Progesterone-induced miR-152 interferes with embryonic implantation by downregulating GLUT3 in endometrial epithelium.

To investigate the role of progesterone-induced micro-RNA (miR)-152 in early embryonic development and implantation by regulating GLUT3 in endometrial epithelium, qRT-PCR was used to detect the expression of miR-152, GLUT1, and GLUT3 in the endometrial epithelial cells of female mice. GLUT1 and GLUT3 proteins were detected by immunohistochemical staining in the mouse endometrial epithelium. Bioinformatics prediction associated with a luciferase assay was performed to determine whether GLUT1 and GLUT3 are target genes of miR-152. Specific miR-152 mimics or inhibitors were transfected into the endometrial epithelial cells to, respectively, overexpress or downregulate miR-152. Next, the glucose concentration of uterine fluid was measured by conducting high-performance liquid chromatography in vivo, and the glucose uptake of the endometrial epithelial cells was observed using a fluorometric assay in vitro. Early embryonic development and implantation were also observed after the miR-152 mimics or inhibitors had been transfected. Embryo transfer was observed after the miR-152 mimic transfection. miR-152 was found to directly target and thereby downregulate GLUT3 expression. The expressions of both miR-152 and GLUT3 in the mouse endometrial epithelium had spatiotemporal characteristics on days 1-4 of pregnancy. miR-152 affected the glucose concentration of uterine fluid and the glucose uptake of endometrial epithelial cells. The transfection of specific miR-152 mimics led to impaired embryonic development and implantation. To conclude, in endometrial epithelial cells, progesterone-induced miR-152 downregulates GLUT3 at the posttranscriptional level to maintain a proper glucose concentration in the uterine fluid, which is necessary for early embryonic development and implantation.

Progesterone-Induced miR-145/miR-143 Inhibits the Proliferation of Endometrial Epithelial Cells.

Our previous study showed that progesterone (P4) can specifically regulate the expression of some microRNAs (miRNAs) in endometrial epithelium. In the present study, we verified the P4-dependent expression of miR-145/miR-143 in endometrial epithelial cells, explored the regulative mechanism of the P4 receptor (PR), and investigated their effects on the proliferation of endometrial epithelial cells. Our results showed that P4 can induce the expression of miR-145/143 in endometrial epithelial cells by acting on the PR A subtype. P4-induced miR-145/143 can inhibit the expression of cyclin D2 by binding to cyclin D2 mRNA 3'UTR. It can also inhibit cell proliferation in mouse endometrial epithelium by arresting the cell cycle during the G1-S checkpoint. Furthermore, miR-145 and miR-143 can inhibit the proliferation of human endometrial cancer cells. In conclusion, P4-induced miR-145/miR-143 is an important regulator in the proliferation of endometrial epithelial cells, and it can also inhibit the proliferation of human endometrial cancer cells. Our study indicates miRNAs are important mechanism of P4 in inhibiting the proliferation of endometrial epithelial cells. And these miRNAs are potential candidates for the diagnosis of endometrial cancer and therapeutic targets.

The Changes of Cytoskeletal Proteins Induced by the Fast Effect of Estrogen in Mouse Blastocysts and Its Roles in Implantation.

It is necessary for estrogen to activate mouse blastocysts, so that they can attach to endometrial epithelium in implantation and in our previous research, we have proved estrogen can induce a fast increase in intracellular calcium of mouse blastocysts through acting on G protein-coupled receptor 30 (GPR30), which further promotes their implantation. Moreover, there has been evidence that cytoskeletal proteins are involved in integrin-mediated adhesion of many kinds of cells, which also plays an important role in implantation. To prove estrogen induces rapidly the changes of cytoskeletal proteins in mouse blastocysts and its roles in implantation, we first used immunofluorescence staining and laser confocal microscopy to investigate the fast effect of estrogen on the expression and localization of cytoskeletal proteins in mouse blastocysts. Second, we used electroporation associated with RNA interference to knock down one of the important cytoskeletal proteins, talin, in the mouse blastocyst cells to investigate the fast effect of estrogen on the localization of integrins and the binding activity of integrins with their ligand fibronectin (FN). At last, mouse blastocysts with different treatments were cultured with FN or uterine epithelial cell line Ishikawa in vitro, respectively, and transferred into the bilateral uterine horns of recipient mice, to study the role of the fast effect of estrogen on cytoskeletal proteins in blastocysts adhesion and implantation. Our results indicated that estradiol (E2), E2 conjugated with bovine serum album (E2-BSA) and G-1 (a GPR30-specific agonist) could induce cytoskeletal protein talin, vinculin, and actin to cluster in the mouse blastocysts, while G15 (a GPR30-specific antagonist) and BAPTA (a calcium chelator) may block this effect induced by E2-BSA. Furthermore, E2-BSA could induce the clustering and relocalization of integrin ß1 and ß3 and increase the FN-binding activity of integrins in blastocyst cells, while E2-BSA could not induce these effects in the blastocysts pretreated with talin-small interfering RNA (siRNA). Meanwhile, the adhesion rate and implantation rate of blastocysts pretreated with talin-siRNA were significantly lower than those pretreated with control-siRNA. We provided the first evidence that the fast effect of estrogen might cause the clustering of the cytoskeletal proteins in mouse blastocyst cells and further induce the changes of localization and functional activity of integrins in the blastocyst cells, which play important roles in blastocyst implantation.

