Activation of rabbit oocytes: the impact of the Ca2+ signal regime on development.

Postfertilization manipulation of mammalian embryos results in various developmental alterations. To determine whether the manipulation of the Ca2+ regime causing oocyte activation is a valuable experimental means in helping understand the biological process by which embryos integrate signals from outside and later regulate gene expression, we linked Ca2+ signal parameters i.e. amplitude, number and frequency, with the efficiency and quality of postimplantation development. Freshly ovulated rabbit oocytes were subjected to repetitive and modulated Ca2+ influx. The results provide three major pieces of information. Firstly, the Ca2+ stimulus is the most efficient signal activating mammalian eggs when it is applied in a repetitive manner, the amplitude being the crucial factor. Secondly, the dynamics of early cleavage does not appear to be determined by either the frequency or the amplitude of modulation of the Ca2+ signal that activates the oocyte. Thirdly, amplitude and temporal modulation of the Ca2+ signal in the early minutes influences the developmental performance and the morphology of the rabbit parthenogenetic conceptus at day 11.5 of pregnancy. The results demonstrate the importance of epigenetic events during postfertilization as well as the possible uses of Ca2+ modulation in studying long term developmental effects.

Electrical activation and in vitro development of human oocytes that fail to fertilize after intracytoplasmic sperm injection.

OBJECTIVE: To determine whether electrically stimulated Ca2+ influx can "rescue" fertilization and early embryogenesis in human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). DESIGN: Prospective, randomized trial of a laboratory procedure. SETTING: A research laboratory at a university medical center. PATIENT(S): Discarded oocytes from ICSI-IVF cycles. INTERVENTION(S): Oocytes (n = 104) that showed no evidence of fertilization 16-24 hours after ICSI were assigned to three treatment groups: group 1 (one direct current electrical pulse at 1.36-1.50 kV/cm for 40-60 micros), group 2 (three pulses every 15-20 minutes), or group 3 (treated the same as group 2 but with no electrical stimulation). MAIN OUTCOME MEASURE(S): After stimulation, the oocytes were cultured in vitro for 3-5 days. Oocytes that displayed two pronuclei and a second polar body within 16 hours were considered to have fertilized normally. Fertilization and embryo cleavage rates were compared between groups. RESULT(S): Fertilization occurred in 26 (70%) of 37 and 38 (78%) of 49 group 1 and 2 oocytes, respectively, but in only 5 (27%) of 18 group 3 oocytes. Within 3 days, group 2 embryos routinely developed beyond the two-cell to four-cell stage (61% versus 13% in group 1); 11% of these oocytes developed to the morula or early blastocyst stage. Sex chromosome analyses indicated 10 male and 8 female embryos. CONCLUSION(S): Oocytes that fail to fertilize by 24 hours after ICSI can resume apparently normal fertilization and early embryonic development in response to electrical stimulation. Moreover, the degree of cytoplasmic activation as determined by the number of pulses applied affects fertilization efficiency and early embryonic development.

The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases.

During fertilization in mouse eggs, the sperm triggers a series of intracellular Ca2+ oscillations that lead to egg activation, as indicated by pronuclear formation. We show that Ca2+ oscillations in fertilized mouse eggs can be inhibited by addition of either the Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) or the heavy-metal-ion chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) plus dithiothreitol (DTT). Both treatments inhibited Ca2+ oscillations, but they had different effects upon egg activation. Blocking Ca2+ oscillations with BAPTA-AM after the occurrence of just two Ca2+ spikes resulted in most eggs forming pronuclei. However, we found that BAPTA-AM-treated fertilizing eggs showed a decreased rate of protein synthesis, which by itself can promote egg activation. In contrast, blocking Ca2+ oscillations with TPEN plus DTT was accompanied by the inhibition of egg activation with no significant effect on protein synthesis. In eggs that were fertilized and then treated with TPEN plus DTT, there was a correlation between the number of Ca2+ spikes and the proportion of eggs that formed pronuclei, as well as between the number of Ca2+ spikes and the time taken for pronuclear formation and the first mitosis to occur. The addition of TPEN plus DTT did not block the generation of Ca2+ spikes or pronuclear formation when eggs were artificially stimulated by electroporation pulses. These data suggest that TPEN plus DTT inhibits pronuclear formation in fertilizing eggs via the inhibition of Ca2+ oscillations and that the number of Ca2+ spikes may regulate egg activation.

