Receptivity In Rats Research Articles

Noninvasive and Local Delivery of Adenoviral-Mediated Herpes Simplex Virus Thymidine Kinase to Treat Glioma Through Focused Ultrasound-Induced Blood-Brain Barrier Opening in Rats.

Adenoviral-mediated gene therapy has been shown great prospects for tumor treatment. However, it is still a great challenge for its application in the glioma. A main cause is the blood-brain barrier (BBB) limits the delivery of adenoviral vectors, greatly compromising their efficacy. Here, we used focused ultrasound (FUS) induced microbubble cavitation to locally and reversibly open BBB for enhancing gene delivery. In this study, Ad-CMV-TK-IRES-EGFP (Ad-HSV-TK-EGFP) carrying the herpes simplex virus thymidine kinase (HSV-TK) and EGFP transgenes was chose as gendicine and Ad-CMV-IRES-EGFP (Ad-EGFP) as the control. The in vitro experiments showed that Ad-HSV-TK-EGFP had a high infection efficiency for C6 glioma cells, producing good tumor cell killing effects when these cells were exposed to more than 10 μg/ml ganciclovir (GCV). Taking advantage of FUS-induced BBB opening, Ad-HSV-TK-EGFP could be effectively delivered into the brain tumors, getting the overexpression of HSV-TK gene in the tumor cells. After exposure to GCV, the significantly stronger anti-tumor efficacy and longer survival time were observed in tumor-bearing mice treated with Ad-HSV-TK-EGFP + FUS than those treated with Ad-EGFP + FUS or only Ad-HSV-TK-EGFP. Histological examination indicated that the reduced expression level of Ki67 proteins and the increased apoptotic tumor cells in tumor xenografts, causing the inhibition of tumor growth. In conclusion, our study provided a new strategy to efficiently and locally deliver recombinant adenoviral vector-mediated HSV-TK gene into the brain to treat glioma.

Effects of non-contingent cocaine on 3 alpha-androstanediol. II. Disruption of lordosis of proestrous rats

Drug use influences sexual behavior, performance, and can be associated with increased sexual risk-taking. Our prior results using an animal model indicate that progestogens contribute to hormonally-mediated changes in sexual behavior of female rodents during acute cocaine exposure. Androgens, such as testosterone, and its metabolite 3ɑ-androstanediol (3α-diol), and estradiol, are known to influence male sexual behavior, but can also alter the expression of sexual behavior of female rodents. As such, we investigated the influence of endogenous androgen and estradiol fluctuations on cocaine-mediated changes in motor behavior and sexual receptivity of rats during diestrous or proestrous phases of the estrous cycle. Female rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug administration, and then sexual responding was assessed for 15min. Cocaine decreased aggressive behavior in response to attempted mounts by a male among non-receptive (diestrous) rats and inhibited sexual behavior among sexually receptive (proestrous) rats. Cocaine dose-dependently altered concentrations of testosterone metabolites (estradiol and 3α-diol), but not testosterone, which correlated to motor and sexual behaviors of diestrous and proestrous rats, respectively. These data suggest that actions of 3α-diol may be involved in female sexual and motor behavior in response to cocaine, in a cycle-dependent manner.

PEGylated PLGA-based phase shift nanodroplets combined with focused ultrasound for blood brain barrier opening in rats

Previous studies have shown that focused ultrasound (FUS) combined with systematic administration of microbubbles (MBs) can open the blood brain barrier (BBB) locally, transiently and reversibly. However, because of the micro size diameters, MBs are restricted in the intravascular space and cannot extravasate into diseased sites through the opened BBB. In this study, we fabricated one kind of nanoscale droplets which consisted of encapsulated liquid perfluoropentane cores and poly (ethyleneglycol) - poly (lactide-co-glycolic acid) shells. The nanodroplets had the capacity to realize liquid to gas phase shift under FUS. Significant extravasation of Evan's blue appeared when acoustic pressure reached 1.0 MPa. Intracerebral hemorrhages and erythrocyte extravasations were observed when the pressure was increased to 1.5 MPa. Prolonged sonication duration could enhance the level of BBB opening and broaden the time window simultaneously. Furthermore, compared with MBs, the distribution of EB extravasation was firmly confined within narrow region in the center of focal zone, suggesting the site of FUS induced BBB opening could be controlled with high precision by this procedure. Our results show the feasibility of serving PEGylated PLGA-based phase shift nanodroplet as an effective alternative mediating agent for FUS induced BBB opening.

