08 Sep. 2013, The Journal of Poultry Science優秀論文賞, Detection of LeptinActivity in Living Cells Expressing Chicken Leptin Receptor and STAT3, 日本家禽学会
Hiromi Adachi;Daisuke Murase;Susumu Atomura;Takeshi Ohkubo

Sep. 2013, 日本家禽学会賞, 日本家禽学会

Sep. 2013, The Journal of Poultry Science優秀論文賞, Detection of LeptinActivity in Living Cells Expressing Chicken Leptin Receptor and STAT3, 日本家禽学会
Hiromi Adachi;Daisuke Murase;Susumu Atomura and Takeshi Ohkubo

Genetic Analysis of HSP70 and HSF3 Polymorphisms and Their Associations with the Egg Production Traits of Bangladeshi Hilly Chickens.
Md Yousuf Ali; Shakila Faruque; Sadequllah Ahmadi; Takeshi Ohkubo, In warm environments, thermoregulation in poultry is controlled by heat shock protein 70 (HSP70), whose expression is controlled by heat shock factor 3 (HSF3). Although the association between genetic polymorphisms in these genes and thermotolerance as well as reproductive traits has been extensively studied in mammals, the association has not yet been studied in poultry. This study aimed to explore the relationship between single-nucleotide polymorphisms (SNPs) in these genes and the egg production traits of Bangladeshi hilly chickens. Sequencing and allele-specific PCR (AS-PCR) were used to detect new SNPs and perform genotyping. We identified two novel SNPs (G-399A and A-68G) in the 5'-flanking regions of HSP70 that were significantly associated with egg numbers (ENs) at 161-190 days and increased egg weight (EW) at 40 weeks of age. Furthermore, three SNPs in HSP70 (A258G, C276G and C1431A) and one SNP in HSF3 (A-1388G) were associated with EN at different ages. The haplotype and combined genotypic effects of these two genes were found to be associated with age at sexual maturity (ASM), EN, EW, and body weight at ASM. The identified SNPs and their corresponding haplotypes may be useful in selective breeding to enhance the productivity of chickens in warm environments.
Animals : an open access journal from MDPI, 10 Dec. 2024

Molecular form identification of anterior pituitary gland-secreted prolactin in chicken
Kansaku N and Ohkubo T, Endocrine changes during bird reproduction are well documented. Prolactin (PRL) exhibits a strong relationship between incubation and broody behavior. The molecular forms of PRL in the anterior pituitary gland during the reproductive cycle have already been previously identified but not those in the secreted form. To identify the molecular forms of secreted PRL during the reproductive cycle, we thus monitored the physiological status and incubation behavior of 10 Silkie hens by a video recording system over 1-2 years. Nine out of ten mature hens exhibited incubation behavior multiple times during the experiment. Ten hens demonstrated two interesting features. In a typical clutch, hens spent 10-15 min in the nest to lay an egg. Once they spent over 1 h in the nest, the nest occupancy increased incrementally. This shift in the nest occupancy occurred 7-10 days before the incubation onset and was highly repeatable. Based on the behavior of the hens, we cultured the anterior pituitary gland during four stages (premature non-laying, laying, trans, and incubation) with physiological PRL-releasing factor, vasoactive intestinal peptide (VIP). Based on our two-dimensional protein analysis, glycosylated PRL (G-PRL) displayed several isoforms with varying isoelectric points (pI), whereas we could detect one primary signal for non-glycosylated PRL (NG-PRL). However, 3-4 NG-PRL isoforms were detected in the anterior pituitary gland. These results suggested that secreted PRL, especially from the trans and incubation stages, contains various isoforms and it is post-translationally glycosylated and phosphorylated., Elsevier
General and Comparative Endocrinology, Jan. 2024, [Reviewed]

〔Major achievements〕Impact of in ovo leptin injection and dietary protein levels on ovarian growth markers and early folliculogenesis in post-hatch chicks (Gallus gallus domesticus)
Ahmadi S; Nemoto Y and Ohkubo T, Corresponding, MDPI
Biology, Jan. 2024, [Reviewed]

Regulation of prolactin release at the end stage of chicken embryogenesis
Kansaku N; Wakui S; Sasanami T; Ohkubo T
Journal of Poultry Science, Oct. 2022, [Reviewed]

〔Major achievements〕Leptin promotes primordial follicle activation by regulating ovarian insulin-like growth factor system in chicken
Ahmadi S; Ohkubo T, Corresponding, Endocrine Society
Endocrinology, Sep. 2022, [Reviewed]

〔Major achievements〕Leptin Modulates the mRNA Expression of Follicle Development Markers in Post-hatch Chicks in an Age-Dependent Manner.
Shaikat AH; Ochiai M; Sasaki A; Takeda M; Arima A and Ohkubo T, Corresponding, Frontiers
Frontiers in Physiology, Jul. 2021, [Reviewed]

Orexin system is expressed in avian liver and regulates hepatic lipogenesis via ERK1/2 activation.
Greene ES; Zampiga M; Sirri F; Ohkubo T; Dridi S
Scientific Reports, Nov. 2020, [Reviewed]

Determination of Polymorphisms in Pituitary Genes of the Native Afghani Naked Neck Chicken　　　　　　　　　　　　　　　
Ahmadi S; Takeda M; Ohkubo T, Corresponding
Journal of poultry Science, Oct. 2019, [Reviewed]

Identification of hypothalamic genes in associating with food intake during incubation behavior in domestic chicken.　　　　　　　　　　　　　　　
Takeda M; Ohkubo T, Corresponding
Animal Science Journal, Sep. 2019, [Reviewed]

AMP-activated protein kinase mediates the effect of leptin on avian autophagy in a tissue-specific manner
Alissa Piekarski; Gurueswar Nagarajan; Peter Ishola; Joshua Flees; Elizabeth S. Greene; Wayne J. Kuenzel; Takeshi Ohkubo; Helena Maier; Walter G. Bottje; Mark A. Cline; Sami Dridi, Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKa1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKa1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner., Frontiers Media S.A.
Frontiers in Physiology, 15 May 2018, [Reviewed]

Gene expression profiling in embryonic chicken ovary during asymmetric development
Amir Hossan Shaikat; Shoko Namekawa; Sadequllah Ahmadi; Misa Takeda; Takeshi Ohkubo, The reproductive system in female birds arises as bilateral asymmetrical anlagen, excluding the birds of prey. Earlier, histological and messenger RNA (mRNA) expression profile studies of several genes related to gonadal sex differentiation in chicken embryos tried to elucidate the query of this asymmetry in a scattered manner. To understand the matter precisely, we have focused on mRNA expression of a cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) in second half of the embryonic days (E10–E18). The established role of leptin in development of the embryo and its expression in the embryonic ovary also drove us to check leptin receptor (LEPR) expression in the ovary. Increased expression of FSHR and CYP19A1 in the left ovary compared with that in the right ovary was identified (P <
 0.05), promoting preferential left ovarian development and functionality. Significant high expression (P <
 0.05) of the apoptotic genes in the right ovary were also involved here. Leptin probably has no direct influence on ovarian asymmetry as no significant variation in gonadal mRNA expression of LEPR was observed within the same experimental days. We propose that asymmetric expression of this cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) leads to the development of dimorphic gonads during embryogenesis., Blackwell Publishing
Animal Science Journal, 01 Apr. 2018, [Reviewed]

Leptin activates chicken growth hormone promoter without chicken STAT3 in vitro
Murase D; Namekawa S; Ohkubo T, Corresponding, Leptin is an adipocyte-derived hormone that not only regulates food intake and energy homeostasis but also induces growth hormone (GH) mRNA expression and release, thereby controlling growth and metabolism in mammals. The molecular mechanism of leptin-induced regulation of GH gene transcription is unclear. The current study investigated the effects of leptin on the chicken GH (cGH) promoter and the molecular mechanism underlying leptin-induced cGH gene expression in vitro. Leptin activated the cGH promoter in the presence of chPit-1 alpha in CHO cells stably expressing the chicken leptin receptor. Promoter activation did not require STAT-binding elements in the cGH promoter or STAT3 activity. However, JAK2 activation was required for leptin-dependent activity. JAK2-dependent pathways include p42/44 MAPK and PI3K, and inhibition of these pathways partially blocked leptin-induced cGH gene transcription. Although CK2 directly activates JAK2, a CK2 inhibitor blocked leptin-dependent activation of the cGH gene without affecting JAK2 phosphorylation. The CK2 inhibitor suppressed Erk1/2 and Akt phosphorylation. Additional data implicate Src family kinases in leptin-dependent cGH gene activation. These results suggest that leptin activates the cGH gene in the presence of chPit-1 alpha via several leptin-activated kinases. Although further study is required, we suggest that the leptin-induced JAK2/p42/44 MAPK and JAK2/PI3K cascades are activated by Src-meditated CK2, leading to CBP phosphotylation and interaction with chPit-1 alpha, resulting in transactivation of the cGH promoter. (C) 2015 Elsevier Inc All rights reserved., ELSEVIER SCIENCE INC
Comparative Biochemistry and Physiology Part B, Jan. 2016, [Reviewed]

