Injection Of Estradiol Research Articles

Effect of inhibiting connexin 43 expression on the expression of rat glial cell growth factors in prolactinomas

Objective To investigate the effect of inhibiting connexin 43 (Cx43) expression on the expression of rat glial cell growth factor in prolactinomas. Methods Forty-eight female rats were randomly assigned to normal, simple estradiol, estradiol+ carbenoxolone, and simple carbenoxolone groups by the completely randomized method (n=12 in each group). A model of rat prolactinoma was induced by estradiol. The normal group did not accept drug treatment; the simple estradiol group was injected estradiol intramuscularly (3 000 mg/kg, once a week, for 13 weeks); 10 weeks after injection of estradiol, the estradiol+ carbenoxolone group injected carbenoxolone via subarachnoid (once a week, for 3 weeks); and the simple carbenoxolone group only injected carbenoxolone for 3 weeks. The changes of body mass of pituitary and histomorphology were observed. Western blot was used to detect the expression levels of Cx43 and glial growth factor (GGF). Results The pituitary weights of the normal, simple estradiol, estradiol+ carbenoxolone and simple carbenoxolone groups were 51.0±0.4 mg, 93.3±1.0 mg, 52.7±2.1 mg and 68.5±1.3 mg, respectively. There were significant differences (F=786.73, P<0.01); the relative expression quantities of Cx43 were 8.0±2.2%, 34.7±4.0, .9.3±3.1%, and 15.3 ±3.8%, respectively, and the relative expression quantities of GGF were 17.7±5.2%, 46.9±1.4%, 15.8±3.6%, and 20.0±2.3%, respectively. There were significant differences (F=1051.51. P<0.01; F=806.58, P<0.01). The pituitary histopathological morphology of the estradiol+ carbenoxolone group was close to the normal group and the simple carbenoxolone group. Conclusions Inhibition of pituitary Cx43 expression may inhibit the GGF expression, thereby inhibiting the growth of pituitary prolactinomas in rats. Key words: Prolactinoma; Gap junction proteins; Gap junction inhibitor; Glial cell growth factor; Rats

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats.

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.

Curiouser and Curiouser: The Evolving Story of the Mechanisms Involved in Puberty.

Sometimes, like a giant jigsaw puzzle, concepts in endocrinology take time to develop, to form a coherent picture of how endocrine systems are integrated to support physiological responses. Such, it can be argued, has been the case with respect to our understanding of the mechanisms underlying puberty. For more than 50 years, it has been known that mammals experience a period of agonadal restraint during development, in which reproductive function is held in check (1). As puberty approaches, this restraint is switched off and pulsatile GnRH secretion is activated. What caused the juvenile period to end remained a matter of conjecture, although a shift in steroid feedback was suggested by observations showing that immature rats had enhanced sensitivity to the negative feedback effects of estradiol (2), whereas the onset of ovarian cyclicity could be advanced by prepubertal injections of estradiol (3). In the past 5 decades, several other important pieces of the puzzle have been filled in. The brain was itself found to be a site of gonadal steroid synthesis (4, 5). Data emerged to indicate that neurosteroid synthesis might contribute to reproductive function (6). Dramatic advances were made in understanding the neurotransmitter and neuropeptide circuitry responsible for regulating the onset of reproductive function, in particular the kisspeptin system (7, 8). However, what actually triggered the change in feedback control of gonadotrophin secretion at puberty remained obscure. A shift from a predominantly inhibitoryhypothalamic environment toone inwhichexcitatory neurotransmitters activated the GnRH neurons was believed to occur (9, 10), but what caused this shift remained unknown. In the current issue of Endocrinology, Kenealy et al (11) develop the novel idea that in female primates, the control of GnRH secretion before and during puberty may actually operate in a relatively similar hypothalamic steroidal milieu: only the source of the estradiol is different. To determine hypothalamic steroid concentrations, perfusates were collected in vivo from the stalk-median eminence (S-ME) region of 32 female rhesus monkeys at different stages of prepubertal and pubertal development. The samples were assayed for estradiol, estrone, testosterone, androstenedione, and progesterone, using a sensitive and specific liquid chromatography-mass spectrometry-based method. Concomitantly, GnRH in the perfusates, as well as LH and steroid levels in circulating serum samples from the same animals, were also assayed. As expected, GnRH and LH levels followed a similar developmental pattern, levels at midpuberty being higher than either prepuberty or early puberty. Circulating estradiol levels also were higher in midpuberty. The steroid measurements in the S-ME perfusates, however, exhibited a strikingly different pattern. Although androstenedione, testosterone, and progesterone did not change significantly in the perfusates across the different stages of puberty, estrone and estradiol were found to be high in the S-ME perfusates from prepubertal animals, declining in midpuberty and early puberty, respectively. Because serum estradiol levels increased over the same period, the S-ME to serum ratio fell dramatically in the midpubertal animals, more than 5-fold in the case of estradiol and more than 60-fold in the case of estrone. These results suggest that although the prepubertal period is indeed characterized by a period of GnRH inhibition that does not depend on ovarian hormone secretion, it is not in fact steroid independent. Rather, it may be mediated at least in part by estradiol produced in situ,

