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Messenger Ribonucleic Acid Expressions of Hepatocyte Growth Factor, Angiopoietins and Their Receptors During Follicular Development in Gilts OAK
SHIMIZU, Takashi; IIJIMA, Koji; SASADA, Hiroshi; SATO, Eimei; 清水, 隆.
Angiogenic factors are associated with angiogenesis during follicular development in the mammalian ovary. The aim of the present study was to determine the relationships between the vascular network and mRNA expressions of angiopoietins (Ang)-1, Ang-2 and hepatocyte growth factor (HGF), and their receptors in follicles at different developmental stages during follicular development. Ovaries in gilts were collected 72 h after equine chorionic gonadotropin (eCG, 1250 IU) treatment for histological observation of the capillary network. Granulosa cells and thecal tissues in small (<4 mm), medium (4-5 mm) or large (>5 mm) individual follicles were collected for detection of mRNA expression of HGF, Ang-1 and Ang-2 in granulosa cells, and HGF receptor...
Palavras-chave: Follicular Development; Angiogenesis; Angiopoietins; HGF; Pig.
Ano: 2003 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4055
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血管増殖因子を用いた排卵誘導法 OAK
清水, 隆; 佐藤, 英明; SHIMIZU, Takashi.
J-stageにて公開
Ano: 2003 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4118
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The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-α Gene on Reproductive Performance and Immune Function in Dairy Cattle OAK
KAWASAKI, Yurie; AOKI, Yuka; MAGATA, Fumie; MIYAMOTO, Akio; KAWASHIMA, Chiho; HOJO, Takuo; OKUDA, Kiyoshi; SHIRASUNA, Koumei; SHIMIZU, Takashi; 宮本, 明夫; 清水, 隆.
The present study aimed to assess the effect of polymorphisms in the tumor necrosis factor α (TNF-α) promoter (A/A, A/G and G/G) and exons (T/T, T/C and C/C) on immune function and reproductive performance in dairy cows. The occurrence of the first postpartum ovulation within 3 weeks in the cows with the TNF-α promoter A/G and G/G genotypes was higher than in the A/A group. Among the different TNF-α exon genotypes, the occurrence of early first postpartum ovulation was higher in the T/C and C/C genotype groups than in the T/T group. Single nucleotide polymorphisms (SNPs) in the TNF-α gene did not affect the rate of artificial insemination (AI) or duration from parturition to next conception (days open). The apoptosis rate of polymorphonuclear leukocytes...
Palavras-chave: Cattle; Immune function; Polymorphism; Reproductive performance; Tumor necrosis factor α (TNF-α).
Ano: 2014 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/3934
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Promotion of Ovarian Follicular Development by Injecting Vascular Endothelial Growth Factor (VEGF) and Growth Differentiation Factor 9 (GDF-9) Genes OAK
SHIMIZU, Takashi; 清水, 隆.
Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a...
Palavras-chave: Follicle development; Gene injection; Growth differentiation factor 9 (GDF-9); Ovary; Vascular endothelial growth factor (VEGF).
Ano: 2005 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4059
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Differential Effect of Follicle-Stimulating Hormone and Estradiol on Expressions of Vascular Endothelial Growth Factor (VEGF) 120, VEGF164 and Their Receptors in Bovine Granulosa Cells OAK
SHIMIZU, Takashi; JAYAWARDANA, Barana C.; TETSUKA, Masafumi; MIYAMOTO, Akio; 清水, 隆; 手塚, 雅文; 宮本, 明夫.
Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. However, whether the transcript of the VEGF120 and VEGF164 isoforms differs during follicular development in the ovary is still unknown. We first examined the gene expression of VEGF120, VEGF164, fms-like tyrosine kinase (Flt-1), and fetal liver kinase (Flk-1) in the granulosa cells (GCs) and theca cells (TCs) of pre-selection and post-selection follicles (PRF and POF respectively) from the bovine ovary. Then we examined the effects of FSH and estradiol (E2) on these factors in cultured bovine GCs. Messenger RNA (mRNA) expression was quantified using real-time PCR...
