Ionotropic Glutamate Receptor AMPA 1 Is Associated with Ovulation Rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy

Expression and Potential Role of GATA6 in Ruminant Trophoblasts during Peri-Implantation Periods

Abstract: Expression of GATA6 has been found during embryonic development in many mammalian species. In mouse embryonic development, GATA6 is a primitive endoderm (PrE) marker. However, the expression and effect of GATA6 in ruminant ungulates has not been well characterized. In this report, the expression of GATA6 mRNA was examined in the uteri of ruminants. GATA6 mRNA was detected in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses and in days 15, 17, and 21 ovine conceptuses. In both cases, GATA6 mRNA increased on day 22 or 21 after conceptus attachment to the uterine epithelium. GATA6 mRNA was also detected in bovine trophoblast CT-1 or F3 cells and ovine trophoblast oTr cells. In transient transfection analyses using the upstream region of the bovine IFNT gene (bIFNT, IFN-tau-c1), GATA6 overexpression was effective in the up-regulation of the bIFNT construct that had been transfected into human choriocarcinoma JEG3 or bovine ear-derived fibroblast EF cells. The observation in which GATA6 increased IFNT transcription suggests that in addition to lineage specification, GATA6 may have acquired ruminant-specific functions involving the control of trophoblast gene expression

ウシα1-3 galactosyltransferaseのCOS-7細胞への導入

αGal抗原は異種抗原として知られ,ヒトおよび旧世界サル以外の生物に広汎に存在し,異種移植における超急性拒絶反応の原因抗原と考えられている.このαGal抗原はα1-3 galactosyltransferase (α1-3GT)によって合成される.異種移植の分野では,α1-3GTを様々な方法で修飾することにより,移植可能な動物の作製が試みられている.本実験では,黒毛和牛からα1-3GTを単離し,これをαGal陰性のCOS-7細胞に導入し,我々のクローニングしたウシα1-3GTが機能するかどうかについて検討した.黒毛和牛末梢血から,RT-PCR法を用いてα1-3GTcDNAをクローニングした.これを,ネオマイシン耐性遺伝子を含むベクターに挿入し,αGal抗原発現ベクターを作製した(N3GA). N3GAをリポフェクション法により,αGal抗原陰性であるCOS-7細胞に導入した.N3GA導入翌日にIB4レクチンを用いた蛍光抗体染色を行った.N3GAを導入したCOS-7細胞(COS(+))では,αGal抗原陽性所見を認めた.また,N3GA導入2週間後に,同じくIB4レクチンを用いて行ったflow cytometryでもCOS(+)ではαGal抗原陽性所見を認めた.以上より,我々が黒毛和牛からクローニングしたα1-3GTは酵素機能を有しており,異種細胞においても機能することが示唆された.The polysaccharide antigen, called aGal epitope is ubiquitously found in all animals except for humans and old world monkeys. An αGal epitope is a main xenoantigen which hampers the success of the clinical xenotransplantation. The αGal epitope is synthesized by an enzyme, α1-3 galactosyltransferase (α1-3GT). Thus, disruption of the α1-3GT gene in large animals is one strategy for the successful xenotransplantation. In the present study, we isolated α1-3GT cDNA of Japanese Black Cattle (JBC), and transfected it into an α1-3GT negative COS-7 cell line to demonstrate that the cDNA was functional. The α1-3GT cDNA of JBC was amplified by a polymerase chain reaction. The α1-3GT cDNA was cloned to an expression vectors, named N3GA and transfected into α1-3GT negative COS-7 cells. Cell surface expression of αGal epitope was examined by immunofluorescent staining and flow cytometry with IB4 lectin. Transfected COS-7 cells were positive for the immunoreactive IB4 lectin examined at 24 hours after the transfection. Then, transfected COS-7 cells were selected with neomycin for 10 days. Flow cytometry of COS-7 cells performed 4 weeks after the transfection demonstrated positive log shift for αGal epitope. In conclusion, we isolated α1-3GT cDNA of JBC and obtained stable cell line expressing the gene