Heat and oxidative stress alter the expression of orexin and its related receptors in avian liver cells
Greene E; Khaldi S; Ishola P; Bottje W; Ohkubo T; Anthony N; Dridi S, Orexins (A and B) or hypocretins (1 and 2) are hypothalamic orexigenic neuropeptides that are involved in the regulation of several physiological processes in mammals. Recently, orexin has been shown to activate the hypothalamic-pituitary-adrenal (HPA) stress axis and emerging evidences identify it as a stress modulator in mammals. However, the regulation of orexin system by stress itself remains unclear. Here, we investigate the effects of heat, 4-Hydroxynonenal (4-HNE) and hydrogen peroxide (H2O2) stress on the hepatic expression of orexin (ORX) and its related receptors (ORXR1/2) in avian species. Using in vivo and in vitro models, we found that heat stress significantly down-regulated ORX and ORXR1/2 mRNA and protein abundances in quail liver and LMH cells. H2O2, however, decreased ORX protein and increased ORX mRNA levels in a dose dependent manner (P < 0.05). The absence of correlation between orexin mRNA and protein levels suggests that H2O2 treatment modulates post-transcriptional mechanisms.4-HNE had a biphasic effect on orexin system expression, with a significant up-regulation at low doses (10 and 20 mu M) and a significant down-regulation at a high dose (30 mu M). Taken together, our data indicated that hepatic orexin system could be a molecular signature in the heat and oxidative stress response. (c) 2015 Elsevier Inc. All rights reserved., ELSEVIER SCIENCE INC
Comparative Biochemistry and Physiology Part A, Jan. 2016, [Reviewed]

黒毛和種繁殖雌牛の成長ホルモン受容体遺伝子多型が子牛市場体重の直接および母性遺伝効果に及ぼす影響について
高橋和裕; 木村 信熙; 田中実; 大久保武, Last, 離乳前の子牛の発育は子牛自身の発育性と母牛の哺育能力に依存し，発育初期の子牛には母牛の哺育能力が非常に重要である．哺育能力に関与する遺伝子が判明されれば，子牛が生産された時点で遺伝的な能力を予測することが可能である．そこで本研究では成長ホルモン受容体遺伝子の肝臓特異的転写制御領域に存在するLINE-1In/Del多型と黒毛和種の直接および母性遺伝効果の育種価との関連性を検討した．直接および母性遺伝効果のそれぞれの育種価はLINE-1配列挿入遺伝子をホモに持つLL型の育種値が13.84kg，4.87kg，LINE-1配列挿入遺伝子と欠損遺伝子をそれぞれ持つLS型の育種値が11.18kg，2.10kg，LINE-1配列欠損遺伝子をホモに持つSS型の育種値が−0.29kg，−8.61kgであり，直接および母性遺伝効果の育種価はともにLL型とSS型，LS型とSS型の間には有意な差が認められた（P<0.01）．このことからGHR遺伝子のLINE-1In/Del多型は子牛自身の発育に関する直接遺伝効果ならびに繁殖雌牛の母性遺伝効果の選抜の一指標となる可能性が推察された．, Japanese Society of Animal Science
日本畜産学会報, 2015, [Reviewed]

Avian blood induced intranuclear translocation of STAT3 via the chicken leptin receptor
Takeshi Ohkubo; Kanako Hirota; Daisuke Murase; Hiromi Adachi; Tsutomu Nozawa-Takeda; Shoei Sugita, Leptin is a multi-functional adipokine in vertebrates. The leptin gene and protein are found in many vertebrates; however, the existence of leptin in birds remains controversial. Here we detected leptin-like activity in avian blood using chicken leptin receptor (chLEPR) and green fluorescent protein (GFP)-fused chicken signal transducer and activator of transcription (chSTAT3) co-expressed in CHO-K1 cells (CHO-chLEPR/STAT3). We validated that rat serum specifically induces intranuclear migration of GFP-fused chSTAT3 (GFP-chSTAT3) in CH-OchLEPR/STAT3 cells, but not in CHO-K1 cells expressing GFP-STAT3 (CHO-chSTAT3) before testing the avian blood samples. Blood of chickens (Gallus gallus), wild jungle crows (Corvus macrorhynchos), and carrion crows (Corvus corone) accumulated the GFP signal into nuclei, and frequency varied in each blood sample. Western blotting showed that chicken and crow blood samples specifically phosphorylated GFP-chSTAT3 in the chLEPR-transfected cells. These results indicate that avian blood contains a leptin-like molecule that specifically binds to LEPR, suggesting that the leptin system is conserved across all vertebrate classes. (C) 2014 Elsevier Inc All rights reserved., ELSEVIER SCIENCE INC
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, Aug. 2014, [Reviewed]

Discovery of the elusive leptin in birds: identification of several 'missing links' in the evolution of leptin and its receptor.
Prokop JW; Schmidt C; Gasper D; Duff RJ; Milsted A; Ohkubo T; Ball HC; Shawkey MD; Mays HL Jr; Cogburn LA and Londraville RL, Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds has remained elusive and controversial. Here we identify leptin sequence from the Peregrine falcon, Falco peregrinus (pfleptin), and identify sequences from two other birds (mallard and zebra finch), and 'missing' vertebrates (elephant shark, alligator, Indian python, Chinese soft-shelled turtle, and coelacanth). The pattern of genes surrounding leptin (snd1, rbm28) is syntenic between the falcon and mammalian genomes. Phylogenetic analysis of all known leptin protein sequences improves our understanding of leptin's evolution. Structural modeling of leptin orthologs highlights a highly conserved hydrophobic core in the four-helix cytokine packing domain. A docked model of leptin with the leptin receptor for Peregrine falcon reveals several conserved amino acids important for the interaction and possible coevolution of leptin with its receptor. We also show for the first time, an authentic avian leptin sequence that activates the JAK-STAT signaling pathway. These newly identified sequences, structures, and tools for avian leptin and its receptor will allow elucidation of the function of these proteins in feral and domestic birds., PUBLIC LIBRARY SCIENCE
PLoS One, 24 Mar. 2014, [Reviewed]

コレシストキニンA受容体遺伝子g.420 C > A 一塩基多型は比内鶏の発育を改善する
力丸宗弘; 武田尚人; 大久保武; 高橋大希; 小松恵; 高橋秀彰, 我々は，既報において，発育が異なる比内鶏系統個体を交配し，作出したF2家系集団において，コレシストキニンA受容体遺伝子(CCKAR)の5'非翻訳領域の特定部位における一塩基多型(SNP，g. 420 C>A)とF2個体の発育形質との間に，有意な関連性を確認し，AアリルはCアリルよりも発育形質に対する効果が優れていることを報告した。本研究では，発育形質が改良されていない保存会系統の比内鶏を用いて，当該SNPによって比内鶏の発育が改善されるか検証を行った。比内鶏各個体における当該SNPの遺伝子型(A/A，A/C，C/C)を既報に従って，4週齢になる前に判定した後，4週齢から14週齢まで育雛ケージで飼育した。4週齢から14週齢まで，2週間おきに各個体の体重を測定し，2週間間隔の平均日増体重を算出した。また，2週おきに飼料摂取量を測定し，さらに平均日増体重と飼料摂取量から，飼料要求率を算出した。その結果，A/A型個体は他の遺伝子型個体より14週齢体重が有意に重く，平均日増体重も有意に優れていた。飼料摂取量では，遺伝子型間で有意差は認められなかった。飼料要求率では，4-10週齢において，A/A型個体がC/C型個体より有意に優れていたが，4-14週齢間では，各遺伝子型間に有意な差は認められなかった。以上の結果から，CCKAR遺伝子のg. 420 C>A SNPによって，比内鶏の発育が実際に改善されることが明らかになった。, 日本万国家禽学会
日本家禽学会誌, 2014, [Reviewed]

Inhibitory Mechanism of Signal Transduction through Chicken Leptin Receptor by Suppressor of Cytokine Signaling 3 (SOCS3)
Hiromi Adachi; Daisuke Murase; Takeshi Ohkubo, Leptin is an adipocyte-derived hormone involved in the regulation of feeding behavior and energy homeostasis in vertebrates. We recently reported that leptin activates the JAK-STAT signaling pathway through the chicken leptin receptor (chLEPR). However, the molecular inhibitory mechanism by suppressor of cytokine signaling 3 (SOCS3), observed in mammalian leptin signaling, has not been elucidated in avian species. Therefore, the role of chicken SOCS3 (chSOCS3) in signal transduction through the chLEPR was analyzed in this study. Leptin increases SOCS3 mRNA expression in chicken hepatoma cells, LMH, and also activates the chSOCS3 gene promoter in the chLEPR-expressing cells. Overexpression of chSOCS3 inhibited leptin-induced signaling by blocking phosphorylation of JAK2 and subsequent activation of STAT3 similar to that observed in mammals. Signaling inhibited by chSOCS3 was not restored in the chLEPR mutated docking site of SOCS3. In addition, mutation of Phe25 in the kinase inhibitory region of chSOCS3 abolished SOCS3 activity via the wild chLEPR. The present study indicates that SOCS3 is a negative feedback regulator of leptin signaling in chickens as well as in mammals. However, the inhibitory mechanism in chickens may differ slightly from that observed in mammals., JAPAN POULTRY SCIENCE ASSOC
JOURNAL OF POULTRY SCIENCE, Jul. 2013, [Reviewed]

