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Verónica M Negrón-Pérez, Yanping Zhang, and Peter J Hansen

The first two differentiation events in the embryo result in three cell types – epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2. Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17, while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN. The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype.

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Qian Chen, Yong Fan, Xiaowei Zhou, Zheng Yan, Yanping Kuang, Aijun Zhang, and Chen Xu

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

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Lan Xiao, Qiong Zhang, Xi Huang, Aihua He, Shi Xie, and Yanping Li

Uterine peristalsis plays a vital role in fertility and female reproductive health. Although uterine peristalsis is thought to be correlated with some hormones and uterine pathologies, the physiological mechanisms underlying uterine peristalsis remain not quite clear. This study aimed to identify changes in miRNA in the endometrium of patients with abnormally high-frequency (hyper-) and low-frequency (hypo-) peristalsis to clarify whether miRNAs regulate uterine peristalsis. We used a miRNA microarray and RT-qPCR to identify changes in miRNA in endometrial tissue, a collagen gel contraction assay on co-cultured human endometrial stromal cells (ESCs) to analyze how the altered regulation of miRNAs influences uterine smooth muscle (USM) contraction, Western blots and other assays to elucidate the potential mechanisms involved. We found that among several differentially regulated miRNAs, miR-29c-3p was overexpressed in endometrial samples from patients with hypoperistalsis; oxytocin receptor (OXTR) expression was low in endometrial samples from patients with hypoperistalsis. Bioinformatic analysis and luciferase assays indicated that OXTR is a target of miR-29c-3p, which attenuates its expression. Additionally, downregulation of miR-29c-3p in ESC cultures increased the expression of aldo-keto reductase family 1, member C3 (AKR1C3) and increased the release of prostaglandin F2 alpha (PGF2α). Co-cultured ESCs overexpressing miR-29c-3p reduced USM cell contractions; the opposite tendency was found when ESCs were transfected with a miR-29c-3p inhibitor. To conclude, miR-29c-3p in endometrial cells regulates uterine contractility by attenuating the expression of OXTR and reducing PGF2α release.