著者

渡邉 剛広 島 麗香 坂原 聖士 高倉 啓 黒谷 玲子 阿部 宏之

出版者

日本繁殖生物学会

雑誌

日本繁殖生物学会 講演要旨集 第106回日本繁殖生物学会大会

巻号頁・発行日

pp.OR1-15, 2013 (Released:2013-09-10)

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【目的】ミトコンドリア内膜に存在する呼吸鎖複合体IV(シトクロムcオキシダーゼ:COX)は電子伝達系の終末酵素である。COXは機能分化した体細胞において詳しい機能解析は行われているが,卵子や初期胚における研究はほとんど進んでいない。本研究では,マウスの初期発生におけるCOXの役割を調べるために,未受精卵子から器官形成期までの初期発生におけるCOX mRNA発現とミトコンドリア呼吸機能を解析した。【方法】ICR系雌マウスからMII期卵母細胞,1細胞期〜8細胞期胚,桑実胚,胚盤胞および胎生8.5日胚(E8.5)を回収した。各発生ステージにおいて,COXを構成する全13サブユニットのmRNAをRT-PCRにより解析した。また,卵母細胞および胚において(1)酸素消費量,(2)活性型ミトコンドリアの局在と相対膜電位,(3)ATP含量を解析した。【結果】ミトコンドリアゲノム由来のCOX1,COX2およびCOX3のmRNAは,全ての発生ステージの卵母細胞および胚において検出された。一方,核ゲノム由来COXの mRNAは,MII期の卵母細胞ではCOX6a,COX7bおよびCOX7cを除く7サブユニットのmRNAが検出されたが,1細胞期から8細胞期の胚においてこれら7サブユニットのmRNAは著しく発現量が低下した。桑実胚および胚盤胞ではCOX7cを除く9サブユニットのmRNAが検出され,E8.5では核ゲノムにコードされる全サブユニットのmRNAが検出された。この核ゲノムCOX mRNAの増加は,酸素消費量の増加とミトコンドリア膜電位活性の上昇と一致した。本研究では,未受精卵子から器官形成期胚に至るマウス初期発生におけるCOX mRNAの発現パターンの解析に初めて成功した。ミトコンドリアゲノムと核ゲノムのCOX mRNAはそれぞれ異なる発現パターンを示し,ミトコンドリア呼吸機能の亢進に核ゲノムCOXサブユニットが重要な役割を果たしていることが示唆された。

著者

皆川 至 高力 宙 佐方 醍 柴田 昌利 与語 圭一郎 河原崎 達雄 高坂 哲也

出版者

日本繁殖生物学会

雑誌

日本繁殖生物学会 講演要旨集 第103回日本繁殖生物学会大会

巻号頁・発行日

pp.81, 2010 (Released:2010-08-25)

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【目的】リラキシン関連因子(RLF)はブタで発見されたinsulin-relaxin gene familyの一つで,マウスでは精巣下降に必須である。多くの動物でcDNAのクローニングがなされ,RLFはA-B-C鎖からなる前駆体(プロRLF)として生合成された後,A-B鎖ヘテロダイマーとして機能すると推測されてきた。しかし,native RLFがダイマーとして精巣に存在するか不明である。加えて,本遺伝子の発現は成熟精巣でも認められているものの,その機能は定かでない。本研究ではブタ精巣よりnative RLFを単離してその構造と特性を明らかにすると共に,分泌後の行方や受容体の分布から作用発現の可能性を究明した。【方法】デュロック種の雄ブタを用いた。各種クロマトを組み合わせ精巣よりRLFを単離した。構造解析はMaldi Tof/Tof MSで行い,生物活性は受容体LGR8を導入したHEK293細胞におけるcAMP産生能で評価した。血中および体液中RLF濃度は時間分解蛍光免疫測定法で,受容体の局在は免疫染色で調べた。【結果】In situ hybridizationと免疫染色よりライディッヒ細胞でRLFの産生を確認した。精巣をゲル濾過,イオン交換FPLCおよび逆相HPLCに供し,RLFを約12kDaの単一ピークとして単離することに成功した。MSMS解析の結果,60%のプロテインカバレッジでA-B-C鎖ドメインが同定され,RLFは前駆体の構造をとることが判明した。単離したRLFはnMオーダーでcAMP産生を刺激し,十分な生物活性を有していることが分かった。一方,産生源のライディッヒ細胞から分泌されたRLFは精巣静脈のほか,精巣間隙,精細管内液および精巣網液で高濃度で検出された。さらに,精細管内上皮細胞とライディッヒ細胞では受容体の免疫局在が観察された。【結論】ブタでは,RLFは生物活性を持った前駆体としてライディッヒ細胞より分泌され,内分泌,傍分泌または自己分泌因子として機能することが示唆された。

