive cells in the preoptic area whereas PR-immunoreactivity appeared in more cells in the anterior part of the ventrolateral nucleus. The numbers of PR-immunoreactive cells in the arcuate nucleus did not differ significantly between males and females. These results show that regionally localized sex differences exist in the induced PR system after 5 days exposure to EB.     

雌激素对卵巢摘除大鼠心内神经节细胞Bcl-2和Bax表达的影响

本实验建立了去卵巢和雌激素替代疗法动物模型,用免疫组织化学SABC法结合图象分析方法,观察大鼠心内神经节细胞中Bcl-2和Bax的变化,探讨雌激素对卵巢摘除大鼠心内神经节细胞中Bcl-2和Bax表达的影响。去卵巢(OVX)大鼠心内神经节细胞中Bcl-2的表达较正常对照组明显减弱(P<0.05),雌激素替代治疗组大鼠心内神经节中Bcl-2的表达与正常对照组相比无显著性差异(P>0.05),而较卵巢摘除组显著增高(P<0.05);卵巢摘除后同时给予特异性雌激素受体阻断剂(TAM)和17β-雌二醇(OVX+TAM+ERT)组大鼠心内神经节中Bcl-2的表达较正常对照组显著减弱(P<0.05),但与OVX组无显著性差异(P>.05)。各组中Bax的表达无显著性差异(P>0.05)。实验结果提示雌激素能上调大鼠心内神经节细胞中Bcl-2的表达,但对Bax的表达无影响。     

Olfactory environment and early mating behavior in the cyclic female rat

The objectives of this study were to examine the effects of olfactory cues from the male and of olfactory bulb removal on early mating behavior in sexually inexperienced diestrous female rats primed with estrogen. Four-day cyclic rats isolated from the male were given either 2.5 micrograms or 10 micrograms estradiol benzoate (EB) and presented to stimulated males in the late afternoon of diestrus 2 between 18:00 and 19:00 for a 10 min sexual behavioral session. Dose-dependent effects of estrogen were observed since 10 micrograms EB significantly increased the proportion of females displaying early mating as compared with those given 2.5 micrograms EB. Olfactory bulb removal prior to estrogen treatment caused a rise in the number of females which mated early with respect to the non bulbectomized controls. Exposing the females to bedding soiled with male urine on diestrus 2 at 10:00 did not affect early mating behavior. By contrast the olfactory stimuli became efficient when 2.5 micrograms EB treated females were given 10 micrograms progesterone (P) by the time of exposure to male urine. The results were discussed with respect to the role played by the olfactory system in the control of lordosis behavior throughout estrous cycle in female rats. P was concluded to be involved in the perception of the olfactory signals from the male which facilitate early mating behavior in diestrous female rats.     

Effect of ER-beta gene disruption on estrogenic regulation of anxiety in female mice

It has been shown that long-term estrogen treatment in gonadectomized female mice increases anxiety levels. On the other hand, a recent study has reported that estrogen may down-regulate the levels of anxiety by acting through estrogen receptor (ER) beta. In the present study, we investigated the role of ER-beta in the regulation of anxiety levels in female mice after long-term estrogen treatment. Gonadectomized ER-beta knockout (betaERKO) female mice and their wild type (betaWT) littermates were implanted several different doses (experiment 1: 2.0 microg/day, experiment 2: 1.0, 0.4, 0.2 or 0.1 microg/day) of an estradiol benzoate (EB) or placebo pellet. Ten days after pellet implant, behavioral tests commenced to measure the anxiety levels (experiment 1: light-dark transition test (LDT), experiment 2: LDT, elevated plus maze test (EPM) and social investigation test (SIT)). We found that, at higher-doses, long-term treatment of EB had anxiogenic effects in both betaWT and betaERKO mice as indicated by a decrease of the time spent in the light side and the number of transitions between two sides during LDT. In contrast, several behavioral measurements indicated that the lower-doses treatment of EB might reduce the anxiety levels possibly through ER-beta. Particularly, the anxiolytic effects of EB in the SIT were more pronounced in betaWT mice than betaERKO mice. Together, the findings in the present study suggest that estrogen may have both anxiolytic and anxiogenic effects in female mice, and that ER-beta gene disruption did not affect anxiogenic regulation by estrogen in female mice, but partially affected anxiolytic regulation.     

