Cleavage rates of diploid and tetraploid mouse embryos during the preimplantation period

Despite the fact that spontaneous tetraploidy is a rare phenomenon in mice, such embryos may be produced experimentally by a variety of means, though only a very limited degree of postimplantation development has been achieved. Despite this apparent limitation, much data on the rate of development of preimplantation tetraploid embryos has been published. However, the findings from these studies has often been conflicting. In the light of the recent successful achievement of advanced postimplantation tetraploid development in our laboratory, we decided it was an opportune time to re-evaluate the preimplantation development of these embryos in as near to optimal conditions as we could achieve. Three groups were studied, namely 1) control (diploid) embryos developing in vivo, 2) control (diploid) embryos that had been isolated at the 2-cell stage, briefly retained in culture, then transferred to the oviducts of pseudopregnant recipients, and 3) tetraploid embryos produced by electrofusion of blastomeres at the 2-cell stage, then transferred to the oviducts of pseudopregnant recipients. Embryos were isolated from females from each group at specific times after the HCG injection to induce ovulation. The total cell number of each embryo was established and the log mean values were plotted against time. From the gradients of the lines it was possible to establish that there was a significant difference between the cell doubling time of the transferred controls (group 2) compared to the in vivo controls (group 1) with cell doubling times of 15.86 +/- 1.45 h and 10.27 +/- 0.24 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.     

Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.     

Developmental potential and chromosome constitution of strontium-induced mouse parthenogenones.

The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2-10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two-pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr(2+)-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr(2+)-induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.     

The histochemical identification of primordial germ cells in diploid parthenogenetic mouse embryos

An attempt has been made to improve the early post-implantation development potential of diploid parthenogenetic mouse embryos by transferring parthenogenetic blastocysts to one uterine horn of a pseudopregnant recipient and a similar number of fertilized embryos to the contralateral horn. In control studies, diploid parthenogenetic embryos were transferred to both uterine horns of appropriate recipients. Unfortunately no obvious advantage appeared to be gained by carrying out the former manoeuvre. A significant improvement in the development potential of the parthenogenones could have indicated that their poor post-implantation survival might have been associated with a deficiency, possibly of hormonal origin, in the functioning of their decidual reaction. However, sufficient somite-containing parthenogenetic embryos were obtained in this study to allow a comparison to be made between them and fertilized embryos that were morphologically at a comparable stage of development. The parthenogenones were found to have a markedly smaller crown-rump length than their fertilized counterparts. A high proportion of both the parthenogenetic and fertilized embryos were subsequently fixed and appropriately stained in order to localize alkaline phosphatase activity. The analysis of this material clearly demonstrated that parthenogenetic mouse embryos are in fact capable of producing primordial germ cells. The latter were recognized by their morphology, histochemical staining appearance, and characteristic location, being found in the early 'turned' embryos within the dorsal mesentery in close proximity to the developing gut tube, and in the more advanced limb-bud stage embryos within the gonadal ridges.     

The cleavage rate of digynic triploid mouse embryos during the preimplantation period

Triploidy is a lethal condition in mammals, with most dying at some stage between implantation and term. In humans, however, a very small proportion of triploids are liveborn but display a wide range of congenital abnormalities. In particular, the placentas of human diandric triploid embryos consistently display “partial” hydatidiform molar degeneration, while those of digynic triploids generally do not show these histopathological features. In mice, the postimplantation development of diandric and digynic triploid embryos also differs. While both classes are capable of developing to the forelimb bud stage, no specific degenerative features of their placentas have been reported. Diandric triploid mouse embryos are morphologically normal while digynic triploid mouse embryos consistently display neural tube and occasionally cardiac abnormalities. Previously it was shown that the preimplantation development of micromanipulated diandric triploid mouse embryos was similar to developmentally matched diploid control embryos. In this study, the preimplantation development of micromanipulated digynic triploid mouse embryos is analysed and compared with that of diandric triploid mouse embryos in order to determine whether there is any difference in cleavage rate between these two classes of triploids. Standard micromanipulatory procedures were used to insert a female or a male pronucleus into a recipient diploid 1-cell stage embryo. The karyoplast was fused to the cytoplasm of the embryo by electrofusion. These tripronucleate 1-cell stage embryos were then transferred to pseudopregnant recipients and, at specific times after the HCG injection to induce ovulation, the embryos were recovered and total cell counts made. These results were plotted and regression lines drawn. An additional control group of embryos was subjected to similar micromanipulatory procedures to those used in the experimental study. These embryos had a single pronucleus removed and this was then reinserted into the perivitelline space. Diploidy was immediately restored by electrofusion. These embryos were transferred to recipients and at specific times after the HCG injection the embryos were recovered and total cell counts made. These results were also plotted and regression lines drawn. The results show that the cell doubling time of the digynic triploid embryos was 14.84 h (± 1.19). This was not significantly different from that of the diandric triploid embryos (13.55 h ± 0.86; P > 0.05) or of the manipulated diploid controls (12.12 h ± 0.79; P > 0.05). © 1993 Wiley-Liss, Inc.     