Palavras-chave: Estradiol; Fetal liver kinase (Flk-1); Fms-like tyrosine kinase (Flt-1); Follicle-stimulating hormone (FSH); Granulosa cell; Vascular Endothelial Growth factor (VEGF).
Ano: 2007 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4057
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Comparison of Bacterial Endotoxin Lipopolysaccharide Concentrations in the Blood, Ovarian Follicular Fluid and Uterine Fluid: A Clinical Case of Bovine Metritis OAK
MAGATA, Fumie; ISHIDA, Yoshikazu; MIYAMOTO, Akio; FURUOKA, Hidefumi; INOKUMA, Hisashi; SHIMIZU, Takashi; 宮本, 明夫; 古岡, 秀文; 猪熊, 壽; 清水, 隆.
We investigated the concentration of the bacterial endotoxin lipopolysaccharide (LPS) in the blood, ovarian follicular fluid and uterine fluid of a clinical case of bovine metritis. A 2-year-old lactating Holstein cow exhibited continuous fever >39.5°C for more than 2 weeks after normal calving. The cow produced a fetid, watery, red-brown uterine discharge from the vagina and was diagnosed with metritis. The LPS concentrations in plasma and uterine fluid were 0.94 and 6.34 endotoxin units (EU)/ml, respectively. One of seven follicles showed an extremely high level of LPS (12.40 EU/ml) compared to the other follicles (0.62–0.97 EU/ml). These results might suggest the presence of high concentration of LPS in follicles in cows with postpartum metritis.
Palavras-chave: Dairy cow; Follicle; Lipopolysaccharide; Metritis.
Ano: 2015 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4043
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Gene Expressions in the Persistent Corpus Luteum of Postpartum Dairy Cows: Distinct Profiles from the Corpora Lutea of the Estrous Cycle and Pregnancy OAK
MAGATA, Fumie; SHIRASUNA, Koumei; STRÜVE, Klaas; HERZOG, Kathrin; SHIMIZU, Takashi; BOLLWEIN, Heinrich; MIYAMOTO, Akio; 清水, 隆; 宮本, 明夫.
Persistence of the corpus luteum (CL) in cattle usually occurs during the puerperium and is associated with interference of prostaglandin (PG) F2α release from the uterus. The objective of the present study was to determine for the first time the gene expressions in the persistent CL compared with the CL of pregnancy and cyclic CL. Three types of CL biopsy samples were collected from 32 lactating Holstein cows: (1) CL persisting for 29 to 33 days after the first ovulation postpartum (persistent CL, n=9), (2) CL between days 29 and 33 of early pregnancy (CL of pregnancy, n=8) and (3) CL between days 10 and 13 of the estrous cycle (cyclic CL, n=27). mRNA expression of 2',5'-oligoadenylate synthetase-1 was upregulated only in the CL of pregnancy, confirming...
Palavras-chave: Dairy cow; Early pregnancy; Persistent corpus luteum; MRNA expression.
Ano: 2012 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/3938
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Expression of Pin1, a peptidyl-prolyl isomerase, in the ovary of eCG/hCG-treated immature female mice. OAK
SHIMIZU, Takashi; AKIYAMA, Hirotada; ABE, Yasuyuki; SASADA, Hiroshi; SATO, Eimei; MIYAMOTO, Akio; UCHIDA, Takafumi; 清水, 隆; 宮本, 明夫.
Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivotal signaling mechanism in diverse cellular processes. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Although much protein is phosphorylated in the ovary, the role of Pin1 in the ovary is still unknown. The purpose of this study is to investigate the effects of gonadotropins on protein and mRNA expression of Pin1 in mice ovaries. Quantitative PCR analysis showed that the expression of Pin1 mRNA significantly increased in the ovaries of equine chorionic gonadotropin (eCG)-treated mice compared with those of untreated mice (P<0.05). However,...