ヒトリンパ球のウシリンパ球に対する免疫学的反応性に関する研究

異種移植最大の障害は超急性拒絶反応(hyper acute rejection; HAR)であるが,近年トランスジェニック動物が開発されHAR回避の端緒が開かれた.HARの克服後にはヒト同種移植と同様,細胞性免疫反応が立ちはだかる.以前より異種移植の有力なドナー候補とみなされてきたブタに比べ,ウシに対するヒトの免疫反応に関する報告は少ない.そこで我々は,ヒト末梢血単核球(peripheral blood mononuclear cells; PBMC)に対するウシPBMCの免疫原性を検討するため,ヒトPBMCをresponder,ヒト(アロ),ウシ,ブタPBMCをstimulatorとしてそれぞれ混合培養し,フローサイトメトリー,リンパ球混合試験(mixed lymphocyte reaction; MLR)を施行し,またヒトPBMCのサイトカイン産生能を測定した.培養開始当日,および5日後のヒトPBMCのフローサイトメトリーではCD3陽性(T)細胞は,ウシに対しては40.3±4.5%から61.6±6.8%へと増加した.CD19陽性(B)細胞には明らかな変化を認めなかったがCD56陽性(NK)細胞は15.9±7.2%から25.2±8.9%へと増加した.これらphenotypeの変化は,stimulatorがヒト,ブタと代わっても大きな差異は認めなかった.MLRにおいては,対ヒトに比較すると対ウシMLRは低値であり,対ブタMLRとほぼ同等であった.また,サイトカイン産生においてはTh1細胞由来のサイトカイン優位であるものの,対ヒトに比べると低値であった.以上,ヒトのウシに対する細胞性免疫反応はブタと同様,ヒト(アロ)に対する反応より弱いことが示唆された.There have not been many studies on the human cellular response to bovine. In this study, we report the first evidence that the human cellular response to bovine is weaker than to allogeneic humans. Human peripheral mononuclear cells were cultured with inactivated bovine peripheral mononuclear cells. Allogeneic mixed lymphocyte reaction (MLR) and human anti-porcine MLR were compared with human anti-bovine MLR. In addition, human cytokine production against bovine was assessed by enzyme-linked immunoabsorbant assay (ELISA) of culture supernatant. The results of ELISA showed that human peripheral mononuclear cells produced preferentially Th1 deviated cytokines (INF-γ and TNF-α) upon stimulation by bovine peripheral mononuclear cells. But the magnitude of the cytokine production was lower than in the allogeneic combination. The examination of MLR showed that human anti-bovine MLR was significantly weaker than allogeneic MLR, and on the same level as the human anti-porcine MLR. These results suggest that the human cellular response to bovine is weaker than to allogeneic humans

