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Biology of Reproduction 64, 647-653 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

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

Tohru Azumaa, Shuntaro Ikedaa, Takuya Kondoa, Hiroshi Imaia, and Masayasu Yamada2,a

a Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

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.

developmental biology, implantation/early development, oocyte development

INTRODUCTION

In mammals, sperm entry into oocytes arrested at the second meiotic metaphase (MII) triggers egg activation to initiate a developmental program that leads to embryogenesis. Sperm-induced egg activation is accompanied by a series of events such as induction of intracellular Ca2+ ([Ca2+]i) oscillations, and resumption and completion of meiotic division followed by formation of male and female pronuclei (PN) [1–4]. The transient increase of [Ca2+]i is critical for egg activation. Increasing the [Ca2+]i concentration of the egg by artificial stimulators such as calcium ionophore A23187 or an electric pulse elicits egg activation events [1, 3, 5–8]. In contrast, decreasing the [Ca2+]i concentration by incubation of the egg with the membrane-permeable Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymetyl ester (BAPTA-AM) or heavy-metal-ion chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) prevents early events of egg activation, such as cortical granule exocytosis and emission of the second polar body (PB) [1, 3, 9, 10]. Although the exact mechanism initiating a transient increase of [Ca2+]i is unclear, a plausible mechanism has now been proposed in which an increase in inositol 1,4,5-trisphosphate, a hydrolyzed product of inositol phospholipids, that is induced immediately after sperm entry or treatment with artificial stimuli, causes the release of [Ca2+]i from intracellular stores [3, 11, 12].

It has been reported that extracellular Ni2+, Zn2+, Cu2+, and La2+, but not all heavy-metal ions, evoke an increase in [Ca2+]i by increased release of intracellular Ca2+ stores in several types of cells such as endothelial cells, fibroblasts, and hepatocytes [13–15]. In Xenopus oocytes, too, metal ions with the potency order Cd2+ > Ni2+ > Zn2+ > Co2+ > Mn2+ > Cr2+ > Sr2+ stimulate release of intracellular Ca2+ stores [16]. Likewise, it is known that externally added Sr2+ induces repetitive Ca2+ increase in mouse oocytes at the MII stage, leading to parthenogenetic activation [1, 17–19]. However, during the course of experiments on the effects of such heavy metal ions on artificial activation of porcine oocytes, contrary to our expectations, it was noted that treatment of germinal vesicle (GV) stage oocytes with the cell membrane-impermeable metal ion chelator EDTA during the maturation period induces formation of the PN instead of formation of the MII plate. Therefore, we attempted to pursue this unexpected observation further.

In the present study, we examined 1) the effects of EDTA on nuclear morphology, especially PN formation of porcine oocytes at the GV stage during the maturation period; 2) the developmental competence of the oocytes treated with EDTA, until the blastocyst stage; and 3) the effects of EDTA saturated with metal ions such as Ca2+, Cu2+, Fe3+, or Zn2+ (Ca-EDTA, Cu-EDTA, Fe-EDTA, and Zn-EDTA, respectively), and of the Ca2+ chelator EGTA on PN formation.

MATERIALS AND METHODS

Collection of Oocytes

Ovaries collected from prepubertal gilts at a local slaughterhouse were transported to the laboratory within 3 h in saline (9 g NaCl/L) at 35–38°C. From small antral follicles (2–6 mm in diameter), oocytes were aspirated with a 21-gauge needle fixed to a 10-ml disposable syringe. Oocytes surrounded by compact cumulus cells and with evenly granulated cytoplasm were selected. Oocytes freed from surrounding cumulus cells (denuded oocytes, DOs) were also prepared by repeated pipetting cumulus cell-enclosed oocytes (CEOs) through a fine-bored pipette in Hepes-buffered Tyrodes solution with 0.1% polyvinyl alcohol (PVA) containing 0.02% hyaluronidase (Sigma Chemical Co., St. Louis, MO). Both CEOs and DOs were washed four times with Hepes-buffered Tyrodes solution with 0.1% PVA before they were used for experiments.

