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H Funahashi

The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO(2)-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N(omega)-nitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

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H. Funahashi and B. N. Day

The effects of porcine follicular fluid (PFF) on sperm penetration of pig oocytes and on prevention of polyspermy were examined and characteristics of spermatozoa exposed to PFF were determined. The addition of PFF at the level of 1 and 10% to the prefertilization and fertilization media decreased penetration rates and the mean number of spermatozoa in penetrated eggs regardless of the origin of PFF. In the presence of BSA, supplementation of 0.1% PFF to prefertilization and fertilization media and 1% PFF to prefertilization media did not decrease the penetration rates but did increase monospermic penetration to 54 and 68%, respectively. When PFF was added to prefertilization media, the number of spermatozoa binding to the zona and the percentage of acrosome-intact spermatozoa decreased with increased PFF concentration (from 43.1 ± 2.8 and 73.1 ± 4.9% to 7.2 ± 1.3 and 15.7 ± 15.4%, respectively). At the end of prefertilization incubation, sperm agglutination was observed and the degree depended on PFF concentration. Supplementation of fetal calf serum to prefertilization and fertilization media blocked the effects of PFF on sperm penetration and binding of spermatozoa to the zona. These results indicate that the prefertilization incubation of porcine spermatozoa in suitable concentrations of porcine follicular fluid will effectively reduce both the number of spermatozoa that attach to the surface of pig eggs and the incidence of polyspermy.

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H. Funahashi and B. N. Day

The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte–cumulus complexes in a medium with PMSG (10 iu ml−1), hCG (10 iu ml−1) and oestradiol (1 μg ml−1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte–cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact and oocytectomized oocyte–cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte–cumulus complexes were improved after the removal of oocyte–cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte–cumulus complexes to hormone were similar to those of intact oocyte–cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

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H. Funahashi, T. Cantley and B. N. Day

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 μg 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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H Funahashi, H Ekwall, K Kikuchi and H Rodriguez-Martinez

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

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K. Miyoshi, H. Funahashi, K. Okuda and K. Niwa

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 μmol phosphate l−1, but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 μmol 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 four-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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N-H. Kim, H. Funahashi, L. R. Abeydeera, S. J. Moon, R. S. Prather and B. N. Day

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.