Extracellular Vesicles From a Three-Dimensional Culture of Perivascular Cells Accelerate Skin Wound Healing in a Rat.

BACKGROUND: Soluble proteins and extracellular vesicles (EVs) are crucial wound repair mediators in cell-based therapy. Previous studies reported that EVs of perivascular cells stimulated migration and proliferation of cell types involved in the dermatological wound healing process. However, these studies only show effects of EVs from perivascular cells (PVCs) for in vitro models. METHODS: EVs were collected from 3D-cultured PVC (PVC-3D-EV) and compared with EVs from 2D-culture PVC (PVC-2D-EV) to investigate effects on wound contraction, angiogenesis, activation of myofibroblast, and collagen deposition. RESULTS: PVC-3D-EV was significantly improved in terms of wound contraction compared with PVC-2D-EV and the control. Activation of myofibroblast and collagen deposition in a rat skin wound model was significantly stimulated by PVC-3D-EV. In addition, angiogenesis and vascular endothelial growth factor expression were also highly stimulated by PVC-3D-EV. These results suggest that PVC-3D-EV was regulated in granulation tissue formation, angiogenesis, and wound contraction in healing of a rat skin wound. These results indicate a pivotal role of PVC-3D-EV in wound healing through multiple mechanisms. CONCLUSIONS: 3D-culture using a polystyrene scaffold is demonstrated to be a better system for providing better physiological conditions than the 2D-culture system, and EVs from 3D-cultured PVC could be a promising option for healing skin wound. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Loss-of-function of EBP50 is a new cause of hereditary peripheral neuropathy: EBP50 functions in peripheral nerve system.

Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50+/- heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.

Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre- and peri-implantation embryonic development in mice.

Recently, many studies have investigated the role of extracellular vesicles (EVs) on reproductive events, including embryo development and death, oviduct-embryo crosstalk, in vitro fertilization and others. The aim of this study was to demonstrate whether outgrowth embryo-derived EVs function as bioactive molecules and regulate mouse embryonic developmental competence in vitro and implantation potential in utero. The EVs from mouse outgrowth embryos on 7.5 days postcoitum were detected and selectively isolated to evaluate the embryotrophic functions on preimplantation embryos. Developmental outcomes such as the percentage of blastocyst formation, hatching, and trophoblastic outgrowth were assessed. Furthermore, the total cell number and apoptotic index of blastocysts, which were incubated with EVs during the culture period, were evaluated by fluorescence microscopy. Implantation potential in utero was investigated following embryo transfer. The EVs from outgrowth embryo-conditioned media have rounded membrane structures that range in diameter from 20 to 225 nm. Incubation with EVs improved preimplantation embryonic development by increasing cell proliferation and decreasing apoptosis in blastocysts. Moreover, the implantation rates following embryo transfer were significantly higher in EV-supplemented embryos compared with the control. Collectively, EVs from outgrowth embryo could enhance the embryonic developmental competence and even implantation potential in mice.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Identification of differentially expressed microRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos in mice.

Recurrent implantation failure (RIF) is one of the main causes for the repeated failure of IVF, and the major reason for RIF is thought to be a miscommunication between the embryo and uterus. However, the exact mechanism underlying embryo-uterus cross-talk is not fully understood. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) among blastocysts, non-outgrowth and outgrowth embryos in mice using microarray analysis. A bioinformatics analysis was performed to predict the potential mechanisms of implantation. The miRNA expression profiles differed significantly between non-outgrowth and outgrowth embryos. In all, 3163 miRNAs were detected in blastocysts and outgrowth embryos. Of these, 10 miRNA candidates (let-7b, miR-23a, miR-27a, miR-92a, miR-183, miR-200c, miR-291a, miR-425, miR-429 and miR-652) were identified as significant differentially expressed miRNAs of outgrowth embryos by in silico analysis. The expression of the miRNA candidates was markedly changed during preimplantation embryo development. In particular, let-7b-5p, miR-200c-3p and miR-23a-3p were significantly upregulated in outgrowth embryos compared with non-outgrowth blastocysts. Overall, differentially expressed miRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos could be involved in embryo attachment, and interaction between the embryo proper and maternal endometrium during the implantation process.

Galectin-1 from conditioned medium of three-dimensional culture of adipose-derived stem cells accelerates migration and proliferation of human keratinocytes and fibroblasts.