Estradiol enhances the acquisition of lithium chloride-induced conditioned taste aversion in castrated male rats.

The present study examined the effects of short-term treatment with ovarian hormones on the acquisition of conditioned taste aversion (CTA). Adult male rats were castrated and randomly divided into LiCl- and saline-treated groups. Nineteen days after castration, all of the animals were subjected to 23.5-h daily water deprivation for seven successive days (day 1 to day 7). On the conditioning day (day 8), the rats received either a 4 ml/kg of 0.15 M LiCl or the same dose of saline injection immediately after administration of a 2 % sucrose solution during the 30-min water session. Starting from day 6, rats in both groups received one of the following treatments: daily subcutaneous injection of (1) estradiol alone (30 μg/kg; estradiol benzoate (E) group), (2) estradiol plus progesterone (500 μg; E + progesterone (P) group), or (3) olive oil. From day 9 to day 11, all of the rats were given daily two-bottle preference tests during the 30-min fluid session. The estradiol and estradiol plus progesterone treatments in the LiCl groups resulted in significantly lower preference scores for the sucrose solution compared with the olive oil treatment groups, but no difference in preference score was seen between these two groups. These results indicate that both the estradiol and estradiol plus progesterone treatments in the LiCl groups enhanced the acquisition of CTA learning and suggest that estradiol affects the acquisition of CTA mediated by an activational effect in male rats, whereas progesterone treatment does not influence the effects of estradiol on the acquisition of CTA.

Mayor experiencia en el tratamiento del útero hipoplásico con estrógeno al cérvix

El autor presenta su experiencia en el tratamiento de la hipoplasia uterina con inyecciones locales de estradiol. Su grupo estaba formado por 66 pacientes que sufren de esterilidad; la media de edad fue de 29 años; 78% de los casos tenía esterilidad primaria, 19% eran del tipo secundario y el 3% correspondió a la infertilidad. Después que se completó el tratamiento hubo 19 embarazos, esto es 28,7%. Los mejores resultados se obtuvieron en esterilidad primaria y la hipoplasia discreta del útero. 27% de los casos presentó algunos efectos secundarios que fueron bien tolerados. El autor cree que algunos casos de esterilidad debido a la hipoplasia del útero pueden ser tratados con éxito con inyecciones locales de estradiol.