著者

若宮 香理 小林 芳彦 Acosta Tomas J. 高橋 昌志 奥田 潔

出版者

日本繁殖生物学会

雑誌

日本繁殖生物学会 講演要旨集 第103回日本繁殖生物学会大会

巻号頁・発行日

pp.69, 2010 (Released:2010-08-25)

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【目的】Prostaglandins(PGs) は,生殖機能の調節に重要な役割を果たしている生理活性物質であり,多岐の作用が知られている。なかでも,PGE2は初期胚の発育環境を最適に保つ因子であることが報告されている一方で,子宮内膜から分泌される PGF2α(PGF) は胚の生存および発育を抑制し,妊娠率を低下させることが知られている。また,PGE2およびPGFはともに,受精卵の移送に重要な卵管の収縮運動に関係していると考えられている。ウシにおいて,夏期の気温上昇による暑熱ストレス (heat stress; HS) は,卵胞の発育異常による排卵障害,初期胚の死滅などを引き起こし,妊娠率を低下させることが報告されている。さらに,HSは初期胚の発育の場である卵管に影響を与え,初期胚の生存性に影響を与える可能性も考えられる。本研究では,卵管の機能に及ぼすHSの影響を明らかにするために,培養ウシ卵管上皮細胞を用いて以下の検討を行った。【方法】排卵後 0-5 日の卵管から単離した卵管上皮細胞を播種し,1)細胞接着後,培養液を交換した時間を 0 日とし,通常の培養温度(37.5℃)を control 区,39℃ (HS39) および 41℃ (HS41) をHS 処理区とした。HS 処理 1-4 日後に細胞を採取し,DNA assay により卵管上皮細胞の増殖を検討した。2) コンフルエントに達した卵管上皮細胞を control 区,HS39およびHS41で培養した。HS 処理 4,24,48 時間後の培養上清中PGE2およびPGF濃度をEIAにより測定した。なお,PGs 濃度は DNA (µg) あたりに換算した。【結果】1) Control 区と比較して HS41 で細胞増殖率が有意に低下した (P<0.05)。2) PGE2濃度は HS 処理 4 時間後に control 区と比較してHS41 で有意に減少し,PGF濃度は HS 処理 24 時間後に control 区と比較して HS39 で増加した(P<0.05)。本研究において,HS は卵管上皮細胞の増殖率を低下させ,卵管内における受精卵移送を困難にするとともに,PGE2 濃度の減少および PGF 濃度を増加することにより,初期胚の発達に悪影響を与え,繁殖率を低下させている可能性が示された。

著者

吉永 浩二 関口 喜一 江藤 〓一 星冬 四郎

出版者

日本繁殖生物学会

雑誌

家畜繁殖研究會誌 (ISSN:04530551)

巻号頁・発行日

vol.7, no.2, pp.85-86, 1961-10-15 (Released:2008-05-15)

参考文献数

12

著者

Carmen RODENAS Inmaculada PARRILLA Jordi ROCA Emilio Arsenio MARTINEZ Xiomara LUCAS

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2014-024, (Released:2014-07-21)

被引用文献数

2 9

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The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.

