Parthenogenetic Activation Of Mouse Oocytes Research Articles

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Dynamic alterations in H4K12 acetylation during meiotic maturation and after parthenogenetic activation of mouse oocytes.

The aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1

In all mammalian species studied to date, the initiation of oocyte activation is orchestrated through alterations in intracellular calcium (Ca2+) signaling. Upon sperm binding to the oocyte plasma membrane, a sperm-associated phospholipase C (PLC) isoform, PLC zeta (PLCζ), is released into the oocyte cytoplasm. PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG), which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which induces the release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores. Subsequent Ca2+ oscillations are generated that drive oocyte activation to completion. Ca2+ ionophores such as ionomycin have been successfully used to induce artificial human oocyte activation, facilitating fertilization during intra-cytoplasmic sperm injection (ICSI) procedures. Early studies have also demonstrated that the PKC activator phorbol 12-myristate 13-acetate (PMA) acts synergistically with Ca2+ ionophores to induce parthenogenetic activation of mouse oocytes. Interestingly, the Ca2+-induced signaling cascade characterizing sperm or chemically-induced oocyte activation, i.e. the “shock and live” approach, bears a striking resemblance to the reactivation of latently infected HIV-1 viral reservoirs via the so called “shock and kill” approach, a method currently being pursued to eradicate HIV-1 from infected individuals. PMA and ionomycin combined, used as positive controls in HIV-1 latency reversal studies, have been shown to be extremely efficient in reactivating latent HIV-1 in CD4+ memory T cells by inducing T cell activation. Similar to oocyte activation, T cell activation by PMA and ionomycin induces an increase in intracellular Ca2+ concentrations and activation of DAG, PKC, and downstream Ca2+-dependent signaling pathways necessary for proviral transcription. Interestingly, AMPK, a master regulator of cell metabolism that is activated thorough the induction of cellular stress (e.g. increase in Ca2+ concentration, reactive oxygen species generation, increase in AMP/ATP ratio) is essential for oocyte maturation, T cell activation, and mitochondrial function. In addition to the AMPK kinase LKB1, CaMKK2, a Ca2+/calmodulin-dependent kinase that also activates AMPK, is present in and activated on T cell activation and is also present in mouse oocytes and persists until the zygote and two-cell stages. It is our hypothesis that AMPK activation represents a central node linking T cell activation-induced latent HIV-1 reactivation and both physiological and artificial oocyte activation. We further propose the novel observation that various compounds that have been shown to reactivate latent HIV-1 (e.g. PMA, ionomycin, metformin, bryostatin, resveratrol, etc.) or activate oocytes (PMA, ionomycin, ethanol, puromycin, etc.) either alone or in combination likely do so via stress-induced activation of AMPK.

Comparison between different combinations of chemical treatment on parthenogenetic activation of mouse oocytes and its subsequent embryonic development

Combination of different chemicals has been an effective method to activate oocytes. The objectives of this study were to evaluate the optimal: (1) concentration (Experiment 1) and duration (Experiment 2) of strontium chloride (SrCl2) in the presence of cytochalasin B (CB), (2) secondary activation agent [6-dimethylaminopurine (6-DMAP), cycloheximide (CHX) and CB] in combination with calcium ionophore (CaI) (Experiment 3) and (3) ethanol (EtOH) concentration in combination with 6-DMAP (Experiment 4). There were no significant (P>0.05) differences in cleavage (86.82–89.06%) and blastocyst (33.42–46.83%) rates when oocytes were treated with various concentrations of SrCl2 (2–10 mM). Three-hour duration generally showed the highest cleavage (93.83%) and blastocyst rates (60.42%) compared to other durations. Comparing with various combinations, CaI +6-DMAP showed the highest cleavage at all stages of development and significantly (P<0.05) higher from other combinations at 8-cell (50.58%), morula (30.29%) and blastocyst (23.28%) stages. When comparing the EtOH concentrations, 8% EtOH gave significantly (P<0.05) highest cleavage rates at 8-cell stage (39.70%), morula (21.50%) and blastocyst (13.86%) stages followed by 9% and 7% EtOH. In conclusion, 10 mM SrCl2+5 µg/ml CB in calcium-free Chatot Ziomek Bavister medium for 3-h incubation duration gave the highest cleavage rates of parthenotes mouse oocytes.

