研究者詳細 - 木村　康二

論文 - 木村　康二

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Links between de novo fatty acid synthesis and leptin secretion in bovine adipocytes 査読

Hiroya Kadokawa, Katsuhiro Aikawa, Koji Kimura, Domique Blache, Ian H. Williams, Graeme B. Martin

JOURNAL OF VETERINARY MEDICAL SCIENCE 69 ( 3 ) 225 - 231 2007年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.

DOI： 10.1292/jvms.69.225

黒毛和種牛における卵胞刺激ホルモン皮下１回投与による過剰排卵誘起法第１報水酸化アルミニウムゲル吸着卵胞刺激ホルモン投与牛における血漿中卵胞刺激ホルモン濃度の推移および過剰排卵誘起効果査読

磯崎良寛, 稲田淳, 浅田研一, 古賀鉄也, 木村康二, 角川博哉, 平子誠

福岡県農業総合試験場研究報告 26 ( 1 ) 61 - 64 2007年

記述言語：日本語掲載種別：研究論文（学術雑誌）

現行の牛過剰排卵誘起（SOV）においては、豚由来卵胞刺激ホルモン（FSH-P）を数日間にわたり多回投与する必要があり、作業者や牛の負担が大きい。そこで、高分子吸着・徐放能を有する水酸化アルミニウムゲル（AG）に吸着させたFSH-P1回投与（ワンショット）によるSOV効果について検討した。30AU（アーマー単位）のFSH-PをAGに吸着（最終アルミニウム濃度：1.3mg／ml）させた後に黒毛和種雌牛の皮下に投与し、その2日後にプロスタグランジンF2α類縁体製剤を投与することによりワンショットSOVを実施した。血漿中FSH-P濃度の推移および胚採取成績について、20AUのFSH-Pを3日間朝夕漸減投与した群と比較した。FSH-Pをワンショットすることにより、漸減投与の場合と同様に72時間以上血漿中FSH-P濃度が高値を持続し、複数の卵胞が発育した。ワンショットおよび漸減投与によりSOVを実施した場合の胚採取総数は、12.0および8.2個／頭とワンショットの方が有意（P＜0.05）に多く、正常胚採取数については、7.8および5.8個／頭とワンショットの方が多い傾向にあった。本研究の結果より、FSH-PをAGに吸着させて牛皮下に1回投与することにより、3日間朝夕漸減投与と同等の過剰排卵誘起効果が得られることが明らかとなった。

Superovulation treatment by a single injection of FSH with aluminum hydroxide gel in Holstein cattle 査読

S. Matoba, M. Tagawa, H. Matsuda, H. Yoshioka, K. Kimura, H. Kadokawa, M. Yonai, K. Imaia

REPRODUCTION FERTILITY AND DEVELOPMENT 19 ( 1 ) 309 - 310 2007年

ウシ胚盤胞期胚のcDNAライブラリー構築法査読

塚本智史, 今市寿史, 木村康二, 今井裕, 南直治郎

日本胚移植学雑誌 = Japanese journal of embryo transfer 28 ( 3 ) 118 - 123 2006年9月

Influence of feeding regime on timing of parturition in beef cattle and the relationship of vaginal temperature to parturition 査読

Mari Aoki, Koji Kimura, Osamu Suzuki

Animal Science Journal 77 ( 3 ) 290 - 299 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

The timing of parturition was recorded for a total of 56 beef cattle (Japanese Black x Holstein Friesian) on different dietary treatments. The rate of calving during daylight hours in cows night-fed (18.00 hours) with a roughage diet was significantly higher than that in cows night-fed with a high concentrate diet (79.2% vs 38.5%, P &lt
0.05). Subsequently, the vaginal temperature (VT) of these cows was analyzed using a cosinor method. When the feeding schedule was changed from twice daily (08.30 and 15.30 hours) to night feeding, the periodicity, the acrophase and the bathyphase, which were the parameters of the cosine curve, were unstable from the first day of night feeding until after day 6 (P &lt
0.05). Prior to parturition, the midline-estimating statistic of rhythm (MESOR) and the amplitude for the cows that were fed a high-roughage diet at night and that calved at night-time were lower and larger, respectively, than that for the other treatments (P &lt
0.01). Based on these results, the time of parturition in most of the beef cows was influenced by feeding time and diet composition. Those cows that calved at night-time in spite of night feeding had lower vaginal temperatures. © 2006 Japanese Society of Animal Science.

