Expression of the Raf-1 protein in rat brain during development and its hormonal regulation in hypothalamus

To study mechanisms involved in the sexual differentiation of the rat brain, the expression of the protein product of the proto-oncogene c-raf-1 (Raf-1) was examined. Biochemical and immunocytochemical analyses localized Raf-1 in embryonic rat brain regions and demonstrated hormonally induced changes in Raf-1 expression. For this study an affinity-purified anti-peptide antiserum specific for Raf-1 (NH-44) was used. Western blots revealed an approximately 77 kD polypeptide isolated in the cytosol of developing rat brains. Raf-1 levels were highest in the embryonic (E) day 22 female hypothalamus (HYP), and approximately twofold higher than levels detected in male HYP at E22 as determined by quantitative protein dot blot and semiquantitative Western blot analyses. Raf-1 levels in HYP were greater than those in either brain stem (BS) or cortex. Immunocytochemical analysis revealed high levels of Raf-1 in selective brain regions (e.g., the ventromedial nucleus in the HYP, the mitral cell layers in the main and accessory olfactory bulbs (OB), and the locus coeruleus) at E22 and postnatal (P) day I. Lower levels of immunoreactivity were observed in many areas of the perinatal neuraxis. To test hormonal regulation of Raf-1, testosterone propionate (TP) was administered to pregnant rats on E17; male and female fetuses were examined on E22. This treatment significantly decreased Raf-1 levels in female HYP, but not in male HYP, as determined by Western blot analysis. No significant sex difference or response to prenatal hormone treatments were observed in either brain stem or cortex. No significant sex difference was noted postnatally, and administration of TP 3 h after birth did not change Raf-1 levels examined 24 h later. In summary, Raf-1 was localized within selective regions of the rat brain, and its expression was altered by exogenous prenatal hormonal stimulation. One role for Raf-1 in signal transduction may be to delimit hormonal critical periods in sexual differentiation of the brain.     

产前束缚应激子代大鼠海马神经颗粒素表达降低

神经颗粒素（neurogranin,NG）是脑特异性突触后蛋白,参与在学习记忆功能中起核心作用的信号转导通路及突触可塑性。本研究旨在探讨产前束缚应激对子代大鼠海马NG表达的影响。连续7d对孕晚期大鼠进行束缚应激,建立产前束缚应激模型,分为对照雌、雄组,应激雌、雄组。采用免疫组化方法观察NG在产前束缚应激子代大鼠海马不同亚区的分布特点;采用蛋白免疫印迹方法检测产前束缚应激子代大鼠海马NG蛋白的表达。结果显示：各组子代大鼠海马各区均有NG蛋白表达,CA1和CA3区表达高于齿状回（dentate gyrus,DG）;应激组雌、雄子代大鼠海马NG的表达明显低于对照组（P〈0．01）,应激组雌性子代比雄性子代减少更显著,对照组雌、雄子代之间无差异。免疫组化与蛋白免疫印迹方法所得结果一致。上述结果表明,NG在产前束缚应激子代大鼠海马表达降低,并且雌性比雄性降低明显,NG对产前束缚应激子代大鼠有差异性调制,NG表达减少可能与产前束缚应激子代大鼠学习记忆能力下降有关。     

Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice

Little is known about the hormonal regulation of sexual behavior and about the pattern of expression in the brain of sex-steroid receptors in the BALB/c AnN strain of mice (Mus musculus). In this study, 8-week old male BALB/c AnN mice were castrated and the temporal course of decline of sexual behavior was studied, as well as the effects of daily treatment with either testosterone propionate (TP), estradiol benzoate (EB), or dihydrotestosterone propionate (PDHT). Castration resulted in rapid decline of sexual behavior, in both control or vehicle-treated mice. TP maintained full sexual behavior of castrated mice, while PDHT or EB did not have this effect. The expression of ER-alpha dropped nearly 50% after castration, and this pattern remained in TP or PDHT-treated mice, while EB increased the ER-alpha mRNA levels to almost the same values as in intact control mice. The same pattern was found when ER-beta mRNA levels were analyzed. The expression of the PR-A/B gene in the different brain regions in intact mice and after castration, or among the differently treated mice, showed significant differences between normal and castrated mice at all times in all brain regions studied, with the exception of the frontal cortex. Castration reduced the expression of AR by 10-fold, as compared to intact control mice, while TP or PDHT treatment returned its expression to the same levels as in intact control mice, in all brain areas studied. The changes are more prominent in POA-HIP than in HYP and CF. These results demonstrated a rapid decline of sexual behavior in this strain of mice after castration, and show that only TP was able to maintain male sexual behavior, with no correlation with the pattern of expression of sex hormone receptors in specific areas of the mouse brain.     