著者

Misuzu YAMASHITA Kazuo YAMAGATA Keiko TSUMURA Tomoko NAKANISHI Tadashi BABA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.53, no.2, pp.255-262, 2007 (Released:2007-05-12)

参考文献数

28

被引用文献数

9 15 14

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To improve assessment of the acrosome reaction of mouse epididymal sperm, we employed anti-Izumo1 antibody instead of antibodies against acrosomal proteins. The acrosomal states among acrosome-intact, spontaneously acrosome-reacted, truly acrosome-reacted, and probably dead and/or membrane-damaged sperm were clearly distinguished by combined application of anti-Izumo1 antibody, DNA dye Hoechst 33342, and monoclonal antibody MN7 to paraformaldehyde-fixed sperm. When the acrosome reaction of capacitated epididymal sperm on the oocyte zona pellucida was examined using anti-Izumo1 antibody, approximately 20% of sperm bound onto the zona pellucida were acrosome-reacted 30 min after insemination. We also observed the moment of the acrosome reaction of live sperm on the zona pellucida by time-lapse monitoring using fluorescein isothiocyanate-conjugated anti-Izumo1 antibody.

著者

Yuki YAMAMOTO Tatsuya YAMAMOTO Natsuki YUTO Thomas B. HILDEBRANDT Imke LUEDERS Gudrun WIBBELT Osamu SHIINA Yasushi MOURI Keisuke SUGIMURA Sayuri SAKAMOTO Saroch KAEWMANEE Kentaro NAGAOKA Gen WATANABE Kazuyoshi TAYA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.1111070413-1111070413, (Released:2011-11-11)

被引用文献数

2 13

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The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of immunoreactive (ir-) PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-prolactin clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4-6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.

著者

Rasoul KOWSAR Nina HAMBRUCH Jinghui LIU Takashi SHIMIZU Christiane PFARRER Akio MIYAMOTO

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2013-036, (Released:2013-06-21)

被引用文献数

12 38

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This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10, IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.

著者

Wichai CHERDSHEWASART Yosaporn KITSAMAI Suchinda MALAIVIJITNOND

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.0701160049-0701160049, (Released:2007-01-17)

被引用文献数

26 34

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The aim of this study was to evaluate the estrogenic activity of tuberous samples of phytoestrogen-rich Pueraria mirifica collected from 25 of 76 provinces in Thailand by vaginal cornification assay. Tuberous powders were prepared and administered to ovariectomized rats for 14 consecutive days at dosages of 10, 100, and 1,000 mg/kg BW respectively, and were compared with a daily treatment with 200 μg/100 g BW 17β-estradiol (E2). Rats treated with 10 mg/kg BW Pueraria mirifica showed no vaginal cornification. Treatment with 100 mg/kg BW Pueraria mirifica from 13 out of 25 plant samples resulted in development of vaginal cornification. The cell count percentages of the vaginal smeared cells for the treatment with the 2 plant samples that exhibited the fastest vaginal cornification revealed large variation in their estrogenic activities. Treatment with 1,000 mg/kg BW Pueraria mirifica from all plant samples produced vaginal cornification with the mean value for the period (day) of first appearance of cornified cells being 4.08 days compared to 2 days with 200 μg/100g BW E2. The overall appearance period (day) of cornified cells during the treatment and post-treatment period with 1,000 mg/kg BW per day Pueraria mirifica was shorter than treatment with 200 μg/100 g BW E2. The results demonstrate that the plant population shows differential estrogenic activity as evaluated by vaginal cornification assay.

著者

Hataitip TRISOMBOON Suchinda MALAIVIJITNOND Wichai CHERDSHEWASART Gen WATANABE Kazuyoshi TAYA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.52, no.4, pp.537-542, 2006 (Released:2006-08-25)

参考文献数

30

被引用文献数

17 19

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To investigate the estrogenic effect of Pueraria mirifica (PM), a Thai herbal plant that contains many phytoestrogens, sexual skin coloration was studied in cynomolgus monkeys. Aged menopausal monkeys were divided into three groups. Each group (n=3) was fed 10, 100, or 1,000 mg of PM daily. The treatment schedule was divided into three periods, a 30-day pre-treatment period, 90-day treatment period, and 60-day post-treatment period. The results show that the sexual skin exhibited reddish coloration within 24 h after PM-treatment and remained this way for the first half of the PM-feeding period. The changes in sexual skin coloration were not dose-dependent. The present results indicate that PM had estrogenic action by increasing reddish sexual skin coloration in aged menopausal monkeys.