Keratinocytes and fibroblasts cells play important roles in the skin-wound healing process and are the cell types activated by trauma. Activated cells participate in epithelialization, granulation, scar tissue formation, wound remodeling, and angiogenesis via a series of cellular activities including migration and proliferation. Previous studies reported that the conditioned medium (CM) of adipose-derived stem cells (ADSCs) stimulated the migration and proliferation of cell types involved in the skin wound healing process; however, these studies only show ADSC-CM effects that were obtained using 2-dimensional (2D) culture. Recently, 3-dimensional (3D) culture has been considered as a more physiologically appropriate system than 2D culture for ADSC cultures; therefore, ADSC-CM was collected from 3D culture (ADSC-CM-3D) and compared with ADSC-CM from 2D culture (ADSC-CM-2D) to investigate the effects on the migration and proliferation of human keratinocytes (HaCaTs) and fibroblasts. The migrations of the HaCaT cells and fibroblasts were significantly higher with ADSC-CM-3D compared with the 2D culture; similarly, the proliferation of HaCaT cells was also highly stimulated by ADSC-CM-3D. Proteomic analyses of the ADSC-CM revealed that collagens and actins were highly expressed in the 3D-culture system. Chitinase 3-like 1 (CHI3L1), tissue inhibitor of metalloproteinases (TIMP), and galectin-1 were specifically expressed only in ADSC-CM-3D. Especially, through antibody neutralization, galectin-1 in ADSC-CM-3D was found to be an important factor for the migration of human keratinocytes. Therefore, these results suggest that ADSC-CM-3D was more effective in the wound healing than ADSC-CM-2D, and galectin-1 in ADSC-CM-3D was could be a promising option for skin-wound healing. Furthermore, the differential expressions of several ADSC-CM proteins between the 2D- and 3D-culture systems may be used as basic information for the development of efficient wound-healing strategies.

Prediction of blastocyst development and implantation potential in utero based on the third cleavage and compaction times in mouse pre-implantation embryos.

Cytokinesis and cell division during pre-implantation embryonic development occur as an orchestrated spatiotemporal program. Cleavage, compaction, and blastulation in pre-implantation embryos are essential for successful implantation and pregnancy. Their alteration is associated with chromosomal imbalance and loss of developmental competence. In this study, we evaluated the time of cleavage and compaction as predictors for in vitro pre- and peri-implantation development and in utero implantation potential by time-lapse monitoring. Mouse 2-cell embryos were collected on 1.5 days post coitum (dpc) and were individually cultured to the outgrowth (OG) stage (7.5 dpc). Developmental stages were classified as 3-cell, 4-cell, 8-cell, morula, blastocyst, and OG. Cut-off times for successful blastocyst development were determined by receiver operating characteristic curve analysis. When cut-off times were set as 9 h for the third cleavage from the 2- to 4-cell stage, and 40 h for compaction from the 2-cell to morula stage, blastocyst and OG development rates, respectively, were significantly higher (P < 0.0001). Embryos were grouped according to the above cut-off time and transferred to the contralateral uterine horn on 3.5 dpc. Implantation rates in utero on 5.5 dpc were significantly higher in early third cleaved (≤ 9 h from 2- to 4-cell) and early compacted embryos (≤ 40 h from 2-cell to morula) than those in delayed embryos (P < 0.05). Therefore, the time of the third cleavage from 2- to the 4-cell stage and compaction from 2-cell to morula stage may be a useful morphokinetic parameter for predicting developmental potential, including successful implantation and pregnancy in human in vitro fertilization-embryo transfer programs.

Melatonin attenuates dextran sodium sulfate induced colitis with sleep deprivation: possible mechanism by microarray analysis.

BACKGROUND: Inflammatory bowel disease is a chronic inflammatory condition of the gastrointestinal tract. It can be aggravated by stress, like sleep deprivation, and improved by anti-inflammatory agents, like melatonin. We aimed to investigate the effects of sleep deprivation and melatonin on inflammation. We also investigated genes regulated by sleep deprivation and melatonin. METHODS: In the 2% DSS induced colitis mice model, sleep deprivation was induced using modified multiple platform water bath. Melatonin was injected after induction of colitis and colitis with sleep deprivation. Also mRNA was isolated from the colon of mice and analyzed via microarray and real-time PCR. RESULTS: Sleep deprivation induced reduction of body weight, and it was difficult for half of the mice to survive. Sleep deprivation aggravated, and melatonin attenuated the severity of colitis. In microarrays and real-time PCR of mice colon tissues, mRNA of adiponectin and aquaporin 8 were downregulated by sleep deprivation and upregulated by melatonin. However, mRNA of E2F transcription factor (E2F2) and histocompatibility class II antigen A, beta 1 (H2-Ab1) were upregulated by sleep deprivation and downregulated by melatonin. CONCLUSION: Melatonin improves and sleep deprivation aggravates inflammation of colitis in mice. Adiponectin, aquaporin 8, E2F2 and H2-Ab1 may be involved in the inflammatory change aggravated by sleep deprivation and attenuated by melatonin.

