No abstract

Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.

In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0Zday of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

No abstract

Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity.

No abstract

Amino acid transporters are the main mediators of nitrogen distribution throughout the plant body, and are essential for sustaining growth and development. In this review, we summarize the current state of knowledge on the identity and biological functions of amino acid transporters in plants, and discuss the regulation of amino acid transporters in response to environmental stimuli. We focus on transporter function in amino acid assimilation and phloem loading and unloading, as well as on the molecular identity of amino acid exporters. Moreover, we discuss the effects of amino acid transport on carbon assimilation, as well as their cross-regulation, which is at the heart of sustainable agricultural production.

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.