Effects of EDTA saturated with Ca2+ (Ca-EDTA) on pig, bovine and mouse oocytes at the germinal vesicle stage during maturation culture and the involvement of chelation of Zn2+ in pronuclear formation induction by Ca-EDTA

Abstract

Preovulatory mammalian oocytes at the germinal vesicle (GV) stage, when removed from follicles, can spontaneously undergo meiotic maturation in culture, resulting in secondary oocytes arrested at metaphase of the second meiotic division (MII). The duration of the period required for the meiotic maturation of mammalian oocytes in culture varies depending on the species: in mice, cattle and pigs it is about 12, 24 and 48 h, respectively. During the meiotic maturation period, there are two distinct successive metaphases: metaphase I (MI) and metaphase II (MII). The interval between the two meiotic divisions, known as interkinesis, is very short and is not accompanied by an S phase (or DNA replication).Meiotic progression is regulated by the activity of metaphase-promoting factor (MPF), a heterodimer composed of a catalytic p34 cdc2 kinase and a cyclin B-regulatory subunit. Entry into metaphase requires an accumulation of MPF activity (reviewed by Nigg, 1993). When the MPF activity of oocytes at the GV stage is inhibited during meiosis I by moderate ectopic expression of Wee1 kinases (Nakajo et al., 2000), or by failure to accumulate cyclin B due to injection of antisense c-mos oligonucleotide (O'Keefe et al., 1989(O'Keefe et al., , 1991, although the oocytes undergo germinal vesicle breakdown (GVBD), interphase nuclear formation can be induced just after meiosis I.It has been reported that treatment of pig oocytes at the GV stage with a cell membrane-impermeable metal ion chelator, EDTA or EDTA saturated with Ca 2+ (Ca-EDTA) at 0.1-2.0 mmol l -1 , induced formation of a pronucleus after 48 h of maturation culture. Moreover, time-course experiments on the nuclear status of oocytes treated with EDTA during the maturation period revealed that the pronuclear formation resulted from the lack of meiotic maturation before the completion of meiosis I (Azuma et al., 2001). Notably, the oocytes with a pronucleus that formed precociously as a result of EDTA treatment could develop parthenogenetically Reproduction (2002) for 48 h to examine the effects of Ca-EDTA treatment on these oocytes during maturation culture. Most (70-86%) of the bovine oocytes that underwent germinal vesicle breakdown matured to the MII stage via the MI phase, regardless of whether Ca-EDTA was present for the first 24 h of culture. However, 61% of oocytes that had been cultured with Ca-EDTA for 48 h formed a pronucleus without a second polar body, whereas oocytes cultured in the absence of Ca-EDTA were not observed to form a pronucleus at any time during culture. However, even when mouse oocytes at the germinal vesicle stage were cultured for up to 48 h in maturation medium containing Ca-EDTA, pronuclear formation was not observed. Finally, when bovine oocytes that had been cultured with 1 mmol Ca-EDTA l -1 for 48 h from the germinal vesicle stage were cultured further in medium without Ca-EDTA that was supplemented with 5% fetal calf serum, only 26% of the oocytes developed to the cleaved stage, and none could develop further.

Effect of dentin etching and chlorhexidine application on metalloproteinase‐mediated collagen degradation

“…The ultrastructural features of collagen in EDTA-and PA-demineralized dentin beams were found to be similar for both groups (17). As EDTA has been shown to be an effective Zn 2+ and Ca 2+ chelator (27), it was expected to inhibit MMP activity. However, EDTA (or Ca-EDTA) has to be present in excess, relative to Zn 2+ , for the chelating efficiency of EDTA to be observed (27).…”

“…As EDTA has been shown to be an effective Zn 2+ and Ca 2+ chelator (27), it was expected to inhibit MMP activity. However, EDTA (or Ca-EDTA) has to be present in excess, relative to Zn 2+ , for the chelating efficiency of EDTA to be observed (27). In our study, most traces of EDTA were removed from dentin by extensive rinsing with water, so there was no residual EDTA in EDTA-demineralized dentin beams to inhibit the activity of MMPs.…”

Zinc-Inhibited MMP-Mediated Collagen Degradation after Different Dentine Demineralization Procedures

“…EDTA-demineralized dentine presented MMP collagen degradation values similar to those of PA-demineralized dentine [Osorio et al, 2011b]. It may be that EDTA (or calcium EDTA) has to be present in excess in order to be an effective zinc and calcium chelator [Azuma et al, 2002]. However, both substances lost their effect after 24 h, probably due to precipitation in the presence of cations derived from simulated body fluids [Di Palma et al, 2003;Zerella et al, 2005].…”

“…Ethylenediaminetetraacetic acid (EDTA) has been shown to be an effective zinc and calcium chelator [Azuma et al, 2002]. However, EDTA-demineralized dentine presented MMP collagen degradation values similar to those of phosphoric acid (PA)-demineralized dentine [Osorio et al, 2011b].…”

Toward Understanding the Genetic Basis of Yak Ovary Reproduction: A Characterization and Comparative Analyses of Estrus Ovary Transcriptiome in Yak and Cattle

“…In addition, the membrane concentration gradient established by the ion transmembrane transport provides the energy source for a series of cell metabolic activities. In many cation transmembrane transport activities, the transport of calcium and zinc ions plays an important role in the regulation of ooctye maturation [ 33 – 35 ]. In this study, the enrichment of cation transmembrane transport-related GO categories confirmed that a different regulation of ion-transmembrane transport exists in yak ovaries compared with cattle ovaries, but its exact function requires further study.…”