Detection of leptin activity in living cells expressing chicken leptin receptor and STAT3
Hiromi Adachi; Daisuke Murase and Takeshi Ohkubo, Corresponding, The chicken leptin receptor (chLEPR) activates Janus kinase (JAK) - signal transducers and activator of transcription (STAT) signaling pathway after leptin stimulation. We have previously developed a bioassay using leptin inducible reporter gene in cultured cells. However, we failed to detect leptin-like activity in chicken blood. In the present study, we expressed green fluorescent protein (GFP) fused chSTAT3 (GFP-chSTAT3) in cells expressing chLEPR and analyzed leptin dependent activation of the chSTAT3. Leptin phosphorylated GFP-chSTAT3 and by which fluorescent signal translocated into nuclei in COS-7 cells transiently expressing GFP-chSTAT3 with chLEPR. Furthermore, we established CHO-K1 cells stably expressing chLEPR and chSTAT3 (CHO-chLEPR/chSTAT3), and in which detected time- and dose-dependent activation of chSTAT3 by leptin. Therefore, the CHO-chLEPR/STAT3 cells would be an excellent tool to detect and monitor leptin-like activity in avian tissues., JAPAN POULTRY SCIENCE ASSOC
Journal of Poultry Science, 20 Jan. 2012, [Reviewed]

Role of chicken Pit-1 isoforms in activating growth hormone gene.
Daisuke Murase; Shusuke Taniuchi; Sakae Takeuchi; Hiromi Adachi; Norio Kansaku; Katsuichiro Okazaki; Takeshi Ohkubo, Corresponding, In the present study, we expressed chicken (ch) Pit-1 alpha (chPit-1 alpha) and chPit-1 gamma in vitro to compare the roles of chPit-1s in the transcription of the chicken growth hormone (chGH) gene. Both green fluorescence protein (GFP)-fused chPit-1 gamma and GFP-fused chPit-1 alpha were localized in the nuclei of COS-7 cells. In a luciferase reporter gene assay, both chPit-1 alpha and chPit-1 gamma transactivated the chGH promoter, and chPit-1 alpha showed a more potent effect than chPit-1 gamma. On the other hand, an increase of cellular cAMP induced by forskolin promoted transactivation of the chGH gene with chPit-1 alpha and chPit-1 gamma to similar extents. These results suggest that chPit-1 gamma may modulate the basal promoter activity of the chGH gene to the same degree as chPit-1 alpha; however, a structural difference observed at the N-terminus transactivation domains in chPit-1 alpha and chPit-1 gamma could be associated with the efficiency of basal activation of the chGH promoter. (C) 2011 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE
General and Comparative Endocrinology, 01 Sep. 2011, [Reviewed]

Central administration of metastin increases food intake through opioid neurons in chicks.
Kahn; M.S.I; Ohkubo; T.; Masuda; N.; Tachibana; T and Ueda; H., Metastin, an RFamide peptide, has been isolated from human placenta and possesses several physiological actions in mammals. However, little is known about this bioactive peptide in avian species. This study was conducted to assess the effect of metastin on feeding behavior of chicks (Gallus gallus). The food intake of chicks is significantly increased by the intracerebroventricular injection of metastin. Beta-funaltrexamine, a mu-opioid receptor antagonist, significantly attenuates metastin-induced food intake in chicks. In contrast, delta- and kappa-opioid receptor antagonists did not show any influence on metastin-induced food intake in chicks. In addition, administration of W-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence metastin-induced food intake. Taken together, this study shows the orexigenic effect of metastin in chicks and suggests that this effect is mediated by mu-opioid receptor. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved., ELSEVIER SCIENCE INC
Comparative Biochemistry and Physiology, Part A: Molecular & Integrative Physiology, 2009, [Reviewed]

黒毛和種における成長ホルモン受容体多型と枝肉形質の遺伝的な能力の比較
高橋和裕; 木村 信熙; 田中実; 大久保武, Corresponding, In the present study, we have compared LINE-1 polymorphism in the growth hormone receptor gene and carcass traits in 348 heads of Japanese Black cattles fatten in Kagawa Prefecture. Gene frequency of GHR gene with LINE-1 (L) or without LINE-1 (S) was 0.529 and 0.479, respectively. The estimated breeding values for longissimus muscle area and rib thick ness were greater in LL population than in LS and SS populations, whereas beef marbling score was not significantly different among the populations. The data in the present study suggested that the LINE-1 polymorphism might associate with body weight gain and/or muscle growth, and introducing L type of GHR might be advantage to increases carcass weight in Japanese Black cattle., Japanese Society of Animal Science
日本畜産学会報, 2009, [Reviewed]

Chicken leptin receptor is functional in activating JAK-STAT pathway in vitro.
Adachi; H.; Takemoto; Y.; Bungo; T. and Ohkubo; T., Corresponding, Leptin is a cytokine-like hormone that regulates food intake and energy homeostasis via its interaction with the leptin receptor (LEPR) located in the target tissues. Leptin-dependent signal transduction pathways have been well characterised in mammals but less is known about them in other vertebrates. In birds, although the existence of the LEM has been confirmed, the identity of the natural ligand for the LEM is controversial and the signalling cascade is not fully understood either. Here, we describe the in vitro expression of chicken LEPR (chLEPR), which can mediate the leptin signal. Murine leptin specifically bound with the chLEPR, which initiated the activation of luciferase in chLEPR-expressing cells. Leptin stimulation led to phosphorylation of signal transducers and activators of transcription 3 (STAT3) via chLEPR, and Janus kinase(-2) (JAK(-2)) inhibitor partially blocked leptin-induced luciferase activation in CHO-K1 cells stably expressing chLEPR(CHO-cliLEPR). RNA interference for chLEPR reduced the induction rate of luciferase activity by leptin in CHO-chLEPR cells. Furthermore, we found that leptin phosphorylated STAT3 and increased luciferase activity in LMH cells, a chicken hepatoma cell line, transiently expressing chLEPR. These results strongly Suggest that the chLEPR is functional in activating the JAK-STAT pathway, which may indicate that the LEPR expressed in chicken tissues is capable of binding endogenous ligand as well as exogenous mammalian leptin, leading to physiological actions., BIOSCIENTIFICA LTD
Journal of Endocrinology, 2008, [Reviewed]

Molecular cloning of Pit-1 cDNA and genomic DNA of the domestic duck (Anas platyrhynchos).
Kansaku; N.; Ohkubo; T.; Guémené; D.; Kuhnlein; U.; and Zadworny; D., The expression of unique isoform of pituitary transcription factor (Pit-1) is reported in the chicken and turkey. However, it is not well known whether this isoform is expressed in the other avian species. In the present study, complementary DNA (cDNA) and genomic DNA, of Pit-1 of domesticated duck, were cloned and sequenced. Duck Pit-1 alpha was found to have 93.3, and 92.4% sequence identity at the cDNA level to Pit-1 alpha of chicken and turkey, respectively. The predicted amino acid sequence had 96.7% similarity with a comparable region of Pit-1 alpha of both chicken and turkey. Based on the cDNA sequence, the genomic structure of the gene was characterized. The duck Pit-1 gene consisted of seven exons and six introns. Sequence analysis of the duck Pit-1 gene allowed for the design of a forward primer for amplification of Pit-1 gamma, which is synthesized from an alternative transcription initiation site in galliforms. Reverse transcription of total RNA followed by polymerase chain reaction successively amplified a 984 bp product of Pit-1 gamma cDNA, which encoded a peptide of 326 amino acids. These results suggest that the expression of Pit-1 isoform originates from a different site of transcription initiation, which is not unique to galliforms but is observed in other avian species including anseriforms., BLACKWELL PUBLISHING
Animal Science Journal, 2007, [Reviewed]

Existence of leptin receptor protein in chicken tissues: isolation of a monoclonal antibody against chicken leptin receptor.
Ohkubo; T.; Nishio; M.; Tsurudome; M.; Ito; M. and Ito; Y.:, Lead, Leptin receptor belongs to the class I cytokine receptor superfamily, which mediates multiple physiological roles in mammals. However, the leptin system is poorly understood in birds, as the evidence for the existence of a natural ligand of the receptor in birds is controversial. As part of a strategy to reveal the physiological significance of leptin in birds, we isolated a monoclonal antibody (mAb) against a chicken leptin receptor (chLEPR). Based on the cDNA sequence for chLEPR, a peptide coding for the cytoplasmic domain of chLEPR was expressed in Escherichia coli and this was used to immunize mice to obtain the mAb. The anti-chLEPR mAb recognized proteins migrated at approximately 180 kDa by Western blot analysis using cellular extracts prepared from COS-7 cells transfected with chLEPR expression vector. By Western blot analysis using the same mAb, an immunoreactive band migrated at 180 kDa was detected in the chicken brain and Leghorn male hepatoma (LMH) cells, and which was similar to the size observed in the in vitro transfection study. Taken together, the chLEPR mAb obtained in the present study cross-reacted, at least, with long isoform chLEPR, suggesting that LEPR mRNA expressed in chicken tissues is likely to be translated. The chLEPR mAb, which has not been described elsewhere, enables us to explore the expression and localization of the receptor in the chicken tissues at the protein level. Therefore, this antibody would be a powerful tool in studying and understanding the regulation and function of leptin and its receptors in birds. (c) 2007 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE
General and Comparative Endocrinology, 2007, [Reviewed]