Intravitreal bevacizumab alters type IV collagenases and exacerbates arrested alveologenesis in the neonatal rat lungs

ABSTRACTPurpose/Aim: Intravitreal bevacizumab (Avastin) is an irreversible vascular endothelial growth factor (VEGF) inhibitor used off-label to treat severe retinopathy of prematurity in extremely low gestational age neonates. VEGF and matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) participate in lung maturation. We tested the hypothesis that intravitreal bevacizumab enters the systemic circulation and has long-lasting effects on lung MMPs. Materials and Methods: Neonatal rats were exposed to: (1) hyperoxia (50% O2); (2) intermittent hypoxia (IH) (50% O2 with brief episodes of 12% O2); or (3) room air (RA) from birth (P0) to P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected into the vitreous cavity of the left eye. A control group received equivalent volume saline. At P23 and P45, lung MMP-2 and MMP-9, and TIMP-1, and TIMP-2 were assessed in the lungs. Results: At P23, Avastin increased MMP-2, MMP-9, and TIMP-1 levels in the hyperoxia group but decreased TIMP-1 levels in the IH group. The ratios of MMP-2/TIMP-1 and MMP-9/TIMP-1 were significantly elevated at P23 in the IH group treated with Avastin. At P45, the levels of MMP-2 and MMP-9 remained elevated in the hyperoxia and IH groups treated with Avastin, while a rebound increase in TIMP-1 levels was noted in the IH group. Conclusions: Avastin treatment in IH has lasting alterations in the balance between MMPs and their tissue inhibitors. These changes may lead to impaired alveologenesis and tissue damage consistent with bronchopulmonary dysplasia/chronic lung disease.

Ocular Adverse Effects of Intravitreal Bevacizumab Are Potentiated by Intermittent Hypoxia in a Rat Model of Oxygen-Induced Retinopathy.

Intravitreal bevacizumab (Avastin) use in preterm infants with retinopathy of prematurity is associated with severe neurological disabilities, suggesting vascular leakage. We examined the hypothesis that intermittent hypoxia (IH) potentiates intravitreal Avastin leakage. Neonatal rats at birth were exposed to IH from birth (P0)–P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected intravitreally into the left eye. Animals were placed in room air (RA) until P23 or P45 for recovery (IHR). Hyperoxia-exposed and RA littermates served as oxygen controls, and equivalent volume saline served as the placebo controls. At P23 and P45 ocular angiogenesis, retinal pathology and ocular and systemic biomarkers of angiogenesis were examined. Retinal flatmounts showed poor peripheral vascularization in Avastin-treated and fellow eyes at P23, with numerous punctate hemorrhages and dilated, tortuous vessels with anastomoses at P45 in the rats exposed to IH. These adverse effects were associated with robust increases in systemic VEGF and in both treated and untreated fellow eyes. Histological analysis showed severe damage in the inner plexiform and inner nuclear layers. Exposure of IH/IHR-induced injured retinal microvasculature to anti-VEGF substances can result in vascular leakage and adverse effects in the developing neonate.

Peripheral and Central Mechanisms Involved in the Hormonal Control of Male and Female Reproduction.

Reproduction involves the integration of hormonal signals acting across multiple systems to generate a synchronised physiological output. A critical component of reproduction is the luteinising hormone (LH) surge, which is mediated by oestradiol (E2 ) and neuroprogesterone interacting to stimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in rats. Recent evidence indicates the involvement of both classical and membrane E2 and progesterone signalling in this pathway. A metabolite of gonadotrophin-releasing hormone (GnRH), GnRH-(1-5), has been shown to stimulate GnRH expression and secretion, and has a role in the regulation of lordosis. Additionally, gonadotrophin release-inhibitory hormone (GnIH) projects to and influences the activity of GnRH neurones in birds. Stress-induced changes in GnIH have been shown to alter breeding behaviour in birds, demonstrating another mechanism for the molecular control of reproduction. Peripherally, paracrine and autocrine actions within the gonad have been suggested as therapeutic targets for infertility in both males and females. Dysfunction of testicular prostaglandin synthesis is a possible cause of idiopathic male infertility. Indeed, local production of melatonin and corticotrophin-releasing hormone could influence spermatogenesis via immune pathways in the gonad. In females, vascular endothelial growth factor A has been implicated in an angiogenic process that mediates development of the corpus luteum and thus fertility via the Notch signalling pathway. Age-induced decreases in fertility involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally, morphological changes in the arcuate nucleus of the hypothalamus influence female sexual receptivity in rats. The processes mediating these morphological changes have been shown to involve the rapid effects of E2 controlling synaptogenesis in this hypothalamic nucleus. In summary, this review highlights new research in these areas, focusing on recent findings concerning the molecular mechanisms involved in the central and peripheral hormonal control of reproduction.