Association of inflammation, myocardial fibrosis and cardiac remodelling in patients with mild aortic stenosis as assessed by biomarkers and echocardiography.

1. The aim of the present study was to investigate the relationships among inflammation, myocardial fibrosis and cardiac remodelling in patients with mild aortic stenosis (AS), as assessed by biomarkers and echocardiography. 2. We evaluated 32 consecutive patients with mild AS, as well as 30 age- and gender-matched healthy individuals with normal aortic valves as control subjects. 3. Baseline echocardiography showed that the left ventricular (LV) mass index (111.3 ± 26.9 vs 94.5 ± 18.2 g/m(2); P = 0.006) and left atrial (LA) volume index (LAVI 27.5 ± 9.0 vs xx.x ± 5.2 mm(3)/mm(2); P = 0.005) were significantly higher in patients with mild AS. 4. Furthermore, LA enlargement (LAVI > 33 mm(3)/mm(2); 32.4% vs 3.3%; P = 0.003) and elevated LV filling pressure (E/e' > 15; 50.0% vs 23.3%; P = 0.036) were higher in patients with mild AS. 5. In patients with mild AS, stepwise, multivariate linear regression analysis revealed that the LV end-diastolic volume index was independently associated with matrix metalloproteinase (MMP)-1 (ß = 0.371; P = 0.015), that the aortic valve mean pressure gradient was independently associated with MMP-2 (ß = 0.19; P = 0.019), that MMP-2 was independently associated with transforming growth factor-ß (ß = 0.95; P < 0.001) and interleukin (IL)-1 (ß = 0.17; P = 0.019) and that IL-1 was independently associated with tissue inhibitor of matrix metalloproteinase-1 (ß = 0.68; P = 0.001). 6. Myocardial fibrosis in mild AS is independently associated with three factors: LV volume overload, aortic valve pressure overload and inflammation.

Synergistic effect of interleukin-6 and hyaluronic acid on cell migration and ERK activation in human keratinocytes.

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-κB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-κB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.

Effect of nicotinamide mononucleotide on osteogenesis in MC3T3-E1 cells against inflammation-induced by lipopolysaccharide.

Objective: Nicotinamide mononucleotide (NMN) is extensively utilized as an anti-aging agent and possesses anti-inflammatory properties. Lipopolysaccharide (LPS) activates Toll-like receptor 4, a process modulated by intracellular signaling pathways such as the Wnt/ß-catenin pathway. This study investigated the impact of NMN on osteogenesis in the presence of LPS. Methods: To elucidate the role of NMN in osteogenesis in the context of Gram-negative bacterial infection after LPS treatment, we cultured a mouse pre-osteoblast cell line (MC3T3-E1) and subsequently incubated it with NMN and/or LPS. We then evaluated osteogenic activity by measuring alkaline phosphatase activity, assessing gene expression and protein levels, and performing Alizarin Red S staining and immunocytochemistry. Results: MC3T3-E1 cells underwent successful differentiation into osteoblasts following treatment with osteogenic induction medium. LPS diminished features related to osteogenic differentiation, which were subsequently partially reversed by treatment with NMN. The restorative effects of NMN on LPS-exposed MC3T3-E1 cells were further substantiated by elucidating the role of Wnt/ß-catenin signaling, as confirmed through immunocytochemistry. Conclusion: This study showed that infection with Gram-negative bacteria disrupted the osteogenic differentiation of MC3T3-E1 cells. This adverse effect was partially reversed by administering a high-dose of NMN. Drawing on these results, we propose that NMN could serve as a viable therapeutic strategy to preserve bone homeostasis in elderly and immunocompromised patients.

Ovastacin: An oolemma protein that cleaves the zona pellucida to prevent polyspermy.