Structure and tissue distribution of cholecystokinin-1 receptor in chicken.
Ohkubo; T.; Shamoto; K. and Ogino; T.:, Lead, Chicken cholecystokinin-1 receptor (chCCK1R) cDNA was isolated from a chicken brain and tissue distribution of the chCCK1R mRNA was determined by RT-PCR. The chCCK1R is composed of 429 amino acid residues and showed approximately 73% and 50% identity with those of mammalian CCK1R and CCK2 receptor (CCK2R) at the amino acid level, respectively. In phylogenetical analysis, the chCCK1R belonged to a cluster of CCK1R and CCK-CHR, another CCK receptor cloned from chicken, was placed into a cluster of CCK2R. By RT-PCR analysis, whilst both chCCK1R and CCK-CHR mRNAs were widely expressed in the chicken tissues, the expression pattern for each receptor was slightly different. Abundant chCCK1R mRNA was observed in multiple tissues, while CCK-CHR mRNA expression was dominantly observed in the brain and proventriculus in the chicken. Taken together, chCCK1R is a potent peripheral CCKR and CCK-CHR cloned and characterized previously is classified as CCK2R in the chicken. Furthermore, mRNA expression for chicken CCKRs is possible to be regulated by tissue specific manner and that may be associated with the diverse roles of CCK and gastrin in the chicken.
Journal of Poultry Science, 2007, [Reviewed]

Bos indicus type of growth hormone receptor gene is retained in Japanese Black cattle.
Ohkubo; T.; Yano; H.; Takahashi; S.; Takahashi; K.; Kimura; N. and Tanaka; M:, Lead, The growth hormone receptor (GHR) gene is responsible for growth and carcass traits, and polymorphisms associated with the variation of meat production are thought to occur in the liver-specific promoter of the GHR gene in cattle. The aim of this study was to analyse the structure of the liver-specific promoter of GHR in Japanese Black cattle, as the relationship between GHR polymorphism and meat production is poorly understood in this breed. Typically in European cattle, the LINE-1 element, a family of retrotransposons, is inserted in the liver-specific promoter. However, a short GHR promoter without the LINE-1 sequence was found in the Japanese Black breed as in Bos indicus cattle. The frequency of the short allele was approximately 60%. In addition, 24 of 29 Holstein/Japanese Black crosses carried the short allele from their sire. The present result suggests that the short allele for GHR may be a candidate marker for improving meat production of Japanese Black cattle., BLACKWELL PUBLISHING
Journal of Animal Breeding and Genetics, 2006, [Reviewed]

Cloning of duck PRL cDNA and genomic DNA.
Kansaku; N.; Ohkubo; T.; Okabayashi; H.; Guémené; D.; Kuhnlein; U.; Zadworny; D. and Shimada; K., Complementary DNA (cDNA) and genomic DNA, including flanking regions of the prolactin (PRL) gene of domesticated duck, were cloned and sequenced. Duck PRL was found to have 92.0, 91.7. and 91.4% sequence identity at the cDNA level to PRL of chicken, turkey, and quail, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (93.4%), turkey (91.3%), and quail (91.3%) PRL. Mature duck PRL contains the consensus sequence for N-linked glycoslylation at position 6 which is not present in either chickens or turkeys. Thus, duck PRL is likely to be post-translationally modified differently from other avian species. Based on the cDNA sequence, the genomic structure of the gene was characterized. The duck PRL gene consists of 5 exons and 4 introns. Moreover, sequence analysis of the proximal region of duck PRL promoter revealed a high degree of similarity to that of chicken and turkey PRL promoter. These results suggest that the mechanisms, which regulate expression of the PRL gene, may be widely conserved in avian species. (C) 2004 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE
General and Comparative Endocrinology, 2005, [Reviewed]

Comparison of mammalian prolactin-releasing peptide and Carassius RFamide for feeding behavior and prolactin secretion in chicks.
Tachibana; T.; Tsukada; A.; Fujimoto; M.; Takahashi; H.; Ohkubo; T.; Boswell; T. and Furuse; M., Prolactin-releasing peptide (PrRP) was named for its originally reported effects as a prolactin (PRL) secretagogue in mammals. Carassius RFamide (C-RFa) is an orthologous PRL secretagogue in fishes and a gene encoding a 20-amino acid peptide of identical sequence is present in the chicken. These facts suggest that C-RFa is a putative chicken PrRP. However, no information is available for the physiological effects of C-RFa in chickens. Therefore, in the present study, we compared the effect of intracerebroventricular (ICV) injection of C-RFa and mammalian PrRP (mPrRP) on feeding behavior and plasma PRL, growth hormone (GH), and corticosterone (CORT) concentrations. ICV injection of C-RFa did not affect feeding behavior of chicks while mPrRP was stimulatory. The injection of C-RFa also did not significantly affect plasma PRL, GH, and CORT concentrations. In contrast, ICV injection of mPrRP exerted similar effects to those reported in mammals by increasing plasma CORT and decreasing GH concentrations. Additionally, the peptide induced an unexpected inhibitory effect on plasma PRL concentrations. Overall, these data suggest that an as yet unidentified peptide that shares some functional similarities with mPrRP is present in birds, but that the physiological role of the avian 20-amino acid C-RFa peptide remains to be determined. (c) 2005 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE
General and Comparative Endocrinology, 2005, [Reviewed]

Characterization of structure and expression of the growth hormone receptor gene of the Japanese flounder (Paralichtys olivaceus).
Nakao; N.; Higashimoto; Y.; Ohkubo; T.; Yoshizato; H.; Nakai; N.; Nakashima; K. and Tanaka; M., Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay., SOC ENDOCRINOLOGY
Journal of Endocrinology, 2004, [Reviewed]

Effect of central administration of prolactin-releasing peptide on feeding in chicks.
Tachibana; T.; Saito; S.; Tomonaga; S.; Takagi; T.; Saito; E.; Nakanishi; T.; Koutoku; T.; Tsukada; A.; Ohkubo; T.; Boswell; T. and Furuse; M., Prolactin-releasing peptide (PrRP) is one of the inhibitory factors in feeding regulation of mammals. However, no information is available for avian species. The present study was done to clarify the effect of intracerebroventricular (ICV) injection of PrRP on feeding in chicks. Firstly, we found that ICV injection of PrRP (94-1500 pmol) significantly increased food intake in chicks. The result was completely different from those obtained in mammals. The orexigenic effect of PrRP was significantly weaker than that of neuropeptide Y (NPY), a potent orexigenic peptide, on an equimolar basis. The orexigenic effect of NPY was further enhanced with coinjection of PrRP. These results suggest the existence of a novel orexigenic mechanism in the chick brain, which might differ from NPY-involved feeding regulatory pathway. In addition, ICV injection of PrRP significantly decreased the rectal temperature, but the effect was weaker than that of NPY, suggesting that PrRP may inhibit energy expenditure in chicks. Taken together, we showed here that PrRP may be involved in the regulation of both feeding behavior and energy metabolism in the chick brain. (C) 2004 Elsevier Inc. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Physiology & Behavior, 2004, [Reviewed]

cDNA cloning of chicken orexin receptor and tissue distribution: sexually dimorphic expression in chicken gonads.
Ohkubo; T.; Tsukada; A. and Shamoto; K., Lead, Orexin-A and -B are known to stimulate food intake in mammals. However, the critical roles of orexins in birds are not fully understood, since orexins have no stimulatory effect on food intake in the chicken. To understand the physiological role(s) of orexins in birds, we have cloned chicken orexin receptor (cOXR) cDNA by RT-PCR, and analysed the tissue distribution of OXR mRNA in the chicken. The cOXR cDNA is 1869 bp long and encodes 501 amino acids. The cloned cDNA for cOXR corresponds to the type 2 OXR in mammals, and shows approximately 80% similarity to those of mammals at the amino acid level. Expression analysis by RNase protection assay revealed OXR mRNA was distributed widely in brain regions, and expression in the cerebrum, hypothalamus and optic tectum were abundant. In peripheral tissues, OXR mRNA was expressed in the pituitary gland, adrenal gland and testis, but no mRNA expression was observed in other tissues examined. Furthermore, we found that the amount of cOXR mRNA was different between testis and ovary, while prepro-orexin mRNA is equally expressed in the gonads of both sexes in the chicken. These data indicate that the orexins have neuroendocrine actions in chickens, which are mediated through hypothalamic receptors as has been observed in mammals. In addition, orexin may have specific role(s) in the regulation of gonadal function in which sex-dependent mechanisms could be involved., SOC ENDOCRINOLOGY
Journal of Molecular Endocrinology, 2003, [Reviewed]