The Development of the Head Direction System before Eye Opening in the Rat

SummaryHead direction (HD) cells are neurons found in the hippocampal formation and connected areas that fire as a function of an animal’s directional orientation relative to its environment [1, 2]. They integrate self-motion and environmental sensory information to update directional heading [3]. Visual landmarks, in particular, exert strong control over the preferred direction of HD cell firing [4]. The HD signal has previously been shown to appear adult-like as early as postnatal day 16 (P16) in the rat pup, just after eye opening and coinciding with the first spontaneous exploration of its environment [5, 6]. In order to determine whether the HD circuit can begin its organization prior to the onset of patterned vision, we recorded from the anterodorsal thalamic nucleus (ADN) and its postsynaptic target in the hippocampal formation, the dorsal pre-subiculum (PrSd), before and after eye opening in pre-weanling rats. We find that HD cells can be recorded at the earliest age sampled (P12), several days before eye opening. However, this early HD signal displays low directional information content and lacks stability both within and across trials. Following eye opening, the HD system matures rapidly, as more cells exhibit directional firing, and the quality and reliability of the directional signal improves dramatically. Cue-rotation experiments show that a prominent visual landmark is able to control HD responses within 24 hr of eye opening. Together, the results suggest that the directional network can be organized independently of visual spatial information while demonstrating the importance of patterned vision for accurate and reliable orientation in space.

Microvascular density and vascular endothelial growth factor and osteopontin expression during the implantation window in a controlled ovarian hyperstimulation rat model.

Vascular endothelial growth factor (VEGF) and osteopontin (OPN) are suggested to facilitate angiogenesis and vascular remodeling in endometrial receptivity. Determination of the endometrial microvascular density (MVD) is the commonest method used to indirectly assess the levels of vasculogenesis and angiogenesis; however, the associations among VEGF, OPN and MVD remain unclear. Controlled ovarian hyperstimulation (COH) with the gonadotrophin-releasing hormone agonist-long protocol may impair endometrial receptivity, and Traditional Chinese Medicine (TCM) may exert therapeutic effects to relieve this impairment. The aim of the present study was to investigate the effects of COH on implantation biology and pregnancy outcome, and to explore the potential therapeutic role of the TCM Zi Dan Yin (ZDY). Female Sprague Dawley rats were divided into four groups: Control, COH, ZDY and COH + ZDY. On days 3, 4 and 5 of pregnancy (D3, D4 and D5, respectively), endometrial MVD was measured with cluster of differentiation 34 immunohistochemical detection, and VEGF and OPN protein and mRNA expression was detected through western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. On D10, the average number of implantation sites was observed. Subsequent to conceiving and bearing newborn rats, the number of live births from each group was recorded. COH was shown to have adverse effects on implantation and pregnancy outcome. The MVD was found to be significantly increased in the COH group compared with that in the control, ZDY and COH+ZDY groups. The results of the protein and RT-qPCR analysis of VEGF and OPN revealed the same trend. Conversely, ZDY reversed the changes in endometrial MVD, VEGF and OPN, and was indicated to improve uterine receptivity and pregnancy outcome. No significant difference was observed among the control, ZDY and COH + ZDY groups. In conclusion, since the results for MVD and VEGF and OPN expression were consistent, MVD could be used as an alternative approach to identify the period of receptivity in rats.

Sociosexual behaviors during the transition from non-receptivity to receptivity in rats housed in a seminatural environment

Female behavioral estrus is defined as the period between the first lordosis displayed during the estrus cycle to the lordosis that is not followed by another within 60min. In a seminatural environment, an estrous female consistently displays lordosis in response to every male mount from the start of behavioral estrus until the end of it. This means that the female suddenly changes from a state of complete non-receptivity to full receptivity and then abruptly changes back to non-receptivity. It is unlikely that these abrupt changes are caused by sudden changes in serum concentration of ovarian steroids. Here, we present the results of a detailed study of sociosexual behaviors during the transition from non-receptivity to receptivity and vice versa. The frequency of male mounting was close to zero before and after estrus. It remained at a constant, high level throughout estrus. Female paracopulatory behavior and male pursuit of the female increased drastically from a very low level before estrus to a high level during estrus. They returned to low levels immediately after estrus. None of the many other behavior patterns registered changed during the transitions. It appears that the sudden increase in male pursuit and female paracopulatory behavior can explain the beginning of behavioral estrus, and their equally sudden disappearance causes it to end. The neurochemical mechanisms behind these almost instantaneous behavioral changes are unknown.

