78 results on '"Mami Miyado"'
Search Results
2. LBODP008 Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Fibroblasts, IPS Cells And Model Mice
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Yasuhiro Naiki, Mami Miyado, Reiko Horikawa, Noriyuki Katsumata, Shuji Takada, Hidenori Akutsu, Masafumi Onodera, and Maki Fukami
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Endocrinology, Diabetes and Metabolism - Abstract
Background Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s. Microsomal P450s include 21-hydroxylase and 17α-hydroxylase/17-lyase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, is the most common cause of CAH and results in underproduction of glucocorticoids and mineralocorticoids, and overproduction of androgens. 11β-hydroxylase deficiency (11β-OHD), which is a defect of a mitochondrial P450, is the second common cause of CAH. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Intravenous adeno-associated virus (AAV)-mediated administration of human CYP21A2 into adult 21-OHD patients has been reported at the ENDO 2021 meeting. In this study, we examined the effects of induction of causative genes with an AAV vector into fibroblasts from patients and iPS cells and adrenal glands of model mice. Methods Fibroblasts from CAH patients were subjected to primary culture. The cells were infected with a serotype-2 AAV vector (AAV2) containing defected genes and cultured in steroid substrate-containing medium for 24 hours. We measured steroid metabolites in the medium by liquid chromatography tandem mass spectrometry and evaluated gene expressions by RT-PCR. In addition, iPS cells were established from fibroblasts of 11β-OHD and differentiated into adrenocortical cells with retinoic acid and cAMP. They were infected with a serotype-9 AAV vector (AAV9) containing CYP11B1 cDNA. Steroid metabolites in the culture medium were measured and gene expressions were evaluated by RT-PCR. 11β-OHD model mice were made by a gene-editing method. We injected AAV9 containing Cyp11b1 cDNA into the adrenal gland and measured serum DOC and corticosterone levels before and every 4 weeks after injection. Results AAV2 infected fibroblasts from 21-OHD and 17α-hydroxylase/17-lyase deficiency can metabolite steroid precursors but failed to convert DOC to corticosterone in 11β-OHD. Differentiation into adrenocortical cells from iPS cells of 11β-OHD was confirmed by expression of CYP21A2. AAV9 gene induction of adrenal cortical cells successfully induced 11β-hydroxylase activity. 11β-OHD mice also showed improved steroid synthesis after AAV9 adrenal induction. Conclusion These results indicate that extra-adrenal induction of CYP21A2 may ameliorate steroid metabolism in 21-OHD patients. In contrast, 11β-OHD needs AAV9 adrenal induction to treat the steroid abnormality. Our results suggest that specific gene therapeutic strategies are needed to be adapted for each type of CAH. Presentation: No date and time listed
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- 2022
3. Adeno-Associated Virus-Mediated Gene Therapy for Patients' Fibroblasts, Induced Pluripotent Stem Cells, and a Mouse Model of Congenital Adrenal Hyperplasia
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Yasuhiro Naiki, Mami Miyado, Miyuki Shindo, Reiko Horikawa, Yuichi Hasegawa, Noriyuki Katsumata, Shuji Takada, Hidenori Akutsu, Masafumi Onodera, and Maki Fukami
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Adrenal Hyperplasia, Congenital ,Induced Pluripotent Stem Cells ,Steroid 17-alpha-Hydroxylase ,Genetic Therapy ,Dependovirus ,Fibroblasts ,Disease Models, Animal ,Mice ,Mutation ,Genetics ,Molecular Medicine ,Animals ,Steroid 11-beta-Hydroxylase ,Steroid 21-Hydroxylase ,Molecular Biology - Abstract
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration of
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- 2022
4. Embryonic β-Catenin Is Required for Priming of the Uterus to Implantation
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Youki Takezawa, Maki Iwai, Yukiko Fujiki, Ryo Yokomizo, Harue Kishigami, Mami Miyado, Natsuko Kawano, Mitsutoshi Yamada, Miyuki Shindo, Miki Suzuki, Ban Sato, Daiki Katano, Shintaro Kamijo, Toshio Hamatani, Mamoru Tanaka, Akihiro Umezawa, Woojin Kang, and Kenji Miyado
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Cell Biology ,Molecular Biology ,Pathology and Forensic Medicine - Published
- 2023
5. Extra-mitochondrial citrate synthase controls cAMP-dependent pathway during sperm acrosome reaction in mice
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Woojin, Kang, Daiki, Katano, Natsuko, Kawano, Mami, Miyado, and Kenji, Miyado
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Sperm must undergo sequential and complex processes in the uterus and oviducts before fertilizing the eggs, including capacitation and acrosome reaction (AR) (Gervasi and Visconti 2016). Adenosine triphosphate (ATP) production through glycolysis and oxidative phosphorylation (OXPHO) is essential for maintaining sperm motility and completing AR (Stival et al. 2016). Citrate synthase (CS) is localized in the mitochondrial matrix, where it catalyzes the reaction between acetyl coenzyme A and oxaloacetate to form citric acid (Surpin and Chory 1997). The extra-mitochondrial form of CS (eCS) is encoded by a separate gene in mice (citrate synthase like (Csl) MGI:1919082) and expressed by alternative splicing of the CS gene in humans (Kang et al. 2020). eCS also has CS activity, implying its involvement in ATP production which is related to sperm function in the extra-mitochondrial cytoplasm (Kang et al. 2020). Citrate serves as an essential substrate in the tricarboxylic acid (TCA) cycle, and its subsequent complete oxidation is the major source of cellular ATP production (Iacobazzi and Infantino 2014). Consequently, the activation of TCA cycle-related enzymes that regulate cellular ATP production may be involved in controlling sperm functions (Zhu et al. 2019). Conceivably, eCS is involved in regulating AR in the sperm head, as citrate is an important substrate in cellular energy metabolism (Iacobazzi and Infantino 2014). However, the role of eCS in AR remains unclear.
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- 2022
6. Trehalose Suppresses Lysosomal Anomalies in Supporting Cells of Oocytes and Maintains Female Fertility
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Woojin Kang, Eri Ishida, Mitsuyoshi Amita, Kuniko Tatsumi, Hitomi Yonezawa, Miku Yohtsu, Daiki Katano, Kae Onozawa, Erika Kaneko, Wakako Iwasaki, Natsuko Naito, Mitsutoshi Yamada, Natsuko Kawano, Mami Miyado, Ban Sato, Hidekazu Saito, Takakazu Saito, and Kenji Miyado
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endocrine system ,Mice ,Nutrition and Dietetics ,Fertility ,oocyte quality ,autophagy ,chloroquine ,trehalose ,lysosomal anomalies ,Oocytes ,Animals ,Humans ,Trehalose ,Chloroquine ,Female ,Lysosomes ,Food Science - Abstract
Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility.
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- 2022
7. Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction
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Yasuhiro Iwao, Yoshiki Hayashi, Kenji Miyado, Mami Miyado, Woojin Kang, Akihiro Umezawa, Hidekazu Saito, Yuichirou Harada, Kenji Yamatoya, Seiya Kanai, Akihiro Nakamura, Yoko Kuroki, Yoshitaka Miyamoto, and Natsuko Kawano
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Male ,0301 basic medicine ,Aging ,media_common.quotation_subject ,Citric Acid Cycle ,Reproductive biology ,Fertility ,Citrate (si)-Synthase ,Mitochondrion ,Article ,Pathology and Forensic Medicine ,Male infertility ,Andrology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Citrate synthase ,Calcium Signaling ,030212 general & internal medicine ,Molecular Biology ,Infertility, Male ,Ovum ,media_common ,chemistry.chemical_classification ,biology ,Chemistry ,Correction ,Cell Biology ,Tricarboxylic acid ,medicine.disease ,Spermatozoa ,Sperm ,Citric acid cycle ,Ageing ,030104 developmental biology ,Enzyme ,Metabolome ,biology.protein ,Female - Abstract
Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age., Citrate synthase is a core enzyme of tricarboxylic acid cycle, but the role of extra-mitochondrial CS (eCS) is less clear. eCS, abundant in the sperm head, triggers a first spike of egg Ca2+ oscillation in a phospholipase C zeta 1 -independent manner. Moreover, the fertility of eCs-knockout male mice dropped with age, indicating that eCS suppresses age-dependent sperm dysfunction by accelerating Ca2+ oscillation.
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- 2020
8. Suppressive Role of Lactoferrin in Overweight-Related Female Fertility Problems
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Ban Sato, Seiya Kanai, Daiki Sakaguchi, Kodai Yajima, Yu Matsumoto, Kazunori Morohoshi, Shinji Kagaya, Nobuo Izumo, Minoru Ichinose, Woojin Kang, Mami Miyado, Kenji Miyado, and Natsuko Kawano
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Nutrition and Dietetics ,Fertility Agents, Female ,Overweight ,Up-Regulation ,Lactoferrin ,Mice ,Fertility ,Diabetes Mellitus, Type 2 ,Animals ,lactoferrin ,overweight ,obesity ,female fertility problems ,Female ,Obesity ,Infertility, Female ,Food Science - Abstract
The secretory glycoprotein lactoferrin (LF) is suggested to ameliorate overweight regardless of non-genetic or genetic mechanisms. Although maternal overweight represents a key predictor of offspring growth, the efficacy of LF on fertility problems in overweight and obese mothers remains unknown. To address this issue, we examined the effect of LF ingestion by analyzing overweight mice (Institute of Cancer Research (ICR) mice with high-fat diets; HF mice) and obese mice (leptin-deficient mice with type II diabetes; ob/ob mice). Plasma insulin, leptin, glucose, and cholesterol levels were measured, and thermal imaging and histological analysis were employed. The litter size of HF females was reduced due to miscarriage, which was reversed by LF ingestion. In addition, LF ingestion suppressed overweight prevalence in their offspring. The component analysis of the maternal blood demonstrated that glucose concentration in both HF females and their offspring was normalized by LF ingestion, which further standardized the concentration of insulin, but not leptin. LF ingestion was unable to reverse female infertility in ob/ob mice, although their obesity and uterine function were partially improved. Our results indicate that LF upregulates female fertility by reinforcing ovarian and uterine functions in females that are overweight due to caloric surplus.