著者

Hataitip TRISOMBOON Suchinda MALAIVIJITNOND Juri SUZUKI Yuzuru HAMADA Gen WATANABE Kazuyoshi TAYA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.50, no.6, pp.639-645, 2004 (Released:2005-01-07)

参考文献数

35

被引用文献数

18 19

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To determine the effect of Pueraria mirifica (PM) on serum parathyroid hormone (PTH) and calcium levels on aged menopausal monkeys (Macaca fascicularis), subjects were treated with 10, 100, or 1,000 mg/day of PM. Blood samples were collected every 5 days for 30, 90, and 60 days during pre-treatment, treatment, and post-treatment periods, respectively. Sera were assayed for PTH, estradiol, and calcium levels. PM-1,000 had the strongest effect on the decrease in PTH (0.001<P≤0.05) and calcium levels (0.001<P≤0.03) during the treatment period. PTH levels remained low for the first 15 days of the post-treatment period (0.01≤P ≤0.05). PM-10 induced a significant decrease in PTH level on day 80 (P=0.02) during the treatment period and a significant decrease in calcium level on day 75 (P<0.01). There were no changes in serum PTH and calcium levels throughout the study period in the PM-100 group. Estradiol levels decreased significantly during the treatment period in all treatment groups. The results suggest that long-term treatment with 1,000 mg/day of PM decreases serum PTH and calcium levels in aged menopausal monkeys, indicating that PM ameliorates bone loss caused by estrogen deficiency.

著者

Thanida SANANMUANG Nawapen PHUTIKANIT Catherine NGUYEN Sukanya MANEE-IN Mongkol TECHAKUMPHU Theerawat THARASANIT

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2012-116, (Released:2013-01-25)

被引用文献数

4 6 1

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Developmental competence and quality of in vitro produced embryos has been demonstrated to be lower than in vivo derived embryos. This study aimed specifically to determine the effects of in vitro culture of feline embryos using various culture densities on developmental competence and expression of stress- and apoptotic-related genes in terms of heat shock protein 70 (HSP70) and apoptotic-related (BAX and BCL-2) gene expressions. In experiment 1, we characterized the inducible form of a feline-specific HSP70 mRNA sequence, as it has not been previously reported. The primers for feline HSP70 mRNA were synthesized and tested on heat-treated cat fibroblasts. In experiment 2, feline embryos were cultured at different culture densities (embryo:culture volume; 1:1.25, 1:5 and 1:20). The developmental competence was determined along with HSP70, BAX and BCL-2 transcript abundances using quantitative RT-PCR. In vivo derived embryos were used as a control group. A partial cat HSP70 mRNA sequence (190 bp) was characterized and exhibited high nucleotide identity (93 to 96%) with other species. Cleaved embryos cultured at high density (1:1.25) developed to blastocysts at a lower rate than those generated from lower densities. Irrespective of the culture densities used, in vitro cultured blastocysts showed increased levels of HSP70 and BAX transcripts compared with in vivo counterparts. Blastocysts derived from the highest culture density (1:1.25) showed higher levels of upregulation of HSP70 and BAX transcripts than those cultured at lower culture densities (1:5 and 1:20). In conclusion, increased levels of pro-apoptotic (BAX) and stress-response (HSP70) transcripts correlated with developmental incompetence of embryos cultured at high embryonic density, indicating that stress accumulated during in vitro embryo culture affected the fate for embryo development and quality.

著者

Narong TIPTANAVATTANA Chommanart THONGKITTIDILOK Mongkol TECHAKUMPHU Theerawat THARASANIT

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2012-130, (Released:2013-01-25)