Impact of progesterone and estradiol treatment before the onset of the breeding period on reproductive performance of Bos indicus beef heifers

The present aimed to develop a hormonal therapy based on the insertion of a progesterone (P4) insert (PI) during 10 d plus an estradiol injection (E2) at PI removal before the onset of breeding of beef heifers. In Exp. 1, the plasma P4 profile of prepubertal heifers showed that the insertion of a PI or 24 d previously used P4 insert (UPI) sustained plasma P4 above 1ng/mL for at least the first 7 d of the treatment. In Exp. 2 there was no positive effect of additional estradiol benzoate (EB) administered at the insertion of UPI on the proportion of heifers with a corpus luteum (CL/Treated) 30 d after UPI removal [UPI+EB=85.3%a (n=134); EB+UPI+EB=80.8%a (n=125)]; however, both were greater (P<0.0001) than the Control group [60.3%b (n=129)]. In Exp. 3, a positive effect (P=0.01) of UPI treatment and both E2 supplementations [EB and estradiol cypionate (EC); P=0.10] at the UPI removal was observed on CL/Treated [Control=42.5%b (n=94); UPI=58.5%a (n=130); UPI+EB=64.0%a (n=128); UPI+EC=67.2%a (n=128)]. However, greater pregnancy per treated heifer (P/Treated) following artificial insemination (AI) upon estrus detection was achieved when EC was applied [Control=20.2%b; UPI=29.2%ab; UPI+EB=26.6%b; UPI+EC=36.7%a]. In Exp. 4, the treatment prior to the timed AI (TAI) tended to improve pregnancy per TAI [P/AI; Control 43.6% (n=298) vs. UPI+EC 51.9% (n=342); P=0.08], but increased P/Treated [26.5% vs. 43.3%; P<0.001]. Thus, UPI+EC treatment were efficient in increasing the CL/Treated, tended to improve the P/AI and consequently enhanced P/Treated of zebu beef heifers.

Effect of Saraca asoca (Asoka) on estradiol-induced keratinizing metaplasia in rat uterus.

Estrogen-mediated uterus endometrium instability is considered as one of the etiological factors in dysfunctional uterine bleeding (DUB) and uterine cancer. Saraca asoca (Family: Fabaceae) and its fermented preparation, Asokarishta, are extensively used as uterine tonic to treat gynecological disorders in Ayurveda. The present study evaluated the effect of S. asoca (Asoka) on estrogen-induced endometrial thickening of rat uterus. Endometrial thickening was induced by intraperitoneal injection of estradiol (20 μg/kg b.wt) to 8-day-old immature rats for alternate 5 days. Methanolic extract (200 mg/kg b. wt) from S. asoca bark was given orally along with estradiol. Uterus endometrial thickening was analyzed histopathologically and serum estrogen level by radioimmunoassay (RIA). Cyclooxygenase (COX-2) expression in rat uterus was also estimated by Western blot. Anti-inflammatory activity of the extract was analyzed by formalin- and carrageenan-elicited paw edema models in mouse. Uterus endometrium proliferation and keratinized metaplasia with seven to eight stratified epithelial layers on day 16 was observed in rats administered with estradiol. Treatment with S. asoca reduced the thickening to two to four layers and the serum estrogen level diminished significantly to 82.9±12.87 pg/mL compared to rats administered with estrogen alone (111.2±10.68 pg/mL). A reduction of formalin- and carrageenan-induced paw edema in mouse by S. asoca extract was observed. Lower level of lipopolysaccharides (LPS)-induced COX-2 enzyme in rat uterus by the extract further confirms its anti-inflammatory activity. Present study reveals the antiproliferative and antikeratinizing effects of S. asoca in uterus endometrium possibly through its anti-estrogenic and anti-inflammatory properties.

Estradiol modulates visceral hyperalgesia by increasing thoracolumbar spinal GluN2B subunit activity in female rats.