Effect of estradiol & testosterone on the pituitary prolactin levels in developing male & female rats

Testosterone priopionate (TP) or estradiol-17 beta (E2) were injected into male and female rats from day of birth to 15 days of age to determine the effect of these steroids on the pituitary prolactin (PRL) level in developing rats. Animals were autopsied on Days 5, 7, 10, 14, 17, 22, 25, 30, 37, 45, 52, and 60 and pituitary PRL estimated by radioimmunoassay. Neonatal administration of TP or E2 markedly increased PRL content in male rats. The peak of PRL was advanced to Days 14 and 23, respectively, in E2- and TP-treated groups. The content of pituitary PRL declined sharply and values increased again by Day 52 in TP-treated rats and Day 37 in E2-treated rats. In the female rat both the steroids advanced the time of PRL peak. Peaks were observed on Days 22 and 30, respectively. Although PRL content declined following these peaks, values remained at a significantly higher level than normal. These results suggest that mechanisms controlling the PRL secretion are functional during the neonatal period. It is also suggested that TP acts to increase PRL levels by 1st being converted to E2.     

Estrogenic feminization of the LH response to orchidectomy: association with prolonged nuclear estradiol receptor retention and induction of cytoplasmic progestin receptors in brain and pituitary

A sex difference in the LH rise after gonadectomy is clearly observable in the rat. While male rats respond with an early (10-12 h) increase in LH after orchidectomy, a delayed response (2-3 days) is recorded after ovariectomy. In this study we tested the hypothesis that the delayed response to gonadectomy in the E2-treated males is due to a more prolonged retention of E2 (when compared with the corresponding male feedback signal, testosterone) within specific central nuclear receptor sites. Orchidectomized (ORDX) animals implanted with either empty or E2-filled Silastic capsules were sacrificed at 0, 24, 48 and 72 h after ORDX or E2-capsule removal. LH levels in ORDX rats rose several-fold by 24 h, whereas E2-treated, ORDX rats, showed no changes in peripheral LH levels until 72 h after E2-capsule removal. At the time of E2-capsule removal (0 h) large increases in nuclear estradiol receptor (NER) levels were seen in anterior pituitary, preoptic area, and hypothalamus (HYP). Twenty-four hours after E2-capsule removal, NER levels were still high in the 3 areas, and by 48 h NER values had returned to control (ORDX) levels, with the exception of HYP where they were slightly but significantly elevated. The increase in NER, as well as the subsequent decline after E2-capsule removal was paralleled by similar changes in cytosolic progestin receptor (CPR) levels in all three regions. Cytosolic testosterone levels were not changed by the E2-treatment. The results indicate that the feminized response to orchidectomy observed in E2-implanted males is related to a prolonged retention of the E2-receptor in nuclear sites. Further, they indicate that E2-treatment in males, as is the case in females, can induce a marked increase in progestin receptor levels within specific brain regions as well as in the pituitary. The reduction in NER and CPR levels to castrate values precedes the first detectable increase in peripheral LH levels. In conclusion, the pattern of LH rise after gonadectomy in the rat is dependent upon the steroidal milieu at the time of removal of the gonads.     

Influence of prenatal stress on the rat hypothalamic-pituitary-adrenal axis activity: the role of the brain glycocorticoid receptors

The effects of prenatal stress on the hypothalamic-pituitary-adrenal (HPA) axis activity and brain glycocorticoid receptors were studied in neonatal male and female offspring, as well as the influence of neonatal glycocorticoid receptors blockade on hormonal stress reactivity of adult rats. The results showed that there were sexual differences in plasma corticosterone level and corticosteroid binding in the cortex and hypothalamus of 5-day old control rats. Prenatal stress increased basal level of corticosterone in female rats, decreased corticosterone binding in hypothalamus and hippocampus of male and female rats, and increased corticosteroid receptor level in the male cortex. Neonatal administration of glycocorticoid receptor antagonist did not change plasma corticosterone level in 5-day old rats, but prolonged hormonal stress response of the HPA axis in adult male rats and increased hormonal stress response in female ones. The character of the IIPA axis activity of male and female rats with neonatal blockade of glycocorticoid receptors correspond to hormonal stress response of prenatal stressed rats. These data suggest that change of brain glycocorticoid receptors function in neonatal period of development might be one of the mechanisms of prenatal stress influence on the HPA axis activity in the adulthood.     