Fasting differentially regulates expression of agouti-related peptide, pro-opiomelanocortin, prepro-orexin, and vasoactive intestinal polypeptide mRNAs in the hypothalamus of Japanese quail.
Phillips-Singh; D.; Li; Q.; Takeuchi; S.; Ohkubo; T.; Sharp; P.J. and Boswell; T., Research in mammals has established the existence of a neuronal network that lies within the hypothalamus and that regulates energy homeostasis. However, it is unknown whether this system has been evolutionarily conserved. The objective of the present study was therefore to examine the influence of the agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), prepro-orexin, and vasoactive intestinal polypeptide (VIP) genes on energy balance in birds by quantifying the effect of a 24-h fast on their expression in the hypothalamus of the Japanese quail. In situ hybridization revealed strong signals for AGRP and POMC mRNAs in the infundibular nucleus (IN), for prepro-orexin in the lateral hypothalamic area (LHy) and periventricular hypothalamic nucleus, and for VIP in the LHy. POMC mRNA was co-localized with alpha-melanocyte-stimulating hormone-like immunoreactivity in individual IN neurons. Compared with the ad-libitum-fed state, a 24-h fast resulted in a 2.2-fold increased expression of AGRP mRNA in the IN. However, fasting did not induce changes in POMC, prepro-orexin, or VIP mRNAs. The results suggest an involvement of the central melanocortin system in the regulation of energy balance in birds, as in mammals. In contrast, orexins in birds may be primarily involved in the control of physiological functions other than energy homeostasis., SPRINGER-VERLAG
Cell and Tissue Research, 2003, [Reviewed]

Up-regulated expression of a novel gene in activated human peripheral blood mononuclear cells that is a truncated paralog of the human system L-amino acid transporter 1.
Ito; M.; Takebayashi; S.-I.; Okumura; K.; Ohkubo; T.; Nishio; M.; Kawano; M.; Komada; H.; Ito; Y. and Tsurudome; M., Introduction and aims: The human system L-amino acid transporter I (hLAT1) is one of the CD98 light chains and its gene has been mapped to chromosome 16q24. Our preliminary findings have indicated that in HeLa S3 cells there are transcripts whose nucleotide sequences are very similar but not identical to that of the amino acid transporter, This study intends to examine whether these novel transcripts have biological significance through elucidating their genetic aspects and expression profiles in human cells. Methods: The expression levels of the transcripts were quantified by real-time PCR analysis, Chromosomal mapping of the gene was performed by fluorescence in situ hybridization (FISH), Results: Three types of transcripts were identified and their nucleotide sequences were aligned with the chromosome 16p12 clone with high identity, They encoded 180- or 190-amino acid proteins, showing 92-94% of amino acid identity to the amino-terminal region of the hLAT1 (507 amino acids). However, their 3' non-coding sequences did not show homology to the nucleotide sequence of the amino acid transporter. Their genes were mapped to chromosome 16p11.2-p13.1 as low-copy repeats (LCRs). The transcription of one of these genes in peripheral blood mononuclear cells was significantly up-regulated when the cells were stimulated with concanavalin A. Conclusion: We have characterized the three truncated paralogs of the hLAT1 gene. It is suggested that the expression of one of these paralogs may play an important role in the activation of peripheral blood mononuclear cells. (C) 2002 Elsevier Science Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
International Journal of Biochemistry and Cell Biology, 2002, [Reviewed]

Tryptophans 286 and 288 in the C-terminal region of protein B23.1 are important for its nucleolar localization.
Nishimura; Y.; Ohkubo; T.; Furuichi; Y. and Umekawa; H., Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein 1323, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology and Biochemistry, 2002, [Reviewed]

Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus.
Ohkubo; T.; Boswell; T. and Lumineau; S., Lead, Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. The predicted amino acid sequence of chicken prepro-orexin cDNA revealed that orexin-A and -B are highly conserved among vertebrate species. In situ hybridization and immunohistochemistry localized orexin-positive cell bodies in the periventricular hypothalamic nucleus extending into the lateral hypothalamic area. Comparisons of orexin gene expression in the brains of 24-h-fasted and ad libitum-fed chickens were made using semi-quantitative RT-PCR. No significant differences in orexin mRNA expression were observed. (C) 2002 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV
Biochimica et Biophysica Acta, 2002, [Reviewed]

Structure and tissue distribution of prolactin receptor mRNA in Japanese flounder (Paralichtys olivaceus): conserved and preferential expression in osmoregulatory organs.
Higashimoto; Y.; Nakao; N.; Ohkubo; T.; Tanaka; M. and Nakashima; K.:, In diadromous and euryhaline teleosts, it has been established that prolactin (PRL) is a major hormone regulating the maintenance of water and electrolyte homeostasis by acting on its receptor (PRLR) expressed in the osmoregulatory organs. To investigate the major physiological role of PRL in a marine teleost, cDNA for the Japanese flounder (Paralichtys olivaceus) prolactin receptor (fPRLR) has been cloned and characterized. The predicted fPRLR is composed of 636 amino acids conserving common structural features, such as the WSXWS motif and box 1, that are observed in the members of the cytokine receptor superfamily. By Northern blot analysis, 3.5-kb transcripts for fPRLR were clearly detected in the gill, kidney, and intestine. By RNase protection assay, similarly high levels of mRNA expression were detected in these osmoregulatory organs and lower expression levels were seen in the brain for both males and females. Interestingly, a distinct expression level of fPRLR mRNA was observed in the testis, but not in the ovary. The present results suggest that PRL may play an important role in the control of water and electrolyte balance through PRLR expressed in the osmoregulatory organs in the marine teleost the Japanese flounder as well as in other teleosts. Furthermore, PRL may differentially regulate gonadal functions in males and females of Japanese flounder. (C) 2001 Academic Press., ACADEMIC PRESS INC
General and Comparative Endocrinology, 2001, [Reviewed]

Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization.
Kansaku; N.; Shimada; K.; Ohkubo; T.; Saito; N.; Suzuki; T.; Matsuda; Y. and Zadworny; D., Chicken vasoactive intestinal polypeptide receptor (VIPR) cDNA was cloned by the reverse transcription-polymerase chain reaction method using primers designed on the basis of other species of VIPR cDNA. The cDNA obtained was sequenced by the dideoxy-mediated chain-termination method, Of the 2227 nucleotides that were sequenced, 84, 855, and 1338 bases represent the 5 ' -untranslated region (UTR), the 3 ' -UTR, and the open reading frame that predicts a peptide of 446 amino acids. The cDNA of the chicken VIPR shows 65% and 60% homologies to human cDNA of VIP1 and VIP2 receptors, respectively. The clone had the expected similarity to highly conserved features of the other G protein-coupled receptors (GPCRs) such as six cysteine residues that are functionally important in the VIPR subfamily. In addition, the seven potential membrane-spanning domains characteristic of the family B group III GPCR superfamily and highly conserved motif within the third cellular loop between transmembrane regions 5 and 6. Northern blot hybridization analysis in this study indicated mRNA expression of VIPRs in the various tissues of the chicken. Strong signal was detected in the brain and anterior pituitary gland. High levels of VIPR mRNA in the brain was consistent with VIP-binding experiments and with the function of VIP in the brain as a neuroendocrine factor or neurotransmitter. Expression of VIPR was detected in the anterior pituitary gland of chick embryos, The expression of VIPR mRNA in the chick anterior pituitary gland may indicate a regulatory function of VIP on prolactin (PRL) production or PRL cell proliferation during embryogenesis. Chicken VIPR shows high homology with mammalian type I VIPR but, in some part, possesses similarity of amino acid sequence. Expression of VIPR in various tissues supports diverse functions for VIP in the chicken., SOC STUDY REPRODUCTION
Biology of Reproduction, 2001, [Reviewed]

Structure and tissue distribution of chicken leptin receptor (cOb-R) mRNA.
Ohkubo; T.; Tanaka; M. and Nakashima; K., Lead, Chicken leptin receptor (cOb-R) cDNA has been cloned, sequenced and characterized. The predicted cOb-R preprotein was composed of 1148 amino acids showing approximately 60% sequence identity with the long isoform of mammalian leptin receptor, and contained a putative signal peptide, a single transmembrane domain and the conserved box 1, 2 and 3 motifs in the cytoplasmic region. High levels of cOb-R mRNA expression were observed in ovary and brain, and less abundant expression of the mRNA was detected in liver, kidney and intestine in juvenile females and sexually matured hens. The expression levels of cOb-R mRNA did not change during sexual maturation in most tissues, but the mRNA level in the intestine was higher in matured hens than in juveniles. Estrogen treatment was found to enhance the Ob-R mRNA expression in the intestine, but not in other tissues. (C) 2000 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV
Biochimica et Biophysica Acta, 2000, [Reviewed]

Molecular cloning of the chicken prolactin gene and activation by Pit-1 and cAMP-induced factor in GH3 cells.
Ohkubo; T.; Tanaka; M. and Nakashima; K., Lead, Transcription of the prolactin (PRL) gene has been reported to be activated by a nuclear factor, Pit-1. However, the precise molecular mechanisms of the Pit-1-mediated PRL gene activation are still unclear. We have cloned the chicken PRL (cPRL) gene and its 5'-flanking region to analyze their structure and transcription-initiating mechanism. In luciferase assay, forskolin activated the proximal promoter region between -248 and -76 to transcribe the cPRL gene in GH3 cells, although there is no canonical cyclic AMP-responsive element in the promoter region. In gel mobility shift assay, a DNA fragment between - 104 and -76 containing a putative Pit-1 binding site was bound by nuclear factors from the GH3 cells. Furthermore, it was observed that Pit-i protein specifically bound to the DNA fragment in the supershift assay. These results indicate that both Pit-1 and cAMP-induced factor(s) associated with the cis element on the proximal promoter region to activate cPRL gene expression in GH3 cells, (C) 2000 Academic Press., ACADEMIC PRESS INC
General and Comparative Endocrinology, 2000, [Reviewed]