We previously reported estrogen modulates spinal N-methyl-d-aspartate (NMDA) receptor processing of colorectal pain through changes in spinal GluN1 subunit phosphorylation/expression. The purpose of this study was to investigate whether spinal GluN2B containing NMDA receptors are involved in estrogen modulation of visceral pain processing. Behavioral, molecular, and immunocytochemical techniques were used to determine spinal GluN2B expression/phosphorylation and function 48h following subcutaneous injection of estradiol (E2) or vehicle (safflower oil, Saff oil) in ovariectomized rats in the absence or presence of colonic inflammation induced by mustard oil. E2 increased the magnitude of the visceromotor response (VMR) to colorectal distention compared to Saff oil in non-inflamed rats. Intrathecal injection of the GluN2B subunit antagonist, Ro 25-6981, had no effect on the VMR in non-inflamed E2 or Saff oil rats. Colonic inflammation induced visceral hyperalgesia in E2, but not Saff oil rats. Visceral hyperalgesia in E2 rats was blocked by intrathecal GluN2B subunit selective antagonists. In inflamed rats, E2 increased GluN2B protein and gene expression in the thoracolumbar (TL), but not lumbosacral (LS), dorsal spinal cord. Immunocytochemical labeling showed a significant increase in GluN2B subunit in the superficial dorsal horn of E2 rats compared to Saff oil rats. These data support the hypothesis that estrogen increases spinal processing of colonic inflammation-induced visceral hyperalgesia by increasing NMDA receptor activity. Specifically, an increase in the activity of GluN2B containing NMDA receptors in the TL spinal cord by estrogen underlies visceral hypersensitivity in the presence of colonic inflammation.

Neonatal estrogen exposure results in biphasic age-dependent effects on the skeletal development of male mice.

Peak bone mass, one of the most important predictors for fracture risk later in life, is attained during puberty and adolescence and influenced by neonatal and pubertal sex-specific gonadal hormones and GH-IGF-I secretion patterns. This study examined the effects of brief neonatal estrogen (NE) exposure on growth and skeletal development in C57BL/6J mice. A single injection of 100-μg estradiol or vehicle was administered on the first day of life. Growth parameters were monitored and skeletal phenotyping performed at 16 weeks in female mice and at 4 and 16 weeks in male mice. NE exposure negatively impacted adult femoral length in both sexes, but adult body weight, areal bone density, and bone strength in female mice were unaffected. In contrast, somatic growth was attenuated in estrogen-exposed male mice throughout the study period. At the prepubertal time point, the estrogen-exposed males exhibited higher bone mineral density, cortical volume, and cortical thickness compared with controls. However, by the time of peak bone mass acquisition, the early skeletal findings had reversed; estrogen-exposed mice had lower bone density with reduced cross-sectional area, cortical volume, and cortical thickness, resulting in cortical bones that were less resistant to fracture. NE exposure also resulted in reduced testicular volume and lower circulating IGF-I. Male mice exposed to estrogen on the first day of life experience age-dependent changes in skeletal development. Prepubertal animals experience greater endocortical bone acquisition as a result of estrogen exposure. However, by adulthood, continued developmental changes result in overall reduced skeletal integrity.

The effect of ghrelin and estradiol on mean concentration of thyroid hormones.

Background:Ghrelin is a novel peptide hormone that has GH releasing activity and also other endocrine and metabolic functions. It can also increase food intake and decrease T3 and T4 concentrations. Several parameters of hypothalamic-pituitary-thyroid (HPT) axis function are modulated by 17β-estradiol (E2).Objectives:The purpose of this study was to investigate the effect of interactions between ghrelin and estradiol (injected via ICV route) on plasma T3 and T4 concentrations in female rats.Materials and Methods:Eighteen Wistar female rats (bodyweight, 200-250 g) were randomly divided into three groups. Group 1 received estradiol, Group 2 received ghrelin and Group 3 received ghrelin and estradiol. Plasma samples were used to assess T3 and T4 concentration by RIA.Results:The results indicated that ghrelin significantly decreased thyroid hormone concentrations, whereas estradiol increased these concentrations. The simultaneous injection of ghrelin and estradiol significantly decreased the inhibitory effect of ghrelin on thyroid hormone concentrations (P < 0.05).Conclusions:According to the results of this study, both ghrelin and estradiol affect the concentration of thyroid hormone but in opposite directions. This difference might be due to different underlying hormonal mechanisms such as HPA and/or HPT axis melanocyte stimulating hormone (MSH) systems could be suggested.