Effects of food deprivation and refeeding on neuropeptide Y (NPY) mRNA levels in goldfish

In mammals, NPY is a key factor in the regulation of feeding behavior. In the present study, the effects of refeeding for 1-3 h in 72-75-h food deprived (FD) goldfish on the levels of NPY mRNA in telencephalon-preoptic (TEL-POA), hypothalamus (HYP) and optic tectum-thalamus (OT-THAL) were examined, using Northern blot analysis. Goldfish FD for 72 h exhibited a significant increase in NPY mRNA levels in all brain regions. At 1 h after 72-h FD (73-h FD), NPY mRNA was significantly increased in TEL-POA and OT-THAL, but remained the same as 72-h FD fish in HYP. At 3 h after 72-h FD (75 h), all brain regions exhibited a significant increase in NPY mRNA levels. However, subsequent refeeding for 1-3 h rapidly and completely reversed the effects of FD in all brain regions, reaching fed levels within 1-3 h of refeeding. Serum GH levels were highest in 72-h FD fish, but decreased significantly over 1-3 h after 72-h FD; whereas, refeeding reversed the increase in serum GH levels only at 3 h after refeeding. Taken together, these results further support that NPY is a physiological brain transducer involved in the regulation of daily appetite and feeding in goldfish.     

Chronic Lead Exposure and Mixed Factors of Gender×Age×Brain Regions Interactions on Dendrite Growth,Spine Maturity and NDR Kinase

NDR1/2 kinase is essential in dendrite morphology and spine formation, which is regulated by cellular Ca²⁺. Lead (Pb) is a potent blocker of L-type calcium channel and our recent work showed Pb exposure impairs dendritic spine outgrowth in hippocampal neurons in rats. But the sensitivity of Pb-induced spine maturity with mixed factors (gender×age×brain regions) remains unknown. This study aimed to systematically investigate the effect of Pb exposure on spine maturity in rat brain with three factors (gender×age×brain regions), as well as the NDR1/2 kinase expression. Sprague–Dawley rats were exposed to Pb from parturition to postnatal day 30, 60, 90, respectively. Golgi-Cox staining was used to examine spine maturity. Western blot assay was applied to measure protein expression and real-time fluorescence quantitative PCR assay was used to examine mRNA levels. The results showed chronic Pb exposure significantly decreased dendritic length and impaired spine maturity in both rat hippocampus and medial prefrontal cortex. The impairment of dendritic length induced by Pb exposure tended to adolescence > adulthood, hippocampus > medial prefrontal cortex and female > male. Pb exposure induced significant damage in spine maturity during adolescence and early adult while little damage during adult in male rat brain and female medial prefrontal cortex. Besides, there was sustained impairment from adolescence to adulthood in female hippocampus. Interestingly, impairment of spine maturity followed by Pb exposure was correlated with NDR1/2 kinase. The reduction of NDR1/2 kinase protein expression after Pb exposure was similar to the result of spine maturity. In addition, NDR2 and their substrate Rabin3 mRNA levels were significantly decreased by Pb exposure in developmental rat brain. Taken together, Pb exposure impaired dendrite growth and maturity which was subject to gender×age×brain regions effects and related to NDR1/2 signal expression.     

An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions

An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.     