Two novel first exons in the prolactin receptor gene are transcribed in a tissue-specific and sexual maturation-dependent manner to encode multiple 5’-truncated transcripts in the testis of the chicken.
Tanaka; M.; Yamamoto; I.; Hayashida; Y.; Nakao; N.; Ohkubo; T.; Wakita; M. and Nakashima; K., Cloning and sequencing of the chicken prolactin receptor (PRLR) gene segment from the transmembrane domain to the box 2 motif revealed the presence of the two testis-specific first exons, TSE-1 and TSE-2, encoding the unique 5'-end sequences of the reported and newly identified multiple 5'-truncated PRLR transcripts containing only the cytoplasmic domain in the testis. TSE-1 was located downstream of the exon encoding the transmembrane domain and TSE-2 presented downstream of the exon encoding the box 1 motif. These findings indicate that the box 1-containing 5'-truncated transcripts are generated by the utilization of TSE-1 as the first exon with distinct splicing donor sites to the box 1-containing exon, and that the utilization of TSE-2 as the first exon and its splicing to the box 2-containing exon results in the generation of the box 1-lacking transcript. Three transcription initiation sites for the box 1-containing 5'-truncated transcripts and two transcription initiation sites for the box 1-lacking transcript were detected by the RNase protection assays. Reverse transcription-polymerase chain reaction analysis showed that the expression levels of all these 5'-truncated PRLR transcripts are simultaneously increased during sexual maturation, accompanying the decrease of the amount of the canonical full-length transcript for PRLR. (C) 2000 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV
Biochimica et Biophysica Acta, 2000, [Reviewed]

Effects of thyroid hormone on serum insulin-like growth factor binding proteins (IGFBPs) and IGFBP-2 mRNA expression in chickens.
Tsukada; A.; Ohkubo; T.; Tanaka; M.; Nakashima; K.; Wakita; M.; and Hoshino; S.
Japanese Poultry Science, 1999, [Reviewed]

cDNA cloning and developmental alterations in gene expression of the two Pit-1/GHF-1 transcription factors in the chicken pituitary.
Tanaka; M.; Yamamoto; I.; Ohkubo; T.; Wakita; M.; Hoshino; S. and Nakashima; K.:, Pit-1/GHF-1 (Pit-1) transcription factors promote the gene expressions for growth hormone (GH), prolactin (PRL), and the beta chain of thyroid-stimulating hormone in vertebrate pituitary glands. The present study analyzed the nature of chicken Pit-1s (cPit-1s) and their developmental expressions in the pituitary. Chicken pituitary expressed two cPit-1 mRNAs encoding cPit-1 alpha and cPit-1 gamma composed of 335 and 327 amino acid residues, respectively They possessed different N-terminal regions and the common C-terminal regions containing a POU-specific domain and a POU homeodomain. Northern blot analysis revealed the pituitary-specific expressions of these Pit-1 mRNAs, and the Pit-1 alpha mRNA expressions were two to three times higher than those for Pit-1 gamma in both cephalic and caudal lobes of the pituitary. The cPit-1 alpha and gamma mRNA expressions simultaneously increased after hatching until 4 weeks and then slightly decreased at 5 weeks. Similar gene expression profiles were observed for GH and PRL during the posthatch developmental period. (C) 1999 Academic Press., ACADEMIC PRESS INC
General and Comparative Endocrinology, 1999, [Reviewed]

Genetic mapping of the chicken prolactin receptor gene: a candidate gene for the control of broodiness.
Dunn; I.C.; McEwan; G.; Okhubo; T.; Sharp; P.J.; Paton; I.R. and Burt; D.W., CARFAX PUBLISHING
British Poultry Science, 1998, [Reviewed]

Prolactin receptor gene expression in the brain and peripheral tissues in broody and nonbroody breeds of domestic hen.
Ohkubo; T.; Tanaka; M.; Nakashima; K.; Talbot; R.T. and Sharp; P.J., Lead, The objective of this study was to establish whether the gene encoding prolactin receptor (PRLR) is expressed in the hypothalamus and peripheral tissues of the domestic chicken and, if so, to determine whether there are breed differences in the structure or expression of the gene which might account for the observation that broodiness does not occur in the White Leghorn hen but does occur in other breeds of domestic hens, including the bantam. A preliminary experiment demonstrated that the absence of broodiness in White Leghorns is not due to a lack of a prolactin response to the avian prolactin-releasing hormone vasoactive intestinal polypeptide. The largest amounts of PRLR mRNA in the brain, which did not differ significantly between laying White Leghorns and bantams, were found in the pituitary gland and basal and preoptic hypothalamus. Small or nondetectable amounts were found in both breeds in the forebrain, cerebellum, and optic lobes. Prolactin receptor mRNA was widely distributed in peripheral tissues in both breeds, in the following descending order of abundance: kidney, leg skin, brood patch, duodenum, intestine > thyroid gland > adrenal gland, liver, ovary much greater than adipose tissue > thymus, spleen > muscle > blood. Southern blotting analysis using four restriction enzymes and a chicken PRLR cDNA probe demonstrated identical digestion patterns for White Leghorn and bantam genomic DNA. Northern blotting analysis identified two sizes of chicken PRLR mRNA transcripts (7.5 and 3.3 kb) in hypothalami from laying White Leghorn and bantam hens. It is concluded that differences in the expression of broodiness in White Leghorn and bantam hens cannot be explained by differences in the amounts of PRLR mRNA in the hypothalamus or in the transcription or gross structure of the PRLR gene. (C) 1998 Academic Press., ACADEMIC PRESS INC
General and Comparative Endocrinology, 1998, [Reviewed]

Relationship between prolactin receptor mRNA in the anterior pituitary gland and hypothalamus and reproductive state in male and female bantams (Gallus domesticus).
Ohkubo; T.; Tanaka; M.; Nakashima; K. and Sharp; P.J.:, Lead, The aim of this study was to test the hypothesis that prolactin may up- and down-regulate prolactin receptor gene expression in the anterior pituitary gland and hypothalamus respectively. Experiments were carried out in bantams (Gallus domesticus). Comparisons were made of concentrations of PRLR mRNA in the anterior pituitary gland and basal and preoptic hypothalamus in adult males and females held on long days (low vs high plasma prolactin); in 3-week-old juvenile male and females on short days (high vs low plasma prolactin); in 8-week-old juvenile male and females on short days (both low plasma prolactin); in adult laying, incubating, and out-of-lay (high, very high, and low plasma prolactin, respectively); in adult cockerels exposed to long or short days (high vs low prolactin); and in adult hens exposed to long or short days (high vs low prolactin). There was a sex difference in anterior pituitary and basal hypothalamic PRLR mRNA, with lower values in both tissues in females than in males. Compared with laying and out-of-lay hens, anterior pituitary and basal hypothalamic PRLR mRNA concentrations in incubating hens were increased and decreased, respectively. In adult birds of either sex held on long or short days, there was no difference in pituitary PRLR mRNA, while basal hypothalamic PRLR mRNA was lower on short days. PRLR mRNA in the preoptic hypothalamus was not affected by sex, reproductive state, or photoperiod. It is concluded that there is no consistent relationship between plasma prolactin, in the physiological range, and the concentration of PRLR mRNA in the anterior pituitary gland, basal hypothalamus, and preoptic hypothalamus. (C) 1998 Academic Press., ACADEMIC PRESS INC
General and Comparative Endocrinology, 1998, [Reviewed]

Cloning and expression of pigeon growth hormone receptor cDNA in COS-7 monkey kidney cells.
Ohkubo T.; Tsukada A.; Tanaka M. and Nakashima K.:, Lead, Pigeon growth hormone receptor (pGHR) cDNA has been cloned, sequenced, characterized and expressed in COS-7 cells. The predicted pGHR preprotein was composed of 611 amino acids and contained a putative signal peptide, a single transmembrane region and the conserved proline-rich box 1 domain in the cytoplasmic region. A canonical polyadenylation signal was found in the extracellular region, as was observed in chicken growth hormone receptor cDNA. Northern blot analysis identified a single species of pGHR mRNA and its expression was widely observed in tissues in the young male pigeon. A pGHR expression vector was constructed and transfected into COS-7 cells. The transfected cells were specifically bound by I-125-labeled chicken GH and the binding was suppressed by the addition of excess amounts of unlabeled chicken GH. (C) 1998 Elsevier Science Inc. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Comparative Biochemistry and Physiology, 1998, [Reviewed]

Tissue-specific regulation of growth hormone receptor and growth hormone binding protein gene expression during pregnancy and lactation in the rat.
Sakaguchi; K.; Tanaka; M.; Ohkubo; T.; Yoshizato; H.; Hanai; Y.; Fujikawa; T.; Kaneko; H. and Nakashima; K.:, JAPAN ENDOCRINE SOCIETY
Endocrine Journal, 1998, [Reviewed]

cDNA cloning and characterization of neuron-specific enolase from chicken.
Tanaka; M.; Taniguchi; T; Ohkubo; T. and Nakashima; K., Chicken gamma-enolase cDNA was cloned and its sequence and tissue-specific expression were analyzed. The cDNA, consisting of 2273 bp of nucleotides, was composed of 86 bp of the 5'-noncoding region, 1305 bp of an open reading frame encoding a protein of 434 amino acids and 882 bp of the 3'-noncoding region. The deduced amino acid sequence showed higher homology (more than 90%) to those of mammalian gamma-enolases than to those of chicken alpha- or beta-enolases. Northern blot analysis has revealed that the mRNA for gamma-enolase (2.3 kb) is expressed in the brain and, to much less but significant extents, in the pituitary and adrenal gland of the chicken. (C) 1998 Elsevier Science B.V., ELSEVIER SCIENCE BV
Biochimica et Biophysica Acta, 1998, [Reviewed]

