Regulation of autophagy during embryo-uterine crosstalk

Hyunjung (Jade) Lim 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

Autophagy means “self-eating” and it is a major catabolic pathway within cells. A basal level of autophagy is required for survival of cells or organisms, but prolonged activation of autophagy may have an adverse effect. In mammalian systems, autophagy is stimulated by nutrient starvation or deprivation of growth factors. Ovariectomy on day 4 of pregnancy in mice to deprive blastocysts of estrogen induces “dormancy” in blastocysts and delay the process of implantation until estrogen is given. Dormant blastocysts maintain a state of low metabolism in utero and survive for many days without initiating implantation under the unfavorable condition of estrogen deficiency. We tested the hypothesis if an autophagic response is operative in dormant blastocysts for prolonged survival in utero during the delayed implantation. We observed that autophagy is highly activated in dormant blastocysts. Interestingly, autophagic activation is more prominent in trophectoderm than in inner cell mass. Activation of blastocysts by estrogen supplementation induces formation of multivesicular bodies and exosomes in the trophectoderm. Dormant blastocysts with longer period of autophagic activation show compromised development after implantation. Thus, autophagy may be a critical cellular mechanism to provide energy source during extended survival of dormant blastocysts. However, prolonged activation of autophagy may compromise developmental outcome of blastocysts with irreparable cellular damage.

Uterine disorders and pregnancy complications: insights from mouse models

Lim, Hyunjung Jade,Wang, Haibin American Society for Clinical Investigation 2010 The Journal of clinical investigation Vol.120 No.4

<P>Much of our knowledge of human uterine physiology and pathology has been extrapolated from the study of diverse animal models, as there is no ideal system for studying human uterine biology in vitro. Although it remains debatable whether mouse models are the most suitable system for investigating human uterine function(s), gene-manipulated mice are considered by many the most useful tool for mechanistic analysis, and numerous studies have identified many similarities in female reproduction between the two species. This Review brings together information from studies using animal models, in particular mouse models, that shed light on normal and pathologic aspects of uterine biology and pregnancy complications.</P>

Autophagy in the uterine vessel microenvironment: Balancing vasoactive factors

Lim, Hyunjung Jade The Korean Society for Reproductive Medicine 2020 Clinical and Experimental Reproductive Medicine Vol.47 No.4

Autophagy, which has the literal meaning of self-eating, is a cellular catabolic process executed by arrays of conserved proteins in eukaryotes. Autophagy is dynamically ongoing at a basal level, presumably in all cells, and often carries out distinct functions depending on the cell type. Therefore, although a set of common genes and proteins is involved in this process, the outcome of autophagic activation or deficit requires scrutiny regarding how it affects cells in a specific pathophysiological context. The uterus is a complex organ that carries out multiple tasks under the influence of cyclic changes of ovarian steroid hormones. Several major populations of cells are present in the uterus, and the interactions among them drive complex physiological tasks. Mouse models with autophagic deficits in the uterus are very limited, but provide an initial glimpse at how autophagy plays a distinct role in different uterine tissues. Herein, we review recent research findings on the role of autophagy in the uterine mesenchyme in mouse models.

Steroid hormonal regulation of the autophagic response in the mouse uterus

Soyoung Choi,Hyunjung Jade Lim 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

Autophagy is a major cellular catabolic pathway and is tightly associated with survival and death of cells. The involvement of autophagy during prolonged survival of blastocysts in the uterus is established and it was assumed that ovarian steroid hormones – estrogen (E2) and progesterone (P4) – play important roles in its regulation. The uterus is a major target organ of E2 and P4. To examine if E2 or P4 modulate autophagy in the mouse uterus in vivo, the following three systems were used. 1) Normal pregnancy model (days 1 to 8); 2) delayed implantation model; 3) ovariectomized (OVX) mice model treated with single steroid hormone. Six-week-old virgin ICR mice were used for pregnancy and OXV. OVX mice received P4 (1 mg/0.1 ml) or E2 (100 ng/0.1 ml) after 12 days of rest. Collected uteri were subjected to Western blotting and immunofluorescence staining using anti-LC3B antibody to monitor autophagy. In pregnant mouse uterus, the autophagic response was downregulated after implantation. In OVX model, either E2 or P4 injection downregulated the autophagic response in the uterus within several hours. To confirm whether hormone-induced downregulation is mediated by classical estrogen receptor (ER) and progesterone receptor (PR), receptor antagonists (ICI 182,780 and RU-486) were co-treated. Antagonist-treated uteri showed recovery of autophagic response, suggesting that ER or PR mediates hormonal effects on autophagy. In oder to determine which signaling pathway is involved in autophagic regulation by E2, rapamycin (5 mg/kg), a mTOR inhibitor, and LY294002 (5 mg/kg), a PI3 kinase inhibitor, were used. Rapamycin and LY294002 were injected just before E2 injection to OVX mice. Western blotting was performed by using anti-phospho-mTOR and anti-AKT antibodies. We observed that rapamycin treatment partially antagonized downregulation of autophagic activation by E2, whereas LY294002 treatment did not have any effect. Therefore, downregulation of autophagy by E2 seems to be partially mediated by mTOR pathway. Collectively, this study suggests that ovarian steroid hormones are upstream controllers of autophagic response in the mouse uterus.