Genistein Stimulates Jejunum Chloride Secretion via an Akt-Mediated Pathway in Intact Female Mice

Background/Aims: We have previously shown that daily subcutaneous injections with the naturally occurring phytoestrogen genistein (600 mg genistein/kg body weight/day, 600G) results in a significantly increased basal intestinal chloride, Cl^-, secretion (I_sc, a measure of transepithelial secretion) in intact C57BL/6J female mice after 1-week of treatment, compared to controls (DMSO vehicle injected). Removal of endogenous estrogen via ovariectomy (OVX) had no effect on the 600G-mediated increase in basal I_sc. Methods: Given the estrogen-like characteristics of genistein, we compared the effects of daily estradiol (E2) injections (10 mg E2/kg body weight/day, 10E2) on basal I_sc in intact and OVX mice. In intact mice, 10E2 was without effect on basal I_sc, however, in OVX mice, 10E2 significantly increased basal I_sc (mimicked 600G). The goal of the current study was to characterize the intracellular signaling pathways responsible for mediating 600G- or 10E2-stimulated increases in basal I_sc in intact female or OVX mice. Results: We measured total protein expression in isolated segments of jejunum using western blot from the following six groups of mice; intact or OVX with; 600G, 10E2 or control. The proteins of interest were: Akt, p-Akt, p-PDK1, p-PTEN, p-c-Raf, p-GSK-3β, rap-1 and ERK1/2. All blots were normalized to GAPDH levels (n = 6-18/group). Conclusion: These data suggest that the presence of the endogenous sex steroid, estrogen, modifies the intracellular signaling pathway required to mediate Cl^- secretion when the intestine is exposed to exogenous 600G or E2. These studies may have relevance for designing pharmacological tools for women with intestinal chloride secretory dysfunctions.

Cyclic estradiol treatment modulates the orexigenic effects of ghrelin in ovariectomized rats

Data from a wide variety of mammalian species indicate that feeding behavior can be influenced by changes in endogenous estrogens and exogenous estrogenic treatments. Ghrelin is an important physiological signal for the regulation of energy balance, and ghrelin treatment increases eating and body weight in male rodents. The following studies evaluated the hypothesis that the inhibitory effects of estradiol on feeding involve interactions with orexigenic peptides by examining the ability of estradiol to modulate the behavioral effects of ghrelin in female rats. In these experiments, adult rats were ovariectomized and assigned to an estradiol benzoate (EB) or an oil (control) group. Three weeks after ovariectomy, animals received two daily subcutaneous injections of EB or the oil vehicle. Animals then received intraperitoneal (ip) injections of ghrelin (6.0 or 12.0nmol) or saline during the nocturnal and diurnal periods three days after the first injection of estradiol or oil. Food intake, meal size, and meal number were determined during the 2-hour period following ghrelin or saline treatments. Ghrelin significantly increased food intake during nocturnal tests in oil-treated but not estradiol-treated rats. The hyperphagic effects of ghrelin on nocturnal food intake were also accompanied by an increase in meal size, and this effect of ghrelin on meal size was attenuated in estradiol-treated females. These findings support the hypothesis that the effects of estradiol on feeding behavior involve an attenuation of orexigenic signals, possibly by modulating the effects of the peripheral ghrelin signal on hypothalamic neuropeptides involved in the control of food intake.

Synchronization of follicular wave emergence following ultrasound-guided transvaginal follicle ablation or estradiol-17β administration in water buffalo (Bubalus bubalis)

