Maturation and fertilization of pig enucleolated oocytes.

ABSTRACT- Fully grown oocytes have a large nucleus, the germinal vesicle (GV), which includes a single large nucleolus. Since the nucleolus is transcriptionally inactive and disappears during oocyte maturation, the role of the nucleolus in oocyte maturation and subsequent fertilization has not been elucidated. In the present study, pig oocytes at the GV-stage were enucleolated (removed the nucleolus by micromanipulation), and subjected to intracytoplasmic sperm injection (ICSI). Enucleolated oocytes underwent germinal vesicle breakdown after 12 hr of culture (21/35). After 30 hr, they matured to metaphase II (MII) with a normal spindle (23/45). Sham-operated oocytes underwent germinal vesicle breakdown after 12 hr (22/35), and matured to MII (28/39) in the similar time course to that of enucleolated oocytes. Activities of Cdc2 kinase and MAP kinase were increased after 12 hr of maturation culture and the high activities were maintained until 30 hr in both enucleolated and sham-operated oocytes. When the matured oocytes were electro-stimulated, Cdc2 kinase activity in both group oocytes decreased to the basal level after 8 hr, and the activity of MAP kinase decreased after Cdc2 kinase inactivation. Enucleolated and matured oocytes were subjected to ICSI. Sham-operated oocytes formed male and female pronuclei (33/73) having the nucleoli (29/33) 24 hr after sperm injection, and the enucleolated oocytes form the male and female procnuclei (36/76) 24 hr after ICSI but no nucleolus was observed in both pronuclei (0/36). Enucleolated oocytes and nucleoli at GV-stage were cultured separately for 26 hr. Enucleolated oocytes matured to MII, while nucleoli were still intact. The cultured nucleoli were put back into enucleolated MII oocytes after 26 hr culture. These oocytes were cultured further 4 hr and subjected to ICSI. The enucleolated MII oocytes returned the nucleoli formed the male and female pronuclei with nucleoli 24 hr after ICSI. In summary, the prominent nucleoli in pig GV oocytes are not required for oocyte maturation and fertilization, and activation and inactivation of Cdc2 kinase and MAP kinase. However, it is indispensable for nucleolus formation in male and female pronuclei. This suggests that nucleoli in pronuclei of fertilized oocytes are maternal origin.[...