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Reproductive Biology and Endocrinology... Jan 2015Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the...
Local uterine Ang-(1-7) infusion augments the expression of cannabinoid receptors and differentially alters endocannabinoid metabolizing enzymes in the decidualized uterus of pseudopregnant rats.
BACKGROUND
Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the endogenous ligand for the CB1 and CB2 receptors (R), interfere with receptivity of the blastocyst. Ang-(1-7) is down-regulated in the implantation site (IS) in normal pregnancy at day 7 of gestation. We determined the effects of intra-uterine angiotensin-(1-7) [Ang-(1-7)] (24 microg/kg/h) or vehicle given into the left uterine horn on the ECs in the decidualized uterus.
METHODS
Ovariectomized rats were sensitized for the decidual cell reaction by steroid treatment and decidualization was induced by a bolus of oil injected into the left horn; the right horn served as a control.
RESULTS
Decidualization increased endometrial permeability (3.1+/-0.2 vs. 7.1+/-0.5 uterus/muscle of cpm of (125)I-BSA, p < 0.0001). VEGF mRNA was increased by the decidualization (1.4-fold, p < 0.05) and by Ang-(1-7) (2.0-fold, p < 0.001). CB1R mRNA was reduced by decidualization (2.7-fold, p < 0.001), but increased by Ang-(1-7) (1.9-fold, p < 0.05). CB2R mRNA was increased by decidualization (4-fold, p < 0.05) and by Ang-(1-7) (2.4-fold, p < 0.001). The enzyme metabolizing AEA, fatty acid amide hydrolase (FAAH), was reduced by decidualization (7.8 fold, p < 0.001) and unchanged by Ang-(1-7) (p > 0.05), whereas the enzyme metabolizing 2-arachidonoylglycerol, monoacyl glycerol lipase (MAGL), was unchanged by decidualization (p > 0.05) and increased by Ang-(1-7) (1.7 fold, p < 0.001).
CONCLUSIONS
These findings report for the first time that Ang-(1-7) augments the expression of CB1R, CB2R and MAGL in the decidualized uterus and thus may interfere with the early events of decidualization.
Topics: Amidohydrolases; Angiotensin I; Animals; Arachidonic Acids; Decidua; Embryo Implantation; Endocannabinoids; Female; Gene Expression Regulation, Enzymologic; Glycerides; Infusion Pumps; Monoacylglycerol Lipases; Peptide Fragments; Pregnancy; Pseudopregnancy; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Up-Regulation; Uterus
PubMed: 25596750
DOI: 10.1186/1477-7827-13-5 -
Fertility and Sterility Mar 2015To determine the potential microRNA (miRNA) regulators of embryo implantation, as a continuation of genomic and proteomic research.
OBJECTIVE
To determine the potential microRNA (miRNA) regulators of embryo implantation, as a continuation of genomic and proteomic research.
DESIGN
Laboratory animal research.
SETTING
University hospital laboratory.
ANIMAL(S)
Adult healthy female C57BL6/J mice (age 6-8 weeks, nonfertile, weighing 18-20 g each).
INTERVENTION(S)
Female mice were mated naturally with fertile males to produce pregnancy. Luminal epithelium was collected by laser-capture microdissection during the implantation period. Mouse models of pseudopregnancy, delayed implantation, and artificial decidualization were established.
MAIN OUTCOME MEASURE(S)
The miRNA profile in luminal epithelium was clarified by microarray analysis and validated by real-time reverse transcription polymerase chain reaction (qRT-PCR) in a series of models. Target genes were predicted and confirmed by luciferase activity assay. The role of miRNA in implantation was examined by loss-of-function and gain-of-function of miRNA in vitro and in vivo.
RESULT(S)
A total of 29 and 15 miRNAs were up- and down-regulated, respectively, during the implantation period; 11 of these miRNAs were validated by qRT-PCR. The profile of miR-451 was clarified in a series of models. A dual-luciferase activity assay showed that Ankrd46 was a target gene of miR-451. Loss-of-function by LV-miR-451 sponge or miR-451 inhibitor led to a reduced number of embryo implantations, but had little effect on fertilization.
CONCLUSION(S)
miR-451 was specifically up-regulated during the implantation period, and it may play a major role in embryo implantation by targeting Ankrd46.
