Faculty Profiles - FUNAHASHI Hiroaki

MISC - FUNAHASHI Hiroaki

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受精促進ペプチド(FPP)およびアデノシンが豚精子の自発的先体反応に及ぼす阻止効果(共著)

15 ( 2 ) S26 1998

Extracorporeal production and artificial operation of fertilized egg of pig.

舟橋弘晃

家畜人工授精 ( 185 ) 14 - 22 1998

豚受精卵の体外生産技術の現状

日本畜産学会関西支部報 ( 138 ) 18 - 20 1998

EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

Journal of Reproduction and Fertility, Abstract Series 21 10 1998

EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

Journal of Reproduction and Fertility, Abstract Series 21 10 1998

Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization

H Funahashi, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 57 ( 1 ) 49 - 53 1997.7

Language：English Publisher：SOC STUDY REPRODUCTION

The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (fetal mean, 82.1 +/- 2.1%) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p < 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p < 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

DOI： 10.1095/biolreprod57.1.49

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Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation International journal

Ken Sawai, Hiroaki Funahashi, Koji Niwa

Biology of Reproduction 57 ( 1 ) 1 - 6 1997.7

Language：English

The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-I) to metaphase II (M-II) and M-II at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-I to M-II stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

DOI： 10.1095/biolreprod57.1.1

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Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation

K Sawai, H Funahashi, K Niwa

BIOLOGY OF REPRODUCTION 57 ( 1 ) 1 - 6 1997.7

Language：English Publisher：SOC STUDY REPRODUCTION

The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-l) to metaphase II (M-II) and M-Il at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-l to M-ll stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

DOI： 10.1095/biolreprod57.1.1

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Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization International journal

H Funahashi, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 57 ( 1 ) 49 - 53 1997.7

Language：English Publisher：SOC STUDY REPRODUCTION

DOI： 10.1095/biolreprod57.1.49

Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations International journal

LR Abeydeera, H Funahashi, NH Kim, BN Day

ZYGOTE 5 ( 2 ) 117 - 125 1997.5

Language：English Publisher：CAMBRIDGE UNIV PRESS

Porcine oocyte-cumulus complexes were cultured in bovine serum albumin (BSA)-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM) 199 containing caffeine (5 mM), fetal calf serum (FCS; 10%) and varying concentrations (26-56 mM) of NaHCO3 for 9 h (experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%) and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p < 0.05) at 36-56 mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46 mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p < 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56 mM NaHCO3, respectively. The fertilisation medium containing 46 mM NaHCO3 and 1% FCS showed a higher penetration rate (84%) with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

DOI： 10.1017/S0967199400003798

Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations

LR Abeydeera, H Funahashi, NH Kim, BN Day

ZYGOTE 5 ( 2 ) 117 - 125 1997.5

Language：English Publisher：CAMBRIDGE UNIV PRESS

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Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes

Z Machaty, H Funahashi, BN Day, RS Prather

BIOLOGY OF REPRODUCTION 56 ( 4 ) 921 - 930 1997.4

Language：English Publisher：SOC STUDY REPRODUCTION

The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP(3)), the InsP(3) receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP(3)-induced Ca2+ release was inhibited by heparin, the InsP(3) receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP(3) and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.

DOI： 10.1095/biolreprod56.4.921

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Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes International journal

Z Machaty, H Funahashi, BN Day, RS Prather

BIOLOGY OF REPRODUCTION 56 ( 4 ) 921 - 930 1997.4

Language：English Publisher：SOC STUDY REPRODUCTION

DOI： 10.1095/biolreprod56.4.921

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro. International journal

H Funahashi, T C Cantley, B N Day

Theriogenology 47 ( 3 ) 679 - 86 1997.2

Language：English

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

DOI： 10.1016/S0093-691X(97)00026-5

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Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

Fifth International Conference on Pig Reproduction 142 1997

Production of hyaluronic acid by porcine oocyte-cumulus cell complexes during in vitro maturation.

Fifth International Conference on Pig Reproduction 139 1997

Effect of tissue inhibitor of metalloproteinase (TIMP-1) on early development of swine oocytes matured and fertilized in vitro

FUNAHASHI H

Theriogenology 47 ( 1 ) 277 - 277 1997

Tris-buffered medium (TBM) is better for in vitro fertilization of porcine oocytes than mM199 which has been traditionally used.

H Funahashi, Y Kajiwara, K Sawai, K Niwa

BIOLOGY OF REPRODUCTION 56 ( Supplementl ) 514 - 514 1997

Advances in in vitro production of pig embryos

H Funahashi, BN Day

JOURNAL OF REPRODUCTION AND FERTILITY Supplement52 271 - 283 1997

Language：English Publisher：J REPROD FERTIL INC

A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

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Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocytes penetrated in vitro

Ken Sawai, Hiroaki Funahashi, Wei Hua Wang, Koji Niwa

Journal of Reproduction and Development 43 ( 1 ) 73 - 80 1997

Language：English Publisher：The Japanese Society of Animal Reproduction (JSAR)

Pig immature oocytes were cultured for 24, 36 and 48 h in serum-free maturation medium with or without 0.57 mM cysteine. The addition of cysteine to the medium was associated with increased glutathione synthesis by oocytes but did not promote nor inhibit nuclear maturation, sperm penetration in vitro, and decondensation of sperm nuclei in penetrated oocytes. The incidence of activation of penetrated oocytes 14 h after insemination in vitro was lower in those cultured both in the presence and absence of cysteine for 24 h than 36 and 48 h. The lower ability of oocytes cultured for 24 h to be activated was not improved by neither the addition of cysteine (0.57 mM) in fertilization medium nor prolonged culture time after insemination. However, male pronuclear formation in activated oocytes after sperm penetration at any stages of maturation was largely accelerated when cysteine was added to maturation medium. An increased concentration of glutathione may induce full decondensation of sperm nuclei in immature pig oocytes penetrated in vitro ensuring transformation of the decondensed sperm nuclei to male pronuclei only in synchronization with oocyte activation.

DOI： 10.1262/jrd.43.73

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