Effects of chemical combinations on the parthenogenetic activation of mouse oocytes

The aim of this study was to identify an optimal method for the parthenogenetic activation of mouse oocytes. Ethanol (EH), strontium chloride (SrCl2) and ionomycin calcium salt were each combined with cytochalasin B to induce the parthenogenetic activation of CD-1® mouse oocytes. Among the EH combination groups, the blastocyst formation and hatching rates of the group that was activated with EH and CB for 5 min were significantly higher compared with those of the groups that were activated for 7 and 10 min (P<0.05). Among the SrCl2 combination groups, the blastocyst formation and hatching rates of the group that was activated with SrCl2 and CB for 30 min were significantly higher compared with those of the groups that were activated for 1 and 2 h (P<0.05). Among the ionomycin calcium salt combination groups, the blastocyst formation and hatching rates of the group that was activated with ionomycin and CB for 3 min were higher compared with those of the groups that were activated for 5 and 7 min (P<0.05). Compared with the other two combinations, the experimental indicators of the EH combination groups were notably superior (P<0.05). For combined activation, simultaneous activation with two substances was significantly more effective than successive activation (P<0.05). For combined activation with EH and cytochalasin B in mouse oocytes, 5 min of parthenogenetic activation had significant advantages with regard to cleavage, blastocyst formation and blastocyst hatching rates. In addition, the activation rate of combined activation was higher than that of single activators. For combined activation, the simultaneous application of two activators has a superior effect.

Parthenogenetic activation of mouse oocytes by strontium chloride: A search for the best conditions

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10 mM SrCl 2 for 0.5–5 h, activation rates (88.4 ± 4.1 to 91.2 ± 2.7%) did not differ (mean ± S.E.; P > 0.2), but rate of blastulation (57.3 ± 3.5%) and cell number per blastocyst (45.0 ± 2.4) were the highest after treatment for 2.5 h. When treated with 1–20 mM SrCl 2 for 2.5 h, the activation rate and cell number per blastocyst were higher ( P < 0.02) after 10 mM SrCl 2 treatment than other treatments. The best activation and development were obtained with Ca 2+–free Sr 2+ medium, but the activation rate was low (37.7 ± 1.6%) in Ca 2+-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher ( P < 0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6 mM SrCl 2, and many lysed, with increased intensity of Sr 2+ treatment. The presence of CB in SrCl 2 medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr 2+ treatment alone, with 34% blastulating, but rates of activation and blastulation increased ( P < 0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less ( P < 0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl 2 treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5 h with 10 mM SrCl 2 in Ca 2+-free medium supplemented with 5 μg/mL of CB.

Pre- and Post-implantation Development of Parthenogenetic Embryos Induced by Progesterone, and Birth of Aggregation Chimeras with Diploids in Mice

We have previously demonstrated that parthenogenetic activation of mouse oocytes can be induced by exposure to 100 μM progesterone for 6 h, and activated eggs developed to the blastocyst stage. Here we examined the pre- and post-implantation development of these parthenogenones in mice, and whether they could be rescued and develop to term by forming aggregation chimeras with in vivo-derived embryos or triploids. When these parthenogenetic embryos were diploidized by 5 μg/ml cytochalasin B, 23% of them developed to the blastocyst stage, and the implantation rate was 51% after transfer to recipients. Live parthenogenetic fetuses were also found on day 10.5 of gestation, but parthenogenones could not survive beyond day 10.5 of gestation. To rescue these lethal parthenogenetic embryos, aggregation chimeras of parthenogenones and in vivo-derived embryos were made at the 4-cell stage. Sixty six percent of chimeric embryos which developed to single blastocysts were transferred to 8 recipients. Two of them delivered a total of nine live pups, and one of them was chimeric. In the present study we confirmed that parthenogenetic mouse embryos, even when activated by progesterone, developed to the mid-gestation stage, and could be rescued by forming chimeras consisted of parthenogenetic and diploid embryos. On the other hand, aggregation chimeras derived from parthenogenetic and triploid embryos were lethal after implantation.