DOI： 10.1111/j.1740-0929.2006.00351.x

Vascular endothelial growth factor has beneficial effect independent of serum components throughout oocyte maturation and early embryonic development in cattle 査読

HL Luo, K Kimura, M Hirako

ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 19 ( 4 ) 495 - 499 2006年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOC

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p < 0.05) and FBS+VEGF (42.1%, p < 0.05) groups than in the PVA group (17.1%). Two-factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

Comparison between the characteristics of follicular fluid and the developmental competence of bovine oocytes

H Iwata, J Inoue, K Kimura, T Kuge, T Kuwayama, Y Monji

ANIMAL REPRODUCTION SCIENCE 91 ( 3-4 ) 215 - 223 2006年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

There are great differences in the developmental competence of oocytes collected from individual cows. Oocytes grow and mature in the follicular fluid (FF). In the present study, characteristics of the FF of each ovary and the developmental competence of enclosed oocytes were investigated, and these data were then compared. A total of 37 pairs of ovaries were collected from beef heifers. The concentration of magnesium (Mg), aspirate aminotransferase (AST), nonesterified fatty acids (NEFA), and lactate dehydrogenase (LDH) in the FF were great compared with serum standard. Several significant correlations among these characteristics were detected. Forty-eight hours after fertilization, the stage of embryo development at an advanced developmental stage (>6 cell stage) is related to the rate of blastulation 8 days after fertilization. In addition, a significantly positive or negative correlation was observed between the developmental competence (the rate of cleavage in the embryo and blastulation) and the concentration of the icterus index (ICT) or blood urea nitrogen (BUN) in the FF. In conclusion, the quality of oocytes is affected by the environment in the follicle, and BUN or ICT is a predictable index of the developmental competence of oocytes. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.anireprosci.2005.04.006

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

H. Iwata, T. Hayashi, H. Sato, K. Kimura, T. Kuwayama, Y. Monji

Zygote 13 ( 4 ) 303 - 308 2005年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35°C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20°C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period. © 2005 Cambridge University Press.

DOI： 10.1017/S0967199405003412

Effects of D-glucose concentration, D-fructose, and inhibitors of enzymes of the pentose phosphate pathway on the development and sex ratio of bovine blastocysts 査読

K Kimura, LD Spate, MP Green, RM Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 72 ( 2 ) 201 - 207 2005年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

D-glucose at 5.6 mM reduces the progression of in vitro-produced (IVP) bovine embryos from the morula to the blastocyst stage and skews sex ratio towards males. Possibly, the presence of two transcriptionally active X-chromosomes compromises female embryo development through imbalance in glucose metabolism. Here, we have determined the threshold of embryo sensitivity to glucose, whether substitution Of D-fructose for glucose reduces the selective loss of female embryos, and whether inhibition of an X-linked gene product, glucose 6-phosphate dehydrogenase (G6PD), normalizes sex ratio among bovine blastocysts. IVP zygotes were cultured in glucose-free medium to 72 hr post-insemination, at which time 8-cell embryos were selected for treatment and cultured until 144 hr post-insemination. Addition of 4 mM glucose at the 8-cell stage reduced (P < 0.05) the number of blastocyst that formed, whereas 2.5 and I mM glucose had no effect. Sex ratio in the presence of 4 and 2.5 mM glucose differed significantly from 0.5, while 1 mM glucose had no effect. D-fructose (5.6 mM) did not compromise development to blastocyst and did not alter the sex ratio of blastocysts that formed. When G6PD inhibitors (dehydroepiandrosterone: DHEA and 6-aminonicotinamide: 6-AN), which effectively inhibit the reduction of the NADPH-sensitive dye, brilliant cresyl blue (BCB) in bovine morulae, were added to the culture medium containing 4 mM glucose, embryo development was moderately reduced, but sex ratio skewing was corrected (with 6-AN) or lowered (with DHEA). In conclusion, glucose above 2.5 mM impairs bovine embryo development and increases sex ratio, most likely as a result of increased pentose-phosphate (PP) pathway activity in female embryos.

DOI： 10.1002/mrd.20342

Comparison of the concentrations of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in maternal and fetal blood, amniotic and allantoic fluids in cattle 査読

M Hirako, M Aoki, K Kimura, Y Hanafusa, H Ishizaki, Y Kariya

REPRODUCTIVE TOXICOLOGY 20 ( 2 ) 247 - 254 2005年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