Palavras-chave: E2F; Follicle development; Gonadotropin; Ovary; Pin1.
Ano: 2006 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4056
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Molecular and cellular mechanisms for the regulation of ovarian follicular function in cows OAK
SHIMIZU, Takashi; 清水, 隆.
Ovary is an important organ that houses the oocytes (reproductive cell). Oocyte growth depends on the function of follicular cells such as the granulosa and theca cells. Two-cell two gonadotropin systems are associated with oocyte growth and follicular cell functions. In addition to these systems, it is also known that several growth factors regulate oocyte growth and follicular cell functions. Vascular endothelial growth factor (VEGF) is involved in thecal vasculature during follicular development and the suppression of granulosa cell apoptosis. Metabolic factors such as insulin, growth hormone (GH) and insulin-like growth factor 1 (IGF-1) also play critical roles in the process of follicular development and growth. These factors are associated not only...
Palavras-chave: Endotoxin; Metabolic factors; Ovarian follicle; Steroid hormone; Vascular endothelial growth factor (VEGF).
Ano: 2016 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4325
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Effect of Suckling on the Reproductive Performance and Metabolic Status of Obese Japanese Black Cattle during the Early Postpartum Period OAK
KAWASHIMA, Chiho; KIDA, Katsuya; MATSUHASHI, Miki; MATSUI, Motozumi; SHIMIZU, Takashi; MATSUNAGA, Nobuyoshi; ISHII, Mitsuo; MIYAKE, Yoh-Ichi; MIYAMOTO, Akio; 川島, 千帆; 木田, 克弥; 松井, 基純; 清水, 隆; 石井, 三都夫; 三宅, 陽一; 宮本, 明夫; 松長, 延吉.
The aim of our study was to investigate the effects of suckling on reproductive performance and metabolic status of obese (mean body condition score of more than 4.0 on a scale of 1-5) maternal Japanese Black cows during early postpartum period. We used 7 postpartum Japanese Black cattle. Four cows were suckled ad libitum (suckled) until completion of their first artificial insemination (AI), while 3 cows were not suckled at all because they were separated from their calves immediately after parturition (non-suckled). Body weight and plasma concentrations of metabolites and hormones were measured from wk 1 to 9 postpartum. Ovarian activity was detected using plasma progesterone concentration, and all cows received their first AI after application of the...
Palavras-chave: Japanese Black cattle; Metabolic status; Obesity; Reproductive performance; Suckling.
Ano: 2008 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2991
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In Vivo Evidence that Local Cortisol Production Increases in the Preovulatory Follicle of the Cow OAK
ACOSTA, Tomas J.; TETSUKA, Masa; MATSUI, Motozumi; SHIMIZU, Takashi; BERISHA, Bajram; SCHAMS, Dieter; MIYAMOTO, Akio; 松井, 基純; 清水, 隆; 宮本, 明夫.
The aim of the present in vivo study was to monitor real-time fluctuations of cortisol (Cr) in the wall of preovulatory follicles using a microdialysis system (MDS) implanted in the theca layer as well as changes in ovarian venous plasma (OVP) and jugular venous plasma (JVP). Seven cows were superovulated using FSH and prostaglandin F2α injections. Dialysis capillary membranes were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system. Fractions of the perfusates were collected from Day -1 (Day 0=LH surge) to Day 3. No difference in the concentrations of Cr between JVP and OVP was detected throughout the experiment. Circulating concentrations of Cr ranged from 20 to 35 ng/ml 8 h after surgery in ovulatory and...
Palavras-chave: Bovine; Cortisol; Follicle; Ovary; Ovulation.
Ano: 2005 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2980
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Nutritional factors that regulate ovulation of the dominant follicle during the first follicular wave postpartum in high-producing dairy cows. OAK
KAWASHIMA, Chiho; MATSUI, Motozumi; SHIMIZU, Takashi; KIDA, Katsuya; MIYAMOTO, Akio; 川島, 千帆; 松井, 基純; 清水, 隆; 木田, 克弥; 宮本, 明夫.
