COCULTURE WITH PIG MEMBRANA GRANULOSA-CELLS MODULATES THE ACTIVITY OFCDC2 AND MAP KINASE IN MATURING CATTLE OOCYTES

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis ( 8h of initial culture) or metaphase I (12h of initial culture), were s ubsequently co-cultured for 6h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determ ined by Western blotting. The alterations in nuclear envelope breakdow n, meiotic spindle formation and the patterns of chromosome condensati on were analysed by immunofluorescence and transmission electron micro scopy. The diakinesis-stage oocytes (initially cultured for 8h) alread y possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. T he ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP k inase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20h of culture without PMG. The metaphase I oocytes, initially cultured for 12h, displayed a fusif orm meiotic spindle and a metaphase array of chromosomal bivalents, ac companied by high levels of both histone H1 and MAP kinase activity. G o-culture of MI oocytes with PMG abolished the activity of both kinase s and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, th e spindle microtubules were depolymerised and the chromosomal bivalent s clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that u nder in vitro conditions membrana granulosa cells can cause a prompt d ecrease in histone H1 and MAP kinase activities, and metaphase I oocyt es. While these events are fully reversible in late diakinesis oocytes , metaphase I oocytes did not complete maturation after release from c o-culture.