Intracerebral sites of action of estrogen on the sleep-wakefulness circadian rhythm in female rats

Effects of estradiol on the sleep-wakefulness circadian rhythm were studied in ovariectomized female rats under a 12: 12 light-dark schedule. In rats that were subcutaneously injected with 20 micrograms of estradiol benzoate (EB) or that had crystalline EB implanted into the third ventricle (III V), the total time of slow wave sleep (SWS) and paradoxical sleep (PS) in the night time was significantly decreased on the third day after the EB administration. The suppression of PS was more remarkable than that of SWS. Slight increases were observed in the total time of SWS and PS in the day time. These effects of estrogen on the appearance patterns of SWS and PS in the night and day time could be elicited in spayed rats after crystalline EB was implanted into the medial preoptic area (MPO) or the medial amygdala (mAMYG), but the implant into the latter proved less effective. Cholesterol implants into IIIV, MPO or mAMYG produced no change in the sleep-wakefulness circadian pattern. Implantations of crystalline EB into the ventromedial hypothalamic nucleus or the dorsomedial thalamic nucleus were ineffective. These results indicate that MPO is probably the most effective site of estrogen on the sleep-wakefulness circadian rhythm of female rats.     

Stereological evaluation and Golgi study of the sexual dimorphisms in the volume, cell numbers, and cell size in the medial preoptic nucleus of the rat

The medial preoptic nucleus (MPN) and the sexually dimorphic nucleus of the preoptic area (SDN-POA) stand out as prominent sexually dimorphic cell groups of the rat brain. However, quantitative data on sex-related differences in these nuclei in the adult rat are confined to their volume. We have used stereological methods and Golgi-impregnated material to examine whether, in young adult rats, the sexual dimorphism in the volume of the MPN, including its divisions, and of the SDN-POA, reflect similar differences in the number and size of their neurons. We found that the total number of neurons in all MPN divisions is higher and the mean somatic volume larger in males than in females. In addition, the total dendritic length of MPN neurons is greater, but the dendritic spine density is smaller, in males than in females. Likewise, in the SDN-POA the total number and size of its neurons is greater in males than in females. The sex differences in all quantitative parameters evaluated accounted for the larger volume of the MPN and SDN-POA in males relative to females. In addition, the MPN neuropil also displays sex-related differences in its volume, and these differences closely match those detected for the volume of each MPN division. It deserves to be emphasised that the numerical density of neurons was the only parameter found to be significantly higher in females than in males in all MPN divisions and in the SDN-POA. Our results show that the MPN and the SDN-POA display sex differences in the volume, total number of neurons, and size of neuronal cell bodies and dendritic trees. Furthermore, they also indicate that the neuropil is critical for the establishment of sexual dimorphism in the size of the MPN.     

The role of estrogen and progesterone in the control of preputial gland sex attractant odors in the female rat

Sexually experienced male rats were used to test the attractiveness of body odors of female rats. The attractiveness of these odors varied with the estrous cycle. Odors from female rats in proestrus were the most attractive to male rats and those from female rats during the darkness hours of diestrus the least attractive. The preputial glands appeared to be the source of these odors for the male rats showed no preference for the odors of proestrous female rats that had been preputialectomized. Administration of 1 μg estrdiol benzoate (EB) for 5 days increased the attractiveness of body odors of ovariectomized rats. A higher dose of EB (5 μg) had the same effect when administered for 1 or 5 days although the increase that occurred after 3 days was not significant. A single dose of progesterone (P) (500 μg) on the other hand, decreased the attractiveness of ovariectomized female odors although no change was seen after 3 days of treatment. A single injection of P also decreased the attractiveness of odors of ovariectomized females that had received EB for 3 days. However, P failed to decrease the attractiveness of odors in ovariectomized females after preputialectomy. We conclude that the preputial glands are an important source of sex attractant odors in the female rat and that the changes in the release of these odors that occur throughout the estrous cycle and pregnancy are controlled by ovarian steroids. While estrogen acts to stimulate the production and release of these odors P appears to inhibit their release.     

