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Artificial activation of bovine oocytes matured in vitro by electric shock or exposure to ionophore A23187
THERIOGENOLOGY
ARTIFICIAL ACTIVATION OF BOVINE OOCYTES MATURED IN VITRO BY ELECTRIC SHOCK OR EXPOSURE TO IONOPHORE A23187 Y.Aoyagi, K.Kameyama and T.Takeda Central Research Institute Zen-Noh, Tsukuba, 300-33, Japan Artificial activation of the recipient ooplasm may be important to the success of nuclear transplantation. This study attemptedto examine the effect of the electric DC pulse (EP) duration or the concentration of ionophore (IA) on the activation and subsequent development of in vitro matured bovine oocytes. Ovaries were obtained at a slaughterhouse, and oocytes were aspirated from 2 to 5 mm follicles. Cumulus-oocyte complexes were cultured in TCM199 + 10% FCS for 30h, then stripped of cumulus cells with 0.1% hyaluronidase. Oocytes possessing the first polar body were selected and allocated to one of the following treatments: (A) 15OV/mm EP for 0, 10, 30, 90, 120 ,usec, (B) IA (0, 0.1, 1, 5 plM) treatments for 5 min. Oocytes were cultured for 12-14h before fixing and staining with 1% aceto-orcein. Oocytes that reached pronuclear formation were considered activated. In the next experiment, oocytes were activated by either EP or IA prior to culturing in TCM 199 + 10% FCS containing cytochalasin D (2.5 pglml) for 1214 h. They were cultured in CR1 (Theriogenology, 1991., 35, 266) for 7 d to examine the development. Activation rates after EP or IA treatment are presented in the tables. Table 1.
Effect of EP duration on activation
EP Duration(,usec) 0 10
30 90 120 Different superscripts 0.05). Table 2.
1 5
No. Activated(%) 2 ( 5.9)a 6 (23.1)a 17 (56.7)b 28 (87.5)c
30 denote a significant
Effect of IA concentration
IA Conc.(,uM) 0 0.1
No. Pulsed 34 26 30 32
rates
27 (90.0)c difference(P<
on activation
rates
No. Examined 25
No. Activated(%) i ( 4.0)a
24 22 29
3 (i2.5)a 16 (72.7)b 27 (93.l)b
'fferent Di 0.05). Both EP (90 psec) and IA (5&M) activated OOCyteS with cytochalasin D treatment had two pronuclei (EP:83% or IA: 87%) and developed into morphological blastocysts (EP:7% or IA:9%). 188
JANUARY
1992 VOL. 37 NO. 1
ARTIFICIAL ACTIVATION OF BOVINE OOCYTES MATURED IN VITRO BY ELECTRIC SHOCK OR EXPOSURE TO IONOPHORE A23187 Y.Aoyagi, K.Kameyama and T.Takeda Central Research Institute Zen-Noh, Tsukuba, 300-33, Japan Artificial activation of the recipient ooplasm may be important to the success of nuclear transplantation. This study attemptedto examine the effect of the electric DC pulse (EP) duration or the concentration of ionophore (IA) on the activation and subsequent development of in vitro matured bovine oocytes. Ovaries were obtained at a slaughterhouse, and oocytes were aspirated from 2 to 5 mm follicles. Cumulus-oocyte complexes were cultured in TCM199 + 10% FCS for 30h, then stripped of cumulus cells with 0.1% hyaluronidase. Oocytes possessing the first polar body were selected and allocated to one of the following treatments: (A) 15OV/mm EP for 0, 10, 30, 90, 120 ,usec, (B) IA (0, 0.1, 1, 5 plM) treatments for 5 min. Oocytes were cultured for 12-14h before fixing and staining with 1% aceto-orcein. Oocytes that reached pronuclear formation were considered activated. In the next experiment, oocytes were activated by either EP or IA prior to culturing in TCM 199 + 10% FCS containing cytochalasin D (2.5 pglml) for 1214 h. They were cultured in CR1 (Theriogenology, 1991., 35, 266) for 7 d to examine the development. Activation rates after EP or IA treatment are presented in the tables. Table 1.
Effect of EP duration on activation
EP Duration(,usec) 0 10
30 90 120 Different superscripts 0.05). Table 2.
1 5
No. Activated(%) 2 ( 5.9)a 6 (23.1)a 17 (56.7)b 28 (87.5)c
30 denote a significant
Effect of IA concentration
IA Conc.(,uM) 0 0.1
No. Pulsed 34 26 30 32
rates
27 (90.0)c difference(P<
on activation
rates
No. Examined 25
No. Activated(%) i ( 4.0)a
24 22 29
3 (i2.5)a 16 (72.7)b 27 (93.l)b
'fferent Di 0.05). Both EP (90 psec) and IA (5&M) activated OOCyteS with cytochalasin D treatment had two pronuclei (EP:83% or IA: 87%) and developed into morphological blastocysts (EP:7% or IA:9%). 188
JANUARY
1992 VOL. 37 NO. 1