- Email: [email protected]
Processing of nuclei transplanted into in vitro matured porcine oocytes
Theriogenology
39:322,
1993
PROCESSING OF NUCLEI TRANSPLANTED INTO IN VITRO MATURED PORCINE OOCYTES T.T. Stumpf, S.L. Terlouw, H. Funahashi, R.S. Prather and B.N. Day Department of Animal Science University of Missouri Columbia, MO 65211 USA When a cleavage stage porcine nucleus is transplanted into an enucleated in uiuo matured oocyte it swells in diameter (Prather et al., J. Exp. Zool. 255:365,1990). Our objectives were to determine if 1) maturational age of the oocyte and 2) activation of oocyte cytoplasm before nuclear transplantation alter processing of a transplanted nucleus. Initiation of pronuclear (PN) formation after electrical activation (EA) of oocytes at two maturational ages was first established Cumulus-oocyte complexes from slaughterhouse ovaries were cultured in TCM 199 containing 10% porcine follicular fluid with supplemental hormones (10 IU/ml PMSG and hCG, 1 pg/mll7/&estradiol) for 20 h and then without supplemental hormones until EA. Oocytes were stri ped of cumulus cells with 0.1% hyaluronidase at h 40 and EA at h 42 or h 64. 6 ocytes were EA (10 s exposure to 4.0 V/mm AC followed by 20 ps pulse of 1.2 kV/cm DC) in a solution of 0.25M mannitol supplemented with 5% TL-Hepes. After EA oocytes were cultured in TL-He es. Oocytes (N=383) were fued prior and at 2 h intervals for 8 h after & Resumption of meiosis was observed in a total of 82.6% (139/168) and 96.0% (182/190) of those oocytes matured to metaphase II (MID at h 42 and h 64, respectively. Initiation of PN formation of MI1 oocytes occurred within 6 h after EA at h 42 (12/31; 38.7%) and within 2 h after EA at h 64 (16/43; 37.2%). In a subsequent nuclear transplantation experiment, the intervals (6h and 2h) to initiation of PN formation were used to establish the interval between EA and fusion. Nuclei from 8-16 cell embryos were transplanted into oocytes which had been matured for 42 or 64 h. Donor nuclei were fused to enucleated recipient oocytes synchronous with EA (42h FA; 64h FA), 6 h after EA of oocytes matured 42 h (42h AF) and 2 h after EA of oocytes matured 64 h (64h AF). Nuclear diameters were determined by staining with 2 pg/ml Hoechst 33342 and visualization with W fluorescence. Measurements for each nucleus were taken before fusion and at 2,4 and 20 h post fusion. Data were analyzed separately by age of recipient oocyte for the influence of treatment. Change in Diameter (tim) of Trausnlanted Nuclei Pooled Recipient Trt” N 2h 20h Std Error Oocvte Ace FA 11 0.79 42h 2th5 20 1 1?6 162 42h AF 16 2012 22.2 24.9 2913 0.66 0.46 FA 8 15.0 17.0 19.4 22.6 64h 0.41 AF 10 15.7 18.5 21.0 24.2 64h “FA, Synchronous fusion of nuclei and EA of recipient oocytes. Al?, Fusion of nuclei 6h (42h) and 2h (64h) after EA of recipient oocytes. All nuclei of the 42h FA group were reduced in diameter by h 20, whereas nuclei of the 42h AF group swelled by h 20 (time x trt interaction, P < 0.01). However, all nuclei of both 64h FA and 64h AF groups swelled (P c 0.01) by h 20 (time x trt interaction, P > 0.05; time, P < 0.01). In summary, processing of a transplanted nucleus in an zn vitro matured pig oocyte is influenced by maturational age of the recipient oocyte and the interval between activation and fusion.
322
Copyright
0 1993 Butterworth-Heinemann
39:322,
1993
PROCESSING OF NUCLEI TRANSPLANTED INTO IN VITRO MATURED PORCINE OOCYTES T.T. Stumpf, S.L. Terlouw, H. Funahashi, R.S. Prather and B.N. Day Department of Animal Science University of Missouri Columbia, MO 65211 USA When a cleavage stage porcine nucleus is transplanted into an enucleated in uiuo matured oocyte it swells in diameter (Prather et al., J. Exp. Zool. 255:365,1990). Our objectives were to determine if 1) maturational age of the oocyte and 2) activation of oocyte cytoplasm before nuclear transplantation alter processing of a transplanted nucleus. Initiation of pronuclear (PN) formation after electrical activation (EA) of oocytes at two maturational ages was first established Cumulus-oocyte complexes from slaughterhouse ovaries were cultured in TCM 199 containing 10% porcine follicular fluid with supplemental hormones (10 IU/ml PMSG and hCG, 1 pg/mll7/&estradiol) for 20 h and then without supplemental hormones until EA. Oocytes were stri ped of cumulus cells with 0.1% hyaluronidase at h 40 and EA at h 42 or h 64. 6 ocytes were EA (10 s exposure to 4.0 V/mm AC followed by 20 ps pulse of 1.2 kV/cm DC) in a solution of 0.25M mannitol supplemented with 5% TL-Hepes. After EA oocytes were cultured in TL-He es. Oocytes (N=383) were fued prior and at 2 h intervals for 8 h after & Resumption of meiosis was observed in a total of 82.6% (139/168) and 96.0% (182/190) of those oocytes matured to metaphase II (MID at h 42 and h 64, respectively. Initiation of PN formation of MI1 oocytes occurred within 6 h after EA at h 42 (12/31; 38.7%) and within 2 h after EA at h 64 (16/43; 37.2%). In a subsequent nuclear transplantation experiment, the intervals (6h and 2h) to initiation of PN formation were used to establish the interval between EA and fusion. Nuclei from 8-16 cell embryos were transplanted into oocytes which had been matured for 42 or 64 h. Donor nuclei were fused to enucleated recipient oocytes synchronous with EA (42h FA; 64h FA), 6 h after EA of oocytes matured 42 h (42h AF) and 2 h after EA of oocytes matured 64 h (64h AF). Nuclear diameters were determined by staining with 2 pg/ml Hoechst 33342 and visualization with W fluorescence. Measurements for each nucleus were taken before fusion and at 2,4 and 20 h post fusion. Data were analyzed separately by age of recipient oocyte for the influence of treatment. Change in Diameter (tim) of Trausnlanted Nuclei Pooled Recipient Trt” N 2h 20h Std Error Oocvte Ace FA 11 0.79 42h 2th5 20 1 1?6 162 42h AF 16 2012 22.2 24.9 2913 0.66 0.46 FA 8 15.0 17.0 19.4 22.6 64h 0.41 AF 10 15.7 18.5 21.0 24.2 64h “FA, Synchronous fusion of nuclei and EA of recipient oocytes. Al?, Fusion of nuclei 6h (42h) and 2h (64h) after EA of recipient oocytes. All nuclei of the 42h FA group were reduced in diameter by h 20, whereas nuclei of the 42h AF group swelled by h 20 (time x trt interaction, P < 0.01). However, all nuclei of both 64h FA and 64h AF groups swelled (P c 0.01) by h 20 (time x trt interaction, P > 0.05; time, P < 0.01). In summary, processing of a transplanted nucleus in an zn vitro matured pig oocyte is influenced by maturational age of the recipient oocyte and the interval between activation and fusion.
322
Copyright
0 1993 Butterworth-Heinemann