Expression of Piwi, MMP, TIMP, and Sox during Gut Regeneration in Holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirotida)

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Modulation of endometrial E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli

The endometrium is a dynamic tissue that undergoes extensive remodelling to attain a receptive state which is further modulated in presence of an embryo for successful initiation of pregnancy. Cadherins are the proteins of junctional complex of which E-cadherin (E-Cad) is crucial for maintaining epithelial cell state and integrity of the epithelial barrier; gain of N-cadherin (N-Cad) in epithelial cells leads to epithelial to mesenchymal transition (EMT). In the present study, we aimed to investigate the expression of E-Cad and N-Cad in the mouse endometrial luminal epithelium and its modulation by estrogen, progesterone and embryonic stimuli. We observed that E-Cad is diffusely expressed in the luminal epithelium of mouse endometrium during the estrus stage and upon estrogen treatment. It is apico-laterally and basolaterally sorted at the diestrus stage and in response to combined treatment of estrogen and progesterone. In 3D spheroids of human endometrial epithelial cells, combined treatment with estrogen and progesterone led to lateral sorting of E-Cad. In the mouse endometrium at the time of embryo implantation, there is loss of E-Cad which was associated with the gain of N-Cad suggestive of EMT in the luminal epithelium. This EMT is possibly driven by embryonic stimuli as treatment with estrogen and progesterone did not lead to gain of N-Cad expression. In conclusion, the present study demonstrates that steroid hormones directly affect E-Cad sorting in the endometrial epithelium.

MAML1: a coregulator that alters endometrial epithelial cell adhesive capacity

Background Abnormalities in endometrial receptivity has been identified as a major barrier to successful embryo implantation. Endometrial receptivity refers to the conformational and biochemical changes occurring in the endometrial epithelial layer which make it adhesive and receptive to blastocyst attachment. This takes place during the mid-secretory phase of woman’s menstrual cycle and is a result of a delicate interplay between numerous hormones, cytokines and other factors. Outside of this window, the endometrium is refractory to an implanting blastocyst. It has been shown that Notch ligands and receptors are dysregulated in the endometrium of infertile women. Mastermind Like Transcriptional Coactivator 1 (MAML1) is a known coactivator of the Notch signaling pathway. This study aimed to determine the role of MAML1 in regulating endometrial receptivity. Methods The expression and localization of MAML1 in the fertile human endometrium (non-receptive proliferative phase versus receptive mid-secretory phase) were determined by immunohistochemistry. Ishikawa cells were used as an endometrial epithelial model to investigate the functional consequences of MAML1 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. After MAML1 knockdown in Ishikawa cells, the expression of endometrial receptivity markers and Notch dependent and independent pathway members were assessed by qPCR. Two-tailed unpaired or paired student’s t-test were used for statistical analysis with a significance threshold of P < 0.05. Results MAML1 was localized in the luminal epithelium, glandular epithelium and stroma of human endometrium and the increased expression identified in the mid-secretory phase was restricted only to the luminal epithelium (P < 0.05). Functional analysis using Ishikawa cells demonstrated that knockdown of MAML1 significantly reduced epithelial adhesive capacity (P < 0.01) to HTR8/SVneo (trophoblast cell line) spheroids compared to control. MAML1 knockdown significantly affected the expression of classical receptivity markers (SPP1, DPP4) and this response was not directly via hormone receptors. The expression level of Hippo pathway target Ankyrin repeat domain-containing protein 1 (ANKRD1) was also affected after MAML1 knockdown in Ishikawa cells. Conclusion Our data strongly suggest that MAML1 is involved in regulating the endometrial adhesive capacity and may facilitate embryo attachment, either directly or indirectly through the Notch signaling pathway.