The effect of Atg7 deletion on the uterine autophagy

Jaekyoung Park,Hyunjung Jade Lim 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

Autophagy is a self-degradative process which accompanies the formation of double-membraned vesicles inside the cell. In the mouse uterus, autophagy is enhanced during steroid hormone deprivation and associated with acute inflammation. There are 17 major Autophagy related genes (Atg). Herein we investigated the role for Atg7 by using uterine cell-specific deletion model of this gene. We crossed Atg7flox/flox (Atg7f/f) mouse and Anti-Mullerian hormone type 2 receptor (Amhr2)-Cre mice (Amhr2-Cre; Atg7f/f). Amhr2 is mainly expressed in stroma and myometrium in the uterus, ovary, and oviduct, during 30 to 60 days. To confirm the region of Cre expression and to monitor whether conditional deletion of Atg7 was by Cre recombinase, we isolated uterine epithelial and stromal cells from 8 and 16 weeks mice by enzymatic digestion and performed RT-PCR. We confirmed that Amhr2-Cre is expressed in stoma and myomotrium, but not in epithelium. Then we examined the uterine histology and embryonic development of day 3 pregnant Amhr2-Cre; Atg7f/f mice. However, there was no specific difference between Atg7f/f (control) and Amhr2-Cre; Atg7f/f mice. To examine the effect of hormone deprivation, we performed western blotting and immunofluorescence staining of p62 (SQSTM1), an indicator of autophagic flux, and LC3B, a marker of autophagic activation, in Amhr2-Cre; Atg7f/f mice ovariectomized (OVX) for 2 weeks. p62 increased dramatically in OVX Amhr2-Cre; Atg7f/f uteri but not in control mice, suggesting that autophagic activation did not occur in the absence of Atg7 in the uterine stroma and that this led to massive accumulation of p62 in this cell type. p62 marks to-be-degraded proteins and target them for autophagic-lysosomal degradation. Thus it is predictable that Atg7-driven uterine autophagy is responsible for degradation of macromolecules during hormone deprivation.

The formation of multivesicular bodies in the trophectoderm is associated with autophagic activation in dormant and activated blastocysts in mice

Hyejin Shin,Soyoung Bang,Hyunjung Jade Lim 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

Induction of autophagy in the vitrified-warmed mouse oocytes

Soyoung Bang,Hyejin Shin,Hyunjung Jade Lim 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

Vitrification uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitrified-warmed oocytes has not been studied. In this work, we investigated if vitrification-warming process induces autophagy in mouse oocytes. Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitification solutions (1.3 M EG+1.1 M DMSO and 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 2.5 min) and warming solutions (0.5 M, 0.25, 0,125, and 0 M sucrose at intervals 2.5 min). Group3: vitrified-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2 weeks). RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitrification-warming process on autophagy in oocytes. In RT-PCR analyses, expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 was examined. Expression of Atg7 and Atg12 was slightly reduced in Group 3 (vitrified-warmed oocytes). The expression levels of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitrified-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Our results suggest that induction of autophagy may serve as an indicator of conditions of vitrification-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efficacy of oocyte cryopreservation.

Etv5, a transcription factor with versatile functions in male reproduction

Eo, Jinwon,Song, Haengseok,Lim, Hyunjung Jade The Korean Society for Reproductive Medicine 2012 Clinical and Experimental Reproductive Medicine Vol.39 No.2

Transcription factors govern diverse aspects of cell growth and differentiation as major switches of gene expression. Etv5, a member of the E26 transformation-specific family of transcription factors, has many stories to share when it comes to reproduction. Etv5 deficient mice show complex infertility phenotypes both in males and females. In males, the infertility phenotype exhibited by Etv5 deficiency is sexually dimorphic, and it involves both somatic cells and germ cells. In $Etv5^{-/-}$ female mice, the problem is more complicated by hormonal involvement. This review synthesizes old and new information on this versatile transcription factor-from the inadvertent discovery of its role in the testes to its newly discovered role in maintaining spermatogonial stem cells.

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

Kwon, Sojung,Shin, Hyejin,Lim, Hyunjung Jade Published for the European Society for Human Repro 2011 Molecular human reproduction Vol.17 No.5

<P>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.</P>

LDP12, a novel cell-permeable peptide derived from L1 capsid protein of the human papillomavirus

Lee, Jong-Eun,Lim, Hyunjung Jade Springer-Verlag 2012 MOLECULAR BIOLOGY REPORTS Vol.39 No.2