Alteration of endometrial receptivity in rats with ovarian hyperstimulation syndrome

It is unclear if the higher pregnancy rate in patients who experience ovarian hyperstimulation syndrome (OHSS) indicates that OHSS is favourable for embryo implantation, or if patients should be maintained in a hyperstimulation state in order to increase the success rate of embryo transplantation. We developed an animal model to determine the endometrial receptivity in rats with OHSS. Endometrial mRNA levels of ER, PR, HOXA10 and LIF were determined by semi-quantitative PCR and ER, PR, HOXA10, LIF and integrin αv β3 protein levels were determined by Western blotting. Development of pinopodes in the hyperstimulation group was slightly delayed, while in the regular stimulation group, development was significantly inhibited. mRNA transcription in the regular stimulation group was lower, while transcription in the hyperstimulation group was not different from controls. Protein expressions were lowest in the regular stimulation. We conclude that OHSS is associated with favourable endometrial receptivity, similar to that seen in a normal cycle, and receptivity that is increased relative to that seen with a routine stimulation protocol.

Platelet activating factor induces transient blood‐brain barrier opening to facilitate edaravone penetration into the brain

The blood-brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra-performance liquid chromatograph combined with a hybrid quadrupole time-of-flight mass spectrometer (UPLC-MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to (125)I-albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule-1, matrix-metalloproteinase-9 and P-glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain. Platelet activating factor (PAF) transiently induces BBB dysfunction and increases BBB permeability, which may be due to vessel contraction and a temporary decline of regional cerebral blood flow (rCBF) triggered by PAF. More importantly, the PAF induced transient BBB opening facilitates neuroprotectant edaravone penetration into brain. The results of this study may provide a new approach to improve drug delivery into the brain.

Bimodal binding and free energy of the progesterone receptor in the induction of female sexual receptivity by progesterone and synthetic progestins

Synthetic progestins (SPs) are used for regulation of fertility, contraception and hormone replacement therapy. The acetylated medroxyprogesterone (MPA), megestrol (MGA) and chlormadinone (CLA) are related to progesterone (P). Other SPs are 19-nortestosterone derivatives such as: norethisterone (NET), norethynodrel (NED) or the 13-ethyl gonane, levonorgestrel (LNG). We studied MPA, NET, NED and LNG in a dose–response manner to induce sexual receptivity in rats. Results showed that MPA, NET and NED act as partial agonists, with similar or lower potency than P. However, LNG is a full agonist. Additionally, the molecules of MPA, MGA, CLA, NET, NED, LNG, and P, were submitted to computer calculations at ab initio quantum mechanics theory, to obtain their electronic structure and molecular properties. The aim was to correlate their behavioral effect with their physicochemical properties. In addition, the crystals of P, NET and LNG bound to the progesterone receptor (PR) were studied. The PR crystallizes as a dimer forming two monomers (mA and mB), in which Gln725 interacts in either of two possible ways with the C3-carbonyl pharmacophore of progestins. P binds differentially to both PR monomers, while NET binds exclusively as mA and LNG binds only as mB in both monomers with no difference. Energetically, binding of LNG and P to mB, is more favorable than that of NET and P to mA. Consequently, this bimodal mechanism increases the action possibilities of SPs on biological systems. Interestingly, progestin potency depends mostly on local molecular structure and electronic features, prevailing over total molecular properties.