To characterize the maternal-fetal transport of lipophilic endocrine disrupting chemicals, concentrations of polychlorinated (2,3,7,8-substituted) dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) were measured in maternal and fetal blood, and amniotic and allantoic fluids in cattle. Total toxicity equivalent quantity (TEQ) was highest in amniotic fluid on a fat-weight basis, whereas it was highest in maternal blood on a total weight basis. TEQ was lowest in allantoic fluid on either basis; 26 of 29 congeners analyzed in this experiment were detected in one or more samples. The largest number of congeners was detected in amniotic fluid. O8CDD, 2,3,4,7,8-P5CDF and 2,3',4,4',5-P5CB were the major congeners in PCDDs, PCDFs and PCBs, respectively. The O8CDD concentration was higher in fetal blood than in maternal blood on a fat-weight basis, whereas concentrations of other congeners were lower in fetal blood than in maternal blood. Furthermore, on a fat-weight basis, the O8CDD concentration was considerably higher in allantoic fluid compared with other samples. Concentrations of major PCB congeners were higher in amniotic fluid than in maternal and fetal blood on a fat-weight basis. In conclusion, it is suggested that lipophilic endocrine-disrupting chemicals contained in maternal blood are all transferred to the fetal circulation via the placenta in cattle. Furthermore, the results of this experiment imply that O8CDD has different transportation systems from other dioxins in the circulation, and that a considerable amount of PCBs is excreted and accumulated in amniotic fluid during the fetal stage in cattle. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2005.01.011

Predicting time of parturition from changing vaginal temperature measured by data-logging apparatus in beef cows with twin fetuses 査読

M Aoki, K Kimura, O Suzuki

ANIMAL REPRODUCTION SCIENCE 86 ( 1-2 ) 1 - 12 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Vaginal temperatures (VT) of crossbred (Japanese Black crossed Holstein-Friesian) beef cows (n = 31) were measured by a data-logging apparatus to obtain serial data from days 0 to 6 before parturition. For both single and twin pregnancies, no significant differences were observed in VT during days 3-6 before parturition. Maternal VT was not affected by maternal weight just after parturition, parity, fetal sex, or total fetal litter weight. Average of twin litter weights for two males (MM) and two females (FF) had the strong positive correlations (r = 0.84; P < 0.05) with maternal VT, whereas twin weights of mixed-gender twins (FM) did not correlate with maternal VT (r = -0.26; P = 0.61). Maternal temperature decreased as weights of the female fetus of FM twins became heavier (r = -0.82; P < 0.05). In contrast, maternal VT of FF and MM twins increased as twin weights increased. We defined when the VT began to decrease before parturition by two different methods. One was the "same hours method" where differences in VT between a particular time of day and the corresponding time of the preceding day were compared when the VT was consistently >= 0.3 or >= 0.5 degrees C for more than 3 h. The second method was the "maximum-minimum method" where decreased in the maximum or the minimum values of the day over >= 0.3 and >= 0.5 degrees C were compared to values of preceding day. Onset of decreased VT before expulsion was not different between singletons and twins. In an attempt to define the critical condition in predicting parturition, we estimated assumable predicting probability using the 31 cows that were collected VT. When the parturition occurred within 60 It in the "same hours method" and 72 h since VT was >= 0.3 degrees C and in the "maximum-minimum method", the assumable probability was 100%. In verification experiment under these condition, the "same hours method" had a higher probability of predicting the time of parturition than the "maximum-minimum method", and it was possible to detect the onset of decreased VT at the correct time by the minutes. We concluded that "same hours method" was useful for predicting parturition time in cattle with single and twin pregnancies by the serial measurement of vaginal temperature. (c) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.anireprosci.2004.04.046

黒毛和種のワンショット過剰排卵処理法の確立に関する研究査読

林登, 加藤誠二, 坂口慎一, 澤田幹夫, 関誠, 竹内昭司, 木村康二, 角川博哉

岐阜県畜産研究所研究報告 5 ( 5 ) 30 - 34 2005年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：岐阜県畜産研究所

牛の過剰排卵処理法の簡易化・省力化を図るため、水酸化アルミニウムゲルを卵胞刺激ホルモン(FSH)の溶解剤として用いたFSHの1回投与(ワンショット法)による過剰排卵処理法の有効性を検討した。黒毛和種経産牛9頭を用い、1頭の牛について水酸化アルミニウムゲルを用いたワンショット法(30AU 1回、筋肉内投与)と従来の漸減投与法(5，5，3，3，2，2，計6回、20AU、筋肉内投与)による過剰排卵処理をそれぞれ1回ずつ実施し、発情の状況、採卵時の黄体数、卵胞数および採卵成績を比較した。1.ワンショット区では9頭中8頭に、漸減投与区では9頭中全頭に明瞭な発情が観察された。2.採胚時の平均黄体数、大卵胞数は、ワンショット区で、それぞれ8.7土5.3個、2.9土1.5個、漸減投与区で9.6土5.7個、1.9土1.7個でいずれも有意な差は認められなかった。3.平均回収卵数、正常卵数は、ワンショット区でそれぞれ6.7±6.4個、4.1±3.4個、漸減投与区で8.6土5.8個、4.1±2.8個でいずれも有意な差は認められなかった。以上の結果から、黒毛和種において、水酸化アルミニウムゲルを用いたFSHの1回投与による過剰排卵処理を行うことにより、従来の漸減投与法と比較し遜色ない採卵成績が得られ、この方法が過剰排卵処理法の節易化、省力化を図るための技術として利用可能であることが示唆された。