The aim of this study was to assess the synchrony in follicular wave emergence and subsequent ovulation following dominant follicle ablation or estradiol-17β administration. Six cycling Murrah buffaloes were sequentially allotted to three groups, that is, control, follicular ablation, and estradiol-17β groups. For the control group, buffaloes at random stages of estrous cycle were examined daily by transrectal ultrasonography for 14 days and the day of wave emergence was recorded. Following induced luteolysis and ovulation (Day 0), these buffaloes were included in the ablation group. All follicles (>5mm) were ablated on Day 3 or 5 or 7 (n=2 each day). Seven days after the ablation, these buffaloes were administered prostaglandin F2α to induce luteolysis and ovulation. Following this, buffaloes were included in the estradiol treatment group with estradiol administered on similar days as for ablation in the ablation group. Luteolysis was induced nine days after the estradiol injection. All animals of the treatment groups were subjected to transrectal ultrasound and blood samplings daily from treatment to induced ovulation. The follicular waves emerged significantly earlier (P=0.001) in both the ablation (2.1±0.79 days) and estradiol (4.0±0.25 days) treatment groups than the control group (8.3±0.88 days). The deviation from mean day of ovulation was greater (P=0.02) for the control group buffaloes (1.66±0.3 day) than those of the treatment groups (ablation, 0.76±0.2 and estradiol, 0.58±0.2 day). In conclusion, both ablation and estradiol resulted in synchronous emergence of a new follicular wave irrespective of stage at which the treatment was given, with greater synchrony of ovulations in water buffalo.

Role of pregnane xenobiotic receptor in the midbrain ventral tegmental area for estradiol- and 3α,5α-THP-facilitated lordosis of female rats

Progesterone and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), have actions in the ventral tegmental area (VTA) that are required for lordosis, a characteristic mating posture of female rodents. 17β-estradiol (estradiol) co-varies with progestogens over natural cycles, enhances production of 3α,5α-THP, and is required for successful reproductive behavior. A question of interest is the role of pregnane xenobiotic receptor (PXR), a nuclear receptor that regulates enzymes needed for the production of 3α,5α-THP, for estradiol-mediated lordosis. The hypothesis tested was that if PXR is involved in estradiol-mediated biosynthesis of 3α,5α-THP and reproductive behavior, knocking down expression of PXR in the VTA of estradiol-primed, but not vehicle-primed, rats should decrease lordosis and midbrain 3α,5α-THP; effects may be attenuated by 3α,5α-THP administered to the VTA. Ovariectomized rats were administered subcutaneous injections of oil vehicle or estradiol. Rats were then administered PXR antisense oligonucleotides (PXR AS-ODNs; which are expected to locally knock down expression of PXR), or control (saline), infusions to the VTA. Rats were administered 3α,5α-THP or vehicle via infusions to the VTA. Reproductive behavior (paced mating task) of rats was determined in addition to exploratory (open field), affective (elevated plus maze), and pro-social (social interaction task) behavior. Reproductive behavior (i.e., increased lordosis) was enhanced with estradiol-priming and infusions of 3α,5α-THP to the VTA. Infusions of PXR AS-ODNs to the VTA attenuated responses in estradiol-, but not vehicle-, primed rats, compared to control infusions. PXR may be involved in a neuroregulatory response involving biosynthesis of 3α,5α-THP in the midbrain VTA of estradiol-primed rats.

MicroRNA Let-7a and dicer are important in the activation and implantation of delayed implanting mouse embryos