Topics: Animals; Embryo Implantation; Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Targeting; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Microarray Analysis; Muscle Proteins; NIH 3T3 Cells; Pregnancy
PubMed: 25542822
DOI: 10.1016/j.fertnstert.2014.11.024 -
Molecular and Cellular Endocrinology Jan 2015To define endometrial stromal-derived paracrine mediators that participate in estradiol-17β (E2)-induced epithelial proliferation, microarray analysis of gene...
To define endometrial stromal-derived paracrine mediators that participate in estradiol-17β (E2)-induced epithelial proliferation, microarray analysis of gene expression was carried out in mouse uterine epithelial-stromal co-culture systems under the condition of E2 or vehicle (control). Our results demonstrated gene alteration by E2: in epithelial cells, we found up-regulation of 119 genes and down-regulation of 28 genes, while in stroma cells we found up-regulation of 144 genes and down-regulation of 184 genes. A functional enrichment analysis of the upregulated epithelial genes implicated them for proliferation, while upregulated stromal genes were associated with extracellular functions. Quantitative RT-PCR and in situ hybridization results confirmed differential gene expression in both cell cultures and ovariectomized uteri after the above treatments. Based on our identification of stromal secretory factors, we found evidence that suppression by siRNA specifically for Bmp8a and/or Fgf10 in the stromal layer caused significant inhibition of proliferation by E2 in the co-culture system, suggesting Bmp8a and Fgf10 act as paracrine mediators during E2-dependent control of uterine proliferation. The localization of receptors and receptor activation signaling in epithelial cells in both the co-culture system and uteri was consistent with their involvement in ligand-receptor signaling. Interestingly, loss of Bmp8a or Fgf10 also caused abrogation of E2-regulated epithelial receptor signaling in co-culture systems, suggesting that stroma-derived Fgf10 and Bmp8a are responsible for epithelial communication. Overall, stromal Fgf10 and Bmp8a serve as potential paracrine factors for E2-dependent regulation of epithelial proliferation in the uterus.
Topics: Animals; Bone Morphogenetic Proteins; Cell Proliferation; Coculture Techniques; Epithelial Cells; Estradiol; Female; Fibroblast Growth Factor 10; Gene Expression Profiling; Gene Expression Regulation; Mice; Microarray Analysis; Ovariectomy; Paracrine Communication; Primary Cell Culture; Pseudopregnancy; RNA, Small Interfering; Signal Transduction; Stromal Cells; Uterus
PubMed: 25451979
DOI: 10.1016/j.mce.2014.11.002 -
Journal of Physiology and Pharmacology... Oct 2014Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most...
Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most visible effects are present in the local compartment, i.e. in uterine draining lymph nodes and the decidua, while peripheral changes are less obvious. In our recent paper we indicated that costimulation phenotype of APCs in spleens of female mice during the preimplantation period of pregnancy differs from mice in pseudopregnancy. However, the effect of differential costimulation in the context of the T lymphocyte function at periphery in early pregnancy is still unknown. For that reason, we decided to investigate global protein expression in splenic CD4(+) lymphocytes in order to identify and validate the most important biomarkers characteristic for the preimplantation period of pregnancy at periphery. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were utilized to analyze the protein expression pattern of magnetically sorted CD4(+) lymphocytes from spleens of pregnant and pseudopregnant females at 3.5 days after mating. The first goal of this study was to create a 2-DE map of the splenic CD4(+) T cells of pregnant mice. As a result, 106 protein spots from 373 were identified using MS. The comparison of lymphocyte protein patterns between pregnant and pseudopregnant mice depicted differential expression of 11 identified proteins belonging to the group of proteins involved in cytoskeletal structure, cell motility and metabolism. Profoundly diminished expression of cofilin-1, F-actin capping protein subunit alpha and malate dehydrogenase proteins in lymphocytes of pregnant mice indicates that preimplantation pregnancy could change the activation state of peripheral CD4(+) lymphocytes.
Topics: Animals; CD4-Positive T-Lymphocytes; Embryo Implantation; Female; Mice; Pregnancy; Proteome; Spleen
PubMed: 25371532
DOI: No ID Found -
Reproductive Sciences (Thousand Oaks,... May 2015Endoplasmic reticulum stress (ERS), which is a novel pathway of regulating cellular apoptosis and the function of ERS during corpus luteum (CL) regression, is explored....
