Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Abstract

Abstract. The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term. Key words: Aged donor, Clawn miniature pig, Cytoskeletal inhibitors, Latrunculin A, Nuclear transfer (J. Reprod. Dev. 58: [398][399][400][401][402][403] 2012) C lawn miniature pigs (inbred strain) have a potential value for biomedical research and xenotransplantation due to their clear genetic background [1][2][3] and few individual differences; thus, they have stimulated investigation of novel strategies directed at generating a genetically modified miniature pig by somatic cell nuclear transfer (SCNT). We have shown how to produce live offspring derived from Clawn miniature pig SCNT embryos; however, the efficiency still remains low [4]. To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15]. SCNT embryos derived from diploid cells are generally treated with cytoskeletal inhibitors, such as cytochalasin B (CB), after activation to prevent extrusion of a pseudo polar body (pPB) and the possible loss of chromosomes maintain normal ploidy [7,16,17]. Several reports have shown that CB treatment of SCNT embryos resulted in increased blastocyst formation in vitro [18][19][20][21]. However, it has been reported that postactivation treatment with CB had no effect on either blastocyst rate or cell number of SCNT embryos [9,22]. CB possess cytotoxicity [23], and its cytotoxicity might have a detrimental effect on the subsequent development of SCNT embryos.Latrunculin A (LatA), a toxin purified from the Red Sea sponge Latrunculia magnifica, is a member of the cytoskel...

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

“…After electric activation of in vitro -matured oocytes, these were cytoplasmically injected with a solution (~2 pL) containing in vitro synthesized hCas9 mRNA (2 ng/μL), gRNA (2 ng/μL; specific to GGTA1 exon 4), and EGFP mRNA (2 ng/μL) and were then cultured in vitro until blastocyst formation for approximately seven days. From a total of 107 oocytes injected, both the cleavage rate (79.4%) and blastocyst formation rate (34.6%) were comparable to those of intact PA oocytes (67.7% and 33.1% for cleavage and blastocyst formation, respectively) described in a previous article [ 44 ]. Thus, the concentration of RNA used does not seem to be harmful for porcine embryonic development.…”

Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes

“…1A, oocytes that were matured in vitro immediately or 6 h after electric activation were injected with a solution containing Cas9 mRNA, gRNA, and EGFP mRNA into the cytoplasm and then cultured in vitro for 7 days until the blastocyst stage. In both groups, the cleavage rate (69.6% vs. 78.8%; Table 1) and blastocyst formation rate (46.6% vs. 48.2%; Table 1) were comparable to those of intact PA oocytes (67.7% for cleavage and 33.1% for blastocyst formation; [32]). Thus, the timing of the RNA injection does not appear to be a critical factor for porcine embryonic development.…”

“…Porcine oocytes were prepared as previously described by Himaki et al [32]. Briefly, ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory.…”

“…For PA oocyte production, denuded oocytes were placed between two wire electrodes 1 mm apart in activation medium (250.3 mM sorbitol, 0.5 mM Ca(CH 3 COO) 2 , 0.5 mM Mg(CH 3 COO) 2 , and 0.1% BSA) [32]. Activation was induced with one direct current pulse of 100 V/mm for 50 μs using an LF 101 Fusion Machine (Nepa Gene Co., Chiba, Japan).…”

“…Successful injection was confirmed by a slight swelling of the oocyte cytoplasm. The injected oocytes were cultured in a drop (50 L) of modified PZM-3 (mPZM-3) medium [33] containing 0.5 M latrunculin A (LatA) (# L5163; Sigma-Aldrich Co.) for 2 h at 38.5 °C to increase their in vitro developmental rate [32]. After washing with mPZM-3 medium three times, these oocytes were cultured in mPZM-3 medium under an atmosphere of 5% CO 2 /5% O 2 /90% N 2 at 38.5 C.…”