Does Let-7a have a functional role in modulating dicer expression to activate dormant mouse blastocysts for implantation? Let-7a post-transcriptionally regulates dicer expression altering microRNA expression to affect the implantation competency of the activated blastocysts. The Let-7a microRNA is up-regulated during blastocyst dormancy and its forced-expression suppresses embryo implantation in vitro and in vivo. Dicer is a Let-7 target, which processes pre-microRNA to mature microRNA. The effects on the expression of Let-7a and dicer in dormant blastocysts during the first 12 h after estradiol-induced activation, and the relationship between Let-7a and dicer in preimplantation embryos were determined. The effects on the microRNA expression and embryo implantation in vivo in dicer-knockdown mouse 5-8 cell embryos and dormant blastocysts at 1 h post estradiol activation were also studied. ICR female mice at 6 weeks of age were ovariectomized on Day 4 of pregnancy to generate the delayed implantation model. Mouse 5-8 cell embryos and/or dormant blastocysts at 1 h after estradiol injection were electroporated with dicer siRNA and Let-7a precursor or Let-7a inhibitor. At 48 h post electroporation, the Let-7a expression, dicer transcripts and proteins in the embryos were determined using qPCR and immunostaining/western blotting, respectively. All experiments were repeated at least three times. Estradiol injection down-regulated Let-7a and up-regulated dicer in the dormant blastocysts during the first 12 h post-activation. Dicer knockdown at 1 h post-activation of blastocysts suppressed EGFR expression, attenuated EGF binding and compromised implantation of the transferred embryos. Let-7a transcriptionally regulated dicer by binding to the 3'-UTR of dicer in trophoblast cells. Dicer knockdown in blastocysts suppressed mature Let-7a expression and compromised implantation. Gain- and loss-of-function approaches were used by analyzing transient expressions of transfected microRNA modulators or genes. The consequence of the Let-7a-dicer interaction on pregnancy remains to be determined. The study used the mouse as a model and the applicability of the observed phenomena in humans warrants further investigation. Our results indicate that the Let-7a-dicer interaction leads to differential microRNA expression in dormant blastocysts after estradiol activation. Because the expression pattern of Let-7a in human blastocysts is similar to that in mouse blastocysts, our observation that the Let-7a-dicer interaction has a role in regulating the implantation potential of the mouse blastocysts could be applicable to humans. This project is supported partly by a research grant from the Research Grant Council to W.S.B.Y. The authors have no competing interests to declare.

Estrogen potentiates the behavioral and nucleus accumbens dopamine response to continuous haloperidol treatment in female rats

Estrogen has been shown to enhance the effects of antipsychotics in humans. To investigate the mechanisms of how this may occur, the current study examined estradiol's effects on dopaminergic transmission and behavior in amphetamine-sensitized and non-sensitized female rats. Sixty-four ovariectomized female Sprague-Dawley rats were used for this study. Half of the rats were sensitized to four once-daily injections of 1mg/kg amphetamine and the other half served as controls. Rats received chronic administration of either low-dose haloperidol (0.25mg/kg/day) or saline vehicle via osmotic minipumps implanted subcutaneously. The groups were further subdivided with respect to estradiol treatment: low chronic estrogen (subcutaneous estradiol implant, 0.36mg/pellet: 90-day release, plus an additional oil vehicle injection every second day) and high pulsatile estrogen (subcutaneous estradiol implant plus an additional 10μg/kg estradiol injection every second day). Motor activity was assessed at day2 and day12 during haloperidol treatment, while nucleus accumbens dopamine availability was assessed via microdialysis 10days into antipsychotic treatment. Haloperidol treatment along with high, but not low, estradiol replacement was effective in reducing amphetamine-induced locomotor activity in sensitized rats. High estradiol treatment also augmented the effects of chronic haloperidol in reducing dopaminergic release in sensitized rats. These data suggest that estradiol levels affect both the behavioral and the dopamine responses to chronic antipsychotic treatment.

Adolescent cannabinoid treatment negatively affects reproductive behavior in female rats

Sex differences in the neurobehavioral effects of chronic cannabinoid exposure suggest that gonadal hormones may modify cannabinoid activity. The current experiment assessed the impact of combined cannabinoid and estradiol treatment in ovariectomized, adolescent female rats on subsequent adult sexual behavior. Female Long-Evans rats were administered daily injections of either the cannabinoid agonist, CP 55,940 (0.4mg/kg), or vehicle from post-natal day (PND) 40–49. Half of each treatment group also received daily injections of estradiol (50μg/kg) or sesame oil vehicle from PND 39–49. Beginning on PND 74, subjects' reproductive behavior, including receptivity, proceptivity, and pacing, was assessed via a fifteen-minute paced mating test. There were significant main effects of estradiol treatment on rate of hop-darts emitted, number of arena crossings, and ejaculations received during the paced mating test. Overall, estradiol-treated females produced higher and more typical levels of sexual behavior. There were significant interactions between hormone and cannabinoid treatment on lordosis quotient, percentage of exits following male sexual stimulation, and contact-return latency for intromissions. Adolescent cannabinoid treatment detrimentally impacted the display of female receptivity and pacing, but only within estradiol-treated subjects. We discuss possible interpretations of these results, including potential mechanisms by which adolescent cannabinoid exposure might disrupt the normal development of female reproductive behavior.