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- 2022
9. Mosaic loss of the Y chromosome and men's health
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Maki Fukami and Mami Miyado
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Reproductive Medicine ,Cell Biology - Abstract
Although Y chromosomal genes are involved in male sex development, spermatogenesis, and height growth, these genes play no role in the survival or mitosis of somatic cells. Therefore, somatic cells lacking the Y chromosome can stay and proliferate in the body.Several molecular technologies, including next-generation sequencing and multiplex PCR-based assays, are used to detect mosaic loss of the Y chromosome (mLOY) in the blood of men.Accumulating evidence suggests that mLOY represents the most common acquired chromosomal alteration in humans, affecting40% of men over 70 years of age. Advanced age, tobacco smoking, and some SNPs in cell cycle genes are known to increase the frequency of mLOY. The developmental process of mLOY in elderly men remains to be clarified, but it possibly reflects recurrent mitotic elimination of Y chromosomes or clonal expansion of 45,X cell lineages. In rare cases, mLOY also occurs in young men and fetuses. MLOY has been associated with early death, cancers, and other disorders in elderly men, infertility in reproductive-aged men, and developmental defects in children.Y chromosomes in men can be lost at every life stage and Y chromosomal loss is associated with various health problems.
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- 2022
10. Intrauterine Hyponutrition Reduces Fetal Testosterone Production and Postnatal Sperm Count in the Mouse
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Yasuko Fujisawa, Hiroyuki Ono, Alu Konno, Ikuko Yao, Hiroaki Itoh, Takashi Baba, Kenichirou Morohashi, Yuko Katoh-Fukui, Mami Miyado, Maki Fukami, and Tsutomu Ogata
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Endocrinology, Diabetes and Metabolism - Abstract
Background Although intrauterine hyponutrition is regarded as a risk factor for the development of “testicular dysgenesis syndrome” (TDS) in the human, underlying mechanism(s) remain largely unknown. Methods To clarify the underlying mechanism(s), we fed vaginal plug-positive C57BL/6N female mice with regular food ad libitum throughout the pregnant course (control females) (C-females) or with 50% of the mean daily intake of the C-females from 6.5 dpc (calorie-restricted females) (R-females), and compared male reproductive findings between 17.5-dpc-old male mice delivered from C-females (C-fetuses) and those delivered from R-females (R-fetuses) and between 6-week-old male mice born to C-females (C-offspring) and those born to R-females (R-offspring). Results Compared with the C-fetuses, the R-fetuses had (1) morphologically normal external genitalia with significantly reduced anogenital distance index, (2) normal numbers of testicular component cells, and (3) significantly low intratesticular testosterone, in association with significantly reduced expressions of steroidogenic genes. Furthermore, compared with the C-offspring, the R-offspring had (1) significantly increased TUNEL-positive cells and normal numbers of other testicular component cells, (2) normal intratesticular testosterone, in association with normal expressions of steroidogenic genes, (3) significantly reduced sperm count, and normal testis weight and sperm motility, and (4) significantly altered expressions of oxidation stress-related, apoptosis-related, and spermatogenesis-related genes. Conclusions The results, together with the previous data including the association between testosterone deprivation and oxidative stress-evoked apoptotic activation, imply that reduced fetal testosterone production is the primary underlying factor for the development of TDS in intrauterine hyponutrition, and that TDS is included in the clinical spectrum of Developmental Origins of Health and Disease.
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- 2021
11. Microexosomes versus exosomes: Shared components but distinct structures
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Natsuko Kawano, Kenji Miyado, Mami Miyado, and Woojin Kang
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Biomaterials ,Text mining ,business.industry ,Commentary ,Biomedical Engineering ,Computational biology ,Biology ,business ,Microvesicles ,Developmental Biology - Published
- 2019
12. Aneuploid rescue precedes X-chromosome inactivation and increases the incidence of its skewness by reducing the size of the embryonic progenitor cell pool
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E. Suzuki, Tsutomu Ogata, Mami Miyado, Kenichi Kinjo, Kenta Matsubara, Masayo Kagami, Hidenori Akutsu, Maki Fukami, Masashi Mikami, and Tomoko Yoshida
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Adolescent ,Embryonic Development ,Aneuploidy ,Biology ,X-inactivation ,Cohort Studies ,Andrology ,Genomic Imprinting ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Pregnancy ,X Chromosome Inactivation ,medicine ,Humans ,Progenitor cell ,Child ,Skewed X-inactivation ,Embryonic Stem Cells ,Preimplantation Diagnosis ,X-linked recessive inheritance ,Cell Size ,030304 developmental biology ,Chromosomes, Human, X ,0303 health sciences ,030219 obstetrics & reproductive medicine ,Incidence ,Rehabilitation ,Infant ,Obstetrics and Gynecology ,Bayes Theorem ,medicine.disease ,Uniparental disomy ,Blastocyst ,Reproductive Medicine ,Child, Preschool ,DNA methylation ,Female ,Trisomy - Abstract
STUDY QUESTIONDo monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)?SUMMARY ANSWERAneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools.WHAT IS KNOWN ALREADYMore than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase.STUDY DESIGN, SIZE, DURATIONWe examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells.PARTICIPANTS/MATERIALS, SETTING, METHODSWe studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1–12 cell pools.MAIN RESULTS AND THE ROLE OF CHANCEA total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive.LIMITATIONS, REASONS FOR CAUTIONThis is an observational study and needs to be validated by experimental analyses.WIDER IMPLICATIONS OF THE FINDINGSThis study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest.TRIAL REGISTRATION NUMBERNot applicable.
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- 2019
13. Endometrial preparation methods for frozen-thawed embryo transfer are associated with altered risks of hypertensive disorders of pregnancy, placenta accreta, and gestational diabetes mellitus
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Naho Morisaki, Minoru Irahara, Maki Fukami, Tomonori Ishikawa, Kazuki Saito, Mami Miyado, Kenji Miyado, Akira Kuwahara, Hidekazu Saito, Osamu Ishihara, and Naoyuki Miyasaka
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0303 health sciences ,medicine.medical_specialty ,Pregnancy ,030219 obstetrics & reproductive medicine ,Assisted reproductive technology ,business.industry ,Obstetrics ,Placenta accreta ,medicine.medical_treatment ,Rehabilitation ,Obstetrics and Gynecology ,Retrospective cohort study ,Odds ratio ,medicine.disease ,Gestational diabetes ,03 medical and health sciences ,Pregnancy rate ,0302 clinical medicine ,Reproductive Medicine ,medicine ,Live birth ,business ,030304 developmental biology - Abstract
STUDY QUESTION What were the risks with regard to the pregnancy outcomes of patients who conceived by frozen-thawed embryo transfer (FET) during a hormone replacement cycle (HRC-FET)? SUMMARY ANSWER The patients who conceived by HRC-FET had increased risks of hypertensive disorders of pregnancy (HDP) and placenta accreta and a reduced risk of gestational diabetes mellitus (GDM) in comparison to those who conceived by FET during a natural ovulatory cycle (NC-FET). WHAT IS KNOWN ALREADY Previous studies have shown that pregnancy and live-birth rates after HRC-FET and NC-FET are comparable. Little has been clarified regarding the association between endometrium preparation and other pregnancy outcomes. STUDY DESIGN, SIZE, DURATION A retrospective cohort study of patients who conceived after HRC-FET and those who conceived after NC-FET was performed based on the Japanese assisted reproductive technology registry in 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS The pregnancy outcomes were compared between NC-FET (n = 29 760) and HRC-FET (n = 75 474) cycles. Multiple logistic regression analyses were performed to investigate the potential confounding factors. MAIN RESULTS AND THE ROLE OF CHANCE The pregnancy rate (32.1% vs 36.1%) and the live birth rate among pregnancies (67.1% vs 71.9%) in HRC-FET cycles were significantly lower than those in NC-FET cycles. A multiple logistic regression analysis showed that pregnancies after HRC-FET had increased odds of HDPs [adjusted odds ratio, 1.43; 95% confidence interval (CI), 1.14–1.80] and placenta accreta (adjusted odds ratio, 6.91; 95% CI, 2.87–16.66) and decreased odds for GDM (adjusted odds ratio, 0.52; 95% CI, 0.40–0.68) in comparison to pregnancies after NC-FET. LIMITATIONS, REASONS FOR CAUTION Our study was retrospective in nature, and some cases were excluded due to missing data. The implication of bias and residual confounding factors such as body mass index, alcohol consumption, and smoking habits should be considered in other observational studies. WIDER IMPLICATIONS OF THE FINDINGS Pregnancies following HRC-FET are associated with higher risks of HDPs and placenta accreta and a lower risk of GDM. The association between the endometrium preparation method and obstetrical complication merits further attention. STUDY FUNDING/COMPETING INTEREST(S) No funding was obtained for this work. The authors declare no conflicts of interest in association with the present study. TRIAL REGISTRATION NUMBER Not applicable.
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- 2019
14. Germline-Derived Gain-of-Function Variants of Gsα-Coding GNAS Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis
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Kenichiro Hata, Yoko Tanaka, Kazuhiro Ogata, Shuji Takada, Keisuke Nagasaki, Goro Sasaki, Masaaki Shiina, Tsutomu Ogata, Miho Terao, Mami Miyado, Maki Fukami, Hirotomo Saitsu, Youhei Masunaga, Yoichi Matsubara, and Kazuhiko Nakabayashi
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0301 basic medicine ,Genetics ,Water transport ,Gs alpha subunit ,biology ,General Medicine ,medicine.disease ,Phenotype ,03 medical and health sciences ,Exon ,030104 developmental biology ,0302 clinical medicine ,Nephrology ,030220 oncology & carcinogenesis ,Arginine vasopressin receptor 2 ,GNAS complex locus ,biology.protein ,medicine ,Pseudopseudohypoparathyroidism ,Exome sequencing - Abstract
Background The stimulatory G-protein α -subunit encoded by GNAS exons 1–13 ( GNAS -Gs α ) mediates signal transduction of multiple G protein–coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS -Gs α variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS -Gs α variants have been detected in McCune–Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. Methods We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. Results Whole-exome sequencing revealed two GNAS -Gs α candidate variants for NSIAD: GNAS -Gs α p.(F68_G70del) in one family and GNAS -Gs α p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS -Gs α alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function–compatible conformational property for p.M255V-Gs α , although such assessment was not possible for p.F68_G70del-Gs α . Both variants had gain-of-function effects that were significantly milder than those of McCune–Albright syndrome–specific somatic Gs α variants. Model mice for p.F68_G70del-Gs α showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gs α exhibited severe failure to thrive. Conclusions This study shows that germline-derived gain-of-function rare variants of GNAS -Gs α exist and cause NSIAD as a novel Gs α -mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS -Gs α ’s gain-of-function effects.