被引用文献数

7 15 3

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Spermatogonial stem cells (SSCs) function to regulate the balance of self-renewal and differentiation of male gametes. SSCs have been successfully isolated and cultured in vitro in several species, but not in feline. Therefore, in this study, we aimed to culture and characterize feline SSCs. In experiment 1, testes (n=5) from different pubertal domestic cats were cryosectioned and fluorescently immunolabeled to examine the expression of SSC (GFRα-1), differentiated spermatogonium (c-kit) and germ cell (DDX-4) markers. In experiments 2 and 3, testicular cells were digested and subsequently cultured in vitro. The resultant presumptive SSC colonies were then collected for SSC identification (experiment 2), or further cultured in vitro on feeder cells (experiment 3). Morphology, gene expression and immunofluorescence were used to identify the SSCs. Experiment 1 demonstrated that varying types of spermatogenic cells existed and expressed different germ cell/SSC makers. A rare population of putative SSCs located at the basement membrane of the seminiferous tubules was specifically identified by co-expression of GFRα-1 and DDX-4. Following enzymatic digestion, grape-like colonies formed by 13-15 days of culture. These colonies expressed GFRA1 and ZBTB16, but did not express KIT. Although we successfully isolated and cultured feline SSCs in vitro, the SSCs could only be maintained for 57 days. In conclusion, this study demonstrates, for the first time, that putative SSCs from testes of pubertal domestic cats can be isolated and cultured in vitro. These cells exhibited SSC morphology and expressed SSC-specific genes. However, long-term culture of these putative SSCs was compromised.

著者

Qing LI Yong FAN Xiaofang SUN Yanhong YU

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2012-109, (Released:2012-11-09)

被引用文献数

2 13

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The ectopic expression of transcription factors for reprogramming human somatic cells to a pluripotent state represents a valuable resource for the development of in vitro-based models for human disease and has great potential in regenerative therapies. However, the majority of studies have used skin fibroblasts to generate induced pluripotent stem cells (iPSCs) that typically require the enforced expression of several transcription factors, thereby posing a mutagenesis risk by the insertion of viral transgenes. To reduce this risk, iPSCs have been generated with OCT4 and KLF4 from human neural stem cells that endogenously express the remaining reprogramming factors. However, human neural stem cells are rare and difficult to obtain. Here, we show that iPSCs can be generated from human amniotic fluid cells (hAFCs) with two transcription factors: OCT4 and KLF4. Furthermore, iPSCs can be readily derived from hAFCs in a feeder-free conditions, thereby eliminating the potential variability caused by using feeder cells. Our results indicate that hAFCs represent an accessible source of cells that can be reprogrammed into pluripotent stem cells with two Yamanaka factors. Therefore, hAFCs may become a preferred cell type in the future for safe reprogramming without any exogenous genetic material.

著者

Ayumi HASEGAWA Kazuya YONEZAWA Akihiko OHTA Keiji MOCHIDA Atsuo OGURA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.58, no.1, pp.156-161, 2012 (Released:2012-03-22)

参考文献数

30

被引用文献数

1 18 4

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The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 μl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.

著者

Hamayun KHAN Ken Takeshi KUSAKABE Shoichi WAKITANI Masato HIYAMA Ai TAKESHITA Yasuo KISO

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.1112200427-1112200427, (Released:2011-12-22)

被引用文献数

6 16

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Nitric oxide synthase (NOS) is a key regulator of angiogenesis and embryogenesis in the mammalian reproductive process. Here, we attempted to clarify the expression and localization of inducible and endothelial NOS (iNOS and eNOS) in the developing rabbit placenta. Real-time RT-PCR analysis indicated that iNOS mRNA was significantly upregulated during pregnancy, and then significantly decreased after d18 (completion of the placentation period). The eNOS mRNA was also enhanced in the pregnant uteri and gradually decreased near the term of pregnancy. Western blot analysis also showed elevation of the iNOS and eNOS protein levels during the course of pregnancy. Immunohistochemical study revealed distinct localizations of iNOS along the radial arteries and eNOS at the spiral arteries and arterial sinuses in the developing placenta. This may reflect that iNOS and eNOS participate in pregnancy success through placentation-specific vascular formation and by supporting adequate blood circulation in the rabbit placenta.