Viable offspring derived from cryopreserved haploid rabbit parthenotes

The female parthenogenetic haploid embryos can be stored long-term by cryopreservation. Briefly, rabbit haploid parthenotes at the 32-cell stage were produced by electroactivation and in vitro culture. At this embryonic stage, parthenotes were individually cryopreserved by a slow-freezing procedure. After thawing, every embryo was disaggregated and blastomeres used as haploid maternal donors of nuclei. These nuclei were transferred to androgenetic haploid hemizygotes, obtained by female pronuclear removal offertilizedova. In the firstexperiment, 38 out of 87 reconstructedheteroparental diploid zygotes reachedthe hatched blastocyst stage invitro. In the second experiment, ourpurpose was toobtain live pups from each frozen-thawed parthenote. Viable offspring (at least one live pup at delivery) were obtained from five out of seven frozen-thawed haploid morula used as donors, with three live hemiclones being the highest number of pups produced from a single thawed parthenote. These results indicate that the rabbit female gametic endowment can be successfully stored by cryopreservation of parthenogenetic haploid embryos.     

THE PRODUCTION OF PARTHENOGENETIC MAMMALIAN EMBRYOS AND THEIR USE IN BIOLOGICAL RESEARCH

1. The eggs of many mammalian species show signs of early parthenogenetic development as they age after ovulation and oocytes may form transplantable terato-carcinomas. These cases of apparently spontaneous parthenogenetic development suggest that the cells of the female germ line have an inherent tendency to divide and differentiate. 2. The ovulated eggs of virgin female mammals may be stimulated to start parthenogenetic development by a wide variety of treatments. Most of these damage the egg so that it does not develop beyond the 4 cell stage. However if the eggs are exposed to electrical activation, hyaluronidase treatment, or temperature shock then in many cases they will develop into blastocysts. 3. These blastocysts may be either haploid or diploid. Haploid blastocysts may be formed either by the egg extruding the nucleus of the second polar body or by the egg dividing in half, so that the female pronucleus is in one cell and the nucleus of the second polar body is in another cell. Diploid blastocysts are formed by the retention of the nucleus of the second polar body within the egg. The way in which the egg develops may be controlled by altering the osmolarity of the culture medium, the age of the egg at the time of activation, or the strain of animal used. 4. The action of the sperm on the egg can be defined by comparing the events of normal fertilization and parthenogenetic activation. Both these stimuli cause the egg to expose binding sites for Concanavalin A to synthesize DNA and to divide. However, the release of cortical granules, which occurs after fertilization, does not appear to be induced by parthenogenetic activation, and it is significant that parthenogenones lack the sperm nucleus and mitochondria. 5. The majority of parthenogenones die soon after implantation. Death at this time occurs with parthenogenones obtained from the activated eggs of both inbred and outbred stocks. Death might be caused by recessive lethal mutations or by extra-genetic effects of the maternal chromosomes. 6. Parthenogenones contain endogenous A-type particles which shows that these bodies are inherited through the female germ line. 7. Parthenogenones may in the future provide both a method for chromosome mapping and a source of haploid cells. At present the use of mammalian parthenogenones in biological research is restricted by the heavy embryonic losses which occur around the time of implantation. This means that the role of the sperm, gene activity and virus expression must be studied during a very limited period. Part of the mortality before implantation is the consequence of the damage which the egg suffers during activation and it should be possible to reduce this loss by improving the techniques for activation. It may also be possible to increase the quantity of cells derived from haploid and diploid mammalian embryos by deriving teratocarcinomas from them.     

The Chromosomes of Turkey Embryos during Early Stages of Parthenogenetic Development

In the early stages of parthenogenetic development in turkey eggs, many blastoderms are mosaics of haploid, diploid and polyploid cells. The genome composition of these blastoderms can be identified by C-banding. They may be generally described as either A-Z/2A-ZZ/nA-nZ or A-W/2A-WW/nA-nW and are found in a nearly 1:1 ratio. The blastoderms showing the W body (W⁺) become lethal within two days of incubation. The haploid cell proportion decreases rapidly during the early stage of development, and, as haploid cells decrease, the proportion of polyploid cells appears to increase. At six days of incubation, various kinds of parthenogenetic development can be observed. Their genome compositions are either diploid (2A-ZZ) or mosaic (A-Z/2A-ZZ). These findings suggest that diploid parthenogenesis occurs by either suppression of meiosis II or chromosome doubling some time after the first cleavage division. The frequent occurrence of mosaic blastoderms indicates that the majority, if not all, of the parthenogenetic embryos initiate their development in haploid ova.     