During recent decades, milk production per cow has increased drastically due to improved management, nutrition, and genetic selection; however, the reproductive performance of high-producing dairy cows has been declining. One of the factors responsible for this low reproductive performance is negative energy balance (NEB). NEB affects the onset of first ovulation in early postpartum cows. It is generally accepted that early first ovulation positively relates to the resumption of normal ovarian function, first service, and conception rate in dairy cows. Hence, delayed first ovulation has a negative impact on subsequent fertility. The metabolic condition of cows in NEB shifts to catabolic metabolism, which in turn causes increased plasma growth hormone and...
Palavras-chave: Β-Carotene; Dairy cow; First follicular wave postpartum; First ovulation; Metabolic hormone.
Ano: 2012 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4041
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Relationship between the degree of insulin resistance during late gestation and postpartum performance in dairy cows and factors that affect growth and metabolic status of their calves OAK
KAWASHIMA, Chiho; MUNAKATA, Megumi; SHIMIZU, Takashi; MIYAMOTO, Akio; KIDA, Katsuya; MATSUI, Motozumi; 川島, 千帆; 清水, 隆; 宮本, 明夫; 木田, 克弥; 松井, 基純.
This study aimed to investigate the effects of insulin resistance (IR) during the close-up dry period on the metabolic status and performance of dairy cows as well as to determine the effects on body weight (BW) and metabolic status of their calves. An insulin tolerance test (ITT) was conducted by administering 0.05 IU/kg BW of insulin to 34 multiparous Holstein cows at 3 weeks prepartum. Blood samples were collected at 0, 30, 45 and 60 min after insulin injection, and cows were divided into two groups based on the time required for glucose to reach the minimum levels [non-IR (NIR), 45 min (n=28); and IR, 60 min (n=6)]. Blood or milk sampling and body condition score (BCS) estimation were performed twice weekly during the experimental period. Blood samples...
Palavras-chave: Dairy cow; Dry period; Insulin tolerance test; Metabolic status; Postpartum performance.
Ano: 2016 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4363
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An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro OAK
LIU, Jinghui; A. MAREY, Mohamed; KOWSAR, Rasoul; HAMBRUCH, Nina; SHIMIZU, Takashi; HANEDA, Shingo; MATSUI, Motozumi; SASAKI, Motoki; HAYAKAWA, Hiroyuki; PFARRER, Christiane; MIYAMOTO, Akio; 羽田, 真悟; 松井, 基純; 佐々木, 基樹; 宮本, 明夫.
We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in...
Palavras-chave: Alpha 1-acid glycoprotein (AGP); Bovine; Oviduct; Phagocytosis; Sperm.
Ano: 2014 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/3946
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Follicular Microvasculature and Angiogenic Factors in the Ovaries of Domestic Animals OAK
SHIMIZU, Takashi; KAWAHARA, Manabu; ABE, Yasuyuki; YOKOO, Masaki; SASADA, Hiroshi; SATO, Eimei; 清水, 隆.
The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate the process of thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation. A recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the process of thecal angiogenesis during follicular development. We are developing a novel technology for the induction of follicular development using the technique of in vivo gene administration. Here, we summarize the recent progress of our research.
Palavras-chave: Follicular development; Microvasculature; Angiogenic factor.
Ano: 2003 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4058
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Prostaglandin F2α Differentially Affects mRNA Expression Relating to Angiogenesis,Vasoactivation and Prostaglandins in the Early and Mid Corpus Luteum in the Cow OAK
SHIRASUNA, Koumei; SASAHARA, Kiemi; MATSUI, Motozumi; SHIMIZU, Takashi; MIYAMOTO, Akio; 白砂, 孔明; 松井, 基純; 清水, 隆; 宮本, 明夫.
Administration of prostaglandin (PG) F2α in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF2α does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF2α administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF2α injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 μg PGF2α analogue or saline as the control...