Mitochondrial function and the effect of oocyte aging on parthenogenetic activation of mouse oocytes.

Mitochondrial function and the effect of oocyte aging on parthenogenetic activation of mouse oocytes.

Parthenogenetic Activation of Mouse Oocytes by Exposure to Strontium as a Source of Cytoplasts for Nuclear Transfer

Cell-cycle phase of the donor and recipient cells at the moment of nuclear transfer influences subsequent development of the reconstituted embryo. In order to study this effect, the precise cell-cycle phase of the recipient oocyte at the time of fusion must be known and this depends on reliable activation of oocytes in a protocol that has a low incidence of spontaneous activation. Mouse oocytes recovered before (8-10 hours post-human chorionic gonadotropin [hCG]) and after ovulation (14 and 18 hours post-hCG) were exposed to strontium ions in calcium magnesium-free M16 culture medium. The effect on development of haploid parthenotes of post-hCG age of the oocyte, the duration of exposure, and strontium concentration in the medium was determined. These experiments established a reliable method of parthogenetic activation of recently ovulated mouse oocytes, involving the culture of oocytes for 60 minutes in 25 mM strontium in a calcium magnesium-free M16 medium. This method of activation was also able to induce activation of preovulatory oocytes after a preincubation period in vitro. Only a low incidence of spontaneous activation was observed if oocytes were recovered before or immediately after ovulation (14 hours after hCG).

Parthenogenetic Activation of Mouse Oocytes by Strontium.

The purposes of this study were to determine the optimal conditions for inducing parthenogenetic activation by strontium and to evaluate the developmental ability of the activated oocytes in mice. MII oocytes collected from B6CBF1 and CD-1 mice were cultured for 2-60 min in medium containing 1.7 mM strontium. The proportion of oocytes activated increased in a time-dependent manner, with an apparent difference between the B6CBF1 and CD-1 strains in sensitivity to strontium. In oocytes derived from B6CBF1 mice more than 90% of oocytes were activated by the treatment with strontium for 30 min, whereas more than 80% of the oocytes from CD-1 mice were activated by the treatment for 5 min. The majority of oocytes formed the second polar body and a single pronucleus in both cases. Of the parthenogenetically activated 1-cell embryos with the haploid genome, around 39% developed to the blastocyst stage in the B6CBF1, and in contrast the developmental ability was low (11%) in the CD-1 strain. The ability to develop to blastocysts was significantly improved in diploid parthenogenetic embryos of both the strains; 93% and 58% were developed to the blastocyst stage, respectively. After transfer of the diploid parthenogenetic embryos to recipient mice, 11% of the embryos developed to day 10 of gestation. The record of intracytoplasmic Ca2+ concentrations showed that the exposure of oocytes to medium containing 1.7 mM strontium induced repetitive intracellular Ca2+ transients. These data clearly showed that strontium is a potent stimulus for inducing parthenogenetic activation and supporting in vitro and in vivo development in mouse oocytes.

Parthenogenetic activation of mouse oocytes by 6-dimethylaminopurine

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca2+-free, Na+-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na+-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.

Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes.

Intracytoplasmic sperm injection (ICSI) in the human is a very effective procedure which allows the fertilization of the majority of oocytes even in cases of extreme oligoasthenoteratozoospermia. Round-headed acrosomeless human spermatozoa, however, form an exception to this rule, because in about half of the couples with globozoospermia all oocytes remain unfertilized after injection. The incapacity of the spermatozoon to activate the oocyte following injection of round-headed spermatozoa could be the underlying mechanism. To investigate this hypothesis, activation rates of mouse oocytes injected with spermatozoa from a patient with globozoospermia were compared with those obtained after injection with normal spermatozoa. Of mouse oocytes surviving the injection with donor spermatozoa, 95% underwent activation, compared to none of the 88 mouse oocytes surviving the injection with round-headed spermatozoa. After fixation, prematurely condensed sperm chromosomes were found in these oocytes. Parthenogenetic activation of mouse oocytes (8% ethanol at 40 min after injection) injected with round-headed spermatozoa led to the activation of 96% of oocytes. These oocytes developed normally to the first mitosis and were fixed for analysis of the sperm karyotypes. The incidence of chromosomal abnormalities of round-headed spermatozoa (6%) was similar to that in spermatozoa from a fertile donor (9%). These data provide further information on the basic defect in cases of globozoospermia and demonstrate that globozoospermia is not associated with sperm karyotype abnormalities.