その他リンク： http://agriknowledge.affrc.go.jp/RN/2010722694

Effects of oxidative stress and inhibitors of the pentose phosphate pathway on sexually dimorphic production of IFN-tau by bovine blastocysts 査読

K Kimura, LD Spate, MP Green, RM Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 68 ( 1 ) 88 - 95 2004年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

Bovine interferon-tau (IFN-tau), the anti-luteolytic factor secreted by conceptuses of pecoran ruminants, is a product of autosomal genes, yet in vitro produced (IVP) female expanded blastocysts (EB) secrete about twice as much IFN-tau as males. Two possible explanations have been tested here. One is that embryos of one sex are differentially susceptible to oxidative stress. The second is that female EB produce more IFN-tau because pentose-phosphate pathway (PPP) activity is elevated as a result of delayed X-chromosome inactivation. IVP bovine zygotes were cultured to the 8-cell stage and placed under conditions designed either to promote oxidative stress (+/-H2O2; 20 vs. 5% O-2), or to inhibit glucose 6-phosphate dehydrogenase (G6PDH) activity (addition of dehydroepiandrosterone, DHEA or 6-aminonicotinamide, 6-AN to the medium). At day 8, blastocysts were cultured individually for a further 48 hr to assess IFN-tau production, and embryo sex determined retrospectively. Blastocyst numbers were reduced (P < 0.05) and their continued development impaired (P < 0.05) in presence of H2O2 (200 muM) and 20% O-2, but neither IFN-tau production nor sexually dimorphic expression of IFN-tau were affected. IFN-tau production was reduced, particularly in females (P < 0.05), and sexual dimorphic differences in production were lost in the presence of both DHEA (100 muM) and 6-AN (1 muM). In the case of 6-AN, these effects were achieved without a significant decline in blastocyst developmental progression, quality, or cell number. The data suggest that the higher production of IFN-tau by female EB is an indirect outcome of the increased activity of the oxidative arm of the PPP pathway. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/mrd.20053

Effects of follicle size and electrolytes and glucose in maturation medium on nuclear maturation and developmental competence of bovine oocytes 査読

H Iwata, S Hashimoto, M Ohota, K Kimura, K Shibano, M Miyake

REPRODUCTION 127 ( 2 ) 159 - 164 2004年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIO SCIENTIFICA LTD

The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0-2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5-3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2-3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.

DOI： 10.1530/rep.1.00084

Sexual dimorphism in interferon-tau production by in vivo-derived bovine embryos 査読

K Kimura, LD Spate, MP Green, CN Murphy, GE Seidel, RM Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 67 ( 2 ) 193 - 199 2004年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

Interferon-tau (IFN-tau) is an antiluteolytic factor responsible for preventing regression of the maternal corpus luteum (CL) during early pregnancy of cattle. In vitro-produced (IVP) bovine embryos first produce IFN-tau as blastocysts. In the present study, we have examined whether sexually dimorphic production of IFN-tau, which is observed among IVP blastocysts, also occurs among in vivo-produced blastocysts, and whether this difference between the sexes persists to day 14 when silencing of one of the X-chromosomes in the trophectoderm is complete. Embryos were flushed from cattle that had been superovulated and bred by AI. Blastocysts (63 male, 62 female) recovered between days 8.5 and 9.5 of pregnancy, were cultured individually. No differences were observed between males and females in either their developmental stage or quality at the beginning, during, and at the end of culture. Female embryos produced more IFN-tau than males by 24 hr (mean values, males: 16.6 +/- 3.7, females: 49.4 +/- 9.0 pg per embryo; P < 0.05) and 48 hr (male: 189.8 +/- 37, 1, female: 410.9 +/- 66.6 pg per embryo; P<0.05). However, the variability in IFN-tau production between individual blastocysts was so great that IFN-tau secretion is unlikely to be of value as a non-invasive means to predict embryo sex. When conceptuses were recovered at day 14, elongating males (n = 25) and females (n = 24) were similar in dimension and did not differ in their IFN-tau production after 4.5 hr (male: 2,550 +/- 607, female: 2,376 +/- 772 ng per conceptus) and 24 hr (male: 12,056 +/- 2,438, female: 8,447 +/- 1,630 ng per conceptus) of culture. Thus, sexual dimorphism in IFN-tau production is observed in both IVP and in vivo-produced expanded blastocysts, but is lost by day 14 of in vivo development. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/mrd.10389

