ABSTRACT
Purpose@#Preimplantation genetic testing for monogenic disorders (PGT-M) has been successfully used to prevent couples with monogenic disorders from passing them on to their child. Charcot–Marie–Tooth Disease (CMT) is a genetic disorder characterized by progressive extremity muscle degeneration and loss of sensory function. For the first time in Korea, we report our experience of applying single nucleotide polymorphism genotyping and karyomapping for PGT-M of CMT disease. @*Materials and Methods@#Prior to clinical PGT-M, preclinical tests were performed using genotypes of affected families to identify informative single-nucleotide polymorphisms associated with mutant alleles. We performed five cycles of in vitro fertilization PGT-M in four couples with CMT1A, CMT2A, and CMT2S in CHA Fertility Center, Seoul Station. @*Results@#From July 2020 through August 2021, five cycles of PGT-M with karyomapping in four cases with CMT1 and CMT2 were analyzed retrospectively. A total of 17 blastocysts were biopsied and 15 embryos were successfully diagnosed (88.2%).Ten out of 15 embryos were diagnosed as unaffected (66.7%). Five cycles of PGT-M resulted in four transfer cycles, in which four embryos were transferred. Three clinical pregnancies were achieved (75%) and the prenatal diagnosis by amniocentesis for all three women confirmed PGT-M of karyomapping. One woman delivered a healthy baby uneventfully and two pregnancies are currently ongoing. @*Conclusion@#This is the first report in Korea on the application of karyomapping in PGT-M for CMT patients. This study shows that karyomapping is an efficient, reliable and accurate diagnostic method for PGT-M in various types of CMT diseases.
ABSTRACT
Objective@#Endometrial fibrosis, the primary pathological feature of intrauterine adhesion, may lead to disruption of endometrial tissue structure, menstrual abnormalities, infertility, and recurrent pregnancy loss. At present, no ideal therapeutic strategy exists for this fibrotic disease. Eupatilin, a major pharmacologically active flavone from Artemisia, has been previously reported to act as a potent inducer of dedifferentiation of fibrotic tissue in the liver and lung. However, the effects of eupatilin on endometrial fibrosis have not yet been investigated. In this study, we present the first report on the impact of eupatilin treatment on transforming growth factor beta (TGF-β)-induced endometrial fibrosis. @*Methods@#The efficacy of eupatilin on TGF-β–induced endometrial fibrosis was assessed by examining changes in morphology and the expression levels of fibrosis markers using immunofluorescence staining and quantitative real-time reverse-transcription polymerase chain reaction. @*Results@#Eupatilin treatment significantly reduced the fibrotic activity of TGF-β–induced endometrial fibrosis in Ishikawa cells, which displayed more circular shapes and formed more colonies. Additionally, the effects of eupatilin on fibrotic markers including alpha-smooth muscle actin, hypoxia-inducible factor 1 alpha, collagen type I alpha 1 chain, and matrix metalloproteinase-2, were evaluated in TGF-β–induced endometrial fibrosis. The expression of these markers was highly upregulated by TGF-β pretreatment and recovered to the levels of control cells in response to eupatilin treatment. @*Conclusion@#Our findings suggest that suppression of TGF-β–induced signaling by eupatilin might be an effective therapeutic strategy for the treatment of endometrial fibrosis.
ABSTRACT
Objective@#Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. @*Methods@#Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. @*Results@#Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. @*Conclusion@#These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.
ABSTRACT
Background@#Extensive eyelid defects are extremely challenging to reconstruct. Although numerous procedures for reconstructing periorbital defects have been proposed, no method is universally used. However, the Tenzel flap is the most commonly used technique to reconstruct eyelid defects affecting one-third to two-thirds of the eyelid. @*Methods@#Recognizing the usefulness of the Tenzel method, we adapted it to reconstruct larger defects around the eyes. Seven patients underwent reconstruction with a modified Tenzel flap with an extended concept after wide excision of a malignant skin lesion. The main difference from the conventional method is that the modified Tenzel flap includes the medial portion of the lower lid defect. The design of a modified Tenzel flap begins as a semicircle at the lateral canthal area, in the same way as a classical Tenzel flap, and extends medially along the subciliary line to cover the defect on the medial lower eyelid. The follow-up time ranged from 3 to 28 months. @*Results@#All flaps survived and healed well, with minimal scarring and natural palpebral outlines. @*Conclusion@#Compared to traditional procedures, the modified Tenzel flap has several advantages, including a one-stage operation, a less noticeable scar, and effective prevention of complications such as lower eyelid ectropion.