Metabolism of energy substrates following fertilization or parthenogenetic activation of mouse oocytes

Extruded polycrystalline pure magnesium (Mg) with fine grain size (~1.2 µm) exhibits ductility of over 100% at room temperature, in spite of the presence of a strong basal texture. In this study, a set of complementary in-situ characterisation techniques over multiple-length scales were utilised to reveal the deformation modes enabling such ductility. Synchrotron X-ray diffraction results show that the elastic lattice strain of fine-grained sample for tensile elongation up to ~55% is 3–10 times lower than that in the coarse-grained sample, indicating the absence of significant strain accumulation inside fine grains and potential inter-granular deformation in bulk. In-situ scanning electron microscopy validates the predominant operation of the inter-granular deformation, and it further reveals that the inter-granular deformation occurs by the relative sliding between groups of grains having similar orientations. The deformation resulting from such sliding is substantial, and it is accommodated by the rotation of grains located between slid grouped grains, from hard to softer orientations to allowing dislocation slip to readily occur. The accommodating mode of dislocation slip is further supported by in-situ transmission electron microscopy observations. Dislocations glide readily to, and annihilate at, grain boundaries. The observations and direct evidences presented herein suggest that the major deformation mode is sliding between grouped grains that is accommodated by grain rotation and dislocation slip, in contrast to dislocation slip in coarse-grained Mg. The coordinated deformation processes postpone the occurrence of localised stress concentration and greatly increases the ductility of pure Mg at room temperature.

Stimulation of Parthenogenesis in Mouse Ovarian Follicles by Inhibitors of Inosine Monophosphate Dehydrogenase1

The effects of hormonal priming and inosine monophosphate (IMP) dehydrogenase inhibitors on the meiotic maturation and parthenogenetic activation of mouse oocytes were examined in this study. In the first series of experiments, unprimed mice or mice primed 24 h with equine chorionic gonadotropin (eCG) received injections of the IMP dehydrogenase inhibitors, bredinin (Br) or mycophenolic acid (MA), followed by histological examination at 24 h, 48 h, and 72 h after drug administration. In both treatment groups, oocytes from nonatretic antral follicles were stimulated to undergo germinal vesicle breakdown by 24 h and became parthenogenetically activated as manifested by pronuclear formation and early cleavage divisions. The parthenotes underwent degeneration by 72 h. In the second part of this study, the effects of priming and drug treatment on parthenogenetic activation and subsequent developmental potential in vitro were examined. Mice were primed with eCG, and 24 or 48 h later received injections of Br or MA. Cumulus cell-enclosed oocytes were isolated 21-22 h later and assessed for maturation; those having undergone germinal vesicle breakdown were cultured and subsequently examined for embryonic development. In mice primed for 24 h, but not 48 h, Br and MA stimulated a significant number of oocytes to resume maturation in vivo; these subsequently underwent activation and developed to blastocysts in vitro. In another series of experiments, germinal vesicle-stage oocytes were isolated from primed or unprimed mice and cultured in vitro to permit spontaneous meiotic maturation. Nine percent of mature ova from 24-h-primed mice developed to 2-cell parthenotes; activation in ova from unprimed and 48-h-primed mice was considerably lower. A time-course experiment demonstrated that the extent of parthenogenetic activation in vivo following Br treatment was related to the period of time between drug injection and isolation of ova, the optimal period being 12 h. Neither Br nor MA had a direct activating effect on the oocytes as evidenced by an inability to induce parthenogenesis in vitro. Simultaneous injection of hCG with either Br or MA stimulated ovulation and prevented the parthenogenetic response. These data are consistent with the idea that conditions within the follicle promote parthenogenetic activation when the oocyte matures in the absence of gonadotropin stimulation.

Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+

The parthenogenetic activation of mouse oocytes by several agents is accompanied by a large rise in the intracellular calcium concentration ( [Ca2+]i). The tumour-promoting phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) activates some cellular processes which are also activated by raised [Ca2+] (ref. 2). We therefore tested TPA on mouse oocytes and found it to be a potent parthenogenetic activating agent. Recent reports suggest that TPA mimics endogenous diacylglycerol3 and can stimulate cells by activating protein kinase C without raising [Ca2+]i (refs 4, 5). We have now measured [Ca2+]i with aequorin in mouse oocytes treated with TPA. Sustained oscillations in [Ca2+]i appeared in those oocytes which were subsequently activated. We also measured [Ca2+]i during fertilization. The response began with [Ca2+]i transients as reported previously, but the transients continued in a regular pattern for 4 h. There was no rise in [Ca2+]i of the type induced by activating agents such as ethanol. One component of the fertilization response appears to be related to the TPA-induced oscillations.

Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol.

AbstractTreatment in vitro of ovulated mouse oocytes for 5–10 min with 4.5–8.6% ethanol or 0.24–0.4% benzyl alcohol was found to induce parthenogenetic activation. Suppression of the second polar body with cytochalasin D and subsequent culture in vitro gave a high yield of parthenogenetic morulae and blastocysts (up to 85% overall). Fifty percent of transferred diploid parthenogenones implanted, 14% developed into egg cylinders, and 8% formed somites and developed as far as the forelimb‐bud stage.

The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment

A technique in which mouse eggs are stimulated to develop parthenogenetically following their brief incubation in 7% ethanol in PBS is described. Very high rates of activation were achieved, and a detailed analysis presented of the class of parthenogenone which develops a single haploid pronucleus following second polar body extrusion. As preliminary studies on presumptive haploid morulae indicated that a proportion of the metaphase spreads examined had an aneuploid chromosome constitution, the incidence of aneuploidy at the first cleavage mitosis was investigated. In the control groups the level of aneuploidy was about 1%, whereas in the ethanol-treated series the incidence ranged from 13 . 6-18 . 8%. Additional pre-treatment of ethanol-activated oocytes in low osmolar medium raised the incidence of aneuploidy to 28 . 3%. Metaphase groups with 18, 19, 21 and 22 chromosomes present were observed in addition to groups with a normal complement of 20 chromosomes. The possible mode of action of ethanol in inducing parthenogenetic activation of mouse oocytes, and a high incidence of aneuploidy, is discussed in relation to previous knowledge of the action of this agent. Preliminary studies using G-banding indicate that the aneuploidy observed appears to arise as a result of non-disjunction which may involve any of the chromosomes of the complement.

Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis

Recently ovulated mouse oocytes at the Metaphase II stage undergo parthenogenetic activation (as indicated by the formation of pronuclei) when incubated for 6 h in the presence of cycloheximide or puromycin; the activation response increases progressively with the concentration of inhibitor. Activation is induced with concentrations of cycloheximide that depress protein synthesis by more than 70%. Pronoculear formation occurs when protein synthesis is almost totally inhibited. Incubation of oocyte in Actinomycin D failed to initiate activation. The results show that the Metaphase II oocyte of the mouse synthesizes protein factor(s) which are necessary for the maintenance of the meiotic block. Other protein(s) having opposite effects and a different rate of turnover may also participate in activation since when the oocytes are treated with a high concentration of cycloheximide (10 microgram ml-1) for varying periods of time, or with varying concentrations for a short period of time (1 h), a more complex activation response curve is obtained. Oocytes activated with cycloheximide are capable of further development, following transfer to the oviducts of pseudopregnant recipients, in a proportion similar to that of oocytes activated in other ways.

Parthenogenetic activation of mouse oocytes following avertin anaesthesia

ABSTRACT Avertin anaesthesia induced mouse eggs to become activated parthenogenetically. An increasing incidence of activation was observed when females were anaesthetized 6·5, 9 and 13 h after ovulation, reaching a maximum of 45·8 %. In a spontaneously ovulating group approximately 12·5 % of all the eggs ovulated, or 27·3 % of all the eggs activated evoked a decidual response, and the presence of implanting embryos was confirmed histologically. These findings demonstrate a new and simple method of inducing post-implantation parthenogenetic development in the mouse, and stress the necessity of taking into account the possible consequences of anaesthesia in the early post-ovulatory period.