Differential responsiveness of LH and prolactin to p-tyramine in male and female rats

The effect of p-tyramine, a natural amine which is found in the rat brain in trace amounts, was evaluated for its capacity to influence LH and prolactin secretion in male and female rats under different hormonal conditions. p-Tyramine (40 mg/kg ip) was ineffective in modifying LH levels in either female or male rats which had been gonadectomized for 2 days, but if the animals were injected with 12.5 micrograms of estradiol benzoate (EB) on the day of castration, p-tyramine was able to release LH in female but not in male rats. To evaluate whether early androgenization of brain structures which control LH secretion was involved in the sexual difference observed, p-tyramine was tested in female androgenized rats (200 micrograms of testosterone propionate on the day of birth), and in male rats castrated at birth. The trace amine was ineffective in altering LH levels in both experimental models, even if rats were pretreated with EB as control females. On the other hand, p-tyramine inhibited prolactin secretion in male rats pretreated with EB, and not in similarly treated female rats. The present results suggest that p-tyramine may be involved not only in prolactin regulation as it has been previously shown, but also in LH control, and that the hormonal response to this amine is sexually differentiated in the rat.     

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen I: site specific suppression of estrogen receptor alpha

Male rat copulation is mediated by estrogen-sensitive neurons in the medial preoptic area (MPO) and medial amygdala (MEA); however, the mechanisms through which estradiol (E(2)) acts are not fully understood. We hypothesized that E(2) acts through estrogen receptor α (ERα) in the MPO and MEA to promote male mating behavior. Antisense oligodeoxynucleotides (AS-ODN) complementary to ERα mRNA were bilaterally infused via minipumps into either brain area to block the synthesis of ERα, which we predicted would reduce mating. Western blot analysis and immunocytochemistry revealed a knockdown of ERα expression in each brain region; however, compared to saline controls, males receiving AS-ODN to the MPO showed significant reductions in all components of mating, whereas males receiving AS-ODN to the MEA continued to mate normally. These results suggest that E(2) acts differently in these brain regions to promote the expression of male rat sexual behavior and that ERα in the MPO, but not in the MEA, promotes mating.     

Microimplants of estradiol in the sexually dimorphic area of the hypothalamus activate ultrasonic vocal behavior in male Mongolian gerbils

The hormonal control of ultrasonic vocal behavior in the male Mongolian gerbil was examined by comparing the behavioral effects of androgen with those of estrogen administered to the preoptic-anterior hypothalamic area (POA-AH) in castrates. By measuring radioactivity released from solid "floating" POA-AH microimplants (mean diameter, 141 microns) of testosterone (3H-T, mean weight, 880 ng) in Experiment 1, we found that the steroid had a concentration gradient which fell rapidly from the edge of the microimplant, suggesting restricted diffusion. Using floating microimplants in Experiment 2, we studied the effects of testosterone propionate (TP, 650 ng), estradiol-17 beta benzoate (EB, 439 ng), or cholesterol (C, 478 ng) on rates of a frequency modulated ultrasonic vocalization emitted during sexual interactions. The effects on the upsweep call were compared with those on sexual mounting. The upsweep rate remained significantly below precastration levels in C implanted males. EB reinstated upsweep calling within 5 days, 3 days earlier than TP microimplants. Mounting in EB implanted males was maintained at precastration levels, whereas TP implantation restored mounting to precastration levels only after 5 days. EB was effective in inducing ultrasonic vocalizations when placed in, or near, the sexually dimorphic area (SDA) in the medial preoptic area (POM). Our results indicate that brain mechanisms underlying both ultrasonic vocalizations and mounting are directly sensitive to estradiol (E2) in the male gerbil. We conclude that E2 affects mechanisms in the SDA associated with ultrasonic calling and suggest that T is likely to act via aromatization products in the brain.     

Antagonism of central estrogen action by intracerebral implants of tamoxifen

In order to determine the neural site(s) of estradiol (E2) priming of receptive behavior in female Long-Evans rats, we attempted to inhibit the behavioral effects of peripheral injections of E2 by administering the E2 antagonist tamoxifen (TX) to particular brain regions. Crystalline TX was administered unilaterally or bilaterally via 28-gauge cannulae into the ventromedial hypothalamic nucleus (VMN), the preoptic area (POA), or the interpeduncular region (IP) 1 hr prior to the first of three daily E2 benzoate injections. Subjects were tested for the presence or absence of behavioral estrus 5 hr after a 200-micrograms progesterone injection given 4 days after the initial hormone treatment. Results of this experiment showed that TX inhibits lordosis when directed toward the VMN, but not when directed toward the POA or IP. The quality of the lordosis response and the proportion of subjects showing solicitation behavior were both lower in VMN subjects treated with TX than in POA or IP subjects given the same treatment. Unilateral implants were as effective as bilateral implants in inhibiting the behavior of VMN subjects. A second experiment measured uptake of radiolabeled E2 by nuclei of hypothalamic (HYP) and POA tissue following bilateral TX administration to the VMN or POA. TX was capable of inhibiting uptake of [3H]E2 into nuclei of cells located near the implant site. Most subjects which showed behavioral inhibition also showed a reduction in uptake of [3H]E2 by HYP tissue. These data support the hypothesis that exposure of the VMN to E2 is necessary for the priming of estrous behavior in the female rat.     

