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Biomedical subjects

Yasunori Yoshimura

Publications and source records attributed to Yasunori Yoshimura.

At least 19 recordsLinked to original sources
PubMed2006

Histone deacetylase inhibitors stimulate cell migration in human endometrial adenocarcinoma cells through up-regulation of glycodelin.

Histone deacetylase inhibitors (HDACIs) have recently emerged as promising anticancer drugs to induce cell cycle arrest, cytodifferentiation, and apoptosis. It is suggested, however, that HDACIs promote cell migration and invasion depending on the cell type. We have reported previously that treatment with HDACIs, including trichostatin A and suberoylanilide hydroxamic acid (SAHA) or progesterone in combination with estrogen, can induce cytodifferentiation of endometrial adenocarcinoma Ishikawa cells through up-regulation of glycodelin, a progesterone-induced endometrial glycoprotein. Given the reported role of glycodelin in cell motility and the migration-modulating potential of HDACIs, we investigated using wound healing assay and transwell migration assay whether ovarian steroid hormones, trichostatin A, or SAHA affects cell migration in endometrial cancer cell lines, Ishikawa and RL95-2. Treatment with ovarian steroid hormones, trichostatin A, and SAHA enhanced cell migration together with up-regulation of glycodelin. SAHA-augmented cell migration was almost completely blocked by gene silencing of glycodelin. Furthermore, overexpression of gycodelin alone resulted in increased cell motility in Ishikawa cells. Our results collectively indicate that glycodelin positively regulates cell motility acting as a mediator of HDACI-enhanced endometrial cell migration, suggesting the involvement of glycodelin in the dynamic endometrial gland morphogenesis during menstrual cycle. Our results raise a possibility that the use of HDACIs in the therapy for glycodelin-inducible endometrial and presumably other gynecological cancers may enhance invasion in cases in which the HDACIs fail to exert differentiation-inducing and/or antiproliferative effects.

Adenocarcinoma↗
PubMed2006

Midkine, a heparin-binding growth factor, selectively stimulates proliferation of definitive zone cells of the human fetal adrenal gland.

CONTEXT: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. OBJECTIVE: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. DESIGN AND SETTING: RNA, cryosections, and primary cell cultures from HFAs (14-24 wk) and adult adrenal RNA were used. MAIN OUTCOME MEASURES: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). RESULTS: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P < 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P < 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P < 0.01) in isolated FZ cells. CONCLUSIONS: MK likely plays a key role in HFA development. MK's selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.

Adrenal Glands↗
PubMed2006

Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

CONTEXT: Matricellular proteins are a group of secreted, multifunctional extracellular matrix glycoproteins that includes thrombospondins (TSPs), tenascin-C, and secreted protein acidic and rich in cysteine (SPARC). They may be implicated in the dynamic developmental processes of the human fetal adrenal (HFA) in which the outer, definitive zone (DZ) cells are postulated to proliferate, migrate centripetally, differentiate, and populate the inner, steroidogenic fetal zone (FZ). OBJECTIVE: The objective of the study was to identify a matricellular molecule that likely plays a major role in HFA development. DESIGN: Studies involved RNA, cryosections, and cell cultures from 14- to 23-wk HFAs and human adult adrenal RNA. MAIN OUTCOME MEASURES: Measures included transcripts encoding matricellular proteins, using real-time quantitative RT-PCR; SPARC localization by immunostaining; and ACTH regulation of SPARC expression and secretion by quantitative RT-PCR and Western blot. RESULTS: SPARC HFA mRNA was 100-, 700-, and 300-fold higher than TSP-1, TSP-2, and tenascin-C mRNA, respectively. HFA SPARC mRNA was 3-fold higher than adult adrenals (P < 0.005), comparable with levels in adult brain (positive control), whereas mRNAs encoding TSP-1 and TSP-2 were lower in fetal than adult adrenals. SPARC immunoreactivity was detected exclusively in the FZ, not DZ. ACTH, a key regulator of HFA growth and function, increased SPARC mRNA (by 1.7-fold at 1 nm, 48 h, P < 0.05) in isolated FZ cells but not DZ cells. ACTH up-regulation of SPARC protein was also detected in FZ cell lysates and culture medium. CONCLUSIONS: Results suggest a possible role for SPARC in development of functional and/or structural zonation of the HFA.

Adrenal Glands↗
PubMed2006

Complete removal of HIV-1 RNA and proviral DNA from semen by the swim-up method: assisted reproduction technique using spermatozoa free from HIV-1.

