scholarly journals Uterine secretome and its modulation in rat (Rattus norvegicus)

Reproduction ◽  
2013 ◽  
Vol 146 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Sumit Bhutada ◽  
R R Katkam ◽  
Tarla Nandedkar ◽  
S M Metkari ◽  
U K Chaudhari ◽  
...  
Keyword(s):  
Protein Profile ◽  
Embryo Implantation ◽  
Pregnant Rats ◽  
Differential Abundance ◽  
Endometrial Epithelial Cell ◽  
Epithelial Cell Lines ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Uterine Fluid ◽  
Liquid Chromatography Tandem Mass ◽  
Culture Supernatants

The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography–tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P<0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P<0.05; t-test) abundance of β-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P<0.05; t-test) levels of UF β-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.

532. SOLUBLE MEDIATORS IN THE HUMAN UTERINE CAVITY: POTENTIAL ROLES IN EMBRYO IMPLANTATION

2009 ◽  
Vol 21 (9) ◽  
pp. 130
Author(s):  
N. Hannan ◽  
K. L. Meehan ◽  
L. J. F. Rombauts ◽  
L. A. Salamonsen
Keyword(s):  
Chemokine Receptors ◽  
Embryo Implantation ◽  
Uterine Cavity ◽  
Cycle Phase ◽  
Human Trophoblast ◽  
Endometrial Epithelial Cell ◽  
Glandular Secretion ◽  
Uterine Fluid ◽  
Human Chemokines ◽  
Insight Into

Embryo implantation requires synchronized dialogue between a receptive endometrium and an activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle there is increased glandular secretion into the uterine lumen. These secretions likely contain important mediators that modulate the endometrium and support the conceptus during implantation. Previously we identified that several chemokines were maximally produced during the MS phase by endometrial glandular epithelium (GE) (1, 2) and the presence of chemokine receptors on GE and human trophoblast (3). Furthermore recombinant human chemokines and endometrial epithelial cell-conditioned media stimulated trophoblast migration; this was attenuated by neutralizing specific chemokines (3). Chemokines also regulate a variety of adhesion and ECM molecules on trophoblast (4). Thus chemokines have important roles during embryo implantation. We hypothesized that chemokines are secreted into the uterine cavity and may act on the implanting blastocyst and the endometrium. This study aimed to identify chemokines in uterine fluid (collected by flushing the uterine cavity with 5mls of saline) from fertile women during the proliferative (non-receptive; n=4) and MS (receptive; n=4) phases of the cycle, and from women with unexplained infertility during the MS phase (n=4). Uterine fluid was analyzed using quantitative MilliplexTM Luminex® 42-plex cytokine/chemokine assays revealing the presence of IL-8, CCL2, CCL4, CCL7, CCL11, CCL22 and CX3CL1 in uterine fluid from all women. Importantly chemokine profiles were altered with both cycle phase and fertility; for example CCL4 and CCL22 levels were lower in the infertile cohort, where as CCL2 levels were higher in uterine fluid collected during the proliferative phase. Identifying the soluble mediators in human uterine fluid may provide potential markers of endometrial receptivity, insight into the unique microenvironment essential for pregnancy and a profile of maternal factors that influence the implanting blastocyst.

2-Methoxyoestradiol impairs mouse embryo implantation via F-spondin

2019 ◽  
Vol 31 (4) ◽  
pp. 689
Author(s):  
Emanuel Guajardo-Correa ◽  
Denisse Mena-Silva ◽  
Patricia Diaz ◽  
Carlos Godoy-Guzmán ◽  
Hugo Cardenas ◽  
...  
Keyword(s):  
Mouse Embryo ◽  
Embryo Implantation ◽  
Target Protein ◽  
Methyl Transferase ◽  
Uterine Fluid ◽  
Protein F

The anti-implantation effects of high oestradiol (E2) concentrations could be mediated by E2 metabolites. Herein, we examined whether 2-methoxyoestradiol (2ME) impairs embryo implantation via its target protein F-spondin. Mice on Day 3 of pregnancy were treated with E2 concomitantly with the cathecol-O-methyl transferase inhibitor OR486 and the number of implanted embryos was recorded 5 days later. The effect of 2ME or 4-methoxyoestradiol (4ME) on embryo implantation was also investigated. Plasma and uterine levels of 2ME were measured 0.5, 1 or 3h after E2 treatment while the mRNA for spondin 1 (Spon1) and F-spondin were determined in the uterus 3, 6, 12 or 24h after 2ME treatment. Finally, the effect of a neutralising F-spondin antibody on the anti-implantation effect of 2ME was explored. OR486 blocked the anti-implantation effect of E2; 2ME, but not 4ME, affected embryo implantation. The 2ME concentration was increased after 0.5 and 1h in plasma and 3h in uterine fluid following E2 treatment. 2ME increased levels of Spon1 at 12 and 24h although F-spondin was increased at 12h. F-spondin antibody blocked the effect of 2ME on embryo implantation. We conclude that 2ME impairs mouse embryo implantation via activation of F-spondin in the uterus.

