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Preimplantation development of embryos produced by intergeneric nuclear transplantation
THERIOGENOLOGY PRE~PLA~ATION DEVELOPMENT INTERGENERIC NUCLEAR
OF EMBRYOS PRODUCED TRANSPLANTATION
BY
B.A. Wolfel, ME. Westhusin2, MJ. Levanduski2, K.R. BondioliZ and D.C. Kraemert lDepartment of Vemrhmry Physiology and Pharmacology Texas Agricultttral Experiment Station Texas A&M University, College Station, TX 77843 USA 2Granada Genetics Inc., 100 Research Parkway, Suite 100 College Station, TX 77840 USA A preliminary study was conducted to exam& the developmental capacity of embryos produced by ihc transfer of nucbi from prehnplantation embryos to enucleated ova from species of varying genetic distance fmm the nuclear donor. Superovulation was induced for the cogection of tmfertiked ova from American bison (n-10). Splunishgoats (n=iS), Syrian hamstas (n=6). and domestic cows @=X2). Ova wete recover& from the oviducts of each species approximately 36h after the onset of esmts in bison, goats, and domestic cows, and 14-16h following human chorionic ~~ injection in hamsmrs Ali ova were recovcrcd and heId in modified phosphate buffered saline solution containing 0.4% bovine serum aIburnin and l(X) IUlml ~ci~~s~ep~mycin (mPBSA). Enucleation of ova was performed in mPBSA + Sug/mi cytochalasin B by cutting the zona pellucida and aspirating away half of the cytoplasm. A singic blastomere from a 4.5 to 5Sday domestic bovine anbryos was pked against each haif-ovum, and the pair was subjectedto electrofusion in Zimmerman cell fusion medium using a BTK Electra Cell Manipulator 200 (Biotechnologies and Experimental Research, Inc.; San Diego, CA). Following lh incubation in PBS + 20% newborncalf serum, electtically treated cells were evaluated for fusion, pooled, embedded in doublclayer agar chips (1.0% and 1.2%). and transferred to the oviducts of Rambouillet ewes for six days. Each ewe received a Synchro-Mate B implant (Ceva Corp.; Overland Park, KS) during the six day culture period. Upon recovery of rhe a8ar chips, &velopment of embryos was evahutted and nuclear division was confirmed in intergeneric embryos by nuclear staining. Control inuaspecifii transfers were conducted in small groups throughout the course of the study. Nuclear donor embryos were collected on the day of manipulation from supemvulated domestic cows in all cases with the exception of 12 bovine-bison nuclear transplant embryos for which the donor embryo was frozen/thawed. No development was observed among these 12 follnwmg in viw cuIture. ElCCnically
Recipient
treated
ovum GllkWS-
cells
Bison Goat Hamster Domestic cow (control)
80 112 63 185
Fused/ Recovered Recovemd ._&I-~~5_16ceu~ 52 (65) 25 (48) 83 (74.1) 33 (39.8) 51 (81) 28 (54.9) 134 (72.4)
67 (50)
Developmentai stage after six-day in viva culture 1% reu)vered1
0 0 0 13 (9.7)
g.6) 3
0 :
0 0
5 (3.7)
l(1.9) 1 (1.2) 0 26 (19.4)
No cleavage was observed when hamster ova were nuclear recipients. Four cleaved bovine-goat nuclc:u transplant embryos contained nucleated cells: one rno~~i~~y normal blastocyst and three 6- to IOcelled embryos A single blastocyst containing approximately 20-25 cells was the only cleavage obscrvcci when bovine nuclei were transferred to bison half-ova. The pteaence of nucleated half-ova of the recipient species in in viva cuhure precludes elimination of the possibility of parthenogenic cleavage. However, the development of biastocysts from the nansfer of domestic bovine embryo nuclei to bison and goat half-ova in this study suggests that mammalian nuclei may be capable of interacting with cytoplasm from oihcr mammalian species to support normal development Confmon of this will be accomplished following transfer of resulting embryos to recipient females of the nuclear donor species. The authors gratefully acknowledge the assistance of S. Dupiantis, R. Powell, C. Long, Dr. D. Davis. ;uttl Dr. R. Simpson.