ABSTRACT
Background@#Basiliximab is used as an alternative to tacrolimus in patients with decreased renal function. However, studies on basiliximab as a maintenance immunosuppressant, particularly in patients with lung transplantation, are limited. Therefore, here, we investigated the efficacy and safety of basiliximab in patients with lung transplantation. @*Methods@#Adult patients with acute kidney injury (AKI) who received lung transplantation at a single general hospital between July 1, 2014 and June 30, 2018, were selected and classified in tacrolimus and basiliximab groups. Both groups received a triple-drug regimen (tacrolimus, mycophenolate mofetil, and steroids). However, tacrolimus was discontinued in the basiliximab group when AKI occurred, and two or more repeat basiliximab doses were administered within 3 months after transplantation. The electronic medical records were analyzed retrospectively. @*Results@#Of the 85 patients who met the selection criteria, 61 and 24 were assigned to the tacrolimus and basiliximab groups, respectively. Significant improvement in renal function was observed in the basiliximab group (p <0.001).However, there were no differences in acute and chronic rejection rates in both the groups. No difference was observed in the incidence rate of complications between the groups, except for chronic kidney disease, which showed higher incidence in the basiliximab group (25.0% vs. 4.9%; p =0.013). @*Conclusions@#We suggest the use of basiliximab as an immunosuppressant alternative to tacrolimus in patients with acute renal failure after lung transplantation. Basiliximab demonstrated effectiveness as an immunosuppressant and improved renal function. Therefore, basiliximab can be used in patients with decreased renal function.
ABSTRACT
Background@#Basiliximab is used as an alternative to tacrolimus in patients with decreased renal function. However, studies on basiliximab as a maintenance immunosuppressant, particularly in patients with lung transplantation, are limited. Therefore, here, we investigated the efficacy and safety of basiliximab in patients with lung transplantation. @*Methods@#Adult patients with acute kidney injury (AKI) who received lung transplantation at a single general hospital between July 1, 2014 and June 30, 2018, were selected and classified in tacrolimus and basiliximab groups. Both groups received a triple-drug regimen (tacrolimus, mycophenolate mofetil, and steroids). However, tacrolimus was discontinued in the basiliximab group when AKI occurred, and two or more repeat basiliximab doses were administered within 3 months after transplantation. The electronic medical records were analyzed retrospectively. @*Results@#Of the 85 patients who met the selection criteria, 61 and 24 were assigned to the tacrolimus and basiliximab groups, respectively. Significant improvement in renal function was observed in the basiliximab group (p <0.001).However, there were no differences in acute and chronic rejection rates in both the groups. No difference was observed in the incidence rate of complications between the groups, except for chronic kidney disease, which showed higher incidence in the basiliximab group (25.0% vs. 4.9%; p =0.013). @*Conclusions@#We suggest the use of basiliximab as an immunosuppressant alternative to tacrolimus in patients with acute renal failure after lung transplantation. Basiliximab demonstrated effectiveness as an immunosuppressant and improved renal function. Therefore, basiliximab can be used in patients with decreased renal function.
ABSTRACT
BACKGROUND/OBJECTIVES: The nutrition of the elderly depends on various factors. Oral health, especially oral dryness, can be an important risk factor. In this study, we attempted to determine whether dry mouth is associated with compromised nutrient intakes.SUBJECTS/METHODS: A total of 120 participants aged 65–86 yrs (mean age: 69 ± 1 y) were included in this study. Demographic and health-related characteristics, living status, meals, number of medications, medical conditions, chewing ability, and quality of life, the Oral Health Impact Profile (the OHIP-14) were assessed. We performed one day 24-hr recall assessment for nutrient analyses. The differences of the means between the dry-mouth and non-dry-mouth groups were analyzed. Elderly subjects with xerostomia-induced dry mouth were classified as those who reported at least one dryness symptom on a questionnaire.RESULTS: A significant difference in population distribution was observed among the elderly who took medications for hypertension, diabetes and osteoporosis and was significantly higher in the dry-mouth group (70.2%) than in the non-dry-mouth group (44.4%) (P = 0.005). Compared with the non-dry-mouth group (50.8%), a significantly higher proportion (73.7%) of participants in the dry-mouth group took multiple medicines (≥ 4 medications) (P = 0.019). The intakes of vegetable fat, vitamin E, folate and water in the dry-mouth group were lower than in the non-dry-mouth group. The intakes of fluoride and ω-3 fatty acids were significantly lower in the dry-mouth group than in the non-dry-mouth group.CONCLUSION: The participants in the dry-mouth group exhibited low nutrient and water intakes. It is recommended that the elderly with dry mouth should drink sufficient water and receive targeted and specific nutritional guidance to prevent malnutrition.