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- 2019
15. DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations
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Atsushi Hattori, Masako Izawa, Hitoshi Yamamoto, Hirohito Shima, Kenichiro Ogushi, Shun Soneda, Akihiro Umezawa, Reiko Horikawa, Hideaki Yagasaki, Erina Suzuki, Tomohiro Ishii, Noriyuki Namba, Mami Miyado, Toshiaki Tanaka, Koji Muroya, Yasuhiro Hirano, Keiko Matsubara, Masayo Kagami, Kimiaki Uetake, and Maki Fukami
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Genetics ,0303 health sciences ,030305 genetics & heredity ,Pseudoautosomal region ,Bisulfite sequencing ,Biology ,medicine.disease ,X-inactivation ,03 medical and health sciences ,CpG site ,DNA methylation ,medicine ,Copy-number variation ,Haploinsufficiency ,Molecular Biology ,Léri–Weill dyschondrosteosis ,Genetics (clinical) ,030304 developmental biology - Abstract
SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.
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- 2019
16. Quantification of androgens and their precursors in full-term human placenta
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Tomoko Yoshida, Mami Miyado, Maki Fukami, Kenji Matsumoto, Haruhiko Sago, Yoshimichi Miyashiro, and Reiko Horikawa
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Adult ,Male ,medicine.medical_specialty ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Placenta ,Biology ,Steroid ,chemistry.chemical_compound ,Endocrinology ,Limit of Detection ,Pregnancy ,Tandem Mass Spectrometry ,Internal medicine ,medicine ,Humans ,Testosterone ,Androstenedione ,Progesterone ,Androsterone ,Adrenal gland ,Infant, Newborn ,Reproducibility of Results ,Dihydrotestosterone ,General Medicine ,Androgen ,medicine.anatomical_structure ,chemistry ,Androgens ,Female ,Steroids ,medicine.drug - Abstract
Introduction The two major androgens in humans are testosterone (T) and dihydrotestosterone (DHT). DHT is produced via the classical, backdoor, and alternative steroidogenic pathways. In addition, recent studies have identified C11-oxy C19 steroids as novel human androgens. Although the placenta is known to be involved in steroid metabolism, androgen levels in full-term placentas have poorly been investigated. Subjects and methods Ten placentas of healthy full-term neonates (five males and five females) were examined. We quantified progesterone, androstenedione (A4), T, allopregnanolone, androsterone, and estradiol, as well as four C11-oxy androgens (11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT)), using liquid chromatography-tandem mass spectrometry. Results In all samples, levels of the ten steroids were above the detection limit. Progesterone was by far most abundant, while levels of T and androsterone were relatively low. Levels of 11KT and 11KA4 were higher than those of T and A4, respectively. There were no differences in steroid levels between male and female samples. Discussion This study demonstrates that full-term placentas contain several steroids in the classical, backdoor, and alternative pathways. Placentas are likely to function as the supplier of progesterone to other steroidogenic tissues. More importantly, we found that placentas comprise relatively large amounts of 11KA4 and 11KT, which may be produced through steroid transfer from the adrenal gland or from the maternal circulation. These results indicate that the placenta participates in a feto-maternal multi-organ network for androgen biosynthesis.
- Published
- 2021
17. Role of Liquid-Liquid Separation in Endocrine and Living Cells
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Yuko Katoh-Fukui, Satoshi Narumi, Maki Fukami, Mami Miyado, Yukihiro Hasegawa, Kei Yoshida, and Kazuhisa Akiba
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sex development ,Endocrinology, Diabetes and Metabolism ,Cellular functions ,Context (language use) ,Paraspeckle ,Enteroendocrine cell ,Biology ,Mini-Review ,paraspeckle ,phase transition ,Liquid liquid ,Endocrine system ,type 2 diabetes ,cellular body ,Neuroscience ,Eukaryotic cell ,Evidence synthesis ,AcademicSubjects/MED00250 - Abstract
ContextRecent studies have revealed that every eukaryotic cell contains several membraneless organelles created via liquid–liquid phase separation (LLPS). LLPS is a physical phenomenon that transiently compartmentalizes the subcellular space and thereby facilitates various biological reactions. LLPS is indispensable for cellular functions; however, dysregulated LLPS has the potential to cause irreversible protein aggregation leading to degenerative disorders. To date, there is no systematic review on the role of LLPS in endocrinology.Evidence acquisitionWe explored previous studies which addressed roles of LLPS in living cells, particularly from the viewpoint of endocrinology. To this end, we screened relevant literature in PubMed published between 2009 and 2021 using LLPS-associated keywords including “membraneless organelle,” “phase transition,” and “intrinsically disordered,” and endocrinological keywords such as “hormone,” “ovary,” “androgen,” and “diabetes.” We also referred to the articles in the reference lists of identified papers.Evidence synthesisBased on 67 articles selected from 449 papers, we provided a concise overview of the current understanding of LLPS in living cells. Then, we summarized recent articles documenting the physiological or pathological roles of LLPS in endocrine cells.ConclusionsThe discovery of LLPS in cells has resulted in a paradigm shift in molecular biology. Recent studies indicate that LLPS contributes to male sex development by providing a functional platform for SOX9 and CBX2 in testicular cells. In addition, dysregulated LLPS has been implicated in aberrant protein aggregation in pancreatic β-cells, leading to type 2 diabetes. Still, we are just beginning to understand the significance of LLPS in endocrine cells.
- Published
- 2021
18. Structural and numerical Y chromosomal variations in elderly men identified through multiplex ligation-dependent probe amplification
- Author
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Erina Suzuki, Kouichi Ozaki, Mami Miyado, Yasuko Ogiwara, Sumpei Niida, and Maki Fukami
- Subjects
0301 basic medicine ,Male ,congenital, hereditary, and neonatal diseases and abnormalities ,endocrine system diseases ,DNA Copy Number Variations ,030105 genetics & heredity ,Biology ,Y chromosome ,Germline ,03 medical and health sciences ,mental disorders ,Genetics ,Humans ,Multiplex ,Digital polymerase chain reaction ,Multiplex ligation-dependent probe amplification ,Genetics (clinical) ,Genetic Association Studies ,Aged ,Chromosome Aberrations ,Azoospermia factor ,Chromosomes, Human, Y ,Genetic Variation ,030104 developmental biology ,Phenotype - Abstract
Human Y chromosomes frequently acquire structural and numerical alterations. Known alterations include germline copy-number variations (CNVs) in the azoospermia factor (AZF) region and somatic mosaic loss of the Y chromosome (mLOY). Here, we explored Y chromosomal variations in 160 Japanese men aged 75-90 years. Multiplex ligation-dependent probe amplification (MLPA) identified ten types of AZF-linked CNVs in 77 men and mLOY of various degrees in 37. Seventeen men carried both a CNV and mLOY. MLOY levels estimated by MLPA were closely correlated with those determined by droplet digital PCR. No association was found between AZF-linked CNVs and the frequency or levels of mLOY. These results emphasize the high frequency and large inter-individual variability of AZF-linked CNVs and mLOY, and demonstrate the usefulness of MLPA in the detection of these variations. More importantly, this study provides the first evidence that AZF-linked CNVs do not increase the risk of aging-related mLOY.
- Published
- 2021
19. MAMLD1 and Differences/Disorders of Sex Development: An Update
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Maki Fukami, Tsutomu Ogata, and Mami Miyado
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Male ,Embryology ,Fetus ,Mutation ,Endocrinology, Diabetes and Metabolism ,Infant, Newborn ,Causative gene ,Nuclear Proteins ,Leydig Cells ,Biology ,Bioinformatics ,medicine.disease ,medicine.disease_cause ,DNA-Binding Proteins ,Testicular function ,medicine ,Ovarian dysfunction ,Humans ,Sex organ ,Disorders of sex development ,Gene ,Developmental Biology ,Transcription Factors - Abstract
MAMLD1 (alias CXorf6) was first documented in 2006 as a causative gene of 46,XY differences/disorders of sex development (DSD). MAMLD1/Mamld1 is expressed in the fetal testis and is predicted to enhance the expression of several Leydig cell-specific genes. To date, hemizygous MAMLD1 variants have been identified in multiple 46,XY individuals with hypomasculinized external genitalia. Pathogenic MAMLD1 variants are likely to cause genital abnormalities at birth and are possibly associated with age-dependent deterioration of testicular function. In addition, some MAMLD1 variants have been identified in 46,XX individuals with ovarian dysfunction. However, recent studies have raised the possibility that MAMLD1 variants cause 46,XY DSD and ovarian dysfunction as oligogenic disorders. Unsolved issues regarding MAMLD1 include the association between MAMLD1 variants and 46,XX testicular DSD, gene-gene interactions in the development of MAMLD1-mediated DSD, and intracellular functions of MAMLD1.
- Published
- 2021
20. Circulating steroids and mood disorders in patients with polycystic ovary syndrome
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Kazuki Saito, Naoyuki Miyasaka, Mami Miyado, Tomonori Ishikawa, Maki Fukami, Keiko Matsubara, Tsuguhiko Kato, Toshihiro Kawamura, and Tomoko Yoshida
- Subjects
Adult ,medicine.medical_specialty ,Neuroactive steroid ,Adolescent ,Clinical Biochemistry ,030209 endocrinology & metabolism ,Hospital Anxiety and Depression Scale ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Internal medicine ,Medicine ,Humans ,Androstenedione ,Molecular Biology ,Depression (differential diagnoses) ,Pharmacology ,business.industry ,Mood Disorders ,Organic Chemistry ,Allopregnanolone ,medicine.disease ,Polycystic ovary ,chemistry ,Mood disorders ,030220 oncology & carcinogenesis ,Androgens ,Anxiety ,Female ,medicine.symptom ,business ,Polycystic Ovary Syndrome - Abstract
Aberrant androgen metabolism is a characteristic feature of polycystic ovary syndrome (PCOS). Various androgens as well as their precursors and metabolites can accumulate in the blood of PCOS patients. Although these steroids include neuroactive steroids, such as allopregnanolone and androstenedione (Δ4A), it remains unknown whether altered blood steroid levels contribute to the high risk of mood disorders in PCOS. In this study, we measured blood levels of 11 steroids in 25 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay, and assessed the psychological status of these patients using the Hospital Anxiety and Depression Scale (HADS) questionnaire. We also examined age and the degree of metabolic abnormalities of each patient. Steroid values of the patients were compared to our previous data from 31 eumenorrheic women. As a result, 20 patients exhibited aberrant blood levels of one or more of the 11 tested steroids. In most cases, Δ4A and allopregnanolone levels were within or close to the reference ranges. Levels of four steroids were negatively correlated with patients' age, while no correlation was observed between steroid values and metabolic conditions. Seven patients showed high HADS scores. HADS scores were correlated with blood Δ4A levels even after stratifying by body mass indexes, but not with the levels of other steroids or clinical data. These results indicate that the high frequency of anxiety and depression in PCOS patients cannot be ascribed to altered blood levels of a specific steroid, although there may be a weak association between circulating Δ4A levels and psychological conditions of the patients.