The Effect of Cyproterone Acetate and Estradiol Benzoate on the Adrenal Gland of Juvenile and Adult Rats: A Morphometrical Study

Little is known about the effect of antiandrogens and estrogens on the adrenal cortex. To investigate their influence the antiandrogen Cyproterone acetate (CP) and the estrogen estradiol benzoate (EB) were applied separately to matched groups of animals. While EB affects the hypothalamic-pituitary axis via a negative feedback mechanism, CP has a competitive influence on peripheral androgen receptors and also affects the central regulatory mechanism through its gestagenic component.48 male rats were divided in 6 groups, two for CP, two for EB, two control, each pair consisting of half young, half adult animals. Animals were killed on the 36th day of the assay. The histologic slides of adrenal tissue were evaluated using microphotographs enlarged by copyprint to 800fold magnification.The effects of the two drugs are recorded separately for the three zones of the Adrenal cortex, Z. glomerulosa (ZG), Z. fasciculata (ZF) and Z. reticularis (ZR): EB provokes atrophy of ZG in young rats, whereas no effects of CP on this zone are registered morphometrically in young nor in adult animals. ZF shows marked regressive transformation and CP, stronger in adult than in young animals. EB causes regressive transformation only in young rats. The effects of CP on ZR in different in young and adult rats, the young ones showing marked atrophy, the adult group demonstrating an increase of cell volume, — EB-treated rats, both young and adult, show a volume increase of reticularis cells.ZG-atrophy under EB is not adequately explained by the morphological methods used in our assay. The regressive transformation of ZF in young and adult rats seems to be due to an additional corticotropic action of CP, whereas the progressive transformation under EB is probably due to the paradoxical effect of estrogens with a positive feedback mechanism. The diversity of ZR reactions to CP is explained by different steroid metabolisms in the groups. The volume increase of reticularis cells under EB may be due to an increased storage of steroid precursors.     

Dopamine release in the medial preoptic area of female rats in response to hormonal manipulation and sexual activity

Dopamine (DA) is responsive to hormonal manipulations and has been implicated in the regulation of female rat sexual behavior. In the present studies, extracellular DA levels were assessed in the medial preoptic area (MPOA) of ovariectomized female rats in response to exogenous ovarian hormones and during sexual activity. In female rats primed with a low dose of estradiol benzoate (2 microg), but not with a higher dose (20 microg), a 500-microg progesterone injection increased extracellular DA and facilitated copulatory behavior. Extracellular DA levels in the MPOA were further augmented during sexual interactions with a male rat in a nonpacing copulatory chamber by either perineal or vaginal stimulation. However, in a pacing chamber, DA efflux did not increase, although the metabolites rose significantly during copulation. Together, these findings suggest that extracellular DA in the MPOA responds to the hormonal state of the female rat and may contribute to her expression of sexual behavior.     

Estrogen influences stimulated water intake by ovariectomized female rats

To further elucidate the influence of estrogen on water consumption, we examined water intake by adult female rats stimulated by water deprivation, injection of hypertonic saline or injection of isoproterenol (ISOP), a beta-adrenergic agonist that activates the renin-angiotensin system (RAS). Rats were ovariectomized (OVX) then injected with estradiol benzoate (EB; 10 microg/0.1 ml oil) or the oil vehicle (OIL; 0.1 ml) for 2 consecutive days. Twenty-four hours after the second injection, rats were deprived of food and water. On the following day, rats were given water and intake was measured after 2 h. EB significantly decreased water intake compared with that by OIL-treated rats following water deprivation. Two additional groups of adult female rats were OVX and treated with EB or OIL. Forty-eight hours after EB or OIL treatment, rats were injected with hypertonic saline (1 ml of 2 M NaCl) or ISOP (30 microg/kg in 0.15 M saline) and water intake was measured after 2 h. EB significantly attenuated water intake following ISOP but not after hypertonic saline. Finally, we examined plasma sodium concentration (pNa) after hypertonic saline and plasma renin activity (PRA) after ISOP in EB- and OIL-treated rats and found no differences in pNa or PRA. These results suggest that the stimuli for water intake after hypertonic saline and ISOP were comparable in EB- and OIL-treated rats. Taken together, these results raise the possibility that EB attenuation of stimulated water intake is specific to water intake elicited by activation of the RAS.     