Cyclooxygenase-2 prostaglandins mediate anandamide-inhibitory action on nitric oxide synthase activity in the receptive rat uterus

Anandamide, an endocannabinoid, prostaglandins derived from cyclooxygenase-2 and nitric oxide synthesized by nitric oxide synthase (NOS), are relevant mediators of embryo implantation. We adopted a pharmacological approach to investigate if anandamide modulated NOS activity in the receptive rat uterus and if prostaglandins mediated this effect. As we were interested in studying the changes that occur at the maternal side of the fetal–maternal interface, we worked with uteri obtained from pseudopregnant rats. Females were sacrificed on day 5 of pseudopregnancy, the day in which implantation would occur, and the uterus was obtained. Anandamide (2ng/kg, i.p.) inhibited NOS activity (P<0.001) and increased the levels of prostaglandin E2 (P<0.001) and prostaglandin F2α (P<0.01). These effects were mediated via cannabinoid receptor type 2, as the pre-treatment with SR144528 (10mg/kg, i.p.), a selective cannabinoid receptor type 2 antagonist, completely reverted anandamide effect on NOS activity and prostaglandin levels. The pre-treatment with a non-selective cyclooxygenase inhibitor (indomethacin 2.5mg/kg, i.p.) or with selective cyclooxygenase-2 inhibitors (meloxicam 4mg/kg, celecoxib 3mg/kg, i.p.) reverted anandamide inhibition on NOS, suggesting that prostaglandins are derived from cyclooxygenase-2 mediated anandamide effect. Thus, anandamide levels seemed to modulate NOS activity, fundamental for implantation, via cannabinoid receptor type 2 receptors, in the receptive uterus. This modulation depends on the production of cyclooxygenase-2 derivatives. These data establish cannabinoid receptors and cyclooxygenase enzymes as an interesting target for the treatment of implantation deficiencies.

Activation of amygdalar metabotropic glutamate receptors modulates anxiety, and risk assessment behaviors in ovariectomized estradiol-treated female rats

Anxiety disorders are more prevalent in females than males. The underlying reasons for this gender difference are unknown. Metabotropic glutamate receptors (mGluRs) have been linked to anxiety and it has been shown that interaction between estrogen receptors and mGluRs modulate sexual receptivity in rats. We investigated the role of mGluRs in anxiety-related behaviors in ovariectomized female rats with (OVX+EB) or without (OVX) estradiol implants. We centrally infused (s)-3,5-dihydroxyphenylglycine (DHPG), a group I mGluR agonist, into the basolateral amygdala (BLA) of OVX+EB and OVX rats at 0.1 and 1.0μM. Male rats that normally have low estradiol levels were used to compare with OVX rats. Generalized anxiety, explorative activity and detection and analysis of threat were analyzed in the elevated plus maze (EPM) and risk assessment behaviors (RABs). DHPG (1.0μM) increased the percentage of time spent in- and entries into- the open arms in OVX+EB, but not in OVX or male rats. Flat-back approaches and stretch-attend postures, two RABs, were significantly reduced by DHPG (0.1 and 1.0μM) in OVX+EB rats only. DHPG did not modulate rearing and freezing, behaviors related to exploration and fear-like behavior, respectively. However, DHPG (1.0μM) increased head dipping and decreased grooming behaviors in OVX rats, suggesting a weak explorative modulation. The effects of DHPG observed in OVX+EB, were blocked by 50μM of (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), a mGluR1 antagonist. AIDA and/or estradiol did not modulate anxiety and or RABs. Our results show that intra-BLA infusion of DHPG exerts an anxiolytic-like effect in OVX+EB, but not in OVX or male rats. This effect seems to depend upon mGluR1 subtype activation. Our findings led us to suggest that the effects observed in OVX+EB rats might be due to an interaction at the membrane level of estrogen receptors with mGlu1 within the BLA.

The Role of Caveolin-1 in Blood–Brain Barrier Disruption Induced by Focused Ultrasound Combined with Microbubbles

This research was designed to determine whether disrupting the blood-brain barrier (BBB) in rats by applying focused ultrasound (FUS) combined with microbubbles induced changes in the density of caveolae and/or the expression of the structural protein caveolin-1. To this end, two approaches were utilized. First, using enhanced magnetic resonance imaging, characteristics of BBB disruption induced by our specific FUS parameters and dose of microbubble were recorded, and the time after treatment when the BBB was the most permeable was determined. Second, rats were treated with FUS or microbubbles alone, both or neither, and a combination of Evans blue (EB) BBB permeability assays, streptavidin-peroxidase (SP) immunohistochemistry, western blot, and transmission electron microscopy (TEM) was employed to detect any changes in caveolae density and caveolin-1 expression at the previously determined time point when the BBB was the most permeable. The first set of studies revealed that our specific FUS parameters and dose of microbubbles were able to induce a transient, targeted, and reversible BBB opening in rats, and that the BBB was the most permeable 1 h after treatment with FUS and microbubbles. In the second set of experiments, the results of the SP immunohistochemistry, western blot, and TEM, taken together, revealed that caveolae and caveolin-1 were primarily localized in the brain microvascular endothelial cells of all of the rats regardless of treatment, and that caveolin-1 expression was highest in the rats treated with both FUS and microbubbles. In summary, treatment with FUS, in combination with a dose of microbubbles, can enhance BBB permeability through a caveolae-mediated transcellular approach by upregulating the expression level of caveolin-1 and, consequently, the amount of caveolae. This caveolin-1-mediated transcellular transport pathway may cooperate with other transport pathways to induce opening of the BBB. This research sheds light on the mechanism of a transient, targeted, and reversible opening of the BBB induced by FUS combined with microbubbles.

