AMINO-ACIDS IN MATURATION MEDIUM AND PRESENCE OF CUMULUS CELLS AT FERTILIZATION PROMOTE MALE PRONUCLEAR FORMATION IN PORCINE OOCYTES MATURED AND PENETRATED IN-VITRO

The present study was conducted to examine the ability of porcine oocy tes to achieve male pronuclear (MPN) formation when they are matured a nd penetrated in vitro under various culture conditions. When cumulus- enclosed oocytes were cultured for 24-48 h in modified Whitten's mediu m (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/m l, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of cultur e. However, there were no differences in the proportions (94-95%) of o ocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM -FG supplemented with 2% (v:v) minimal essential medium (MEM) essentia l amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v :v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-9 7% of oocytes were penetrated regardless of the presence of amino acid s during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The a ddition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not aff ect sperm penetration in vitro, while it promoted MPN formation (76-83 %) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumul us cells after culture in mWM-FG, sperm penetration rates were not dif ferent between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) th an in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indica te that MPN formation in porcine oocytes is promoted by the addition o f amino acids and/or cysteine in simple maturation medium and by the p resence of cumulus cells at fertilization in vitro.