Progesterone-Induced miR-152 Inhibits the Proliferation of Endometrial Epithelial Cells by Downregulating WNT-1.

Progesterone (P4) is an important ovarian hormone that inhibits estrogen-dependent proliferation of endometrial epithelial cells (EECs). miR-152 has been reported to be a cell cycle regulator. In this study, we first demonstrated that P4 induced the expression of miR-152 in ovariectomized mice and Ishikawa cell. miR-152 was detected in the human endometrial cell lines that were stably transfected with P4 receptor. Results showed that P4 induced its expression through its receptor B subtype. Then, using the specific miRNA mimic and inhibitor, we proved that miR-152 impeded G1/S transition in the cell cycle of EECs and inhibited cellular proliferation via downregulating WNT-1 in mice and human endometrial cancer cell lines (Ishikawa, HEC-1-b, and KLE). miR-152 induced by P4 is an important inhibitor for the proliferation of EECs. miR-152 may be an important tumor suppressor microRNA in endometrial cancer.

Identification and characterization of progesterone- and estrogen-regulated MicroRNAs in mouse endometrial epithelial cells.

In endometrial epithelial cells, progesterone (P4) functions in regulating the cell structure and opposing the effects of estrogen. However, the mechanisms of P4 that oppose the effects of estrogen remain unclear. MicroRNAs (miRNAs) are important posttranscriptional regulators that are involved in various physiological and pathological processes. Whether P4 directly induces miRNA expression to antagonize estrogen in endometrial epithelium is unclear. In this study, total RNAs were extracted from endometrial epithelium of ovariectomized mice, which were treated with estrogen alone or a combination of estrogen and P4. MicroRNA high-throughput sequencing with bioinformatics analysis was used to identify P4-induced miRNAs, predict their potential target genes, and analyze their possible biological functions. We observed that 146 mature miRNAs in endometrial epithelial cells were significantly upregulated by P4. These miRNAs were extensively involved in multiple biological processes. The miRNA-145a demonstrated a possible function in the antiproliferative action of P4 on endometrial epithelial cells.

Optical dissection of stimulus-evoked retinal activation.

Better understanding of stimulus-evoked intrinsic optical signals (IOSs) in the retina promises new methodology for study and diagnosis of retinal function. Using a flood-illumination near infrared (NIR) light microscope equipped with high-speed CCD (80 Hz) and CMOS (1000 Hz) cameras, we validated depth-resolved enface imaging of fast IOSs in isolated retina of leopard frog. Both positive (increasing) and negative (decreasing) IOSs were observed at the photoreceptor and inner layers of the retina. The distribution of IOSs with opposite polarities showed a center-surround pattern. At the photoreceptor layer, negative IOSs dominated the center area illuminated by the stimulus light spot, while positive signals dominated the surrounding area. In contrast, at inner retinal layers, positive IOSs dominated the center area covered by the stimulus light spot, and negative IOSs were mainly observed in the surrounding area. Fast CMOS imaging disclosed rapid IOSs within 5 ms after the stimulus onset, and both ON and OFF optical responses were observed associated with a step light stimulus.

Intrinsic optical imaging of stimulus-modulated physiological responses in amphibian retina.

We demonstrated near-infrared light imaging of stimulus-modulated physiological activities in an isolated frog retina using transient intrinsic optical responses (TIORs). While low-strength visible-light stimuli evoked TIORs with positive polarity, strengthened stimuli elicited additional negative TIORs. Our experimental study and physiological analysis suggest that the negative TIORs are associated with photoreceptor response, and the positive TIORs result primarily from dynamic changes of postphotoreceptor response during retinal activation. In coordination with the sophisticated design of visible-light stimulation protocols, optical imaging of TIORs promises an important application for noninvasive assessment of retinal photoreceptor and inner neurons.

Light propulsion of microbeads with femtosecond laser pulses.

Milli-Joule, femtosecond laser pulses have been used to propel microbeads for the first time. The microbeads of three different materials (iron, glass, and polystyrene) are used, weighting from 0.84 mg to 1.4 mg with a diameter range of 0.7-1.1 mm. Experimental parameters such as focused beam spot diameter, pulse energy, and pulse width are carefully varied to investigate their respective influences on the specific ablative laser propulsion. It is found that both the momentum coupling efficiency and the overall energy conversion efficiency from light energy to kinetic energy are greater for shorter laser pulses. A typical value of the momentum coupling efficiency of 5.0 dyne/W for iron beads is obtained. It is also evident that for metallic and non-metallic microbeads the momentum coupling efficiency has different variation tendencies versus the focused beam spot diameter.