Experimental Design

In experiment 1, to examine the dose effects of EDTA disodium salt (Na-EDTA; Wako Pure Chemical Industries, Ltd., Osaka, Japan) on PN formation in oocytes at the GV stage, CEOs were cultured in TCM199 with Earles balanced salt solution (Nissui, Tokyo, Japan) supplemented with 18 mM NaHCO3, 3.05 mM glucose, 0.91 mM sodium pyruvate, 10 IU/ml hCG (Sankyo Inc., Tokyo, Japan), 10 IU/ml eCG (Isei Inc., Yamagata, Japan), and 0.1% PVA (Sigma), referred to as IVM medium, in the presence or absence of Na-EDTA at concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 mM for 48 h at 38.5°C under 5% CO2 in air. To evaluate the nuclear morphology of oocytes after the culturing, oocytes were stripped from surrounding cumulus cells by repeated pipetting and then mounted on slides. The mounted oocytes were fixed in 25% (v/v) acetic alcohol for 2 days, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and then examined under a phase-contrast microscope. Oocytes that formed PN were considered activated.

In experiment 2, effects of treatment time on nuclear morphology were examined. The CEOs were cultured in IVM medium with or without 1 mM Na-EDTA for 0, 18, 23, 28, 33, 38, 43, or 48 h. At the end of the culture periods, the nuclear morphology of the oocytes was examined according to the method described above.

In experiment 3, to examine the effects of Na-EDTA on DOs, DOs were cultured with 1 mM Na-EDTA for 48 h. Oocytes were then mounted on slides and the nuclear morphology was examined according to the method described above.

In experiment 4, the developmental ability of Na-EDTA-treated oocytes was examined. After CEOs had been cultured with 1 mM Na-EDTA for 48 h, oocytes freed from surrounding cumulus cells were further cultured in North Carolina State University (NCSU) 23 medium [20] supplemented with 0.4% BSA (Sigma) for 48 or 144 h at 38.5°C under 5% CO2 in air.

In experiment 5, to examine if the effects of Na-EDTA on the induction of PN formation in oocytes at the GV stage are due to the chelation of the extracellular metal ions Ca2+, Cu2+, Fe2+, or Fe3+ and Zn2+, we used Ca-EDTA, Cu-EDTA, Fe-EDTA, Zn-EDTA, and EGTA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at a concentration of 1.0 mM instead of Na-EDTA to induce PN formation in oocytes at the GV stage. After culture for 48 h, the nuclear morphology of oocytes was examined by the method described above.

Statistical Analysis

Experiments were repeated three (experiments 1, 2, 3, and 5) or four times (experiment 4). Data were analyzed by ANOVA and Fisher's protected least significant difference test using the Statview program (Abacus Concepts, Inc., CA). Percentage data were subjected to arcsine transformation before statistical analysis. A value of P < 0.05 was considered to be an indication of statistical significance.

RESULTS

Experiment 1

The effects of various concentrations of Na-EDTA on the nuclear maturation of CEOs cultured for 48 h are summarized in Table 1. When oocytes at the GV stage were cultured in IVM medium alone, 66.2% of them reached the MII stage, and the others remained at immature stages such as GV, germinal vesicle breakdown (GVBD), or first meiotic metaphase (MI). However, adding Na-EDTA at various concentrations (0.1–2 mM) into the IVM medium induced some of the cultured oocytes to form nuclei that were very similar to a PN in morphological appearance and size (see Fig. 1). The percentage of oocytes with a PN increased with an increasing concentration of Na-EDTA, from 16.3% at 0.1 mM to 63.1% at 1 mM. However, the PN formation rates were markedly decreased at 2 and 5 mM (26.7% and 0%, respectively) compared to 1 mM. Among oocytes cultured with 0.1–2 mM Na-EDTA, 4.4%–21.9% of oocytes were fragmented or had unidentifiable nuclear status. Those oocytes were classified as degenerated. All oocytes cultured with 5 mM Na-EDTA became degenerated.


