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The effect of pig follicular fluid fractions on cumulus expansion and male pronucleus formation in porcine oocytes matured and fertilized in vitro

The aim of this study was to analyse the substance(s) present in porcine follicular fluid that promotes cumulus expansion and male pronucleus formation in pig oocytes matured and fertilized in vitro. The follicular fluid was separated into four fractions by ultracentrifugation at 220 000 g at 10°C for 48 h. Oocyte–cumulus-cell complexes obtained from prepubertal gilts were cultured in vitro for 48 h in each of the fractions after reconstitution with a modified Krebs–Ringer bicarbonate solution. Each fraction containing oocyte–cumulus complexes was then fertilized in vitro with frozen–thawed and preincubated epididymal boar spermatozoa. After 24 h of culture, oocytes that matured in fraction I showed a marked expansion of the surrounding cumulus, as did those matured in pig follicular fluid. However, complexes cultured in the remaining fractions exhibited very little or no expansion. No difference was observed in the degree of expansion caused by follicular fluid collected from small, medium or large follicles. Further analysis of fraction I by HPLC gave several subfractions, one of which (22) induced a significantly greater expansion of the cumulus than did others (P < 0.01). Fraction 1 also induced higher rates of male pronuclei formation (62%) than did fractions 2, 3 and 4 (22%, 35% and 30%, respectively). The rate of male pronuclei formation in pig follicular fluid from large follicles was significantly lower (33%) than in fluid from small or medium follicles (89% and 78%, respectively). Two subfractions (22 and 23) derived from HPLC of fraction 1 produced the highest rate of male pronuclei formation compared with the other subfractions (which showed very minimal rates of male pronuclei formation). The active factor(s) responsible for cumulus expansion was heat stable at 100°C for 15 min, resistant to freezing and thawing, and did not lose its activity completely when subjected to proteinase K digestion. The molecular mass of the active factor(s) was < 6.5 kDa, as measured by HPLC.

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