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): Multiple mRNA species encode a single short mature IGF-I.
Tanaka; M.; Taniguchi; T.; Yamamoto; I.; Sakaguchi; K; Yoshizato; H.; Ohkubo; T. and Nakashima; K., To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus, The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb, By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486, In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724, The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression., MARY ANN LIEBERT INC PUBL
DNA and Cell Biology, 1998, [Reviewed]

Thyroid hormones are involved in insulin-like growth factor-I (IGF-I) production by stimulating hepatic growth hormone receptor (GHR) gene expression in the chicken.
Tsukada; A.; Ohkubo; T.; Sakaguchi; K.; Tanaka; M.; Nakashima; K.; Hayashida; Y.; Wakita; M. and Hoshino; S., Effect of thyroid status on IGF-I production in growing chickens was studied. Serum concentrations of GH were not affected by propylthiouracil (PTU) or thyroxine (T-4) treatments, whereas serum IGF-I levels were significantly decreased in PTU-treated chickens. The lowered serum IGF-I levels in the PTU-treated group were completely restored to the control levels by T-4 injections. In the liver, the messenger RNA (mRNA) expressions both for GH receptor (GHR) and IGF-I were significantly repressed by PTU treatment, and were restored again by T-4 replacement. In addition, the results of analysis on radiolabelled GH binding to the liver membrane were consistent with the levels of hepatic GHR mRNA expression. Serum concentrations of IGF-I were positively correlated with hepatic IGF-I mRNA and GHR mRNA expressions. The correlation coefficient between serum T3 levels and hepatic IGF-I mRNA expressions was also significant. These results indicate that thyroid hormones regulate IGF-I production in the chicken by affecting hepatic GHR expression. (C) 1998 Churchill Livingstone., CHURCHILL LIVINGSTONE
Growth Hormone and IGF Research, 1998, [Reviewed]

Structures of mollusc shell framework proteins.
Sudo; S.; Fujikawa; T.; Nagakura; T.; Ohkubo; T.; Sakaguchi; K.; Tanaka; M.; Nakashima; K. and Takahashi; T.:, Nature Publishing Group
Nature, 1997, [Reviewed]

Influence of recombinant chicken prolactin on thyroid hormone metabolism in the chick embryo.
6. Kühn; E.R.; Shimada; K.; Ohkubo; T.; Vleurick; L.M.; Berghman; L.R. and Darras; V.M., The influence of a recombinant chicken prolactin (rcPRL) preparation on thyroid function was studied in 18- and 19-day-old chicken embryos. Displacement studies on hepatic microsomes indicate that this preparation does not compete with radiolabeled chicken growth hormone (cGH) for hepatic GH-receptor binding. In a first series of experiments rcPRL or immunoaffinity-purified cGH was injected intravenously in 19-day-old chicken embryos. After 2 hr, cGH increased plasma T3 in a dose-dependent way by inhibiting hepatic inner ring type III deiodination (IRD-III) and consequently T3 degradation. Outer ring deiodination (ORD-I) was not influenced confirming previous results. The rcPRL preparation (2 and 10 mu g) did not influence plasma T3, but depressed T-4 and raised hepatic IRD-III activity simultaneously, whereas no influence on hepatic ORD-I activity could be found. In a second experiment on 18-day-old embryos, it could be demonstrated that the effect of 2.5 mu g cGH on plasma T-3 and liver IRD-III lasted up to 6 hr after injection, whereas 2.5 mu g cPRL affected plasma T-4 and Liver IRD-III up to 2 hr. Both rcPRL and cGH depressed rT(3) up to 6 hr, whereas an injection of rcPRL, but not of cGH, elevated plasma concentrations of corticosterone. These results indicate that prolactin may have a role, together with GH, in controlling peripheral thyroid hormone metabolism. (C) 1996 Academic Press, Inc., ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS
General and Comparative Endocrinology, 1996, [Reviewed]

Molecular cloning and characterization of the yellowtail GH gene and its promoter: a consensus sequence for teleost and avian Pit-1/GHF-1 binding sites.
Ohkubo; T.; Araki; M.; Tanaka; M.; Sudo; S. and Nakashima; K., Lead, The gene and 5' flanking promoter region for yellowtail (Seriola quinqueradiata) GH (yGH) have been cloned, sequenced and characterized. The yGH gene spans approximately 4.6 kb and consists of six exons and five introns, as has been observed with rainbow trout, Atlantic salmon and tilapia GH genes. This result suggests that the structure of six exons and five introns is a dominant form in fish GH genes. A typical TATA box exists 26 bp upstream from the transcription start site, and Pit-1/GHF-1 (Pit-1) binding site-homologous regions were found in the promoter region of the yGH gene. In a gel shift assay, however, a single shifted band was detected with the fragments containing a region from - 128 to - 90 of the yGH 5' flanking region when they were incubated with yellowtail pituitary nuclear extracts. The bound fragments contained an octamer base sequence similar, but not identical, to mammalian consensus Pit-1 binding element. A consensus octamer sequence is also proposed in this report for the binding of teleost and avian Pit-1 transcription factors., J ENDOCRINOLOGY LTD
Journal of Molecular Endocrinology, 1996, [Reviewed]

Induction of brain prolactin receptor long-form mRNA expression and maternal behavior in pup-contacted male rats: promotion by prolactin administration and suppression by female contact.
Sakaguchi; K.; Tanaka; M.; Ohkubo; T.; Doh-ura; K.; Fujikawa; T.; Sudo; S. and Nakashima; K., Prolactin (PRL) is considered to induce maternal behavior toward foster young in female rats. In the present study, we studied the relationship between pup contact-induced maternal behavior and serum PRL concentrations and brain PRL receptor (PRL-R) mRNA expression in male rats. Both intact and castrated male rats exposed to foster pups gradually developed caretaking behavior such as crouching and licking, but their exhibitions of other maternal behavior components, retrieval/grouping and nest building, were incomplete. However, in the male rats displaying crouching and licking, the concomitant increases in serum PRL concentration and brain mRNA expression for long-form PRL-R were observed. The expression of short-form PRL-R mRNA in the brain was not stimulated by pup contact. Administration of PRL, remarkably promoted the onset of those maternal responses in male rats. On the other hand, when an intact male rat was housed in a cage where a lactating female rat and her pups were living, his scores in maternal behavior tests toward pups were lowered. And, concomitantly, increases in serum PRL concentration and brain expression of long-form PRL-R mRNA were reduced. In castrated male rats, however, the ratings of maternal behavior toward foster young, serum PRL, concentration increase, or brain long-form PRL-R mRNA expression were not reduced at all by cohabitation with a female and her pups. These findings indicated that maternal behavior was triggered and maintained in pup-contacted male rats through elevated serum PRL levels and induced brain long-form PRL-R., KARGER
Neuroendocrinology, 1996, [Reviewed]

Growth hormone-independent expression of insulin-like growth factor I messenger ribonucleic acid in extrahepatic tissues of the chicken.
Tanaka; M.; Hayashida; Y.; Sakaguchi; K.; Ohkubo; T.; Wakita; M.; Hoshino; S. and Nakashima; K.:, The sex-linked dwarf (SLD) chicken, which lacks GH receptor (GHR), and its normal littermates provide a useful experimental system to investigate OH-dependent cellular responses. The GH dependence of insulin-like growth factor I (IGF-I)expression in tissues was examined in SLD and normal chickens of the Gifu 20 strain. Four weeks after hatching, the most abundant expression of IGF-I messenger RNA (mRNA) was observed in liver of normal chickens, whereas no IGF-I mRNA expression was detected in that organ of dwarf chickens. On the contrary, in extrahepatic tissues such as spleen, lung, brain, kidney, heart, intestine, thymus, and muscle, IGF-I mRNA expression was equally observed in normal and GHR-lacking dwarf chickens. In the testis, expression of IGF-I mRNA was enhanced by about 5-fold in dwarf chickens, showing an expression level comparable to that in normal liver. On day 16 in the embryonic stage, IGF-I mRNA was expressed in muscle, brain, eye, heart, and lung in both normal and SLD chick embryos. However, no IGF-I mRNA expression was observed in liver or kidney of normal and dwarf chick embryos. These results suggest that in chicken, IGF-I mRNA is expressed in liver in a GH-dependent manner after hatching, whereas in other tissues, mRNA expression is independent of GH and GHR before and after hatching, except for testis, in which GH seems to inhibit IGF-I mRNA expression., ENDOCRINE SOC
Endocrinology, 1996, [Reviewed]