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  		TABLE 1. Effect of Na-EDTA on nuclear status of porcine GV oocytes.*



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  		FIG. 1. Typical nuclear morphologies of porcine oocytes treated with 0.5 mM (b) or 1 mM Na-EDTA (a, c, d, and e) for 48 h. a) Pronucleus without a PB; b) one PN and one PB; c) two PNs without a PB; d) two PNs with one PB; and e) three PNs without a PB. Bars = 15 µm

Oocytes that had formed PN(s) during the 48-h treatment with Na-EDTA at concentrations of 0.1–2 mM from the GV stage were then classified according to numbers of PNs and PBs formed (Table 2 and Fig. 1). Most of the oocytes (72.2%–95.3%) formed 1 PN at all concentrations of Na-EDTA: although 21.2%–35.7% of oocytes cultured with lower concentrations of Na-EDTA (0.5 mM or less) extruded 1 PB, others had no PB. In all experimental groups, oocytes with two or three PNs were observed at very low rates: some oocytes with two PNs cultured with 0.5 mM or less Na-EDTA extruded one or two PBs. No oocytes with two PNs cultured with 1 mM or more Na-EDTA or oocytes with more than three PNs cultured with Na-EDTA at any concentration (0.1–1 mM) formed PBs.


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  		TABLE 2. [cb9]Classification of oocytes that formed PN after Na-EDTA treatment according to the numbers of PN and PB.*

Experiment 2

To determine the timing of PN formation in oocytes during the 48 h of culturing in IVM medium with 1 mM Na-EDTA, the nuclear status of oocytes was evaluated at 18 h after commencement of culturing and then every 5 h by examination under the microscope after fixation and staining (Fig. 2). In control oocytes without Na-EDTA treatment, a small proportion of oocytes cultured reached the MII stage at 28 h of culturing, and then the proportion of oocytes at the MII stage greatly increased at 38 h of culturing. A total of 86.3% of oocytes reached the MII stage after 43 h in culture. In oocytes cultured with Na-EDTA, although GVBD had started at 18 h of culturing, the proportions of GVBD increased more gradually than in control oocytes. Formation of PN was first observed after 33 h in culture, and thereafter the proportion greatly increased by 48 h of culture. Oocytes at the MI or anaphase I/telophase I (AnaI/TeloI) stages were mainly observed at 33 and 38 h of culturing, at the rates of 32.0% and 24.3%, respectively. After that, the proportion decreased by less than 5% at 48 h of culturing. From these results, it seems likely that most of the oocytes cultured with 1 mM Na-EDTA for 48 h might undergo PN formation directly from interphase between GVBD and AnaI/TeloI, not via MII stage.



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  		FIG. 2. Time course experiments on Na-EDTA effects on nuclear status of oocytes during the course of maturation in culture. Oocytes at the GV stage were cultured for 48 h in IVM medium without (A) or with (B) 1 mM Na-EDTA. Black diagonal bars, GV; dotted bars, GVBD; stippled bars, MI; white diagonal bars, AnaI/TeloI; horizontal bars, MII; black bars, PN; white bars, unidentifiable, and fragmented oocytes. *Numbers in parentheses represent the number of oocytes examined. The values on the histograms are the means of three replicates

Experiment 3

To ascertain if Na-EDTA affects oocytes directly or via cumulus cells surrounding oocytes, DOs at the GV stage were cultured in IVM medium with or without 1 mM Na-EDTA for 48 h. As shown in Table 3, Na-EDTA induced PN formation in DOs at a rate (64.6%) similar to that (66.6%) in CEOs.