Palavras-chave: Corpus luteum; Cow; Luteolysis; Prostaglandin F2α.
Ano: 2010 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2812
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Expression of mRNA for Cell Adhesion Molecules in the Bovine Corpus Luteum During the Estrous Cycle and PGF2α-Induced Luteolysis OAK
SHIRASUNA, Koumei; WATANABE, Sho; NAGAI, Kaya; SASAHARA, Kiemi; SHIMIZU, Takashi; RICKEN, Albert M.; SPANEL-BOROWSKI, Katharina; MIYAMOTO, Akio; 白砂, 孔明; 清水, 隆; 宮本, 明夫.
Cell-to-cell interaction via cell contact-dependent pathway is essentially important for maintenance and regulation of corpus luteum (CL) integrity and its physiological actions. The objective of the present study was to evaluate the mRNA expression of the cell adhesion molecules (CAMs) that are constituent factors of gap junctions [connexin (Cx) 43] and adherence junctions (VE-, E-, N-cadherin) in two types of endothelial cells from the mid CL and in CL tissue during the estrous cycle and PGF2α-induced luteolysis in the cow. Specific mRNA expression for Cx43 and N-cadherin was detected in cytokeratin-positive (CK+) and cytokeratin-negative (CK-) luteal endothelial cells (EC) and fully luteinized granulosa cells (LGC). E-cadherin mRNA was expressed in...
Palavras-chave: Cadherin; Connexin; Corpus luteum; Cow; Luteolysis.
Ano: 2007 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2988
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Changes in Plasma Metabolic Hormone Concentrations During the Ovarian Cycles of Japanese Black and Holstein Cattle OAK
KAWASHIMA, Chiho; KIDA, Katsuya; HAYASHI, Ken-Go; AMAYA MONTOYA, Carlos; KANEKO, Etsushi; MATSUNAGA, Nobuyoshi; SHIMIZU, Takashi; MATSUI, Motozumi; MIYAKE, Yoh-Ichi; SCHAMS, Dieter; MIYAMOTO, Akio; 川島, 千帆; 木田, 克弥; 清水, 隆; 松井, 基純; 三宅, 陽一; 宮本, 明夫.
The aim of the present study was to investigate the changing profiles of plasma metabolic hormones during the ovarian cycles of beef and dairy cattle. We used 16 non-pregnant, non-lactating Japanese Black beef cattle (6 heifers and 10 cows; parity=2.3 ± 0.8) and 12 multiparous Holstein dairy cows (parity=3.0 ± 0.3). Blood samples for hormonal analysis (growth hormone, GH; insulin-like growth factor-I, IGF-1; insulin; and progesterone, P4) were obtained twice weekly for 40 days before artificial insemination for Japanese Black cattle and from 50 to 100 days postpartum for Holstein cows. Luteal phases were considered normal if the P4 concentrations for at least 3 time points over the course of 7 days remained above 1 ng/ml and at least 2 of the time points...
Palavras-chave: Cattle; Insulin-like growth factor-I; Ovarian cycle; Progesterone.
Ano: 2007 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2984
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Changes in the Messenger RNA Expressions of the Endothelin-1 and Angiotensin Systems in Mature Follicles of the Superovulated Bovine Ovary OAK
SHIMIZU, Takashi; BERISHA, Bajram; SCHAMS, Dieter; MIYAMOTO, Akio; 清水, 隆; 宮本, 明夫.
The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at...
Palavras-chave: Angiotensin; Bovine; Endothelin-1; Gonadotrophin releasing hormone (GnRH); Ovulation.
Ano: 2007 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/2987
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Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response OAK
KOWSAR, Rasoul; HAMBRUCH, Nina; LIU, Jinghui; SHIMIZU, Takashi; PFARRER, Christiane; MIYAMOTO, Akio; 清水, 隆; 宮本, 明夫.
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...
Palavras-chave: Bovine oviduct epithelial cell; Ovarian steroids; TLR-4; Th1/Th2 response.
Ano: 2013 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/3937
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