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Animal Reproduction Science 59 2000 99–107 www.elsevier.comrlocateranireprosci
Influence of sequence duration and number of electrical pulses upon rabbit oocyte activation and
parthenogenetic in vitro development
M.J. Escriba , F. Garcıa-Ximenez
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Laboratorio de Reproduccion, Departamento de Ciencia Animal, UniÕersidad Politecnica de Valencia,
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Camino de Vera 14, 46071 Valencia, Spain Received 15 April 1999; received in revised form 20 October 1999; accepted 14 December 1999
Abstract
Electroactivation of in vivo mature young rabbit oocytes was investigated here. The effects of four or eight electrical pulse treatment over 90, 150 or 270 min upon oocyte activation frequency
and type, and even upon their subsequent in vitro development, were studied. The lowest Ž
. activation frequency was observed after applying four-pulses over 90 min 54 . However,
extending four-pulse treatment duration over 150 or 270 min induced more oocytes to activate Ž
. Ž
. from 84 to 100 , as did the eight pulsing treatments from 91 to 97 . With eight pulses,
Ž .
extending treatment duration improved the normal activation rates from 47 to 76; P - 0.05 . Nevertheless, the haploid morulae and blastocyst rates decreased significantly with extended eight
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pulsing treatment duration morulae: from 94 to 41 and blastocysts: from 31 to 0 . q 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords: Rabbit; Oocyte electroactivation; Parthenogenetic development
1. Introduction
The genetic content of the mammalian female gamete could be amplified by parthenogenetic haploid development. In mouse, blastomeres from haploid embryos at
the two to eight cell stage provided donor nuclei which, when combined with the paternal nuclear counterpart, would reconstruct the normal heteroparental ploidy and
Corresponding author. Tel.: q34-96-387-9433; fax: q34-96-387-7439. Ž
. E-mail address: mescribadca.upv.es M.J. Escriba .
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0378-4320r00r - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. Ž
. PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 0 6 9 - 5
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derive into viable offspring Surani et al., 1987; Renard et al., 1991 . Thus, under this perspective, an oocyte activation treatment which maximises the haploid activation and
subsequent in vitro haploid development, at least up to the 8-, 16- or 32-cell stage, would be desirable.
In a previous report, we have observed that a higher number of pulses applied to recently ovulated rabbit eggs over 90 min increased the activation frequencies and
subsequent parthenogenetic in vitro development, but reduced haploid egg production by Ž
. suppression of the second polar body extrusion Escriba and Garcıa-Ximenez, 1999 . On
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Ž the other hand, in this same work, the weakest treatment based on four pulses over 90
. min , induced low global activation and developmental rates, but excellent normal
activation rates. These results coincide with those reported by other authors in several Ž
species Onodera and Tsunoda, 1989; Collas and Robl, 1990; Ozil, 1990; Collas et al., .
1993, 1995 . Following results from our earlier work, and in order to improve global haploid egg
production, two strategies would be planned. In the case of the four-pulse sequences, the global stimulation level would have to be enhanced, whereas in the eight-pulse se-
quences, the over-stimulation level would have to be reduced, but without compromising Ž
the beneficial effects of multiple pulses. Extension of total sequence duration without .
altering number of applied pulses would conciliate both latter strategies. The reason is that in lengthened four-pulse sequences, oocytes would be more responsive to stimula-
Ž tion presumably by their in vitro ageing: Stice and Robl, 1988; Ware et al., 1989;
Collas and Robl, 1990; Yang et al., 1991; Fissore and Robl, 1992; Collas et al., 1993; .
Prochazka et al., 1993; Adenot et al., 1997 , whereas in eight-pulse treatments, as
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Ž sequence duration extends, the pulsing frequency pulse numberrtotal treatment dura-
. Ž
tion is reduced, which may determine a reduction on over-stimulation level Collas et .
al., 1995 . Mature oocytes are arrested at metaphase stage, probably by cytoplasmic MPF
Ž .
maturationrmitosisrmeiosis promoting factor activity. Prolonged MPF inactivation would allow oocytes to progress on meiosis and culminate it with the PB2 extrusion
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Meyerhof and Masui, 1977; Shibuya and Masui, 1988; Fissore and Robl, 1992 . In mammalian fertilisation, calcium oscillations were observed over an extended time
Ž period, decaying at the pronuclear stage in rabbits, reached stage at 4–5 h following
. efficient activating stimulus: Oh and Brackett, 1975; McCulloh et al., 1983 and finally
Ž disappearing at the pronuclear apposition stage Cuthbertson and Cobbold, 1985; Fissore
. and Robl, 1993 . On the other hand, an electrical pulse evokes a single intracellular
2q
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2q
Ca transient in mature oocytes Fissore and Robl, 1992 ; this Ca
stimulation induces a transient decline in MPF activity which regains the metaphase levels rapidly
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estimated at 90 min in rabbits, Collas et al., 1995 . Besides their role in MPF inactivation, calcium rises also act as regulatory signals for entry into the cell cycle and
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2q
stimulation of DNA synthesis Whitacker and Patel, 1990 . Multiple Ca transients are
therefore likely to have additional beneficial effects, such as membrane depolarisation Ž
. changes McCulloh et al., 1983 and initiation of DNA replication, on parthenogenetic
embryo development. In rabbits, all of the above delimits both a 90-min interval for pulses, at the
Ž maximum, to avoid MPF reactivation and a 4- to 5-h total treatment duration corre-
. sponding to estimated time from activation display to start of the pronuclear stage ,
therefore the minimal number of pulses will be four. However, extending pulse sequence is likely to affect development. Thus, as the time sequence enlarges, oocytes age being
more sensitive to calcium stimulus, which would compromise their ability to develop to
Ž .
Ž .
blastocyst Stice and Robl, 1988; Collas and Robl, 1990 . Moreover, Kaufman 1978 indicated the detrimental effect that culture and handling conditions to which eggs were
submitted during the first hour following activation would exert upon mouse parthenote development. In our conditions, this fact may be aggravated by using a sub-optimal
Ž .
culture medium Brackett and Oliphant’s defined medium over longer activating time period.
Thus, the objectives of this work are to test the effects of extending four and eight pulse sequences upon activation rate, type and even subsequent parthenogenetic devel-
opment, in order to define an efficient activating procedure which would increase the haploid embryo production.
2. Materials and methods
