Male Nuclei Research Articles

176 CYTOCHALASIN PREVENTS SPERM HEAD INCORPORATION INTO FERTILIZED PIG OOCYTES

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.

Early life stress disrupts peripubertal development of aggression in male mice

To investigate the effects of early life stress on the development of social behaviors in male mice, we examined behavioral responses toward same sex stimulus mice in the social investigation test and aggressive behaviors in peripubertal male mice exposed to maternal separation (MS) during the first 2 weeks of life. MS suppressed aggressive behaviors from 5-9 weeks of age, but had no effect on social investigative behaviors in the social investigation test. Investigation of neuroendocrine bases of behavioral effects of MS showed that MS reduced plasma testosterone levels and decreased arginine vasopressin and increased oxytocin immunoreactivity in the paraventricular nucleus of peripubertal males. These results collectively suggest that early life stress disrupts the development of male aggressive behaviors and associated neuroendocrine systems.

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague–Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABAB receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.

Study of chromatin decondensation factors in human spermatozoids by flow cytometry

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.

Post-plasmogamic pre-karyogamic sexual selection: mate choice inside an egg cell

Intersexual selection is often categorized as pre-copulatory or post-copulatory mate choice by individuals of one sex over showy individuals of the other sex. We extend the framework of post-copulatory choice to include post-plasmogamic pre-karyogamic sexual selection. That is, selection of haploid nuclei within the microcosm of a single fertilized egg cell after sperm has entered an egg cell but before fusion of their nuclear membranes, in which an egg nucleus chooses a sperm nucleus with which to fuse. The role of sexual selection at this nuclear level is probably small in monospermic out-crossers, but large in polyspermic out-crossers. Post-plasmogamic pre-karyogamic sexual selection may also explain how the sperm nucleus is rejected in gynogens, and how the egg nucleus is rejected in androgens. We propose that male and female nuclei behave independently within an egg cell, and that post-plasmogamic pre-karyogamic mate choice is largely based on either (1) a pre-existing sensory bias for certain characteristics of the sperm pronuclear mem-brane, or (2) detection of good genes through signal (mRNA) detection.

Fashioning sperm in vitro

Fashioning sperm in vitro

An Analysis of Histone Modifications in Relation to Sex-Specific Chromatin Organization in the Mealybug Maconellicoccus hirsutus

The male-specific heterochromatization of the paternal genome, observed in coccids, is an example of both genomic imprinting and differential regulation of homologous chromosomes. We observed a highly nuclease-resistant chromatin (NRC) organization of a part of the paternal genome in males of Maconellicoccus hirsutus as reported earlier in Planococcus lilacinus. The nuclease resistance of NRC is correlated with nuclear matrix association and is lost when NRC is dissociated from the matrix. We carried out a comparative analysis of epigenetic modifications of histones in matrix associated chromatin of male and female mealybugs by ELISA. We detected H3K27me3, H4K20me3, H3K9me2 and H3K4me3 in both males and females and observed significant enrichment of H3K27me3 in the nuclear matrix of males compared to that of females. To further examine the presence of NRC in the germ line, nuclei were sorted based on chromatin compaction. The analysis of sorted nuclei indicates the presence of NRC in nuclei with different DNA content including the haploid nuclei from males. We discuss these results in the light of the presence of NRC exclusively in male nuclei and the retention of the maternal genome in sperm nuclei of mealybugs.

Co-localization of Sorting Nexin 2 and androgen receptor in the song system of juvenile zebra finches

Mechanisms regulating sexual differentiation of the zebra finch song system appear to include both genetic and hormonal factors. Sorting Nexin 2 (SNX2), which is involved in trafficking proteins between cellular membranes, and androgen receptor (AR) mRNA are both increased in song control nuclei of juvenile males compared to females. Here, in situ hybridization for SNX2 and immunohistochemistry for AR were used to evaluate these sexual dimorphisms in more detail. Estimates of the total number of HVC cells expressing SNX2 and AR, individually as well as together, were greater in 25-day-old males compared to females. The densities of these types of cells were generally also increased in males compared to females in HVC and Area X (or the equivalent portion of the medial striatum in females). On average, more than half of the AR+ cells co-expressed SNX2 in both brain regions. The potential, therefore, exists for both AR and SNX2 to be involved in masculinization of these two brain regions. One possibility is that they, either separately or in conjunction, enhance the action of trophic factors within the brain.

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.