著者

金 金 八重樫 朋祥 澤田 建 斉藤 隼人 後藤 由希 中嶋 侑佳 澤井 健 橋爪 力

出版者

日本繁殖生物学会

雑誌

日本繁殖生物学会 講演要旨集 第103回日本繁殖生物学会大会

巻号頁・発行日

pp.101, 2010 (Released:2010-08-25)

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【目的】本研究は,日長や温度変化が反芻家畜の成長ホルモン分泌(GH)に及ぼす影響を明らかにするために,ヤギを人工気象室内で飼養し,日長や温度を変化させた時のGH分泌の変化を調べた。 【方法】成熟雌シバヤギを室温20°Cに設定した人工気象室内で,8時間明,16時間暗(8L-16D), 12時間明,12時間暗(12L-12D)及び16時間明,8時間暗(16L-8D)の人工照明下で飼養した。また12L-12Dの一定照明下で室温を25°C又は5°Cに設定した人工気象室内でもヤギを飼養した。それぞれの条件下で飼養したヤギの頸静脈内にカテーテルを留置し,15分間隔で4時間採血して血中GH濃度の変化を調べた。また各期に成長ホルモン放出ホルモン(GHRH: 0.25 µg/kg b.w.)を頸静脈内投与してGHの放出反応も調べた。GHパルスの解析はEllisら方法に従った。 【結果】(1) 照明を変化させた時,GHのパルス頻度には有意な変化は見られなかった。パルスの振幅は8L-16D区に比べ12L-12D区と16L-8D区では高くなる傾向が見られた。平均GH濃度は8L-16D区に比べ12L-12D区と16L-8D区は有意に高かった(P<0.05)。(2)温度を変化させた時,パルス頻度と平均GH濃度には有意な変化は見られなかった。GHパルスの振幅は25°C区に比べ5°C区の方が高い傾向にあった。 (3) GHRH投与実験においては,16L-8D区は8L-16D区に比べGH放出反応が高まる傾向が見られた。温度変化による差は見られなかった。 本研究の結果は,ヤギのGH分泌は温度変化より,日長変化により修飾されることを示唆する。

著者

難波 陽介 中務 桂佑 説田 章平 大石 真也 藤井 信孝 若林 嘉浩 岡村 裕昭 上野山 賀久 束村 博子 前多 敬一郎 大蔵 聡

出版者

日本繁殖生物学会

雑誌

日本繁殖生物学会 講演要旨集 第103回日本繁殖生物学会大会

巻号頁・発行日

pp.54, 2010 (Released:2010-08-25)

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【目的】キスペプチンは,Kiss1遺伝子にコードされ,性腺刺激ホルモン放出ホルモン(GnRH)分泌刺激因子として注目されている神経ペプチドである。我々はウシKiss1遺伝子のcDNA塩基配列を同定し,ウシ型キスペプチンが53アミノ酸残基からなると推定した(第101回日本繁殖生物学会大会)。また,黒毛和種成熟雌ウシにおいて,ウシ型キスペプチンC末端部分ペプチドの末梢投与が黄体形成ホルモン(LH)および卵胞刺激ホルモン(FSH)の分泌を刺激することを示した(第102回日本繁殖生物学会大会)。本研究では,ウシにおけるキスペプチンの生理作用についてさらなる知見を得るため,全長ウシ型キスペプチン(bKp-53)の末梢投与によるLHおよびFSH分泌に対する効果と,第1卵胞発育波における主席卵胞の発育刺激効果を検討した。【方法】動物は黒毛和種成熟雌ウシを供試した(n=5)。プロスタグランジンF2α投与により誘起した発情日をDay 0とした。Day 5に,bKp-53 (0.2 nmol/kg)またはGnRH (0.2 nmol/kg)を静脈内投与した。投与前4時間から投与後6時間まで10分間隔で採血し,血漿中LHおよびFSH分泌動態を調べた。また,実験期間を通じて毎日,主席卵胞の直径を超音波診断装置により測定した。【結果】bKp-53の静脈内投与により,LHおよびFSH分泌の一過性の亢進がみられたが、主席卵胞は排卵しなかった。一方,GnRHの静脈内投与により,LHおよびFSH分泌は顕著に亢進し、投与後30時間から42時間までに主席卵胞の排卵を確認した。以上より,GnRHによる強力な性腺刺激ホルモン分泌刺激効果とは異なり、ウシにおいてキスペプチンはLHおよびFSH分泌を緩やかに亢進させる可能性が示唆された。本研究は生研センター「新技術・新分野創出のための基礎研究推進事業」の一部として実施した。