The objective of this study was to determine whether coculture of stallion spermatozoa and mare oviductal (uterine tubal) epithelial cells induced sperm cell capacitation in vitro. Capacitation as determined by zona binding and chlortetracycline staining of the sperm cells was compared for stallion spermatozoa: (1) incubated with medium alone (negative control), (2) treated with calcium ionophore A23187 (positive control) or (3) cultured with mare oviductal epithelial cells (OEC) for 4 h. Chlortetracycline staining patterns of sperm cells bound to the zonae were used to group spermatozoa as uncapacitated, capacitated or acrosome reacted. The zonae and attached spermatozoa were stained for evaluation after initial binding (pulse) and after 1 h of co-incubation (chase). More sperm cells in the ionophore and OEC treatments bound to the zonae at both the pulse and chase than in control medium (P < 0.001). More bound sperm cells were capacitated at the pulse, and acrosome reacted at the chase, for the ionophore and co-culture groups than for the controls (P < 0.001). Staining patterns for sperm cells not bound to the zona pellucida in each of the treatments differed (P < 0.05) from the population of sperm cells that bound to the zona pellucida. There was a higher percentage of capacitated spermatozoa and a lower percentage of acrosome-reacted spermatozoa bound to the zonae at the pulse than were represented in the treatment suspensions of sperm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immature pig oocytes cultured for 36 h in a modified tissue culture medium 199B were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescence illumination, Ricinus communis agglutinin (RCA-I) and Lens culinaris agglutinin (LCA) bound to all oocytes, with the strongest fluorescence in either the outer region of the ZP (RCA-I) or throughout the ZP (LCA). However, Bandeiraea simplicifolia lectin-I (BS-I) and Ulex europaeus agglutinin-I (UEA-I) bound with either strong or weak intensity mainly to the outer and inner regions of the ZP, respectively, in ~70-90% of oocytes examined. Bandeiraea simplicifolia lectin-II (BS-II) bound to various regions of the ZP with different intensities in only 60% of oocytes examined. At 2 h after insemination in vitro, significantly fewer spermatozoa were bound to the ZP of lectin-treated oocytes than untrearted control oocytes. Of the lectins, RCA-I and LCA most inhibited sperm binding. At 12 h after insemination, penetration rates were similar in control oocytes and those treated with BS-I or BS-II, but penetration rates were lower in oocytes treated with UEA-I than those in untreated controls, and an almost complete block of sperm penetration was observed in oocytes treated with RCA-I or LCA. The incidence of monospermy was similar in untreated oocytes and those treated with BS-I or UEA-I, but it was higher in oocytes treated with BS-II. These results suggest that β-D-galactose and α-D-mannose residues in the pig ZP may act as primary sperm receptors and/or inducers of the sperm acrosome reaction and β-D-N-acetylglucosamine residues may be involved in the block of polyspermy. The variability in binding of BS-II to the ZP, which correlated with monospermy/polyspermy may reflect differences in the maturation state of individual oocytes. In future, this lectin might provide a means of monitoring maturation in vitro, leading to the development of improved culture systems. Key words: Pig, Oocyte, Zona pellucida, Lectin-binding, Gamete interaction.The zona pellucida (ZP), a relatively thick extracellular coat that surrounds mammalian oocytes, is known to mediate critical steps in fertilization including speciesspecific binding of spermatozoa, inducing the sperm acrosome reaction and preventing penetration of excess spermatozoa (reviewed in [1,2]). The mammalian ZP typically consists of a few glycoproteins which have different polypeptide chains and oligosaccharides [3][4][5][6][7][8] and evidence indicates that these oligosaccharides play an important role in sperm-oocyte recognition in all mammalian species (reviewed in [9][10][11]). Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity [12]. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the ZP of several mammalian species [13][14][15][16][17] including...

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Abstract. Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.

In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.