- Published
- 2020
21. Random X chromosome inactivation in patients with Klinefelter syndrome
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Erina Suzuki, Yoshitomo Kobori, Tsutomu Ogata, Mami Miyado, Maki Fukami, Tomoko Yoshida, Kenichi Kinjo, and Hiroshi Okada
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DNA methylation ,030219 obstetrics & reproductive medicine ,Skewed inactivation ,Short Communication ,Aneuploidy ,Locus (genetics) ,Karyotype ,Biology ,medicine.disease ,X-inactivation ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,47,XXY ,medicine ,Blastocyst ,Klinefelter syndrome ,Sex chromosome ,030217 neurology & neurosurgery ,X chromosome - Abstract
Background X chromosome inactivation (XCI) is an indispensable process in the development of human female embryos. Reportedly, XCI occurs when a blastocyst contains 10–12 embryonic progenitor cells. To date, it remains unclear whether XCI ratios are normally preserved in Klinefelter syndrome (KS) patients with 47,XXY karyotype. Methods We examined XCI ratios in 18 KS patients through DNA methylation analysis for the polymorphic trinucleotide locus in the AR gene. The results of the KS patients were compared to previous data from healthy young women. Results XCI ratios in KS patients followed a normal distribution. Skewed XCI was observed in two patients, one of whom exhibited extremely skewed XCI. The frequencies of skewed and extremely skewed XCI in the KS cohort were comparable to those in healthy women. Conclusion This study confirmed the rarity of skewed XCI in KS patients. These results indicate that the presence of a supernumerary X chromosome during the cleavage and early blastocyst stages does not affect the developmental tempo of embryos. Our data deserve further validation.
- Published
- 2020
22. Degradation of phosphate polymer polyP enhances lactic fermentation in mice
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Mami Miyado, Natsuko Kawano, Maki Iwai, Maito Hanai, Kei Motomura, Kenji Miyado, Yoshitaka Miyamoto, Toshio Hamatani, Takakazu Saito, Hidekazu Saito, Kiyoshi Sekiguchi, Woojin Kang, Akihiro Umezawa, Mitsutoshi Yamada, Akio Kuroda, Mamoru Tanaka, and Akihiro Nakamura
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Polymers ,Cell Respiration ,030106 microbiology ,Mice, Transgenic ,Saccharomyces cerevisiae ,Biology ,Phosphates ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Polyphosphates ,Escherichia coli ,Genetics ,Animals ,Lactic Acid ,Exopolyphosphatase ,Cell Biology ,Phosphate ,biology.organism_classification ,Acid Anhydride Hydrolases ,Mitochondria ,Lactic acid ,030104 developmental biology ,chemistry ,Biochemistry ,Fermentation ,Oocytes ,Adenosine triphosphate ,Intracellular ,Lactic acid fermentation ,Bacteria - Abstract
In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.
- Published
- 2018
23. Autophagy-disrupted LC3 abundance leads to death of supporting cells of human oocytes
- Author
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Woojin Kang, Akihiro Umezawa, Natsuko Kawano, Kuniko Tatsumi, Maki Iwai, Kazuki Saito, Hidekazu Saito, Eri Ishida, Toshio Hamatani, Mami Miyado, Yoshitaka Miyamoto, Kenji Yamatoya, Kenji Miyado, Akihiro Nakamura, and Takakazu Saito
- Subjects
Cell death ,0301 basic medicine ,endocrine system ,Programmed cell death ,Cumulus cells ,Granulosa cell ,Biophysics ,Cell parts ,Biology ,Immunoglobulin light chain ,Biochemistry ,lcsh:Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Human fertilization ,Autophagy ,LC3 ,medicine ,lcsh:QD415-436 ,lcsh:QH301-705.5 ,chemistry.chemical_classification ,Human granulosa cells ,Oocyte ,Cell biology ,030104 developmental biology ,Enzyme ,medicine.anatomical_structure ,lcsh:Biology (General) ,chemistry ,030220 oncology & carcinogenesis ,embryonic structures ,biological phenomena, cell phenomena, and immunity ,ATG7 ,Research Article - Abstract
Autophagic recycling of cell parts is generally termed as the opposite of cell death. Here, we explored the relation between cell death and autophagy by examining granulosa cell layers that control oocyte quality, which is important for the success of fertilization. Granulosa cell layers were collected from infertile women and morphologically divided into four types, viz., mature (MCCs), immature (ICCs), and dysmature cumulus cells (DCCs), and mural granulosa cells (MGCs). Microtubule-associated protein light chain 3 (LC3), which is involved in autophagosome formation, was expressed excessively in DCCs and MGCs, and their chromosomal DNA was highly fragmented. However, autophagy initiation was limited to MGCs, as indicated by the expression of membrane-bound LC3-II and autophagy-related protein 7 (ATG7), an enzyme that converts LC3-I to LC3-II. Although pro-LC3 was accumulated, autophagy was disabled in DCCs, resulting in cell death. Our results suggest the possibility that autophagy-independent accumulation of pro-LC3 proteins leads to the death of human granulosa cells surrounding the oocytes and presumably reduces oocyte quality and female fertility., Graphical abstract fx1, Highlights • LC3 expression was remarkably high in MGCs and DCCs. • Typical autophagy was seen only in MGCs. • ATG7 expression and number of lysosomes were low in DCCs. • Distinct modes of cell death occurred in MGCs and DCCs.
- Published
- 2018
24. Ubiquitin-activating enzyme E1 inhibitor PYR-41 retards sperm enlargement after fusion to the egg
- Author
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Woojin Kang, Hidekazu Saito, Mami Miyado, Akihiro Umezawa, Natsuko Kawano, Keiichi Yoshida, Maki Iwai, Kenji Miyado, Akihiro Nakamura, Toshio Hamatani, Yoshitaka Miyamoto, and Maito Hanai
- Subjects
Male ,0301 basic medicine ,Immunocytochemistry ,Mice, Transgenic ,Fertilization in Vitro ,Ubiquitin-Activating Enzymes ,Toxicology ,Benzoates ,03 medical and health sciences ,Ubiquitin ,Meiosis ,Animals ,Furans ,beta Catenin ,Ovum ,Sperm-Ovum Interactions ,biology ,Chemistry ,fungi ,Ubiquitination ,Embryo ,Spermatozoa ,Sperm ,Spindle apparatus ,Cell biology ,030104 developmental biology ,Sperm Motility ,biology.protein ,Pyrazoles ,PYR-41 ,Spermatogenesis - Abstract
The ubiquitin-proteasome system, which is initiated by a single ubiquitin-activating enzyme E1 (UBE1), is involved in male reproduction via spermatogenesis and function in mammals. Here we explored the influence of UBE1-specific inhibitor, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (pyrazone-41 or PYR-41) in female reproduction. UBE-1 was detected by immunoblotting and immunocytochemistry in mouse eggs and was localized mainly under the egg plasma membrane. PYR-41 pretreatment suppresses the development of eggs into two-cell embryos. Specifically, pretreatment retarded sperm enlargement and meiotic chromosomal division after sperm-egg fusion. PYR-41 pretreatment disturbed β-catenin, a well-known target protein for ubiquitination, localization under the egg plasma membrane and on spindle microtubules in wild-type eggs. Otherwise, PYR-41 treatment had no effect on the two-cell development of eggs lacking β-catenin. Our results raise the possibility that inhibition of the ubiquitin-proteasome system suppresses sperm enlargement through impaired β-catenin-mediated mechanism.
- Published
- 2018
25. Expression of Xenobiotic Biomarkers CYP1 Family in Preputial Tissue of Patients with Hypospadias and Phimosis and Its Association with DNA Methylation Level of SRD5A2 Minimal Promoter
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Toshiki Aiba, Maki Fukami, Seiichiroh Ohsako, Yoshiyuki Kojima, Yutaro Hayashi, Kentaro Mizuno, Tsutomu Ogata, and Mami Miyado
- Subjects
Male ,0301 basic medicine ,medicine.medical_specialty ,Adolescent ,Health, Toxicology and Mutagenesis ,CYP1B1 ,Foreskin ,Preputial gland ,Biology ,Toxicology ,Article ,Xenobiotics ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,3-Oxo-5-alpha-Steroid 4-Dehydrogenase ,Internal medicine ,Cytochrome P-450 CYP1A1 ,medicine ,Humans ,RNA, Messenger ,Child ,Promoter Regions, Genetic ,Hypospadias ,Infant ,Membrane Proteins ,Promoter ,General Medicine ,Methylation ,DNA Methylation ,Phimosis ,medicine.disease ,Pollution ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Receptors, Androgen ,Child, Preschool ,SRD5A2 ,Cytochrome P-450 CYP1B1 ,DNA methylation ,CpG Islands ,Biomarkers ,030217 neurology & neurosurgery - Abstract
Several epidemiological studies have suggested that the incidence of male reproductive organ malformations, including hypospadias or cryptorchidism, has increased due to fetal-stage exposure to environmental pollutants. However, the association of chemical exposure with the expression of target regulatory genes in the tissues of patients has not yet been reported. Because experimental approaches or clinical trials in human studies are limited, especially those using fetal and/or infants, it is difficult to obtain clear physiological evidence of mechanisms underlying male reproductive malformations. Thus, the lack of physiological evidence makes this issue controversial. We analyzed preputial tissues from patients with hypospadias (n = 23) and phimosis (n = 16). The atypical CYP1 family genes, CYP1A1 and CYP1B1, are potential biomarkers of environmental chemical exposure. We then compared the expression levels of CYP1A1 and CYP1B1 between hypospadias and phimosis samples by quantitative RT-PCR analysis. The mRNA expression levels of SRD5A2 and AR also were measured, because the androgen-related genes involved in the onset of disorders of male reproductive system. A significantly higher CYP1B1 expression level and a lower AR expression level were observed in the hypospadias groups than in the phimosis group. Positive correlations (P
- Published
- 2017
26. Next-generation sequencing for patients with non-obstructive azoospermia: implications for significant roles of monogenic/oligogenic mutations
- Author
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Hiromichi Ishikawa, A. Yoshida, Kohji Okamura, Kazuhiko Nakabayashi, Kazuki Saito, Yoichi Matsubara, A. Nakamura, Shigeru Nakamura, Yoshitomo Kobori, Tsutomu Ogata, Mami Miyado, Yoko Tanaka, Hiroshi Okada, H. Ogata, Momori Katsumi, Maki Fukami, Kenichirou Hata, and Hideo Nakai
- Subjects
Male ,0301 basic medicine ,Multifactorial Inheritance ,DNA Copy Number Variations ,endocrine system diseases ,Chromosomal Proteins, Non-Histone ,Sequence analysis ,Urology ,Endocrinology, Diabetes and Metabolism ,DNA Mutational Analysis ,Gene Dosage ,Cell Cycle Proteins ,Biology ,urologic and male genital diseases ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Japan ,Predictive Value of Tests ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,Humans ,Missense mutation ,Genetic Predisposition to Disease ,Gene ,X chromosome ,Azoospermia ,Genetics ,Chromosomes, Human, X ,Comparative Genomic Hybridization ,Chromosomes, Human, Y ,Polymorphism, Genetic ,030219 obstetrics & reproductive medicine ,Autosome ,Splice site mutation ,High-Throughput Nucleotide Sequencing ,medicine.disease ,Fertility ,Phenotype ,030104 developmental biology ,Reproductive Medicine ,Mutation ,Genome-Wide Association Study ,Comparative genomic hybridization - Abstract
Azoospermia affects up to 1% of adult men. Non-obstructive azoospermia is a multifactorial disorder whose molecular basis remains largely unknown. To date, mutations in several genes and multiple submicroscopic copy-number variations (CNVs) have been identified in patients with non-obstructive azoospermia. The aim of this study was to clarify the contribution of nucleotide substitutions in known causative genes and submicroscopic CNVs in the genome to the development of non-obstructive azoospermia. To this end, we conducted sequence analysis of 25 known disease-associated genes using next-generation sequencing and genome-wide copy-number analysis using array-based comparative genomic hybridization. We studied 40 Japanese patients with idiopathic non-obstructive azoospermia. Functional significance of molecular alterations was assessed by in silico analyses. As a result, we identified four putative pathogenic mutations, four rare polymorphisms possibly associated with disease risk, and four probable neutral variants in 10 patients. These sequence alterations included a heterozygous splice site mutation in SOHLH1 and a hemizygous missense substitution in TEX11, which have been reported as causes of non-obstructive azoospermia. Copy-number analysis detected five X chromosomal or autosomal CNVs of unknown clinical significance, in addition to one known pathogenic Y chromosomal microduplication. Five patients carried multiple molecular alterations. The results indicate that monogenic and oligogenic mutations, including those in SOHLH1 and TEX11, account for more than 10% of cases of idiopathic non-obstructive azoospermia. Furthermore, this study suggests possible contributions of substitutions in various genes as well as submicroscopic CNVs on the X chromosome and autosomes to non-obstructive azoospermia, which require further validation.