Sequence of the flounder (Paralichthys olivaceus) growth hormone encoding gene and its promoter region.
Tanaka; M.; Toma; Y.; Ohkubo; T.; Sudo; S. and Nakashima; K., The flounder (Paralichthys olivaceus) growth hormone (GH)-encoding gene (fGH) and its promoter region were cloned and sequenced following amplification of genomic DNA by the polymerase chain reaction. The fGH gene is 2.1-kb long and consists of six exons and five introns. In the 5'-flanking region of the determined transcription start point, a potential TATA box is located at -24, and Pit-1/GHF-1-binding site candidates are located in the -70 to -53 and -133 to -141 regions., ELSEVIER SCIENCE BV
Gene, 1995, [Reviewed]

ブリ及びヒラメ成長ホルモン遺伝子のクローニング　　　　　　　　　　　　　　　
中島邦夫; 田中実; 大久保武
月刊海洋, 1995, [Reviewed]

Differential regulation of prolactin receptor mRNA expression in rat liver and kidney by testosterone and oestradiol.
Sakaguchi; K.; Ohkubo; T.; Sugiyama; T.; Tanaka; M.; Ushiro; H. and Nakashima; K., Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat Liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen., J ENDOCRINOLOGY LTD
Journal of Endocrinology, 1994, [Reviewed]

High-level expression of biologically active chicken prolactin in E. coli.
Ohkubo; T.; Tanaka; M.; Nakashima; K.; Shimada; K.; Saito; N. and Sato; K., Lead, 1. A large quantity of chicken prolactin (cPRL) was produced by manipulating the cPRL cDNA clone and an expression vector pKK223-3. To augment the production of the hormonal protein in E. coli, in addition to the potent tac promoter, a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron sequence was inserted into adjacent 5'-region of the coding region.
2. In sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, recombinant cPRL protein exhibited a molecular mass of 23 kDa.
3. The recombinant cPRL showed equivalent binding kinetics to an antiserum raised against turkey PRL. Also, this product increased the weight of pigeon crop sac mucosa to a degree comparable to that induced by turkey PRL.
4. These results indicate that this recombinant cPRL has immunological and biological activities identical to those of authentic avian PRL., PERGAMON-ELSEVIER SCIENCE LTD
Comparative Biochemistry and Physiology, 1993, [Reviewed]

Effects of removal of chicks from hens on concentrations of prolactin, luteinizing hormone and oestradiol in plasma of brooding Gifujidori hens
Kuwayama; T.; Shimada; K.; Saito; N.; Ohkubo; T.; Sato; K.; Wada; M. and Ichinoe; K, Gifujidori hens were allowed to repeat a breeding cycle in one season. In the first breeding cycle the duration of the brooding (raising chicks) stage was limited to 3 weeks, whereas in the second breeding cycle it was limited to 1 week by removing all chicks from mother hens. In the first breeding cycle, plasma prolactin (PRL) was high during the incubation period, but rapidly decreased on the day of hatching and reached minimum values about 1 week after hatching. In contrast, plasma luteinizing hormone (LH) concentrations were low during the incubation period, but after hatching they gradually increased and reached peak values immediately after removal of chicks. Concentrations of oestradiol in plasma were low in the incubation and brooding stages but increased significantly immediately after removal of chicks. In the second breeding cycle, changes in PRL and LH concentrations were similar to those observed in the first breeding cycle except that even greater increases in plasma LH and oestradiol concentrations were observed one week after hatching when the chicks were removed. These results suggest that coexistence of newly hatched chicks may suppress LH secretion from the pituitary of the hen in the natural breeding cycle., J REPROD FERTIL INC
Journal of Reproduction and Fertility, 1992, [Reviewed]

Double antenna structure of chicken prolactin receptor deduced from the cDNA sequence.
M TANAKA; K MAEDA; T OKUBO; K NAKASHIMA, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS
Biochemical and Biophysical Research Communications, 1992, [Reviewed]

A Bird's-Eye Overview of Leptin and Female Reproduction -with Mammalian Comparisons.
Sadequllah Ahmadi; Takeshi Ohkubo
Journal of Poultry Science, Feb. 2025, [Reviewed], [Invited]

Recent progress in avian leptin research
Takeshi Ohkubo
Journal pf Poultry Science, 2014, [Reviewed]

Leptin signaling and action in birds.
Ohkubo; T. and Adachi; H.
Journal of Poultry Science, 2008, [Reviewed]
Lead

Cloning and characterization of chicken cholecystokinin receptor.　　　　　　　　　　　　　　　
Ohkubo; T. and Shamoto; K.
Proceedings of the Fifth Congress of the Asia and Oceania Society for Comparative Endocrinology in Conjunction with the Annual Meeting of the Japan Society for Comparative Endocrinology, 2004, [Reviewed]
Lead

Cloning and regulation of chicken cholesterol 7α-hydroxylase.　　　　　　　　　　　　　　　
Yamamoto; Y.; Tsukada; A.; Ohkubo; T.; Umekawa; H.; Takahashi; T and Furuichi; Y.:
Proceedings 6th Asian Pacific Poultry Congress, 1998, [Reviewed]

Neuroendocrine Control of Broodiness　　　　　　　　　　　　　　　
Ohkubo; T, Contributor
Springer, 29 Jun. 2017

ニワトリの科学　　　　　　　　　　　　　　　
Joint work
朝倉書店, 10 Jul. 2014

The molecular biology of prolactin　　　　　　　　　　　　　　　
Shimada; K.; Ohkubo; T.; Saito; N.; Talbot; R.T.; Sharp; P.J, Joint work
1993

Does leptin have a role in asymmetrical development of chicken ovary?　　　　　　　　　　　　　　　
SHAIKAT AMIR; Namekawa Shoko; Ahmadi Sadequllah; Takeda Misa; Ohkubo Takeshi
日本畜産学会第122回大会, 27 Mar. 2017

Action of leptin for pituitary hormone gene expression in the chick　　　　　　　　　　　　　　　
Shoko Namekawa and Takeshi Ohkubo
World's Poultry Congress 2016, 10 Sep. 2016

Expression of Pigeon Leptin in vitro with Determination of Biological Activity　　　　　　　　　　　　　　　
Sadequllah Ahmadi; Shoko Namekawa and Takeshi Ohkubo
17th AAAP Animal Science Congress, 23 Aug. 2016

Interaction of STAT3 and STAT5 in the leptin signal transduction　　　　　　　　　　　　　　　
Shoko Namekawa; Daisuke Murase and Takeshi Ohkubo
17th AAAP Animal Science Congress, 23 Aug. 2016

ニワトリの下垂体ホルモン遺伝子発現に対するレプチンの作用　　　　　　　　　　　　　　　
滑川晶子; 大久保武
日本家禽学会２０１６年度春季大会, 30 Mar. 2016

鶏の就巣性の制御について　　　　　　　　　　　　　　　
大久保武
平成27年度関東甲信越地域鶏試験研究推進協議会全体会議, 08 Oct. 2015, [Invited]

レプチン情報伝達における STAT3 と STAT5 の相互作用　　　　　　　　　　　　　　　
滑川晶子; 大久保武
日本家禽学会２０１５年度秋季大会, 10 Sep. 2015

Change of appetite-associated genes in the chicken hypothalamus during incubation behavior　　　　　　　　　　　　　　　
Takeshi Ohkubo; Takahiro Kodama; Rumi Ohnuki
10th Asia Pacific Poultry Conference, 21 Oct. 2014

抱卵行動がニワトリ視床下部の摂食関連遺伝子の発現に及ぼす影響　　　　　　　　　　　　　　　
児玉孝弘; 大貫瑠美; 大久保武
2014年度日本家禽学会春季大会, 29 Mar. 2014, 日本家禽学会

コレシストキニン A受容体遺伝子の一塩基多型は比内鶏発育を改善する　　　　　　　　　　　　　　　
力丸宗弘; 武田尚人; 小松恵; 高橋大希; 大久保武; 高橋秀彰
2013年度日本家禽学会秋季大会, 08 Sep. 2013, 日本家禽学会

Role of transactivation domain of Pit-1 is different in the transcriptional regulation of GH gene by leptin in chicken and mouse　　　　　　　　　　　　　　　
Daisuke Murase; Kaori Ogawa and Takeshi Ohkubo
17th International Congress on Comparative Endocrinology, 16 Jul. 2013

鳥類血清中のレプチン様活性の検出　　　　　　　　　　　　　　　
村瀬大輔; 廣田佳菜子; 安達洋泉; 竹田努; 杉田昭栄; 大久保武
2013年度家禽学会春季大会, 29 Mar. 2013

Leptin regulates Pit-1α-dependent chicken GH gene expression in mammalian cells　　　　　　　　　　　　　　　
10th International Symposium on Avian Endocrinology, 06 Jun. 2012, Daisuke Murase, Susumu Atomura and Takeshi Ohkubo

Effect of chicken STAT5b mutant on prolactin signal transduction　　　　　　　　　　　　　　　
Susumu Atomura; Hiromi Adachi; Daisuke Murase; Takeshi Ohkubo
10th International Symposium on Avian Endocrinology, 06 Jun. 2012

ニワトリGH遺伝子のレプチン応答におけるPit-1転写活性領域の機能解析　　　　　　　　　　　　　　　
村瀬大輔; 後村進; 大久保武
2012年度家禽学会春季大会, 30 Mar. 2012

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