Nonpregnant Endometria Research Articles

The mRNA and lncRNA landscape of the non-pregnant endometrium during the oestrus cycle in dairy goat

Cyclic changes in the endometrium are essential for embryo implantation in mammals; many studies report that such changes constitute a complex process involving numerous molecular mediators. In the present study, goat endometria at oestrus Day 5 and oestrus Day 15 were selected to systematically analyse the transcriptome using strand-specific Ribo-Zero RNA sequencing. Over 120 million high-quality paired-end reads were generated and 440400 transcripts were identified in the endometrial tissue of dairy goats. In total, 489 differentially expressed mRNAs and 854 differentially expressed long non-coding RNAs were identified when comparing the endometrium at goat endometria at oestrus Day 5 and oestrus Day 15. Neurotensin was found to play a potentially important role in the non-pregnant goat endometrium during the oestrus cycle. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses of the cis-target genes of the differentially expressed long non-coding RNAs showed that GO:0005198 (structural molecule activity) and ko04510 (focal adhesion) might be involved in cyclic endometrial changes. Taken together, the resulting transcriptomic profiles elucidate global trends in mRNA and lncRNA expression in non-pregnant endometria during the oestrus cycle in dairy goats.

Comparison of the Effects of Early Pregnancy with Human Interferon, Alpha 2 (IFNA2), on Gene Expression in Bovine Endometrium1

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

83 EFFECTS OF HUMAN INTERFERON-α ON GENE EXPRESSION IN THE BOVINE ENDOMETRIUM IN COMPARISON TO DAYS 15 AND 18 OF PREGNANCY

Interferon-τ (IFNT), a Type-I interferon (IFN), is the pregnancy recognition signal produced by the ruminant conceptus (Godkin et al. 1984; Hansen et al. 1985; Helmer et al. 1987; Spencer et al. 2007). In addition to these specific functions of IFNT in ruminants, many studies suggest that IFNs play a general role in establishment of pregnancy and conceptus attachment/implantation in most mammalian species (Bazer et al. 2009; Bazer et al. 2010; Johnson et al. 2009; Roberts et al. 2008). To characterise the effects of prototype Type-I IFNs on bovine endometrium, in experiment one, Simmental heifers were treated from Day 14 to Day 16 of the oestrous cycle with a rod-shaped intrauterine device releasing human interferon-α (IFNA) or placebo lipid extrudates or PBS only as controls (n = 4 each). Lipid formulation and concentration of human IFNA were adjusted to release 8–9 × 107 IU of IFNA over a period of 2 days in in vitro release experiments. On Day 16, endometrial biopsy samples were collected after flushing the uterus. In experiment 2, endometrial tissue samples were obtained on Day 12, 15 and 18 post-mating from nonpregnant or pregnant heifers. All samples from both experiments were analysed with an Affymetrix Bovine Genome Array (Santa Clara, CA). In experiment one, IFNA treatment resulted in differential gene expression in the bovine endometrium. Significant differences were found between the IFNA group and both control groups, whereas no differences were observed between the placebo and the PBS control group. In experiment 2, differentially expressed genes were found between pregnant and nonpregnant endometria on Day 15 and 18, but not on Day 12, with many of them known IFN-stimulated genes. The comparison of the data sets from both experiments showed very similar gene expression changes for most of the typical IFN-stimulated genes. In addition, several genes were identified which were differentially expressed after IFNA treatment but not different at Day 15 or 18 of pregnancy compared with nonpregnant animals. Conversely, some genes were found as differentially expressed during pregnancy but not after IFNA treatment. Differential expression of selected genes was verified by quantitative real-time PCR and 4 genes, namely jumonji C domain containing histone demethylase 1 homologue D (JHDM1D), indoleamine 2,3-dioxygenase 1 (IDO1), fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3) and dickkopf homologue 1 (DKK1), were selected for localization of mRNA expression in endometrial tissue sections. The findings of this study suggest that there may be differential effects of bovine IFNT compared with human IFNA and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. This study was supported by the German Ministry for Education and Research (BMBF, FUGATO-plus, COMPENDIUM) and the German Research Foundation (DFG FOR478). The authors are part of the European Union COST action GEMINI.