350
JANUARY 1990 VOL. 33 NO. 1
OF EMBRYOS PRODUCED TRANSPLANTATION
BY
B.A. Wolfel, ME. Westhusin2, MJ. Levanduski2, K.R. BondioliZ and D.C. Kraemert lDepartment of Vemrhmry Physiology and Pharmacology Texas Agricultttral Experiment Station Texas A&M University, College Station, TX 77843 USA 2Granada Genetics Inc., 100 Research Parkway, Suite 100 College Station, TX 77840 USA A preliminary study was conducted to exam& the developmental capacity of embryos produced by ihc transfer of nucbi from prehnplantation embryos to enucleated ova from species of varying genetic distance fmm the nuclear donor. Superovulation was induced for the cogection of tmfertiked ova from American bison (n-10). Splunishgoats (n=iS), Syrian hamstas (n=6). and domestic cows @=X2). Ova wete recover& from the oviducts of each species approximately 36h after the onset of esmts in bison, goats, and domestic cows, and 14-16h following human chorionic ~~ injection in hamsmrs Ali ova were recovcrcd and heId in modified phosphate buffered saline solution containing 0.4% bovine serum aIburnin and l(X) IUlml ~ci~~s~ep~mycin (mPBSA). Enucleation of ova was performed in mPBSA + Sug/mi cytochalasin B by cutting the zona pellucida and aspirating away half of the cytoplasm. A singic blastomere from a 4.5 to 5Sday domestic bovine anbryos was pked against each haif-ovum, and the pair was subjectedto electrofusion in Zimmerman cell fusion medium using a BTK Electra Cell Manipulator 200 (Biotechnologies and Experimental Research, Inc.; San Diego, CA). Following lh incubation in PBS + 20% newborncalf serum, electtically treated cells were evaluated for fusion, pooled, embedded in doublclayer agar chips (1.0% and 1.2%). and transferred to the oviducts of Rambouillet ewes for six days. Each ewe received a Synchro-Mate B implant (Ceva Corp.; Overland Park, KS) during the six day culture period. Upon recovery of rhe a8ar chips, &velopment of embryos was evahutted and nuclear division was confirmed in intergeneric embryos by nuclear staining. Control inuaspecifii transfers were conducted in small groups throughout the course of the study. Nuclear donor embryos were collected on the day of manipulation from supemvulated domestic cows in all cases with the exception of 12 bovine-bison nuclear transplant embryos for which the donor embryo was frozen/thawed. No development was observed among these 12 follnwmg in viw cuIture. ElCCnically
Recipient
treated
ovum GllkWS-
cells
Bison Goat Hamster Domestic cow (control)
80 112 63 185
Fused/ Recovered Recovemd ._&I-~~5_16ceu~ 52 (65) 25 (48) 83 (74.1) 33 (39.8) 51 (81) 28 (54.9) 134 (72.4)
67 (50)
Developmentai stage after six-day in viva culture 1% reu)vered1
0 0 0 13 (9.7)
g.6) 3
0 :
0 0
5 (3.7)
l(1.9) 1 (1.2) 0 26 (19.4)
No cleavage was observed when hamster ova were nuclear recipients. Four cleaved bovine-goat nuclc:u transplant embryos contained nucleated cells: one rno~~i~~y normal blastocyst and three 6- to IOcelled embryos A single blastocyst containing approximately 20-25 cells was the only cleavage obscrvcci when bovine nuclei were transferred to bison half-ova. The pteaence of nucleated half-ova of the recipient species in in viva cuhure precludes elimination of the possibility of parthenogenic cleavage. However, the development of biastocysts from the nansfer of domestic bovine embryo nuclei to bison and goat half-ova in this study suggests that mammalian nuclei may be capable of interacting with cytoplasm from oihcr mammalian species to support normal development Confmon of this will be accomplished following transfer of resulting embryos to recipient females of the nuclear donor species. The authors gratefully acknowledge the assistance of S. Dupiantis, R. Powell, C. Long, Dr. D. Davis. ;uttl Dr. R. Simpson.
350
JANUARY 1990 VOL. 33 NO. 1