Sex differences in the effect of prepubertal GALP infusion on growth, metabolism and LH secretion

The hypothalamic neuropeptide, galanin-like peptide (GALP), is known to have an effect on energy expenditure and reproduction in adult male rats, but little work has been done on prepubertal rats. We hypothesized that hypothalamic GALP is involved in physiological changes associated with the onset of puberty. To test this hypothesis, we first determined the postnatal ontogeny of GALP gene expression via in situ hybridization of developing male and female rat pups through adulthood. GALP gene expression was not observed in either male or female rat pups until after postnatal day (PND) 10 and did not reach adult-like levels until after weaning (PND25). To determine if exogenous GALP could induce the onset of puberty, PND25 male and female rats were implanted with lateral ventricular cannulas connected to an osmotic minipump that delivered either GALP or vehicle. GALP infusion significantly (p<0.05) increased body weight, food intake, and metabolic rate in male but not female rats compared to control infusion. After 2 weeks, GALP infusion had no significant effect on the onset of puberty, percent body fat, nor plasma levels of insulin, FSH or gonadal steroids in either sex; however, GALP did significantly (p<0.05) increase plasma levels of LH and leptin in male but not female rats and increased plasma growth hormone (GH) in both sexes. Our observations further demonstrate a sex difference in GALP responsiveness in prepubertal rats. These data suggest that GALP may be involved with the prepubertal increase in circulating leptin, LH, and GH resulting in an increase in metabolic rate and lean growth associated with puberty.     

罗布麻颗粒对化疗型卵巢早衰大鼠Bax和Bcl-2蛋白表达的影响

目的： 探讨罗布麻颗粒对化疗型卵巢早衰大鼠Bax 和Bcl-2 蛋白表达的影响。方法：性成熟SD雌鼠随机分 为对照组、造模组,腹腔注射（ 顺铂1.5 mg/kg）制备卵巢早衰模型,随机分为卵巢早衰模型组和罗布麻颗粒高、 中、低剂量治疗组。H-E 染色观察卵巢形态;ELISA 检测各组大鼠血清Bax、Bcl-2 和雌二醇、促黄体生成素（LH）、 促卵泡激素（FSH））的含量,免疫组织化学法检测卵巢Bax 与Bcl-2 蛋白表达。结果：卵巢早衰模型组大鼠中初 级卵泡减少,闭锁卵泡增多,罗布麻颗粒高剂量治疗组大鼠可见初级卵泡,闭锁卵泡较卵巢早衰模型组减少;卵 巢早衰模型组大鼠血清中雌二醇及Bcl-2 含量降低,FSH、LH、Bax 的含量升高; 各药物治疗组雌二醇、Bcl-2 含 量升高,FSH、LH、Bax 的含量降低;Bax 蛋白主要表达在颗粒层细胞及卵泡液中,卵巢早衰模型组大鼠较对照 组表达显著升高, 各药物治疗组在不同程度上能够降低其表达;Bcl-2 蛋白在各组主要表达在颗粒层细胞中,卵 巢早衰模型组大鼠较对照组表达显著下降, 各药物治疗组在不同程度上能够升高其表达。结论：顺铂能够导致雌 性大鼠血清中性激素水平的紊乱及卵巢组织形态的改变,可能与细胞凋亡因子Bax 与Bcl-2 在血清及卵巢组织中 表达水平的高低有关;罗布麻颗粒可能通过下调Bax 蛋白、上调Bcl-2 蛋白从而改善顺铂致卵巢早衰大鼠的卵巢 功能。     

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.