Expression of stage-specific genes during zygotic gene activation in preimplantation mouse embryos

The expression of mouse two-cell stage specific genes was studied using the modified DDRT-PCR method, which overcame the paucity of the experimental materials of preimplantation embryos. Embryo tissues equivalent to that of four blastomeres are sufficient for amplification of target genes as visualized using polyacrylamide gel. Sequence analyses and reverse Northern blots indicate that the genes of ATPase 6 and Ywhaz are expressed specifically in two-cell embryos. ATPase 6 is essential for one-cell to two-cell transition and plays an important role in establishment of oxidative phosphorylation, while Ywhaz is related to initiating cellular communication system.     

Molecular origin of mitotic aneuploidies in preimplantation embryos

Mitotic errors are common in human preimplantation embryos. The occurrence of mitotic errors is highest during the first three cleavages after fertilization and as a result about three quarters of human preimplantation embryos show aneuploidies and are chromosomally mosaic at day three of development. The origin of these preimplantation mitotic aneuploidies and the molecular mechanisms involved are being discussed in this review.At later developmental stages the mitotic aneuploidy rate is lower. Mechanisms such as cell arrest, apoptosis, active correction of the aneuploidies and preferential allocation of the aneuploid cells to the extra-embryonic tissues could underlie this lower rate.Understanding the mechanisms that cause mitotic aneuploidies in human preimplantation embryos and the way human preimplantation embryos deal with these aneuploidies might lead to ways to limit the occurrence of aneuploidies, in order to ultimately increase the quality of embryos and with that the likelihood of a successful pregnancy in IVF/ICSI. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

Inhibition of phosphorylated Ser473‐Akt from translocating into the nucleus contributes to 2‐cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis

Phosphorylated Ser473‐Akt (p‐Ser473‐Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2‐cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1‐cell embryos and found that p‐Ser473‐Akt acquired prominent nucleus localization in 2‐cell embryos physiologically. Akt specific inhibitors API‐2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2‐cell arrest at certain concentrations. 2‐cell embryos exposed to 2.0 μmol/L API‐2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p‐Ser473‐Akt in the nucleus. Simultaneously, qRT‐PCR results revealed that 2.0 μmol/L API‐2 treatment significantly downregulated the mRNA pattern of MuERV‐L and eIF‐1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p‐Ser473‐Akt during major ZGA, and Akt specific inhibitors API‐2 and MK2206 which led to 2‐cell arrest inhibited p‐Ser473‐Akt from translocating into the nucleus of 2‐cell embryos with defective ZGA as well, implying p‐Ser473‐Akt may be a potential player in the major ZGA of 2‐cell mouse embryos.     

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94–99%) and blastocyst (90–96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94–98%) and blastocysts (91–93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.     

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.     

Decompaction and recompaction of mouse preimplantation embryos

Summary Early (non-compacted) and late (compacted) 8-cell embryos were observed after few hours of culture in vitro. The former embryos underwent compaction and the latter embryos were found decompacted. Cell counting suggested that decompaction preceded fourth cleavage division of any blastomere and lasted until the blastomeres divided.About one third of mouse morulae, which had about twenty cells, were found non-compacted upon obtaining from females. After few hours of culture in vitro these embryos underwent recompaction and cavitation. Increasing the contributions of mitosis-arrested and cytokinesisarrested cells within the morulae by culture with nocodazole and cytochalasin B respectively, did not delay recompaction.The data show that periods of decompaction and recompaction alternate in preimplantation development.     

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.     

Glycine transport in mouse eggs and preimplantation embryos

In pre-compaction embryos glycine was taken up by the glycine-specific gly-system, which is concentrative, weakly exchangeable and dependent on Na+. After compaction glycine uptake increased, apparently due to the expression of the A-transport system and its reactivity with glycine. Studies of the metabolic fate of carbon from glycine indicated conversion to serine and alanine. These changes are interpreted to show that glycine could provide carbon for intermediary energy metabolism, resulting in CO2, as well as for macromolecular synthesis.     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Short-term rescue by RNA injection of a mitotic arrest mutation that affects the preimplantation mouse embryo

The mutation oligosyndactyly results in syndactyly, abnormal fusion and insertion of certain limb muscles, and diabetes insipidus in heterozygous mice. When homozygous the mutation is lethal; beginning at the blastocyst stage, the homozygous cells arrest in metaphase with intact spindles. The mutant phenotype cannot be corrected by forming aggregation chimeras with wild-type cells, suggesting that the mutation results in a cell autonomous lethal condition. Short-term rescue of the homozygous-induced mitotic arrest can be achieved, however, by cytoplasmic injection of polyadenylated RNA obtained from a rapidly dividing embryo-derived stem cell line.     

In vitro exposure of preimplantation mouse embryos to cocaine and benzoylecgonine inhibits subsequent development.

Cocaine is used by over 20% of women of reproductive age. Although there have been numerous studies focusing on its effects on reproductive processes, none has evaluated its direct effect on preimplantation development. We have investigated the effect of cocaine and its major metabolite, benzoylecgonine, on in vitro preimplantation mouse embryogenesis. One-cell embryos were exposed at the one-, two-, four-, or eight-cell stage for 24 hr to medium containing 0-400 micrograms/ml cocaine or benzoylecgonine and then cultured to the blastocyst stage. Cocaine had its strongest inhibitory effect at the earliest stages of development. At the one- and two-cell stages, there was a significant inhibition of blastocyst formation following exposure to cocaine concentrations of 25-400 micrograms/ml, and at the four-cell stage there was an inhibitory effect at 100 and 400 micrograms/ml cocaine. Benzoylecgonine inhibited the development of embryos to blastocyst only at the one- and two-cell stages, at concentrations of 100-400 micrograms/ml. These findings suggest that cocaine is capable of blocking preimplantation embryogenesis, particularly following exposure at the earliest stages, and that this toxicity may abate as cocaine is biotransformed to benzoylecgonine.