Faculty Profiles - FUNAHASHI Hiroaki

Papers - FUNAHASHI Hiroaki

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Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine Reviewed

Hiroaki Funahashi, Takashi Nagai

Molecular Reproduction and Development 58 ( 4 ) 424 - 431 2001.4

Involvement of oviduct in sperm capacitation and oocyte development in pigs Invited Reviewed

H Rodriguez-Martinez, P Tienthai, K Suzuki, H Funahashi, H Ekwall, A Johannisson

CONTROL OF PIG REPRODUCTION VI 58 ( 58 ) 129 - 145 2001

Language：English Publisher：SOC REPRODUCTION FERTILITY

An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for < 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

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Zona Reaction in Porcine Oocytes Fertilized In Vivo and In Vitro as Seen with Scanning Electron Microscopy1 Reviewed

Hiroaki Funahashi, Hans Ekwall, Heriberto Rodriguez-Martinez

Biology of Reproduction 63 ( 5 ) 1437 - 1442 2000.11

Modulation of the Function of Boar Spermatozoa via Adenosine and Fertilization Promoting Peptide Receptors Reduce the Incidence of Polyspermic Penetration into Porcine Oocytes1 Reviewed

Hiroaki Funahashi, Toshimitsu Fujiwara, Takashi Nagai

Biology of Reproduction 63 ( 4 ) 1157 - 1163 2000.10

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro Reviewed

Hiroaki Funahashi, Atsushi Asano, Toshimitsu Fujiwara, Takashi Nagai, Koji Niwa, Lynn R. Fraser

Molecular Reproduction and Development 55 ( 1 ) 117 - 124 2000.1

Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes. Reviewed

Hiroaki FUNAHASHI, Takashi NAGAI

Journal of Reproduction and Development 46 ( 5 ) 319 - 324 2000

Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes Reviewed

Hiroaki Funahashi, Naoya Bandoh, Shinobu Nakahira, She-Hoon Oh, Seiji Tsuboi

Zygote 7 ( 4 ) 301 - 305 1999.11

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Cambridge University Press (CUP)

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 ± 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 ± 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 ± 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 ± 74.1 nmol/egg in unfertilised eggs, 146.0 ± 50.0 nmol/egg in DSH eggs and 39.7 ± 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

DOI： 10.1017/s0967199499000702

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DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes Reviewed

Syahruddin Said, Hiroaki Funahashi, Koji Niwa

Zygote 7 ( 3 ) 249 - 254 1999.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Cambridge University Press (CUP)

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 ± 0.16) and testicular spermatozoa (4.03 ± 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 ± 0.03). However, the ratio was not different among corpus (0.69 ± 0.01), cauda epididymis (0.68 ± 0.11) and ejaculated spermatozoa (0.63 ± 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 ± 0.16) through epididymis (caput, 2.72 ± 0.08; corpus, 1.07 ± 0.03; cauda, −0.05 ± 0.05; ejaculated, 0.08 ± 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

DOI： 10.1017/s0967199499000635

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Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells1 Reviewed

Kwang-Wook Park, Sun-Ho Choi, Xue-Xiong Song, Hiroaki Funahashi, Koji Niwa

Biology of Reproduction 61 ( 1 ) 298 - 304 1999.7

Co-culture of Cumulus-Enclosed Bovine Oocytes with Theca Cells Induces the Meiotic Arrest but does not Inhibit Germinal Vesicle Development. Reviewed

Kwang-Wook Park, Hiroaki Funahashi, Koji Niwa

Journal of Reproduction and Development 45 ( 3 ) 223 - 231 1999

The Presence of Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) During Meiosis Improves Porcine 'Oocyte Competence' as Determined by Early Embryonic Development After In-vitro Fertilization. Reviewed

Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

Journal of Reproduction and Development 45 ( 4 ) 265 - 271 1999

Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo Culture Medium Containing a High Sodium Chloride Concentration and Bovine Serum Albumin Maintain Developmental Ability to the Blastocyst Stage Reviewed

She-Hoon Oh, Kazuchika Miyoshi, Hiroaki Funahashi

Biology of Reproduction 59 ( 4 ) 884 - 889 1998.10

Effects of Cumulus Cells on the Ability of Pig Oocytes to Utilize Cysteine or Cystine During Maturation In Vitro. Reviewed

Ken SAWAI, Hiroaki FUNAHASHI, Koji NIWA

Journal of Reproduction and Development 44 ( 2 ) 161 - 168 1998

Stage-Specific Requirement of Cysteine during in Vitro Maturation of Porcine Oocytes for Glutathione Synthesis Associated with Male Pronuclear Formation1 Reviewed

Ken Sawai, Hiroaki Funahashi, Koji Niwa

Biology of Reproduction 57 ( 1 ) 1 - 6 1997.7

Synchronization of Meiosis in Porcine Oocytes by Exposure to Dibutyryl Cyclic Adenosine Monophosphate Improves Developmental Competence Following in Vitro Fertilization1 Reviewed

Hiroaki Funahashi, Thomas C. Cantley, Billy N. Day

Biology of Reproduction 57 ( 1 ) 49 - 53 1997.7

Chlortetracycline fluorescence patterns andin vitrofertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations Reviewed

Lalantha R. Abeydeera, Hiroaki Funahashi, Nam-Hyung Kim, Billy N. Day

Zygote 5 ( 2 ) 117 - 125 1997.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Cambridge University Press (CUP)

Summary

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: 10IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22h. 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 (5mM), fetal calf serum (FCS; 10%) and varying concentrations (26–56 mM)of NaHCO₃for 9h(experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO₃concentrations. 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 36–56mM NaHCO₃. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46mM 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 56mM NaHCO₃, respectively. The fertilisation medium containing 46mM NaHCO₃and 1% FCS showed a higher penetration rate (84%)with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO₃stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

DOI： 10.1017/s0967199400003798

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Developmental Changes in the Intracellular Ca 2+ Release Mechanisms in Porcine Oocytes1 Reviewed

Zoltán Macháty, Hiroaki Funahashi, Billy N. Day, Randall S. Prather

Biology of Reproduction 56 ( 4 ) 921 - 930 1997.4

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

H Funahashi, TC Cantley, BN Day

THERIOGENOLOGY 47 ( 3 ) 679 - 686 1997.2

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 10% 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. (C) 1997 by Elsevier Science Inc.

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Effects of Cysteine in Serum-Free Maturation Medium on Male Pronuclear Formation of Maturing Pig Oocytes Penetrated In Vitro. Reviewed

Ken SAWAI, Hiroaki FUNAHASHI, Wei-Hua WANG, Koji NIWA

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

gamma-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs Reviewed International journal

H Funahashi, Z Machaty, RS Prather, BN Day

MOLECULAR REPRODUCTION AND DEVELOPMENT 45 ( 4 ) 485 - 490 1996.12

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

The presence of gamma-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pi of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 +/- 1.4%)was not different(P > 0.05) from HEPES-injected controls (24.9 +/- 1.3%) at 6 h after injection. Injected GTP-gamma-S, however, activated 76.0 +/- 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 +/- 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-gamma-S injected oocytes (4.2 +/- 0.7 pmol/oocyte) and noninjected oocytes (4.0 +/- 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 +/- 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 +/- 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pi of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that(1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-gamma-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes. (C) 1996 Wiley-Liss, Inc.

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