Maternal recognition of pregnancy in the horse: a mystery still to be solved

Maternal recognition of pregnancy in the horse is the sum of events leading to maintenance of pregnancy; in a narrow sense, maternal recognition of pregnancy refers to the physiological process by which the lifespan of the corpus luteum is prolonged. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. The presence of the conceptus reduces pulsatile prostaglandin F(2α) secretion by the endometrium during early gestation in the mare, partly attributed to the reduced expression of cyclooxygenase-2. Cyclooxygenase-2 has therefore been suggested as one of the regulators of endometrial prostaglandin F(2α) release modified by the antiluteolytic factor secreted by the conceptus. In addition, altered oxytocin responsiveness has been implicated in the adjustment of prostaglandin release in pregnant mares. While conceptus mobility has proven to be essential for establishment of pregnancy, conceptus-derived oestrogens and prostaglandins, principally prostaglandin E(2), have not been confirmed as the critical antiluteolytic factor. Various ways to induce prolonged luteal function in the non-pregnant mare will be highlighted in the current review, specifically, how they may pertain to the process of maternal recognition of pregnancy. Furthermore, recently published microarray experiments comparing the transcriptome of pregnant and non-pregnant endometria and different stages of conceptus development will be reviewed. Findings include the prevention of conceptus adhesion, the provision of nutrients to the conceptus and the avoidance of immunological rejection, among others.

Polyunsaturated fatty acids differentially alter PGF 2α and PGE 2 release from bovine trophoblast and endometrial tissues during short-term culture

Two studies tested the hypothesis that eicosapentaenoic (20:5ω3; EPA), docosahexaenoic acids (22:6ω3; DHA) or linoleic acid (C18:2ω6; LIN) reduced bovine endometrial and trophoblast prostaglandin F 2α (PGF 2α) and prostaglandin E 2 (PGE 2) release during short-term culture. In Study 1, endometrial tissues were collected from non-lactating, non-pregnant cows and endometrial plus trophoblast tissues from pregnant cows 16 days post-insemination. In Study 2, endometrial and trophoblast tissues were collected on day 17 of pregnancy, from cows synchronised using a double prostaglandin (PG) or Ovagen™ synchronisation. Tissues were incubated in medium only (M) or media supplemented with fatty acids: eicosapentaenoic (20:5ω3; EPA), docosahexaenoic acids (22:6ω3; DHA) or linoleic acid (C18:2ω6; LIN). In Study 1, PGE 2 release from ‘pregnant’ endometria was higher ( P = 0.094) than from ‘non-pregnant’ endometria, while PGF 2α concentrations were similar. Fatty acids treatment had no effect on PGF 2α or PGE 2 release from either pregnant or non-pregnant endometria. Individual fatty acid treatments had no effect on the ratio of PGF 2α to PGE 2 from trophoblast tissues, but when the data from the 3 fatty acid treatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF 2α to PGE 2 was reduced ( P = 0.026) when compared to medium only. In Study 2, PGE 2 concentrations were higher ( P = 0.013) from the trophoblast collected from Ovagen™ cows as compared to that of the PG synchrony group. When the data from the 3-omega fatty acids were combined (DHA and EPA treatment groups), the 3-omega treatments decreased ( P < 0.05) PGE 2 biosynthesis from both endometrial and trophoblast tissues from animals synchronised following PG synchrony but not Ovagen™ synchrony. Short-term culture with low concentrations of 3-omega fatty acids tended to reduce prostaglandin release from trophoblast collected 16 days after insemination, with the type of synchrony modifying PGE 2 production from the trophoblast tissues collected 17 days after insemination. The ability of exogenous fatty acids to modify embryonic prostaglandin release needs to be examined in the context of supplementing dairy cows with different sources of fats. Synchronisation method altered trophoblast PGE 2 release, highlighting the importance of the hormonal environment in modifying embryonic prostaglandin synthesis and release.

