Colcemid treatment during oocyte maturation improves preimplantation development of cloned pig embryos by influencing meiotic progression and cytoplasmic maturation

Abstract

The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98.4%) than in controls not exposed to colcemid (68.7%). The proportion of metaphase-II (MII) oocytes after 30 hr of IVM was higher in control (79.6%) than in COL0-22 oocytes (61.7%); overall nuclear progression to the MII stage was not influenced by colcemid treatment by the end of the IVM period (93.8, 86.7, 86.8, and 84.8% for control, COL0-22, COL38-42, and COL0-22/38-42, respectively). COL0-22 oocytes showed higher intra-oocyte glutathione content (1.7 vs. 1.0-1.3 pixels/oocyte) and increased blastocyst formation after PA (68.7% vs. 42.5-52.2%) and SCNT (39.4% vs. 16.3-28.6%) than control, COL38-42, and COL0-22/38-42 oocytes. Colcemid treatment for 0-22 and 0-22/38-42 hr of IVM also stimulated the expression of cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNAs. Our results thus demonstrate that the presence of colcemid during the early stage of IVM stimulates preimplantation development of PA and SCNT porcine embryos by improving the cytoplasmic microenvironment.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

“…Demecolcine is a microtubule‐depolymerizing agent that has been shown to efficiently induce partial enucleation of oocytes (Yin et al, ) and has been used for post‐activation of SCNT oocytes (Song, Hyun, Shin, & Lee, ) and in vitro oocyte maturation (Lee et al, ) in pigs. Demecolcine disturbs microtubule polymerization by binding tightly to tubulin dimers, preventing the formation of spindle microtubules by depolymerization (Ibáñez, Albertini, & Overström, ).…”

Choosing a culture medium for SCNT and iSCNT reconstructed embryos: from domestic to wildlife species

“…In pigs, supplementation of in vitro maturation (IVM) culture with lanosterol improved preimplantation development of SCNT embryos, possibly by elevating lipid content of oocytes or regulating genes involved in the cholesterol biosynthetic pathway in cumulus cells or those involved in lipid metabolism and apoptosis, with a positive effect on blastocyst development [ 54 ]. Furthermore, the addition of rapamycin [ 55 ], colcemide [ 56 ], or coculture with denuded oocytes and their oocyte secreted factors [ 57 , 58 ] were also tested during IVM in order to stimulate preimplantation development in SCNT embryos.…”

Padrão de metilação do gene XIST e da ICR/H19 em ovócitos imaturos e maturados in vitro de porcas cíclicas e marrãs pré-púberes

“…A aquisição dessa competência ocorre, in vivo, durante a sua fase de crescimento, ou seja, simultaneamente ao desenvolvimento do folículo. In vitro, a maturação espontânea dos ovócitos causa uma assincronia entre a maturação nuclear e citoplasmática levando a uma diminuição da competência (Lee et al, 2015).…”

“…Na maturação citoplasmática há uma reorganização de estruturas para a periferia do ovócito e na maturação molecular o acúmulo de nutrientes, substratos e mRNA que são necessários para o término da maturação, fecundação e para o futuro desenvolvimento embrionário inicial (Watson, 2007;Gottardi e Mingoti, 2009). Nesse período, é importante que haja uma sincronização entre a maturação nuclear e citoplasmática para que se obtenha uma boa eficiência da PIVE (Lee et al, 2015).…”