Par3/Integrin β1 regulates embryo adhesion via changing endometrial luminal epithelium polarity

Objective To investigate the pathophysiological significance of Par3 and integrin β1 with regards to the functionality of the endometrial luminal epithelium (LE). Design Laboratory study. Setting University research laboratory. Patients Analysis involved endometrial aspirates and human endometrial epithelial cells (HEC-1A cells and RL95–2 cells). Interventions We first examined the expression and localization of Par3 and integrin β1 in HEC-1A cells and RL95–2 cells. Then we knocked down Par3 and integrin β1 in HEC-1A cells and RL95–2 cells respectively and found that Par3/integrin β1 affected embryo adhesion by regulating the intercellular tight junctions (TJs) structure and thus the polarity of endometrial LE. These findings were also confirmed in endometrium specimens from human and mice. Main Outcome Measures The expression and localization of Par3 and integrin β1 in endometrial epithelial cell lines and endometrium specimens. The regulations of Par3 and integrin β1 on TJs, polarity and embryo adhesion. Results Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin β1 in RL95–2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin β1 in HEC-1A cells. Conclusions Par3/integrin β1 regulates embryo adhesion by regulating intercellular TJs structure and polarity of endometrial LE under the action of ovarian hormones.

Integrating LCM-Based Spatio-Temporal Transcriptomics Uncovers Conceptus and Endometrial Luminal Epithelium Communication that Coordinates the Conceptus Attachment in Pigs

Attachment of conceptus to the endometrial luminal epithelium (LE) is a critical event for early placentation in Eutheria. Since the attachment occurs at a particular site within the uterus, a coordinated communication between three spatially distinct compartments (conceptus and endometrial LE from two anatomical regions of the uterus to which conceptus attaches and does not attach) is essential but remains to be fully characterized. Using the laser capture microdissection (LCM) technique, we firstly developed an approach that can allow us to pair the pig conceptus sample with its nearby endometrial epithelium sample without losing the native spatial information. Then, a comprehensive spatio-temporal transcriptomic profile without losing the original conceptus-endometrium coordinates was constructed. The analysis shows that an apparent difference in transcriptional responses to the conceptus exists between the endometrial LE from the two anatomically distinct regions in the uterus. In addition, we identified the communication pathways that link the conceptus and endometrial LE and found that these pathways have important roles in conceptus attachment. Furthermore, a number of genes whose expression is spatially restricted in the two different anatomical regions within the uterus were characterized for the first time and two of them (SULT2A1 and MEP1B) may cooperatively contribute to establish conceptus attachment in pigs. The results from our study have implications in understanding of conceptus/embryo attachment in pigs and other large polytocous species.

410 Steroids Regulate SLC2A1 and SLC2A3 to Deliver Glucose into Trophectoderm for Metabolism via Glycolysis

The conceptuses (embryo/fetus and placental membranes) of pigs require energy to support elongation and implantation, and amounts of glucose and fructose increase in the uterine lumen during the peri-implantation period. Conceptuses from Day 16 of pregnancy were incubated with either 14C-glucose or 14C-fructose and amounts of radiolabeled CO2 released from the conceptuses measured to determine rates of oxidation of glucose and fructose. Both glucose and fructose transport into conceptuses, and glucose is preferentially metabolized in the presence of fructose, while fructose is actively metabolized in the absence of glucose and to a lesser extent in the presence of glucose. Endometrial and placental expression of glucose transporters SLC2A1, SLC2A2, SCL2A3 and SLC2A4 were determined. SLC2A1 mRNA and protein, and SLC2A4 mRNA were abundant in the uterine luminal epithelium of pregnant compared to cycling gilts, and increased in response to progesterone and conceptus-secreted estrogen. SLC2A2 mRNA was expressed weakly by conceptus trophectoderm on Day 15 of pregnancy, while SLC2A3 mRNA was abundant in trophectoderm/chorion throughout pregnancy. Therefore, glucose can be transported into the uterine lumen by SLC2A1, and then into conceptuses by SLC2A3. On Day 60 of gestation, the cell-specific expression of these transporters was more complex, suggesting that glucose and fructose transporters are precisely regulated in a spatial-temporal pattern along the uterine-placental interface of pigs to maximize hexose sugar transport to the pig conceptus/placenta.