- Published
- 2017
27. Increased incidence of post-term delivery and Cesarean section after frozen-thawed embryo transfer during a hormone replacement cycle
- Author
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Toshiro Kubota, Takakazu Saito, Mami Miyado, Hidekazu Saito, Kazuki Saito, Tomonori Ishikawa, Kenji Miyado, Maki Fukami, Eisuke Inoue, Akira Kuwahara, and Kenji Yamatoya
- Subjects
medicine.medical_specialty ,Reproductive Techniques, Assisted ,Offspring ,medicine.medical_treatment ,Reproductive medicine ,Hormone replacement ,Fertilization in Vitro ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Pregnancy ,Genetics ,medicine ,Humans ,Term delivery ,In patient ,030212 general & internal medicine ,Assisted Reproduction Technologies ,Genetics (clinical) ,Cryopreservation ,030219 obstetrics & reproductive medicine ,Assisted reproductive technology ,Cesarean Section ,Obstetrics ,business.industry ,Incidence (epidemiology) ,Pregnancy Outcome ,Obstetrics and Gynecology ,General Medicine ,Embryo Transfer ,Embryo transfer ,Reproductive Medicine ,Female ,business ,Live Birth ,Developmental Biology - Abstract
This study aimed to clarify the risks of adverse pregnancy outcomes in patients who conceive singletons after frozen embryo transfer (FET) during a hormone replacement cycle and their offspring.A retrospective cohort study was conducted in patients who conceived after FET, based on the Japanese-assisted reproductive technology registry for 2013. The perinatal outcomes in cases with live-born singletons achieved through natural ovulatory cycle FET (NC-FET) (n = 6287) or hormone replacement cycle FET (HRC-FET) (n = 10,235) were compared. Multiple logistic regression analyses were performed to determine the potential confounding factors.The frequencies of macrosomia (1.1% in NC-FET and 1.4% in HRC-FET; P = 0.058) were comparable between patients after NC-FET and HRC-FET. The proportions of post-term delivery (0.2% in NC-FET and 1.3% in HRC-FET; P 0.001) and Cesarean section (33.6% in NC-FET and 43.0% in HRC-FET; P 0.001) were higher in patients after HRC-FET than in patients after NC-FET. The risks of post-term delivery (adjusted odds ratio (AOR) 5.68, 95% confidence interval (CI) 3.30-9.80) and Cesarean section (AOR 1.64, 95% CI 1.52-1.76) were also higher in patients after HRC-FET than in patients after NC-FET.Patients who conceived singletons after HRC-FET were at increased risk of post-term delivery and Cesarean section compared with those who conceived after NC-FET.
- Published
- 2017
28. Reply: Artificial cycle 'per se' or the specific protocol of endometrial preparation as responsible for obstetric complications of frozen cycle?
- Author
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Naho Morisaki, Tomonori Ishikawa, Maki Fukami, Hidekazu Saito, Kenji Miyado, Minoru Irahara, Osamu Ishihara, Naoyuki Miyasaka, Akira Kuwahara, Mami Miyado, and Kazuki Saito
- Subjects
Gynecology ,Pregnancy ,medicine.medical_specialty ,Placenta accreta ,business.industry ,Rehabilitation ,Obstetrics and Gynecology ,Hypertension, Pregnancy-Induced ,Placenta Accreta ,medicine.disease ,Endometrium ,Embryo Transfer ,Embryo transfer ,Diabetes, Gestational ,medicine.anatomical_structure ,Reproductive Medicine ,Diabetes mellitus ,medicine ,Gestation ,Pregnancy induced ,Humans ,Female ,business - Published
- 2019
29. Publisher Correction: Calaxin is required for cilia-driven determination of vertebrate laterality
- Author
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Ryuji Yanase, Mami Miyado, Shuji Takada, Akihiro Nakamura, Hiroshi Yamaguchi, Masahide Kikkawa, Masahito Ikawa, Daisuke Shibata, Mami Nomura, Yuhkoh Satouh, Natsuko Kawano, Kogiku Shiba, Kenji Miyado, Keita Sasaki, Hironori Ueno, Motohiro Morikawa, Tadashi Baba, Shigenori Nonaka, Kei Jokura, and Kazuo Inaba
- Subjects
lcsh:Biology (General) ,biology.animal ,Cilium ,Laterality ,Medicine (miscellaneous) ,Vertebrate ,Biology ,General Agricultural and Biological Sciences ,lcsh:QH301-705.5 ,Neuroscience ,General Biochemistry, Genetics and Molecular Biology - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2019
30. Losing maleness: Somatic Y chromosome loss at every stage of a man's life
- Author
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Mami Miyado and Maki Fukami
- Subjects
Infertility ,Cancer Research ,Y chromosome ,Physiology ,Turner syndrome ,Cancer ,Karyotype ,Disease ,Hypothesis ,Biology ,medicine.disease ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Short stature ,karyotype ,somatic mosaicism ,lcsh:Biology (General) ,medicine ,Molecular Medicine ,Disorders of sex development ,medicine.symptom ,infertility ,lcsh:QH301-705.5 ,Demography - Abstract
Mosaic loss of Y chromosome (LOY) is assumed to be among the most common acquired genetic variations in elderly people. Recent studies have linked aging‐related mosaic LOY to the risk of Alzheimer's disease, cancer, and early death. Here, we propose that mosaic LOY can present in men at any age. Mosaic LOY appears to be associated with disorders of sex development and Turner syndrome at birth, short stature from childhood, and spermatogenic failure at reproductive age, in addition to shortened survival after 60 years of age.
- Published
- 2019
31. SHOX far-downstream copy-number variations involving cis-regulatory nucleotide variants in two sisters with Leri-Weill dyschondrosteosis
- Author
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Hirohito Shima, Kenichiro Ogushi, Koji Muroya, Mami Miyado, Tomoko Jinno, and Maki Fukami
- Subjects
0301 basic medicine ,Male ,Adolescent ,DNA Copy Number Variations ,Genotype ,Single-nucleotide polymorphism ,Dwarfism ,Haploinsufficiency ,030105 genetics & heredity ,Biology ,Osteochondrodysplasias ,Polymorphism, Single Nucleotide ,Chromosome conformation capture ,03 medical and health sciences ,Gene Frequency ,Short Stature Homeobox Protein ,Genetics ,medicine ,Humans ,Gene Regulatory Networks ,Copy-number variation ,Indel ,Child ,Léri–Weill dyschondrosteosis ,Genetics (clinical) ,Growth Disorders ,Siblings ,medicine.disease ,Idiopathic short stature ,Pedigree ,Minor allele frequency ,030104 developmental biology ,Gene Expression Regulation ,Haplotypes ,Child, Preschool ,Mutation ,Female - Abstract
SHOX haploinsufficiency leading to Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature typically results from intragenic mutations or copy-number variations (CNVs) involving SHOX and/or its putative enhancer regions that are distributed in the genomic interval between 400 kb and 840 kb from Xpter/Ypter. Here, we report two sisters with LWD, who carried a deletion in the far-downstream region of SHOX. The 0.62 Mb deletion contained 50 single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels), whose genotypes were linked to SHOX expression levels in the Genotype-Tissue Expression portal. Notably, most of these SNPs/indels accumulated within a ~20 kb interval that was positioned ~900 kb away from Xpter/Ypter. These SNPs/indels showed similar minor allele frequencies, indicating that they reside within a haplotype block. The ~20 kb interval was not evolutionarily conserved; however, it was associated with the previously determined peak of chromosome conformation capture profiling (4C)-seq. Importantly, the deletion in the present cases partially overlapped with CNVs of three previous cases with skeletal deformity and/or short stature. The results indicate that far-downstream CNVs constitute rare genetic causes of SHOX haploinsufficiency. These CNVs possibly impair SHOX expression through copy-number changes of a human-specific cis-regulatory haplotype block. This notion awaits further validation.
- Published
- 2019
32. Dihydrotestosterone induces minor transcriptional alterations in genital skin fibroblasts of children with and without androgen insensitivity
- Author
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Yoshiyuki Kojima, Kenji Matsumoto, Kanako Tanase-Nakao, Mariko Hara, Yoichi Matsubara, Maki Fukami, Maki Igarashi, Kentaro Mizuno, Yutaro Hayashi, and Mami Miyado
- Subjects
Male ,medicine.medical_specialty ,Apolipoprotein D ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,030209 endocrinology & metabolism ,Biology ,urologic and male genital diseases ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Internal medicine ,medicine ,Humans ,Genital tubercle ,Partial androgen insensitivity syndrome ,Microarray analysis techniques ,Infant ,Dihydrotestosterone ,Androgen-Insensitivity Syndrome ,Fibroblasts ,Androgen ,medicine.disease ,Androgen receptor ,Gene Expression Regulation ,Receptors, Androgen ,Tissue Array Analysis ,030220 oncology & carcinogenesis ,Androgens ,Androgen insensitivity syndrome ,medicine.drug ,Penis - Abstract
Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.