Blastocyst-mediated induction of endometrial connexins: an inflammatory response?

As a prerequisite for successful embryo implantation in mammals, before implantation ovarian hormones regulate the transformation of the endometrium into the receptive phase. During the implantation process, gene expression in the receptive endometrium is additionally modulated by the presence of a blastocyst. During this complex differentiation process, in humans as well as in rodents, gap junction connexin 26 (Cx26) is suppressed in the uterine epithelium and Cx43 is suppressed in the endometrial stromal cells during the receptive phase. In rodents, a blastocyst-mediated induction of Cx26 takes place locally in the uterine epithelial cells of the implantation chamber surrounding the blastocyst, followed by an increase in Cx43 in the cells of the developing decidua. The Cx26 induction is dependent on the presence of a blastocyst and occurs even before adhesion and invasion of the trophoblast takes place. The signal cascades involved in this blastocyst-mediated connexin induction are still elusive. The process of implantation is considered as a proinflammatory response, and inflammatory factors have been shown to be involved in the implantation process. In fact, Cx26 expression can be induced in the receptive rat endometrium by mediators of the inflammatory cascade including prostaglandin-F2α and IL1β by an ER-independent pathway similar to the blastocyst-mediated connexin induction at the time of implantation. Thus, in the receptive endometrium induction of connexin expression may also be induced by mediators of the inflammatory signaling cascade, and the implantation-related induction of intercellular communication may in part be due to an inflammatory response.

ICAM-1 Is Involved in Uterine Receptivity in Rats.

The uterine epithelium undergoes extensive molecular changes during early pregnancy to become receptive to an implanting embryo, and this is tightly regulated by ovarian hormones. Many adhesion molecules are known to reorganize during early pregnancy in order to facilitate embryo adhesion to the apical membrane of uterine epithelial cells and play an important role in uterine receptivity. This study examined the changes in distribution, expression and hormonal control of ICAM-1 in the rat uterus during early pregnancy, in ovariectomised rats, pseudo pregnant rats and cultured preimplantation embryos. The use of ICAM-1 specific vivo-morpholino antisense oligonucleotides was employed to discover the role of ICAM-1 in embryo development and implantation. ICAM-1 was visualized in the rat uterus using indirect immunofluorescence microscopy and was absent in the uterine epithelium on day 1 (pre-receptive) and day 3 of pregnancy. By day 6 (receptive) and day 7 of pregnancy at the time of implantation in the rat, ICAM-1 is present in the uterine epithelium and is localized to the apical region. ICAM-1 then disappears in uterine epithelial cells by day 9 (post receptive) of pregnancy. ICAM-1 thus is likely an important molecule in the adhesion of an implanting embryo to the apical membrane of uterine epithelial cells in rats. ICAM-1 is absent in the uterine epithelium of ovariectomised rats treated with oestrogen alone (non-receptive) and apical in the uterine epithelium of rats treated with progesterone alone, or combined with estrogen (receptive); and was apical in day 6 pseudo pregnant animals. Apical ICAM-1 staining in the uterine epithelium, on day 6 of pregnancy and in ovariectomised rats treated with progesterone and in pseudo pregnant rats always coincides with an increase in ICAM-1 monomeric expression on western blots. Dimeric expression of ICAM-1 did not alter from day 1 to day 6 of pregnancy and corresponds to the ICAM-1 protein found in endothelium. ICAM-1 was however present in endothelial cells of blood vessels found in the endometrial stroma at all stages of pregnancy. This indicates that ICAM-1 in uterine epithelial cells is under progesterone control and that endothelial ICAM-1 is not at all influenced by ovarian hormones. Previous studies have suggested that ICAM-1 might be up regulated by cytokines released by implanting embryos; however the use of pseudo pregnant and ovariectomised rats has shown that this is not the case. ICAM-1 is also present in pre-implantation embryos and may be necessary in facilitating embryo adhesion. Moreover, early day 3 embryos exposed to ICAM-1 vivo-morpholino antisense oligonucleotides fail to develop into blastocysts. In summary ICAM-1 is likely an important molecule in the receptivity of uterine epithelial cells and is under progesterone control. ICAM-1 is also a necessary molecule in embryo development. (platform)