ABSTRACT
BACKGROUND: Although clinicians, nurse specialists, pharmacists, and nutritionists expend significant time and resources in optimizing care for patients with diabetes, the effectiveness of integrated diabetes care team approach remains unclear. We assessed the effects of a multidisciplinary team care educational intervention on glycated hemoglobin (HbA1c) levels among diabetes patients. METHODS: We conducted a matched case-control study in Korean patients with type 2 diabetes, comparing the propensity scores pertaining to the effectiveness in reducing HbA1c levels between a group receiving an educational intervention and a control group. We included 40 pairs of patients hospitalized between June 2014 and September 2016. HbA1c values measured at baseline, 3 months, and 6 months were compared between the two groups. RESULTS: The educated group showed an improvement in HbA1c levels compared to the control group at 3 months (6.3 ± 2.3% vs. 9.5 ± 4.0%; P = 0.020) and at 6 months (7.5 ± 1.5% vs. 9.6 ± 3.0%; P = 0.106). There was a significant difference in the change in mean HbA1c from baseline to 3 months between the two groups (−35.7 ± 26.1% vs. −9.1 ± 20.5%; P = 0.013). CONCLUSION: A multidisciplinary team care education intervention was advantageous for improving glucose control in patients with type 2 diabetes, and may help to optimize glycemic control in clinical practice.
Subject(s)
Humans , Case-Control Studies , Diabetes Mellitus , Education , Glucose , Health Education , Glycated Hemoglobin , Nurse Clinicians , Nutritionists , Pharmacists , Propensity Score , SpecializationABSTRACT
A 25-years-old woman with mandibular prognathism underwent a mandibular setback by way of mandibular sagittal split ramus osteotomy (MSSRO). After 2 days of operation, she developed difficulty of closing her right eye. The blink reflex test and motor nerve conduction study of the right orbicularis oris muscle were revealed right facial neuropathy of unknown origin and House-Brackmann facial nerve grading system (HBFNGS) grade V. For treatment, we initially prescribed oral prednisolone and nimodipine including physical therapy. The samples consisted of 11 facial nerve palsy patients caused by MSSRO and were analysed about onset of facial nerve palsy, postoperative HBFNGS, final HBFNGS, treatment method and recovery time. At 10 weeks of treatment of nimodipine, she had completely regained normal function (HBFNGS grade I) of the right facial nerve. The clinical results lead to assume a fast recovery of facial nerve function by the nimodipine medication, whereas average time of recovery is 16.32 weeks in references. Despite of the limited one patient treated, the result was very promising with respect to a faster recovery of the facial nerve function. Considering the use of nimodipine treatment for peripheral facial nerve palsy following a surgical approach with an anatomically preserved nerve can be recommended.