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  		TABLE 3. Pronuclei formation by Na-EDTA treatment in porcine CEOs and DOs.*

Experiment 4

Whether oocytes cultured with 1 mM Na-EDTA for 48 h from the GV stage could undergo mitotic cleavage and then differentiate into blastocysts was examined. After 48 h culture in IVM medium with or without Na-EDTA, all the oocytes that had been freed from surrounding cumulus cells as described in Materials and Methods were cultured for 48 or 144 h in NCSU23 medium containing 0.4% BSA (Table 4 and Fig. 3). In oocytes cultured with Na-EDTA, 48 h after commencement of the culture, 23.6% of the oocytes had undergone cleavage, and at 144 h of culture, the proportion of embryos at the blastocyst stage was 10.4%. The mean total number of cells per blastocyst was 40 (data not shown), which was similar to the number of cells in blastocysts derived from oocytes that were cultured in the same medium for 144 h after in vitro maturation and fertilization. In control oocytes not cultured with Na-EDTA, only 2.5% of the oocytes underwent cleavage, and they did not develop further. These results strongly suggest that Na-EDTA treatment allowed maturing (MI) oocytes to form PNs and subsequent entrance into the mitotic cell cycle, leading to development to the blastocyst stage.


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  		TABLE 4. Developmental ability of porcine oocytes after treatment with Na-EDTA.*



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  		FIG. 3. Photomicrographs of a) cleaved oocytes and b) blastocysts developed in in vitro culture for 48 and 144 h, respectively, from 1 mM Na-EDTA-treated oocytes. Bars = 100 µm

Experiment 5

To determine how Na-EDTA induces PN formation in oocytes during the meiotic maturation period, we examined the effects of Ca-EDTA, Cu-EDTA, Fe-EDTA, Zn-EDTA, and EGTA on the nuclear maturation of oocytes. The CEOs were cultured for 48 h in IVM medium with or without Ca-EDTA, Cu-EDTA, Fe-EDTA, Zn-EDTA, or EGTA at a concentration of 1 mM, stained, and observed to determine their nuclear status (Table 5). The EDTA is a cell membrane-impermeable chelator that has a higher affinity for Ca2+ than for Mg2+, but it has a much higher affinity for Zn2+ than for either Ca2+ or Mg2+ (log stability constants for EDTA-metal complexes at pH 7 are 5.4 for Mg2+, 7.3 for Ca2+, 10.9 for Fe2+, 13.1 for Zn2+, and 15.5 for Cu2+). Thus, Ca-EDTA binds extracellular Zn2+ without reducing extracellular Ca2+ [21, 22] or causing much reduction of extracellular Mg2+. Thus, Ca-EDTA is used for experiments attempting to clarify the biological functions of extracellular Zn2+, such as Zn2+-induced oxidative neuronal death [22–24]. In oocytes cultured in IVM medium alone (control), similar to the results in Table 1, 60.7% of oocytes reached the MII stage. On the other hand, when oocytes were cultured with Ca-EDTA, 81.2% of the oocytes formed PNs, and among the oocytes more than 90% had 1 PN without any PB (data not shown). However, neither the non-Zn2+ chelator Zn-EDTA nor the Ca2+ chelator EGTA caused any PN formation, although most oocytes cultured with Zn-EDTA or EGTA reached the MII stage (74.6% and 77.4%, respectively). Because Ca-EDTA is not necessarily specific for Zn2+ and can also chelate endogenous Fe2+ and Cu2+ [21, 22], we also examined the effects of Fe-EDTA and Cu-EDTA. As shown in Table 5, treatment of oocytes with Fe-EDTA and Cu-EDTA at 1 mM also induced PN formation; however, the rates were lower (28.3% and 10.9%, respectively) than that (81.2%) induced by Ca-EDTA. Unlike Zn-EDTA- and EGTA-treated oocytes, Fe-EDTA- and Cu-EDTA-treated oocytes that did not form PN became degenerated or remained at immature stages, suggesting that Fe-EDTA and Cu-EDTA at 1 mM may exert some toxic effects on porcine oocytes. These results suggested that extracellular chelation of Zn2+ by EDTA may be involved in induction of PN formation in porcine oocytes during the maturation period.