C-fos and c-jun expression in human endometrium and myometrium

Estrogen is a mitogen in human endometrium and is considered to be responsible also for myometrial cell proliferation. Signalling pathways of estrogen action in these tissues are not known. In various other estrogen responsive cells, estrogen induces transient expressions of c-fos and c-jun mRNAs. We examined c-fos and c-jun mRNA expressions by Northern blotting in paired samples of endometrium, myometrium and leiomyoma tissues obtained from women under various hormonal environments as well as of endometrium and myometrium at term pregnancy. In nonpregnant endometria, strong expressions of c-fos (2.2 kb) and of c-jun (2.7 kb and 3.2 kb) were detected both in the follicular and luteal phase of the menstrual cycle, and the c-fos expression was significantly stronger in proliferative phase endometrium than in the adjacent myometrium. In most of the myometrial and leiomyoma tissue samples the signals for both protooncogenes were weak, and there were no systematic differences in the expressions between normal myometrium and myomatous tissue. In pregnant endometrium and myometrium, both the c-fos and c-jun mRNA expressions were nearly undetectable, and in pregnant endometrium expressions were significantly lower than those in nonpregnant endometrium. Also in late pregnancy myometria, the expression of c-jun was significantly lower than in nonpregnant tissues. These data suggest that c-fos and c-jun activation may be a part of estrogen-induced signal transduction in the endometrium, and that in term pregnancy endometrium this signalling pathway is inhibited. Due to the strong expression of c-jun and c-fos both in the proliferative and secretory phase endometrium, it is likely that these protooncogenes are related to functions other than epithelial cell proliferation in human endometrium. The weak expressions of c-fos and c-jun in the myometrium and in leiomyomata suggest that signalling pathways mediating steroid hormone action in endometrium and myometrium are different.

Pseudopregnancy-dependent accumulation of cholesterol sulfate due to up-regulation of cholesterol sulfotransferase and concurrent down-regulation of cholesterol sulfate sulfatase in the uterine endometria of rabbits.

The uterine endometria of rabbits induced into pseudopregnancy by intramuscular injection of 17 beta-estradiol, followed by intravenous injection of human chorionic gonadotropin, expressed cholesterol sulfate at a significantly high concentration. The highest concentration of cholesterol sulfate was observed 4 days after the injection of gonadotropin for formation of the corpus luteum, being 10 times higher than that in nonpregnant endometria, and 15.2% of the total cholesterol in the endometrium was converted to the sulfated form, whose percentage in nonpregnant endometrium was 3.2%. However, no significant change in the concentration of gangliosides was observed during the period of pseudopregnancy. In the pseudopregnant endometria, the activity of cholesterol sulfotransferase, a cytosolic thiol enzyme, was increased thirtyfold over that in the nonpregnant endometria, whereas cholesterol sulfate sulfatase, a microsomal enzyme, exhibited approximately one-tenth of the activity in nonpregnant endometria. Arylsulfatase C, but not arylsulfatases A and B, exhibited the same change in activity as cholesterol sulfate sulfatase. Thus, the striking increase in cholesterol sulfate after induction of pseudopregnancy was found to be due to the activation of cholesterol sulfotransferase and the simultaneous inhibition of cholesterol sulfate sulfatase.

Galactose-binding lectins as markers of pregnancy-related glycoproteins

Protein extracts from pregnant mouse endometria were compared with those obtained from non-pregnant and pseudopregnant mice to detect early pregnancy-specific galactose-rich glycoproteins. Gradient gel electrophoresis combined with lectin overlay and lectin histochemistry were used to identify Ricinus communis I (RCA-I), R. communis II (RCA-II) and Cytisus scoparius (CSA) lectin binding glycoproteins. Using this approach, galactose-rich glycoproteins were identified that were maximally expressed in the estrus phase of non-pregnant endometria and also those that had peak expression in pregnancy. Lectin histochemistry revealed pregnancy related changes in three portions of mouse endometrium: endometrial glands, luminal epithelium and its basement membrane. Two major glycoproteins (RCA-I reactive 64 kDa and RCA-II reactive 35 kDa) were specifically expressed in peri-implantation endometrium on days 3 and 4 of pregnancy. The appearance of these glycoproteins during the period of the implantation window in mouse suggests that they could serve as markers of uterine receptivity to the implanting blastocyst.