Application of Contrast-Enhanced Ultrasound in the Differential Diagnosis of Different Molecular Subtypes of Breast Cancer

To explore the value of contrast-enhanced ultrasound (CEUS) in the differential diagnosis of molecular subtypes of breast cancer. Sixty-two cases of breast cancer were divided into luminal epithelium A or B subtype (luminal A/B), Her-2 over-expression subtype and triple negative subtype (TN). CEUS and routine ultrasonography were performed for all patients before surgery. (1) The luminal epithelium subtype contrast enhancement pattern was more likely to present with radial edge (76.92%, p < 0.05) and low perfusion (69.23%, p < 0.05). The maximum intensity (IMAX) was lower in the luminal epithelium subtype ( p < 0.05). (2) The Her-2 over-expression subtype contrast enhancement pattern was more likely to present with centripetal enhancement (93.75%, p < 0.05) and perfusion defect (75.0%, p < 0.05), and the time to peak (TTP) was shorter (80.0%, p < 0.05). (3) The contrast enhancement pattern of the triple negative subtype was shown to have a clear boundary. Compared to the other two subtypes, the triple negative subtype did not have significantly different perfusion parameters ( p > 0.05). (4) Our study showed that the areas under the ROC curve for radial edge, low perfusion and IMAX for the luminal epithelium subtype breast lesions were 76.5%, 75.6%, and 82.1%, respectively. Additionally, the areas under the ROC curve for centripetal enhancement, perfusion defect and TTP for the Her-2 over-expression subtype breast lesions were 68.6%, 92.4%, and 97.8%, respectively. The sensitivity, specificity, and diagnostic accuracy of clear boundaries in detecting triple negative subtype breast lesions were 90.5%, 80.0%, and 91.9%, respectively.

Integrins and OPN localize to adhesion complexes during placentation in sheep

Integrins and OPN are potential mediators of blastocyst attachment to the endometrium to initiate implantation. The goals were to examine the temporal/spatial pattern of expression of integrins at the endometrial–placental interface of sheep encompassing Days 9 through 80 of gestation and determine if OPN co-localizes with integrins. Results show the following: (1) αv, α4, β1, β3 and β5 integrins at the apical surface of endometrial luminal epithelium (LE) from Days 11 through 16 of pregnancy that indicate a role for these integrins during implantation; (2) large, intermittent aggregates of αv, α4, α5, β1 and β5 integrins at the endometrial–placental interface from Days 20 through 55, suggesting adaptation to a localized tissue remodeling stage of placentation; and (3) integrin adhesion complexes (IACs) containing αv, α4, α5, β1 and β5 integrins precisely distribute at the apical surfaces of apposed endometrial LE and chorion along expanses of the interplacentomal endometrial–placental interface between Days 60 and 80 of gestation, suggesting engagement of these integrins with the ECM to stabilize adhesion between endometrial LE and chorion in response to the increasing mechanical stress on this interface by the increasing size of the fetus and volumes of fetal fluids. An advancement is the clear co-localization of OPN and integrins at the endometrial–placental interface throughout gestation in sheep. The comprehensive nature of these results provide evidence that integrins potentially interact with OPN to play key roles in the mechanisms required for implantation and placentation throughout pregnancy in sheep and have implications concerning implantation and placentation in other species.

Anatomical and cellular heterogeneity in the mouse oviduct-- its potential roles in reproduction and preimplantation development

ABSTRACTThe oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive tissue that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are basic coiling patterns and turning points in the coiled mouse oviduct can serve as reliable landmarks for luminal morphological regionalities. We identified previously unrecognized anatomical structures in the isthmus and uterotubal junction (UTJ) that likely play important roles in reproduction. Interestingly, during ovulation, the isthmus was transiently plugged by a thick mucus, keeping the oocytes within the ampulla. Preimplantation embryos travelled along the oviduct and formed a queue within small compartments of the UTJ before uterine entry. Taken together, the oviduct luminal epithelium had highly diverse luminal structures with distinct cell populations reflecting its complex functions in reproduction.