Monospermy occurs in the process of normal fertilization where a single sperm fuses with the egg, resulting in the formation of a diploid zygote. During the process of fertilization, the sperm must penetrate the zona pellucida (ZP), the outer layer of the egg, to reach the egg's plasma membrane. Once a sperm binds to the ZP, it undergoes an acrosomal reaction, which involves the release of enzymes from the sperm's acrosome that help it to penetrate the ZP. Ovastacin is one of the enzymes that is involved in breaking down the ZP. Studies have shown that ovastacin is necessary for the breakdown of the ZP and for successful fertilization to occur. However, the activity of ovastacin is tightly regulated to ensure that only one sperm can fertilize the egg. One way in which ovastacin helps to prevent polyspermy (the fertilization of an egg by more than one sperm) is by rapidly degrading the ZP after a sperm has penetrated it. This makes it difficult for additional sperm to penetrate the ZP and fertilize the egg. Ovastacin is also thought to play a role in the block to polyspermy, a mechanism that prevents additional sperm from fusing with the egg's plasma membrane after fertilization has occurred. In summary, the role of ovastacin in monospermic fertilization is to help ensure that only one sperm can fertilize the egg, while preventing polyspermy and ensuring successful fertilization.

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity.

OBJECTIVE: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. METHODS: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. RESULTS: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. CONCLUSION: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

Non-invasive evaluation of embryo quality for the selection of transferable embryos in human in vitro fertilization-embryo transfer.

The ultimate goal of human assisted reproductive technology is to achieve a healthy pregnancy and birth, ideally from the selection and transfer of a single competent embryo. Recently, techniques for efficiently evaluating the state and quality of preimplantation embryos using time-lapse imaging systems have been applied. Artificial intelligence programs based on deep learning technology and big data analysis of time-lapse monitoring system during in vitro culture of preimplantation embryos have also been rapidly developed. In addition, several molecular markers of the secretome have been successfully analyzed in spent embryo culture media, which could easily be obtained during in vitro embryo culture. It is also possible to analyze small amounts of cell-free nucleic acids, mitochondrial nucleic acids, miRNA, and long non-coding RNA derived from embryos using real-time polymerase chain reaction (PCR) or digital PCR, as well as next-generation sequencing. Various efforts are being made to use non-invasive evaluation of embryo quality (NiEEQ) to select the embryo with the best developmental competence. However, each NiEEQ method has some limitations that should be evaluated case by case. Therefore, an integrated analysis strategy fusing several NiEEQ methods should be urgently developed and confirmed by proper clinical trials.

Effects and safety of COVID-19 vaccination on assisted reproductive technology and pregnancy: A comprehensive review and joint statements of the KSRM, the KSRI, and the KOSAR.

Humanity is in the midst of the coronavirus disease 2019 (COVID-19) pandemic, and vaccines-including mRNA vaccines-have been developed at an unprecedented speed. It is necessary to develop guidelines for vaccination for people undergoing treatment with assisted reproductive technology (ART) and for pregnancy-related situations based on the extant laboratory and clinical data. COVID-19 vaccines do not appear to adversely affect gametes, embryos, or implantation; therefore, active vaccination is recommended for women or men who are preparing for ART. The use of intravenous immunoglobulin G (IVIG) for the treatment of immune-related infertility is unlikely to impact the effectiveness of the vaccines, so COVID-19 vaccines can be administered around ART cycles in which IVIG is scheduled. Pregnant women have been proven to be at risk of severe maternal and neonatal complications from COVID-19. It does not appear that COVID-19 vaccines harm pregnant women or fetuses; instead, they have been observed to deliver antibodies against severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) to the fetus. Accordingly, it is recommended that pregnant women receive COVID-19 vaccination. There is no rationale for adverse effects, or clinical cases of adverse reactions, in mothers or neonates after COVID-19 vaccination in lactating women. Instead, antibodies to SARS-CoV-2 can be delivered through breast milk. Therefore, breastfeeding mothers should consider vaccination. In summary, active administration of COVID-19 vaccines will help ensure the safe implementation of ART, pregnancy, and breastfeeding.

Ovarian Tissue-Based Hormone Replacement Therapy Recovers Menopause-Related Signs in Mice.

