Ethylenediamine-N,N,N′,N′-Tetraacetic Acid Induces Parthenogenetic Activation of Porcine Oocytes at the Germinal Vesicle Stage, Leading to Formation of Blastocysts1

Abstract

The present study showed that treatment with a cell membrane-impermeable metal ion chelator, EDTA, of porcine oocytes at the germinal vesicle (GV) stage collected from follicles 2-6 mm in diameter induced artificial activation followed by formation of a pronucleus (PN). When the oocytes were cultured for 48 h in medium containing 0.1 to 2 mM EDTA disodium salt (Na-EDTA), they were activated to form PN, and the maximum PN formation rate (63%, n = 68) was achieved in oocytes cultured with 1 mM Na-EDTA. More than 90% of oocytes activated by 1 mM Na-EDTA treatment formed 1 PN without emission of the first and the second polar bodies (PB). This result suggests that EDTA at 1 mM may force the maturing (meiosis I) oocytes to form a PN without chromosome segregation. When oocytes at the GV stage that had been cultured with 1 mM Na-EDTA for 48 h were further cultured in 0.4% BSA-containing NCSU23 medium for 144 h, blastocysts that appeared to be morphologically normal were formed at the rate of 10%, whereas no blastocysts were formed from oocytes that had not been cultured with Na-EDTA. Next we examined the effects of Ca2+, Zn2+, Fe3+, or Cu2+-saturated EDTA (Ca-EDTA, Zn-EDTA, Fe-EDTA, and Cu-EDTA, respectively), and a Ca2+-specific chelator, EGTA, at a concentration of 1 mM. The Ca-EDTA, Fe-EDTA, and Cu-EDTA, but not Zn-EDTA or EGTA, had the ability to activate the oocytes. From these results, it is suggested that extracellular chelation of Zn2+ with EDTA of maturing (meiosis I) porcine oocytes results in parthenogenetic activation of the oocytes, which induces PN formation followed by development to blastocysts.

The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

“…Historically, Azuma et al . [ 3 ] were the first to report that porcine GV-stage oocytes can be parthenogenetically activated by treatment with Na-EDTA during maturation culture, inducing PN formation, followed by blastocyst formation. Moreover, they demonstrated that Ca-EDTA treatment could also activate porcine oocytes and induce precocious PN formation during oocyte meiosis [ 3 , 4 ].…”

“…[ 3 ] were the first to report that porcine GV-stage oocytes can be parthenogenetically activated by treatment with Na-EDTA during maturation culture, inducing PN formation, followed by blastocyst formation. Moreover, they demonstrated that Ca-EDTA treatment could also activate porcine oocytes and induce precocious PN formation during oocyte meiosis [ 3 , 4 ]. They suggested that the treatment of porcine oocytes with Ca-EDTA leads to a deficiency of extracellular Zn 2+ , alters the composition of the membrane and modifies intracellular conditions, eventually resulting in the promotion of parthenogenetic development.…”

“…COCs were matured in maturation medium (IVM medium), as described by Azuma et al . [ 3 ] with minor modifications. Briefly, IVM medium consisted of tissue culture medium (TCM) 199 (Earle’s salts) with 25 mM HEPES buffer (TCM 199; Life Technologies, Carlsbad, CA, U.S.A.) supplemented with 0.1% polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO, U.S.A.), 3.05 mM D-glucose (Wako, Osaka, Japan), 0.91 mM sodium pyruvate (Life Technologies), 10 IU/m l equine chorionic gonadotropin (Kawasaki-Mitaka K.K., Kawasaki, Japan), 10 IU/m l human chorionic gonadotropin (Kawasaki-Mitaka) and 50 µ g/m l gentamicin (Sigma-Aldrich).…”

“…In this case, PN formation starts directly from interphase, between the germinal vesicle breakdown (GVBD) and anaphase I/telophase I (AT) stages, and not via the MII stage. [ 3 , 4 ]. Ca-EDTA at a concentration of 1 mM has also been shown to induce PN formation from porcine and bovine GV-stage oocytes after 48 hr of maturation culture; however, mouse oocytes were not activated by this treatment [ 4 ].…”

Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

“…Artificial stimuli that elevate the cytoplasmic levels of calcium ions can induce activation of oocytes even without penetration by sperm (For review see KAUFMAN, 1983). A wide spectrum of activating stimuli, e.g.including ionophore, ethanol, electrical pulses, and other chemicals, is used to induce artificial activation of mammalian eggs (PRATHER et al, 1997;GRUPEN et al, 1999;AZUMA et al, 2001;JILEK et al, 2001;). However, the search for other suitable methods continues The present study was designed 1) to determine the effectiveness of two oocyte maturation media, 2) to compare the effect of two fertilization media on the developmental competence of pig oocytes, and 3) to test the activation ability of pig oocytes matured in the two maturation media, electroactivated, and cultured in mSOFaa.…”