Dietary ω-3 fatty acids alter cardiac mitochondrial phospholipid composition and delay Ca 2+-induced permeability transition

Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA + DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA + DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation ( n = 10–14), and fed a standard diet, or supplemented with EPA + DHA (2.3% of energy intake) for 12 weeks. EPA + DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA + DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA + DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA + DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.

Expression of α Vβ 3 integrin in rat endometrial epithelial cells and its functional role during implantation

The α Vβ 3 integrin as a marker of endometrial receptivity has been well established in human and other mammalian species; however, its expression is still not known in rats. Our objective was to establish the expression of α Vβ 3 integrin as a marker of endometrial receptivity in rat and to further prove its role in implantation by function-blocking studies in this species. Immunocytochemical, immunohistochemical and flow-cytometric studies were performed in rat endometrial epithelial cells (EEC) to demonstrate the expression of α Vβ 3 integrin during non-receptive, pre-receptive and receptive phases of the uterus. Results revealed positive immunocytochemical staining for α v and β 3 subunits on the surface of EEC of days 4 and 5 p.c. (post-coitum), but the intensity was higher in cells of day 5 p.c. Flow-cytometric study revealed higher level of α Vβ 3 on day 5 p.c. as compared to day 4 p.c. and non-pregnant animals. Immunohistochemical analysis of uterine tissue also revealed that the α Vβ 3 expression in LE was higher on day 5 p.c. morning as compared to that observed on day 4 p.c. In addition, the expression of β 3 subunit was not evident in rats receiving ormeloxifene, an agent known to inhibit the uterine receptivity. Immunoblotting experiments also revealed higher expression of uterine β 3 on day 5 p.c. On day 6, expression of β 3 was high in implantation sites than on inter-implantation sites. In immature ovariectomized rats, α Vβ 3 was up-regulated by progesterone and by a combination of estrogen and progesterone. The expression of α Vβ 3 was also up-regulated in EEC co-cultured with blastocysts. All the agents used for function-blocking studies showed significant reduction in the number of implantation sites in treated horn as compared to sham control horn. The present study has successfully demonstrated the expression of α Vβ 3 in rat EEC as a marker of endometrial receptivity and showed that this molecule is indispensable for the process of implantation in this species.

Increasing 3α,5α-THP following inhibition of neurosteroid biosynthesis in the ventral tegmental area reinstates anti-anxiety, social, and sexual behavior of naturally receptive rats

The progesterone metabolite and neurosteroid, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), has actions in the midbrain ventral tegmental area (VTA) to modulate lordosis, but its effects on other reproductively relevant behaviors are not well understood. Effects on exploration, anxiety, and social behavior resulting from inhibition of 3alpha,5alpha-THP formation, as well as 3alpha,5alpha-THP enhancement, were investigated in the midbrain VTA. Naturally sexually receptive, female rats (n=8-10/group) received infusions aimed at the midbrain VTA of vehicle, PK11195 (an inhibitor of neurosteroidogenesis), and/or indomethacin (an inhibitor of 3alpha,5alpha-THP formation from prohormones), and were subsequently infused with vehicle or FGIN 1-27 (a neurosteroidogenesis enhancer). The rats were then assessed in a behavioral battery that examined exploration (open field), anxiety (elevated plus maze), social (social interaction), and sexual (paced mating) behavior. Inhibition of 3alpha,5alpha-THP formation decreased exploratory, anti-anxiety, social, and sexual behavior, as well as midbrain 3alpha,5alpha-THP levels. Infusions of FGIN 1-27 following 3alpha,5alpha-THP inhibition restored these behaviors and midbrain 3alpha,5alpha-THP levels to those commensurate with control rats that had not been administered inhibitors. These findings suggest that 3alpha,5alpha-THP formation in the midbrain VTA may influence appetitive, as well as consummatory, aspects of mating behavior.