ウシα1-3 galactosyltransferaseのCOS-7細胞への導入

αGal抗原は異種抗原として知られ,ヒトおよび旧世界サル以外の生物に広汎に存在し,異種移植における超急性拒絶反応の原因抗原と考えられている.このαGal抗原はα1-3 galactosyltransferase (α1-3GT)によって合成される.異種移植の分野では,α1-3GTを様々な方法で修飾することにより,移植可能な動物の作製が試みられている.本実験では,黒毛和牛からα1-3GTを単離し,これをαGal陰性のCOS-7細胞に導入し,我々のクローニングしたウシα1-3GTが機能するかどうかについて検討した.黒毛和牛末梢血から,RT-PCR法を用いてα1-3GTcDNAをクローニングした.これを,ネオマイシン耐性遺伝子を含むベクターに挿入し,αGal抗原発現ベクターを作製した(N3GA). N3GAをリポフェクション法により,αGal抗原陰性であるCOS-7細胞に導入した.N3GA導入翌日にIB4レクチンを用いた蛍光抗体染色を行った.N3GAを導入したCOS-7細胞(COS(+))では,αGal抗原陽性所見を認めた.また,N3GA導入2週間後に,同じくIB4レクチンを用いて行ったflow cytometryでもCOS(+)ではαGal抗原陽性所見を認めた.以上より,我々が黒毛和牛からクローニングしたα1-3GTは酵素機能を有しており,異種細胞においても機能することが示唆された.The polysaccharide antigen, called aGal epitope is ubiquitously found in all animals except for humans and old world monkeys. An αGal epitope is a main xenoantigen which hampers the success of the clinical xenotransplantation. The αGal epitope is synthesized by an enzyme, α1-3 galactosyltransferase (α1-3GT). Thus, disruption of the α1-3GT gene in large animals is one strategy for the successful xenotransplantation. In the present study, we isolated α1-3GT cDNA of Japanese Black Cattle (JBC), and transfected it into an α1-3GT negative COS-7 cell line to demonstrate that the cDNA was functional. The α1-3GT cDNA of JBC was amplified by a polymerase chain reaction. The α1-3GT cDNA was cloned to an expression vectors, named N3GA and transfected into α1-3GT negative COS-7 cells. Cell surface expression of αGal epitope was examined by immunofluorescent staining and flow cytometry with IB4 lectin. Transfected COS-7 cells were positive for the immunoreactive IB4 lectin examined at 24 hours after the transfection. Then, transfected COS-7 cells were selected with neomycin for 10 days. Flow cytometry of COS-7 cells performed 4 weeks after the transfection demonstrated positive log shift for αGal epitope. In conclusion, we isolated α1-3GT cDNA of JBC and obtained stable cell line expressing the gene

ヒトリンパ球のウシリンパ球に対する免疫学的反応性に関する研究

異種移植最大の障害は超急性拒絶反応(hyper acute rejection; HAR)であるが,近年トランスジェニック動物が開発されHAR回避の端緒が開かれた.HARの克服後にはヒト同種移植と同様,細胞性免疫反応が立ちはだかる.以前より異種移植の有力なドナー候補とみなされてきたブタに比べ,ウシに対するヒトの免疫反応に関する報告は少ない.そこで我々は,ヒト末梢血単核球(peripheral blood mononuclear cells; PBMC)に対するウシPBMCの免疫原性を検討するため,ヒトPBMCをresponder,ヒト(アロ),ウシ,ブタPBMCをstimulatorとしてそれぞれ混合培養し,フローサイトメトリー,リンパ球混合試験(mixed lymphocyte reaction; MLR)を施行し,またヒトPBMCのサイトカイン産生能を測定した.培養開始当日,および5日後のヒトPBMCのフローサイトメトリーではCD3陽性(T)細胞は,ウシに対しては40.3±4.5%から61.6±6.8%へと増加した.CD19陽性(B)細胞には明らかな変化を認めなかったがCD56陽性(NK)細胞は15.9±7.2%から25.2±8.9%へと増加した.これらphenotypeの変化は,stimulatorがヒト,ブタと代わっても大きな差異は認めなかった.MLRにおいては,対ヒトに比較すると対ウシMLRは低値であり,対ブタMLRとほぼ同等であった.また,サイトカイン産生においてはTh1細胞由来のサイトカイン優位であるものの,対ヒトに比べると低値であった.以上,ヒトのウシに対する細胞性免疫反応はブタと同様,ヒト(アロ)に対する反応より弱いことが示唆された.There have not been many studies on the human cellular response to bovine. In this study, we report the first evidence that the human cellular response to bovine is weaker than to allogeneic humans. Human peripheral mononuclear cells were cultured with inactivated bovine peripheral mononuclear cells. Allogeneic mixed lymphocyte reaction (MLR) and human anti-porcine MLR were compared with human anti-bovine MLR. In addition, human cytokine production against bovine was assessed by enzyme-linked immunoabsorbant assay (ELISA) of culture supernatant. The results of ELISA showed that human peripheral mononuclear cells produced preferentially Th1 deviated cytokines (INF-γ and TNF-α) upon stimulation by bovine peripheral mononuclear cells. But the magnitude of the cytokine production was lower than in the allogeneic combination. The examination of MLR showed that human anti-bovine MLR was significantly weaker than allogeneic MLR, and on the same level as the human anti-porcine MLR. These results suggest that the human cellular response to bovine is weaker than to allogeneic humans