- Published
- 2019
33. DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations
- Author
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Kenichiro, Ogushi, Atsushi, Hattori, Erina, Suzuki, Hirohito, Shima, Masako, Izawa, Hideaki, Yagasaki, Reiko, Horikawa, Kimiaki, Uetake, Akihiro, Umezawa, Tomohiro, Ishii, Koji, Muroya, Noriyuki, Namba, Toshiaki, Tanaka, Yasuhiro, Hirano, Hitoshi, Yamamoto, Shun, Soneda, Keiko, Matsubara, Masayo, Kagami, Mami, Miyado, and Maki, Fukami
- Subjects
Adult ,Adolescent ,DNA Copy Number Variations ,Genetic Diseases, X-Linked ,Sequence Analysis, DNA ,DNA Methylation ,Osteochondrodysplasias ,Chondrocytes ,Short Stature Homeobox Protein ,Case-Control Studies ,Child, Preschool ,Humans ,CpG Islands ,Female ,Child ,Cells, Cultured ,Growth Disorders - Abstract
SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.
- Published
- 2018
34. IdenticalNR5A1Missense Mutations in Two Unrelated 46,XX Individuals with Testicular Tissues
- Author
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Kei Takasawa, Maki Fukami, Yoichi Matsubara, Akiko Hakoda, Kenichi Kashimada, Shuji Takada, Tsutomu Ogata, Mami Miyado, Maki Igarashi, Kenichiro Hata, Ken Ichirou Morohashi, Takashi Baba, Ryohei Sekido, Toshihiro Tajima, Junko Kanno, and Kazuhiko Nakabayashi
- Subjects
Male ,Models, Molecular ,0301 basic medicine ,endocrine system ,Genotype ,Protein Conformation ,DNA Mutational Analysis ,Karyotype ,Disorders of Sex Development ,Mutation, Missense ,030209 endocrinology & metabolism ,SOX9 ,Biology ,Steroidogenic Factor 1 ,medicine.disease_cause ,XY gonadal dysgenesis ,03 medical and health sciences ,0302 clinical medicine ,Testis ,Genetics ,medicine ,Humans ,Missense mutation ,Disorders of sex development ,Gonads ,Gene ,Alleles ,Genetics (clinical) ,Mutation ,Wild type ,Infant ,medicine.disease ,Phenotype ,030104 developmental biology ,Amino Acid Substitution ,Female ,Biomarkers - Abstract
The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wildtype NR5A1, the mutant protein was less sensitive to NR0B1-induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations in NR5A1 underlie testicular development in genetic females. This article is protected by copyright. All rights reserved
- Published
- 2016
35. Steroidogenic pathways involved in androgen biosynthesis in eumenorrheic women and patients with polycystic ovary syndrome
- Author
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Toshiro Kubota, Keiko Homma, Minoru Irahara, Hidekazu Saito, Kazuki Saito, Maki Fukami, Tsutomu Ogata, Mami Miyado, Eisuke Inoue, Takeshi Iwasa, Tomonobu Hasegawa, Toshiya Matsuzaki, and Yoshimichi Miyashiro
- Subjects
0301 basic medicine ,medicine.medical_specialty ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Androstanedione ,Internal medicine ,medicine ,Androstenedione ,Molecular Biology ,Testosterone ,030219 obstetrics & reproductive medicine ,Androsterone ,Allopregnanolone ,Cell Biology ,Androgen ,Polycystic ovary ,030104 developmental biology ,chemistry ,Dihydrotestosterone ,Molecular Medicine ,medicine.drug - Abstract
The conventional Δ5 and Δ4 steroidogenic pathways mediate androgen production in females. While multiple non-conventional pathways to dihydrotestosterone (DHT) have recently been postulated in humans, the functional significance of these pathways remains to be elucidated. The aim of this study was to clarify the origin of androgens in healthy women and in patients with polycystic ovary syndrome (PCOS), a multifactorial disorder characterized by androgen overproduction. We measured 13 steroids in blood samples of 31 eumenorrheic females and 28 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay. We found that 17-hydroxy (17-OH) progesterone (17-OHP), androstenedione (Δ4A), testosterone, androstanedione, androsterone, and androstanediol levels were higher in the patient group than in the eumenorrheic group, while levels of other steroids were comparable between the two groups. In the eumenorrheic group, DHT levels were correlated with testosterone, androstanedione, and androstanediol. Quantitative correlations were also observed among 17-OH allopregnanolone, androsterone, androstanediol, and DHT, and among Δ4A, androstanedione, androsterone, and androstanediol. In the patient group, DHT levels were correlated with testosterone levels, but not with androstanedione or androstanediol levels. Δ4A and testosterone paralleled 17-OHP. Androstanedione, androsterone, androstanediol, and 17-OH allopregnanolone were quantitatively correlated. In both groups, multivariable linear regression analyses suggested relationships between androsterone and androstanedione, as well as between androsterone and 17-OH allopregnanolone. These results indicate that multiple androgen biosynthesis pathways are operating in eumenorrheic females and PCOS patients. In PCOS patients, excessive androgens are produced primarily via the conventional pathways, while two alternative pathways; i.e., an androstanedione-mediated pathway and a so-called backdoor pathway, likely serve as sources of a weak androgen and potential precursors of DHT.
- Published
- 2016
36. Copy Number Variations of the Azoospermia Factor Region and SRY Are Not Associated with the Risk of Hypospadias
- Author
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Maki Fukami, Masafumi Kon, Kimihiko Moriya, Nobuo Shinohara, Katsuya Nonomura, Maki Igarashi, Kazuki Saito, Tsutomu Ogata, Mami Miyado, and Takahiko Mitsui
- Subjects
Male ,0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Embryology ,Adolescent ,DNA Copy Number Variations ,endocrine system diseases ,Endocrinology, Diabetes and Metabolism ,Biology ,Y chromosome ,03 medical and health sciences ,0302 clinical medicine ,Asian People ,mental disorders ,Gene duplication ,medicine ,Humans ,Multiplex ligation-dependent probe amplification ,Copy-number variation ,Child ,Azoospermia ,Genetics ,Hypospadias ,Azoospermia factor ,Chromosomes, Human, Y ,030219 obstetrics & reproductive medicine ,Infant, Newborn ,Infant ,medicine.disease ,030104 developmental biology ,Testis determining factor ,Child, Preschool ,Developmental Biology - Abstract
We investigated the frequency of copy number variations (CNVs) in the Y chromosome of Japanese children with hypospadias. We analyzed the copy number of the azoospermia factor (AZF) region and SRY, using multiplex ligation-dependent probe amplification. Four AZF-linked CNVs, including one novel simple duplication, were identified in 39 of 89 patients, at a frequency comparable to that of those in unaffected individuals. SRY-linked CNVs were absent in our patients. The results imply that CNVs in the AZF region and SRY are not associated with the risk of hypospadias in the Japanese population, although the pathogenicity of the AZF-linked simple duplication remains to be elucidated.
- Published
- 2016
37. Extra-adrenal induction of Cyp21a1 ameliorates systemic steroid metabolism in a mouse model of congenital adrenal hyperplasia
- Author
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Noriyuki Katsumata, Yasuhiro Naiki, Songya Pang, Reiko Horikawa, Maki Fukami, Tsutomu Ogata, Mami Miyado, and Masafumi Onodera
- Subjects
0301 basic medicine ,medicine.medical_specialty ,endocrine system diseases ,medicine.drug_class ,Steroid 21-Hydroxylase ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Genetic enhancement ,urologic and male genital diseases ,Viral vector ,Steroid ,03 medical and health sciences ,Endocrinology ,Internal medicine ,medicine ,Congenital adrenal hyperplasia ,business.industry ,Extra-Adrenal ,nutritional and metabolic diseases ,medicine.disease ,030104 developmental biology ,Mineralocorticoid ,business ,Glucocorticoid ,medicine.drug - Abstract
Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (21-OH) deficiency (21-OHD) is an autosomal recessive disorder, in which CYP21A2 mutations or deletions result in underproduction of glucocorticoid and mineralocorticoid, and overproduction of androgens. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Previously, adenovirus-mediated administration of human CYP21A2 to adrenal glands rescued the phenotype of a mouse model of 21-OHD. In this study, we examined whether transduction of murine Cyp21a1 in extra-adrenal tissues could rescue steroid metabolism in 21-OHD mice. We transduced primary fibroblasts obtained from 21-OHD mice with a retroviral vector containing Cyp21a1. In vitro assays demonstrated that Cyp21a1-expressing fibroblasts can uptake progesterone from the culture media, convert it to deoxycorticosterone (DOC), and subsequently release DOC back into the media. Autotransplantation of Cyp21a1-expressing fibroblasts into the subcutaneous tissues of the back resulted in a significant reduction in the serum progesterone/DOC ratio in four of six 21-OHD mice at 4 weeks after injection. We also directly injected an adeno-associated viral vector containing Cyp21a1 into the thigh muscles of 21-OHD mice. Serum progesterone/DOC ratios were markedly reduced in all four animals at 4 weeks after injection. These results indicate that extra-adrenal induction of Cyp21a1 ameliorates steroid metabolism in 21-OHD mice. This study suggests a novel therapeutic strategy for congenital adrenal hyperplasia, which warrants further investigations.