Binge-pattern alcohol exposure during puberty induces sexually dimorphic changes in genes regulating the HPA axis

Maternal alcohol consumption during critical periods of fetal brain development leads to devastating long-term consequences on adult reproductive physiology, cognitive function, and social behaviors. However, very little is known about the long-term consequences of alcohol consumption during puberty, which is perhaps an equally dynamic and critical period of brain development. Alcohol abuse during adulthood has been linked with an increase in clinically diagnosed anxiety disorders, yet the etiology and neurochemical mechanisms of alcohol-induced anxiety behavior is unknown. In this study, we determined the effects of binge ethanol exposure during puberty on two critical central regulators of stress and anxiety behavior: corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP). Our results showed that ethanol increased plasma corticosterone (CORT) levels in both sexes, yet binge-treated animals had significantly lower CORT levels than animals exposed to a single dose, suggesting that the hypothalamo-pituitary-adrenal (HPA) axis habituated to the repeated stressful stimuli of ethanol. Binge ethanol exposure also significantly increased CRH and AVP gene expression in the paraventricular nucleus of males, but not females. Overall, our results demonstrate that binge ethanol exposure during puberty changes the central expression of stress-related genes in a sex-specific manner, potentially leading to permanent dysregulation of the HPA axis and long-term behavioral consequences.

Memoirs of an insult: sperm as a possible source of transgenerational epimutations and genetic instability

Male transgenerational epigenetic effects have been discovered in the discipline of mouse radiation genetics, using genetic and non-genetic readouts. The mechanism to explain the origin of the transmission of epigenetic and genetic instability is still unknown. In a search for a hypothesis that could satisfy the data, we propose that regulation of chromosome structure in the germline, by the occupancy of matrix/scaffold associated regions, contains molecular memory function. The male germline is strikingly dynamic as to chromatin organization. This could explain why experience of irradiation stress leaves a persistent mark in the male germline only. To be installed, such memory requires both S-phase and chromatin reorganization during spermatogenesis and in the zygote, that likely also involves reorganization of loop domains. By this reorganization, another layer of information is added, needed to accommodate early embryonic development. Observations point at the involvement of DNA repair as inducer of transgenerational epigenetic modulation. Nuclear structure, chromatin composition and loop domain organization are aspects of human sperm variability that in many cases of assisted reproduction is increased due to inclusion of more incompletely differentiated/maturated sperm nuclei. Adjustment of loop domains in early embryo development can be anticipated and zygotic and cleavage stage S-phase repair activity will have to deal with potential paternal DNA lesions. Therefore, by changing male nucleus structure due to reproduction from impaired spermatogenesis, the transgenerational information content could be changed as well. We discuss aspects of male reproductive performance in the context of this hypothesis.

Neonatal maternal separation alters the development of glucocorticoid receptor expression in the interpositus nucleus of the cerebellum

Adverse early experience impairs adult learning and memory. Previously, we showed that neonatal maternal separation impaired eyeblink conditioning in adult male rats. This impairment was correlated with increases in glucocorticoid receptor expression in the posterior region of the cerebellar interpositus nucleus, a key structure in the neural circuitry controlling eyeblink conditioning. To begin to establish how separation results in altered glucocorticoid receptor expression in adulthood, we assessed the developmental pattern of glucocorticoid receptor expression in the interpositus nucleus in controls versus rats that had undergone maternal separation for 1h per day on postnatal days 2–14. Rat pups were exposed to either standard rearing (control) or maternal separation and glucocorticoid receptor expression was assessed at postnatal day 15, postnatal day 21, and adulthood. In control males, glucocorticoid receptor expression in the interpositus nucleus declined between postnatal days 15 and 21, then increased into adulthood. On postnatal day 15, there was less glucocorticoid receptor expression in the interpositus nucleus in males that were maternally separated than in controls. However, neonatal separation significantly attenuated the normal decline in the third postnatal week, resulting in significantly greater glucocorticoid receptor expression in the interpositus in separated males than in control rats at postnatal day 21. The developmental pattern of glucocorticoid receptor expression was not altered by maternal separation in female rats. Thus, maternal separation may impair learning and memory in adult males by altering normal developmental changes in glucocorticoid receptor expression.

Coordinated Regulation of Heterochromatic Genes inDrosophila melanogasterMales

Dosage compensation modifies the chromatin of X-linked genes to assure equivalent expression in sexes with unequal X chromosome dosage. In Drosophila dosage compensation is achieved by increasing expression from the male X chromosome. The ribonucleoprotein dosage compensation complex (DCC) binds hundreds of sites along the X chromosome and modifies chromatin to facilitate transcription. Loss of roX RNA, an essential component of the DCC, reduces expression from X-linked genes. Surprisingly, loss of roX RNA also reduces expression from genes situated in proximal heterochromatin and on the small, heterochromatic fourth chromosome. Mutation of some, but not all, of the genes encoding DCC proteins produces a similar effect. Reduction of roX function suppresses position effect variegation (PEV), revealing functional alteration in heterochromatin. The effects of roX mutations on heterochromatic gene expression and PEV are limited to males. A sex-limited role for the roX RNAs in autosomal gene expression was unexpected. We propose that this reflects a difference in the heterochromatin of males and females, which serves to accommodate the heterochromatic Y chromosome present in the male nucleus. roX transcripts may thus participate in two distinct regulatory systems that have evolved in response to highly differentiated sex chromosomes: compensation of X-linked gene dosage and modulation of heterochromatin.