Subject(s)
Female , Humans , Blinking , Facial Nerve Diseases , Facial Nerve , Facial Paralysis , Mandible , Methods , Neural Conduction , Nimodipine , Osteotomy, Sagittal Split Ramus , Paralysis , Prednisolone , PrognathismABSTRACT
BACKGROUND/AIMS: The controlled attenuation parameter (CAP) implemented in FibroScan(R) is reported to be a non-invasive means of detecting steatosis (>10% steatosis). We aimed to evaluate the usefulness of CAP in detecting steatosis among health checkup examinees and to assess its correlation with ultrasonography (US). METHODS: Consecutive CAP results were retrospectively collected. A total of 280 subjects were included. RESULTS: Fatty liver was detected in 119 subjects (42.5%) by US, whereas it was detected in 160 subjects (57.1%) by the CAP. The numbers of subjects with S0:S1:S2:S3 steatosis according to the CAP value were 120:59:58:43, respectively. The mean CAP values were 203.34+/-28.39 dB/m for S0, 248.83+/-6.14 dB/m for S1, 274.33+/-8.53 dB/m for S2, and 322.35+/-22.20 dB/m for S3. CAP values were correlated with body weight (r=0.404, p<0.001), body mass index (r=0.445, p<0.001), and the fatty liver grade by US (r=0.472, p<0.001). Among the 161 subjects with normal US findings, steatosis was detected in 65 subjects (40.4%) using the CAP. CONCLUSIONS: The CAP seems to be useful for detecting very low-grade hepatic steatosis in health checkup examinees. Its role in predicting subjects with a risk of metabolic derangement needs to be evaluated.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Body Mass Index , Body Weight , Elasticity Imaging Techniques/methods , Fatty Liver/diagnostic imaging , Liver/diagnostic imaging , Physical Examination/methods , Predictive Value of Tests , ROC Curve , Retrospective Studies , Severity of Illness IndexABSTRACT
BACKGROUND/AIMS: The controlled attenuation parameter (CAP) implemented in FibroScan(R) is reported to be a non-invasive means of detecting steatosis (>10% steatosis). We aimed to evaluate the usefulness of CAP in detecting steatosis among health checkup examinees and to assess its correlation with ultrasonography (US). METHODS: Consecutive CAP results were retrospectively collected. A total of 280 subjects were included. RESULTS: Fatty liver was detected in 119 subjects (42.5%) by US, whereas it was detected in 160 subjects (57.1%) by the CAP. The numbers of subjects with S0:S1:S2:S3 steatosis according to the CAP value were 120:59:58:43, respectively. The mean CAP values were 203.34+/-28.39 dB/m for S0, 248.83+/-6.14 dB/m for S1, 274.33+/-8.53 dB/m for S2, and 322.35+/-22.20 dB/m for S3. CAP values were correlated with body weight (r=0.404, p<0.001), body mass index (r=0.445, p<0.001), and the fatty liver grade by US (r=0.472, p<0.001). Among the 161 subjects with normal US findings, steatosis was detected in 65 subjects (40.4%) using the CAP. CONCLUSIONS: The CAP seems to be useful for detecting very low-grade hepatic steatosis in health checkup examinees. Its role in predicting subjects with a risk of metabolic derangement needs to be evaluated.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Body Mass Index , Body Weight , Elasticity Imaging Techniques/methods , Fatty Liver/diagnostic imaging , Liver/diagnostic imaging , Physical Examination/methods , Predictive Value of Tests , ROC Curve , Retrospective Studies , Severity of Illness IndexABSTRACT
OBJECTIVE: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. METHODS: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). RESULTS: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. CONCLUSION: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.
Subject(s)
Female , Humans , Centrifugation, Density Gradient , Embryonic Structures , Fertilization , Fertilization in Vitro , Infertility , Oocytes , Pregnancy Rate , Reproductive Techniques , Reproductive Techniques, Assisted , Retrospective Studies , Semen , SpermatozoaABSTRACT
Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.
Subject(s)
Animals , Female , Mice , Pregnancy , Cell Lineage , Embryonic Development , Embryonic Structures , Fertility , Oocytes , Oogenesis , Organelles , Transcriptional ActivationABSTRACT
BACKGROUND: The glucose transporters (GLUTs) exhibit different tissue-specific expression. This study aimed to investigate the types of GLUTs expressed in human granulosa cells (GCs) obtained from women with polycystic ovary syndrome (PCOS) and their relationship with insulin resistance (IR) and the outcomes of in vitro maturation (IVM) of immature oocytes. METHODS: Expression of GLUTs was evaluated in GCs from women with PCOS with or without IR. Thirty-six women with PCOS undergoing an IVM program were included. Differential gene expression between the insulin sensitive (IS) and IR group was measured by reverse transcription polymerase chain reaction. RESULTS: Expression of GLUTs 1, 3, 5, 8, and 13 was constitutive, whereas expression of GLUTs 2 and 7 was not observed in human GCs. The remaining GLUTs, 4, 6, 9, 10, 11, and 12, were differentially expressed among patients according to metabolic status, such as insulin sensitivity. A higher number of GCs from patients with IR (92%) expressed GLUT6 than GCs from IS PCOS patients (46.3%). Logistic regression showed that expression of GLUTs 9, 11, and 12 correlates with rates of IVM at 48 hours, fertilization, and implantation, respectively. CONCLUSION: This is the first report describing the expression pattern of all 13 members of the GLUT family in human GCs. Results of the present study suggest that patients' insulin sensitivity regulates GLUT expression in GCs in PCOS patients, and this may control oocyte quality for IVM and subsequent processes such as fertilization and implantation in patients taking part in an in vitro fertilization program.
