Sojung Kwon

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy.

Abstract.  Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.

Dynamic interaction of formin proteins and cytoskeleton in mouse oocytes during meiotic maturation

Formin-2 (Fmn2) nucleates actin filaments required for spindle migration during the metaphase of meiosis I in mouse oocytes. While recent studies showed that Fmn2 is involved in the formation of a dynamic actin meshwork on meiotic spindle and the migration of chromosomes, the precise location and the mechanism of action of Fmn2 in the mouse oocyte is not known. In this work, we show that Fmn2 is colocalized with spindle during metaphase I (MI) and this pattern is lost in nocodazole-treated oocytes. Fmn2 directly interacts with polymerized microtubules (MTs) in vitro via a well-conserved domain called formin homology 2 (FH2). Microinjection of mRNA encoding formin homology 1 (FH1)FH2 domains of Fmn2 into Fmn2-/- oocytes partially rescued the defect of polar body extrusion, while mRNAs encoding FH2 domain alone could not rescue the defect. mDia1 and mDia2, Diaphanous (Dia) subfamily of formin proteins, exhibit unique patterns of expression in mouse oocytes. While mDia1 is localized on meiotic spindle, mDia2 localization is confined in spindle poles similar to γ-tubulin. Collectively, our results suggest that the ability of Fmn2 to directly interact with MTs and to polymerize actins via the conserved FH1FH2 domains is crucial for chromosomal migration in MI oocytes. We also show that mDia1 and mDia2 are dynamic components of meiotic spindle and pole complex during meiotic maturation of oocytes.

Uncovering a role for endocannabinoid signaling in autophagy in preimplantation mouse embryos

Endocannabinoid signaling plays various roles in directing reproductive processes. Mouse embryos are shown to express high levels of CB1 receptor (CB1R). Low concentrations of anandamide stimulate embryo growth and implantation but at higher concentrations it adversely affects implantation. We tested the hypothesis that high levels of endocannabinoids cause autophagic activation and cell death in preimplantation mouse embryos. We used methanandamide (METH), a selective CB1R agonist, to examine the effect of heightened endocannabinoid signaling on autophagy in mouse embryos. Western blotting, immunofluorescence staining, transmission electron microscopy and TUNEL analysis were performed. We observed that METH treatment in vitro or in vivo up-regulated autophagic response in preimplantation mouse embryos. In blastocysts, apoptosis was also increased after METH injections. At 28 nM, which is considered a high physiological dose to embryonic cells, METH up-regulated autophagic activation in trophoblast stem cells. This work demonstrates for the first time that blastocysts respond to higher than normal levels of endocannabinoid by increasing autophagic activation and apoptosis.

Complex ovarian defects lead to infertility in Etv5-/- female mice

Etv5 is a member of the Etv4 subfamily of Ets transcription factors. In female mice, Etv5 was previously shown to be expressed in the mouse ovary. In this work, we show that Etv5-/- female mice are infertile due to a complex reproductive phenotype. Defects in the ovarian tissue architecture were noted as early as 2 weeks of age in Etv5-/- mice. Adult Etv5-/- female mice show decreased ovulation and no interest in mating even after gonadotrophin treatment. Histological abnormalities were also noted in Etv5-/- ovaries. Injection of 17β-estradiol to gonadotrophin-treated Etv5-/- mice significantly increased ovulation, mating and fertilization rates. However, 2-cell embryos of Etv5-/- females show compromised development, suggesting a role for Etv5 in the developmental competence of embryos. Expression of aromatase (CYP11a1), 17α-hydroxylase/17,20 lyase/17,20 desmolase (CYP17a1), side-chain-cleaving enzyme (CYP19a1) and luteinizing hormone/choriogonadotropin receptor mRNAs was not significantly altered in Etv5-/- ovaries. Collectively, our results suggest that Etv5 is important for the developmental competence of germ cells and the regulation of responses to steroid hormones in female mice.

Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers

The maturation process of mammalian oocytes accompanies an extensive rearrangement of the cytoskeleton and associated proteins. As this process requires a delicate interplay between the cytoskeleton and its regulators, it is often targeted by various external and internal adversaries that affect the congression and/or segregation of chromosomes. Asymmetric cell division in oocytes also requires specific regulators of the cytoskeleton, including formin-2 and small GTPases. Recent literature providing clues regarding how actin filaments and microtubules interact during spindle migration in mouse oocytes are highlighted in this review.

The Transcription Factor Egr3 Is a Putative Component of the Microtubule Organizing Center in Mouse Oocytes

The early growth response (Egr) family of zinc finger transcription factors consists of 4 members. During an investigation of Egr factor localization in mouse ovaries, we noted that Egr3 exhibits a subcellular localization that overlaps with the meiotic spindle in oocytes. Using Egr3-specific antibodies, we establish that Egr3 co-localizes with the spindle and cytosolic microtubule organizing centers (MTOCs) in oocytes during meiotic maturation. Notably, the Egr3 protein appears to accumulate around γ-tubulin in MTOCs. Nocodazole treatment, which induces microtubule depolymerization, resulted in the disruption of spindle formation and Egr3 localization, suggesting that Egr3 localization is dependent on the correct configuration of the spindle. Shortly after warming of vitrified oocytes, growing arrays of microtubules were observed near large clusters of Egr3. An in vitro microtubule interaction assay showed that Egr3 does not directly interact with polymerized microtubules. Egr3 localization on the spindle was sustained in early preimplantation mouse embryos, but this pattern did not persist until the blastocyst stage. Collectively, our result shows for the first time that the Egr3 a transcription factor may play a novel non-transcriptional function during microtubule organization in mouse oocytes.

Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12-EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12-EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12-EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP-LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP-LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.