MISC - FUNAHASHI Hiroaki

USE OF LOW-SALT CULTURE-MEDIUM FOR IN-VITRO MATURATION OF PORCINE OOCYTES IS ASSOCIATED WITH ELEVATED OOCYTE GLUTATHIONE LEVELS AND ENHANCED MALE PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION   51 ( 4 )   633 - 639   1994.10

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The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supple ments; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl(59.3 +/- 8.1 and 70.9 +/- 2.0%,respectively) than in medium containing 68.49 mM NaCl (33.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

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USE OF LOW-SALT CULTURE-MEDIUM FOR IN-VITRO MATURATION OF PORCINE OOCYTES IS ASSOCIATED WITH ELEVATED OOCYTE GLUTATHIONE LEVELS AND ENHANCED MALE PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION International journal

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION   51 ( 4 )   633 - 639   1994.10

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The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supple ments; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl(59.3 +/- 8.1 and 70.9 +/- 2.0%,respectively) than in medium containing 68.49 mM NaCl (33.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

DOI： 10.1095/biolreprod51.4.633

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DEVELOPMENTAL ABILITY OF PORCINE OOCYTES MATURED AND FERTILIZED IN-VITRO

H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

THERIOGENOLOGY   41 ( 7 )   1425 - 1433   1994.5

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The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17 beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42/71) and with monospermic penetration and male and female pronuclei (32%, 23/71) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18/71 and 8%, 6/71, respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28/288 and 13%, 41/318, respectively) were also higher (P < 0.05) than in controls (2%, 6/284 and 6%, 16/248, respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9/288 in PFF-treated group and 2%, 6/284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3/100) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

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IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION   50 ( 5 )   1072 - 1077   1994.5

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Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p < 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p < 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (P < 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p < 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p < 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (P < 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes) as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

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DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO International journal

H FUNAHASHI, T CANTLEY, BN DAY

JOURNAL OF REPRODUCTION AND FERTILITY   101 ( 1 )   159 - 165   1994.5

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Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml(-1), hCG (10 iu ml(-1)) and oestradiol (1 mu g ml(-1)) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 +/- 72.6 ng ml(-1)) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 +/- 13.3 ng ml(-1)), during the first 20 h of culture (42.8 +/- 19.4 ng ml(-1)) and also during the second 20 h period of culture without hormonal supplements (0.925 +/- 0.544 ng ml(-1)). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 +/- 0.2 ng ml(-1) at 0 h to 6.9 +/- 1.5 ng ml(-1) after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.

DOI： 10.1530/jrf.0.1010159

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DEVELOPMENTAL ABILITY OF PORCINE OOCYTES MATURED AND FERTILIZED IN-VITRO International journal

H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

THERIOGENOLOGY   41 ( 7 )   1425 - 1433   1994.5

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The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17 beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42/71) and with monospermic penetration and male and female pronuclei (32%, 23/71) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18/71 and 8%, 6/71, respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28/288 and 13%, 41/318, respectively) were also higher (P < 0.05) than in controls (2%, 6/284 and 6%, 16/248, respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9/288 in PFF-treated group and 2%, 6/284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3/100) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

DOI： 10.1016/0093-691X(94)90193-M

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IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION International journal

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION   50 ( 5 )   1072 - 1077   1994.5

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Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p < 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p < 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (P < 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p < 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p < 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (P < 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes) as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

DOI： 10.1095/biolreprod50.5.1072

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DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO

H FUNAHASHI, T CANTLEY, BN DAY

JOURNAL OF REPRODUCTION AND FERTILITY   101 ( 1 )   159 - 165   1994.5

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Language：English   Publisher：J REPROD FERTIL INC  

Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml(-1), hCG (10 iu ml(-1)) and oestradiol (1 mu g ml(-1)) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 +/- 72.6 ng ml(-1)) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 +/- 13.3 ng ml(-1)), during the first 20 h of culture (42.8 +/- 19.4 ng ml(-1)) and also during the second 20 h period of culture without hormonal supplements (0.925 +/- 0.544 ng ml(-1)). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 +/- 0.2 ng ml(-1) at 0 h to 6.9 +/- 1.5 ng ml(-1) after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.

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DEVELOPMENT OF RAT ONE-CELL EMBRYOS IN A CHEMICALLY-DEFINED MEDIUM - EFFECTS OF GLUCOSE, PHOSPHATE AND OSMOLARITY

K MIYOSHI, H FUNAHASHI, K OKUDA, K NIWA

JOURNAL OF REPRODUCTION AND FERTILITY   100 ( 1 )   21 - 26   1994.1

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Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l(-1) (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-(1). This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l(-1) and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mu mol phosphate l(-1), but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mu mol phosphate l(-1). In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l(-1) (67%) and 10.0 mmol glucose l(-1) (60%) were not statistically different from the percentage at 5.0 mmol glucose l(-1) (46%), but was significantly greater than the percentage at 2.5 mmol l(-1) (33%). When osmolarity of the medium with 5.0 mmol glucose l(-1) was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the few-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

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DEVELOPMENT OF RAT ONE-CELL EMBRYOS IN A CHEMICALLY-DEFINED MEDIUM - EFFECTS OF GLUCOSE, PHOSPHATE AND OSMOLARITY

K MIYOSHI, H FUNAHASHI, K OKUDA, K NIWA

JOURNAL OF REPRODUCTION AND FERTILITY   100 ( 1 )   21 - 26   1994.1

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Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l(-1) (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-(1). This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l(-1) and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mu mol phosphate l(-1), but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mu mol phosphate l(-1). In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l(-1) (67%) and 10.0 mmol glucose l(-1) (60%) were not statistically different from the percentage at 5.0 mmol glucose l(-1) (46%), but was significantly greater than the percentage at 2.5 mmol l(-1) (33%). When osmolarity of the medium with 5.0 mmol glucose l(-1) was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the few-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

DOI： 10.1530/jrf.0.1000021

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Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science   72 ( Supplement 1 )   71   1994

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HISTONE-H1 KINASE (H1K) ACTIVITY AND GLUTATHIONE (GSH) CONTENT OF PORCINE OOCYTES DURING PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   128 - 128   1994

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Histone H1 kinase activity after electrical activation of in vitro matured porcine oocytes.(共著)

Journal of Animal Science   72 ( Supplement 1 )   74   1994

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Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science   72 ( Supplement 1 )   71   1994

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Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science   72 ( Supplement 1 )   75   1994

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MICROTUBULE ORGANIZATION OF PORCINE OOCYTES DURING IN-VITRO FERTILIZATION

NH KIM, H FUNAHASHI, TT STUMPF, BN DAY

BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   146 - 146   1994

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受精卵あるいは卵子輸送装置.(共著)

特許公報   特公平6-94401   1994

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MICROTUBULE ORGANIZATION OF PORCINE OOCYTES DURING IN-VITRO FERTILIZATION

NH KIM, H FUNAHASHI, TT STUMPF, BN DAY

BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   146 - 146   1994

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science   72 ( Supplement 1 )   75   1994

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In vitro maturation/in vitro fertilization of porcine oocytes.(共著)

Proceeding for Annual Meeting of the Society for Theriogenology   206 - 214   1994

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