BACKGROUND: Use of antiretroviral drugs has reduced the mortality rate for HIV infection and many HIV-discordant couples wish to have children. It is possible for an HIV-infected man to father children without risk of HIV transmission if HIV-free spermatozoa can be obtained from his semen. METHODS: An improved swim-up method was used to collect HIV-free spermatozoa from the semen of HIV-positive males. Diluted semen was layered over a Percoll solution with a continuous density gradient of 30-98%, and then centrifuged. The bottom layer was collected by cutting the end from the tube and the sperm suspension was collected using the swim-up method. Spermatozoa were tested by nested polymerase chain reaction (PCR) for HIV-1 RNA and DNA, with a detection limit of one copy. Spermatozoa were used for assisted reproduction in 43 couples. RESULTS: HIV-1 RNA and proviral DNA were not detected by nested-PCR assay in all 73 of the collected spermatozoa samples from 52 patients. The HIV-1-negative sperm was used for in vitro fertilization in 12 couples and for intracytoplasmic sperm injection in 31 couples. No detection of HIV-1 RNA or proviral DNA in the culture medium of the fertilized eggs was confirmed again before embryo transfer. Of the 43 female partners, 20 conceived and 27 babies were born. HIV antibodies, HIV RNA and proviral DNA were negative in all of the females and babies. CONCLUSIONS: HIV-negative spermatozoa could be obtained from semen of HIV-positive men. The method involves no risk of HIV transmission to female partners and their children.

Adult↗
PubMed2006

Human endometrial cytodifferentiation by histone deacetylase inhibitors.

Abstract Human uterine endometrium repeats proliferation, differentiation (decidualization) and tissue breakdown during the menstrual period. Appropriate secretion of ovarian steroid hormones regulates these sequential endometrial remodeling cycles. While progesterone replacement therapy is adopted for endometrial dysfunction of differentiation, including recurrent impairment of implantation, no obvious effective results are obtained. Histone reversible acetylation, regulated by histone acetyltransferases and histone deacetylases plays a pivotal role in gene transcription. Although, in cells cultured with histone deacetylase inhibitors (HDACI), the expression of only about 2% of expressed genes is changed twofold or more compared with untreated control cells. Numerous previous works have demonstrated that HDACI affect cell proliferation/apoptosis in a variety of types of cells. To date, several HDACI are in phase I or phase II clinical trials as anticancer drugs. However, no reports have been found that HDACI is useful for transdifferentiation in human endometrium. Recently, we reported that HDACI could induce the expression of differentiation marker proteins, morphological change and functional cytodifferentiation in both human endometrial stromal and epithelial cells. In this review, we summarize the effect of HDACI against the human endometrial cytodifferentiation, indicating the possibility that HDACI can be used not only as an anticancer drug, but also as a transdifferentiation reagent, based on a new strategy.

Acetylation↗
PubMed2006

Bioethical aspects of regenerative and reproductive medicine.

The birth announced in 1997 of Dolly, the lamb cloned from the somatic mammary cells of an adult ewe, and the discovery of human embryonic stem cells in 1998 have been the most exciting developments in the biological sciences in the past decade. Reproductive somatic cell nuclear transfer (SCNT) in additional species has been inefficient in that relatively few births, harmful side effects and high fetal and neonatal death rates have resulted from many attempts. Ongoing debates about the ethics of reproductive SCNT have revealed that some researchers regard human reproductive SCNT as morally unacceptable in all circumstances, others see merit in reproductive SCNT in certain circumstances and others await more information before making judgment about the ethical status of the procedure. Regenerative medicine and emerging biotechnologies started to revolutionize the practice of medicine. Advances in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential reality. Mammal cloning experiments have provided new impetus to the prospect of regenerative medicine through stem cell research. The procedure of SCNT could be used to create the raw material to replace defective or senescent tissue as a natural extension of the biology of stem cells. Researchers working in reproductive medicine should consider the potential hope given to many patients against the requisite and ethically contentious creation of human blastocysts for therapeutic intent.

Animals↗
PubMed2006

Global gene expression profiling of preimplantation embryos.

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 microm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

Animals↗
PubMed2005

Fine resolution of human sperm nucleoproteins by two-dimensional electrophoresis.

Human sperm nucleoproteins consist of protamines and histones. Changes in composition of these proteins are thought to correlate with spermatogenesis and may be involved in some instances of male infertility. We sought to separate sperm nucleoproteins including variants of protamine using an improved two-dimensional electrophoretic method, with the aim of comprehensively analysing all sperm nucleoprotein constituents. After extracting nuclear basic proteins from the sperm of normal volunteers, we analysed these proteins on a gel sheet by a radical free, highly reducing method based on Kaltschmidt and Whittmann's two-dimensional electrophoresis. Basic proteins from sperm nuclei were separated clearly into 12 spots. By amino acid sequence analysis, these spots corresponded to protamine 1 (P1)- (five spots), protamine 2 (P2)-related proteins (six spots) and testis-specific histone H2B (one spot). The N-terminal amino acid sequences of the six P2-related proteins were compatible with those of HPI1, HPI2, HPS1, HPS2, HP2 and HP3, and quantitative comparison could be performed. In conclusion, human sperm nucleoproteins including all P2-related variants could be analysed quantitatively with high resolution on a single electrophoretic gel.

Amino Acid Sequence↗
PubMed2005

Histone deacetylase inhibitors induce differentiation of human endometrial adenocarcinoma cells through up-regulation of glycodelin.

