The L–H transition in a helical-axis heliotron, Heliotron J, is investigated. For electron cyclotron heating (ECH), neutral beam injection (NBI) heating and ECH + NBI combination heating plasmas, the confinement quality of the H-mode is examined with an emphasis on its magnetic configuration dependence. The vacuum edge rotational transform, ι(a)/2π, is chosen as a label for the magnetic configuration where ι/2π is the rotational transform and a is the average plasma minor radius in metres. The experimental ι(a)/2π dependence of the enhancement factor over the L-mode confinement reveals that specific configurations exist where high-quality H-modes (1.3 < HISS95 < 1.8) are attained. is the experimental global energy confinement time and is the confinement time scaling from the international stellarator database given as . R is the plasma major radius in metres, is the line-averaged plasma density in 1019 m−3, PL is the power loss in megawatts that accounts for the time derivative of the total plasma energy content and Bt is the toroidal magnetic field strength in tesla (Stroth U. et al 1996 Nucl. Fusion 36 1063). The ι (a)/2π ranges for these configurations are near values that are slightly less than those of the major natural resonances of Heliotron J, i.e. n/m = 4/8, 4/7 and 12/22. To better understand this configuration dependence, the geometrical poloidal viscous damping rate coefficient, Cp, is calculated for different values of ι(a)/2π and compared with the experimental results. The threshold line-averaged density of the H-mode, which depends on the configuration, is in the region of 0.7–2.0 × 1019 m−3 in ECH (0.29 MW) + NBI (0.57 MW) operation. As for the edge plasma characteristics, Langmuir probe measurements have shown a reduced fluctuation-induced transport in the region that begins inside the last closed flux surface (LCFS) and extends into the scrape-off layer. In addition, a negative radial electric field Er (or Er-shear) is simultaneously formed near the LCFS at the transition.

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.

Abstract.To elucidate the beneficial effects of follicular fluid (FF) added to maturation medium on nuclear maturation and postfertilization development of bovine oocytes in vitro, we attempted to separate largely bovine FF (bFF) collected from small follicles into heparin-binding and -nonbinding fractions (HBF and HNF, respectively) by heparin affinity chromatography. Each fraction was dialyzed (molecular weight cut off 1,000 Da) and lyophilized, then dissolved in a half original bFF volume of modified synthetic oviduct fluid (mSOF). Experiment 1: Cumulus-oocyte-complexes (COCs) were matured in mSOF in the presence or absence (control) of whole bFF (wFF), HBF or HNF at a concentration of 10% (v/v). After in vitro fertilization, oocytes were freed from COCs and then cultured in mSOF with fetal calf serum until Day 8. Nuclear maturation rates were not different among wFF, HBF, HNF and control (69.8, 75.6, 63.6 and 72.9%, respectively). However, HBF favored postfertilization development, especially to the blastocyst stage (27.2%) compared with the control (19.0%), whereas HNF showed inhibitory effects, although wFF had no effect. Experiment 2: To determine whether the effects of HBF and HNF would become more evident by high-dose supplementation, HBF or HNF was added to in vitro maturation (IVM) medium at concentrations of 10,20 or 40% (v/v). HBF at all concentrations examined resulted in significantly higher (p<0.05) cleavage rates (80.5-86.2%) and blastocyst yield (31.1-44.2%) than the control(cleavage: 70.1%, blastocyst: 22.4%), whereas high dosages of HNF ( 20%) markedly inhibited embryonic development. These results indicated that heparin-binding fraction of bFF when added to the IVM medium was highly effective for enhancement of developmental competence of bovine oocytes in vitro.

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.

Midkine (MK) is known to be a member of a new family of heparin-binding growth/differentiation factors, together with pleiotrophin, and to be quite rich in bovine follicular fluid. To examine whether treatment with MK during in vitro maturation (IVM) of bovine granulosa-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine granulosa-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 h in IVM medium without (control) or with various concentrations (1-500 ng/ml) of MK, followed by in vitro fertilization (IVF) and culturing. Although the MK treatment during IVM did not affect the rate of nuclear maturation or the postfertilization cleavage of oocytes, MK at > or = 10 ng/ml significantly (P: < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared with the case of the control. Next, the effects of various glycosaminoglycans (heparin, heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid) preincubated with MK at 50 ng/ml were examined. The enhancing activity of MK was completely suppressed by heparin at 600 ng/ml but not by the other compounds. The effects of MK during IVM were also tested on oocytes freed from granulosa cells (GCs). When the denuded oocytes were cultured in IVM medium, no blastocyst formation after IVF was observed, regardless of MK supplementation. However, coculture of the denuded oocytes with isolated GC pellets enhanced the cleavage rates and the blastocyst yield, and these effects were more pronounced with MK supplementation. These results indicate that the presence of MK during IVM of bovine granulosa-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that the enhancing effects might be mainly mediated by GCs.