Endoplasmic reticulum stress (ERS), which is a novel pathway of regulating cellular apoptosis and the function of ERS during corpus luteum (CL) regression, is explored. Early-luteal stage (day 2), mid-luteal stage (day 7), and late-luteal stage (day 14 and 20) were induced, and the apoptosis of luteal cells was detected by a terminal 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay. The apoptotic cells were increased with the regression of CL, especially during the late-luteal stage. The ERS markers glucose-regulated protein 78 (Grp78), CCAAT/enhancer-binding protein homologous protein (CHOP), X-box binding protein 1 (XBP1), activating transcription factor 6α (ATF6α), eukaryotic initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α), caspase 12, and apoptosis marker caspase 3 were analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry, in agreement with the results of the TUNEL assay; the expression levels of CHOP, caspase 12, and caspase 3 were increased during the process of CL regression. Luteal cells were isolated and cultured in vitro, and the apoptosis of luteal cells was induced by prostaglandin F2α. The ERS was attenuated by the ERS inhibitor tauroursodeoxycholic acid, and the apoptotic rate was analyzed by flow cytometry. The ERS markers Grp78, CHOP, XBP1s, ATF6α, eIF2α, IRE1α, caspase 12, and apoptotic execute marker caspase 3 were analyzed by real-time PCR and immunofluorescence, and the results suggested that the expression of CHOP, caspase 12, and caspase 3 were increased, and there was increased apoptosis of luteal cells. But the expression of IRE1α/XBP1s and eIF2α was not detected. Taken together, the ERS is involved in the CL regression of rats through the CHOP and caspase 12 pathway.
Topics: Activating Transcription Factor 6; Animals; Apoptosis; Caspase 12; Caspase 3; Cells, Cultured; Corpus Luteum; DNA-Binding Proteins; Dinoprost; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Eukaryotic Initiation Factor-2; Female; Gene Expression Regulation; Heat-Shock Proteins; Immunohistochemistry; In Situ Nick-End Labeling; Luteal Cells; Luteal Phase; Luteolysis; Mice; Multienzyme Complexes; Protein Serine-Threonine Kinases; Pseudopregnancy; Rats; Real-Time Polymerase Chain Reaction; Regulatory Factor X Transcription Factors; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor CHOP; Transcription Factors; X-Box Binding Protein 1
PubMed: 25332219
DOI: 10.1177/1933719114553445 -
Journal of the American Association For... Sep 2014Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the...
Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the cause of facial alopecia, tachypnea, dyspnea, and sudden death. Pneumonia and soft-tissue abscesses were observed, and Pasteurella pneumotropica biotype Jawetz was consistently isolated from the upper respiratory tract, lung, and abscesses. Epidemiologic investigation within the facility revealed presence of this pathogen in mice generated or rederived by the intramural Genetically Engineered Mouse Model (GEMM) Core but not in mice procured from several approved commercial vendors. Epidemiologic data suggested the infection originated from female or vasectomized male ND4 mice obtained from a commercial vendor and then comingled by the GEMM Core to induce pseudopregnancy in female mice for embryo implantation. Enrofloxacin delivered in drinking water (85 mg/kg body weight daily) for 14 d was sufficient to clear bacterial infection in normal, breeding, and immune-deficient mice without the need to change the antibiotic water source. This modified treatment regimen was administered to 2400 cages of mice to eradicate Pasteurella pneumotropica from the facility. Follow-up PCR testing for P. pneumotropica biotype Jawetz remained uniformly negative at 2, 6, 12, and 52 wk after treatment in multiple strains of mice that were originally infected. Together, these data indicate that enrofloxacin can eradicate P. pneumotropica from infected mice in a less labor-intensive approach that does not require breeding cessation and that is easily adaptable to the standard biweekly cage change schedule for individually ventilated cages.
Topics: Animal Husbandry; Animals; Animals, Laboratory; Anti-Bacterial Agents; Enrofloxacin; Female; Fluoroquinolones; Male; Mice, Inbred NOD; Pasteurella Infections; Pasteurella pneumotropica; Rodent Diseases
PubMed: 25255075
DOI: No ID Found -
PloS One 2014Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea...
Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.
Topics: Animals; Corpus Luteum; Corpus Striatum; Dinoprost; Dinoprostone; Dopamine; Female; Gene Expression; Ovary; Progesterone; Protein Transport; Pseudopregnancy; Rabbits; Receptors, Dopamine D1; Receptors, Dopamine D3; Reproduction
PubMed: 25148384
DOI: 10.1371/journal.pone.0104797 -
Journal of the American Association For... May 2014In the past decade, the use of genetically engineered rats has increased exponentially; therefore, the ability to perform embryo transfer (ET) in rats to rederive,...
In the past decade, the use of genetically engineered rats has increased exponentially; therefore, the ability to perform embryo transfer (ET) in rats to rederive, reanimate, or create mutant rat lines is increasingly important. However, the successful generation of pseudopregnant female rats for ET represents a limiting factor. We here evaluated the subcutaneous administration of 40 μg luteinizing hormone releasing hormone agonist (LHRHa) for estrus synchronization during the development and implementation of a rat ET program. Our first experiment assessed endogenous estrus cycling patterns by examining vaginal cytology without administration of LHRHa in 5-wk-old peripubertal Sprague-Dawley female rats. These rats then received LHRHa at approximately 7 wk of age; 57% of the rats were synchronized in proestrus or estrus as assessed by vaginal cytology 96 h later. In a second experiment, 8-wk-old virgin, unmanipulated Sprague-Dawley female rats received LHRHa; 55% were synchronized in proestrus or estrus 96 h later. Copulatory plugs were confirmed in 28% and 82% of the rats that had been synchronized in the first and second experiments, respectively, and mated with vasectomized male rats. Embryo transfer surgery was performed, and live pups were born from both fresh and cryopreserved transgenic rat embryos. Our results indicate that subcutaneous administration of 40 μg LHRHa followed by examination of vaginal cytology 96 h later is an effective technique to generate multiple pseudopregnant recipient rats for use in an ET program.
Topics: Animals; Embryo Transfer; Estrus; Female; Gonadotropin-Releasing Hormone; Male; Pseudopregnancy; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms
PubMed: 24827564
DOI: No ID Found -
Journal of Visualized Experiments : JoVE Feb 2014The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic...
The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.
Topics: Animals; Embryo Transfer; Female; Male; Mice; Models, Animal; Pregnancy; Pseudopregnancy; Uterus; Vasectomy
PubMed: 24637845
DOI: 10.3791/51214 -
PloS One 2014Felids generally follow a poly-estrous reproductive strategy. Eurasian lynx (Lynx lynx) display a different pattern of reproductive cyclicity where physiologically...
Felids generally follow a poly-estrous reproductive strategy. Eurasian lynx (Lynx lynx) display a different pattern of reproductive cyclicity where physiologically persistent corpora lutea (CLs) induce a mono-estrous condition which results in highly seasonal reproduction. The present study was based around a sono-morphological and endocrine study of captive Eurasian lynx, and a control-study on free-ranging lynx. We verified that CLs persist after pregnancy and pseudo-pregnancy for at least a two-year period. We could show that lynx are able to enter estrus in the following year, while CLs from the previous years persisted in structure and only temporarily reduced their function for the period of estrus onset or birth, which is unique among felids. The almost constant luteal progesterone secretion (average of 5 ng/ml serum) seems to prevent folliculogenesis outside the breeding season and has converted a poly-estrous general felid cycle into a mono-estrous cycle specific for lynx. The hormonal regulation mechanism which causes lynx to have the longest CL lifespan amongst mammals remains unclear. The described non-felid like ovarian physiology appears to be a remarkably non-plastic system. The lynx's reproductive ability to adapt to environmental and anthropogenic changes needs further investigation.
Topics: Animals; Corpus Luteum; Corpus Luteum Maintenance; Estrogens; Estrus; Female; Longitudinal Studies; Lynx; Pregnancy; Prostaglandins; Reproducibility of Results; Seasons; Ultrasonography
PubMed: 24599348
DOI: 10.1371/journal.pone.0090469