Role of calcium oscillations in mammalian egg activation: experimental approach.

Biological rhythms are everywhere; the pulsatility of intracellular signals appears to maximise the cellular processes better than constant signaling. The aim of this paper is, firstly, to review the cellular mechanisms that modulate calcium oscillator activity during fertilisation and, secondly, to describe recent results we have obtained by artificially imposing rhythmical calcium stimulation on fertilised rabbit eggs during in vitro culture. The key finding in these experiments is that the egg appears to be sensitive to repetitive signalling during a period that goes far beyond the time of meiosis reinitiation. When delivered at the proper rhythm transient signalling can optimise developmental processes.

[Physiological mechanisms of oocyte activation]. / Mécanismes physiologiques de l'activation de l'ovocyte.

In all species, eggs are activated at fertilization by an increase in intracellular free Ca2+. In frogs, fishes and echinoderms the increase in cytoplasmic free calcium takes the form of a single explosive wave while, in mammals the spermatozoon triggers a series of calcium oscillation that last for several hours. A major unresolved is determination of the mechanism that the spermatozoon uses to trigger this calcium increase in the egg at fertilization. This review will recall the various hypothesis on the signal transduction system and present the recent argument for a soluble sperm cytosolic factor.

Stimulation of repetitive calcium transients in mouse eggs.

1. We have combined cell membrane electroporation by electrical field (EF) stimulation with a rapid perfusion system in order to stimulate repetitive increases in cytoplasmic free [Ca2+] ([Ca2+]i) in mouse eggs. [Ca2+]i was monitored by ratio fluorescent measurements of intracellular indo-1 on individual eggs. The conditions required to cause different types of [Ca2+]i increases were established and the effects of these [Ca2+]i changes upon egg activation examined. 2. The rapid perfusion of non-ionic medium caused a single [Ca2+]i increase. However, to generate repetitive [Ca2+]i increases, eggs were exposed to EF pulses in the presence of Ca2+ and then washed rapidly with culture medium. Sequential EF pulse application led to prolonged elevation of [Ca2+]i levels and eventual cell lysis unless rapid reperfusion with culture medium was achieved. Transient increases in [Ca2+]i in eggs could also be generated by EF pulses in the presence of inositol 1,4,5-trisphosphate (InsP3). 3. In response to EF stimulation fertilized eggs showed [Ca2+]i increases that were enhanced relative to unfertilized eggs. The responses in these fertilized eggs were often followed by repetitive [Ca2+]i oscillations, despite the fact that the [Ca2+]i oscillations associated with sperm penetration had ceased by this stage. 4. In unfertilized mouse eggs the [Ca2+]i increases appeared to be due to direct cation influx since repeated EF pulses caused repeated influx of Mn2+ as monitored by quenching of fluorescence of fura-2 loaded eggs. 5. Under conditions that stimulated reproducible patterns of [Ca2+]i transients we found that a single large [Ca2+]i transient did not cause significant egg activation, but that inducing repetitive [Ca2+]i transients was effective in activating eggs. The speed of activation as judged by the rate of pronuclear formation was also dependent upon the frequency of pulse application. 6. These data show that combining EF pulses with a rapid and precise sequential perfusion system can be used to manipulate [Ca2+]i levels in mammalian eggs. This provides a means of artificial mimicry of the [Ca2+]i transients seen after fertilization. It appears that Ca2+ influx during EF pulses does not cause significant Ca2+ release from internal stores in unfertilized eggs, but after fertilization Ca2+ influx does induce Ca2+ release. It is also apparent that mouse eggs are more successfully activated by repetitive [Ca2+]i increases than by single large [Ca2+]i rises. We suggest that our data provide direct evidence for the hypothesis that a cellular response to oscillations of intracellular [Ca2+]i can be distinct from that to monotonic rises in [Ca2+]i.

Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation.

Freshly ovulated (12 hr post hCG) F1 (C57BL/6 x CBA) hybrid mouse oocytes were parthenogenetically activated by repetitive elevation of Ca2+ induced by carefully controlled electrical pulses. Different patterns of stimulation were employed to examine the role of repetitive calcium changes on meiotic resumption and pronuclear development. In the first series of experiments oocytes received 33 electrical pulses of 1.8 kV/cm delivered every 4 min. The pulse duration decreased according to a negative exponential equation from a 900-microseconds first pulse to give a total pulse duration of 18.721 msec. The strength of calcium stimuli was varied by changing the concentration of CaCl2 in the medium. Ninety-eight percent of the oocytes stimulated with 12 microM calcium extruded the second polar body by the end of treatment and 92% completed pronuclear formation between 3.5 and 8 hr after the first pulse. For higher or lower Ca2+ concentrations the proportion of oocytes developing pronuclei decreased; the timing of pronuclear formation was retarded and the majority of oocytes failed to form a pronucleus after extrusion of the second polar body. In the second series of experiments, the strength of the calcium stimuli was modulated by changing the duration of the 33 electrical pulses given in the presence of 12 microM calcium. By increasing the total pulse duration to 33.958 msec, 100% of the oocytes activated and completed pronuclear formation between 3 and 5 hr after the first electric pulse. Stimulation protocols of lower total pulse duration (less than 18.721 msec) gave rise to high rates of partial activation (up to 95%). Examination of these partially activated oocytes showed metaphases with haploid sets of chromatids characteristic of third meiotic metaphase arrest. The results indicate that repetitive calcium stimuli can regulate the rate and extent of meiotic resumption and the time course of pronuclear formation during mouse oocyte activation. They suggest that meiotic resumption in mammalian oocytes is regulated by the amplitude and frequency of cytosolic calcium oscillations induced by the activating stimulus.

De novo formation of centrioles in parthenogenetically activated, diploidized rabbit embryos.

In rabbit oocytes activated parthenogenetically by repetitive electric pulses, centrioles develop de novo in blastocysts. Centrioles were not observed in earlier stages of development, not until the blastocoele is formed. Up to the morula stage (between 8-32 cells), a filamentous, electron-dense material develops and aggregates with a small vesicle fraction within the well developed Golgi apparatus. A spherical to ovoid electron dense mass forms, which is comparable to the deuterosome or to the blepharoplast. The quantity of the electron dense material enlarges and it seems to give rise to the centriole "generating complex". Centrioles arise in all three differentiated cell types of the blastocysts, the mural and polar trophoblasts and the embryonal cell mass at the same time. Some of the forming centrioles in parthenotes have a co-linear arrangement, as in control blastocysts. It is not yet known whether the co-linearly arranged centrioles represent a maturation phase, prior to the formation of the usual diplosome, with centrioles oriented perpendicularly to each other. Nor is it known whether the forming centrioles are functioning as the polar organizer of the mitotic spindle or if they can perform any other centriolar function.

The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation.