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  		TABLE 5. Effect of treatment of porcine GV oocytes with Zn-EDTA, Ca-EDTA, Fe-EDTA, Cu-EDTA, and EGTA for 48 h on their nuclear status

DISCUSSION

In the present study, we show for the first time that porcine oocytes at the GV stage were parthenogenetically activated during the maturation stage of culture by treatment with Na-EDTA at various concentrations of 0.1–2 mM, leading to PN formation followed by mitotic cell divisions. Cytological examination (Table 2) revealed that most oocytes cultured with Na-EDTA at 0.1–2 mM for 48 h formed 1 PN: although a fraction (21.2%–35.7%) of oocytes cultured with Na-EDTA at 0.5 mM or less extruded 1 PB, others had no PB, suggesting that most oocytes activated with Na-EDTA at 0.1–2 mM formed 1 PN before completion of MI. Moreover, it was found that a small proportion of oocytes cultured with Na-EDTA at all concentrations tested for 48 h showed several different types of nuclear status: 2 PNs, 2 PNs + 1 PB, 2 PNs + 2 PBs, and 3 PNs.

When oocytes cultured with 1 mM Na-EDTA for 48 h were further cultured in NCSU23 medium containing 0.4% BSA for 144 h, about 10% of the oocytes could develop to blastocysts. Because 95.3% of oocytes activated by 1 mM Na-EDTA treatment for 48 h had 1 PN without PB (Table 2), it seems likely that the blastocysts obtained were derived from the activated oocytes whose karyotype was supposed to be tetraploid.

It is known so far that in general, parthenogenetic activation is induced in MII-arrested oocytes by artificial stimuli such as treatment with ethanol [25, 26], calcium ionophore, A23187 [7, 8], protein kinase inhibitors [26–29], or electric pulses [5, 6]. The oocytes treated with such stimuli are released from MII arrest by inducing an increase of [Ca2+]i and then inactivation of maturation promoting factor (MPF) by ubiquitin-dependent proteolysis of cyclin B [30–32]. The MPF is a heterodimeric protein kinase that consists of a 34-kDa catalytic subunit (p34cdc2) and a regulatory subunit (cyclin B), whose activity is maintained at elevated levels by preventing cyclin degradation with cytostatic factor during MII arrest [33]. In activated oocytes, inactivation of MPF results in formation of a female PN with a second PB emission in the activated oocytes. Recently, several lines of experiments revealed precociously regulated events concerning PN formation in oocytes after completion of MI by perturbing meiosis-specific cell cycle regulation. In Xenopus oocytes, Wee1 kinase, a universal mitotic inhibitor, is down-regulated specifically during oogenesis and becomes detectable only after MI or during MII. A moderate level of ectopic expression of Wee1 in oocytes during MI after GVBD was reported to be able to induce interphase nucleus reformation and DNA replication just after MI, indicating that the presence of Wee1 during MI converts the meiotic cell cycle into a mitotic-like cell cycle with an S phase [34]. In mouse oocytes, Mos protein kinase is believed to play an important role in the accumulation of cyclin B in the oocytes after completion of MI, leading to reactivation of MPF [35, 36]. It was reported that the failure of antisense c-mos oligonucleotide-injected oocytes to accumulate cyclin B results in formation of a nucleus containing decondensed chromatin after MI [37, 38]. Likewise, it was also reported that when oocytes at the GV stage from c-mos-deficient mice generated by gene targeting were cultured in vitro, the oocytes underwent GVBD and extrusion of both the first and the second PB, followed in some cases by formation of a PN and progression into cleavage [39–41].