Subject(s)
Female , Humans , Fertilization , Fertilization in Vitro , Gene Expression , Glucose Transport Proteins, Facilitative , Glucose , Granulosa Cells , Insulin , Insulin Resistance , Logistic Models , Oocytes , Polycystic Ovary Syndrome , Polymerase Chain Reaction , Reverse TranscriptionABSTRACT
Homeobox genes play essential roles in embryonic development and reproduction. Recently, a large cluster of homeobox genes, reproductive homeobox genes on the X chromosome (Rhox) genes, was discovered as three gene clusters, alpha, beta, and gamma in mice. It was found that Rhox genes were selectively expressed in reproduction-associated tissues, such as those of the testes, epididymis, ovaries, and placenta. Hence, it was proposed that Rhox genes are important for regulating various reproductive features, especially gametogenesis in male as well as in female mammals. It was first determined that 12 Rhox genes are clustered into alpha (Rhox1-4), beta (Rhox5-9), and gamma (Rhox10-12) subclusters, and recently Rhox13 has also been found. At present, 33 Rhox genes have been identified in the mouse genome, 11 in the rat, and three in the human. Rhox genes are also responsible for embryonic development, with considerable amounts of Rhox expression in trophoblasts, placenta tissue, embryonic stem cells, and primordial germ cells. In this article we summarized the current understanding of Rhox family genes involved in reproduction and embryonic development and elucidated a previously unreported cell-specific expression in ovarian cells.
Subject(s)
Animals , Female , Humans , Male , Mice , Pregnancy , Rats , Embryonic Development , Embryonic Stem Cells , Epididymis , Gametogenesis , Genes, Homeobox , Genome , Germ Cells , Mammals , Multigene Family , Ovary , Placenta , Reproduction , Stem Cells , Testis , Trophoblasts , X ChromosomeABSTRACT
OBJECTIVE: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. METHODS: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. RESULTS: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. CONCLUSION: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.
Subject(s)
Fluorescence , Genes, Homeobox , Glass , Meiosis , OocytesABSTRACT
OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.
Subject(s)
Cell Cycle , Maturation-Promoting Factor , Meiosis , Metaphase , Oocytes , Pentose Phosphate Pathway , Protein Kinases , Ribosemonophosphates , RNA Interference , RNA, Double-Stranded , TransketolaseABSTRACT
OBJECTIVE: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. METHODS: The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. RESULTS: According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. CONCLUSION: Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.
Subject(s)
Animals , Humans , Mice , Chimera , Down-Regulation , Embryonic Stem Cells , Genes, Homeobox , Genome , Neuropeptide Y , Neuropeptides , Oligonucleotide Array Sequence Analysis , Receptors, Neuropeptide Y , RNA , RNA Interference , RNA, Small Interfering , Statistics as TopicABSTRACT
OBJECTIVE: We found previously that interferon regulatory factor (Irf)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of Irf-1 in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of Irf-1 and the mouse oocyte maturation. METHODS: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured in vitro for 16 hours in the presence of varying concentrations of RA (0-10 microM). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 microM). With 100 nM RA treatment, lowest level of Irf-1 mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-alpha, macrophage inflammatory protein-1beta, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. CONCLUSION: We concluded that the maturation of oocytes and Irf-1 expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes in vitro by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for in vitro oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.
Subject(s)
Animals , Female , Humans , Mice , Apoptosis , Cumulus Cells , Cytokines , DNA Nucleotidylexotransferase , Gene Expression , In Vitro Oocyte Maturation Techniques , Interferon Regulatory Factor-1 , Interferons , Interleukin-12 , Macrophages , Mental Competency , Metaphase , Oocytes , Reproductive Techniques, Assisted , RNA, Messenger , Tretinoin , Tumor Necrosis Factor-alphaABSTRACT
OBJECTIVE: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of Obox4 in oocyte maturation by evaluating downstream signal networking. METHODS: The Obox4 dsRNA was prepared by in vitro transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by in vitro maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix GeneChip(R) Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. RESULTS: Total 424 genes were up (n=80) and down (n=344) regulated after Obox4 RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by Obox4 RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. CONCLUSION: From the results of this study, it is concluded that Obox4 is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.