Effect of growth hormone at various periods of development on developmental competence of bovine embryo and its interaction with glucose

Iwata H, Ohota M, Hashimoto S, Kimura K, Isaji M, Miyake M

Journal of Reproduction and Development 49 6巻（2003) 493-499 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

胚の体外培養を改善するため、FCSやBSAの血清蛋白を用いた培養条件でGrowth Hormon の添加とグルコースの濃度の胚発生に及ぼす影響について発生ステージごとに検討した。FCS添加条件下でGHは桑実胚以降において発生を促進しすること、またGHの効果は培養条件中のグルコースの濃度と相互効果が見られること、桑実胚以降のグルコース濃度の上昇は胚発生促進には効果がないこと等を示した。

Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro 査読

H Iwata, M Ohota, S Hashimoto, K Kimura, M Isaji, M Miyake

JOURNAL OF REPRODUCTION AND DEVELOPMENT 49 ( 6 ) 493 - 499 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.

DOI： 10.1262/jrd.49.493

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air

S Hashimoto, K Kimura, H Iwata, R Takakura

JOURNAL OF REPRODUCTION AND DEVELOPMENT 49 ( 1 ) 61 - 66 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The effects of the medium (TCM 199 or SCFaa) and temperature (20 or 39 Q during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 mug/ml CHX in a 50-mul droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 mu l HSOFaa or as a group (40-50 oocytes) with 170-200 mu l HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-mu l droplet of HSCFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-mu l droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

Vascular endothelial growth factor (VEGF) promotes the early development of bovine embryo in the presence of cumulus cells 査読

HL Luo, K Kimura, M Aoki, M Hirako

JOURNAL OF VETERINARY MEDICAL SCIENCE 64 ( 11 ) 967 - 971 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

Three experiments were conducted to determine the effect of Vascular Endothelial Growth Factor (VEGF) on bovine embryonic development in vitro. Human recombinant VEGF(165) was employed at 5 ng/ml in modified synthetic oviduct fluid. In Exp. 1, bovine cumulus oocyte complexes were matured with or without VEGF for 22 hr, inseminated without VEGF for 6 hr, then cultured with or without VEGF for 42 hr. The cleavage rate and the development rate to 4- to 8-cell were higher (P<0.05) in groups with VEGF during in vitro maturation (IVM, 71.4% and 59.6%), in vitro culture (IVC, 70.3% and 62.3%), and both IVM and IVC (75.9% and 67.8%) than in the group cultured thoroughly without VEGF (49.9% and 38.4%, respectively). In Exp. 2, 4- to 8-cell embryos produced in vitro without VEGF were removed from cumulus cells at 48 hr post-insemination (Pi) and cultured with or without VEGF for 144 hr. The development rates to blastocyst at 96 hr (D6), 120 hr (D7) and 144 hr (D8) were similar (P>0.05) in both groups. In Exp. 3, cumulus cells were removed from presumptive embryos produced by IVM and IVF without VEGF at 10 hr Pi. Denuded embryos were cultured with or without VEGF for 38 hr or 182 hr. The cleavage rate and the development rates to 4- to 8-cell at 48 hr Pi and to blastocyst on D6, D7 and D8 were similar (P>0.05) in all groups. These results suggest that VEGF has a beneficial effect on the initial development of bovine embryo through surrounding cumulus cells.

DOI： 10.1292/jvms.64.967

Glucose-6-phosphate dehydrogenase activity of IVM/IVF/IVC bovine embryos is related to the sex and developmental competence under suboptimal gas condition

Iwata H, Kimura K, Hashimoto S, Oota M, Tominaga T, Minami N

Journal of Reproduction and Development 5 447 - 453 2002年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Brilliant Cresyl Blueを用いて発生各ステージの体外受精卵の還元力の違いと性比との関係について検討した。その結果桑実胚において還元力の差が雄と雌との間で異なること、8細胞期や胚盤胞期では差がないことを示した。このことより桑実期胚ではX染色体上にあるG6PDの活性に差があり雌雄判別に利用できることやX染色体の不活化が胚盤胞期胚以降で起こっていることを示唆した。

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