Search

Summary.Pig oocytes were matured in vitroin a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8·3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0·75–1·65 kV cm−1lasting 30 or 100 μs activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0·45 and 0·60 kV cm−1for 30 μs) frequently produced oocytes that remained in pre–pronuclear stages of activation (29·4 and 42·3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88·2% among the activated oocytes). The strongest stimulations used (1·05–1·65 kV cm−1for 100 μs) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56·3%). Under optimal stimulation (0·75 kV cm−1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase II at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5–6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32–36 h of culture) overlapped with the period in which the oocytes complete meiosis (28–38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7·3 intact cortical granules per 10 μm of overlying cytoplasmic membrane was found. This number dropped to 1·5 in 10 μm within 30 min after the pulse.Keywords:pig; oocyte; in vitromaturation; pronucleus; cortical granules

Summary.Oocyte–cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0·01–1·0 μg/ml) or forskolin (50–100 μmol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0·1 μg/ml) or forskolin (100 μmol/l) increased the contents of intracellular adenosine 3′,5′-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0·1 μg/ml) or forskolin (100 μmol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P< 0·01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.Keywords: FSH; cumulus expansion; hyaluronidase; oocytectomy; pig