In Vitro Development of Porcine Nuclear Transfer Embryos Reconstructed by Intracytoplasmic Microinjection of Cumulus Cell Nuclei into In Vitro Matured Oocytes.

Abstract

Abstract. Porcine nuclear transfer embryos were reconstructed by intracytoplasmic nuclear injection of cumulus cell nuclei into recipient oocytes matured in vitro by two types of method (NCSU23-based and TCM199-based methods). Remodelling of the transferred nuclei and in vitro development of the reconstructed embryos were investigated. Intracytoplasmic nuclear injection did not induce activation of the recipient oocytes regardless of the type of maturation method (NCSU23-based: 61/ 61, 100%, TCM199-based: 49/50, 98%). When the reconstructed embryos were examined 1 h after nuclear injection many of them (NCSU23-based: 22/29, 76%; TCM199-based: 19/23, 83%) possessed condensed chromosomes or a chromosome array resembling the maternal metaphase plate. The proportion of the reconstructed embryos possessing condensed chromosomes arranged in a scattered fashion showed a tendency to increase, when they were examined 2-5 h after nuclear injection. Reconstructed embryos were electrically activated either 2-2.5 h or 3.5-4 h after nuclear injection, followed by fixation/staining to examine formation of pronucleus-like nuclei (pseudo-nuclei). Regardless of the nuclear injection/activation interval tested, 68% (36/53) of the reconstructed embryos developed pseudo-pronuclei ranging in number between 1 and 4. When the reconstructed embryos were cultured for 7 days to assess their developmental competence, 5 (5/98) to 11% (12/111) developed to blastocysts regardless of the type of the recipient oocyte and nuclear injection/activation intervals. These results show that intracytoplasmic injection of porcine cumulus cell nuclei achieves a high rate of nuclear remodelling and that the reconstructed embryos are capable of developing into blastocysts.

Bovine Nucleus Transplantation by Intracytoplasmic Injection.

“…ICI, on the other hand, does not have the capability to activate oocytes [11,12], so it can be intentionally used for NT to introduce a donor nucleus into a cytoplasm at the second metaphase, or to prolong the time from nuclei injection to oocyte activation [12,20]. Exposure of somatic cell nuclei into enucleated oocytes at the M2 stage is considered to be the most important factor for developmental potency of NT mouse [12] and pig embryos [13,20]. ICI may also prove useful for elucidating factors governing developmental potency of NT embryos.…”

“…Intracytoplasmic nuclear injection was carried out as previously described using the so-called Honolulu method [11,12,20]. Briefly, the inner surface of the injection pipette was coated by washing several times with PB1 containing 12% polyvinylpyrrolidone (Sigma).…”

Development of pig cloning studies: past, present and future

“…Somatic cell nuclear transfer was used to produce a total of 90 live piglets from in vitro cultured: foetal fibroblast cells (Onishi et al, 2000;Park et al, 2001c;Boquest et al, 2002;Dai et al, 2002;De Sousa et al, 2002;Lai et al, 2002a,b;Walker et al, 2002;Hyun et al, 2003;Ramsoondar et al, 2003;Yin et al, 2003), skin fibroblasts (Bondioli et al, 2001), ear-derived fibroblasts (Park et al, 2002;Lee et al, 2003b), mural granulosa cells (Polejaeva et al, 2000), cells of an indefinite phenotype isolated from foetuses and their genital ridges (Betthauser et al, 2000), and heart cells (probably cardiomyocytes; Yin et al, 2002). In the experiments aimed at determining the in vitro developmental abilities of reconstructed porcine embryos to compact morula and blastocyst stages, foetal fibroblasts (Tao et al, 1999a,b;Koo et al, 2000;Uhm et al, 2000;Verma et al, 2000;Kuhholzer et al, 2000Kuhholzer et al, , 2001Lai et al, 2001;Park et al, 2001b) or cumulus/ mural granulosa cells (Cheong et al, 2000;Uhm et al, 2000;Park et al, 2001a;Martinez Diaz et al, 2002;Nagashima et al, 2002;Lee et al, 2003b;Samiec et al, 2003a,b) as well as ear skin fibroblasts of adult animals (Miyoshi et al, 2001;Roh and Hwang, 2002) were most often used as a source of donor nuclei.…”

“…In the second system cell nuclei at G1, G1/G0 or G2/M stages are introduced into Met II ooplasts, which are activated 30 minutes to several hours (3-4 hours at the most) after nuclear transfer (Betthauser et al, 2000;Kuhholzer et al, 2000;Onishi et al, 2000;Bondioli et al, 2001;Lai et al, 2001;Park et al, 2001a;Boquest et al, 2002;De Sousa et al, 2002;Hyun et al, 2003;Lee et al, 2003b;Ramsoondar et al, 2003;Samiec et al, 2003a,b). Microsurgical transfer of somatic nuclei can be an alternative method for clonal nuclear-cytoplasmic hybrid reconstruction (Tao et al, 1999;Kuhholzer et al, 2000;Onishi et al, 2000;Uhm et al, 2000;Park et al, 2001a;Nagashima et al, 2002;Samiec et al, 2003a,b) prior to cell fusion induced in the electric field. In terms of the molecular mechanisms of nuclear chromatin rearrangement, this has beneficial influences on epigenetic reprogramming and structural remodeling of exogenous genetic material.…”

Association Between Embryonic Loss and Damage to the Zona Pellucida by Invasive Micromanipulation During Oviductal Transfer of Early-Stage Embryos in Pigs

“…Among these technologies, somatic cell nuclear transfer (SCNT) is an important technology that enables the production of cloned and transgenic pigs. SCNT involves introduction of a donor nucleus into ooplasm, which can be performed by either electrofusion or cytoplasmic injection [2][3][4][5][6]. Both of these methods inevitably damage the zona pellucida during enucleation and i n s e r t i o n o f t h e d o n o r n u c l e u s b y m i c r omanipulation.…”