The unusual estrogen-binding protein (UEBP) of rat liver: the role of sex steroids and hypophysis in its regulation

The number of estradiol (E2) binding sites of rat liver unusual estrogen-binding protein (NUEBP) was measured, using a novel modification of the quantitative method of specific UEBP determination. In liver cytosol of mature male and female rats, NUEBP amounted to 6.83 +/- 0.49 and less than 0.05 pmol/mg protein, respectively. Neonatal administration of testosterone-propionate (TP) and TP injections at later periods of ontogenesis increased NUEBP in female rat liver in a similar fashion. The elevated NUEBP was found in the liver of mature ovariectomized females 30 days after cessation of TP injections. Hypophysectomy (but not adrenalectomy or thyroidectomy) prevented TP induction of elevated NUEBP in pubertal females. E2 injections reversibly decreased NUEBP in the liver of all animals under study except of hypophysectomized males. A stimulating regulatory effect of TP on NUEBP in male rat liver was observed only in the case of endogenous androgen deficiency and low NUEBP. TP prevented the E2-dependent decrease of NUEBP upon their simultaneous injections and increased the E2-reduced NUEBP when injected after E2. Hypophysectomy led to a decrease of NUEBP in pubertal males but only slightly affected that in castrated animals. After TP injections to hypophysectomized males, NUEBP returned to a level next to the initial one. It was concluded that estrogen-androgen regulation of the UEBP level led to the maintenance of sex differences in the UEBP content.     

Alterations in NMDA receptor subunit levels in the brain regions of rats chronically administered typical or atypical antipsychotic drugs

It has been hypothesized that glutamatergic neurotransmission is related to the therapeutic effect of antipsychotic drugs. To test this hypothesis, we measured by use of the Western blot technique the polypeptide levels of NMDA receptor subunits, that is, NMDAR1, 2A, 2B, and 2C, in several regions of the rat brain after chronic treatment with haloperidol (HPD) or clozapine (CLZ). Each rat was intraperitoneally injected with HPD or CLZ at 10:00 h daily for 14 days. The brain regions examined were frontal cortex, striatum, nucleus accumbens, hippocampus, and cerebellum. Decreases in the polypeptide level of NMDAR2B were seen in hippocampus (but not in other brain regions) following the treatment with HPD or CLZ. Altered levels in NMDAR1-, 2A-, and 2C were not detected in any brain regions examined. We infer that an alteration in NMDAR2B in hippocampus is related to therapeutic effects of antipsychotic drugs.     

奥利亚罗非鱼DMRT1和DMRT4抗体制备及组织表达谱分析

DMRT1和DMRT4是DMRT基因家族的成员,该家族成员与果蝇的性别决定基因和线虫性别决定基因一样,所编码的蛋白质都包含一个具有DNA结合能力的保守基序,即DM结构域,并以锌指结构与特异DNA序列相结合,在性别决定和分化发育中起调控作用。采用RT-PCR方法分别从奥利亚罗非鱼卵巢和精巢中扩增克隆出DMRT1和DMRT4全长cDNA片段,构建表达载体,在大肠杆菌中表达了BMP-DMRT4和BMP-DMRT1蛋白。经Xa切割、Amylose-sepharose柱层析纯化后作为抗原免疫新西兰白兔制备了DMRT1和DMRT4多克隆抗体,并进行纯化。对纯化多抗进行Western blot分析,结果表明获得了高特异性的DMRT1和DMRT4抗体。为了观察DMRT1和DMRT4在组织中的表达谱,首先,我们通过实时荧光定量RT-PCR检测雌雄奥利亚罗非鱼多种组织mRNA的表达,仅在卵巢和脑中检测到DMRT4,在精巢中检测到DMRT1;其次,制备了多种组织匀浆蛋白,使用纯化的抗体进行Western blot分析,仅分别在卵巢和精巢中检测到DMRT4和DMRT1蛋白的表达;制备多种奥利亚罗非鱼组织切片,使用纯化的DMRT4和DMRT1多抗进行免疫组织化学分析,发现DMRT4仅在卵巢表达,而DMRT1仅在精巢表达。这些结果有助于阐明DMRT4和DMRT1的功能及在鱼类性别调控中的作用。     