scholarly journals Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Human Keratinocytes ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Adhesion ◽  
Culture Supernatants

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

6 Decelerating embryo development? Characterisation of the uterine environment in European roe deer (Capreolus capreolus) during diapause

2019 ◽  
Vol 31 (1) ◽  
pp. 128
Author(s):  
V. A. van der Weijden ◽  
A. R. Vegas ◽  
V. Milojevic ◽  
A. B. Rüegg ◽  
J. T. Bick ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Embryo Development ◽  
Developmental Stages ◽  
Roe Deer ◽  
Embryonic Diapause ◽  
Chromatography Tandem Mass Spectrometry ◽  
Uterine Fluid ◽  
Liquid Chromatography Tandem Mass ◽  
Uterine Environment ◽  
Differentially Abundant Proteins

The early developing embryo faces a continuously changing microenvironment to supports its growth. In the European roe deer, this environment accompanies embryonic diapause, a period of up to 4 months in which fertilization and subsequent implantation are decoupled. Diapause is characterised by a deceleration of embryonic growth. In most ruminants such as cattle and sheep, interferon tau (IFNt) plays a major role in maternal recognition of pregnancy. Uniquely to ruminants, the roe deer embryo does not secrete IFNt. The roe deer was used as a model species to gain insights into the changing uterine environment devoid of IFNt that supports prolonged decelerated embryo development, resumption of developmental velocity, and subsequent implantation. Uterine fluid samples from 188 female does were collected during regular huntings between September and January, and 4 developmental stages-blastocysts at early, mid, and late diapause and elongated embryos (16, 57, 97, and 18 does per developmental stage, respectively)-were defined. The developmental stages were assigned based on morphological characteristics of the embryo and the embryonic genomic DNA content. For the analysis of amino acids (AA), all 188 uterine fluid samples were subjected to targeted liquid chromatography-tandem mass spectrometry. Almost all AA increased over the course of embryo development. Although most AA showed developmental stage-specific concentration peaks, serine, glycine, alanine, glutamate, and glutamine were most abundantly present irrespective of the developmental progression. For the analysis of the protein abundances in the uterine fluid in a selected subset of samples (n=5 per developmental stage), holistic liquid chromatography-tandem mass spectrometry identified and quantified a total of 819 proteins with a false discovery rate of &lt;1%. Comparison between the developmental stages revealed 106 differentially abundant proteins. Most changes in protein abundance that occurred related to embryo elongation. Interestingly, 713 proteins remained stable during embryo development, indicating that these proteins may contribute to prolonged embryo survival during embryonic diapause. The differentially abundant proteins were clustered with DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). The most enriched clusters were cell-cell adhesion, biosynthesis of AA and carbon metabolism, microtubule, structural molecule activity, and chaperone binding. The ongoing detailed identification of stably abundant proteins will advance our basic understanding of the embryos’ needs for sustained survival during prolonged decelerated development. In addition, a comparison with the protein abundances around the time of maternal recognition of pregnancy in other species could advance our knowledge on conserved proteins that support embryo development and establishment of pregnancy in mammals. Our findings may contribute to defining optimal in vitro embryo culture conditions in a species-independent manner and potentially identify factors capable of halting embryo development.

scholarly journals MiR-184 Combined with STC2 Promotes Endometrial Epithelial Cell Apoptosis in Dairy Goats via RAS/RAF/MEK/ERK Pathway

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1052
Author(s):  
Jiuzeng Cui ◽  
Xiaorui Liu ◽  
Lichun Yang ◽  
Sicheng Che ◽  
Hongran Guo ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Embryo Implantation ◽  
Animal Husbandry ◽  
Dairy Goat ◽  
Marker Genes ◽  
Erk Pathway ◽  
Dairy Goats ◽  
Endometrial Epithelial Cell ◽  
Forkhead Box M1 ◽  
Epithelial Cell Apoptosis

The endometrium undergoes a series of complex changes to form a receptive endometrium (RE) that allows the embryo to be implanted. The inability to establish endometrial receptivity of livestock causes embryo implantation failure and considerable losses to animal husbandry. MicroRNAs (miRNAs) are a class of noncoding RNAs. Studies have found that miRNAs can regulate many critical physiological processes, including the establishment of RE during embryo implantation. miR-184 is highly expressed in the endometrial receptive period of dairy goats. This study aimed to explore the effect of miR-184 on endometrial epithelial cell (EEC) apoptosis and RE establishment. Stanniocalcin2 (STC2) is a direct target of miR-184, and miR-184 decreases the expression of STC2 in dairy goat EECs. miR-184 can activate EECs apoptosis through the RAS/RAF/MEK/ERK pathway. Additionally, miR-184 increases the expression levels of RE marker genes, such as forkhead box M1 (FOXM1) and vascular endothelial growth factor (VEGF). These findings indicate that miR-184 promotes the apoptosis of endometrial epithelial cells in dairy goats by downregulating STC2 via the RAS/RAF/MEK/ERK pathway, and that it may also regulate the establishment of RE in dairy goats.