- Published
- 2016
38. Correction: Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction
- Author
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Akihiro Nakamura, Yoko Kuroki, Kenji Yamatoya, Yoshiki Hayashi, Akihiro Umezawa, Hidekazu Saito, Kenji Miyado, Seiya Kanai, Mami Miyado, Woojin Kang, Yuichirou Harada, Natsuko Kawano, Yoshitaka Miyamoto, and Yasuhiro Iwao
- Subjects
medicine.medical_specialty ,biology ,Chemistry ,Age dependent ,Cell Biology ,Sperm ,Pathology and Forensic Medicine ,Endocrinology ,Calcium oscillation ,Internal medicine ,medicine ,biology.protein ,Citrate synthase ,Molecular Biology - Published
- 2020
39. 11-oxygenated C19 steroids as circulating androgens in women with polycystic ovary syndrome
- Author
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Minoru Irahara, Kazuki Saito, Takeshi Iwasa, Tomoko Yoshida, Maki Fukami, Tsutomu Ogata, Mami Miyado, Yoichi Matsubara, and Toshiya Matsuzaki
- Subjects
0301 basic medicine ,Adult ,medicine.medical_specialty ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,030209 endocrinology & metabolism ,Overweight ,Body Mass Index ,03 medical and health sciences ,chemistry.chemical_compound ,Young Adult ,0302 clinical medicine ,Endocrinology ,Tandem Mass Spectrometry ,Internal medicine ,medicine ,Humans ,Clinical significance ,In patient ,Obesity ,business.industry ,Androgen ,medicine.disease ,Polycystic ovary ,030104 developmental biology ,Normal weight ,chemistry ,Androgens ,11-Ketotestosterone ,Female ,medicine.symptom ,business ,Chromatography, Liquid ,Polycystic Ovary Syndrome - Abstract
11-oxygenated C19 steroids (11oxC19s) are newly specified human androgens. Although median serum levels of 11oxC19 were reported to be higher in patients with polycystic ovary syndrome (PCOS) than in unaffected women, inter-individual variations in androgen levels among PCOS patients have poorly been investigated. Here, we quantified four 11oxC19s, i.e., 11-ketotestosterone (11KT), 11β-hydroxytestosterone (11OHT), 11β-hydroxyandrostenedione (11OHΔ4A), and 11-ketoandrostenedione (11KΔ4A), in blood samples of 28 PCOS patients and 31 eumenorrheic women using liquid chromatography-tandem mass spectrometry. We referred to our previous data of classic androgens in these individuals. We found that 11OHT levels were higher in the PCOS group than in the eumenorrheic group. Moreover, although the median values of 11KT, 11KΔ4A, and 11OHΔ4A were comparable between the two groups, these steroids were markedly increased in some patients. Of the 28 patients, 8 had high levels of both 11oxC19s and classic androgens, whereas 4 had an increase only in 11oxC19 levels, and 12 had an increase only in classic androgen levels. Intragroup variations in androgen levels were relatively large in the PCOS group. Levels of 11OHT and 11KT were significantly higher in overweight/obese patients than in normal weight patients and correlated with body mass indexes. These results highlight the clinical significance of 11oxC19s as circulating androgens in PCOS patients and indicate that the accumulation of 11oxC19s and/or classic androgens is an essential feature of PCOS. The profiles of circulating androgens appear to vary among patients. In particular, overweight/obesity likely enhances the 11oxC19s accumulation in PCOS, although this notion awaits further validation.
- Published
- 2018
40. Membrane protein CD9 is repositioned and released to enhance uterine function
- Author
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Mitsutoshi Yamada, Maki Iwai, Takakazu Saito, Hidekazu Saito, Seiya Kanai, Akihiro Umezawa, Natsuko Kawano, Mami Miyado, Toshio Hamatani, Yoshitaka Miyamoto, Akihiro Nakamura, Kenji Miyado, Noriko Yoshii, Woojin Kang, Mamoru Tanaka, and Yasushi Odawara
- Subjects
0301 basic medicine ,Bodily Secretions ,Uterus ,Estrous Cycle ,Mitochondrion ,Exosomes ,Tetraspanin 29 ,Pathology and Forensic Medicine ,Cell Line ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Tetraspanin ,Extracellular ,medicine ,Animals ,Humans ,Induced pluripotent stem cell ,Molecular Biology ,Chemistry ,Cell Biology ,Microvesicles ,Cell biology ,Mitochondria ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Membrane protein ,Cell culture ,030220 oncology & carcinogenesis ,embryonic structures ,Female ,Infertility, Female - Abstract
Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.
- Published
- 2018
41. Calaxin is required for cilia-driven determination of vertebrate laterality
- Author
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Mami Nomura, Natsuko Kawano, Shuji Takada, Motohiro Morikawa, Mami Miyado, Kogiku Shiba, Akihiro Nakamura, Tadashi Baba, Masahide Kikkawa, Masahito Ikawa, Daisuke Shibata, Hiroshi Yamaguchi, Kenji Miyado, Shigenori Nonaka, Kei Jokura, Hironori Ueno, Kazuo Inaba, Ryuji Yanase, Yuhkoh Satouh, and Keita Sasaki
- Subjects
Movement ,Dynein ,Medicine (miscellaneous) ,Flagellum ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Animals, Genetically Modified ,Ciliogenesis ,Ependyma ,medicine ,otorhinolaryngologic diseases ,Animals ,Cilia ,lcsh:QH301-705.5 ,Zebrafish ,Primary ciliary dyskinesia ,Cilium ,Calcium-Binding Proteins ,Brain ,Zebrafish Proteins ,biology.organism_classification ,medicine.disease ,Publisher Correction ,Cell biology ,Mice, Inbred C57BL ,Trachea ,Situs inversus ,Cytoskeletal Proteins ,lcsh:Biology (General) ,Flagella ,General Agricultural and Biological Sciences ,NODAL ,Ciliary Motility Disorders - Abstract
Sasaki, K., Shiba, K., Nakamura, A. et al. Calaxin is required for cilia-driven determination of vertebrate laterality. Commun Biol 2, 226 (2019). https://doi.org/10.1038/s42003-019-0462-y, Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.
- Published
- 2018
42. Somatically Acquired Isodicentric Y and Mosaic Loss of Chromosome Y in a Boy with Hypospadias
- Author
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Maki Fukami, Koji Muroya, Masafumi Kon, Mami Miyado, Kazuki Saito, and Momori Katsumi
- Subjects
0301 basic medicine ,Male ,Karyotype ,Sex Chromosome Disorders ,Biology ,Y chromosome ,Germline ,03 medical and health sciences ,Genetics ,medicine ,Humans ,Limited evidence ,Multiplex ligation-dependent probe amplification ,Molecular Biology ,Genetics (clinical) ,Hypospadias ,Chromosomes, Human, Y ,Mosaicism ,Chromosome ,medicine.disease ,Isodicentric y ,030104 developmental biology ,Child, Preschool ,Chromosome Deletion - Abstract
Isodicentric Y chromosome [idic(Y)] represents a relatively common subtype of Y chromosomal rearrangements in the germline; however, limited evidence supports the postzygotic occurrence of idic(Y). Here, we report a boy with hypospadias and somatically acquired idic(Y). The 3.5-year-old boy has been identified in our previous study for patients with hypospadias. In the present study, cytogenetic analysis including FISH revealed a 45,X[5]/46,X,idic(Y)[7]/46,XY[8] karyotype. MLPA showed a mosaic deletion involving PPP1R12BP1 and RBMY2DP. The idic(Y) was likely to have been formed through aberrant recombination between P1 palindromes and subsequently underwent mosaic loss. The patient's phenotype was attributable to deletion of some Y chromosomal genes and/or mosaic loss of chromosome Y (mLOY). The results suggest that idic(Y) can originate in postzygotic cells via palindrome-mediated crossovers. Moreover, our data indicate that somatically acquired idic(Y) can trigger mLOY, which usually appears as an aging-related phenomenon in elderly men.
- Published
- 2018
43. STX2 is a causative gene for nonobstructive azoospermia
- Author
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Hideo Nakai, Maki Fukami, Hiromichi Ishikawa, Yoshihiko Ueda, Tsutomu Ogata, Mami Miyado, Yoshitomo Kobori, Kazuki Saito, Shigeru Nakamura, Yoko Tanaka, Atsumi Yoshida, Akie Nakamura, Momori Katsumi, and Hiroshi Okada
- Subjects
0301 basic medicine ,Infertility ,Adult ,Male ,Loss of Heterozygosity ,Syntaxin 1 ,Biology ,urologic and male genital diseases ,Frameshift mutation ,Andrology ,03 medical and health sciences ,Mice ,fluids and secretions ,0302 clinical medicine ,Multinucleate ,STX2 ,hemic and lymphatic diseases ,Complementary DNA ,Testis ,Genetics ,medicine ,Animals ,Humans ,Spermatogenesis ,Genetics (clinical) ,Azoospermia ,030219 obstetrics & reproductive medicine ,Wild type ,bacterial infections and mycoses ,medicine.disease ,030104 developmental biology ,Mutation ,Mutation testing ,bacteria - Abstract
STX2 encodes a sulfoglycolipid transporter. Although Stx2 nullizygosity is known to cause spermatogenic failure in mice, STX2 mutations have not been identified in humans. Here, we performed STX2 mutation analysis for 131 Japanese men clinically diagnosed with nonobstructive azoospermia. As a result, we identified a homozygous frameshift mutation [c.8_12delACCGG, p.(Asp3Alafs*8)] in one patient. The mutation-positive patient exhibited loss-of-heterozygosity for 58.4 Mb genomic regions involving STX2, suggesting possible parental consanguinity. The patient showed azoospermia, relatively small testes, and a mildly elevated follicle stimulating hormone level, but no additional clinical features. Testicular histology of the patient showed universal maturation arrest and multinucleated spermatocytes, which have also been observed in mice lacking Stx2. PCR-based cDNA screening revealed wildtype STX2 expression in various tissues including the testis. Our results indicate that STX2 nullizygosity results in nonsyndromic maturation arrest with multinucleated spermatocytes, and accounts for a small fraction of cases with nonobstructive azoospermia.
- Published
- 2017
44. Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds
- Author
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Kazuo Fujita, Mami Miyado, Yusuke Hori, Akiko Tonoike, Akitsugu Konno, Miho Inoue-Murayama, Maki Fukami, Takefumi Kikusui, Kazutaka Mogi, and Miho Nagasawa
- Subjects
Genetics ,Wolves ,DNA Copy Number Variations ,business.industry ,Zoology ,Sequence Analysis, DNA ,General Medicine ,Breeding ,Biology ,Evolution, Molecular ,Dogs ,Japan ,Species Specificity ,Crop production ,Amylases ,Animals ,Animal Science and Zoology ,Livestock ,Rice farming ,Genetic Change ,Copy-number variation ,Domestication ,business ,Gene - Abstract
A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.
- Published
- 2015
45. Testicular dysgenesis/regression without campomelic dysplasia in patients carrying missense mutations and upstream deletion of <scp>SOX</scp> 9
- Author
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Yoichi Matsubara, Yuko Katoh-Fukui, Maki Igarashi, Ken Ichirou Morohashi, Erina Suzuki, Kazuhiko Nakabayashi, Keisuke Nagasaki, Tsutomu Ogata, Mami Miyado, Toshiro Nagai, Kenichiro Hata, Shuji Takada, Takashi Baba, Keiko Hayashi, Takayoshi Tsuchiya, Arisa Igarashi, Reiko Horikawa, and Maki Fukami
- Subjects
Genetics ,endocrine system ,Mutation ,Original Articles ,SOX9 ,testis ,Sex reversal ,Biology ,medicine.disease ,medicine.disease_cause ,Campomelic dysplasia ,Dysplasia ,Cancer research ,medicine ,Missense mutation ,Original Article ,deletion ,enhancer ,mutation ,Haploinsufficiency ,Molecular Biology ,Exome ,Genetics (clinical) - Abstract
SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1–3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C‐terminal domain but not in other known 46,XY DSD causative genes. These substitutions were absent from ~120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited ~491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short‐nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C‐terminal missense substitutions which lead to target‐gene‐specific protein dysfunction, and enhancer‐containing upstream microdeletions mediated by nonhomologous end‐joining.