Freshly ovulated rabbit oocytes were activated parthenogenetically by periodically repeated calcium stimuli generated by electric field pulses applied onto the plasma membrane. Electric field pulses of 1.8 kV cm-1 were delivered every 4 min for 1 h 30 min (22 double pulses) in a specially designed chamber. Before each pulse, the culture medium was replaced by an isotonic glucose solution containing 10 microM Ca2+. The effects of modulating the ionic stimuli (by changing the duration of EF pulse) on a postactivation reaction, and/or on the pre- and postimplantation development, were studied. The rate of activation increased progressively as the pulse duration lengthened. For 22 pulses of 200 microseconds, 13% of oocytes were activated versus 100% for 1200 microseconds. The uniformity of the parthenogenetic response was obtained when oocytes were exposed to a series of pulses within which the reduction of pulse duration followed a negative exponential law. The influence of such activating treatment on the preimplantation development was tested using two treatments of 22 pulses with a total pulse duration equal to 14,868 and 11,228 microseconds, respectively. For the weaker treatment, a lower proportion of embryos underwent compaction and those that compacted were irregular. In contrast, the majority of embryos resulting from the stronger treatment compacted and developed into blastocysts. The most significant result that emerges from this study is that the level of stimulation affects in vitro developmental potency after the third cleavage division. The postimplantation viability of parthenogenetic eggs was tested and the results showed that parthenogenetic rabbit embryos died at a similar stage of development to the parathenogenetic mouse embryos. But, in the present series, high implantation rates and embryonic development (66%) till day 10-11 of pregnancy were obtained after the appropriate pulsatile EF treatment of oocytes. The parthenogenetic fetuses were of smaller size than the controls, but the development of the trophoblast tissue was proportional to the development of the fetuses. Anomalies of fetuses were also observed. This study reveals that activation is not a time-limited event and that the type of activating treatment has a marked effect on the ability of the resulting parthenogenetic embryos to develop to the early postimplantation stages. The sustained alteration of the cytoplasmic activity provides a useful tool to study the function of embryonic or somatic nuclei introduced during the earliest stages of activation.

Effects of electric field on fusion rate and survival of 2-cell rabbit embryos.

Electric-field-induced blastomere fusion was studied in 2-cell rabbit embryos. Field strengths (1 to 3kV cm-1) and durations (35 to 1000 microseconds) were chosen so as to provide the right balance between fusion rate, viability and developmental capacity of embryonic cells. Maximum plasma membrane tolerance of 2-cell rabbit embryos was observed at about 3kV cm-1 for 1000 microseconds. All surviving 'fused' embryos were able to develop in vitro and most of them formed expanded blastocysts. Observation of 'fused' embryos immediately after fusion and during the whole cell cycle showed that 27.7% of the two diploid nuclei remained separated in the hybrid cell. More than one metaphase plate was formed at the onset of mitosis causing direct cleavage into three or four 'cells'. In the remaining embryos the two diploid nuclei seemed to form a common metaphase plate and cleaved into two equal blastomeres. After transfer to recipient does, 54.4% of these tetraploid embryos developed beyond implantation. Between day 11 and 20, ten live and morphologically fully normal embryos were recovered. Nine embryos were uniformly tetraploid and one recovered on day 18 was a diploid/tetraploid mosaic. The remaining implantation sites contained either abnormal, very retarded embryos or indefinable embryo remnants. After transfer of 'nonfused' embryos treated with 3kV cm-1, 49% gave birth to normal live young. These results suggest that the electric field can be applied successfully in a relatively wide strength and duration range without causing any visible teratogenic effect on treated embryos. Thus, tetraploid embryos can develop normally at least until two-thirds of pregnancy, but the question whether they are able to survive till term remains open.

Production of identical twins by bisection of blastocysts in the cow.

