Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC NEPHROLOGY  

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

DOI： 10.1681/ASN.2017090993

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on the brain dopamine system are controversial and not fully defined. To address this question, we analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control of the glial Gfap promoter. Compared with wild-type, the GDNF transgenic mice contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However, there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite homovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in transgenic mice with no apparent signs of neurodegeneration. In agreement with these neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux were decreased in the striatum of transgenic mice. We also explored the influences of GDNF overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and rearing in a novel environment compared with wild-type mice. These results suggest that chronic overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine metabolism and neurotransmission. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.neulet.2017.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY BIOLOGISTS LTD  

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposonderived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/ Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

DOI： 10.1242/dev.114520

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

DOI： 10.1016/j.neuron.2013.12.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Extracellular factors that inhibit axon growth and intrinsic factors that promote it affect neural regeneration. Therapies targeting any single gene have not yet simultaneously optimized both types of factors. Chondroitin sulphate (CS), a glycosaminoglycan, is the most abundant extracellular inhibitor of axon growth. Here we show that mice carrying a gene knockout for CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, recover more completely from spinal cord injury than wild-type mice and even chondroitinase ABC-treated mice. Notably, synthesis of heparan sulphate (HS), a glycosaminoglycan promoting axonal growth, is also upregulated in TI knockout mice because HS-synthesis enzymes are induced in the mutant neurons. Moreover, chondroitinase ABC treatment never induces HS upregulation. Taken together, our results indicate that regulation of a single gene, T1, mediates excellent recovery from spinal cord injury by optimizing counteracting effectors of axon regeneration-an extracellular inhibitor of CS and intrinsic promoters, namely, HS-synthesis enzymes.

DOI： 10.1038/ncomms3740

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

DOI： 10.1038/nn.3525

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: Roles of the syntaxin-1ACaMKII interaction are not physiologically understood in vivo.Results: A point mutation in syntaxin-1A caused abnormal plasticity, recycling, and behaviors in mice. Conclusion: The CaMKII/syntaxin-1A interaction is essential for maintenance of neuronal plasticity. Significance: Syntaxin-1A is involved in regulatory pathways in higher brain functions.Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

DOI： 10.1074/jbc.M113.504050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

DOI： 10.1017/S0967199410000481

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Language：English   Publishing type：Research paper (scientific journal)  

Purpose: 129 inbred mice show poor reproductive ability, as evidenced by small litters; however, the exact cause of this is unknown. In the present in vivo study we examined fertility and subsequent post-implantation development in an attempt to clarify the cause of small litter size in 129 mice. Methods: 129 or C57BL/6J females that displayed vaginal plugs 1 day after mating with males of the same strain were examined for the presence of fertilized eggs. Reciprocal matings were also performed between 129 and C57BL/6J mice. Subsequent post-implantation development of fertilized eggs was examined by dissecting females 18-19 days after the vaginal plugs were found. Results: Mean numbers of recovered eggs were 7.9 and 8.0 in 129 and C57BL/6J mice, respectively. Half of the recovered eggs were unfertilized in 129 mice, whereas all were fertilized in C57BL/6J mice. Mean numbers of live fetuses 18-19 days after mating were significantly lower in 129 mice (4.7) than in C57BL/6J mice (7.3). In different types of pairings using both strains of mice, the fertility was significantly lower whenever 129 females were used. Conclusions: The small litter size in 129 mice is caused by low fertility resulting from female factors.

DOI： 10.1007/s12522-009-0024-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca2+), phosphate (PO43-) and lactate. In all media containing no Call, including medium lacking Ca2+, lacking Ca2+ and PO43- lacking Ca2+ and lactate and lacking Ca2+, PO43- and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca2+ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca2+. In conclusion, preincubation of thawed sperm in medium containing no Ca2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca2+ is practical for use in cryopreserved C57BL/6J sperm.

DOI： 10.1262/jrd.20163

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Mice with maternal duplication of proximal Chromosome 11 (MatDp(prox11)), where Meg1/Grb10 is located, exhibit pre- and postnatal growth retardation. To elucidate the responsible imprinted gene for the growth abnormality, we examined the precise structure and regulatory mechanism of this imprinted region and generated novel model mice mimicking the pattern of imprinted gene expression observed in the MatDp(prox11) by deleting differentially methylated region of Meg1/Grb10 (Meg1-DMR). It was found that Cobl and Ddc, the neighboring genes of Meg1/Grb10, also comprise the imprinted region. We also found that the mouse-specific repeat sequence consisting of several CTCF-binding motifs in the Meg1-DMR functions as a silencer, suggesting that the Meg1/Grb10 imprinted region adopted a different regulatory mechanism from the H19/Igf2 region. Paternal deletion of the Meg1-DMR (+/Delta DMR) caused both upregulation of the maternally expressed Meg1/Grb10 Type I in the whole body and Cobl in the yolk sac and loss of paternally expressed Meg1/Grb10 Type II and Ddc in the neonatal brain and heart, respectively, demonstrating maternalization of the entire Meg1/Grb10 imprinted region. We confirmed that the +/Delta DMR mice exhibited the same growth abnormalities as the MatDp(prox11) mice. Fetal and neonatal growth was very sensitive to the expression level of Meg1/Grb10 Type I, indicating that the 2-fold increment of the Meg1/Grb10 Type I is one of the major causes of the growth retardation observed in the MatDp(prox11) and +/Delta DMR mice. This suggests that the corresponding human GRB10 Type I plays an important role in the etiology of Silver-Russell syndrome caused by partial trisomy of 7p11-p13.