- Published
- 2015
46. SOX3 Overdosage Permits Normal Sex Development in Females with Random X Inactivation
- Author
-
Momori Katsumi, Yoko Izumi, Maki Fukami, Hitoshi Mikami, Maki Igarashi, Tsutomu Ogata, and Mami Miyado
- Subjects
Male ,Embryology ,Gonad ,Endocrinology, Diabetes and Metabolism ,Gene Dosage ,Biology ,Gene dosage ,Genome ,X-inactivation ,chemistry.chemical_compound ,X Chromosome Inactivation ,Gene duplication ,medicine ,Humans ,Disorders of sex development ,Gene Rearrangement ,Genetics ,Genome, Human ,SOXB1 Transcription Factors ,Sexual Development ,medicine.disease ,Phenotype ,Molecular biology ,medicine.anatomical_structure ,chemistry ,Child, Preschool ,Female ,DNA ,Developmental Biology - Abstract
Submicroscopic duplications involving SOX3 and/or its flanking regions have been identified in 46,XX individuals both with and without disorders of sex development, raising the question whether SOX3 overdosage is sufficient to induce testicular development in genetically female individuals. Here, we report a mother-daughter pair with female phenotypes and random X inactivation. The individuals carry complex X chromosomal rearrangements leading to a copy number gain of genomic regions involving SOX3 and its upstream region. The amplified DNA fragments were detected at Xq27. These results provide evidence that SOX3 overdosage permits normal sex development in 46,XX individuals with random X inactivation.
- Published
- 2015
47. Gain-of-function mutations in G-protein-coupled receptor genes associated with human endocrine disorders
- Author
-
Maki Igarashi, Tsutomu Ogata, Mami Miyado, Maki Fukami, and Erina Suzuki
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,030209 endocrinology & metabolism ,Biology ,medicine.disease_cause ,Endocrine System Diseases ,G Protein-Coupled Receptor Gene ,Germline ,Frameshift mutation ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Internal medicine ,medicine ,Humans ,Receptor ,Gene ,G protein-coupled receptor ,Mutation ,Phenotype ,030104 developmental biology ,Gain of Function Mutation - Abstract
The human genome encodes more than 700 G-protein-coupled receptors (GPCRs), many of which are involved in hormone secretion. To date, more than 100 gain-of-function (activating) mutations in at least ten genes for GPCRs, in addition to several loss-of-function mutations, have been implicated in human endocrine disorders. Previously reported gain-of-function GPCR mutations comprise various missense substitutions, frameshift mutations, intragenic inframe deletions and copy-number gains. Such mutations appear in both germline and somatic tumour cells, and lead to various hormonal abnormalities reflecting excessive receptor activity. Phenotypic consequences of these mutations include distinctive endocrine syndromes, as well as relatively common hormonal abnormalities. Such mutations encode hyperfunctioning receptors with increased constitutive activity, broadened ligand specificity, increased ligand sensitivity and/or delayed receptor desensitization. Furthermore, recent studies proposed a paradoxical gain-of-function mechanism caused by inactive GPCR mutants. Molecular diagnosis of GPCR activating mutations serves to improve the clinical management of mutation-positive patients. This review aims to introduce new aspects regarding gain-of-function mutations in GPCR genes associated with endocrine disorders.
- Published
- 2017
48. Microhomology-Mediated Microduplication in the Y Chromosomal Azoospermia Factor a Region in a Male with Mild Asthenozoospermia
- Author
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Momori Katsumi, Tsutomu Ogata, Mami Miyado, Kazuhiko Nakabayashi, Yoichi Matsubara, Kazuki Saito, Hiromichi Ishikawa, Yoko Tanaka, Hiroshi Okada, Yoshitomo Kobori, Hidekazu Saito, and Maki Fukami
- Subjects
Adult ,Male ,Genetics ,Azoospermia factor ,Chromosomes, Human, Y ,DNA Copy Number Variations ,Pseudogene ,Non-allelic homologous recombination ,Biology ,Minor Histocompatibility Antigens ,Chromosome Breakpoints ,USP9Y ,Asthenozoospermia ,Gene Duplication ,Gene duplication ,Humans ,Human genome ,Copy-number variation ,Ubiquitin Thiolesterase ,Molecular Biology ,Gene ,Pseudogenes ,Genetics (clinical) - Abstract
Y chromosomal azoospermia factor (AZF) regions AZFa, AZFb and AZFc represent hotspots for copy number variations (CNVs) in the human genome; yet the number of reports of AZFa-linked duplications remains limited. Nonallelic homologous recombination has been proposed as the underlying mechanism of CNVs in AZF regions. In this study, we identified a hitherto unreported microduplication in the AZFa region in a Japanese male individual. The 629,812-bp duplication contained 22 of 46 exons of USP9Y, encoding the putative fine tuner of spermatogenesis, together with all exons of 3 other genes/pseudogenes. The breakpoints of the duplication resided in the DNA/TcMar-Tigger repeat and nonrepeat sequences, respectively, and were associated with a 2-bp microhomology, but not with short nucleotide stretches. The breakpoint-flanking regions were not enriched with GC content, palindromes, or noncanonical DNA structures. Semen analysis of the individual revealed a normal sperm concentration and mildly reduced sperm motility. The paternal DNA sample of the individual was not available for genetic analysis. The results indicate that CNVs in AZF regions can be generated by microhomology-mediated break-induced replication in the absence of known rearrangement-inducing DNA features. AZFa-linked microduplications likely permit production of a normal amount of sperm, although the precise clinical consequences of these CNVs await further investigation.
- Published
- 2014
49. Extra-adrenal induction of Cyp21a1 ameliorates systemic steroid metabolism in a mouse model of congenital adrenal hyperplasia
- Author
-
Yasuhiro, Naiki, Mami, Miyado, Reiko, Horikawa, Noriyuki, Katsumata, Masafumi, Onodera, Songya, Pang, Tsutomu, Ogata, and Maki, Fukami
- Subjects
Mice, Inbred C57BL ,Disease Models, Animal ,Mice ,Adrenal Hyperplasia, Congenital ,Gene Transfer Techniques ,Animals ,Humans ,Female ,Mice, Transgenic ,Genetic Therapy ,Steroid 21-Hydroxylase ,Glucocorticoids ,Up-Regulation - Abstract
Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (21-OH) deficiency (21-OHD) is an autosomal recessive disorder, in which CYP21A2 mutations or deletions result in underproduction of glucocorticoid and mineralocorticoid, and overproduction of androgens. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Previously, adenovirus-mediated administration of human CYP21A2 to adrenal glands rescued the phenotype of a mouse model of 21-OHD. In this study, we examined whether transduction of murine Cyp21a1 in extra-adrenal tissues could rescue steroid metabolism in 21-OHD mice. We transduced primary fibroblasts obtained from 21-OHD mice with a retroviral vector containing Cyp21a1. In vitro assays demonstrated that Cyp21a1-expressing fibroblasts can uptake progesterone from the culture media, convert it to deoxycorticosterone (DOC), and subsequently release DOC back into the media. Autotransplantation of Cyp21a1-expressing fibroblasts into the subcutaneous tissues of the back resulted in a significant reduction in the serum progesterone/DOC ratio in four of six 21-OHD mice at 4 weeks after injection. We also directly injected an adeno-associated viral vector containing Cyp21a1 into the thigh muscles of 21-OHD mice. Serum progesterone/DOC ratios were markedly reduced in all four animals at 4 weeks after injection. These results indicate that extra-adrenal induction of Cyp21a1 ameliorates steroid metabolism in 21-OHD mice. This study suggests a novel therapeutic strategy for congenital adrenal hyperplasia, which warrants further investigations.
- Published
- 2016
50. Mamld1 Deficiency Significantly Reduces mRNA Expression Levels of Multiple Genes Expressed in Mouse Fetal Leydig Cells but Permits Normal Genital and Reproductive Development
- Author
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Ken Ichirou Morohashi, Shinichiro Sano, Tsutomu Ogata, Mami Miyado, Michiko Nakamura, Eiko Nagata, Maki Fukami, and Kenji Miyado
- Subjects
Male ,endocrine system ,medicine.medical_specialty ,Cell type ,Time Factors ,In situ hybridization ,Biology ,Models, Biological ,Mice ,Endocrinology ,Internal medicine ,Testis ,medicine ,Animals ,RNA, Messenger ,Genital tubercle ,Mice, Knockout ,Models, Genetic ,Leydig cell ,Gene Expression Profiling ,Gene Expression Regulation, Developmental ,Leydig Cells ,Sertoli cell ,DNA-Binding Proteins ,Mice, Inbred C57BL ,Phenotype ,medicine.anatomical_structure ,Reproduction-Development ,CYP17A1 ,HSD3B1 ,Germ cell ,Transcription Factors - Abstract
Although mastermind-like domain containing 1 (MAMLD1) (CXORF6) on human chromosome Xq28 has been shown to be a causative gene for 46,XY disorders of sex development with hypospadias, the biological function of MAMLD1/Mamld1 remains to be elucidated. In this study, we first showed gradual and steady increase of testicular Mamld1 mRNA expression levels in wild-type male mice from 12.5 to 18.5 d postcoitum. We then generated Mamld1 knockout (KO) male mice and revealed mildly but significantly reduced testicular mRNA levels (65-80%) of genes exclusively expressed in Leydig cells (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3) as well as grossly normal testicular mRNA levels of genes expressed in other cell types or in Leydig and other cell types. However, no demonstrable abnormality was identified for cytochrome P450 17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B) protein expression levels, appearance of external and internal genitalia, anogenital distance, testis weight, Leydig cell number, intratesticular testosterone and other steroid metabolite concentrations, histological findings, in situ hybridization findings for sonic hedgehog (the key molecule for genital tubercle development), and immunohistochemical findings for anti-Müllerian hormone (Sertoli cell marker), HSD3B (Leydig cell marker), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (germ cell marker) in the KO male mice. Fertility was also normal. These findings imply that Mamld1 deficiency significantly reduces mRNA expression levels of multiple genes expressed in mouse fetal Leydig cells but permits normal genital and reproductive development. The contrastive phenotypic findings between Mamld1 KO male mice and MAMLD1 mutation positive patients would primarily be ascribed to species difference in the fetal sex development.
- Published
- 2012
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