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Influence of sequence duration and number of
electrical pulses upon rabbit oocyte activation and
parthenogenetic in vitro development
M.J. Escriba
´
), F. Garcıa-Ximenez
´
´
Laboratorio de Reproduccion, Departamento de Ciencia Animal, Uni´ Õersidad Politecnica de Valencia,´
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
Žfrom 84% to 100% , as did the eight pulsing treatments from 91% to 97% . With eight pulses,. Ž .
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
Ž .
pulsing treatment duration morulae: from 94% to 41% and blastocysts: from 31% to 0% .q2000 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: mescriba@dca.upv.es M.J. Escriba .´
0378-4320r00r$ - see front matterq2000 Published by Elsevier Science B.V. All rights reserved. Ž .
Ž .
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
´
´
´
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
´
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
Žmaturationrmitosisrmeiosis promoting factor activity. Prolonged MPF inactivation.
would allow oocytes to progress on meiosis and culminate it with the PB2 extrusion
ŽMeyerhof 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 Ž . 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
Žestimated 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
Ž . 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
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
2.1. Oocyte collection and preparation
Mature mixed-breed female rabbits were tested for sexual receptivity and treated with
Ž .
25 IU of hCG Coriogan, Lab. Ovejero, Spain . The does were not superovulated. Oocytes were recovered from oviducts 14 h after endovenous hCG treatment by flushing
Ž .
in Dulbecco’s phosphate-buffered saline DPBS immediately after euthanasia of the donor females. The cumulus-enclosed oocytes were briefly placed in Ca2q-free DPBS
Ž
supplemented with 1 mgrml bovine testes hyaluronidase Cat. No.: H4272. Type IV-S,
.
Sigma, Spain , then gently pipetted through a small-bore pipette to free them from corona cells. After treatment, oocytes were selected for the presence of a clearly visible
Ž .
first polar body PB1 and for general healthy appearance, then cultured at 398C in
Ž .
defined medium DM; Brackett and Oliphant, 1975 in a 7% CO2 humidified atmo-sphere.
2.2. Oocyte actiÕation
Oocyte activation was accomplished by electrical stimulation initiated at 15–15.5 h post-hCG. Handling, pulse parameters and pulsing medium characteristics have been
Ž .
described Escriba and Garcıa-Ximenez, 1999 . Briefly, oocytes were placed between
´
´
´
two stainless-steel round wire electrodes approximately 0.5 mm apart and overlaid with 0.18 M mannitol containing 100 mM CaCl2 and 100 mM MgCl . Oocytes were2 electrically stimulated with four or eight square electrical DC pulses of 0.6 kVrcm for 60ms each over 90, 150 and 270 min, at regular time.intervals. Pulses were delivered byŽ .
Ž .
with an oscilloscope TDS 320, Tektronix, Spain . Oocytes were transferred from mannitol to DM between pulses.
2.3. Embryo culture
Ž
Following electrical treatment, oocytes were cultured in DM without
CCB-diploidis-. Ž .
ing treatment for 6–7 h and were assessed for second polar body PB2 extrusion
ŽOnodera and Tsunoda, 1989; Ozil, 1990 . After final checking PB2 extrusion, the. Ž .
oocytes were cultured in Ham’s F-10 medium plus 20% vrv homologous-doe serum
Žs-Ham’s under a 7% CO. 2 atmosphere at 398C. After the first 24 h of culture, non-divided oocytes were removed from culture. Embryo development was checked
Ž .
every 24 h until end of culture 5 days .
2.4. Assessment of actiÕation types
The different degrees of activation were established by checking PB2 extrusion
Ž
andror further cleavage. Three types of parthenogenetic eggs were produced Kaufman,
.
1978; Ozil, 1990; Collas et al., 1993; Escriba and Garcıa-Ximenez, 1999 . The first type
´
´
´
had one pronucleus plus the first and second polar bodies and further cleavageŽPB1qPB2 and divided ; it conduces to an haploid condition and is named normal.
activation. The second type had two or more pronuclei plus the first polar body and
Ž .
cleaved PB1 and divided , named as abnormal activation which usually determines a diploid condition. The third type had the first and second polar bodies but no subsequent
Ž .
cleavage PB1qPB2 and non-divided , named incomplete activation.
At least three replicates were performed per group. The results were analysed using a Chi-square test. When a single degree of freedom was involved, the Yate’s correction for continuity was carried out.
3. Results
Activation results are summarised in Table 1. With four pulses, the activation
Ž
frequency significantly increase when treatment duration was extended from 54% to
.
100% . Regardless of treatment duration, eight pulses induced oocytes to activate
Ž .
efficiently 91% to 97%; P)0.05 . The effect of number of pulses is only statistically significant at 90-min treatment duration: more pulses induced greater oocyte activation
Žeight: 92% vs. four: 54%; P-0.05 ..
