Gene Expression in Early Expanded Parthenogenetic and In Vitro Fertilized Bovine Blastocysts

Abstract

Abstract. Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66 shc and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66 shc within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling. Key words: Bovine, Embryo, Gene expression, Parthenote (J. Reprod. Dev. 55: [607][608][609][610][611][612][613][614] 2009) arthenogenesis, a form of reproduction not spontaneous in mammals, is a process by which the oocyte develops without the male gamete. Mammalian parthenotes are unable to develop to term in utero (developing for a maximum of 48 days in the cow; [1]), and this is thought to be due to alterations in their genomic imprinting, a mechanism that provides complementary, but not equivalent, specific expression between maternal and paternal genomes [2]. During fertilization, the spermatozoon triggers multiple and rhythmic oscillations of intracellular free calcium that release the oocyte from this blocked stage [3]. These intracellular calcium patterns can be mimicked by a number of chemicals that activate the oocyte without sperm, yielding parthenogenetic embryos [4]. In cattle, production of reconstructed embryos by somatic cell nuclear transfer (SCNT) [5] and intracytoplasmic sperm injection (ICSI) [6] depends on successful oocyte activation. Therefore, procedures involving SCNT and ICSI could benefit from a better knowledge of mechanisms that differ between embryos and parthenotes.Contrary to the research in mice and humans, most studies have failed to detect putatively imprinted genes in bovine early development [7,8], suggesting that monoallelic expression may not develop until after Day 21 of development, as occurs in ovine species [9]. In fact, a recent study found imprinted expression of bovine Mest-1 in Day-21 but not Day-14 embryos, while 7 other putatively imprinted genes were shown to be not imprinted at the two stages analyzed [10]. However, gene expression does differ between blastocysts from embryos and parthenotes in domestic species [11...

Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation

“…In cow, biopsies of embryos resulting in calf delivery contained more PTGS2, which suggests an important role in the implantation process mediated by embryo-uterus interaction (El-Sayed et al 2006). PTGS2 was also found to be more abundant in Day-7 and Day-8 parthenotes compared with fertilised embryos (Gomez et al 2009a(Gomez et al , 2009b, which supports our microarray data, although quantitative RT-PCR did not quite confirm the overrepresentation of this transcript (P ¼ 0.0552). However, it has been reported that parthenotes contain fewer TE cells than fertilised embryos, but the number of cells in the ICM were reported to be unaltered (Gomez et al 2009b).…”

Expression of placenta-specific 8 in human oocytes, embryos, and models of in vitro implantation

“…PLAC8 expression has been reported in many species, such as zebrafish (3), mice (1), bovines (4)(5)(6)(7)(8)(9), and rabbits (16). However, little is known regarding PLAC8 expression in human oocytes and embryos.…”

“…These findings suggest that PLAC8 plays a crucial role in normal embryo development (3). In the evaluation of bovine embryo quality, PLAC8 is a biomarker that is used to predict pregnancy outcome (4)(5)(6)(7)(8)(9). Two recent reports investigated bovine embryos derived in vitro (4) and in vivo (10), and they demonstrated that PLAC8 was highly expressed in cells from embryos that were capable of developing into calves.…”

Effect of zona pellucida removal on early development of in vitro produced bovine embryos

“…In previous studies it has been demonstrated that a change in the expression pattern of pluripotency markers such as OCT4 is related with the regulation of the expression and the balance of pro and anti apoptotic proteins. For instance, an increase in the BAX/ BCL2 ratio is consistent with the reduction of the OCT4 expression in bovine blastocysts produced by parthenogenesis (Gómez et al 2009) while pig parthenotes treated with melatonin showed a decrease of BAX expression and increased of BCL2L1 and OCT4 expression (Choi et al 2008). In the experiments presented here, there was no drift in the expression of OCT4 as a function of the apoptotic activity as judged by the expression of the studied genes.…”

“…Potentially the absence of zona pellucida during embryo culture could provoke a lack of contact between the blastomeres, thus it might be expected an increase of cell death by apoptosis and consequently an induction of apoptotic mechanisms. Apoptosis occurs during early embryo development stages both in in vitro and in vivo produced embryos; however, the incidence of cell death by apoptosis might increase as a consequence of the procedures for in vitro embryo production (Gómez et al 2009).…”