Developmental potential of bovine androgenetic and parthenogenetic embryos: a comparative study

In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.     

The incidence of aneuploidy after single pulse electroactivation of mouse oocytes

A brief electric pulse often produces a high rate of activation of recently ovulated oocytes. Some other efficient parthenogenetic stimuli, such as alcohol, however, disrupt the spindle apparatus and increase the incidence of aneuploidy. In this paper, we have determined whether electroactivation per se increases the incidence of chromosomal segregation errors in haploid parthenogenones as evidenced at first cleavage mitosis. Superovulated F1 hybrid female mice were killed at 15.5, 18.5, 22.5, and 25 h after the HCG injection. Batches of 10–12 cumulus-denuded oocytes were transferred to an electroactivation chamber containing mannitol which was connected to a high voltage pulse stimulator and the pulse was triggered once. A high proportion of oocytes activated following this treatment, but only the single-pronuclear haploid parthenogenones were incubated overnight in medium containing colcemid, to determine the incidence of aneuploidy as evidenced at first cleavage mitosis. “Sham” electroactivation groups were also examined for evidence of activation and aneuploidy as described above. In these cases, cumulus-denuded oocytes were put through the electroactivation chamber but the pulse was not triggered. A further group of oocytes was studied to determine the effect of handling and exposure to hyaluronidase on activation frequency and parthenogenetic pathways. Finally, the spontaneous rate of aneuploidy was examined in fertilised embryos of F1 hybrid female mice × Rb(1.3)1Bnr male mice at first cleavage mitosis. The results show that single pulse electroactivation does not increase the level of aneuploidy in single-pronuclear parthenogenones compared to the “sham” group or the spontaneous rate observed in 1-cell fertilised embryos, nor does aneuploidy appear to increase with postovulatory age. The developmental pathways observed in the electroactivation group are significantly different to those observed in the “sham” group, and the level of activation observed in both groups is increased through handling of oocytes and their exposure to hyaluronidase. © 1993 Wiley-Liss, Inc.     

Two maternally derived X chromosomes contribute to parthenogenetic inviability

In certain extraembryonic tissues of normal female mouse conceptuses, X-chromosome-dosage compensation is achieved by preferential inactivation of the paternally derived X. Diploid parthenogenones have two maternally derived X chromosomes, hence this mechanism cannot operate. To examine whether this contributes to the inviability of parthenogenones, XO and XX parthenogenetic eggs were constructed by pronuclear transplantation and their development assessed after transfer to pseudopregnant recipients. In one series of experiments, the frequency of postimplantation development of XO parthenogenones was much higher than that of their XX counterparts. This result is consistent with the possibility that two maternally derived X chromosomes can contribute to parthenogenetic inviability at or very soon after implantation. However, both XO and XX parthenogenones showed similar developmental abnormalities at the postimplantation stage, demonstrating that parthenogenetic inviability is ultimately determined by the possession of two sets of maternally derived autosomes.     

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae)

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.     

Cleavage rate of diandric triploid mouse embryos during the preimplantation period.

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)     

The first cycle of DNA replication in parthenogenetic mouse embryos

Dynamics of the first cell cycle in parthenogenetic mouse embryos derived from ethanol-activated eggs was studied using 3H-thymidine. DNA synthesis starts within 5 h and is terminated within 10 h after activation: it lasts ca. 6 h. Changes in the intensity of 3H-thymidine incorporation and in the distribution of radioactive label between haploid and diploid parthenogens were observed. 3H-thymidine was shown to incorporate into pronucleoli of diploid parthenogens and late-labeled heterochromatin blocks were bound in both diploid and haploid pronuclei. The structure of the first cell cycle in parthenogenetic and normal embryos is discussed.     

Effect of 5-azadeoxycytidine and retinoic acid on expression of genomic imprinting in parthenogenetic mouse embryos

The action of two types of substances has been studied: 5-azadeoxycytidine and retinoic acid, which have a demethylation effect on DNA in the development process of diploid parthenogenetic mouse embryos. The effect of 5-azadeoxycytidine on hybrid mice (CBA × C57BL/6)F1 in vitro for 6 h, in the presence of single cell parthenogenetic embryos during the S-phase of the cell cycle has been studied. After developing to the blastocyst stage in vitro, parthenogenetic embryos were transplanted into the uterus of false pregnant females. It has been determined that a concentration of 0.1 μM 5-azadeoxycytidine activates embryonic development in the preimplantation period until the blastocyst stage (69% in experiment; 61% in the control) and during postimplantation, it increases the number of available space in the uterus for implantation (76% in experiment; 63% in the control).     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.