Ž
The type of activation is also shown in Table 1. Incomplete activation PB1qPB2
.
and non-divided was present in all treatments at low frequency, following no specific trend.
Ž .
After four pulses, normal activation rates PB1qPB2 and divided were not affected
Ž .
by treatment duration from 51% to 80%; P)0.05 . However, haploid frequency was
Ž
significantly enhanced by extending total eight-pulsing treatment duration 90 min: 47%;
.
Table 1
Effect of number of pulses and pulsing-sequence duration on rabbit oocyte activation1 2 Ž .
Number of Treatment Number of oocytes Type of activation %
pulses duration Pulsed Activated %Ž . PB1qPB2 and PB1qPB2 and PB1 and
Žmin. non-divided divided divided
c a a,b
Numbers with different superscripts within a column differ P-0.05 . 1
Three replicates per group were performed. Pulses of 0.6 kV cmy1 lasting 60ms were applied at regular time intervals.
2
PB1qPB2 and non-divided: oocytes with the first and second polar bodies and one pronucleus but no
Ž . Ž .
subsequent cleavage incomplete activation ; PB1qPB2 and divided: oocytes with PB2 one pronucleus and
Ž . Ž
further cleavage normal activation ; PB1 and divided: oocytes with suppressed extrusion of the PB2 two or
. Ž .
more pronuclei and further cleavage abnormal activation .
Ž
number of applied pulses only when 90-min treatments were performed four pulses:
.
80% vs. eight pulses: 47%; P-0.05 .
Ž .
In abnormal activation rates PB1 and divided , significant differences were only
Ž .
detected between the four-pulsed over 270 min group 22% and the eight-pulsed over
Ž .
90 min group 50% .
Parthenogenetic embryo development is shown in Table 2 In normally-activated
Ž .
oocytes haploid condition , the rate of compacted morula was significantly increased
Žfrom 69% to 94% when the total duration of four-pulse treatment was increased but,.
with the same number of pulses, the haploid-derived blastocyst rate was not affected by
Ž .
treatment duration ranging from 15% to 37%; P)0.05 . After applying eight pulses, a significant contrary effect due to increase treatment duration was observed on
haploid-Table 2
Effect of number of pulses and pulsing-sequence duration on the subsequent in vitro development of haploid and diploid parthenogenetic eggs
1 1
PB1qPB2 and divided PB1 and divided
Number of Treatment Number Number Number Number Number Number
Ž . Ž . Ž .
pulses duration min cultured compacted blastocysts % cultured compacted blastocysts %
Ž . Ž .
Numbers with different superscripts within a column differ significantly P-0.05 . 1
Ž . Ž
derived morulae rates from 94% to 41% and also in the blastocyst rates from 31% to
.
0%; P-0.05 .
Ž .
Diploid embryos derived from abnormally activated oocytes PB1 and divided did not show in vitro developmental differences up to compacted morula among the applied
Ž .
treatments, ranging from 82% to 100% Table 2; P)0.05 . Moreover, during in vitro diploid development up to the blastocyst stage, no trend has clearly emerged from either the number of applied pulses or the total duration of the sequence. However, from all assayed combinations, only after applying eight pulse treatment over 90 min was the
Ž .
diploid in vitro development to blastocyst significantly higher 65% than that observed
Ž .
after eight pulses over 150 min 17% .
Global data from every treatment were retrospectively grouped and showed that diploid eggs, exclusively obtained by PB2 retention but not by diploidising treatments,
Ž .
are able to develop to the morula and blastocyst stages, reaching an 88% 59r67 and a
Ž .
34% 23r67 , respectively. However, global haploid development up to the morula and
Ž .
blastocyst stage was slightly lower 78%; 102r130 and 21%; 28r130, respectively .
Ž .
The highest global haploid compacted morula rate per pulsed oocytes 70%: 32r46 was obtained after four-pulse over 270-min treatment. These data were not shown in the tables.
4. Discussion
Ž .
This work confirms that in short-length activating procedures 90 min , a higher
Ž .
number of pulses here: eight improved the activation rates of young rabbit oocytes
ŽCollas and Robl, 1990; Ozil, 1990; Collas et al., 1995 but induced lower rates of.
Ž .
normal activation haploids: Escriba and Garcıa-Ximenez, 1999; this work .
´
´
´
Ž .
After applying four pulses, extending sequence duration from 90 to 150 or 270 min improved global activation rates without penalising normal activation frequency. This effect would be due to either enhanced oocyte sensitivity from their in vitro ageing
ŽStice and Robl, 1988; Ware et al., 1989; Collas and Robl, 1990; Fissore and Robl, .
1992; Collas et al., 1993; Prochazka et al., 1993; Adenot et al., 1997 , or to inhibition of
´
Ž
MPF re-activation over a longer time period Fissore and Robl, 1992; Vitullo and Ozil,
.
1992 . Effectively, one electrical pulse promotes intracellular calcium rise which, after
Ž
reaching a critical amplitude level, reduces the MPF activity temporarily Fissore and
.