Histone reversible acetylation, which is controlled by histone acetyltransferases and deacetylases, plays a fundamental role in gene transcription. Histone deacetylase inhibitors (HDACIs), such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have been characterized not only as anticancer drugs, but also as cytodifferentiation-inducing agents. In human endometrium, postovulatory production of progesterone directs estrogen-primed endometrial glandular cells to differentiate and thereby produce a number of unique bioactive substances, including glycodelin, that are critical for implantation at the secretory phase of the menstrual cycle. In this study, we show that TSA and SAHA, belonging to the hydroxamic acid group of HDACIs, can induce the phenotype of a human endometrial adenocarcinoma cell line, Ishikawa (originally derived from the glandular component of the endometrium), to differentiate to closely resemble normal endometrial epithelium in a time- and dose-dependent manner, as determined by morphological changes, synthesis of glycogen, and expression of secretory phase-specific proteins, including glycodelin. The proliferation- and differentiation-modulating effects elicited by TSA and SAHA at their optimal concentrations were comparable or more potent than those exerted by combined treatment with progesterone and estradiol. Furthermore, the gene silencing of glycodelin by small interference RNA resulted in the blockade of HDACI-induced differentiation in Ishikawa cells, suggesting the requirement for glycodelin for endometrial epithelial differentiation. Our results collectively indicate that TSA and SAHA are potent differentiation inducers for endometrial glandular cells, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting glycodelin.

Adenocarcinoma↗
PubMed2005

Impairment of decidualization in SRC-deficient mice.

Many signaling events induced by ovarian steroid hormones, cytokines, and growth factors are involved in the process of decidualization of human and rodent endometrium. We have reported previously that tyrosine kinase activation of SRC functionally participates in decidualization of human endometrial stromal cells. To address its essential role in decidualization, we examined, using wild-type and Src knockout mice, whether the process of decidualization was impaired in the absence of SRC. Immunohistochemistry using an antibody specific for the active form of SRC revealed that the active SRC was expressed prominently in the decidualizing stromal cells of the pregnant wild-type mouse. Moreover, the active SRC was upregulated in the uterine horn with artificially stimulated decidual reaction. In comparison with wild-type and Src heterozygous mice, the uterus of Src null mice showed no apparent decidual response following artificial stimulation. Ovarian steroid-induced decidualization in vitro, as determined by morphological changes and expression of decidual/trophoblast prolactin-related protein and prostaglandin-endoperoxide synthase 2 (also known as Cox2), both of which are decidualization markers, did not occur in a timely fashion in endometrial stromal cells isolated from the uteri of SRC-deficient mice compared to those from wild-type and Src heterozygous mice. Our results collectively suggest that SRC is an indispensable signaling component for maximal decidualization in mice.

Animals↗
PubMed2005

Gonadotrophin therapy in Kallmann syndrome caused by heterozygous mutations of the gene for fibroblast growth factor receptor 1: report of three families: case report.

Gonadotrophin therapy (GT) is frequently used to induce fertility in Kallmann syndrome (KS). We studied the effects and the consequences of GT in autosomal dominant KS caused by heterozygous FGFR1 mutations. Three Japanese families were examined. In family A, an adult male received GT and had two sons. In family B, an adult female received GT and gave birth to dizygotic male and female twins. In family C, an adult female received GT and produced a son and a daughter. Direct sequencing was performed for FGFR1, and clinical assessment was carried out for KS features. The father and the elder son of family A had P745S mutation, the mother and the female twin of family B had G687R mutation, and the mother and the two children of family C had S107X mutation. KS phenotype was detected for the mutation-positive subjects, except for the elder son of family A who had apparently normal phenotype. GT in FGFR1 mutations is effective in acquiring fertility but has a risk of transmitting the mutation and the disease phenotype to the next generation.

Adult↗
PubMed2005

A reduction in intraovarian arterial blood flow resistance after ovulation is necessary to achieve pregnancy in natural cycle.

PURPOSE: Color Doppler imaging permits the accurate localization of vessels and high-frequency pulsed Doppler ultrasonography has improved the resolution of flow velocity waveforms. In this study, intraovarian arterial blood flow before and after follicle rupture in the natural cycle was examined using transvaginal color flow Doppler imaging and changes in intraovarian arterial resistance in relation to the outcome of infertility treatment was analyzed. METHODS: In a prospective study, 227 spontaneous cycles in 118 infertile patients who were undergoing infertility treatment at the division of Reproductive Medicine in our center were recruited in this study. The impedance to flow in intraovarian vessels was measured by means of transvaginal color flow Doppler imaging during the periovulatory period in the natural cycle of all patients. The pulsatility index (PI) of intraovarian arterial blood flow and pregnancy rate was evaluated. RESULTS: On the basis of PI values before and after follicular rupture, 227 cycles were classified into severely decreased (113 cycles) and not-severely decreased groups (114 cycles). The pregnancy rate per cycle in the severely decreased group was 18.6% (21/113), significantly higher than that in the not-severely decreased group (7/114; 6.1%, p = 0.004). The miscarriage rate was similar in the two groups. CONCLUSIONS: A reduction in intraovarian blood vessel resistance is necessary to achieve pregnancy in a natural cycle.

Blood Flow Velocity↗
PubMed2005

The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.

PURPOSE: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." METHODS: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. RESULTS: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. CONCLUSION: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Blastomeres↗