Effect of subcutaneous injection of estradiol on feeding and drinking behaviors and body weight in basolateral amygdaloid lesioned rats.

Estradiol is known to inhibit food intake (FI), water intake (WI) and body weight (BW) across the species including women and it is most evident in rats. Ovariectomy in rats and menopause in women produce hyperphagia and obesity. Estradiol substitution in ovariectomized (OVX) rats and hormone replacement in women reverses these changes suggesting that lack of estradiol causes eating related disorders. However, the neurobiological target/s for estradiol mediating effects remains largely unknown. While lesions of basolateral amygdala (BLA) also produce hyperphagia, polydipsia and obesity in female rats suggesting BLA normally inhibits these behaviors. Since ovariectomy is a useful model to study postmenopausal obesity in women, we have investigated the role of BLA in ovariectomy induced ingestive behaviors. Ovariectomy and stereotaxic lesions in experimental group (n = 6) whereas sham operations in control group (n = 6) were carried out in female rats. Estradiol was injected subcutaneously (s.c) before and after lesions in experimental group and vehicle was injected in control group. Data from the present study shows that there was an additional increase in FI, WI and BW in OVX animals following BLA lesions, but this additive effect was small compared to sham operated controls. Conversely, OVX rats with lesions have shown small but significant reductions in FI, WI, and lost less BW, following s.c injection of estradiol compared to rats with intact BLA. These findings suggest that ovariectomy and estradiol induced changes on ingestive behaviors and body weight are partly mediated via BLA.

Estrogenic regulation of NADPH-diaphorase in the supraoptic and paraventricular nuclei under acute osmotic stress

Estrogen receptors (ERs) α and β are involved in the regulation of the nitrergic system in the supraoptic (SON) and paraventricular (PVN) nuclei under basal conditions. In this study we have assessed whether ERs are also involved in the modulation of the nitrergic system in the SON and PVN under acute systemic hypertonic conditions. Adult ovariectomized rats received a single injection of either estradiol, a selective ERα agonist, a selective ERβ agonist, a selective ERα antagonist, a selective ERβ antagonist or vehicle. Twenty-four hours later, animals received one i.p. injection of 1.5M NaCl to induce osmotic stress and were sacrificed after two additional hours. The number of NADPH-diaphorase-positive cells in the SON and PVN was determined. Their number in the SON was not affected by NaCl administration, whereas in the four PVN subdivisions it was decreased after NaCl administration. Estradiol and the ERα agonist prevented the action of NaCl in the four subdivisions of the PVN. In contrast, the inhibition of ERα enhanced the effect of NaCl, inducing a further decrease in the number of NADPH-diaphorase-positive cells. Moreover, the ERβ agonist enhanced and the ERβ antagonist blocked the effect of NaCl on the number of NADPH-diaphorase-positive neurons in the SON and in the medial magnocellular subdivision of the PVN. These findings indicate that estradiol regulates the nitrergic system in the SON and PVN under acute osmotic stress conditions, but the effects specifically depend on the anatomical subregions and different ERs.

Role of calbindin-D28K in estrogen treatment for Parkinson's disease.

Studies have shown that estrogen has neuroprotective effects on the nigrostriatal system. The present study established a Parkinson's disease model in C57BL/6 mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrapyridine. The mice were subjected to 17β estradiol injection into the lateral ventricle. Immunofluorescence double staining showed that estrogen increased tyrosine hydroxylase and calbindin-D28K expression and co-expression in dopaminergic neurons of midbrain substantia nigra pars compacta of model mice. Behavior experiments showed that estrogen improved swimming and hanging behaviors in this mouse model of Parkinson's disease.