Correlation of Donor Leukocyte Chimerism With Pulmonary Allograft Survival After Immunosuppressive Drug Withdrawal in a Porcine Model

This study was designed to analyze the role of postoperative donor cell chimerism for the induction and maintenance of transplantation tolerance in a porcine lung transplantation model. Left-sided single lung transplantation from major histocompatibility mismatched male donors was performed in 27 female minipigs. All received a 28-day course of pharmacologic immunosuppression using various agents, some in combination with preoperative irradiation. Groups for eventual analysis were strictly defined by outcome, that is, pigs with acute rejection before postoperative day 178 (n=16) were allocated into one group, long-term surviving animals (n=11) into the other. Peripheral blood chimerism was monitored by flow cytometry and real-time polymerase chain reaction. Intragraft chimerism was detected from bronchoalveolar lavage fluid (BALF) by fluorescent in situ hybridization. Blood chimerism peaked 1 hour after transplantation and was significantly higher in the group of long-term survivors at that time. Thereafter chimerism rapidly decreased, but tended to remain higher in long-term survivors. In case of acute rejection donor cells were lost, but remained detectable for up to 36 postoperative months in tolerant animals. In BALF, the percentage of male nuclei was equally high under immunosuppression in both groups. Rejecting animals showed a rapid decrease of Y-bearing cells in BALF after drug withdrawal and an almost complete loss when acute rejection occurred. In tolerant pigs, intragraft chimerism remained detectable throughout the follow-up. This study demonstrates a clear correlation of donor leukocyte chimerism with long-term allograft survival in a porcine allogeneic lung transplantation model.

Spatial working memory learning in young male and female rats: Involvement of different limbic system regions revealed by cytochrome oxidase activity

Sex differences have been found in the spatial memory which involve several regions of the limbic system. The Morris water maze (MWM) is one of the most widely used tasks in behavioral neuroscience to explore spatial and episodic memory in rats. We evaluated the oxidative metabolic activity of the prefrontal cortex, dorsal hippocampus, anterior thalamic nuclei and mammillary region following the acquisition of a spatial working memory (WM) task in young rats (30 days) of both sexes using quantitative histochemistry of the cytochrome oxidase (COx). The rats were trained until they achieved the learning criteria in the MWM using a hidden escape platform with different daily locations, and two control groups were added to evaluate the oxidative metabolism not specific to the learning task. We found a delay in the acquisition of the WM in females. A significant decrease in COx activity was found in the prefrontal cortex in both sexes. Also, changes were found in the dentate gyrus and lateral mammillary nucleus in males, whereas females showed changes in the anterodorsal thalamus and CA3. These results suggest a sex difference in the contribution of brain limbic structures to the WM process during postnatal development.

Effects of estradiol on incorporation of new cells in the developing zebra finch song system: Potential relationship to expression of ribosomal proteins L17 and L37

Mechanisms regulating masculinization of the zebra finch song system are unclear; both estradiol and sex-specific genes may be important. This study was designed to investigate relationships between estrogen and ribosomal proteins (RPL17 and RPL37; sex-linked genes) that exhibit greater expression in song control nuclei in juvenile males than females. Four studies on zebra finches were conducted using bromodeoxyuridine (BrdU) injections on posthatching days 6-10 with immunohistochemistry for the ribosomal proteins and the neuronal marker HuC/D at day 25. Volumes of brain regions were also assessed in Nissl-stained tissue. Most BrdU+ cells expressed RPL17 and RPL37. The density and percentage of cells co-expressing BrdU and HuC/D was greatest in Area X. The density of BrdU+ cells in Area X (or its equivalent) and the percentage of these cells that were neurons were greater in males than females. In RA and HVC, total BrdU+ cells were increased in males. A variety of effects of estradiol were also detected, including inducing an Area X in females with a masculine total number of BrdU+ cells, and increasing the volume and percentage of new neurons in the HVC of females. The same manipulation in males decreased the density of BrdU+ cells in Area X, total number of BrdU+ cells in RA, and density of new neurons in HVC and RA. These data are consistent with the idea that RPL17, RPL37, and estradiol might all influence sexual differentiation, perhaps with the hormone and proteins interacting, such that an appropriate balance is required for normal development.