Robl, 1992; Collas et al., 1993, 1995 . In rabbits, this transient reduction does usually
Ž . Ž
spend 90 min Collas et al., 1995 . Thus, repetitive pulsing at every 90 min as with
.
four-pulse and 270-min sequences would maintain the MPF activity at basal levels
Ž
which is required for oocyte progression into early interphase stages Kubiak, 1989;
.
Fissore and Robl, 1992; Collas et al., 1995; Wu et al., 1998 .
After applying eight pulses, extent of sequence duration increased haploid egg production by increasing normal activation rates. Within a pulsing sequence, when a pulse becomes efficient, the meiosis is resumed and, following a certain time period,
Ž
culminates with the PB2 extrusion following 90 min in rabbits: Oh and Brackett, 1975;
.
Ozil, 1990 . During that period, application of electrical pulses, and their characteristics,
Ž
.
Prochazka et al., 1993; Escriba and Garcıa-Ximenez, 1999 . The PB2 retention due to
´
´
´
´
the over-stimulation applied over said critical period is more frequent the greater theŽ
pulsing frequency number of pulsesrtotal treatment duration; Ozil, 1990; Escriba and
´
.
Garcıa-Ximenez, 1999 . This fact would explain why in eight-pulse sequence, a lower
´
´
pulsing frequency, determined by long-length sequences, does not modify the global activation rates, which are always elevated, but increases the normal activation onesŽlower over-stimulation level . In earlier reports, following either 8- or 12-pulse se-.
Ž .
quences applied over 90 min high pulsing frequency , the global activation rates were maximal, whereas normal activation rates were reduced or zero, and some oocytes even
Ž .
lysed Escriba and Garcıa-Ximenez, 1999 .
´
´
´
Parthenogenetic rabbit embryo development is usually referred to in diploid embryos
ŽOnodera and Tsunoda, 1989; Ozil, 1990; Pinto-Correia et al., 1995; Mitalipov et al., .
1999 . However, in our knowledge, few works on haploid embryo development have
Ž .
been reported for this species Escriba and Garcıa-Ximenez, 1999; this work . In the
´
´
´
present work, the in vitro embryo developmental ability of both haploid and diploid parthenogenotes was verified, obtaining high developmental rates to the compactedŽ .
morula stage for diploids suppression of PB2-derived , and slightly lower for haploids. Previous reports have indicated that insufficient, or, conversely, excessive activation
Ž
stimulus impairs parthenogenetic diploid embryo development Ozil, 1990; Collas et al.,
.
1995; Pinto-Correia et al., 1995 . In our work, both these types of detrimental effects were detected, but only on haploid development. This would be due to different diploid
Ž .
origin in our work derived from oocytes with the PB2 non-extruded and those used by
Ž .
other authors in all cases, diploidised by CCB-treatment .
Haploid embryo development was impaired after applying the weakest assayed
Ž .
treatment four pulses over 90 min . Extension of four-pulse sequences improved activation rates and further development. In contrast, after applying an eight-pulse sequence distributed over 270 min, the haploid embryo development was limited, being evident at the morula, and even at the blastocyst stage. This would probably be due to the culture and handling conditions to which eggs were submitted during the first hour
Ž
following activation, with the osmolarity of the media exerting a main effect Kaufman,
. Ž .
1978 , and even the initial sub-optimal culture medium defined medium . Moreover, in eight-pulse and 270-min sequences, more pulses would have been performed over the post-activation stage, when the oocyte is already at the interphase. In normal fertilisation process, calcium-stimulus amplitude decreases as fertilisation progresses, not being
Ž .
detected at the pronuclear apposition stage Fissore and Robl, 1993 . Perhaps a higher number of calcium stimuli, applied over the pronuclear stage, would be responsible for observed penalisation on subsequent embryo development. In fact, excessively high intracellular calcium levels have been shown to be detrimental to certain cell functions
Ž .
or structures Izant, 1983 . Whether these post-activating pulses are required or not for further parthenogenetic development will be the subject of subsequent research.
5. Conclusions
Thus, either eight pulses over any of studied treatment durations, or four pulses over
Ž .
longer treatment 4.5 h induced oocytes to activate efficiently.
Normal activation rate was enhanced after extending eight-pulse sequences over 270 min or after reducing the number of applied pulses to four over any total treatment duration, but the in vitro haploid development was only improved after eight-pulse over 90 min or four-pulse over 270-min treatments.
The greatest amplification of the female gamete genetic content was achieved following four-pulse over 270-min treatment and further haploid culture.
Acknowledgements
The authors thank Mr. Luis Garcıa Valero for his excellent technical assistance in the
´
laboratory and animal care, and Mr. Neil Macowan for revising the English version.Ž .
This work was supported by CICYT AGF 97-0803 and by Consellerıa de Educacion y
´
´
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