Inconsistently with those findings, as shown in the present experiments, precocious formation of PN in most of porcine maturing oocytes cultured with Na-EDTA resulted from escaping from meiotic division before completion of MI, although a small proportion of oocytes activated with a lower concentration (0.5 mM or less) of Na-EDTA extruded one PB. Thus, it seems likely that the mechanisms by which Na-EDTA-treated porcine oocytes are induced to form PN without completion of MI may be different from those of precocious formation of nuclei induced by ectopic expression of Wee1 kinase activity or inactivation of Mos protein kinase activity. Recently, Lee et al. [42] reported, however, that MPF inactivation by tyrosine phosphorylation of p34cdc2 of MII-arrested porcine oocytes resulted not only in exit from MII arrest but also in formation of an interphase nucleus without chromosome segregation. Thus, the precocious PN formation of porcine oocytes at the GV stage induced with Na-EDTA may be explained by a mechanism related to inactivation of MPF by tyrosine phosphorylation of p34cdc2 rather than degradation of cyclin B, as MPF is known to be activated along with Mos protein in maturing (MI) oocytes [36].

As shown in Table 5, an extracellular zinc chelator, Ca-EDTA, was able to induce formation of the PN at a rate similar to that induced by Na-EDTA, whereas Zn-EDTA did not induce PN formation at all, although most oocytes cultured with Zn-EDTA developed to the MII stage. Treatment of oocytes with Fe-EDTA and Cu-EDTA also induced PN formation at lower rates than that induced by Ca-EDTA treatment. However, unlike Zn-EDTA, most Fe-EDTA- or Cu-EDTA-treated oocytes except for those that formed PN were not able to develop to the MII stage. Because it is known that Fe-EDTA and Cu-EDTA catalyze radical generation through Fenton chemistry [43, 44], such a developmental limitation to PN formation or the MII stage in Fe-EDTA- or Cu-EDTA-treated oocytes may be due to radical toxicity. Thus, taken together, our findings suggest that extracellular chelation of Zn2+ by EDTA is an important regulatory event in precocious PN formation and parthenogenetic activation of porcine oocytes at the GV stage.

Zinc ion is an essential component in many biological systems such as spermatogenesis [45] and conception [46]. In terms of the potential function of Zn2+, it has been found that a substantial amount of Zn2+ per unit of protein is localized in the plasma membranes of cells, and that Zn2+ in the membrane has a stabilizing or protective effect on the structure and function of the membrane [47]. Interestingly, it was reported that Zn2+ affects the motility and capacitation of sperm cells in many species [47–49]. Moreover, treatment of ejaculated spermatozoa with EDTA elicits decondensation of sperm nuclear chromatin, and this effect is reversibly inhibited by Zn2+ supplementation [50]. A clear understanding of the mechanism whereby treatment with the cell membrane-impermeable chelator EDTA of porcine oocytes at the GV stage induces precocious formation of PN and parthenogenetic activation has not yet been achieved at this time. However, these findings suggest that alteration of zinc-coordinated structures of oocytes, which is likely induced by zinc removal by EDTA, may play a pivotal role in the EDTA effects on porcine oocytes. This possibility should be examined further.

In conclusion, EDTA treatment of porcine oocytes at the GV stage results in activation of the oocytes, as measured by precocious formation of PNs and the preimplantation development of the oocytes to the blastocyst stage. It is especially noteworthy that developmental competency to the blastocyst stage could be acquired by the oocytes that passed over the second meiotic maturation period from the interphase between GVBD and completion of MI due to EDTA treatment. The present experiments did not reveal mechanisms responsible for parthenogenetic activation of porcine oocytes by EDTA treatment, but only that extracellular chelation of Zn2+ may be involved.

ACKNOWLEDGMENTS

We are grateful to Prof. J.E. Fléchon for critical reading of the manuscript.

FOOTNOTES

First decision: 6 September 2000.

1 This work was supported in part by grants-in-aid from the Ito Foundation and the Association of Livestock Technology (Japan). Back

2 Correspondence: Masayasu Yamada, Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. FAX: 81 75 753 6329; yamada{at}jkans.jkans.kais.kyoto-u.ac.jp Back

Accepted: October 2, 2000.

Received: July 31, 2000.

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