Progesterone receptor gene and protein expression in the anterior preoptic area and hypothalamus of defeminized rats

Progesterone receptor (PR) plays an important role during sexual differentiation of the rat brain. The objective of the present study was to determine PR protein and gene expression pattern in preoptic-anterior hypothalamic area (POA-AHA) and hypothalamus (HYP), after estradiol or testosterone treatment during the postnatal critical period of sexual differentiation of the rat brain (defeminized animals). Three-day-old female rats were subcutaneously (s.c.) injected with a single dose of 17beta-estradiol (200 microg), or testosterone enanthate (200 microg), or vehicle (corn oil). POA-AHA and HYP were dissected 3 h, 24 h, and 14 days, as well as on the day of vaginal opening (VO) after treatments. Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; POA-AHA and HYP were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR and tissue slices were prepared for protein detection by immunohistochemistry. We observed that PR mRNA expression was increased in POA-AHA and HYP of the animals treated with estradiol or testosterone 3 hours after treatments, compared with the vehicle-treated control group. We also found a significant increase in PR mRNA and protein expression in POA-AHA and HYP on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute administration of estradiol on the day of VO (VO + E(2)) did not increase PR mRNA or protein expression in POA-AHA and HYP of either estradiol or testosterone defeminized animals, as opposed to the marked induction observed in the intact animals of the control group. The overall results suggest that estradiol and testosterone treatment during the postnatal critical period of sexual differentiation of the brain modifies the regulation of the PR mRNA and protein expression during early onset of maturity.     

Sex Differences in Iron Status and Hepcidin Expression in Rats

Studies have shown that men and women exhibit significant differences regarding iron status. However, the effects of sex on iron accumulation and distribution are not well established. In this study, female and male Sprague-Dawley rats were killed at 4 months of age. Blood samples were analyzed to determine the red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), and mean red blood cell volume (MCV). The serum samples were analyzed to determine the concentrations of serum iron (SI), transferrin saturation (TS), ferritin, soluble transferrin receptor (sTfR), and erythropoietin (EPO). The tissue nonheme iron concentrations were measured in the liver, spleen, bone marrow, kidney, heart, gastrocnemius, duodenal epithelium, lung, pallium, cerebellum, hippocampus, and striatum. Hepatic hepcidin expression was detected by real-time PCR analysis. The synthesis of ferroportin 1 (FPN1) in the liver, spleen, kidney, and bone marrow was determined by Western blot analysis. The synthesis of duodenal cytochrome B561 (DcytB), divalent metal transporter 1 (DMT1), FPN1, hephaestin (HP) in the duodenal epithelium was also measured by Western blot analysis. The results showed that the RBC, Hb, and Hct in male rats were higher than those in female rats. The SI and plasma TS levels were lower in male rats than in female rats. The levels of serum ferritin and sTfR were higher in male rats than in female rats. The EPO levels in male rats were lower than that in female rats. The nonheme iron contents in the liver, spleen, bone marrow, and kidney in male rats were also lower (56.7, 73.2, 60.6, and 61.4 % of female rats, respectively). Nonheme iron concentrations in the heart, gastrocnemius, duodenal epithelium, lung, and brain were similar in rats of both sexes. A moderate decrease in hepatic hepcidin mRNA content was also observed in male rats (to 56.0 % of female rats). The levels of FPN1 protein in the liver, spleen, and kidney were higher in male rats than in female rats. There was no significant change in FPN1 expression in bone marrow. Significant difference was also not found in DcytB, DMT1, FPN1, and HP protein levels in the duodenal epithelium between male and female rats. These data suggest that iron is distributed differently in male and female rats. This difference in iron distribution may be associated with the difference in the hepcidin level.