Sexual differentiation of the zebra finch song system: potential roles for sex chromosome genes

BackgroundRecent evidence suggests that some sex differences in brain and behavior might result from direct genetic effects, and not solely the result of the organizational effects of steroid hormones. The present study examined the potential role for sex-biased gene expression during development of sexually dimorphic singing behavior and associated song nuclei in juvenile zebra finches.ResultsA microarray screen revealed more than 2400 putative genes (with a false discovery rate less than 0.05) exhibiting sex differences in the telencephalon of developing zebra finches. Increased expression in males was confirmed in 12 of 20 by qPCR using cDNA from the whole telencephalon; all of these appeared to be located on the Z sex chromosome. Six of the genes also showed increased expression in one or more of the song control nuclei of males at post-hatching day 25. Although the function of half of the genes is presently unknown, we have identified three as: 17-beta-hydroxysteroid dehydrogenase type IV, methylcrotonyl-CoA carboxylase, and sorting nexin 2.ConclusionThe data suggest potential influences of these genes in song learning and/or masculinization of song system morphology, both of which are occurring at this developmental stage.

Persistence of male hematopoietic CD34 + cells in the circulation of women does not affect prenatal diagnostic techniques

We aimed to verify whether fetal microchimerism, because of persisting fetal hematopoietic CD34(+) cells from previous pregnancies, could interfere with the development of genetic tests based on using these cells, isolated from maternal blood for the diagnosis of fetal aneuploidies. CD34(+) cells, isolated from blood of parous women with at least 1 son and nulliparous women, were analyzed by using qualitative polymerase chain reaction (PCR), quantitative PCR, and fluorescence in situ hybridization (FISH) to establish whether these molecular techniques are concurrently capable of detecting circulating male DNA. By qualitative PCR, male DNA was found both in parous and nulliparous women, whereas by quantitative PCR and FISH analyses, no male DNA or male nuclei were revealed except in 1 cultured CD34(+) sample from a nulliparous woman. Fetal hematopoietic CD34(+) cells can be used in the noninvasive prenatal testing of fetal aneuploidies because the presence of fetal microchimerism does not affect fetal diagnosis in current pregnancies.

Sex differences and central protective effect of 17β-estradiol in the development of aldosterone/NaCl-induced hypertension

The present study tested the hypotheses that male and female rats respond differently to subcutaneous infusions of aldosterone (Aldo; 1.8 microg.kg(-1).h(-1), 1% NaCl to drink; 28 days) and that central estrogen plays a protective role against the development of hypertension. In rats with blood pressure (BP) and heart rate (HR) measured by Data Sciences International telemetry, chronic Aldo/NaCl treatment induced a greater increase in BP in males (Delta25.4 +/- 2.4 mmHg) than in females (Delta7.1 +/- 2.2 mmHg). Gonadectomy augmented Aldo/NaCl-induced hypertension in females (Delta18.2 +/- 2.0 mmHg) but had no effect in males (Delta23.1 +/- 2.9 mmHg). Immunohistochemistry for Fra-like activity was higher in the paraventricular nucleus of intact males, castrated males, and ovariectomized (OVX) females compared with intact females after 28 days of Aldo/NaCl treatment. In intact males, central 17beta-estradiol (E(2)) inhibited the Aldo/NaCl increase in BP (Delta10.5 +/- 0.8) compared with that in central vehicle plus systemic Aldo/NaCl (Delta26.1 +/- 2.5 mmHg) rats. Combined administration of E(2) and estrogen receptor antagonist ICI182780 (ICI) blocked the protective effect of E(2) (Delta23.2 +/- 2.4 mmHg). In intact females central, but not peripheral, infusions of ICI augmented the Aldo/NaCl (Delta20.4 +/- 1.8 mmHg) BP increase. Finally, ganglionic blockade after Aldo infusions resulted in a smaller reduction in BP in intact females (-23.9 +/- 2.5 mmHg) and in central estrogen-treated males (-30.2 +/- 1.0 mmHg) compared with other groups (intact males, -39.3 +/- 3.4; castrated males, -41.8 +/- 1.9; intact males with central E(2) + ICI, -42.3 +/- 2.1; OVX females, -40.3 +/- 3.3; and intact females with central ICI, -39.1 +/- 1.3 mmHg). Chronic Aldo infusion produced increases in NaCl intake and decreases in HR that were both similar in all groups. Taken together, the results indicate that central estrogen plays a protective role in the development of Aldo/NaCl-induced hypertension and that this may result from reduced sympathetic outflow.