Day-8 embryos were recovered by a non-surgical method from superovulated crossbred heifers. Normal expanded blastocysts with a distinct inner cell mass and a trophoblast were released from the zona pellucida and bisected along a sagittal plane into two 'half' blastocysts. Each 'half' blastocyst was replaced in an empty zona pellucida and cultured for 2 h in B2 medium. After culture the 'half' blastocysts were directly transferred to recipient heifers via the cervix. From 11 blastocysts, 11 monozygotic 'half' blastocyst pairs were transferred to 11 recipients: 8 recipients became pregnant, 4 carried twins and one delivered a normal calf and an acardiacus amorphus monster consisting of disorganized embryonic tissues. A further 11 'half' blastocysts were transferred as singletons to 11 recipients. Five recipients were apparently pregnant at Day 42. One returned to oestrus at Day 45, 3 were carrying normal fetuses and 1 a pair of normal twin fetuses when slaughtered at Day 128. It is concluded that even after the first irreversible cellular differentiation which occurs at the blastocyst stage it is still possible to produce identical cattle twins by bisection of the Day-8 blastocyst.

Production of monozygotic twins by micromanipulation and cervical transfer in the cow.

To develop the practical production of monozygotic twins in cattle, the ability of day 6 to 7 cow embryos to survive after they were split into two halves was tested. Day 6 to 7 embryos were recovered by the cervical method from superovulated Charolais heifers. Only normal embryos were selected at the advanced compaction stage when the cavity appears. Each embryo was cut into two halves and each half was replaced in an empty zona pellucida. After micromanipulation, the half embryos were directly transferred to recipient heifers via the cervix. From 16 normal embryos manipulated, 14 monozygotic half embryo pairs were obtained which were all transferred to 14 recipients (pregnancy rate 64.2 per cent); six recipients carried twins (twinning rate 66.6 per cent). Thus 15 fetuses were recorded from the 14 monozygotic half embryo pairs transferred.

Cervical transfer of deep frozen cattle embryos.

Cattle blastocysts were collected from 29 donors 7-8 days after estrus and frozen and stored in liquid nitrogen up to several months. Two procedures were used for freezing and thawing: -- procedure A: slow cooling to -60 degrees C (0.3 degrees C/min to -60 degrees C) and slow thawing (12 degrees C/min); -- procedure B: slow cooling to -30 degrees C (0.3 degrees C/min to -30 degrees C) and rapid thawing in a water bath at 37 degrees C. After thawing, the embryos were cultured from 8 to 12 hours before transfer; 36% of the embryos continued normal development during culture; both procedures resulted in a high pregnancy rate (procedure A: 10/15; procedure B: 11/15) after single cervical transfer of the frozen thawed embryos which developed normally in vitro. However the overall survival rate was low (25%) and varied between donors, indicating that progress must be made before the technique of freezing can be extended to applied conditions.

Importance of gestation losses after non-surgical transfer of cultured and non-cultured bovine blastocysts.

Several techniques, including progesterone assay at days 21, 28 and 35, heat detection twice daily, rectal palpation at day 60 or planned slaughter, were combined to determine the importance of gestation losses after a single cervical transfer of 128, 10 to 12-day-old bovine embryos. Using non-cultured embryos (n = 83) transferred within six hours after recovery, 29.3 per cent of the initiated gestations were lost before day 60. Few abortions occurred after that stage. More than 50 per cent of the losses concerned preimplantation embryos from day 21 to day 35. When embryos cultured for 24 hours at 37 degrees C were used, there was a marked increase in the gestation loss (46.3 per cent) occurring mainly at the time of implantation. This suggests that the culture had a detrimental effect on the embryonic disc cells.

An instrument for transcervical recovery of embryos from heifers.

A non-surgical system of embryo recovery is described. It consists of a rigid probe of small diameter (4 mm) for recovering the embryos from young heifers (10 to 12 days after estrus). An average of 6.35 eggs were recovered per donor from 64 heifers 15 to 22 month-old having more than two palpable corpora lutea after superovulation. Forty donors were slaughtered after recovery to determine the number of ovulations, the state of the uterus and to do a post-mortem perfusion. The average recovery rate of embryos was 56.4 % ; an additional 9 % were recovered after slaughter. We compared two recovery methods differing in mode of liquid return ; no significant differences were found. Twenty-four animals not slaughtered after recovery returned to heat a mean 23.8 days after induced estrus (control cycle).