PURPOSE: In women, menopause manifests with a variety of symptoms related to sex-hormone deficiency. Supplementing steroid hormones with pharmacological drugs has been widely practiced. However, considering the possible complications associated with artificial hormone therapy, studies have been conducted to find an alternative to pharmacological hormone replacement therapy. Accordingly, this study aimed to evaluate the efficacy of tissue-based hormone replacement therapy (tHRT) for treating post-menopausal signs and symptoms. MATERIALS AND METHODS: CD-1 mice were ovariectomized, and the ovaries were cryopreserved. Following artificial induction of post-menopausal osteoporosis, cryopreserved ovaries were subcutaneously autografted, and indexes related to bone health were monitored for 12 weeks. Bone mineral density (BMD), bone mineral contents (BMC), total bone volume (BV), and body fat mass were measured by dual energy X-ray absorptiometry. Uterine atrophy was assessed histologically, and bone microstructures were imaged by micro-computed tomography analysis. RESULTS: Regardless of the number of grafted ovaries, the BMC, BMD, and BV values of mice that underwent ovary transplantation were better than those that did not undergo transplantation. The uteruses in these mice were thicker and heavier after auto-transplantation. Furthermore, the bone microstructure recovered after tHRT. CONCLUSION: Recovery of menopause-related bone loss and uterine atrophy was achieved through tHRT. Ovarian tissue cryopreservation and transplantation may be applicable not only in patients wanting to preserve fertility but also in sex hormone-deficient post-menopausal women.

Peripubertal requirement of Tsg101 in maintaining the integrity of membranous structures in mouse oocytes.

OBJECTIVE: As a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte-specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age-dependent oocyte demise in mice. MATERIALS AND METHOD: Tsg101 floxed mice (Tsg101f/f ) were bred with Zp3cre transgenic mice to examine oocyte-specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101. RESULTS: The death of oocytes from Zp3cre /Tsg101f/f (Tsg101d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin-releasing hormone antagonist injections improved the survival rate of oocytes from 5-week-old Tsg101d/d mice. Maturation of oocytes from prepubertal Tsg101d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101d/d mice until 9 h. The depletion of tsg-101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity. CONCLUSIONS: Collectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.

Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice.

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction.

BACKGROUND: Mitochondria are the main sites for fatty acid oxidation and play a central role in lipotoxicity and nonalcoholic steatohepatitis. AIMS: We investigated whether carnitine prevents free fatty acid (FFA)-induced lipotoxicity in vitro and in vivo. METHODS: HepG2 cells were incubated with FFA, along with carnitine and carnitine complexes. Mitochondrial ß-oxidation, transmembrane potential, intracellular ATP levels and changes in mitochondrial copy number and morphology were analysed. Otsuka Long-Evans Tokushima Fatty and Long-Evans Tokushima Otsuka rats were segregated into three experimental groups and fed for 8 weeks with (i) normal chow, (ii) a methionine choline-deficient (MCD) diet or (iii) an L-carnitine-supplemented MCD diet. RESULTS: Carnitine prevented FFA-induced apoptosis (16% vs. 3%, P < 0.05). FFA treatment resulted in swollen mitochondria with increased inner matrix density and loss of cristae. However, mitochondria co-treated with carnitine had normal ultrastructure. The mitochondrial DNA copy number was higher in the carnitine treatment group than in the palmitic acid treatment group (375 vs. 221 copies, P < 0.05). The carnitine group showed higher mitochondrial ß-oxidation than did the control and palmitic acid treatment groups (597 vs. 432 and 395 ccpm, P < 0.05). Carnitine treatment increased the mRNA expression of carnitine palmitoyltransferase 1A and peroxisome proliferator-activated receptor-γ, and carnitine-lipoic acid further augmented the mRNA expression. In the in vivo model, carnitine-treated rats showed lower alanine transaminase levels and lesser lobular inflammation than did the MCD-treated rats. CONCLUSIONS: Carnitine and carnitine-lipoic acid prevent lipotoxicity by increasing mitochondrial ß-oxidation and reducing intracellular oxidative stress.

CD44 disruption attenuates murine hepatic ischemia/reperfusion injury.

Neutrophil adhesion and migration are critical in hepatic ischemia/reperfusion (I/R) injury. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy in reperfusion injury. Therefore, the aim of this study was to assess the role of CD44 in neutrophil infiltration and liver injury from hepatic I/R. In this study, using a partial hepatic ischemic model in vivo, we determined the potential role of CD44 in neutrophil infiltration and liver injury from I/R. Reperfusion caused significant hepatocellular injury as it was determined by plasma ALT levels and liver histopathology. The injury was associated with a marked neutrophil recruitment and CD44 expression into the ischemic livers. Administration of anti-CD44 antibody to mice reduced the infiltration of neutrophil into the ischemic tissue, associated with liver function preservation. These results support crucial roles of CD44 in neutrophil recruitment and infiltration leading to liver damage in hepatic I/R injury. Moreover, they provide the rationale for targeting to CD44 as a potential therapeutic approach in liver I/R injury.