Mitochondrial Dysfunction Induced by High Estradiol Concentrations in Endometrial Epithelial Cells

2019 ◽  
Vol 105 (1) ◽  
pp. 126-135 ◽  
Author(s):  
Chia-Hung Chou ◽  
Shee-Uan Chen ◽  
Chin-Der Chen ◽  
Chia-Tung Shun ◽  
Wen-Fen Wen ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Embryo Implantation ◽  
Ros Production ◽  
Vitro Fertilization ◽  
Endometrial Epithelial Cell ◽  
Dna Contents ◽  
Equine Chorionic Gonadotropin ◽  
In Vivo Effects

Abstract Context A supraphysiological estradiol (E2) concentration after ovarian stimulation is known to result in lower embryo implantation rates in in vitro fertilization. Endometrial epithelial cell (EEC) apoptosis occurs after the stimulation with high E2 concentrations, and mitochondria play important roles in cell apoptosis. Objective To investigate the mitochondrial function in EECs after the stimulation with high E2 concentrations. Materials and Methods Human EECs were purified and cultured with different E2 concentrations (10-10, 10–9, 10–8, 10–7 M) in vitro, in which 10–7 M is supraphysiologically high. Eight-week-old female mouse endometrium was obtained 5.5 days after the injection of 1.25 IU or 20 IU equine chorionic gonadotropin, roughly during the embryo implantation window, to examine the in vivo effects of high E2 concentrations on mouse EECs. Results In vivo and in vitro experiments demonstrated decreased mitochondrial DNA contents and ATP formation after EECs were stimulated with supraphysiologically high E2 concentrations than those stimulated with a physiologic E2 concentration. Less prominent immunofluorescence mitochondrial staining, fewer mitochondria numbers under electron microscopy, lower 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide aggregate/monomer ratio, and greater reactive oxygen species (ROS) production were found after EECs were stimulated with supraphysiologically high E2 concentrations. The high E2-induced ROS production was reduced when EECs were pretreated with N-acetyl-cysteine in vitro, but remained unchanged after the pretreatment with coenzyme Q10. Conclusion High E2 concentrations increase extramitochondrial ROS production in EECs and subsequently result in mitochondrial dysfunction.

Expression and function of Toll-like receptors in human endometrial epithelial cell lines

2010 ◽  
Vol 84 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Wedad Aboussahoud ◽  
Reza Aflatoonian ◽  
Chris Bruce ◽  
Sarah Elliott ◽  
Jon Ward ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Toll Like Receptors ◽  
Endometrial Epithelial Cell ◽  
Epithelial Cell Lines ◽  
And Function

scholarly journals Shot-gun proteome and transcriptome mapping of the jujube floral organ and identification of a pollen-specific S-locus F-box gene

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3588 ◽  
Author(s):  
Ruihong Chen ◽  
Guoliang Chen ◽  
Jian Huang
Keyword(s):  
Protein Profile ◽  
Floral Organ ◽  
Floral Biology ◽  
Reproductive Organ ◽  
S Locus ◽  
Lipid Transfer ◽  
Flowering Season ◽  
New Information ◽  
Liquid Chromatography Tandem Mass ◽  
Systematic Strategy

The flower is a plant reproductive organ that forms part of the fruit produced as the flowering season ends. While the number and identity of proteins expressed in a jujube (Ziziphus jujuba Mill.) flower is currently unknown, integrative proteomic and transcriptomic analyses provide a systematic strategy of characterizing the floral biology of plants. We conducted a shotgun proteomic analysis on jujube flowers by using a filter-aided sample preparation tryptic digestion, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, transcriptomics analyses were performed on HiSeq2000 sequencers. In total, 7,853 proteins were identified accounting for nearly 30% of the ‘Junzao’ gene models (27,443). Genes identified in proteome generally showed higher RPKM (reads per kilobase per million mapped reads) values than undetected genes. Gene ontology categories showed that ribosomes and intracellular organelles were the most dominant classes and accounted for 17.0% and 14.0% of the proteome mass, respectively. The top-ranking proteins with iBAQ >1010 included non-specific lipid transfer proteins, histones, actin-related proteins, fructose-bisphosphate aldolase, Bet v I type allergens, etc. In addition, we identified one pollen-specificity S-locus F-box-like gene located on the same chromosome as the S-RNase gene. Both of these may activate the behaviour of gametophyte self-incompatibility in jujube. These results reflected the protein profile features of jujube flowers and contributes new information important to the jujube breeding system.

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