DOI： 10.1093/hmg/ddp049

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Language：Japanese   Publisher：新潟医学会  

【目的】生殖工学的操作はマウスの生産に広く活用されているが、私は、生殖工学的操作で欠かせない体外培養が初期胚の発生および個体発生に及ぼす影響を明確にするため、体外培養した初期胚の発生の早さ・細胞数・遺伝子発現の解析、および体外培養胚から生産した個体と自然交配で得た個体の主要な臓器重量の比較等の解析を行った。【方法と結果】マウス胚を組換えヒトアルブミン(r)を加えたrKSOMaa培地またはrMW培地で培養を行ったものを体外培養(In vitro)胚とし、偽妊娠マウスに移植を行ったものを母体内発生(In vivo)胚として解析を行った。In vitro胚とIn vivo胚の形態を比較した結果、胚盤胞期胚の形態の違いが観察され、In vitro rKSOMaa区およびIn vivo区では高率で胚盤胞期胚へと発生することが分かった。胚盤胞期胚への発生時期を解析したところ、In vitro rKSOMaa区はIn vitro rMW区より胚盤胞期胚への発生が早いことが観察され、培養培地の違いによっても発生率に違いが見られた。免疫抗体法を用いて簡便に媒精後96時間における内部細胞塊(ICM)および栄養外胚葉(TE)の細胞数を計数した結果、In vitro rKSOMaa区では、In vivo区に比較して、ICM数は少ない(p<0.05)が、TE数は多く(p<0.05)、総細胞数は同等であった。TUNEL法を用いてアポトーシス細胞数を計数した結果、In vitro rKSOMaa区およびIn vivo区と比較しIn vitro rMW区で多かった(p<0.05)。胚移植を行い、妊娠19.5日目における産仔への発生数と総着床数を計数した。In vitro rKSOMaa区では総着床数が多いにもかかわらず、In vivo区よりも産仔への発生数が低かった。In vitro rMW区は産仔率、総着床率共に低かった(p<0.05)。体重および臓器重量の比較した結果、出生後52週ではIn vitro rKSOMaa区で体重が重く、メスでは内臓以外の重量がオスでは内臓重量が多かった。インプリント遺伝子と分化マーカー遺伝子の発現解析では、In vitro rKSOMaa区ではIn vivo区に対して、インプリント遺伝子であるH19の発現は低かったが、一方、栄養外胚葉の分化マーカーであるCdxと未分化細胞マーカーであるNanogは有意に高い値を示した。【考察】マウス初期胚は体外培養を行うと、母体内培養を行ったものと比較し、胚の形態、発生時期並びに細胞数に差を生じ、産仔率の低下と成熟後の体重の増加という個体への発生に大きく影響を受ける事が示された。また、体外培養下で胚盤胞期におけるインプリント遺伝子の発現低下が確認された。このことは体外培養によりインプリント遺伝子のメチル化状態がリセットされていない部分があることを示唆している。【結論】体外培養胚は、母体内発生胚に比べて胚盤胞期胚へ高効率で早く発生するが、インプリント遺伝子および分化マーカー遺伝子の発現に変化が見られ、産仔率に差があった。また、体外培養胚由来の個体は、自然交配由来の個体に比べ、成熟期の体重・臓器重量が増加していた。本研究により、自然交配と同等の動物生産に向けて体外培養を最適化する指標が明らかとなった。(著者抄録)

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2014&ichushi_jid=J00990&link_issn=&doc_id=20150512080005&doc_link_id=%2Fdg3nigta%2F2014%2F012812%2F005%2F0635-0646%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdg3nigta%2F2014%2F012812%2F005%2F0635-0646%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：(一社)日本神経学会  

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2012&ichushi_jid=J05623&link_issn=&doc_id=20120517210008&doc_link_id=%2Fct6mamma%2F2012%2Fs02902%2F008%2F5008-5008%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fct6mamma%2F2012%2Fs02902%2F008%2F5008-5008%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese  

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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Language：Japanese   Publisher：日本発生生物学会  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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